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https://zenodo.org/record/1240751/files/5218aeij01.pdf
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FEASIBILITY STUDY OF ORKNEY'S WINDFARM DEVELOPMENT FOR SELF-SUFFICIENT FOR ENERGY IN ELECTRICITY DEMAND AND ROAD TRANSPORT SECTORS
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Zenodo (CERN European Organization for Nuclear Research)
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Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 KEYWORDS feasibility study, windfarm, Orkney, road transport ABSTRACT In Orkney islands, a number of wind energy projects have been established due to its potential for wind
energy development especially as cluster developments on hilltop and moorland. This Self-sufficient
Orkney Wind Energy (SOWE) project is trying to follow this pattern of development and has designed as a
simple linear development of medium-large scale wind turbines which avoids dominating the landscape,
while providing diversification required for the energy security, along with strong economic benefits to the
local community. The developer completed preliminary designs and landscape assessment, resulting in the
detailed design and the development of 27 wind turbine project for the expectation to generated electricity
of 549.2 GWh annually. he SOWE project has been designed and selected the use of the Enercon E44 - 900 kW and Norex N80 -
5 MW wind turbines. These machines represent the maximum scale, respectively, as; •
the site layout and all modelling have used a 45m, and 60m turbine tower height; •
the site layout and all modelling have used a 45m, and 60m turbine tower height; •
a rotor diameter of 44 m, and 80 m; and •
The blade length is 22 m and 40 m. has been found that the SOWE project provides a promising contribution towards the Scottish
overnment renewable energy production and carbon reduction targets. Prattana Kaewpet1, Colin Bullen2 1Corresponding Author: Department of Electrical Engineering, Faculty of Engineering,
King Mongkut’s Institute of Technology Ladkrabang, Chalongkrung 1 Rd., Ladkrabang,
Bangkok, Thailand, 10520. 2International Center for Island Technology, School of Energy, Geoscience, Infrastructure
and Society, Heriot-Watt Univeristy, Old Academy, Back Road, Stromness, Orkney,
KW16 3AW, Scotland. FEASIBILITY STUDY OF ORKNEY’S WINDFARM
DEVELOPMENT FOR SELF-SUFFICIENT FOR
ENERGY IN ELECTRICITY DEMAND AND ROAD
TRANSPORT SECTORS Prattana Kaewpet1, Colin Bullen2 1.INTRODUCTION Wind energy is the transformation of kinetic energy that is obtained from the moving air. Because
the amount of power and energy output substantially increase as the wind speed increases, the
most cost-effective wind turbines are located in the windiest areas. Wind energy can be used for
either mechanical application: mainly (water pumping) (see Figure 1) or electricity generation. Wind energy has a number of benefits which make it an ideal renewable energy, as can be
described as follows: 1 DOI : 10.5121/aeij.2018.5201 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 • Wind energy is a sustainable form of energy as it is pollution-free. • It does not create greenhouse gases, toxic or radioactive waste. • Any fuel is not required. • Any fuel is not required. Figure 1: The component of wind turbines to pump water for agricultural purpose (DEDE (Department of
Alternative Energy Development and Efficiency), 2018) Figure 1: The component of wind turbines to pump water for agricultural purpose (DEDE (Department of
Alternative Energy Development and Efficiency), 2018) However, producing energy from wind using wind machines can cause some negative effects on
environment such as on wild bird populations and the visual impact on the landscape (Office of
Energy efficiency & Renewable Energy, 2018). Wind turbines may also cause noise and aesthetic
pollution (An-Najah National University, 2018). 1.1 Electricity Transmission Lines and Wind Speed in Orkney A grid connection application will be expected to be accepted by Scottish and Southern Energy,
and it is intended that the turbines will operate under the innovative Registered Power Zone
scheme. There are 11 kV and 33 kV electricity transmission lines in the area. And the turbines are
to be connected to the local distribution system via a switchgear. The average wind speed at the
proposed locations is investigated via http://www.bwea.org/noabl/. In this project, the average
wind speed applied is 9.3 m/s or 20.7 mph according to (Orkney Sustainable Energy Ltd, n.d.)
and (Assessment, 2010). Based on manufacturer’s predictions, 27 wind turbines located on 8 sites
proposed will have a capacity factor around 45% and will produce 549.2 GWh of renewable
capacity per annum equivalent to the Orkney electricity requirement for both electricity and road
transport sectors. Approximately, the production of 268.5 thousand tonnes of carbon dioxide per
annum (Carbon Trust) can be deducted from the production of electricity from renewable
resources. 2 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 1.2 Energy Requirement in Orkney The resource quantified by Aquatera shows that, in the future, Orkney possesses very significant
sources of renewable energy. These sources include a number of wind turbines, in which
technology Orkney has been a pioneer for many years. This is the reason the developer has been
introducing the project to generate electricity from wind power to sustain the electricity
production within year 2030 timeframe. There are 46 of 1 MW units that receive medium level of
acceptability barriers while the current electricity consumption in Orkney is given the number of
around 35 MW at maximum (Orkney Islands Council, 2009). Given these goals, target level of wind energy generation in Orkney can be linked to levels of
both electricity and electrical energy usage for transport sector. The amount of wind energy
capacity would be required to meet these equivalence goals, based on 40% efficiency level
according to the report provided by OIC. The installed capacity of wind energy for electricity use
equivalent would be of 50 MW. With this installed capacity required, the type of electrical
infrastructure required would be Active management of existing grid (Orkney Islands Council,
2009). As the electricity use equivalence of 50 MW does not need new grid connection (Orkney
Islands Council, 2009), it is expected to be achieved in Orkney in a relatively short time scale
with active management of the grid. However, there is additional energy required to meet the
total road transport by establishing a 75:25 mix of electric and hydrogen vehicles and supporting
infrastructures to be considered. Therefore, the new 200 MW grid connection is required. To develop wind farms for electricity production in Orkney (see an example of wind farm in
Orkney in Figure 2), some necessary documents are studied. Based on Scottish National Heritage
(SNH) guidance for strategic planning of wind farms (“Landscape Assessment for Potential
Strategic Wind Energy Development in Orkney,” n.d.), it is recommended that the landscape
capacity for wind energy should be assessed on the basis that a degree of change to landscape
character should be accepted. In general, landscape and visual resources should not be affected,
and some of the more sensitive landscapes should be protected from wind energy development
(“Landscape Assessment for Potential Strategic Wind Energy Development in Orkney,” n.d.). 2.1 Scale of Wind farms Orkney is a remote rural island community, with no gas network, it has over recent decades had a
high dependency on imported oil and coal as its main sources of energy. Orkney suffers high
rates of fuel poverty due to the cool and windy climate as well as the higher costs of fuel. The
costs of fuel in Orkney are higher than in other parts of the UK owning to the transportation costs
involved in delivering. Because of those factors affecting Orkney’s community well-being, a
cost-effective way of harvesting the energy needed from renewable energy is attractive. However,
due to the limitation of the grid capacity, the amount of renewable energy that Orkney can
accommodate will require further upgrade of the grid as mentioned previously (in 1.1). This
section gives an overview of the energy usage in Orkney. Electricity can be imported /exported to
and from Orkney via two 33kV (20 MVA) submarine cables. Renewables such as wind are now
the predominant potential source of electricity in Orkney (ORKNEY.COM, n.d.). Energy use in Orkney can be categorised into three main energy uses (Aquatera Ltd, 2014)
comprising Buildings and Utilities, Transport, and Residual fuel use, which encompasses all
other terrestrial energy. In this windfarm development project, we are interested in the supplying
annual energy requirements which include (1) electricity consumption and (2) energy requirement
in road transport sector by considering their variability. Orkney’s electricity consumption is
equivalent to around 35 MW at maximum. The levels of potential wind energy is very substantial,
which is totally around 256 MW installed capacity with medium and low acceptability from the
community, respectively (Orkney Islands Council, 2009)]. To cover the equivalent electricity
needed in road transport sector which is around 109 GWh (Aquatera Ltd, 2014), the calculation
for installed capacity in addition to the maximum electricity need from the electricity sector (35
MW), the load factor of wind energy is required. Plant load factor is the average hourly quantity
of electricity supplied during the year, expressed as a percentage of the average output capability
at the beginning and the end of year (Department for Business, 2016). Alternatively, it can be
called as annual load factor which is the load factor of generating station over a period of a year
of approximately 8,760 hours (Science Dictionary, 2017). Onshore wind speeds in 2015 in the UK were the highest in the last fifteen years. 2.WIND FARM DEVELOPMENT The Scottish Ministers have set a target of generating 40% (since quantified s 6 GW) of
Scotland’s electricity from renewable source by 2020 and confirmed that this target should not be
regarded as a cap. It is stated that the importance of using clean and sustainable energy from
renewable sources will continue to increase a s a result of global goals to tackle climate change
and the requirement to ensure and diverse energy supplies. As a part of Scotland, Orkney has
been supported by the Scottish Ministers regarding the full range of renewable generation
technologies to realise its considerable wind energy potential. Accordingly, this project will
provide electricity from renewable source, wind energy to support Orkney’s electricity demands
(Series, n.d.). 1.2 Energy Requirement in Orkney Figure 2: Hammars Hill Wind Farm viewed from Evie School (Orkney Sustainable Energy Ltd., 2017) Figure 2: Hammars Hill Wind Farm viewed from Evie School (Orkney Sustainable Energy Ltd., 2017) 3 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 Load factor
L. F.
% =
!"
! #! $! !% & 2.1 Scale of Wind farms This resulted in
the highest onshore wind load factor at 29.4 per cent, and an increase of 3.0 percentage points on
2014, reflecting the higher wind speeds and technological advances in newer sites. (Department
for Business, 2016). But in Orkney, L.F. is assessed to be as high as 0.45 (Assessment, 2010). Assuming the onshore wind load factor at 45% (Assessment, 2010), & ' 100% 4 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 The substitute the number, The substitute the number,
45 = ,
109 x 10 / kWh
the total number of kWh at its maximum continuous rating < ' 100%
the total number of kWh at its maximum continuous rating = ,109 x 10 / kWh
45
< ' 100%
= 242 × 10/ kWh = 242 × 10/ kWh This results in the need of the installed capacity of wind farm for additional equivalent electri p
y
q
y
for road transport of 27.7 MW. To sum up, the island’s equivalent annual electricity requirements
in two key sectors by 2030 – electricity demand and road transport: Energy required from wind
farms = 35 MW + 27.7 MW = 62.7 MW. So, in order to provide the energy by the construction of
sufficient wind farms and appropriate infrastructures, the scale of wind farm suggested would be
around 62.7 MW. 2.4 Determining wind turbine sizes A range of alternative wind turbine models has been considered for the proposed
locations in this project. It is intended that the project should attempt to maximise
generation within the identified constraints, with ecological and visual impact, noise
impact, transportation and grid capacity. It was found that as the equivalent electricity
needed for Orkney is around 62.7 MW, 24 wind turbines of 2.5 MW and 3 wind turbines
of 900 kW have been selected. 2.2 Landscape assessment 2, April 2018 Accordingly, the wind farm developments in each area are recommended in this feasibility study
as follows: •
A single turbine (50-80m) at Low Island Pastures, Golta = 1 turbine of
•
A single turbine (50-80m) at West Flotta Low Moorland =1 turbine of
•
A small array (x3 of 50-80m) at Hoy Moorland Hills, Lyness = 3 turbines of
•
A small array (x3 of 50-80m) at Brough Ness, South Ronaldsay = 3 turbines of
•
A small array (x3 of 50-80m) at South Mainland Moorland Hills = 3 turbines of
•
A small windfarm (x6 of 50-80m) at North Mainland Moorland Hills (Burgar Hill) = 6
turbines of •
A single turbine (50-80m) at Low Island Pastures, Golta = 1 turbine of •
A single turbine (50-80m) at Low Island Pastures, Golta = 1 turbine of
•
A single turbine (50-80m) at West Flotta Low Moorland =1 turbine of
•
A small array (x3 of 50-80m) at Hoy Moorland Hills, Lyness = 3 turbines of
•
A small array (x3 of 50-80m) at Brough Ness, South Ronaldsay = 3 turbines of
•
A small array (x3 of 50-80m) at South Mainland Moorland Hills = 3 turbines of
•
A small windfarm (x6 of 50-80m) at North Mainland Moorland Hills (Burgar Hill) = 6
turbines of
•
A small windfarm (x5 of 125m) at Rothiesholm, Stronsay = 5 turbines of and •
A small windfarm (x5 of 125m) at Rothiesholm, Stronsay = 5 turbines of and •
A small windfarm (x5 of 125m) at Sanday (Loth, Spur Ness) = 5 turbines of •
A small windfarm (x5 of 125m) at Sanday (Loth, Spur Ness) = 5 turbines of 2.3 Project description The average wind speed in different parts of Orkney can be investigated via
http://www.bwea.org/noabl/. In this project, the average wind speed applied is 9.3 m/s or
20.7 mph according to (Orkney Sustainable Energy Ltd, n.d.) and (Assessment, 2010). Based on manufacturer’s predictions, the wind turbines located on these sites will have a
capacity factor of around 45%. The project aims to produce around 549.2 GWh of
renewable electricity per annum. Electricity produced from renewable resources avoids
the emission of pollution, and the production of around 549.2 GWh of wind electricity is
projected to avoid the production of around 268.5 thousand tonnes of carbon dioxide per
annum (Orkney Sustainable Energy Ltd, n.d.). 2.2 Landscape assessment Areas with Greatest Capacity for Wind Energy Development in Orkney identified include
(“Landscape Assessment for Potential Strategic Wind Energy Development in Orkney,” n.d.): •
Moorland Hill (ORK 20) •
Low Moorland (ORK 17) •
Low Island Pastures (ORK 14) •
Undulating Island Pastures (ORK 5) Six areas in Orkney considered the highest underlying capacity for wind energy development
which has been taking into consideration based on a combination of one or more factors including
suitable larger scale simple landforms, landscape patterns, existing development/ land use
affecting character, lower visual sensitivity and lower landscape value (“Landscape Assessment
for Potential Strategic Wind Energy Development in Orkney,” n.d.) are: •
Brough Ness, South Ronaldsay; •
Flotta, Fara and the landscape around Lyness to the south west of Scapa Flow; •
The south facing slopes of the Moorland Hills of West Mainland overlooking Scapa
Flow; •
The northern Moorland Hills of East Mainland; •
Rothiesholm on Stronsay; and •
Spur Ness, southern Sanday. (6a): Undulating Island Pastures According to the study of strategic wind energy development in Orkney: it can be concluded th •
The greatest opportunities of strategic wind energy development in Orkney arise on
Stronsay, south west Scapa Flow and Sanday, with developments of turbines up to 150m
in height potentially acceptable on Stronsay and Sanday. •
The greatest opportunities of strategic wind energy development in Orkney arise on
Stronsay, south west Scapa Flow and Sanday, with developments of turbines up to 150m
in height potentially acceptable on Stronsay and Sanday. •
There is the potential for larger scale strategic developments on the Moorland Hills of
north Mainland. Due to the scale of recommended wind farms (62.7 MW), this project only considers wind
turbines of 50m or greater in terms of their potential to generate electricity in the areas for wind
farm development and their contribution to cumulative issues. In general, turbines with the
dimension of smaller than 50m in height have limited landscape and visual effects. 5 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 3.1 Low carbon vehicle technologies Only the road transport sector energy usage is applied for the calculation of the future
implementation of electric vehicles and fuel cell vehicles that will be required up to year 2030. Using the data from the feasibility study of the possible levels of EVs adoption in Orkney, in
moderate scenario (Lane, Tyne, & Johnstone, 2015), based on an analysis of Orkney’s
population, vehicle ownership trends, and EVs growth projections from previous studies, in
combination with the amount of energy produced for each sectors in road transport sector to
estimate the numbers of EVs that could be adopted in Orkney by 2030. It can be recalculated
from the number of EVs and the energy generated in MWh that for 1 Electric Vehicle will require
around 2.8MWh per annum (2,549 EVs will generate 7,136 MWh). Therefore, as the energy
consumption of EVs is 2.8 times less than that of fossil fuel cars, the total numbers of EVs are
9,674. The details of each type of EVs for different road transport sector are displayed in Tables
3.1- 3.3. The total wind capacity remaining from the use of EVs for road transport sector will be 6 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 around 6MW. In this case, it could be recommended that for the developer to either reduce the
wind capacity or increase the number of EVs. Alternatively, the remaining capacity can be used
to produce hydrogen as a fuel for fuel cell vehicles. around 6MW. In this case, it could be recommended that for the developer to either reduce the
wind capacity or increase the number of EVs. Alternatively, the remaining capacity can be used
to produce hydrogen as a fuel for fuel cell vehicles. 3.1 Low carbon vehicle technologies Table 1: Wind energy required and produced in each sectors of road transport sector in Orkney at the load
factor (L.F.) at 0.45
Sector
Total RE
capacity
(MW)
L.F.*
Energy
produced per
annum (kWh)
Energy required
for EVs (kWh)
Number
of EVs
Domestic
11.64
0.45
45,884,880
16,387,457
5853
Commercial &
Industrial
7.44
0.45
29,328,480
10,474,457
3741
Public
Administration
1.72
0.45
6,780,240
2,421,514
81
total
20.8
9674
* (A
L d 2014) able 1: Wind energy required and produced in each sectors of road transport sector in Orkney at the load
factor (L.F.) at 0.45 Table 2: Numbers of FCVs required to fulfil the need for road transport sector in addition to EVs
Sector
Total RE capacity
(MW)
L.F. Energy produced per annum
(MWh)
Number of
FCVs
FCVs
6.9
0.45
27298
5223 Table 2: Numbers of FCVs required to fulfil the need for road transport sector in addition to EVs To summarise, the numbers of EVs and FCVs that will be required for road transport sectors in
Orkney for all application sectors are equivalent to 9,674 EVs and 5,223 FCVs. Table 3: Conclusion of the number of EVs and FCVs and their energy consumption
Vehicle Types
Numbers of
Vehicles
Energy consumed
(MWh)
Energy
remaining
(MWh)
RE Capacity
remained
(MW)
EVs
9674
29283.4*
52710.17
6
FCVs
5223
9749.4**
17548.9
2
Total
14897
39032.8
70259.1
8 Table 3: Conclusion of the number of EVs and FCVs and their energy consumption * Assumed that Electric Vehicles is 2.8 times higher efficient than Fossil Fuel Vehicles (Lane et
al., 2015)
** Assumed that FCVs is 1.5 times higher efficient than Electric Vehicles (Blamire, 2015) Assumed that Electric Vehicles is 2.8 times higher efficient than Fossil Fuel Vehicles (Lane et
al., 2015)
** Assumed that FCVs is 1.5 times higher efficient than Electric Vehicles (Blamire, 2015) ,
)
** Assumed that FCVs is 1.5 times higher efficient than Electric Vehicles (Blamire, 2015) 3.2 Infrastructure provision A key focus in the promotion of electric vehicles is the provision of recharging infrastructure. Easy access and availability to this infrastructure can influence the convenience and confidence
associated with using the EVs (Lane et al., 2015). The majority of travel in Orkney is short
journeys hence an ideal county to promote and encourage the use of EVs and FCVs due to their
shorter range than comparable petrol/diesel vehicles at present. Existing electric charge points in
Orkney are located at; Kirkwall: East Kirk Car Park, St Rognvald’s House, and Great Western 7 7 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 Road Car Park; St Margaret’s Hope: Braeburn Court Care Home; Dounby: Dounby Primary
School Car Park; and Stormness: Old Academy Business Car Park. Road Car Park; St Margaret’s Hope: Braeburn Court Care Home; Dounby: Dounby Primary
School Car Park; and Stormness: Old Academy Business Car Park. In the future, it is envisaged that a number of charge points will be installed specifically for the
use of commercial vehicles and the remaining units will be placed in accessible locations for
public use. The Council as the Planning Authority will support the inclusion of charge points and
other associated infrastructure for larger developments such as hospitals, schools, leisure facilities
and new housing developments where practicable (Engineering, 2012). Figure 3: Likely split of charging points in Orkney (Engineering, 2012) Figure 3: Likely split of charging points in Orkney (Engineering, 2012) Overnight recharging at home should be seen as the most convenient and cost effective method to
charge an electric vehicle. Future provision of public charge points may, depend on the potential
to realise sufficient revenue from this infrastructure. Due to limitations and constraints on using
charging points or limit of the duration of parking and recharging in some locations, it is
therefore, envisaged that the majority of recharging will be completed at homes and to a lesser
extent workplace areas. The commercial element should be charge points which are especially for
commercial business use either by the local authority or private businesses, as shown in Figure 3. Slow and Fast charge points are more suitable for workplace locations where the EVs can be left
to charge for a prolonged period of time. 3.2 Infrastructure provision EVs for commercial use may require Non-rapid charge
points, in which vehicles are only used at specific times. Rapid charge points provide around 80%
charge in 30 minutes while fast charge points take 3-4 hours to fully charge a battery (see more
details in Figure 4). Rapid charge points are more expensive than fast or slow charge points,
however, it is recommended for key locations of Stromness and Kirkwall in the first instance such
as harbour areas, Kirkwall Airport, key tourist sites, recreational and/or shopping areas. The
numbers of rapid charge points that can be extended will be subjected to external funding and
suitable infrastructure. Overnight recharging at home should be seen as the most convenient and cost effective method to
charge an electric vehicle. Future provision of public charge points may, depend on the potential
to realise sufficient revenue from this infrastructure. Due to limitations and constraints on using
charging points or limit of the duration of parking and recharging in some locations, it is
therefore, envisaged that the majority of recharging will be completed at homes and to a lesser
extent workplace areas. The commercial element should be charge points which are especially for
commercial business use either by the local authority or private businesses, as shown in Figure 3. 8
Figure 4: Charge Point Types (Engineering, 2012) Figure 4: Charge Point Types (Engineering, 2012) 8 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 3.3 Infrastructure requirements Orkney comprises a total area of 990 sq.km. Terrain on the islands is mainly low lying with some
hilly areas and cliffs around the coastal regions. Map of Orkney and their relation to mainland
Scotland is present in Figure 4.6. There are two charging networks that operate across the
Islands: Charge Your Car (CYC) which also includes Charge Place Scotland (CPS), and Zero
Net. Details such as charge point locations, charging network, charger type, and number of
available connectors are summarised elsewhere (Lane et al., 2015) Nevertheless, to expand infrastructure for future increasing use of EVs, factors such as the types
of chargers to be installed, and their advantages and disadvantages need to be considered. For
example, Fast chargers can meet a balance between the lower cost of slow chargers and the
convenience of rapid chargers but they still require a minimum of 3 hours to fully charge and thus
ill require longer parking stay. An additional charging plug and socket configuration of rapid
charging make it more complicated than fast charging. Where Type 1 and Type 2 configurations
cover both slow and fast charging, either CHAdeMO or the CCS configuration are required for
rapid DC charging. Nonetheless, rapid charging still offers a significant advantage in terms of
shorter charging times and is much more suitable for top-up charging to extend journeys, 3.4 Fuel cell vehicle system Although EVs are more technological mature, FCVs are expected to be one of the most promising
technologies for road-transport sectors. To establish the future infrastructure of FCVs filling
station system (see example of wind to hydrogen facility in Figure 5), some key components have
to be studied further as the safety and operational issues are still considerably important. Therefore, it is necessary to consider FCV system components as presented below: •
Fuel Cell Stack: the multicell stack uses hydrogen and oxygen to generate electrical
energy. The number of cells can be varied to provide the desired output; •
Fuel Cell Stack: the multicell stack uses hydrogen and oxygen to generate electrical
energy. The number of cells can be varied to provide the desired output; •
Hydrogen gas storage: the hydrogen storage canisters serve as a vehicle gas tank and are
being fuelled using the hydrogen produced by the hydrogen filling station. The water
absorber is needed to prevent the fuel cell from being flooded with distilled water; •
Fan: the fan help improve the flow of oxygen into the fuel cell stack assisting to regulate
or boost its performance; •
Hydrogen Filling Station: the H2 station comprises a number of double-membrane
electrolysers that utilise electricity from wind energy to decompose water into oxygen
and hydrogen. The hydrogen generated is stored inside the hydrogen canisters and will be
used as a fuel for FCVs; •
Energy Management Centre: this central control unit will allow the system navigation. The navigation could help regulating the vehicle velocity, the electrolyser, as well as data
measurement and acquisition in the initial stages of the infrastructure development. 9 Advanced Energy: An International Journal (AEIJ), Vol. 5, No. 2, April 2018 Figure 5: Wind-to-hydrogen facility at NREL (Gasteiger & Markovic, 2003) Figure 5: Wind-to-hydrogen facility at NREL (Gasteiger & Markovic, 2003) 6. CONCLUSION This work has resulted in the detailed design and the development of 27 wind turbine project for
the expectation to generated electricity of 549.2 GWh annually. The SOWE project has selected
the use of the Enercon E44 - 900 kW and Norex N80 - 2.5 MW wind turbines. A grid connection
application will be expected to be accepted by Scottish and Southern Energy, and it is intended
that the turbines will operate under the innovative Registered Power Zone scheme. There are 11
kV and 33 kV electricity transmission lines in the area, and the turbines are to be connected to the
local distribution system via a switchgear. The average wind speed at the proposed locations is
9.3 m/s. Based on manufacturer’s predictions, 27 wind turbines located on 8 sites proposed will
have a capacity factor around 45% and will produce 549.2 GWh of renewable capacity per annum
equivalent to the Orkney electricity requirement for both electricity and road transport sectors. Approximately, the production of 268.5 thousand tonnes of carbon dioxide per annum (Carbon
Trust) can be deducted from the production of electricity from renewable resources. The total
number of both EVs (75% of energy required) and FCVs (25% of energy required) combined that
are predicted in this work for Orkney Island road users are 14,897 units which can be divided as
for EVs of 9,674 and 5,223 units, respectively. ACKNOWLEDGEMENT I sincerely thank to Colin Bullen, a lecturer at Heriot-watt University Orkney campus, UK, for his
encouragement and suggestions. This report would not have been possible without continuous
moral and technical support from the Ministry of Energy, Thailand, and financial support from
the Chevening, UK. [2] Aquatera Ltd. (2014). Orkney-wide energy audit 2014: Executive Summary. Orkney Renewable
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Suleiman, (July). [11] Office of Energy efficiency & Renewable Energy. (2018). Application of wind energy : 1-
Mechanical application : mainly ( water pumping ) Multi-blade windmill used for water pumping
shown below : 2- [12] Electricity generation :Wind turbine types : 1- Vertical axis wind turbine ( i . e . Darius wind turbine ). [13] ORKNEY.COM.(n.d.).Energy of Orkney Retrieved http://www.orkney.com/invest/sectors/energy [14] Orkney Islands Council. (2009). A Sustainable Energy Strategy for Orkney, (December). [15] Orkney Sustainable Energy Ltd. (n.d.). Hammars Hill Wind Energy Project Technical Description
and Environmental Studies. [16] Orkney Sustainable Energy Ltd. (2017). Hammars Hill Energy. Retrieved March 3, 2018, from
http://www.orkneywind.co.uk/hammars-hill-energy.html Science Dictionary. (2017). What is
ANNUAL LOAD FACTOR? definition of ANNUAL LOAD [17] FACTOR(Science Dictionary).Retrieved March 8, 2017, from http://thesciencedictionary.org/annual-
load-factor/ Series, P. (n.d.). Spp. 11
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Mesothelioma: Do asbestos and carbon nanotubes pose the same health risk?
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Particle and fibre toxicology
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Abstract Carbon nanotubes (CNTs), the product of new technology, may be used in a wide range of
applications. Because they present similarities to asbestos fibres in terms of their shape and size, it
is legitimate to raise the question of their safety for human health. Recent animal and cellular studies
suggest that CNTs elicit tissue and cell responses similar to those observed with asbestos fibres,
which increases concern about the adverse biological effects of CNTs. While asbestos fibres'
mechanisms of action are not fully understood, sufficient results are available to develop
hypotheses about the significant factors underlying their damaging effects. This review will
summarize the current state of knowledge about the biological effects of CNTs and will discuss to
what extent they present similarities to those of asbestos fibres. Finally, the characteristics of
asbestos known to be associated with toxicity will be analyzed to address the possible impact of
CNTs. lated asbestos utilization, the increasing development and
uses of CNTs have triggered concern about their potential
toxicity [4-8]. BioMed Central BioMed Central Received: 28 February 2009
Accepted: 12 June 2009 Published: 12 June 2009 Published: 12 June 2009
Particle and Fibre Toxicology 2009, 6:16
doi:10.1186/1743-8977-6-16 Particle and Fibre Toxicology 2009, 6:16
doi:10.1186/1743-8977-6-16 This article is available from: http://www.particleandfibretoxicology.com/content/6/1/16 © 2009 Jaurand et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2009 Jaurand et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Ac
Review
Mesothelioma: Do asbestos and carbon nanotubes pose the same
health risk?
Marie-Claude F Jaurand*1,2, Annie Renier1,2 and Julien Daubriac1,2 Open Access Received: 28 February 2009
Accepted: 12 June 2009 Introduction Carbon nanotubes (CNTs) have unique chemical and
physical characteristics as a result of their nanostructure. CNTs may be used in a wide range of applications, in
fields as diverse as electronics and medicine [1,2]. Due to
their widespread use, it is important to determine the
safety of CNTs for the protection of ecological systems and
human health. Research to investigate the biological
effects of CNTs is advancing today in order to foresee and
prevent their potentially harmful effects. CNTs have fibre-
like characteristics in terms of their elongated shape,
dimensions and aspect ratio. As particles with at least one
dimension of less than 100 nm, they correspond to High
Aspect Ratio Nanoparticles (HARN) [3]. In light of the
health impact of mineral fibres, especially the fibrogenic
and carcinogenic potency of asbestos fibres, and the
health and socio-economical tragedies caused by unregu- In recent years, several publications have reported the
effects of CNTs. Most studies have concerned animal and
cell responses, focusing primarily on respiratory diseases,
especially the inflammatory effects in the lung. However,
while inhalation is one important probable route of con-
tamination, it must be kept in mind that there are other
relevant routes of exposure. A severe primary cancer,
malignant mesothelioma (MM), has been closely linked
to asbestos exposure [9,10]. Epidemiological and animal
studies have shown that asbestos fibres are not the only
fibres to be associated with a risk of MM development. Epidemiological studies have demonstrated a higher inci-
dence of MM in populations exposed to asbestiform and Page 1 of 14
(page number not for citation purposes) Page 1 of 14
(page number not for citation purposes) Particle and Fibre Toxicology 2009, 6:16 http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 non-asbestos fibres [11-14]. Some manmade vitreous
fibres have caused MM in animal experiments [15]. The
question of whether CNTs might potentially be linked to
MM development justifies further research in this area. Moreover, on the basis of the literature, CNTs have
already shown effects in animals and in cell systems that
are similar to those observed with asbestos fibres
[1,2,5,7]. Two recent studies showed the occurrence of
MM in genetically-modified cancer-sensitized mice and in
conventional Fischer 344 rats exposed to CNTs by intra-
peritoneal or intrascrotal administration respectively
[16,17]. These initial results underline the urgent need for
information to further our knowledge about CNTs' poten-
tial to cause MM. whether purified or used as produced. Introduction The effects of CNTs
have been investigated following in vivo exposure of
rodents, and on several types of cells in culture. Most stud-
ies concerned pulmonary toxicity [1,2,5]. Animal experi-
ments mainly focused on inflammatory responses after
exposure by intratracheal instillation or aspiration, or
intraperitoneal injection. In vitro cell systems with several
types of mammalian cells have been used to study inflam-
matory responses and genotoxicity. A few in vivo and in
vitro studies were related to dermal toxicity, and some in
vitro studies focused on neurons [2]. Toxicity test systems
on procaryotes were also used to assess genotoxicity. Here
our focus will be on respiratory effects. Biological effects on mesothelial cells
In vivo effects on mesothelial cells Biological effects on mesothelial cells
In vivo effects on mesothelial cells
Six recently-published studies concerned CNTs' effects on
mesothelial cells. Three reported findings from animal
experiments and three from cell system studies. One ani-
mal experiment concerned the mesothelial cell inflamma-
tory response and pathological changes after intra-
peritoneal injection [37]. The authors exposed C57Bl/6
mice to four samples of MWCNTs of different sizes and
aggregation states. There was one sample of "short" MWC-
NTs (from NanoLab, Inc; mean diameter: 14.8 ± 0.5 nm;
mean length: 1–5 μm); two samples of "long" MWCNTs
(Long1, from Mitsui & Co.; mean diameter: 84.9 ± 1.9
nm; mean length: 40–50 μm [24% > 15 μm of length];
Long2 from Univ. Manchester; mean diameter: 165 ± 4.7 Page 2 of 14
(page number not for citation purposes) Biological effects of CNTs
Translocation MM is a primary tumour of the serosas caused by the neo-
plastic transformation of mesothelial cells. In populations
exposed to asbestos fibres, MM mainly occurs in the
pleura, and to a lesser extent in the peritoneum and peri-
cardium. MM is considered to be highly specific to asbes-
tos exposure, and is found in from 60% to over 80% of
cases [18-23]. In France, the calculated risk of MM attrib-
utable to occupational asbestos exposure was estimated at
83.2% (95% CI 76.8 to 89.6) in men, and 38.4% (95% CI
26.8 to 50.0) in women [24]. Many studies carried out to
investigate pleural and mesothelial cell response to asbes-
tos fibres have made it possible to reach sound hypotheses
about the mechanism of action of asbestos fibres in neo-
plastic mesothelial cell transformation. Translocation Biodistribution of CNTs after deposition in the lung or via
other routes has been poorly investigated. A translocation
of SWCNTs in various organs has been reported by several
authors [25-29]. In a recent study, MWCNTs deposited by
intratracheal instillation in rats revealed clearance due to
macrophage uptake and the lymphatic system without
evidence of crossing the pulmonary barrier, six months
after instillation [29]. It can be noted that macrophage
and lymphatic clearance was also demonstrated following
administration or exposure to asbestos fibres [30-33]. Erdely et al. [30] suggest that the release of soluble inflam-
matory factors could circulate to the vascular blood com-
partment after lung deposition of CNTs. The release of
circulating factors must be taken into consideration to
account for fibre effects. While asbestos fibres have been
detected in the pleura, soluble molecules could also
account for the pleural response [34], and genotoxicity
may be due to clastogenic factors [35,36]. Additional
studies are needed to determine the pharmacokinetics of
CNTs. Regarding the numerous varieties of CNTs associ-
ated with a broad scale of physical and physico-chemical
properties, fundamental studies will be necessary to estab-
lish the parameters leading the translocation process. The aim of the present review is to explore whether our
knowledge of the mechanism of action of asbestos fibres
could offer a useful paradigm to provide a warning or pre-
dict the risk of CNTs, to interpret data on animal and cel-
lular responses, and to evaluate their potential health
effects. For the purposes of our discussion, we consider
three points: (i) the fate of asbestos fibres following expo-
sure; (ii) their effects on mesothelial cells and the biolog-
ical mechanism associated with the cell response; (iii) the
nature of the fibre parameters involved in the harmful
effects, and their similarities with CNT characteristics. We
begin with a summary of current knowledge on the toxi-
cology of CNTs, then look at asbestos fibres' mechanisms
of action, focusing on carcinogenic effects at the pleural
level. Finally, we address the similarities between asbestos
and CNTs. Effects on mesothelial cells in vitro Effects on mesothelial cells in vitro
To the best of our knowledge, four studies have reported
in vitro effects on mesothelial cells. DNA breakage and
DNA repair were found in both human normal and
malignant mesothelial cells exposed to SWCNTs, as well
as cell activation via AP-1, NF-κB and Akt [44]. Another
study concluded that there was alteration of cell viability
and decreased cell proliferation in human mesothelioma
cells exposed to SWCNTs [45]. Three studies reported
cytotoxicity on human normal mesothelial cells, malig-
nant mesothelioma cell line, and on largeTSV40-trans-
formed mesothelial cells (Met-5A) [44,46,47]. It is
noteworthy that the same raw CNT material with different
degrees of dispersion exerted different cytotoxicity on a
human mesothelioma cell line [47]. In this study, the tox-
icity of CNT-bundles (well-dispersed material with a bun-
dle diameter of around 20 nm) was less than that of CNT-
agglomerates (densely roped aggregates with a rope diam-
eter in the micron-range). CNTs appear to be taken up by
different cell types and diverse in vitro effects have been
associated with CNTs uptake [2,45]. However, the cellular
uptake of CNTs is controversial. Both absence and signif-
icant uptake have been reported, as recently discussed [1]. Uptake is likely dependent on interactions between cellu-
lar receptors and cell surface functions, and CNTs surface
reactivity. A variety of cell surface functions may be found,
depending on the cell type. CNTs may also carry diverse
reactive groups. Different sorts of chemicals and biologi-
cal molecules are currently used to disperse CNTs that
may modify the CNTs surface. Hence cell-CNT interac-
tions are dependent on a number of intrinsic and extrinsic
parameters. It should be recalled that modification of the
surface of asbestos fibres modulates the cell responses
[48-52]. In macrophages, the scavenger receptor with col-
lagenous structure (MARCO) seems to play an important
role in pulmonary damage induced by inorganic particles
[53] and may be involved in interaction between MWC-
NTs and plasma membrane of macrophages [54]. In mes-
othelial cells, integrin receptors were reported to interact
with asbestos fibres [50,55]. Recently, no particle internal-
isation was evidenced in largeTSV40-transformed mes-
othelial cells (MeT-5A) exposed to MWCNTs, despite
cytotoxicity [46]. Further studies are necessary to clarify A long-term study was performed by Takagi et al. [17] who
inoculated MWCNTs (MWCNTs-7 from Mitsui; diameter:
100 nm; length: 27% > 5 μm) in the peritoneal cavity of
C57Bl/6 p53+/- mice. Page 3 of 14
(page number not for citation purposes) Toxicology of CNTs
Context of toxicological studies on CNT gy
Context of toxicological studies on CNT Various kinds of CNTS have been the focus of toxicologi-
cal studies. CNTs are heterogeneous in terms of their
structure, impurities and physico-chemical properties. Both single-walled (SWCNTs) and multi-walled (MWC-
NTs) CNTs have been examined in toxicological studies,
including commercial and laboratory-made CNTs, Page 2 of 14
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and one sample of more tangled MWCNTs (from
NanoLab, Inc.; mean diameter: 10.4 ± 0.3 nm; mean
length: 5–20 μm), as well as carbon black. At the same
time, two samples of amosite fibres were tested; these
were short fibres (4.5% > 15 μm of length) and long fibres
(50.4% > 15 μm of length) known to be differently path-
ogenic in rodents. In prior experiments, inhalation and
intraperitoneal exposure in rats to long amosite fibres
revealed greater pathogenicity than short fibres in terms of
fibrosis and cancer [38,39]. In the study reported by
Poland et al. [37], inflammation was assessed after injec-
tion of 50 μg of MWCNTs/mouse, after 24 h and seven
days. The end points were quantification of inflammation
in peritoneal lavage and histology of diaphragm. Only
long samples of MWCNTs and of amosite produced
inflammation and granulomas. Histological analyses
revealed the occurrence of "frustrated phagocytosis" by
macrophages. These results thus demonstrated some sim-
ilarities between the responses to the long forms of
amosite and MWCNTs. Several of the effects of asbestos
were also found with CNTs. There were higher inflamma-
tory responses with samples of long fibres. Only the sam-
ples that contained long fibres caused granulomas and
"frustrated phagocytosis". controls and 7 UICC crocidolite-treated rats (470 μg/rat)
were also studied. The overall incidence of mesotheliomas
was 86% in MWCNT-treated rats while no mesothelioma
was found in vehicle- or crocidolite-treated rats. This
method of exposure of mesothelial cells is not usually
used to assess a carcinogenic potency of fibres. However,
injury at the scrotal mesothelium is used as a method to
investigate the repair mechanism of peritoneal mesothe-
lium [42,43]. Further data are clearly needed to improve
our knowledge of the effects of these MWCNTs on mes-
othelial cells in vivo. Effects on mesothelial cells in vitro Because these mice have a mutation
in one allele of the Trp53 gene, they are prone to develop
cancer. Crocidolite fibres were inoculated as positive con-
trol. Mesotheliomas were found after exposure to both
MWCNTs and crocidolite. This study has been discussed
on several points, including concern about the type of
mice, inappropriate exposure methods, high exposure
dose, underestimation of the number of particles of
MWCNTs and poorly-illustrated histology [40,41]. Details can be found in the different papers but some of
the authors' replies can be summarized here. It is recog-
nized that Trp53+/- mice are more prone to develop can-
cers, and that the response using high doses by the
intraperitoneal route of exposure provides different infor-
mation regarding hazard potency. However, spontaneous
excess of mesotheliomas has not been reported in this
type of mice, and the injection method is applicable to the
hazard approach for mesothelial cells in the absence of
human data. Concerning the dose, the authors mentioned
that other experiments using lower doses are in progress,
giving similar responses [40]. More recently, MWCNTs-7
were administered by a single intrascrotal injection in 7
Fischer 344 rats (240 μg/rat) maintained for an observa-
tion period of 52 weeks [16]. Dimensions were 82% of the
MWCNTs with a diameter between 70–110 nm, and
72.5% between 1–4 μm in length. Five vehicle-treated Page 3 of 14
(page number not for citation purposes) Page 3 of 14
(page number not for citation purposes) http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 Particle and Fibre Toxicology 2009, 6:16 or rats exposed via intratracheal instillation or inhalation. Several reviews may be consulted for more details [1,2,4-8]. Some recent data are summarized in Table 1[30,56-59]. these controversial results, as fibre internalisation is an
important process accounting for the adverse cellular
effects of particles, and more data are needed to determine
the interactions of CNT with mesothelial cells. Regarding the large applications of CNTs and the known
adverse effects of fine particulate matter, the potential
effects of CNTs have also been investigated on systems
other than respiratory [1,2]. A recent study suggests that
deposition of both SWCNTs and MWCNTs produce a sys- Several studies have investigated the inflammatory
response provoked by CNT exposure, conducted on mice Table 1: Summary of recent in vivo experiments carried out with CNTs
Type of CNT
System
Summary results
Reference
SWCNTs
(Carbon nanotech Inc. Tx). Fate of asbestos fibres Both in vivo and in vitro effects of CNTs suggest a possible
genotoxic effect, related to inflammatory responses and
production of reactive oxygen species, as persistent
inflammation is considered to increase the carcinogenic
risk [60]. Regarding industrial uses and commercial applications of
asbestos fibres, the main risk of contamination is linked
to the inhalation route. In general, particle deposition
depends on aerodynamic considerations. Several authors
have studied the mechanism of fibre deposition and
retention in the lungs [75-78]. Once deposited in the
lung, asbestos fibres may be translocated into different
organs and tissues, including the pleura. This was demon-
strated in animals following inhalation or intratracheal
deposition [79], and in humans by investigation of fibre
retention in different body compartments including the
pleura [80-82]. A recent paper discusses the translocation
pathways of asbestos fibres to the pleura [83]. Transloca-
tion appears to be due to trans-cell migration and lym-
phatic circulation. These authors propose that fibres
deposited in the alveolar space can be translocated to the
interstitium, down the gradient of physiological water
absorption. This transfer is facilitated when the epithelial
layer is damaged. Once in the interstitium, fibres can be
distributed to different organs. Fibres can be cleared from
the interstitium via the lymphatic system and enter the
capillaries as inflammation increases the interstitial pres-
sure, allowing the fibres to migrate and be distributed
throughout the whole body. Therefore, fibres can reach
the pleura via the capillary system and transfer through
the visceral pleura. The parietal pleural has pores of rela-
tively large diameter (about 150–200 nm), and the pleu-
ral fluid drainage goes through stomatas where particles
are found to be concentrated. In vivo, a mutation of the K-ras oncogene was observed in
mice exposed to SWCNTs by inhalation, and chromo-
somal aberrations were detected in type II pneumocytes
after intratracheal deposition of MWCNTs in mice
[58,61]. Several in vitro studies have reported a genotoxic
potency
using
different
cell
types
(Table
2)
[44,46,54,57,61-70]. Activation of DNA repair processes
and mutagenesis of the adenine phosphoribosyl trans-
ferase gene was found in mouse embryonic stem cells
[69]. Genotoxicity as assessed by the cytokinesis-block
micronucleus test, was found in rat lung epithelial cells
exposed to MWCNTs [57]. Micronuclei formation
occurred in human epithelial cells (MCF-7) treated with
MWCNTs, and a pancentrometric probe analysis demon-
strated both chromosome breakage and chromosome loss
[61]. Fate of asbestos fibres DNA damage was also reported in SWCNT-treated
mouse embryo fibroblasts and in CNT-treated bronchial
epithelial BEAS 2B cells [63,67]. No mutation or DNA
breakage was found in a FE1-Mutatrade markMouse lung
epithelial cell line exposed to SWCNTs but purine oxida-
tion was detected with the Comet assay [71]. Investigations of the mutagenic potency of MWCNTs
using bacterial test systems did not reveal mutagenic activ-
ity [72,73]. These bacterial assays may not be fully rele-
vant to evaluate genotoxicity of particles. Previous results
with bacterial cells were generally not or only moderately
positive with asbestos fibres [74]. Translocation of CNTs to the pleura can be assumed, as
asbestos fibres are not the only particles to be translocated
to this site. Migration was observed after inhalation of
refractory ceramic fibres and NMVF10a fibreglass in ham-
sters and rats, and anthracotic areas ("black spots") con-
taining particulate matter are present in human pleura
[81,84-87]. One important point for the study of CNT tox-
icity is therefore to determine their ability to be distrib-
uted in the body and to reach the pleura. It is likely that
the CNT aggregation state will modulate the rate of trans-
location. Recent experiments comparing inhalation and
tracheal or pharyngeal deposition of CNTs concluded that
the different effects were likely related to a difference in
the dispersion and aggregation state of the CNTs [58,59]. It can also be assumed that the CNT pre-treatments used
for particle dispersion will also influence the biodistribu-
tion of these particles. Moreover, it must be kept in mind
that CNT exposure takes place via routes other than inha-
lation, which ought to be investigated. temic response, which may affect the cardiovascular sys-
tem [30]. temic response, which may affect the cardiovascular sys-
tem [30]. involve "frustrated phagocytosis." The biopersistence of
fibres is linked to both their dimensions and stability in
the biological milieu. http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 Particle and Fibre Toxicology 2009, 6:16 temic response, which may affect the cardiovascular sys-
tem [30]. Effects on mesothelial cells in vitro Ø: 0.8–1.2 nm
L: 0.1–1 μm
Pharyngeal deposition in C57Bl/6 mice lung (40
μg/mouse). Observation 4 hours post exposure. Gene expression in lung and blood:
Upregulation of genes involved in
inflammation, oxidative stress, coagulation,
tissue remodeling. Increased percentage of
polymorphonuclear leucocytes (PMN) in
blood and bronchoalveolar lavage (BAL). [30]
SWCNTs. 240 nm
(mode, aerodynamic diameter; in
number)
Inhalation (4 days) in mice – 5 mg/m3
Short and mean term responses (1, 7, 28 days)
Lung analysis: Inflammation – Granulomas –
Fibrosis – Mutation of K-ras
[58]
4.2 μm
(mode, aerodynamic diameter; in
mass)
Laryngeal deposition (10 μg/mouse). Short and mean term responses (1, 7, 28 days)
Lung analysis: Inflammation – Granulomas –
Fibrosis -
No mutation of K-ras. Lower effects
compared to inhalation. MWCNTs. Ø: 40–60 nm
L:0.5–500 μm
Intratracheal deposition in rats. One to 7 mg/kg. Short/mean term responses
(1 to 90 days)
Inflammation; dose-dependent thickening of
the alveolar lining
Particles still present after 3 months
[56]
MWCNTs grinded, unheated,
heated to 600°C, 2400°C; 2400°C
then grinded. Ø: 20–50 nm
L: 0.7 ± 0.07 μm
Intratracheal deposition in rats, 2 mg/rat. Short-
term response (3 days); mean-term (60 days)
Inflammation (3 days). Granulomas (60
days). Effects of heated CNTs lower than
unheated. Grinding restored the effects. [57]
MWCNTs. Ø: 40–60 nm
L:0.5–500 μm
Intratracheal deposition in rats. One to 7 mg/kg. Short/mean term responses (1 to 90 days)
Inflammation; dose-dependent thickening of
the alveolar lining
Particles still present after 3 months
[56]
MWCNTs
(Mitsui & Co., LDT)
Ø: ≅ 80 nm
L: 10–20 μm
Pharyngeal deposition in C57Bl/6 mice lung (40
μg/mouse). Observation 4 hours post exposure. Gene expression in lung and blood:
Upregulation of genes involved in
inflammation, oxidative stress, coagulation,
tissue remodeling. Increased percentage of
polymorphonuclear leucocytes (PMN) in
blood and bronchoalveolar lavage (BAL). [30]
MWCNTs
Shenzhen nanotech
Ø: 500 nm;
L: 10 μm
Inhalation (≈ 32 mg/m3) in mice for 5, 10, 15 days;
deposition ≈ 0.07, 0.14; 0.24 μg/mouse. Short-
term response (8, 16, 24 days)
Small aggregates entering the alveolar wall
Cell proliferation and thickening of alveolar
walls
[59]
Tracheal deposition: 50 μg/mouse
Eight and 16 days: clumps deposited on
lining wall of bronchi, no inflammation – 24
days: inflammation. Clumps in the alveoli destruction of alveolar
structure Table 1: Summary of recent in vivo experiments carried out with CNTs Asbestos fibres' mechanism of action
The asbestos legacy Numerous publications on the mechanism of action of
asbestos fibres have emphasized several responses associ-
ated with the mechanism of toxicity at the serosal level. They make it clear that two aspects must be considered:
the biological response and the particle status. The first
depends on several factors that include the fate of asbestos
fibres following inhalation, i.e., their ability to reach the
pleura. It is well know that deposition, clearance and
translocation of fibres are dependent on biological mech-
anisms and partners (mucociliary transport, phagocytic
cells), but also on fibre parameters, especially fibre
dimensions. Short fibres are more easily internalised by
macrophages than long fibres, and long fibres possibly Page 5 of 14
(page number not for citation purposes) http://www.particleandfibretoxicology.com/content/6/1/16 Particle and Fibre Toxicology 2009, 6:16 http://www.particleandfibretoxicology.com/content/6/1/16 Table 2: Summary of recent in vitro experiments carried out with CNTs
Type of CNT
System
Summary results
Reference
SWCNTs (HiPco), (CNI Inc.). Ø: 0.4–1.2 nm
L: 1–3 μm
Lung hamster fibroblasts (V79)
Cytotoxicity (time and dose dependent)
DNA breakage (comet assay)
No significant enhancement of micronuclei
[62]
SWCNTs (50% SWCNT, about 40%
other nanotubes). Ø: 1.1 nm, L: 0.5–100 μm
BEAS 2B human bronchial epithelial
cells
Dose-dependent decrease in cell viability. Dose-
dependent DNA damage. No formation of
micronuclei
[63]
SWCNTs (NIST)
Ø: 1.4 nm, L:2–5 μm
Normal human mesothelial cells and
human mesothelioma cell line
Cell death. DNA lesions
Stress response activation
[44]
SWCNTs. Folate conjugated. Ø: 1–3 nm, L: 100 – 200 nm
HepG2 cells (express folate receptor)
No toxicity if < 50 μg/ml. Dose-dependent
apoptosis. Kinetics of SWCNT internalisation: Mb →
cytoplasm → extracellular
[64]
SWCNTs (HiPco)
Human lung epithelial cells A549 and
immortalised NHBE
Decreased inflammatory response in TNF alpha-
stimulated cells
[65]
SWCNTs Mitsui & Co., Ltd
Size unspecified
Human aortic endothelial cells
Internalisation: CNTs identified in the cytoplasm. Cytotoxicity. IL-8 release. Actin filament and
Ecadherin disruption. Reduced tubule formation. [66]
SWCNTs
Mouse embryo fibroblasts
Low cytotoxicity. DNA damage (comet assay)
Oxidative stress
[67]
MWCNTs. Ø: 67 nm
Mouse macrophages (J774.1). No MAPKs activation; no apoptosis. Interaction with membrane receptors (MARCO) and
plasma membrane destruction
[54]
MWCNTs. Ø:11.3 nm
L:0.7 μm
Human epithelial cells (MCF-7)
Chromosomal aberrations (micronuclei) showing
chromosome breakage and loss of whole
chromosomes
[61]
MWCNTs (C100, Arkema). Ø: 12 nm,
L: 0.1–13 μm
Human epithelial (A549) and Large T
SV40 transformed mesothelial (Met-
5A) cells
Decrease in cell viability (mitochondrial alteration)
without apoptosis. http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 Particle and Fibre Toxicology 2009, 6:16 Gene expression in asbestos-treated mesothelial cells Gene expression in asbestos-treated mesothelial cells A few studies have investigated gene expression in asbes-
tos-treated mesothelial cells using microarray analysis
(Table 3) [109-111]. They confirmed results obtained in
studies focusing on given types of damages. Modulation
of several biological processes were observed. They were
associated with inflammatory, proliferation, DNA repair
and cell adhesion pathways. Further studies comparing
the cell response to CNTs and to the different types of
asbestos fibres are likely to be informative in order to
approach the possible effects of CNTs. Biological and genomic effects of asbestos fibres on
mesothelial cells 100]. These may be due to ROS/RNS production and to
the mesothelial cells' ability to phagocytise asbestos
fibres. Fibre uptake does not abolish the mitotic process as
some fibres are found in dividing mesothelial cells. More-
over, extensive chromosome damage was described. A list
of chromosome abnormalities has been reported by dif-
ferent authors. Asbestos fibres produce structural chromo-
some alterations; significant enhancement of aneuploid
cells, abnormal anaphases and telophases [101-105]. Induction of micronuclei by all types of asbestos in pri-
mary cultures of human mesothelial cells has been
reported by Poser et al. [106]. Other studies have shown
genomic alterations in asbestos-treated human mesothe-
lial cells. Loss of heterozygosity was detected as asbestos-
induced mutations in a human mesothelioma cell line
[107]. Using 3D reconstruction, Cortez et al. recently
reported mitotic abnormalities, centrosome amplification
and aneuploid cell formation in lung carcinoma cells,
even with long periods of recovery post-treatment [108]. These findings are similar to earlier reports concerning rat
pleural mesothelial cells and using less powerful meth-
ods. Inflammation and mesothelial cell activation Many authors have described the inflammatory processes
occurring in the lung and in the pleura, and shown that
fibres can interact with mesothelial cells in culture condi-
tions. Fibre deposition in the lung is followed by the
recruitment of inflammatory cells, which produce several
factors: ROS (reactive oxygen species), RNS (reactive
nitrogen species), clastogenic factors and cytokines that
may stimulate and/or damage neighbouring mesothelial
cells. Fibres also may produce ROS. Moreover, mesothe-
lial cells respond by fibre internalisation according to a
phagocytic process associated with oxidative reactions
[34,88-93]. In this situation, mesothelial cells adapt to the oxidative
environment by oxidative stress, increasing oxidant
defences and decreasing natural ROS and RNS produc-
tion. At the same time, several regulatory pathways are
activated: signalling pathways (MAPKs) associated with
cell proliferation and apoptosis, and DNA repair and con-
trol of cell cycle progression in response to DNA damage
[94,95]. These different reactions are the consequence of
2 types of interactions: between cells (inflammatory cells/
mesothelial cells) and between cells and fibres. As neo-
plastic transformation is linked to genetic damage and
requires proliferation steps, comparison between the gen-
otoxic effects of asbestos and CNTs might provide clues
making it possible to develop hypotheses about the
potential effects of CNTs. Asbestos fibres' mechanism of action
The asbestos legacy No oxidative stress. No
MWCNT internalisation
[46]
MWCNTs grinded, unheated, heated
to 600°C, 2400°C; 2400°C then
grinded. Ø: 20–50 nm; L: 0.7 ± 0.07 μm
Rat lung epithelial cells. Chromosomal aberrations (micronuclei)
Lower effects with 2400°C sample in comparison to
600°C and unheated
[57]
MWCNTs. Ø: 100–200 nm, L:a few μm
Human epithelial cells (A549)
DNA breakage (comets). No oxidative DNA lesions
[68]
MWCNTs
(Tsinghua & Nananfeng, Cine)
Mouse embryonic cells (ES)
P53 activation. Induction of DNA repair. Mutations (adenine phosphoribosyl transferase)
[69]
MWCNTs Mitsui & Co., Ltd
Size unspecified
Human aortic endothelial cells
Cytotoxicity. IL-8 release. Actin filament and
Ecadherin disruption. Reduced tubule formation. [66]
MWCNTs
Human pneumocytes A549
Decrease in cell viability
Internalisation
[70] Table 2: Summary of recent in vitro experiments carried out with CNTs Page 6 of 14
(page number not for citation purposes) Page 6 of 14
(page number not for citation purposes) Surface reactivity Surface reactivity is an important parameter in asbestos-
related effects. The production of ROS and RNS was men-
tioned above. It is interesting to note that some studies
indicate that, in contrast to asbestos, CNTs quench ROS in
an acellular system generating hydroxyl radicals [115]. While asbestos fibres are hydrophilic, CNTs, unless func-
tionalized, are hydrophobic. As a result, CNTs are often
treated with dispersing agents prior to exposing cells or
animals to CNTs suspended in aqueous medium. Histologically, very similar tumours were observed, and
the genomic alterations in the tumour suppressor genes
investigated were very close to those observed in human
MM. These gene are involved in the control of cell cycle
and junction stability. Regarding the function of the gene,
it might be of interest to determine the consequences of
CNT exposure on cell cycle progression and cell architec-
ture. Asbestos fibre characteristics related to disease
If one looks at fibre parameters, several features appear to
be shared by CNTs and asbestos fibres. To compare CNTs
and asbestos fibres in relation to toxic potential, we
should focus on the asbestos characteristics modulating
asbestos toxicity. Shape, size, chemistry and surface reac-
tivity are all related to cell and tissue responses to asbestos
fibres. Asbestos fibres' ability to adsorb biological molecules is
another fibre parameter to take into consideration. Asbes-
tos adsorbs proteins and phospholipids, which has conse-
quences on cell-fibre interactions. An enhancement of
biological effects can be observed (particle internalisa-
tion, cytotoxicity), as well as a reduction of toxicity [116-
119]. Asbestos bodies are structures found in the lung of asbes-
tos-exposed subjects. They consist of an asbestos fibre core
surrounded by a complex coat produced by the cell and
tissue reaction; they are made of apatite mineralization
and protein aggregation (hemosiderin, ferritin). These
structures are more likely formed on amphiboles rather
than on chrysotile. They are not specific to asbestos, as
they have been reported in other fibrous and non-fibrous
particles. It would be of interest to know whether these
structures could be formed on CNTs [120-122]. http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 Particle and Fibre Toxicology 2009, 6:16 depended on the sample, but generally averaged several
micrometers. However, there was a significant range in
length and a small percentage of fibres longer than 10 μm
were generally present. identification of these changes can provide insight into
the molecular mechanism of action of asbestos on mes-
othelial cells. MM cells exhibit frequent alterations in
tumour suppressor genes found at the INK4 locus, and
often the type of alteration is deletions. NF2 is another fre-
quently inactivated tumour suppressor gene in MM cells. Germinal mutations in NF2 are responsible for type 2
neurofibromatosis, but NF2 patients are not prone to
develop mesothelioma. TP53 is mutated less often in MM
cells. identification of these changes can provide insight into
the molecular mechanism of action of asbestos on mes-
othelial cells. MM cells exhibit frequent alterations in
tumour suppressor genes found at the INK4 locus, and
often the type of alteration is deletions. NF2 is another fre-
quently inactivated tumour suppressor gene in MM cells. Germinal mutations in NF2 are responsible for type 2
neurofibromatosis, but NF2 patients are not prone to
develop mesothelioma. TP53 is mutated less often in MM
cells. Chemistry
M
l Metals are considered to be important elements to
account for fibre toxicity. Iron content, either structural or
as contaminant, may be linked to the formation of ROS
and RNS. Depending on the method of production, CNTs
may contain metals as contaminants; moreover, they can
be functionalized to acquire specific properties. Data in
the literature show a wide qualitative and quantitative
diversity of metal contaminants in the chemical composi-
tion of CNT samples, emphasizing the importance of
using well-defined samples for toxicological analyses [7]. To investigate whether genetic alterations in mesotheli-
oma might be relevant to the effect of asbestos fibres, ani-
mal models of human MM are developed. Mesotheliomas
develop following exposure, by intraperitoneal injection,
of hemizygous NF2 mice to asbestos fibres [112,113]. This made it possible to compare characteristics of mouse
and human mesotheliomas. Shape
h CNTs have a thin and elongated shape compatible with a
fibre, according to the WHO fibre definition of a particle
with parallel edges and an aspect ratio (length/diameter)
greater than three. It seems that CNTs are prone to form
aggregates, ropes and clumps, a feature that is not fully
similar to asbestos, which forms bundles of rather well-
organized structures. The length of CNTs may vary, reach-
ing up to several micrometers or longer [7,114]. Accord-
ingly, "frustrated phagocytosis" was observed in cells
engulfing long CNTs [37]. Biopersistence While biopersistence is not an intrinsic particle parameter,
it has received attention for the evaluation of the carcino-
genic potency of manmade vitreous fibres (MMVFs)
[123,124]. Biopersistence in the lung is the result of a
clearance mechanism and the behaviour of fibres in the
biological medium. Clearance depends on particle uptake
by scavenger cells; it is then modulated by the fibre size Genotoxicity Many investigations have focused on DNA damage pro-
voked by asbestos fibres in mesothelial cells. Several stud-
ies have demonstrated different types of DNA damage
(DNA breakage, base oxidation), and perturbation of the
mitotic process [94,95], showing that base oxidation and
DNA breakage (single strand and double strand breaks)
were detected in asbestos-treated mesothelial cells [95- Gene alterations in mesothelioma Epidemiological studies have shown that MM is a conse-
quence of asbestos exposure in a majority of cases [18-24]. This led us to assume that genomic alterations found in
MM could be linked to the effect of asbestos fibres. The Page 7 of 14
(page number not for citation purposes)
Table 3: Summary of in vitro experiments related to gene expression in crocidolite-treated mesothelial cells
System
Summary results
Reference
Human mesothelial cells (LP9/TERT-1) exposed to low and high
concentrations (15 and 75 μm2/cm2 per dish) for 8 or 24 h
Oligonucleotide microarray analysis
ATF3-dependent modulation of inflammatory cytokines and
growth factor production
[109]
Human SV40-immortalized pleural mesothelial (MeT-5A) cells
exposed to 1 μg/cm2 dish for 1–48 h
Oligonucleotide microarray analysis
1 h: upregulation of nucleosome assembly, translational initiation,
transcription, I-kappaB kinase/NF-kappaB cascade, survival
48 h: downregulation of cytoskeletal anchoring, transcription,
survival
[110]
Normal rat pleural mesothelial cells exposed to 5 μg/cm2 dish for
24 h
Oligonucleotide microarray analysis
Induction of fra-1-linked cd44 and c-met expression
[111] Table 3: Summary of in vitro experiments related to gene expression in crocidolite-treated mesothelial cells
System
Summary results
Reference
Human mesothelial cells (LP9/TERT-1) exposed to low and high
concentrations (15 and 75 μm2/cm2 per dish) for 8 or 24 h
Oligonucleotide microarray analysis
ATF3-dependent modulation of inflammatory cytokines and
growth factor production
[109]
Human SV40-immortalized pleural mesothelial (MeT-5A) cells
exposed to 1 μg/cm2 dish for 1–48 h
Oligonucleotide microarray analysis
1 h: upregulation of nucleosome assembly, translational initiation,
transcription, I-kappaB kinase/NF-kappaB cascade, survival
48 h: downregulation of cytoskeletal anchoring, transcription,
survival
[110]
Normal rat pleural mesothelial cells exposed to 5 μg/cm2 dish for
24 h
Oligonucleotide microarray analysis
Induction of fra-1-linked cd44 and c-met expression
[111] Table 3: Summary of in vitro experiments related to gene expression in crocidolite-treated mesothelial cells Human mesothelial cells (LP9/TERT-1) exposed to low and high
concentrations (15 and 75 μm2/cm2 per dish) for 8 or 24 h
Oligonucleotide microarray analysis Oligonucleotide microarray analysis http://www.particleandfibretoxicology.com/content/6/1/16 Discussion CNTs are valuable industrial products with multiple
applications in the field of nanotechnologies, yet legiti-
mate concerns about their potential adverse effects on
human health need to be addressed. The risk of MM, a pri-
mary pleural carcinoma linked to asbestos exposure, must
be examined in light of the physical nature of CNTs,
which are elongated and ultrafine, and the fact that
human beings may be exposed to CNTs through inhala-
tion. While not yet definitive, data are now available pro-
viding information on the pulmonary and cellular effects
of CNTs, which may be compared to those of asbestos
fibres. Moreover, the asbestos fibre characteristics
involved in the toxic processes may be compared to those
of CNTs to determine their similarities. These compari-
sons make it possible to develop hypotheses about com-
mon and different mechanisms of action. A summary of
comparisons between CNTs and asbestos is provided in
Tables 4 and 5. Shape, structure and chemistry Both CNTs and asbestos particles share fibrous morphol-
ogy, and their dimensions are in the same range. CNTs are
manufactured in two main forms, SWCNTs and MWC-
NTs. A SWCNT is a single-layer graphene sheet rolled up
in a cylindrical shape, whereas a MWCNT contains several
layers [125]. The structure of chrysotile presents similari-
ties with MWCNT. CNT samples may have much higher
length than asbestos fibrils and form clumps resulting in
different presentation and tissue penetration. One role of
CNT sample dispersion to modulate biological effects is
suggested by the results reported from in vivo experiments
studying inhalation and intratracheal deposition. Size The diameters of asbestos fibres fall in the nanosize range. If one refers to the dimensions of the UICC samples,
which have been used in a number of animal and cell
studies, the diameter of chrysotile fibres was less than
about 100 nm, and 200 nm for crocidolite. Length Page 8 of 14
(page number not for citation purposes) Page 8 of 14
(page number not for citation purposes) Particle and Fibre Toxicology 2009, 6:16 http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 and toxicity (short particles are eliminated following
uptake by macrophages; cytotoxic particles impair the
process). The behaviour of the fibres is also size-depend-
ent (fibre dimensions govern the mechanism and site of
deposition in the lung), as well as dependent on the fibre
structure and chemistry (these parameters modulate the
stability of the particles in the biological medium). Some
chemical elements may dissolve and reduce fibre strength,
breaking the fibres into smaller fragments. Finally, biop-
ersistence modulates the amount of fibre retained in the
lung and the time it remains in the lung. To date, CNTs
have been considered biopersistent, but further studies are
needed to determine the relevance of this parameter in the
context of human exposure to CNTs [57]. The reader will find in this quoted review additional infor-
mation on other HARN (nanowires, nanorods) and the
proposal for a research strategy to determine the potential
toxicity of HARN. Biodistribution
l
l
b Similarly to asbestos fibres, CNTs may be deposited and
retained in the lung after inhalation. So far, there is no
definitive data on their migration and long-term reten-
tion, and on their translocation to the pleura. Interaction
with mesothelial cells is likely important to account for
asbestos pathogenicity; however, distant effects after dep-
osition in the lung have been reported. As already men-
tioned, CNTs are the subject of scientific interest for a
large number of already mature or potential applications. One paradox is that biological studies with CNTs are
designed to investigate both adverse (exposure to toxic
dust) and beneficial (nanomedicine) effects. These differ-
ent types of studies show that MWCNTs are concentrated
in the lymph nodes after deposition in the lung, and that
functionalized MWCNTs also accumulate in lymph nodes
after subcutaneous injection [29,126]. CNT biodistribu-
tion has been studied following intraperitoneal or intrave-
nous injection in mice. CNTs are distributed throughout
the entire body and cleared via urine excretion. McDevitt
et al. found an accumulation of labelled SWCNTs in the
kidney, liver, spleen and, to a lesser extent, in bone [127]. A paradigm for the health effects of HARN has emerged
from toxicology studies of industrial fibres, including
asbestos. A recent report reviewed state-of-the-art knowl-
edge of the toxicity of asbestos and HARN [3]. This clearly
suggest a community of toxicological features and con-
cern between HARN of different origin and composition. Table 4: Comparison between physical and chemical parameters of asbestos and CNTs
Parameter
Comparison
Shape
Both are elongated particles; fibre shaped. Dimensions
Asbestos fibre diameter: range of 100 nm. Chrysotile fibrils: ≅ 50 nm of diameter. Same order as MWCNTs. Structure
Chrysotile: multi-layered rolled sheets of brucite (MgOH2) and silicon oxide (SiO2). Important aggregation with CNTs, which
may form more entangled bundles, ropes, than asbestos. Chemistry
Different chemistry. Possibility of metal impurities in both asbestos and CNTs. Surface reactivity
Both show sorptive properties to biological molecules. ROS production: no definitive answer for CNTs. Table 4: Comparison between physical and chemical parameters of asbestos and CNTs
Parameter
Comparison
Shape
Both are elongated particles; fibre shaped. Dimensions
Asbestos fibre diameter: range of 100 nm. Chrysotile fibrils: ≅ 50 nm of diameter. Same order as MWCNTs. Structure
Chrysotile: multi-layered rolled sheets of brucite (MgOH2) and silicon oxide (SiO2). Important aggregation with CNTs, which
may form more entangled bundles, ropes, than asbestos. Chemistry
Different chemistry. Biodistribution
l
l
b Possibility of metal impurities in both asbestos and CNTs. Surface reactivity
Both show sorptive properties to biological molecules. ROS production: no definitive answer for CNTs. Table 4: Comparison between physical and chemical parameters of asbestos and CNTs Page 9 of 14
(page number not for citation purposes) Particle and Fibre Toxicology 2009, 6:16 http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 Table 5: Comparison between biological effects of asbestos and CNTs
Cell/tissue response
Comparison
Particle uptake
Demonstrated with both types. Conflicting results with CNTs. Exocytosis found with CNTs, so far not
investigated with asbestos. Cytotoxicity
Both cytotoxic. DNA damage, mutation, gene interaction
Found with both asbestos and CNTs. Transfection
Gene transfer is with asbestos. CNT gene knockdown. Biodistribution
Both types are cleared via the lymphatic system and found in different organs
Inflammation, granulomas, fibrosis
Found with both asbestos and CNTs. Both types show dependence of biological effects with fibre
dimensions: bioactivity of long fibres. Cancer
MM found with both asbestos and CNTs by peritoneal exposure. Table 5: Comparison between biological effects of asbestos and CNTs
Cell/tissue response
Comparison
Particle uptake
Demonstrated with both types. Conflicting results with CNTs. Exocytosis found with CNTs, so far not
investigated with asbestos. Cytotoxicity
Both cytotoxic. DNA damage, mutation, gene interaction
Found with both asbestos and CNTs. Transfection
Gene transfer is with asbestos. CNT gene knockdown. Biodistribution
Both types are cleared via the lymphatic system and found in different organs
Inflammation, granulomas, fibrosis
Found with both asbestos and CNTs. Both types show dependence of biological effects with fibre
dimensions: bioactivity of long fibres. Cancer
MM found with both asbestos and CNTs by peritoneal exposure. Table 5: Comparison between biological effects of asbestos and CNTs There is to date no reason to exclude the possibility of
CNT translocation to the serosa. treatment or when MWCNTs of lower purity were used
(97%) [131]. Acute pulmonary toxicity and genotoxicity
of MWCNTs were reduced upon heating but restored
upon grinding, in relation with surface defects[57] treatment or when MWCNTs of lower purity were used
(97%) [131]. Acute pulmonary toxicity and genotoxicity
of MWCNTs were reduced upon heating but restored
upon grinding, in relation with surface defects[57] In a recent paper, Hankin et al. [128] summarized the
research required into the mechanism of translocation of
nanoparticles across the respiratory epithelium, and the
resulting possible effects in and beyond the lung. The relationship between structure and biological effects The relationship between structure and biological effects
Based on our present knowledge, a comparison between
cell responses to SWCNTs and MWCNTs cannot be estab-
lished. This is partly due to the limited number of investi-
gations carried out with both types of nanotubes in the
same assays. Nevertheless, both types are able to induce
biological responses in one or several cell types, and in the
lung. While studying the biodistribution of MWCNTs fol-
lowing intraperitoneal injection, Guo et al. compared dif-
ferent results obtained with both SWCNTs and MWCNTs
[129]. These authors suggest that toxic responses observed
in the kidney in some studies may depend on whether
CNTs are functionalized, a procedure that may improve
the biocompatibility. Surface reactivity of asbestos fibres has been largely
advanced as a key parameter accounting for their toxicity
in terms of ROS production and sorptive abilities. ROS
production is associated with cytotoxicity, cell activation,
and chromosome and DNA damage. Conflicting data are
found with CNTs, as both production and scavenging of
ROS were described. CNTs are a large family regarding
their method of generation, treatment and functionaliza-
tion. Hence the surface reactivity of CNTs towards biolog-
ical systems will be largely dependent on the type of
nanotubes. This may be maximized by treatments to dis-
perse CNTs prior to use for biological studies. Different
CNTs samples may have more heterogeneous surface
activities than asbestos. Surface functionalization, purity and treatment of CNTs
appear to modulate the biological response, as found in
different studies [115,130,131]. The surface modifica-
tions of CNTs developed in the field of nanomedicine
studies are of interest to learn about interactions between
CNTs and cells or organelles. It is already known that sur-
face changes influences cell responses. Viability of neu-
roblastoma cells was not affected by pure MWCNTs (99%
purity). Viability and proliferation were reduced after acid Biodistribution
l
l
b The
authors provide recommendations to develop research on
translocation and penetration of nanoparticles that take
into consideration the parameters allowing a robust inter-
pretation of the data. Studies carried out with asbestos have demonstrated that
long and thin fibres are more toxic than short fibres, with-
out excluding potential toxicity for short fibres. Limits of
4 μm or 8 μm in length have been proposed, mainly based
on in vivo experiments. CNTs can fulfil these length crite-
ria, and similarly to asbestos, long CNTs were more active
than short CNTs [37]. More data on size-dependent bio-
logical effects of CNTs will be of great interest. Authors' contributions MCJ contributed to the design and drafting of the manu-
script. AR participated in the acquisition of data on asbes-
tos on mesothelial cells and the genetics of mesothelioma
cells. JD analysed the data in the literature on gene expres-
sion. Competing interests The authors declare that they have no competing interests. References 1. Shvedova AA, Kisin ER, Porter D, Schulte P, Kagan VE, Fadeel B, Cas-
tranova V: Mechanisms of pulmonary toxicity and medical
applications of carbon nanotubes: Two faces of janus? Phar-
macol Ther 2009, 121:192-204. 2. ,
2. Helland A, Wick P, Koehler A, Schmid K, Som C: Reviewing the
environmental and human health knowledge base of carbon
nanotubes. Environ Health Perspect 2007, 115:1125-1131. nanotubes. Environ Health Perspect 2007, 115:1125 1131. 3. Tran CL, Hankin SM, Ross B, Aitken RJ, Jones AD, Donaldson K,
Stone V, Trantra R: An outline scoping study to determine
whether high aspect ratio nanoparticles (harn) should raise
the same concerns as do asbestos fibres. Report on project
CB0406 2008 [http://www.safenano.org/Uploads/HARN.pdf]. 4. Stern ST, McNeil SE: Nanotechnology safety concerns revis-
ited. Toxicol Sci 2008, 101:4-21. 5. Kolosnjaj J, Szwarc H, Moussa F: Toxicity studies of carbon nan-
otubes. Adv Exp Med Biol 2007, 620:181-204. 6. Boczkowski J, Lanone S: Potential uses of carbon nanotubes in
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2007, 2:407-410. p
3. Tran CL, Hankin SM, Ross B, Aitken RJ, Jones AD, Donaldson K,
Stone V, Trantra R: An outline scoping study to determine
whether high aspect ratio nanoparticles (harn) should raise
the same concerns as do asbestos fibres. Report on project
CB0406 2008 [http://www.safenano.org/Uploads/HARN.pdf]. 4. Stern ST, McNeil SE: Nanotechnology safety concerns revis-
ited. Toxicol Sci 2008, 101:4-21. 5. Kolosnjaj J, Szwarc H, Moussa F: Toxicity studies of carbon nan-
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6. Boczkowski J, Lanone S: Potential uses of carbon nanotubes in
the medical field: How worried should patients be? Nanomed
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45:379-387. 10. Wagner JC, Sleggs CA, Marchand P: Diffuse pleural mesotheli-
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the manuscript. properties of CNTs are likely to take into consideration in
the occurrence of this physiological process. to CNTs thus appears mandatory to focus clearly on the
safety and health issues. Exposure by inhalation for aero-
solized CNTs must take into consideration both lung and
pleural diseases. Animals and cell systems exposed to CNTs appear to
develop several responses similar to those observed with
asbestos. There is some evidence that CNTs produce
inflammation and mesothelioma when inoculated in the
peritoneal cavity of mice. This result is consistent with
inflammatory potency after inhalation or intratracheal
instillation. Several CNT-exposed mammalian cells
respond in culture conditions by inflammatory reactions
and oxidative stress. Genotoxic potency has been reported
in different cell types, including mesothelial cells, as well
as in studies conducted in vivo. Hence CNTs used in exper-
imental systems fulfil several criteria to elicit tissue injury,
including the mesothelium. Conclusion The link between asbestos effects and mesothelioma has
been attributed to several mechanisms. This link has been
investigated using different animal models and cell types,
and substantial similarities exist in the responses of the
different cell types. Several fibre properties have been
linked to adverse effects. Shape, dimension and surface
reactivity are all important parameters. Reactive species
may produce DNA lesions (base oxidation, breaks); their
origin is related both to fibre surface reactivity and phago-
cytosis. Inflammation contributes to the production of
ROS/RNS. Chromosome damage appears to be of major
importance to account for the significant effects of asbes-
tos. Gene deletions and recombinations might result from
these effects. Integrity of some cell processes may be criti-
cal in the response to asbestos: dynamics of the cell mem-
brane (fibre uptake, cell division) and control of cell
division (check points, chromosome segregation proc-
esses, repair of DNA breakage, cytokinesis). In view of
findings of a much less hazard of short than some long/
non-biopersistent HARN, more information is needed on
the CNTs' characteristics and conditions to which we may
be exposed. Based on available data in the literature and
knowledge of the mechanisms of action of asbestos fibres,
it appears that CNTs may elicit responses that are similar
to those caused by asbestos fibres. http://www.particleandfibretoxicology.com/content/6/1/16 http://www.particleandfibretoxicology.com/content/6/1/16 Particle and Fibre Toxicology 2009, 6:16 properties of CNTs are likely to take into consideration in
the occurrence of this physiological process. properties of CNTs are likely to take into consideration in
the occurrence of this physiological process. 15.
IARC: Man-made mineral fibres. IARC Monographs on the Evalua-
tion of Carcinogenic Risks to Humans 2002, 81:1-381. Biological effects Available data in the literature concerning the effects of
CNTs on mesothelial cells remain limited. Several effects
of asbestos fibres, especially genetic damage, are related to
fibre internalisation. While asbestos fibres are clearly
internalized by mesothelial cells, there is no definitive
data on CNT uptake by these cells. The physico-chemical Page 10 of 14
(page number not for citation purposes) Page 10 of 14
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expression in mesothelioma. Cancer Res 2003, 63:3539-3545. 112. Altomare DA, Vaslet CA, Skele KL, De Rienzo A, Devarajan K,
Jhanwar SC, McClatchey AI, Kane AB, Testa JR: A mouse model
recapitulating molecular features of human mesothelioma. Cancer Res 2005, 65:8090-8095. Publish with BioMed Central and every
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BioMedcentral Publish with BioMed Central and every
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scientist can read your work free of charge 113. Fleury-Feith J, Lecomte C, Renier A, Matrat M, Kheuang L,
Abramowski V, Levy F, Janin A, Giovannini M, Jaurand MC: Hemizy-
gosity of nf2 is associated with increased susceptibility to
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of carbon-based nanomaterials for cancer therapy. Expert
Opin Drug Deliv 2008, 5:331-342. 115. Fenoglio I, Greco G, Tomatis M, Muller J, Raymundo-Pinero E, Beguin
F, Fonseca A, Nagy JB, Lison D, Fubini B: Structural defects play a
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otubes: Physicochemical aspects. Chem Res Toxicol 2008,
21:1690-1697.
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1 Moscow Pedagogical State University.
1/1 Malaya Pirogovskaya st., Moscow, 119991, Russia.
2 Immanuel Kant Baltic Federal University.
14 Aleksandra Nevskogo st., Kaliningrad, 236016,
Russia.
Received: 16.12.2020.
doi: 10.5922/0207-6918-2021-2-4 1 Московский педагогический государственный уни
верситет.
119991, Москва, ул. Малая Пироговская, д. 1, стр. 1.
2 Балтийский федеральный университет им. И. Канта.
236016, Калининград, ул. Александра Невского, д. 14.
Поступила в редакцию 16.12.2020 г.
doi: 10.5922/0207-6918-2021-2-4
К
в
й б
2021 Т 40 №2 С 95
130 УДК 1(091) УДК 1(091) Публикация, предисловие и комментарии
Н. А. Дмитриевой1,2 Публикация, предисловие и комментарии
Н. А. Дмитриевой1,2 ФИЛОСОФИЯ ГЕРМАНА КОГЕНА A. L. Sakketti ARCHIVE ARCHIVE АРХИВ A. L. Sakketti А. Л. Саккетти Kantian Journal, 2021, vol. 40, no. 2, pp. 95-130. Предисловие был «переведен на службу в Министерство на
родного просвещения с причислением к оно
му и откомандированием в Ученый комитет
сего Министерства для исполнения обязанно
стей делопроизводителя VII класса. 20 февраля
[1912 г.] произведен за выслугу лет в титуляр
ные советники» (Формулярный список…, 1914). С 1914 г. — приват-доцент, в 1918 г. — профес
сор по теории права и истории политических
учений в Петроградском университете. Даль
нейшая деятельность Саккетти была связана с
Советской Россией: в начале 1920-х гг. он рабо
тал в высших учебных заведениях Костромы и
Иваново-Вознесенска; в 1923 г. был приглашен
в Москву консультантом при Совете народных
комиссаров РСФСР и Наркомате юстиции; с
1923 по 1927 г. был активным членом Философ
ского отделения Государственной академии ху
дожественных наук; начиная с 1930-х гг. много
занимался переводами с немецкого, француз
ского и латинского языков, в частности тракта
тов Гуго Гроция (Савенко, 2002, с. 237—241). Первоначальное название публикуемого тек
ста предположительно — «Жизненный подвиг and courses in legal subjects he attended lec
tures on philosophical and historical sciences
at the History and Philology Department”
(Sakketti, 1914). Furthermore, Sakketti spent
two training periods at Neo-Kantian centres in
Germany: in the summer of 1905 he “attend
ed one semester of lectures at the Heidelberg
University (on state law by Jellinek and on
philosophy by Windelband” (ibid.) and in the
summer of 1910 in Marburg he attended Her
mann Cohen’s lectures on the history of new
philosophy and P. Natorp’s lectures on logic
(cf. Dmitrieva, 2007, pp. 201-202). While pre
paring for his magisterial examinations Sakket
ti also worked at the Governing Senate Office
of the First Department (from 17 December
1908) and on 6 September 1911 was “trans
ferred as staff member to the Ministry of Pub
lic Education and seconded to the Ministry
Academic Council to perform the duties of fil
ing clerk VII class. On 20 February [1912] he
was promoted to the rank of titular council
lor” (Anon., 1914). From 1914 he was privatdoz
ent (senior lecturer) and from 1918 professor
of the theory of law and history of political
doctrines at the Petrograd University. 3 Здесь и далее в цитатах для событий до 1917 г. при
водятся даты по старому стилю. Предисловие The text by the Russian Neo-Kantian Alek
sandr L. Sakketti (Saссhetti, born: 13 (25) Au
gust 1881, St. Petersburg, died: 15 February
1966, Moscow) which is published here was
discovered at the Manuscript Department of
the Russian National (formerly Public) Library
of St. Petersburg in the Ernest L. Radlov collec
tion. It is an authorised typewritten copy dated
8 July 1918 (Sakketti, 1918). Публикуемый текст русского неоканти
анца Александра Ливериевича Саккетти
(13[25].08.1881, Санкт-Петербург — 15.02.1966,
Москва) был обнаружен в Отделе рукописей
Российской национальной (ранее — публич
ной) библиотеки Санкт-Петербурга в фонде
Э. Л. Радлова и представляет собой авторизо
ванную машинописную копию, датирован
ную 8 июля 1918 г., объемом 30 л. увеличенно
го формата (35,0 × 22,0) (Саккетти, 1918). y
(
)
Aleksandr Sakketti was the son of Live
rii Sakketti, well-known in the early twenti
eth century as a music scholar and professor
at the St. Petersburg Conservatory. In 1901
A. Sakketti finished the First Classic Gymnasi
um in St. Petersburg and enrolled at the Law
Department of the St. Petersburg University. Upon graduating with a First Class Diploma
in 1908 he was retained at the State Law Sec
tion to prepare for a professorship (cf. Saven
ko, 2002, pp. 232-233). During his studies at
the university Sakketti “was a member of the
law philosophy circle conducted by Professor
Petrazhitsky and took part in practical classes
in the logic of social science under Academy
member A. S. Lappo-Danilevsky (theoretical
philosophy of Kant and Hegel and practical
philosophy of Kant). In addition to lectures А. Л. Саккетти — сын известного в нача
ле ХХ в. музыковеда, профессора Петербург
ской консерватории Л. А. Саккетти. В 1901 г. А. Л. Саккетти окончил Первую классическую
гимназию в Санкт-Петербурге и поступил на
юридический факультет Санкт-Петербургско
го университета, обучение в котором завершил
в 1908 г. с дипломом 1-й степени и был остав
лен на кафедре государственного права для
приготовления к профессорскому званию (см.:
Савенко, 2002, с. 232—233). В студенческие годы
Саккетти «принимал участие в занятиях круж
ка философии права проф. Л. И. Петражицко
го, членом которого состоял, а также участво
вал в практических занятиях по логике обще
ствоведения академика А. С. Лаппо-Данилев
ского (теоретическая философия Канта и Ге
геля и практическая философия Канта). Кро Kantian Journal, 2021, vol. 40, no. 2, pp. 95-130. Кантовский сборник. 2021. Т. 40, № 2. С. 95—130. А. Л. Предисловие Саккетти ме лекций и курсов по предметам юридиче
ским посещал также лекции по философским
и историческим наукам на историко-филоло
гическом факультете» (Саккетти, 1914). Кро
ме того, Саккетти дважды стажировался в не
окантианских центрах: летом 1905 г. «прослу
шал один семестр лекций в университете в го
роде Гейдельберге (по государственному праву
у Еллинека и по философии у Виндельбанда)»
(Там же), а летом 1910 г. в Марбурге посещал
лекции Г. Когена по истории новой философии
и П. Наторпа по логике (см.: Дмитриева, 2007,
с. 201—202). В годы подготовки к магистерским
испытаниям Саккетти также служил по ведом
ству Министерства юстиции в канцелярии
Первого департамента Правительствующего
сената (с 17 ноября 1908 г.)3, 6 сентября 1911 г. был «переведен на службу в Министерство на
родного просвещения с причислением к оно
му и откомандированием в Ученый комитет
сего Министерства для исполнения обязанно
стей делопроизводителя VII класса. 20 февраля
[1912 г.] произведен за выслугу лет в титуляр
ные советники» (Формулярный список…, 1914). С 1914 г. — приват-доцент, в 1918 г. — профес
сор по теории права и истории политических
учений в Петроградском университете. Даль
нейшая деятельность Саккетти была связана с
Советской Россией: в начале 1920-х гг. он рабо
тал в высших учебных заведениях Костромы и
Иваново-Вознесенска; в 1923 г. был приглашен
в Москву консультантом при Совете народных
комиссаров РСФСР и Наркомате юстиции; с
1923 по 1927 г. был активным членом Философ
ского отделения Государственной академии ху
дожественных наук; начиная с 1930-х гг. много
занимался переводами с немецкого, француз
ского и латинского языков, в частности тракта
тов Гуго Гроция (Савенко, 2002, с. 237—241). Первоначальное название публикуемого тек
ста предположительно
«Жизненный подвиг ме лекций и курсов по предметам юридиче
ским посещал также лекции по философским
и историческим наукам на историко-филоло
гическом факультете» (Саккетти, 1914). Кро
ме того, Саккетти дважды стажировался в не
окантианских центрах: летом 1905 г. «прослу
шал один семестр лекций в университете в го
роде Гейдельберге (по государственному праву
у Еллинека и по философии у Виндельбанда)»
(Там же), а летом 1910 г. в Марбурге посещал
лекции Г. Когена по истории новой философии
и П. Наторпа по логике (см.: Дмитриева, 2007,
с. 201—202). В годы подготовки к магистерским
испытаниям Саккетти также служил по ведом
ству Министерства юстиции в канцелярии
Первого департамента Правительствующего
сената (с 17 ноября 1908 г.)3, 6 сентября 1911 г. Предисловие Sakket
ti’s further activities were associated with So
viet Russia: in the early 1920s he worked at
the institutions of higher education in Kostro
ma and Ivanovo-Voznesensk; in 1923 he was
invited to Moscow to work as adviser to the
RSFSR Council of People’s Commissars and
at the People’s Commissariat of Justice; from
1923 until 1927 he was an active member of the
Philosophical Department of the State Acade
my of Art Sciences; beginning in the 1930s he
translated copiously from German, French and
Latin, including the treatises of Hugo Grotius
(cf. Savenko, pp. 237-241). The supposed initial title of the text here Первоначальное название публикуемого тек
ста предположительно — «Жизненный подвиг
Германа Когена»: первые два слова в названии
вымараны чернилами, но распознаются, над The supposed initial title of the text here
published was “The Life Achievement of Her
mann Cohen”. The first two significant words
were blocked out in ink, but are visible. On 96 96 A. L. Sakketti ними вписано слово «Философия». Появление
этого текста, вероятно, связано с работой Саккет
ти в петроградской книгопродавческой и кни
гоиздательской трудовой артели «Наука и Шко
ла», действительным членом правления которой
он был с самого ее основания, то есть с 22 марта
1918 г. (Резниченко, 2017, с. 97). На обороте л. 16
сохранилась пометка от руки: «67/1928 Рукопись
передана в Р[укописный] О[тдел] В. А. Беляевым
по поручению Э. Л. Радлова, 15 августа 1928 г.». Виктор Адрианович Беляев (6[18].01.1883 — по
сле 1953) до революции был приват-доцентом
логики и метафизики Санкт-Петербургской ду
ховной академии, в 1910 г. посещал занятия по
философии в Марбургском университете (Дми
триева, 2007, с. 169), в 1919—1921 гг. был профессо
ром философии Петроградского университета
и членом Философского общества (см.: Шилов,
1999). С 1922 по 1929 г. работал в Публичной би
блиотеке Петрограда (Ленинграда), и, вероятно,
через него профессор философии Петербург
ского (Петроградского, Ленинградского) уни
верситета и в 1918—1924 гг. директор Публич
ной библиотеки Эрнест Львович Радлов (20.11
[2.12].1854 — 28.12.1928) передавал в отдел руко
писей свой архив. С апреля 1918 г. Радлов был
членом той же книгоиздательской артели «На
ука и Школа», что и Саккетти (см.: Резниченко,
2017, с. 97—98), и тесно сотрудничал с Беляевым
не только в рамках своей работы в Публичной
библиотеке, но и в Философском обществе при
Петроградском университете (1921—1922) (см.:
Резвых, 2014, с. 480). top of them is written the word “Philosophy”. Предисловие The appearance of this text was probably con
nected with Sakketti’s work for the Petrograd
book-selling and publishing artel “Science and
School” of which he was a full member of the
board from its foundation on 22 March 1918
(Reznichenko, 2017, p. 97). A surviving hand
written note on the back of page 16 reads:
“67/1928 Manuscript conveyed to M[anu
script] D[epartment] by V. A. Belyaev at the
request of E. L. Radlov, 15 August 1928.” Vic
tor A. Belyaev (6 January 1883 — after 1953)
was before the Revolution a privatdozent (senior
lecturer) of logic and metaphysics at the St. Pe
tersburg Religious Academy, attended philoso
phy lectures at Marburg University (Dmitrieva,
2007, p. 169), in 1919-1921 was a philosophy
professor at the Petrograd University and a
member of the Philosophical Society (cf. Shilov,
1999). From 1922 to 1929 he worked at the Pub
lic Library of Petrograd (Leningrad) and it was
apparently through him that Ernest L. Radlov
(20 November [2 December] 1854 — 28 Decem
ber 1928), professor of philosophy at the Peters
burg (Petrograd, Leningrad) University and
Director of the Public Library, conveyed his
archive to the Manuscript Department. From
April 1918 Radlov was a member of the “Sci
ence and School” book-publishing artel (cf. Reznichenko, 2017, pp. 97-98) and closely col
laborated with Belyaev not only at the Public
Library but also in the Philosophical Society at
the Petrograd University (1921–1922) (cf. Rez
vykh, 2014, p. 480). ними вписано слово «Философия». Появление
этого текста, вероятно, связано с работой Саккет
ти в петроградской книгопродавческой и кни
гоиздательской трудовой артели «Наука и Шко
ла», действительным членом правления которой
он был с самого ее основания, то есть с 22 марта
1918 г. (Резниченко, 2017, с. 97). На обороте л. 16
сохранилась пометка от руки: «67/1928 Рукопись
передана в Р[укописный] О[тдел] В. А. Беляевым
по поручению Э. Л. Радлова, 15 августа 1928 г.». Виктор Адрианович Беляев (6[18].01.1883 — по
сле 1953) до революции был приват-доцентом
логики и метафизики Санкт-Петербургской ду
ховной академии, в 1910 г. посещал занятия по
философии в Марбургском университете (Дми
триева, 2007, с. 169), в 1919—1921 гг. был профессо
ром философии Петроградского университета
и членом Философского общества (см.: Шилов,
1999). С 1922 по 1929 г. работал в Публичной би
блиотеке Петрограда (Ленинграда), и, вероятно,
через него профессор философии Петербург
ского (Петроградского, Ленинградского) уни
верситета и в 1918—1924 гг. 4 Записи лекций и исследовательские материалы Ко
гена считаются утраченными. Предисловие директор Публич
ной библиотеки Эрнест Львович Радлов (20.11
[2.12].1854 — 28.12.1928) передавал в отдел руко
писей свой архив. С апреля 1918 г. Радлов был
членом той же книгоиздательской артели «На
ука и Школа», что и Саккетти (см.: Резниченко,
2017, с. 97—98), и тесно сотрудничал с Беляевым
не только в рамках своей работы в Публичной
библиотеке, но и в Философском обществе при
Петроградском университете (1921—1922) (см.:
Резвых, 2014, с. 480). )
Для какого издания мог предназначать
ся публикуемый текст, установить не удалось. Это один из ранних откликов в России на из
вестие о смерти основателя Марбургской шко
лы неокантианства Германа Когена (ум. 4 апре
ля 1918 г.). Несколько некрологов, вышедших в
апреле 1918 г. в русской периодической печати
(см.: Колеров, 2018), представляли собой в основ
ном лишь краткие заметки в память о немецком
мыслителе. Саккетти же стремился дать доста
точно подробный научно-популярный очерк It is unknown for what publication the text
was intended. It is one of the early reactions
in Russia to the news of the death (on 4 April
1918) of Hermann Cohen, the founder of the
Marburg school of Neo-Kantianism. Several
obituaries, published in April 1918 in Russian
periodicals, were mainly brief notes in mem
ory of the German thinker. Sakketti aimed to
give a detailed popular science review of Co 97 97 А. Л. Саккетти hen’s philosophy, a kind of introduction to his
philosophical system, which was in line with
the tasks of the publishing artel (cf. Reznichen
ko, 2017, pp. 102-103). It is notable that, along
with logic, ethics and aesthetics, Sakketti in
cludes psychology as the fourth part of the sys
tem: during several semesters in Marburg and
then in Berlin, shortly before his death, Cohen
delivered lectures on that discipline, defining it
as “the encyclopaedia of philosophy” and “the
teaching on the unity of cultural conscious
ness” (Dmitrieva, 2007, p. 101).3 However, a
year after Cohen’s death, instead of the book
on psychology, his book on the philosophy of
religion was published — Religion of Reason out
of the Sources of Judaism. Another feature of the
text published here is that Sakketti, in render
ing Cohen’s philosophy, mentions the objec
tions raised by Russian religious philosophers
(see parts I.1 and I.3) and tries to clear up the
“misunderstanding”. философии Когена, своего рода введение в его
философскую систему, что в целом соответ
ствовало задачам издательской артели (см.: Рез
ниченко, 2017, с. 102—103). 3 Cohen’s notes of these lectures and his research
materials on psychology are deemed to have been lost. References Дмитриева Н. А. Русское неокантианство: «Мар
бург» в России. Историко-философские очерки. М. :
РОССПЭН, 2007. Anon., 1914. Formular List of the Service of Alexan
der Liverievich Sakketti, a titular councillor attached to the
Ministry of National Education. Compiled to 17 May 1914. [manuscript] Ministry of Public Education Collection,
733. Inventory 155, Folder 1087, Sheet 92. St. Petersburg:
Russian State Historical Archive (RGIA) (In Rus ) Anon., 1914. Formular List of the Service of Alexan
der Liverievich Sakketti, a titular councillor attached to the
Ministry of National Education. Compiled to 17 May 1914. [manuscript] Ministry of Public Education Collection,
733. Inventory 155, Folder 1087, Sheet 92. St. Petersburg:
Russian State Historical Archive (RGIA) (In Rus ) Колеров М. А. Русский Коген: некрологи 1918 года //
Кантовский сборник. 2018. Т. 37, № 2. С. 58—63. doi:
10.5922/0207-6918-2018-2-4. Russian State Historical Archive (RGIA). (In Rus.) Резвых Т. М. Петербургское философское общество
и журнал «Мысль» (1921—1923): новые документы // Ис
следования по истории русской мысли [10]: Ежегодник
за 2010—2011 год / под ред. М. А. Колерова, Н. С. Плот
никова. М. : Модест Колеров, 2014. С. 479—492. Dmitrieva, N. A., 2007. Russkoe neokantianstvo:
“Marburg” v Rossii. Istoriko-filosofskie ocherki [Russian
Neo-Kantianism: “Marburg” in Russia. Historical and
Philosophical Essays]. Moscow: ROSSPEN. (In Rus.) Kolerov, M. A., 2018. Hermann Cohen: Russian
Obituaries from 1918. Kantian Journal, 37(2), pp. 58-63. http://dx.doi.org/10.5922/0207-6918-2018-2-4. Резниченко А. И. «Симфоническая личность» и
«трудовая артель»: Л. П. Карсавин в 1918 — 1922 гг. //
Гуманитарная наука в России и перелом 1917 года : эк
зистенциальное измерение / под ред. О. А. Довгополо
вой, А. А. Каменских. СПб. : Алетейя, 2017. С. 97—108. Rezvykh, T. M., 2014. Petersburg Philosophical Socie
ty and the Journal “Mysl” (1921—1923): New Documents. In: M. A. Kolerov and N. S. Plotnikov, eds. 2014. Studies
in Russian Intellectual History [10]. Yearbook for 2010—2011. Moskau: Modest Kolerov, pp. 479-492. (In Rus.) Савенко Г. В. Александр Ливериевич Саккетти //
Известия вузов. Правоведение. 2002. № 5. С. 232—241. Саккетти А. Л. Curriculum vitae [1914] // Россий
ский государственный исторический архив (РГИА). Ф. 733. Оп. 155. Д. 1087. Л. 96. Reznichenko, A. I., 2017. “Symphonic Personality”
and “Labour Artel”: L. P. Karsavin in 1918—1922. In:
O. A. Dovgopolova and A. A. Kamenskikh, eds. 2017. Gumanitarnaja nauka v Rossii i perelom 1917 goda: ekzisten
cial’noe izmerenie [Humanitarian Science in Russia and the
Turning Point of 1917: An Existential Dimension]. St. Pe
tersburg: Aletheia, pp. 97-108. 5 Вставка вместо зачеркнутого слова «текущий». References (In Rus.) Саккетти А. Л. Философия Германа Когена [1918] //
Отдел рукописей Российской национальной библиоте
ки. Ф. 626. Д. 126. Формулярный список о службе причисленного к
Министерству народного просвещения титулярно
го советника Александра Ливериевича Саккетти. Со
ставлен по 17 мая 1914 г. [1914] // РГИА. Ф. 733. Оп. 155. Д. 1087. Л. 92. Savenko, G. V., 2002. Aleksandr Liverievich Sakket
ti. Proceedings of Higher Educational Institutions. Pravove
denie, 5, pp. 232-241. (In Rus.) Шилов Л. А. Беляев Виктор Адрианович // Сотруд
ники РНБ — деятели науки и культуры. Биогр. сло
варь. СПб. : РНБ, 1999. Т. 2. С. 91—94. URL: http://nlr.ru/
nlr_history/persons/info.php?id=20 (дата обращения:
25.08.2020). Шилов Л. А. Беляев Виктор Адрианович // Сотруд
ники РНБ — деятели науки и культуры. Биогр. сло
варь. СПб. : РНБ, 1999. Т. 2. С. 91—94. URL: http://nlr.ru/
nlr_history/persons/info.php?id=20 (дата обращения:
25.08.2020). Sakketti, A. L., 1914. Curriculum vitae. [manuscript]
Ministry of Public Education Collection, 733. Inven
tory 155, Folder 1087, Sheet 96. St. Petersburg: Russian
State Historical Archive (RGIA). (In Rus.) Предисловие 98 98 A. L. Sakketti Предисловие Примечательно, что в
качестве четвертой части этой системы, наряду
с логикой, этикой и эстетикой, Саккетти ука
зывает психологию: в течение нескольких семе
стров в Марбурге, а затем в Берлине, незадолго
до своей кончины, Коген читал лекции по этой
дисциплине, определяя ее как «энциклопедию
философии» и «учение о единстве культурно
го сознания» (Дмитриева, 2007, с. 101)4. Но вме
сто книги по психологии через год после смер
ти Когена состоялось издание его книги по фи
лософии религии — «Религия разума из источ
ников иудаизма». Еще одна особенность пу
бликуемого текста заключается в том, что Сак
кетти при рассмотрении когеновской филосо
фии упоминает те возражения, которые были
высказаны в ее адрес русскими религиозными
философами (см. ч. I.1 и I.3), и пытается разъяс
нить возникшее «недоумение». у
В машинописи все зачеркивания и встав
ки сделаны от руки чернилами. Немецкие, ла
тинские и древнегреческие слова и выражения
вписаны от руки, в публикуемом тексте пере
даются курсивом. Прерывистые подчеркива
ния, встречающиеся в машинописи, также пе
реданы курсивом. Кавычки в названиях фи
лософских текстов и в некоторых цитатах, где
они были пропущены, добавлены публикато
ром по умолчанию. Текст написан в дорефор
менной орфографии, публикуется полностью
в современной орфографии и пунктуации. В
публикации сохранены прописные буквы в
словах, которые автору представлялись особо
значимыми, оставлены ударения над отдель
ными словами, поставленные от руки. Много
точия, встречающиеся в тексте, — авторские. Все конъектуры даны публикатором в квадрат
ных скобках. Комментарии автора в отличие от
комментариев публикатора отмечены специ
альным указанием в скобках. g
In the typewritten text all the deletions and
insertions are made by hand in ink. German,
Latin and Greek words and expressions are in
serted by hand and are printed in italics in this
text. Dotted underlining in the typewritten text
is also conveyed in italics. Quotation marks
in the titles of philosophical texts and in some
quotations, where they are missing, have been
added by the publisher by default. The publi
cation retains upper case in the words deemed
particularly important by the author. The text is
published in full. The suspension dots are the
author’s. All conjectures are given in square
brackets by the publisher. The author’s com
ments, unlike those of the publisher, are indi
cated by a special note in brackets. The English
translation omits the publisher’s comments in
dicating the initial variants of corrected and au
thorised words and expressions. 4 Записи лекций и исследовательские материалы Ко
гена считаются утраченными. I. Философия и наука Sakketti, A. L., 1918. Philosophy of Hermann Cohen. [manuscript] E. L. Radlov Collection, 626. Folder 126. 30
sheets oversized (35,0 × 22,0). Moscow: Russian Nation
al Library, Manuscript Department. (In Rus.) 6 Вставка вместо зачеркнутого слова «направления».
7 Вставка вместо зачеркнутого слова «мечи».
8 Далее зачеркнуто: «нечестивого».
9 Вставка вместо зачеркнутого слова «последний».
10 Кн. Е. Трубецкой. Метафизические предположе
ния познания. Изд. автора. М. 1917 г. (примеч. авт.).
11 Далее зачеркнуто: «в краткой посмертной заметке». [1] Shilov, L. A., 1999. Belyaev Viktor Andrianovich. In:
Sotrudniki RNB — deyateli nauki i kul’tury. Biografich
eskii slovar’ [Russian National Library Staff — Figures in
Science and Culture. Biographical Dictionary], Volume 2. St. Petersburg: RNB, pp. 91-94. Available at: Russian
National Library <http://nlr.ru/nlr_history/persons/
info.php?id=20> (Accessed 25 August 2020). (In Rus.) 19185 год, столь обильный крушениями и
утратами, унес в могилу Германа Когена. Угас
выдающийся мыслитель, создатель движения
в области духа, основатель марбургской школы
научного идеализма, духовный вождь религи 99 А. Л. Саккетти 4 Prince Evgeny N. Trubetskoy. Metafizicheskie predpolo
zheniia poznaniia. Opyt preodoleniia Kanta i kantianstva
[Metaphysical Presuppositions of Knowledge. An Attempt
to Overcome Kant and Kantianism]. Author’s publication.
Moscow, 1917. [In Rus.] (author’s note).
5 For an analysis of this book see: Krouglov, A. N., 2016.
Evgeny N. Trubetskoy and Overcoming the Neo-Kan
tian Kant. Russian Studies in Philosophy, 54(5), pp. 408-
421. doi: http://dx.doi.org/10.1080/10611967.2016.129
0417 4 Prince Evgeny N. Trubetskoy. Metafizicheskie predpolo
zheniia poznaniia. Opyt preodoleniia Kanta i kantianstva
[Metaphysical Presuppositions of Knowledge. An Attempt
to Overcome Kant and Kantianism]. Author’s publication.
Moscow, 1917. [In Rus.] (author’s note). I. Philosophy and Science озного протестантства6 в иудаизме. При жизни
Коген являл собою величественный образ под
линного атлета человеческой мысли. В наше
время эпигонов, изобилия всякого рода школ
и направлений, учителей мудрости и профес
соров философии, наставников любомудрия
и мудролюбия Герман Коген, как едва ли кто
другой, воскрешал память о подлинных кори
феях философской мысли. Вокруг имени Коге
на в последнее время кипела борьба, скрещи
вались копья7, звенели клинки. В России про
тив него, как вождя8 «имманентизма», были на
правлены острия полемики блюстителей пра
воверия трансцендентной метафизики, как это
показывает и недавний9 замечательный труд
кн. Е. Трубецкого10, этот полновесный вклад в
сокровищницу философской критики. Все это
показывает, что не улеглись еще страсти вокруг
жизненного подвига Г. Когена, что для него
еще не настало потомство. озного протестантства6 в иудаизме. При жизни
Коген являл собою величественный образ под
линного атлета человеческой мысли. В наше
время эпигонов, изобилия всякого рода школ
и направлений, учителей мудрости и профес
соров философии, наставников любомудрия
и мудролюбия Герман Коген, как едва ли кто
другой, воскрешал память о подлинных кори
феях философской мысли. Вокруг имени Коге
на в последнее время кипела борьба, скрещи
вались копья7, звенели клинки. В России про
тив него, как вождя8 «имманентизма», были на
правлены острия полемики блюстителей пра
воверия трансцендентной метафизики, как это
показывает и недавний9 замечательный труд
кн. Е. Трубецкого10, этот полновесный вклад в
сокровищницу философской критики. Все это
показывает, что не улеглись еще страсти вокруг
жизненного подвига Г. Когена, что для него
еще не настало потомство. ,
[
] (
)
5 For an analysis of this book see: Krouglov, A. N., 2016.
Evgeny N. Trubetskoy and Overcoming the Neo-Kan
tian Kant. Russian Studies in Philosophy, 54(5), pp. 408-
421. doi: http://dx.doi.org/10.1080/10611967.2016.129
0417 [1] The year 1918, replete with disasters and
losses, carried away the life of Hermann Co
hen. An outstanding thinker, creator of a
movement in the realm of the spirit, founder
of the Marburg school of scientific idealism,
the spiritual leader of religious Protestantism
in Judaism passed away. In his lifetime Co
hen was a magnificent image of a true athlete
of human thought. In our time of epigones,
an abundance of various schools and trends,
teachers of wisdom and professors of philos
ophy, mentors of love of wisdom, Hermann
Cohen, perhaps like no one else, brought
back memories of true titans of philosophi
cal thought. In recent times the struggle was
raging, lances were broken and swords were
crossed over Cohen’s name. In Russia, he, as
the leader of “immanentism”, has been the
target of polemical arrows of the guardians
of orthodox transcendental metaphysics, as
witnessed by the recent remarkable work of
Prince Trubetskoy,4 that weighty contribu
tion to the treasure-house of philosophical cri
tique.5 All this shows that passions around the
life achievement of H. Cohen have not subsid
ed and that he has not receded into the past. Коген сошел в могилу маститым семидеся
типятилетним старцем, выполнив в главных
чертах свой жизненный подвиг, т. е. значитель
ную часть системы философии; доныне вышли
в свет три части системы: «Логика чистого по
знания», «Этика чистой воли» и «Эстетика чи
стого чувства». Не напечатана до сих пор послед
няя, четвертая часть системы — Психология. р
Имя Германа Когена, несомненно, принад
лежит истории и будущему, сейчас же при
оценке дéла его жизни неизбежны «ошиб
ки перспективы»: для одних Коген еще не вы
рос во весь свой духовный рост, для иных же,
вследствие близости расстояния, Коген неиз
бежно вырастает чрезмерным гигантом. Попы
таемся же11 воздать должное памяти Когена, не
умаляя его жизненного подвига и не возводя
ему незаслуженного мавзолея... Cohen descended into the grave as a seven
ty-five-year-old Grand Old Man, having largely
fulfilled his life achievement, i. e. producing a
significant part of a philosophical system, three
parts of which have already appeared: The Logic
of Cognition, The Ethics of Pure Will and The Aes 100 A. L. Sakketti Свою научно-литературную деятельность
Коген начал давно, в шестидесятых12 годах ми
нувшего столетия, сотрудничеством с гербар
тианцами Лацарусом и Штейнталем. Тогда же
появились, частью на страницах основанного
последними журнала13, и первые любопытные
работы будущего знаменитого философа, по
священные психологии мифотворчества. [1] Вся
последующая работа его мысли может быть в
известном смысле определена как постепен
ное и настойчивое освобождение, эмансипа
ция от психологизма в познании и преодоле
ние мифа, мифологии в области религии. Ре
шительный и окончательный поворот в обра
зе мыслей и в направлении дальнейшего науч
но-философского творчества Когена связан с
его обращением к изучению и истолкованию
философии Канта, чтó произошло в семидеся
тых годах14 минувшего столетия. thetics of Pure Feeling. The final fourth part of
the system — Psychology — has yet to be pub
lished. The name of Hermann Cohen undoubted
ly belongs to history and the future, but today,
in assessing his life’s accomplishment, “errors
of perspective” are inevitable; for some, Cohen
has yet to attain his full spiritual stature while
for others, at close range, Cohen inevitably be
comes a towering giant. Let us try to give Co
hen’s memory its due without belittling his
life’s achievement or erecting an undeserved
mausoleum to him... Cohen started his scholarly and literary ac
tivities a long time ago in the 1860s, in co
operation with the Herbartians Lazarus and
Steinthal. It was at that time, partly in the pages
of a journal founded by the latter,6 that the first
interesting works of the future famous philos
opher were published, devoted to the psychol
ogy of myth-making. The subsequent work of
his thought can be in some sense described as
gradual and relentless liberation, emancipation
from psychologism in cognition and the over
coming of myth and mythology in the field of
religion. The decisive and final turn in Cohen’s
mode of thought and the direction of his fur
ther scientific and philosophical work occurred
in the 1870s when he undertook the study and
interpretation of Kant’s philosophy. С этого момента Коген становится в ряды пе
редовых вождей и бойцов могучего движения
«назад к Канту», на долгие годы определивше
го судьбы и наложившего неизгладимую пе
чать на ход развития философской мысли по
следних десятилетий XIX века не только в Гер
мании, но также за пределами ее. 6 Zeitschrift für Völkerpsychologie und Sprachwissenschaft
(founded in 1859) (author’s note). 12 Вставка вместо зачеркнутого слова «семидесятых».
13 Zeitschrift für Völkerpsychologie und Sprachwissen
schaft (основан в 1859 г.) (примеч. авт.).
14 Вставка вместо зачеркнутых слов «в половине семи
десятых годов». (
) ( р
)
14 Вставка вместо зачеркнутых слов «в половине семи
десятых годов». [1] С этих пор
Коген неизменно стоит в рядах передовых бой
цов кантианства с тою только разницею, что,
тогда как другие сторонники этого направ
ления, как Риль, Шуппе, Ремке, впоследствии
приблизились к Юму и позитивизму, другие,
как Виндельбанд, Риккерт, Ласк, обратились к
Фихте, пытливая мысль Когена не удовлетво
рялась этими ближайшими этапами развития
европейской философской мысли и, отыскивая
более глубокие источники и корни современ
ного культурного сознания, восходила от Кан
та к лучезарнейшим и подлиннейшим свето
чам мировой мысли — Лейбницу, Декарту и, From that moment onward Cohen joined
the ranks of advanced leaders and fighters of
the powerful “Back to Kant” movement which
determined the fate of, and left an indelible
imprint on, the development of philosophical
thought in the last decades of the nineteenth
century in and outside Germany. Since that
time Cohen was constantly in the front ranks
of the champions of Kantianism with this dif
ference that, while other followers of this trend
such as [Alois] Riehl, [Wilhelm] Schuppe, and 101 А. Л. Саккетти наконец, к величайшему создателю философ
ского идеализма, божественному Платону. наконец, к величайшему создателю философ
ского идеализма, божественному Платону. [Johannes] Rehmke later drew closer to Hume
and Positivism, and others, like [Wilhelm] Win
delband, [Heinrich] Rickert and [Emil] Lask
turned to Fichte, Cohen’s inquisitive mind was
not content with these nearest stages in the de
velopment of European philosophical thought
and, searching for deeper sources and roots of
contemporary cultural consciousness, ascend
ed from Kant to the most radiant and most
genuine luminaries of world thought — Leib
niz, Descartes and, finally, the great creator of
philosophical idealism, the divine Plato. Лозунг «назад к Канту» был соответственно
видоизменен Когеном и заменен более дерзно
венным призывом «назад к Платону». Призыв «назад» и «вспять» вместе с тем зву
чал как призыв «вперед», ибо разыскание исто
рических источников и корней культурного со
знания должно было вместе с тем дать основу
и опору для развития и движения вперед, от
крыть бесконечные дали грядущего. Постепен
но вокруг Г. Когена сосредоточивается группа
союзников, единомышленников, как Пауль На
торп, Август Штадлер, Форлендер, Кассирер,
Гёрланд, из более молодых — Н. Гартман, Ланц
и Гавронский15, образуется философская шко
ла, создается сильное движение, в сфере вли
яния которого оказываются Курд Лассвиц, Ру
дольф Штаммлер и Франц Штаудингер. 7 The last three names are those of Russians who studied
under Cohen in Marburg and later made scholarly
careers in the West: Hartmann in Germany, Lanz in the
USA and Gawronsky in Switzerland. 15 Три последних имени принадлежат выходцам из
России, учившимся у Когена в Марбурге и позднее
сделавшим научную карьеру на Западе: Н. А. Гарт
ман в Германии, Г. Э. Ланц в США, Д. О. Гавронский
в Швейцарии. ц р
16 Было «старо-кантианского», зачеркнуто: «старо-». 17 Далее зачеркнуто: «и объективный».
18 Этого слово внесено от руки.
19 Очевидно, имеются в виду три книги Коге
на — «Kants Theorie der Erfahrung» (1871), «Kants
Begründung der Ethik» (1877) и «Kants Begründung der
Aesthetik» (1889), — посвященные трем кантовским
«Критикам».
20 Н. О. Лосский. Введение в философию. Ч. I. Изд.
2[-е]. СПб. 1915. Стр. 128—231 (примеч. авт.).
21 Союз «и» внесен от руки.
22 Далее зачеркнуто: «и достижения Вечной Истины». [1] Accordingly, Cohen altered the slogan “Back
to Kant” replacing it with a more daring call,
“Back to Plato.” The call “Back” and “Return” simultane
ously sounded as a call “Forward” because the
search for historical sources and roots of cultur
al consciousness had at the same time to pro
vide the basis and support for the development
and forward movement and to open infinite
vistas of what was to come. Gradually, a group
of allies and like-thinking scholars gathered
around Cohen: [Paul] Natorp, [August] Stadler,
[Karl] Vorländer, [Ernst] Cassirer, [Albert]
Görland and, among younger ones, N[icolai]
Hartmann, [Henry] Lanz and [Dmitri] Gawron
sky,7 a philosophical school was formed and a
powerful movement emerged which brought
Kurd Laßwitz, Rudolf Stammler and Franz
Staudinger into its fold. 2 Обращение Когена к изучению и толкова
нию Канта встретило весьма различную, по
рою прямо противоположную и противоречи
вую оценку. Одни видели в нем не более как
«кантианского талмудиста», глоссатора и ком
ментатора трех «Критик» Канта, этих фило
софских дигест кёнигсбергского законодателя
Чистого Разума. Напротив, блюстители кан
тианского16 правоверия усмотрели в толкова
нии Канта Когеном новую ересь и схизму, про
поведь и провозглашение «очищенного» Кан
та, исправленного и дополненного, лишенно
го присущих ему исторически выявленных
свойств, противоречий и недостатков, прида
ющих его образу подлинную конкретность и
живость. 2 Cohen’s turning to the study and interpreta
tion of Kant met with a mixed, sometimes op
positional and controversial assessment. Some
saw him as no more than “a Kantian Talmud
ist”, a compiler of a glossary and commentator
on Kant’s three Critiques, these philosophical 102 A. L. Sakketti Разлад Когена с обычным кантианским тол
кованием и преданием столь значителен, что
такой осторожный17 мыслитель, как Н. О. Лос
ский, в его «Введении в философию» предпо
читает давать своим читателям двоякое изло
жение учения Канта: общепринятое, поддер
жанное господствующим преданием, и дру
гое18, «очищенное» истолкованием Когена, в
его комментарии19 на три «Критики»20. digests of the Königsberg legislator of Pure
Reason. By contrast, the guardians of Kantian
orthodoxy saw Cohen’s interpretation of Kant
as a new heresy and schism, a preaching and
declaration of a “purified” Kant, corrected and
enlarged, devoid of his historically established
properties, contradictions and shortcomings
that lend genuine authenticity and vitality to
his image. i g
So significant is the difference from the tra
ditional Kantian interpretation and legend that
such a cautious thinker as N. O. 8 Apparently this refers to three of Cohen’s books, Kants
Theorie der Erfahrung (1871), Kants Begründung der Ethik
(1877) and Kants Begründung der Aesthetik (1889), dealing
with the three Kantian Critiques.
9 N. O. Lossky. Vvedeniye v filosofiyu [An Introduction
to Philosophy]. Part I. 2nd edition. St. Petersburg, 1915,
pp. 128-231. [In Rus.] (author’s note). 17 Далее зачеркнуто: «и объективный». [
]
р
21 Союз «и» внесен от руки. ру
22 Далее зачеркнуто: «и достижения Вечной Истины». 8 Apparently this refers to three of Cohen’s books, Kants
Theorie der Erfahrung (1871), Kants Begründung der Ethik
(1877) and Kants Begründung der Aesthetik (1889), dealing
with the three Kantian Critiques.ii 3 3 3 Принцип и норма трансцендентального ме
тода, согласно известному определению Коге
на, заключается в следующей скромной, про
стой мысли, der schlichte Gedanke: «те элементы
сознания суть элементы познающего сознания,
которые достаточны и необходимы для обо
снования и укрепления факта науки... Элемен
ты сознания должны обнаружить свою плодот
ворность в качестве основ науки, и предпосыл
ки науки должны получить значение основных
черт познающего сознания» (H. Cohen. Kants
Theorie der Erfahrung. 2[-е] изд. 1885. стр. 77—78). Что же означает этот своеобразный транс
цендентальный метод, или ориентирование
познания в факте науки? — Не что иное, как
умозаключение от факта науки и, в частности,
от математического естествознания, к необходи
мым и достаточным условиям, к основным пред
положениям24 науки, каковы время, число, про
странство, величина, субстанциальность, при
чинность, систематическая взаимообусловлен
ность и т. д., т. е. к тому, что нередко именуют
основами, или принципами, научного знания. Дело представляется, таким образом, в следу
ющем виде. Среди различного рода фактов, яв
лений особо своеобразное положение занимает
факт существования и последовательного раз
С Принцип и норма трансцендентального ме
тода, согласно известному определению Коге
на, заключается в следующей скромной, про
стой мысли, der schlichte Gedanke: «те элементы
сознания суть элементы познающего сознания,
которые достаточны и необходимы для обо
снования и укрепления факта науки... Элемен
ты сознания должны обнаружить свою плодот
ворность в качестве основ науки, и предпосыл
ки науки должны получить значение основных
черт познающего сознания» (H. Cohen. Kants
Theorie der Erfahrung. 2[-е] изд. 1885. стр. 77—78). The principle and norm of the transcenden
tal method, according to a well-known defini
tion of Cohen, consists in the following modest,
simple idea, der schlichte Gedanke: “[...] among
the elements of consciousness the elements of
cognising consciousness are those which are
sufficient and necessary for grounding and
strengthening the fact of science. […] The ele
ments of consciousness must be effective as the
foundations of science and the prerequisites
of science must be clearly recognised as the
characteristics of the cognising consciousness”
(H. Cohen. Kants Theorie der Erfahrung. 2nd ed. 1885, p. 77-78). Что же означает этот своеобразный транс
цендентальный метод, или ориентирование
познания в факте науки? — Не что иное, как
умозаключение от факта науки и, в частности,
от математического естествознания, к необходи
мым и достаточным условиям, к основным пред
положениям24 науки, каковы время, число, про
странство, величина, субстанциальность, при
чинность, систематическая взаимообусловлен
ность и т. д., т. е. к тому, что нередко именуют
основами, или принципами, научного знания. [1] Lossky in his
Introduction to Philosophy chose to offer his read
ers two versions of Kant’s teaching: the gener
ally accepted one, anchored in the dominant
legend, and another one, the “purified” inter
pretation offered by Cohen in his commentary8
to the three Critiques.9 This striking fact is very
naturally and easily explained. Cohen’s inter
pretation of Kant was not slavish, verbal or
literal as, for example, the very useful “philo
logical” commentary by Hans Vaihinger; not
a dogmatic description of the inner fabric and
composition of Kant’s teaching, taken by itself
and independent of other teachings and phi
losophemes; but a critical, uninhibited and cre
ative explication and reproduction of Kant’s
teaching as a stage of development among oth
er similar attempts, efforts and achievements of
the human spirit in quest of the unfading Most
Perfect Truth. Not surprisingly, Cohen’s inter
pretation and explanation of Kant’s teaching
gradually and quietly turned into an overcom
ing of Kant, which we find in Cohen’s System of
Philosophy in which his creative thought soars
freely on its own wings. Этот поразительный факт весьма естествен
но и21 просто объясняется тем, что истолкова
ние Канта Когеном было не рабским, словес
ным или буквальным, каков, например, полез
нейший «филологический» комментарий Ган
са Файгингера; не догматическим раскрытием
внутренней ткани и состава учения Канта, взя
того само по себе и независимо от других уче
ний и философем; но критическим, свобод
ным и творческим раскрытием и воспроизве
дением учения Канта как ступени развития22
в ряду других однородных попыток, дерзнове
ний и достижений человеческого духа на пу
тях разыскания немеркнущей Всесовершен
ной Истины. Немудрено поэтому, что истолко
вание и раскрытие учения Канта у Когена по
степенно и незаметно превратилось в прямое
преодоление Канта, чтó мы и находим в «Си
стеме философии» самого Когена, где творче
ская мысль его свободно воспаряет на своих
собственных крыльях. Но если Когену и удалось преодолеть Канта
как вполне законченное и условно ограничен
ное явление, временное событие в истории раз
вития человеческой мысли, тем не менее Коге However, although Cohen managed to over
come Kant as a complete and limited phenom 103 А. Л. Саккетти ну посчастливилось23 вскрыть у Канта некото
рое непреходящее достижение, некоторую ча
стицу немеркнущей Истины. Эта частица не
преходящей Истины открыта Когеном у Канта
в виде принципа трансцендентального метода,
или ориентирования философии в факте науки. р
у
уд
24 Приставка «пред-» вписана сверху от руки. 23 Вместо зачеркнутого слова «удалось». [1] enon, a temporal event in the history of the
development of human thought, nevertheless
Cohen was lucky enough to discover in Kant
an intransient achievement, a particle of the un
fading Truth. The particle of intransient Truth
Cohen discovered in Kant is the principle of the
transcendental method or the orientation of philoso
phy in the fact of science. 3 возможны синтетические суждения априори, т. е.
суждения математики и механики Ньютона. how mathematical natural science is possible
or, in the historical perspective found in the
Critique of Pure Reason, How are synthetic judg
ments a priori possible?, i. e. the judgements of
mathematics and Newton’s mechanics. Как видно из сказанного, и предмет, т. е. факт научного знания, и постановка вопро
са, и путь решения его, т. е. метод, совершен
но своеобразны и коренным образом отличны
от предмета, задач и методов отдельных поло
жительных наук, занятых в конце концов клас
сификациею и объяснением многообразных
фактов действительности методами анализа
и синтеза, индукции и дедукции. И т[ак] к[ак]
проблема обоснования наук не может входить
в область какой-либо частной науки, например
обоснование математики, физики или химии
не входит ни в математику, ни в физику, ни в
химию и т. д., то очевидно, что проблема самой
науки должна входить в какую-то иную об
ласть. Коген относит ее к области философии. As has been seen from the above, the object,
i. e. the fact of scientific knowledge, and the for
mulation of the question and the way of solv
ing it, i. e. the method, are totally original and
radically different from the object, tasks and
methods of individual positive sciences, which
are ultimately engaged in classification and ex
planation of the diverse facts of reality by the
methods of analysis and synthesis, induction
and deduction. Since the problem of ground
ing sciences cannot belong to the domain of
any particular science, e. g. the grounding of
mathematics, physics or chemistry is not part of
mathematics, or physics or chemistry, etc., it is
obvious that the problem of science itself must
belong to some other domain. Cohen refers it to
philosophy. Но тут неизбежно возникает недоумение. Допустим, что проблема науки не есть пред
мет самой науки и входит в область филосо
фии. Этим, однако, отнюдь не очерчена и не
ограничена область самой философии в це
лом. Было бы жестоким догматизмом прикре
плять философию к решению проблемы нау
ки. Это создало бы новое закрепощение и ка
балу для философии. Подобно тому как сред
невековая схоластика превращала философию
в служанку богословия, так новая схоластика в
лице Когена грозит превратить философию в
служанку науки. Да к чему, в самом деле, фи
лософу ограничивать свой кругозор рамками
научного знания! Разве перед его взором не от
крыт бесконечно разнообразный и неисчерпа
емо богатый мир реальной действительности? 3 What is the meaning of this original tran
scendental method or orientation of cognition
in the fact of science? None other than inference
from the fact of science, in particular, from mathe
matical natural science to the necessary and suffi
cient conditions, the main presuppositions of science
such as time, number, space, extent, substanti
ality, causality, systemic mutual conditioning,
etc., i. e. to what is often referred to as the foun
dations or principles of scientific knowledge. Дело представляется, таким образом, в следу
ющем виде. Среди различного рода фактов, яв
лений особо своеобразное положение занимает
факт существования и последовательного раз
вития науки, научного знания. Существование
и развитие науки, научного знания создает свое
образную проблему, задачу: объяснить, как воз
можна вообще наука, как возможно математи
ческое естествознание или, в исторической по
становке вопроса «Критики чистого разума»: как Thus, the picture that emerges is as follows. Among the various facts and phenomena the
fact of the existence and consistent develop
ment of science, of scientific knowledge oc
cupies a special position. The existence and
development of science, of scientific knowl
edge creates a distinctive problem, the task of
explaining how science in general is possible, 104 A. L. Sakketti возможны синтетические суждения априори, т. е.
суждения математики и механики Ньютона. К чему же ему, не в пример прочим простым
смертным, отворачиваться от жизни и действи
тельности и замыкаться в мире безжизненных
построений и сухих понятий, вместо того что
бы обратить открытый и ясный взор на мир и
жизнь, вооружаться искусственными очками,
всегда более или менее преломляющими лучи
света, или, тем паче, вместо созерцания самой
действительной жизни заниматься «рассмо But this inevitably leads to misunderstand
ing. Suppose the problem of science is not the
object of science itself and pertains to philoso
phy. That, however, does not delineate or lim
it the area of philosophy as a whole. It would
be rigid dogmatism to bind philosophy to the
solution of the problem of science. That would
create a new serfdom and bondage for philoso
phy. Just as medieval scholasticism turned phi
losophy into a maidservant of theology, so the
new scholasticism of Cohen threatens to turn
philosophy into a maidservant of science. But
why indeed should the philosopher confine his
horizon to scientific knowledge! Is not the in
finitely diverse and inexhaustibly rich world of
reality open to his gaze? Why should he, unlike
ordinary mortals, turn away from life and real
ity and withdraw into a world of lifeless con
structions and dry concepts instead of turning
an open and clear eye to the world and life;
why should he arm himself with artificial spec 105 А. Л. Саккетти трением очков»[?] Поистине, пресловутое «от
крытие» Когеном трансцендентального метода
представляет какой-то сомнительный «дар да
найцев», от которого не мешает как можно ско
рее отказаться... tacles which always more or less refract rays of
light or even more, instead of contemplating
real life, “contemplate the spectacles”? Truly,
Cohen’s notorious “discovery” of the transcen
dental method is a dubious Greek gift which is
best given up as soon as possible… Нам думается, что подобные недоумения
являются следствием недоразумения, и мысль
Когена об ориентировании философии в нау
ке имеет более глубокий и полновесный смысл,
нежели это может показаться. We believe that such misunderstandings
are the result of misapprehension, and that
Cohen’s idea of the orientation of philosophy
in science carries a more profound and robust
meaning than meets the eye. Подобно тому как всякое объяснение фактов
предполагает ту или иную классификацию са
мых явлений, так и обоснование научного зна
ния в философии Когена обусловлено извест
ным различением знания от того, что не есть
знание, но легко может быть с ним смешано. возможны синтетические суждения априори, т. е.
суждения математики и механики Ньютона. Далее, опыт хотя и на
правлен непосредственно на многообразное,
единичное и конкретное, но результаты опыта
дают отвлечение, абстракцию. Ибо общие пред
ставления содержат «преодоление» бесконеч
ного неисчерпаемого многообразия действи
тельности, если говорить словами Г. Риккерта. А преодоление бесконечного многообразия со
стоит не в чем ином, как в забвении самой бес
конечности, в удержании и сохранении, «изо
лировании» отдельных сторон и черт явлений. Так, ценою «изолирования», выделения получа
ется упрощение и обеднение богатства много
образной действительности, отвлечение, аб
страгирование ее отдельных моментов от всей
многосложной ткани. Наблюдение и опыт, не
посредственно направленные и устремленные
на единичное, многообразное и конкретное,
на самом деле дают мертвые отвлечения, пу
стые обобщения, которые, как известно из фор
мальной логики, чем шире по объему, тем бед
нее содержанием. Наконец, наблюдение и опыт
не дают сами по себе чего-либо цельного, еди
ного. Результаты опыта — единичные сведе
ния, производные сочетания самостоятельных
данных, бледные, отраженные тени, безжиз
ненные, бескровные схемы. Правда, наблюде
ние, сравнение и обобщение редко даны в та
ком чистом виде: «чистый опыт» есть весьма ис
кусственный препарат, результат весьма томи
тельной дистилляции, надуманной «критики»
(Авенариус). На самом деле наблюдение и опыт
обыкновенно руководствуются известным на
правлением, точкой зрения, или целью, ради
которой предпринимаются эти наблюдения и
обобщения. Таким образом, уже в простом на
блюдении, сравнении и обобщении обыкно than the combination of separate perceptions,
observations or the generalised representations
of separate features scattered across a multitude
of phenomena. Perceptions, observations and
general representations belong to the realm of
mental experiences, i. e. these are mental phe
nomena and facts. Further, although experience
is directed towards the manifold, the singular
and the concrete, the results оf experience yield
an аbstraction. For general representations con
tain the “overcoming” of the infinite and inex
haustible diversity of reality, to use H. Rickert’s
words. The overcoming of the infinite diversity
consists precisely in forgetting infinity, in cap
turing and preserving, “isolating” particular
aspects and features of phenomena. Thus, isola
tion simplifies and impoverishes the richness of
manifold reality and abstracts some of its par
ticular aspects from the entire intricate fabric. Observation and experience, directly targeting
the singular, the diverse and the concrete, ac
tually yields dead abstractions, empty general
isations which, as is known from formal logic,
are the more lacking in content the broader
they are. Finally, observation and experience in
themselves do not yield anything whole. возможны синтетические суждения априори, т. е.
суждения математики и механики Ньютона. Just as any explanation of facts implies a
classification of the phenomena themselves, so
the grounding of scientific knowledge in Co
hen’s philosophy presupposes a differentiation
of knowledge from what is not knowledge but
can easily be mistaken for it. 4 4 В полном согласии и внутреннем созвучии с
великими учителями философского идеализ
ма, Платоном, Кантом и Гегелем, Коген прово
дит строгое различие между восприятием, на
блюдением, обобщающем отвлечением, с од
ной стороны, и знанием, Erkenntnis — с дру
гой. Многообразие отдельных единичных яв
лений, вещей дается, развертывается в опыте,
т. е. в единичном восприятии и наблюдении;
данные восприятий подвергаются дальней
шей обработке путем сравнения, отвлечения
и обобщения. Сообщаемый опытом матери
ал фактов, явлений ограничен исключитель
но пределами самого опыта, т. е. теми именно
данными, которые сообщаются восприятием, а
оно лишь доводит до сведения сознания, meldet
an, о тех или иных явлениях — и только. Пред
ставление лишь воспроизводит и разнообразно
сочетает, комбинирует данные непосредствен
ного восприятия. Обобщение данных восприя
тия лишь устанавливает путем сопоставления,
сравнения и отвлечения общие черты данных
восприятия, т. е. отдельных явлений, фактов. Таким образом, результат опыта есть не что
иное, как совокупность отдельных восприя In full accordance and inner consonance
with the great teachers of philosophical ide
alism, Plato, Kant and Hegel, Cohen draws a
rigorous distinction between perception, obser
vation and generalising abstraction on the one
hand, and cognition, Erkenntnis, on the other. The diversity of singular phenomena or things
is given and unfolds in experience, i. e. in singu
lar perception and observation; the data of per
ceptions are further processed by comparison,
abstraction and generalisation. The material of
facts and phenomena delivered by experience
is confined to experience, i. e. to the data which
are provided by perception, which merely re
ports, meldet an, phenomena to consciousness —
and no more. Representation only reproduces
and variously compounds or combines the data
of spontaneous perception. Generalisation of
the data of perception merely uses juxtaposi
tion, comparison and abstraction to establish
general features of the data rendered by per
ception, i. e. individual phenomena and facts. Thus, the result of experience is nothing more 106 A. L. Sakketti тий, наблюдений или обобщенные представле
ния отдельных черт, рассеянных во множестве
явлений. Восприятия, наблюдения и общие
представления принадлежат к миру душевных
переживаний, т. е. это есть известные психиче
ские явления, факты. А. Л. Саккетти Напротив, познание не есть душевное пере
живание, вообще не есть явление, не есть не
что сущее, по словам Платона. Понятие, Ло
гос, по учению Сократа, есть прежде всего во
прос [«]что есть?[»]. Неутомимо отыскивая ис
тину, Сократ останавливает на улице знакомо
го прохожего и забрасывает его испытующими
вопросами: напр., кто есть муж государствен
ный? Так в разыскании истинных понятий воз
никает майевтика Сократа, повивальное искус
ство мысли. Но недостаточно поставить, задать
вопрос; необходимо дать ему и разрешение, от
вет. Необходимо дать отчет в понятии, учит
божественный Платон. Мальчику, несведуще
му в геометрии, задана задача: удвоить пло
щадь квадрата. После неудачных попыток и
колебаний ответ найден: необходимо постро
ить новый квадрат на диагонали данного. Ква
драт диагонали есть ответ на вопрос и отчет в
понятии. Итак, знание есть целая мысль, от на
чала, т. е. от вопроса, задания, до конца, т. е. до
ответа, решения. Всякое понятие, всякое по
ложение, суждение есть некоторое целое един
ство, охватывающее многообразные моменты,
порождаемые, вытекающие из него. something existing, according to Plato. The con
cept, Logos, according to the teaching of Soc
rates, is first and foremost the question, “What
is X?” In tirelessly searching for the truth Soc
rates stops an acquaintance in the street and
showers him with probing questions, e. g. Who
is a statesman? In his quest of true concepts
Socrates arrives at the concept of maieutics,
likening thought to midwifery. However, it is
not enough to ask a question; it is also neces
sary to provide a solution, an answer. It is nec
essary to give an account of the concept, as the
divine Plato teaches. A boy, ignorant of geom
etry, is given the task of doubling the area of
a square. After several unsuccessful attempts
and some hesitation the answer is found: it is
necessary to build a new square on the diago
nal of the given one. The square of the diagonal
is the answer to the question and the account
of the concept. Thus, knowledge is a complete
thought, from the beginning, i. e. the question,
the task, to the end, i. e. the answer, the solu
tion. Every concept, every proposition, judge
ment is a whole unity spanning the manifold
features it engenders. Словом, знание есть не что иное, как рас
крытие единства в многообразии и многообразия
в единстве, тождество в различии, согласие про
тивоположного, или синтез единства. 10 H. Cohen. System der Philosophie. Erster Theil. Logik der
reinen Erkenntnis. Berlin, 1902, pp. 23-24 (author’s note). 25 H. Cohen. System der Philosophie. Erster Theil. Logik der
reinen Erkenntnis. Berlin. 1902. Стр. 23 и 24 (примеч. авт.). возможны синтетические суждения априори, т. е.
суждения математики и механики Ньютона. The
results of experience are isolated data, deriv
ative combinations of independent data, pale
shadows, lifeless and anemic schemes. True,
observation, comparison and generalisation are
seldom given in such a pure form: “pure expe
rience” is a very artificial preparation, the result
of tedious distillation and speculative “critique”
(Avenarius). In reality, observation and expe
rience usually follow a certain guideline, have
a point of view or a goal for the sake of which
these observations and generalisations are un
dertaken. Thus, even the simple observation,
comparison and generalisation usually have
something that goes beyond immediate experi
ence, a certain direction or goal. B
t
t
iti
i
t
t l тий, наблюдений или обобщенные представле
ния отдельных черт, рассеянных во множестве
явлений. Восприятия, наблюдения и общие
представления принадлежат к миру душевных
переживаний, т. е. это есть известные психиче
ские явления, факты. Далее, опыт хотя и на
правлен непосредственно на многообразное,
единичное и конкретное, но результаты опыта
дают отвлечение, абстракцию. Ибо общие пред
ставления содержат «преодоление» бесконеч
ного неисчерпаемого многообразия действи
тельности, если говорить словами Г. Риккерта. А преодоление бесконечного многообразия со
стоит не в чем ином, как в забвении самой бес
конечности, в удержании и сохранении, «изо
лировании» отдельных сторон и черт явлений. Так, ценою «изолирования», выделения получа
ется упрощение и обеднение богатства много
образной действительности, отвлечение, аб
страгирование ее отдельных моментов от всей
многосложной ткани. Наблюдение и опыт, не
посредственно направленные и устремленные
на единичное, многообразное и конкретное,
на самом деле дают мертвые отвлечения, пу
стые обобщения, которые, как известно из фор
мальной логики, чем шире по объему, тем бед
нее содержанием. Наконец, наблюдение и опыт
не дают сами по себе чего-либо цельного, еди
ного. Результаты опыта — единичные сведе
ния, производные сочетания самостоятельных
данных, бледные, отраженные тени, безжиз
ненные, бескровные схемы. Правда, наблюде
ние, сравнение и обобщение редко даны в та
ком чистом виде: «чистый опыт» есть весьма ис
кусственный препарат, результат весьма томи
тельной дистилляции, надуманной «критики»
(Авенариус). На самом деле наблюдение и опыт
обыкновенно руководствуются известным на
правлением, точкой зрения, или целью, ради
которой предпринимаются эти наблюдения и
обобщения. Таким образом, уже в простом на
блюдении, сравнении и обобщении обыкно
венно заложено нечто такое, что выходит и вы
водит за пределы наличного непосредственно
го опыта, то или иное направление, или цель. By contrast, cognition is not a mental expe
rience, and in general is not a phenomenon, not 107 А. Л. Саккетти А. Л. Саккетти «Синтез
есть синтез единства», — учит Коген в «Логике
чистого познания»; «синтез хотя и направлен к
единству, но имеет своею предпосылкою мно
жество»25. «Фиксационною точкою мыслящего
сознания — а только мыслящее сознание явля
ется сознанием в полном смысле слова, — пояс
няет П. Наторп мысль Когена, — служит един
ство, то единство, в котором временно-различ
ное объединяется именно тогда, когда мысль
не рассеивается уже по разбросанному во вре
мени, но концентрируется сама в себе и вме
сте с тем концентрирует в себе разрозненные In short, knowledge is nothing else than
the uncovering of unity in diversity and diver
sity in unity, identity in difference, harmony
of opposites or synthesis of unity. “Synthesis is
synthesis of unity,” Cohen writes in the Logic
of Pure Cognition; “synthesis, although direct
ed towards unity, is premised on multiplici
ty.”10 Elaborating Cohen’s thought, P. Natorp
writes: “The fixing point of the thinking con
sciousness — and only thinking consciousness
is consciousness in the full sense of the word
— is unity, i. e. unity in which the temporari
ly different is united precisely when thought
is not scattered across what is scattered in
time, but is concentrated within itself and at 108 A. L. Sakketti the same time concentrates in itself the scat
tered elements of its content; it is only then
that it combines them in some true content,”
“consciousness is unity of the manifold, iden
tity of what must at the same time differ.”11
“Science,” R. Stammler explains, “is any con
sciousness that is directed towards unity and
is completed in transformation towards uni
ty. The striving for unity distinguishes science
from mere awareness (Kunde). It is called pure
science or theory in the best meaning of the
word if the unity pursued is unconditional and
represents an idea of perfection liberated from
material. By contrast, it is a technical area of
knowledge if it is content with such cognition
that can at any moment be limited by a certain
finite material.”12 Thus, consciousness ascends
from mosaic-like and fragmented experiential
perception to the unity of cognition thought of
not as a derivative, not as a mere combination
of separate perceptions, or an abstract gener
alisation of independent observations, but as
the main, basic and therefore creative unity, as
the origin of the emergence of manifold, Ursprung
der Erzeugung. “For we know,” Cohen teaches,
“that on the issue of thought, i. e. 11 P. Natorp. Sotsial’naya pedagogika [Social Pedagogics].
Translated by A. A. Grombach. St. Petersburg, 1911,
pp. [19-]20, 21. [In Rus.] (author’s note).
12 R. Stammler. Die Lehre von dem richtigen Rechte. Berlin,
1902, p. 5 (author’s note).
13 Logik der reinen Erkenntnis, p. 26 (author’s note).
14 Ibid., § 8, p. 28ff (author’s note). 26 П. Наторп. Социальная педагогика. Перев. А. А. Гром
баха. СПб. 1911. Стр. [19—]20 и 21 (примеч. авт.).
27 В машинописи: «оно». Сверено по немецкому
источнику.
28 R. Stammler. Die Lehre von dem richtigen Rechte. Ber
lin. 1902. Стр. 5 (примеч. авт.).
29 Logik der reinen Erkenntnis. Стр. 26 (примеч. авт.). 15 P. Natorp. Sotsial’naya pedagogika, p. 21. Already
Aristotle distinguished the first for us and the first by nature,
πρότερον πρòς ἡμᾶς and πρότερον τῆ φύσει (author’s note). 30 Там же, § 8 стр. 28 и след. (примеч. авт.).
31 П. Наторп. Социальная педагогика. Стр. 21. Так,
еще Аристотель различал первое для нас и первое по при
роде, πρότερον πρòς ἡμᾶς и πρότερον τῆ φύσει (примеч. авт.). А. Л. Саккетти creation, we
deal with unity. Hence, multiplicity, which is
the prerequisite of unity provided such a pre
requisite is valid, must itself be created (erzeu
gt), i. e. must itself be thought as a unity. This
eliminates, at least for the time being, the view
that the manifold can be given to thought from
outside.”13 элементы своего содержания; тогда лишь она
объединяет их в некотором истинном содержа
нии», «сознание — это единство многообразно
го, тождество того, что в то же время должно
различаться»26. «Наука, — поясняет Р. Штаммлер, — есть
всякое сознание, направленное к единству и
завершаемое в преобразовании к единству. Стремлением к единству наука отличается от
простой осведомленности (Kunde). Она27 зовет
ся чистой наукою, или теориею в лучшем зна
чении слова, если преследуемое единство без
условно и представляет идею освобожденно
го от материала совершенства. Она, напротив,
есть техническая отрасль знания в том случае,
если довольствуется таким познанием, которое
в любой момент ограничено определенным ко
нечным материалом»28. Так от мозаичного и отрывочного опытного
восприятия сознание поднимается к единству
познания, которое мыслится не как зависимое
производное, не как простая совокупность от
дельных восприятий, или отвлеченное обоб
щение самостоятельных наблюдений, но как
основное, коренное и потому созидательное,
творческое единство, как источник возникнове
ния многообразия, Ursprung der Erzeugung. «Ибо
мы знаем, — учит Коген, — что в вопросе о
мышлении, т. е. о создании, дело идет о един
стве. А отсюда следует, что множество, служа
щее предпосылкою для единства, если толь
ко такая предпосылка может быть целесоо
бразна, само должно быть создано (erzeugt), т. е. тоже само должно быть мыслимо как некото
рое единство. Взгляд, что многообразие может
быть дано мышлению откуда-то извне, тем са
мым сразу же устраняется»29. Therefore pure logic is the logic of the origin,
Logik des Ursprungs.14 “Unity, identity serves as
an earlier expression of consciousness itself,” 109 А. Л. Саккетти А. Л. Саккетти writes Natorp, “the manifold, i. e. multiplicity
and difference, expresses what is juxtaposed
to consciousness, its general object, phenome
non. The true starting point of cognition is in
the former and not in the latter, but we should
give thought to this starting point, and this re
flection, which was not yet cognition, but only
sought access to it, proceeded [...] from tracing
the manifold, from experience. This is precisely
the meaning of Kant’s words that experience is
the beginning of cognition, but not the source
from which it originates.”15 (The manifold of
singular things, phenomena is, as it were, giv
en in advance in the perception of experience
and unity in this manifold is the required, the
task. However, after the path from the given
manifold of phenomena to its all-embracing
unity has been traversed, it will readily be seen
that the manifold of singular phenomena com
bined in a whole unity is in turn a no less im
portant problem than the uniting principle or,
indeed, the same task, only given from the oth
er side). Чистая логика поэтому есть логика первона
чала, Logik des Ursprungs30. «Единство, тождество
служит более изначальным выражением само
го сознания, — пишет Наторп, — многообра
зие же, т. е. множественность и различие, слу
жит выражением того, чтó сознанию противо
поставляется, — выражением его общего объ
екта, явления. Истинный отправной пункт по
знания находится в первом, а не во втором, но
нам надо было еще поразмыслить над этим от
правным пунктом, и это размышление, которое
еще не было познанием, а только искало досту
па к нему, исходило […] от прослеживания мно
гообразного, от опыта. Таков именно смысл слов
Канта, что опыт есть начало познания, но не
источник, из которого оно происходит»31. (Мно
гообразие единичных вещей, явлений пред
ставляется наперед как бы налично данным в
восприятии опыта, и единство в этом многооб
разии представляется искомым, заданным. Од
нако после того, как пройден путь от налично
го многообразия явлений к объемлющему его
единству, нетрудно убедиться в том, что мно
гообразие единичных явлений, связанных в це
лостное единство, в свою очередь, представля
ет не менее важную проблему, нежели объеди
няющее начало или, по правде сказать, — ту же
самую задачу, но лишь поставленную с другой
стороны.) 5 Thus, in accordance with the accepted as
sumption, knowledge is a holistic unity of the
manifold, not a mere derivative sum of indepen
dent terms, not a combination of separate giv
en perceptions or their abstract generalisation,
but a unity of a whole, while the manifold is a
derivative of subsequent differentiation, dis
memberment and development of the whole. Thus, from a generic concept are “generated”
specific and particular concepts, from laws
and propositions, e. g. axioms, “flow” conse
quences, e. g. various theorems. In general,
all the singular and particular is embedded in
some whole, grows out of it, as it were, like or 16 N. Lossky. Vvedeniye v filosofiyu. Part 1, St. Petersburg,
1911, p. 7 (author’s note).
17 N. Lossky. Mir kak organicheskoye tseloye [The World
as an Organic Whole]. Moscow, 1917, pp. 14-15. [In Rus.]
(author’s note). 5 5 Итак, знание, согласно принятому допуще
нию, есть целостное единство многообразного —
не простая производная сумма независимых
слагаемых, не совокупность отдельных данных
восприятий или их отвлеченное обобщение,
но единство целого, многообразие же — произ
водный результат последующего различения,
расчленения и развития целого. Так, из родово 110 A. L. Sakketti gans in a living organism grow out of an ini
tially simple whole, the cell, by division and
development. Sometimes such whole unity is
termed living organic in contradistinction to a
combination of independent terms or mechanis
tic aggregate. “Unity (of a combination of sev
eral elements) has an inorganic character if its
constituent parts existed before the whole and
formed the whole only after encountering one
another and, consequently, after external in
teraction.” “By contrast, unity has an organic
character if the whole exists before its constit
uent parts such that the parts arose from the
whole through division (differentiation) of the
whole.”16 N. O. Lossky recognizes that “one of
the valuable contributions to philosophy made
by Cohen’s school” was the doctrine of the
uninterrupted character of the object of knowl
edge, of the holistic generation of its elements
and not a mosaic synthesis of disparate А, В,
Сs. This understanding of the object of knowl
edge, as N. O. Lossky rightly concludes, “in
evitably leads to the concept of the object as a
system in the sense of organic perception of the
world.”17 The unity of cognition can only be
recognised as a creative origin of the manifold,
a living organic whole if it is unconditionally
all-embracing and completely full and adequate to
its object. And only infinity is unconditionally
full. Therefore a creative whole must be infinite. Every derivative unity, mechanical combina
tion, is inevitably finite although it may not be
completed and represents an open-ended se
quence. A creative whole must furthermore be
uninterrupted: only on this condition does such
a whole possess unconditional wholeness of
all the allowable options, only then is it truly
all-embracing. Creative wholeness and infinite
uninterruptedness of knowledge is what Cohen го понятия «порождаются» видовые и частные
понятия, из законов и положений, напр. акси
ом, «вытекают» следствия, напр. различные те
оремы. Вообще все единичное и частное укоре
нено в каком-либо целом, как бы вырастает из
него подобно тому, как органы в живом организ
ме путем расчленения и развития вырастают из
первоначально простого целого, клетки. 5 Ино
гда такое целостное единство именуется живым
органическим в противоположность совокупно
сти самостоятельных слагаемых, или механиче
скому агрегату. «Единство (сочетания несколь
ких элементов) имеет неорганический характер
в том случае, если части, входящие в него, су
ществовали раньше целого и образовали целое
только после встречи друг с другом и, следова
тельно, после внешнего взаимодействия». «Нао
борот, единство имеет органический характер в
том случае, если целое существует раньше вхо
дящих в него частей, так что части возникли из
целого путем расчленения (дифференцирова
ния) целого»32. Н. О. Лосский признает «одною
из заслуг школы Когена» учение о сплошности
предмета знания, о цельном порождении его мо
ментов, а не о мозаичном синтезе из разрознен
ных А, В, С. Это понимание предмета знания,
по справедливому заключению Н. О. Лосского,
«неизбежно ведет к понятию предмета как си
стемы в смысле органического миропонимани
я»33. Целостное единство познания тогда только
может быть признано творческим источником
многообразного, живым органическим целым,
если оно безусловно всеобъемлюще и совершенно
полно, адекватно своему предмету. А безуслов
ная полнота присуща только бесконечности. По
этому творческое целое должно быть бесконеч
но. Всякое производное единство, механическая
совокупность необходимо конечна, хотя может
не быть завершена и представляет собою неза
конченный ряд. Творческое целое должно быть
сверх того и непрерывно: лишь при этом условии
32 Н. Лосский. Введение в философию. Ч. 1, СПб. 1911. Стр 7 (примеч авт ) го понятия «порождаются» видовые и частные
понятия, из законов и положений, напр. акси
ом, «вытекают» следствия, напр. различные те
оремы. Вообще все единичное и частное укоре
нено в каком-либо целом, как бы вырастает из
него подобно тому, как органы в живом организ
ме путем расчленения и развития вырастают из
первоначально простого целого, клетки. Ино
гда такое целостное единство именуется живым
органическим в противоположность совокупно
сти самостоятельных слагаемых, или механиче
скому агрегату. «Единство (сочетания несколь
ких элементов) имеет неорганический характер
в том случае, если части, входящие в него, су
ществовали раньше целого и образовали целое
только после встречи друг с другом и, следова
тельно, после внешнего взаимодействия». «Нао
борот, единство имеет органический характер в
том случае, если целое существует раньше вхо
дящих в него частей, так что части возникли из
целого путем расчленения (дифференцирова
ния) целого»32. Н. О. Лосский признает «одною
из заслуг школы Когена» учение о сплошности
предмета знания, о цельном порождении его мо
ментов, а не о мозаичном синтезе из разрознен
ных А, В, С. Это понимание предмета знания,
по справедливому заключению Н. О. 32 Н. Лосский. Введение в философию. Ч. 1, СПб. 1911.
Стр. 7 (примеч. авт.).
33 Н. Лосский. Мир как органическое целое. М. 1917.
Стр. 14—15 (примеч. авт.). 5 In the system of knowledge i e the system y g
,
p
interruptedness of individual differences, em
braces the infinity of species, particulars and
singular features. Every scientific law, every
scientific proposition, e. g. axioms of geome
try or the laws of mechanics, cover an infini
ty of consequences, theorems, phenomena and
facts. Knowledge itself, owing to its uninter
ruptedness and infinity, is swept by an irresist
ible inner flow of movement. No systematics
of phenomena offered by this or that classifi
cation of concepts, no system of laws of nature
reveal the inexhaustible fullness and infinite
diversity of phenomena, giving only a remote
approximation to perfect and complete knowl
edge. Daily experience discovers an influx of
ever new phenomena which must be included
in a system, classified, and the emergence of
fresh facts prompts a search for an exhaustive
explanation thereof in the laws of nature. The
discovery of new phenomena and facts dictates
a revision, rearrangement and modification of
concepts and propositions, the replacement of
narrow and tight concepts with broader and
more all-embracing ones; this in turn makes it
possible to subordinate new phenomena and
varieties to them. Differentiation and adjust
ment of knowledge goes hand-in-hand with its
broadening and generalisation. Thus, univer
sal unity is preserved in the individual and par
ticular through infinite uninterruptedness. The
immersion of thought in the individual and sin
gular constitutes at the same time a return of
the universal to itself: the whole finds itself in
the singular, becomes aware of itself in the in
dividual and thus arrives at the self-conscious
ness in the human person. Каждое родовое, общее понятие содержит в
себе непрерывность индивидуальных разли
чий, объемлет бесконечность видовых частно
стей и единичных особенностей34. Каждый на
учный закон, каждое научное положение, на
пример аксиомы геометрии, законы механики,
охватывают бесконечность следствий, теорем,
явлений, фактов. При этом само знание в силу
своей непрерывности и бесконечности охвачено
неудержимым внутренним потоком движения. Никакая систематика явлений, данная в той или
иной классификации понятий, никакая система
законов природы не раскрывают неисчерпаемой
полноты и бесконечного разнообразия явлений,
но дают лишь некоторое отдаленное прибли
жение к совершенному, полному знанию. Еже
дневный опыт обнаруживает приток все новых
и новых явлений, которые должны быть вклю
чены в систему, классифицированы, появление
новых фактов побуждает искать исчерпываю
щее объяснение им в законах природы. 34 Вставка вместо слова «особей». 5 Лосского,
«неизбежно ведет к понятию предмета как си
стемы в смысле органического миропонимани
я»33. Целостное единство познания тогда только
может быть признано творческим источником
многообразного, живым органическим целым,
если оно безусловно всеобъемлюще и совершенно
полно, адекватно своему предмету. А безуслов
ная полнота присуща только бесконечности. По
этому творческое целое должно быть бесконеч
но. Всякое производное единство, механическая
совокупность необходимо конечна, хотя может
не быть завершена и представляет собою неза
конченный ряд. Творческое целое должно быть
сверх того и непрерывно: лишь при этом условии
32 Н. Лосский. Введение в философию. Ч. 1, СПб. 1911. 111 А. Л. Саккетти такое целое содержит в себе безусловную полно
ту всех допустимых возможностей, лишь тогда
оно поистине всеобъемлюще. Творческая пол
нота и бесконечная непрерывность знания и
есть то, что Коген называет чистотою, Reinheit. Every generic, common concept contains un
interruptedness of individual differences, em
braces the infinity of species, particulars and
singular features. Every scientific law, every
scientific proposition, e. g. axioms of geome
try or the laws of mechanics, cover an infini
ty of consequences, theorems, phenomena and
facts. Knowledge itself, owing to its uninter
ruptedness and infinity, is swept by an irresist
ible inner flow of movement. No systematics
of phenomena offered by this or that classifi
cation of concepts, no system of laws of nature
reveal the inexhaustible fullness and infinite
diversity of phenomena, giving only a remote
approximation to perfect and complete knowl
edge. Daily experience discovers an influx of
ever new phenomena which must be included
in a system, classified, and the emergence of
fresh facts prompts a search for an exhaustive
explanation thereof in the laws of nature. The
discovery of new phenomena and facts dictates
a revision, rearrangement and modification of
concepts and propositions, the replacement of
narrow and tight concepts with broader and
more all-embracing ones; this in turn makes it
possible to subordinate new phenomena and
varieties to them. Differentiation and adjust
ment of knowledge goes hand-in-hand with its
broadening and generalisation. Thus, univer
sal unity is preserved in the individual and par
ticular through infinite uninterruptedness. The
immersion of thought in the individual and sin
gular constitutes at the same time a return of
the universal to itself: the whole finds itself in
the singular, becomes aware of itself in the in
dividual and thus arrives at the self-conscious
ness in the human person. 5 С обна
ружением новых явлений и фактов приходится
производить пересмотр, перестановку и видоиз
менение понятий и положений, заменять более
узкие и тесные понятия более широкими и объ
емлющими, а это, в свою очередь, дает возмож
ность подчинить им новые явления и разновид
ности. Расчленение и уточнение знания идет
рука об руку с его расширением и обобщени
ем. Так, в частном и особенном сохраняется все
общее единство через бесконечную непрерыв
ность. Погружение мысли в частное и единич
ное есть вместе с тем возвращение всеобщего к
самому себе: целое находит себя в единичном, со
знает себя в индивиде и таким образом прихо
дит к самосознанию в человеческой личности. В системе знания, т. е. в системе научных по
нятий и положений, постепенно раскрывается In the system of knowledge, i. e. the system
of scientific concepts and propositions, the ho
listic unity of being is gradually revealed. Cohen
quotes from memory the ancient dictum of Par
menides on the identity of thought and being. To В системе знания, т. е. в системе научных по
нятий и положений, постепенно раскрывается В системе знания, т. е. в системе научных по
нятий и положений, постепенно раскрывается 112 A. L. Sakketti целостное единство бытия. Коген приводит на
память древнее изречение Парменида о тожде
стве мышления и бытия. Разумеется, однако, та
кое тождество знания и бытия присуще лишь со
вершенному, полному знанию, которое есть бес
конечное задание, предел, высшая цель, идеал
адекватного знания, вполне совпадающего со сво
им предметом, бытием. Для знания же, развива
ющегося исторически, такой идеал может слу
жить лишь немеркнущей путеводной звездой,
указывающей направление и путь непрестанно
го развития знания, и вместе с тем полагать ме
рило истинности всякого достигнутого знания,
или идею, как ее мыслил божественный Платон. Идея как бесконечный предел целостного един
ства знания и его предмета, системы мышления
и бытия, таким образом, есть и животворящий
источник возникновения, основоначало всякого
знания, ἀρχή, Ursprung, и вместе с тем его задание,
конечная цель, τέλος. Идея одновременно есть ис
тина знания, самое бытие, и вместе с тем развер
тывается как последовательный ход или путь по
знания в направлении его конечной цели, или
метод познания. Начало, цель и путь — все это
внутренне связанные моменты единого непре
рывного живого знания, в своем целостном един
стве совпадающего с самим бытием. 18 The word “truth” has differing meanings in different
languages: thus, in Russian istina means both searching
and the searched for, in Greek ἀλήθεια means uncovering,
revealing and that which is revealed, and in German
Wahrheit means preservation (author’s note). 35 Слово [«]истина[»] на разных языках имеет различное
и неодинаковое значение: так, по-русски истина значит
одновременно искание и искомое, по-гречески ἀλήθεια оз
начает раскрытие, откровение и то, что раскрывается,
по-немецки Wahrheit означает сохранение (примеч. авт.). 36 [Сноска вычеркнута:] Слово философия, φιλοσοφία,
сложилось в среде Сократа — Платона и означает лю
бовь, стремление к мудрости. 19 [Crossed out footnote:] The word philosophy, φιλοσοφία,
came into use in the Socrates-Plato milieu. It means love
of and a striving towards wisdom. 5 Такова, например, возникшая срав
нительно недавно физиология, объясняющая
биологические процессы органической жизни
физическими и химическими причинами (фи
зико-химические процессы питания, кровоо
бращения, процессы в мускульной и нервной
ткани) и как бы перекидывающая мост между
органическим и неорганическим миром явле
ний. Наконец, не следует забывать, что все от
расли современного знания исторически коре
нятся в единой всеобъемлющей и нерасчленен
ной сфере знания, в древности получившей как
раз наименование софии, мудрости, а потом —
философии36, откуда они постоянно выделя
лись, развивались путем расчленения, диффе
ренцирования. Это обстоятельство указыва
ет на внутреннюю непрерывность, сродство и
целостное единство знания, которое так или
иначе должно быть восстановлено. Восстанов
ление целостного единства знания может быть
достигнуто не иначе как строго методически, а
именно постепенным приведением всех отрас
лей знания в единую цельную систему знания. of their discreteness, there is a certain mutu
al cooperation, connection and solidarity be
tween them. Some branches of knowledge play
an auxiliary role of material to other branches
while enjoying the same status with regard to
other branches. In addition to this, the devel
opment of science from time to time sees the
birth of new mediating branches of knowl
edge. An example in point is the relatively re
cent birth of physiology which explains the
biological processes of organic life by physical
and chemical causes (physical-chemical pro
cesses of nutrition, blood circulation, processes
in the muscle and nerve fabric) and throws, as
it were, a bridge between the organic and inor
ganic worlds of phenomena. Finally, it should
be kept in mind that all branches of modern
knowledge are historically rooted in the unified
all-embracing and integral sphere of knowl
edge which in ancient times was called sophia,
or wisdom, and then philosophy,19 from which
they constantly became isolated through di
vision and differentiation. This circumstance
points to the inner uninterruptedness, kinship
and holistic unity of knowledge which must in
any case be restored. Holistic unity of knowl
edge can only be restored in a rigorously me
thodical way, namely by gradually bringing all
the branches of knowledge into a unified inte
gral system of knowledge. 5 be sure, such identity of knowledge and being
is characteristic only of perfect and complete
knowledge which is an infinite task, the limit,
the supreme end, an ideal of adequate knowledge
fully coinciding with its object, with being. For
knowledge evolving historically such an ideal
can only be an ever bright lodestar showing the
direction and path of the constant development
of knowledge and at the same time providing
the measure of the truth of all achieved knowl
edge, or the idea, as the divine Plato thought it. The idea as the infinite limit of the whole uni
ty of knowledge and its subject, the system of
thought and being is thus a life-giving origin of
the emergence, the first beginning of all knowl
edge, ἀρχή, Ursprung, and at the same time its
task, the ultimate end, τέλος. The idea is at the
same time the truth of knowledge, being itself
and yet it unfolds as the progressive course or
path of cognition towards its ultimate end, or
the method of cognition. The beginning, end and
path are all inherently interconnected aspects of
the unified, uninterrupted and living knowl
edge which in its whole unity coincides with
being itself. 6 6 6 It may now be clear why Cohen orients phi
losophy in the fact of knowledge. To orient
philosophy in the fact of knowledge means
nothing else than to develop the inner essence
of knowledge, its inner infinity, fullness and
completeness, to reveal the truth,18 the idea, i. e. the system which coincides with being itself. Indeed, in spite of the fragmentariness of cer
tain areas of knowledge such as mathematics,
physics, chemistry and biology and in spite Теперь может стать понятно, почему Коген
ориентирует философию в факте знания. Ори
ентировать философию в факте знания ведь
значит не что иное, как развивать внутреннюю
сущность знания, его внутреннюю бесконеч
ность, полноту и целостность, раскрывать его
истину35, идею, т. е. систему, совпадающую с са
мим бытием. В самом деле, несмотря на отры
вочность отдельных отраслей знания, каковы
математика, физика, химия, биология, несмо 113 А. Л. Саккетти тря на их прерывность, между ними наблюда
ется определенное взаимное сотрудничество,
связь и солидарность. Одни отрасли знания по
отношению к другим играют вспомогательную,
служебную роль материала, пользуясь тем же со
стороны других. Развитие науки, сверх того, от
времени до времени показывает нарождение
новых посредствующих промежуточных отрас
лей знания. 1 1 1 Методическое объединение всех отраслей
знания в систему осуществимо путем приве
дения к единству основных начал, или принци
пов, наук. Таким образом, трансценденталь
ный метод, или ориентирование философии в
факте науки, приводит к системе науки и тем Methodical unification of all the branches
of knowledge in a system can be carried out
by bringing about a unity of the main fundamen
tals, or principles of sciences. Thus, the transcen
dental method, or orienting philosophy in the 114 A. L. Sakketti самым — к раскрытию целостного единства
предмета знания, бытия. При этом, согласно
мысли Когена, такое раскрытие единства бы
тия должно происходить строго методически
и заключается в планомерном и систематиче
ском развитии основных начал самых наук. Та
ким образом, завоевания и приобретения нау
ки, научного знания отнюдь не утрачиваются
для философии и вместе с тем сохраняется за
конный и плодотворный союз науки с филосо
фиею. Путь восхождения мысли через науку к
философии есть, так сказать, восхождение по
ступеням бесконечности. fact of science, leads to a system of science and
thereby to the discovery of the holistic unity
of the object of knowledge, being. In Cohen’s
view, the unity of being should be revealed in
a strictly methodical way and should consist in a
planful and systemic development of the main
principles of the sciences themselves. Thus, the
gains and acquisitions of science, of scientific
knowledge are by no means lost for philosophy
and at the same time the legitimate and fruitful
alliance between science and philosophy is pre
served. The ascent of thought through science
to philosophy is, so to speak, an ascent up the
steps of infinity. Ибо ведь научные понятия и положения,
системы понятий и научных законов, объемля
необозримые множества отдельных явлений,
фактов, представляют собою как бы бесконеч
ности первого порядка. При всем том область
любой науки, в свою очередь, есть как бы бес
конечность второго порядка. Ведь каждая нау
ка, как, например, геометрия, механика, пред
ставляет безграничное поле связанных меж
ду собой положений, аксиом, теорем, законов
или, как отрасли биологии, ботаника, зооло
гия, — безгранично разветвляющееся древо
понятий. Ни в один исторически данный мо
мент развитие науки не может быть признано
окончательно завершенным; всегда возможно
открытие новых явлений и фактов, образова
ние новых понятий и научных законов, как бо
лее специальных, так и все более общих. 1 Тем не
менее вся бесконечная внутри сфера каждой
науки объемлется одним основным понятием,
или принципом, идеею; вся безграничная сфе
ра геометрии объемлется единой идеею про
странства, вообще сфера математики — идеею
количества, область физики — идеею законо
мерности движения, область биологии — иде
ею органической целесообразности и т. д. Идеи
числа, пространства, движения, органической
целесообразности содержат отчет научного со
знания в своих собственных существенных,
конститутивных принципах, т[ак] к[ак] оно, Indeed, scientific concepts and propositions,
systems of concepts and scientific laws, span
ning as they do the vast multitude of phenom
ena and facts are, as it were, infinities of the
first order. However, the area of any science is
in turn, as it were, an infinity of the second or
der. For every science, as for example geome
try and mechanics, offers a boundless field of
interconnected propositions, axioms, theorems,
laws or, like the subdivisions of biology, bot
any and zoology, are an infinitely branching
tree of concepts. At no historical moment can
the development of science be declared to be fi
nally completed; there is always the possibili
ty of discovering new phenomena and facts,
forming new concepts and scientific laws, both
more specialised and more general. Even so,
the whole inwardly infinite sphere of every
science is informed with one basic concept,
or principle, idea; the whole infinite sphere
of geometry is embraced by the single idea of
space, the sphere of mathematics in general by
the idea of quantity, the sphere of physics by
the idea of the law-governed movement, the
sphere of biology by the idea of organic pur
posiveness, etc. The ideas of number, space,
movement, organic purposiveness contain a
report of scientific consciousness regarding 115 А. Л. Саккетти its own essential constitutive principles, since
this consciousness finds these ideas in all as
pects of the corresponding branches of knowl
edge. These main ideas in turn are, as it were,
infinites of the second order. Finally, separate
branches of knowledge and their main princi
ples do not exhaust the whole area of knowl
edge because the history of the development of
science attests to the birth of ever new branch
es of knowledge, such as mathematical analy
sis, biology, physiology, psychology and other
intermediate branches. This presents the chal
lenge of finding a life-giving principle of the
whole sphere of knowledge, the whole system
of science. 1 Such a life principle in outlining an
infinite “circle of knowledge” discovers the
law of gradual building and determination of
the object of knowledge. сознание, находит эти идеи во всех моментах
соответствующих отраслей знания. В свою оче
редь, эти основные идеи представляют собою
как бы бесконечности второго порядка. Нако
нец, отдельные отрасли знания и их основные
принципы не исчерпывают всей области зна
ния, т[ак] к[ак] история развития науки свиде
тельствует о нарождении новых и новых отрас
лей знания, как математический анализ, био
логия, физиология, психология и другие про
межуточные. Поэтому возникает задача оты
скания животворящего принципа всей сферы
знания, всей системы науки. Такой жизненный
принцип, очерчивая бесконечный «круг зна
ния», вместе с тем открывает закон постепенно
го построения и определения самого предмета
знания, объекта. Так предмет постепенно раскрывается37
в чисто количественных, потом в простран
ственных определениях, потом в известных ме
ханических, химических, биологических соот
ношениях. Так, прежде всего предмет есть гео
метрическое тело, т. е. место точек, затем систе
ма центров сил, равновесие энергий, далее —
организм, т. е. система органов и функций, со
вокупность живых организмов объединяется
в «надорганическую» группу, или обществен
ное «тело». В этой последовательной лестнице
определений низшая ступень отнюдь не отме
няется, не упраздняется, но, напротив, сохра
няется в высшей ступени, как превзойденный,
подчиненный, но необходимый момент. Таким
же точно образом вся сфера отдельных отрас
лей знания, наук, в свою очередь, сохраняется,
содержится как подчиненный, превзойденный
момент в философии, которая в науке почерпа
ет свое многообразно специальное содержание
и сохраняет в ней твердую почву. Так взаимно
оплодотворяют друг друга философия и нау
ка, связанные неразрывными узами: наука со
общает философии удостоверенное исследова
нием положительное содержание, философия — Thus the object is gradually revealed in
purely quantitative, then in spatial definitions
and finally in certain mechanical, chemical
and biological correlations. Thus, an object is
above all a geometrical body, i. e. the place of
dots, then a system of force centres, balance of
energies, then an organism, i. e. a system of or
gans and functions; a combination of living or
ganisms is combined to form a “supra-organic”
group or the social “body”. On this successive
ladder of definitions the lower stage, far from
being cancelled or abolished is, on the contrary,
retained in the higher stage as a superseded,
subordinate but necessary element. 37 Вставка вместо зачеркнутого «является». 2 2 An objection may be raised, however. Let
us assume that the orientation of philosophy
in science mutually pollinates science and phi
losophy, let us assume that one of the tasks of
philosophy is to develop the life principle and
inner essence of science, to reveal the system
of scientific knowledge and at the same time
of objective being as the true object of science
as a whole. And yet the world is not identical
with nature alone, which constitutes the object
of science, theoretical knowledge in the nar
row sense. In addition to nature and the science
of it there is also the infinitely rich and varied
world of the human being’s spiritual and cul
tural life. In addition to nature and science
there is the world of human activity and so
cial relations, the world of creative art, pleasure
and religious service. The human being’s living
consciousness is not limited to scientific knowl
edge alone; in addition to intellect the human
being has the striving of the will and tension,
and finally the inexhaustibly rich and profound
world of feelings, aspirations and hopes. Is it
possible that philosophy, so thoroughly and
honestly oriented in science and theoretical
knowledge, would ignore such essential and
important interests and needs of the human
spirit, shut its eyes to them and pass them over
in silence as an annoying illusion? Нам могут, однако, возразить: допустим, что
ориентирование философии в науке оплодот
воряет взаимно и науку, и философию, допу
стим, что одна из38 задач философии заключа
ется в развитии жизненного принципа и вну
тренней сущности науки, в раскрытии систе
мы научного знания и вместе — самого пред
метного бытия как подлинного объекта науки
в целом. Тем не менее ведь мир не тождествен
только природе, составляющей предмет науки,
теоретического знания в тесном смысле. Ведь
кроме природы и науки о ней существует еще
бесконечно богатый и разнообразный мир ду
ховной, культурной жизни человека. Ведь кро
ме природы и науки, существует мир челове
ческой деятельности и общественных отноше
ний, мир художественного творчества, наслаж
дения и религиозного служения. Ведь живое
сознание человека не исчерпывается только на
учным знанием; ведь кроме интеллекта чело
веку присуще волевое устремление и напряже
ние, наконец — неисчерпаемо богатый и глубо
кий мир чувств, чаяний и упований. 38 Вставка двух слов: «одна из». 1 Similar
ly, the whole sphere of separate branches of
knowledge, the science, in turn, is preserved,
contained as a subordinate, sublated element in
philosophy which draws from science its var
iously specialised content and preserves sol
id ground in it. Thus philosophy and science
cross-pollinate, being linked by inseparable
bonds: science provides philosophy with pos
itive content certified by investigation and phi
losophy discovers the perspectives for science, 116 A. L. Sakketti lends it inner meaning and value, reports on its
tasks, object and method. This is the deep and
underlying meaning of the “orientation” of phi
losophy in science. открывает науке ее перспективы, сообщает ей
внутренний смысл и ценность, дает отчет в ее
задачах, предмете и методе. Вот глубокий и
полновесный смысл «ориентирования» фило
софии в науке. открывает науке ее перспективы, сообщает ей
внутренний смысл и ценность, дает отчет в ее
задачах, предмете и методе. Вот глубокий и
полновесный смысл «ориентирования» фило
софии в науке. 2 2 Ориентирование философии
есть лишь первый, наиболее скромный шаг ее,
за которым должны последовать другие, бо
лее смелые и решительные. Мало того, имен
но для того философия Когена и ориентиру
ется сначала в факте научного знания, чтобы
тем свободнее развернуть более глубокие осно
вы культурной жизни человека. Первый шаг на
пути обеспечения самостоятельных, неотчуж
денных прав культурного сознания составля
ет признание Когеном, вслед за Кантом, границ
научного, т. е. теоретического в тесном смысле,
познания природы, границ человеческого ин
теллекта. и дать отчет во всех разнообразных направле
ниях и гранях человеческого сознания. Мало
того, неуспех ее в этом деле свидетельствовал
бы о полной несостоятельности самого исход
ного начала и указывал бы на необходимость
для построения системы иных начал и осно
ваний философии, нежели ориентирование в
факте науки. И в самом деле, философия Ко
гена никогда не обнаруживала притязаний
на то, чтобы ограничить и замкнуть кругозор
философского творчества рамками научного
«панметодизма». Ориентирование философии
есть лишь первый, наиболее скромный шаг ее,
за которым должны последовать другие, бо
лее смелые и решительные. Мало того, имен
но для того философия Когена и ориентиру
ется сначала в факте научного знания, чтобы
тем свободнее развернуть более глубокие осно
вы культурной жизни человека. Первый шаг на
пути обеспечения самостоятельных, неотчуж
денных прав культурного сознания составля
ет признание Когеном, вслед за Кантом, границ
научного, т. е. теоретического в тесном смысле,
познания природы, границ человеческого ин
теллекта. В самом деле, теоретическое познание чело
века, наука о природе строго ограничена и об
условлена пределами опыта. Правда, границы
эти, как мы видели, подвижны и текучи; ка
ждое достижение научного знания связано с
объемом наличного опыта, восприятия, и по
тому ограничено и условно. Every concept, every scientific law is a con
ditional supposition, a hypothesis which stands
or falls together with the phenomena and facts
that it unifies. Discovery of new phenomena
and facts calls for new concepts and propo
sitions. Scientific knowledge lives, as it were,
from day to day, moving step by step along the
road of partial achievements and relative truths. Purely common knowledge, idea in science un
folds gradually, step by step and is always pre
sented in the shape of a separate achievement,
always bears the mark of being conditional and
limited, is always a hypothesis; the universal
manifests itself in the particular and individ Каждое понятие, каждый научный закон
представляет условное предположение, гипоте
зу, которая держится или рушится вместе с яв
лениями и фактами, которые объединяет. 2 Неужели
же философия, столь основательно и добросо
вестно ориентированная в науке, в теоретиче
ском знании, оставит без внимания эти столь
существенные и важные интересы и потребно
сти человеческого духа, закроет на них глаза и
умолчит о них, как о досадной иллюзии? Напротив, философия, ориентированная
в научном знании, не только не игнорирует
общественной, художественной и религиоз
ной жизни человека, но, наоборот, настоятель
нейшим образом призвана раскрыть основы On the contrary, philosophy oriented in sci
entific knowledge, far from ignoring the social,
artistic and religious life of the human being, is
urgently called upon to reveal the foundations
and give a report on the diverse areas and fac
ets of human consciousness. Moreover, lack of 117 А. Л. Саккетти success in this matter would attest to the total
failure of the starting foundation and spell the
need to build a system of principles and foun
dations of philosophy different from the ori
entation in the fact of science. Indeed, Cohen’s
philosophy never sought to circumscribe the
horizon of philosophical creativity and limit it
to scientific “pan-methodism”. Orientation of
philosophy is but its first and the most modest
step which should be followed by other, bolder
and more decisive steps. Indeed, Cohen’s phi
losophy first orients itself in the fact of scientif
ic knowledge precisely in order to have more
freedom in revealing the deeper foundations
of the human being’s cultural life. The first
step towards asserting independent and inher
ent rights of cultural consciousness is Cohen’s
recognition, after Kant, of the limits of scientif
ic (i. e. “theoretical” in the narrow sense) cogni
tion of nature, the limits of the human intellect. Indeed, the human being’s theoretical cogni
tion, the science of nature is strictly limited to
and conditioned by experience. True, these lim
its, as we have seen, are fluid; every achieve
ment of scientific knowledge depends on the
amount of available experience and perception,
and is therefore limited and relative. и дать отчет во всех разнообразных направле
ниях и гранях человеческого сознания. Мало
того, неуспех ее в этом деле свидетельствовал
бы о полной несостоятельности самого исход
ного начала и указывал бы на необходимость
для построения системы иных начал и осно
ваний философии, нежели ориентирование в
факте науки. И в самом деле, философия Ко
гена никогда не обнаруживала притязаний
на то, чтобы ограничить и замкнуть кругозор
философского творчества рамками научного
«панметодизма». 2 От
крытие новых явлений и фактов требует новых
понятий и положений. Научное знание живет,
т[ак] сказ[ать], изо дня в день, движется шаг за
шагом по пути частичных достижений и услов
ных истин. Чисто всеобщее знание, идея в на
уке развертывается постепенно, шаг за шагом,
всегда дана в виде частного достижения, всег 118 A. L. Sakketti да сохраняет пятно условной ограниченности,
всегда есть гипотеза; всеобщее раскрывается в
особенном и частном, каковы видовые понятия
и специальные научные законы. Идея безуслов
ного всецелого знания таким образом прежде
всего показывает, что не есть действительное
исторически данное научного знание, факт на
уки, и вместе с тем указывает, чем знание долж
но быть на самом деле. Целостное единство и
полнота бытия для научного знания есть «вещь
в себе», обнять, схватить которую во всей полно
те знание стремится, никогда не будучи в состо
янии вполне достигнуть искомой цели. Итак,
теоретическое знание, наука о природе всегда
есть ограниченная, условная и частичная исти
на, границы которой, однако, подвижны. ual, such as species, concepts and specialized
scientific laws. The idea of the uncondition
al universal knowledge thus indicates above
all what is not real historically given scientific
knowledge, a fact of science, and at the same
time shows what knowledge ought to be in re
ality. For scientific knowledge holistic unity
and completeness of being is “a thing in itself”
which knowledge seeks to embrace and cap
ture in all its completeness, never being able to
quite achieve the coveted end. Thus, theoreti
cal knowledge, the science of nature is always a
limited, relative and partial truth whose limits,
however, are fluid. 3 3 The idea of integral and complete unity,
however, is not only a negative limit of scientific
knowledge, but at the same time a positive el
ement. Man does not live only in the moment;
he looks into the future, sets goals and makes
plans and implements them through purposive
and planful activity, personal and conjoint efforts. From the viewpoint of execution, human activ
ity is taking apart and combining the forces of
nature; it has long been common knowledge
that the human being cannot create new forces
in nature, he can merely combine them in var
ious ways and achieve new results through
these combinations. All the technical wonders
that enable man to dominate the forces of na
ture are conditioned by his purposive planful
activity. 2 Идея как
норма должного, долженствующего бытия есть
предмет второй части системы философии,
этики чистой воли, ориентированной не толь
ко в науке и в логике чистого познания, но бли
жайшим образом — в праве и в науке права. Нередко (напр., Е. В. Спекторский) подвергали
сомнению существование науки права как дей
ствительной систематической и наукообразной
дисциплины. Другие (Б. А. Кистяковский) по
лагают, что этика может быть ориентирована
единственно в юридической догматике. Одна
ко при этом нередко упускают из вида то об
стоятельство, что современная наука проводит
грань между общей и особенной частью отраслей
права. Тогда как особенная часть есть приклад activity of many people replenishes the limit
ed power of each and enables them to achieve
unheard-of success, as witnessed by the entire
diverse and intricate fabric of the cultural life
of human societies. Left to personal arbitrari
ness, the human being’s activity inevitably
leads to the domination of spontaneous forces
and blind aspirations, the chaos of the “natu
ral state”, the struggle of all against all, anar
chy in which shameless impudence and crude
force have an advantage. In contrast, in a com
munity of others a person meets not only with
constraint and narrowing of his activity but,
on the contrary, with a broadening, enhance
ment and deepening of one’s personality. In
the person of “the other” the human being for
the first time finds a reflection of his self, finds
himself in the other and becomes aware of his
personality. Order and holistic unity are intro
duced into the mutual relations among peo
ple by the idea of the being of “the ought”, Sein
des Sollens, norms of law and state which regu
late and guide the multifarious conjoint activi
ties of people and create the normative order of
cultural being, the state and social being of the hu
man being. Idea as a norm of what ought to be
is the subject of the second part of the system
of philosophy, the ethics of pure will, orient
ed not only in science and in the logic of pure
cognition, but more immediately in law and
in the science of law. The existence of the sci
ence of law as a real systemic and science-like
discipline has often been questioned (e. g. by
E[vgeniy] A. Spektorsky). Others (B[ogdan]
A. Kistyakovsky) believe that ethics can be
oriented only in legal dogma. 2 Thus, goals and plans of the human
being create a new order of nature, new com
binations of natural forces. Finally, the hu
man being’s activity is limited by the activity
of other human beings; independent activity
of a separate human being, his freedom, clash
es with the activity of other people, meets with
obstacles on their part, joint and coordinated Идея всецелого и полного единства, одна
ко, не есть только отрицательная граница на
учного знания, но вместе с тем и начало поло
жительное. Человек живет не только данною
минутою, сегодняшним днем; он заглядывает
в будущее, ставит цели и строит планы и осу
ществляет их целесообразной и планомерной
деятельностью, личными и совместными усили
ями. Со стороны осуществления, выполнения
деятельность людей представляет собою разъе
динение и соединение сил природы; давно из
вестно, что человек не может создать в приро
де новых сил и может лишь разнообразно со
четать, комбинировать наличные силы при
роды и этими комбинациями достигать новых
результатов. Все чудеса техники, создающие го
сподство человека над силами природы, обу
словлены его целесообразной, планомерной де
ятельностью. Так цели и планы человека тво
рят новый порядок естества, создают новые со
четания сил природы. Наконец, деятельность
человека встречает границу и предел со сто
роны деятельности другого человека; самосто
ятельная деятельность отдельного человека, 119 А. Л. Саккетти его свобода сталкивается с деятельностью дру
гих людей, встречает с их стороны помеху, пре
пятствие, совместная и согласная деятельность
многих людей восполняет ограниченные силы
каждого и дает возможность достигать неслы
ханных успехов, свидетельством чего является
вся разнообразная и сложная ткань культурной
жизни человеческих обществ. Предоставлен
ная личному произволу деятельность человека
неизбежно приводит к господству стихийных
сил и слепых стремлений, к хаосу «естествен
ного состояния», к борьбе всех против всех, к
анархии, в которой перевес имеет беззастенчи
вая дерзость и грубая сила. Напротив, в сообще
стве других человек встречает не только огра
ничение, сужение своей деятельности, но, на
оборот, расширение, восполнение и углубле
ние своей личности. Впервые в лице «другого»
человек находит отражение своего я, находит
себя в другом, сознает свою личность39. Поря
док и целостное единство во взаимные отноше
ния людей вносит идея должного бытия, Sein des
Sollens, нормы права и государства, регулирую
щие, направляющие разнообразную совмест
ную деятельность людей и созидающие нор
мативный порядок культурного бытия, государ
ственно-общественный быт человека. 39 Это и предыдущее предложения внесены в текст от
руки. 2 However, at the
same time, the fact that modern science draws
a distinction between general and specialised
areas of law is often overlooked. Meanwhile
th
i li
d
t i
th
li d t
h i
l 120 A. L. Sakketti in revealing and developing the inner constitu
tion and content of the norms of effective law
in accordance with the rules of formal logic to
solve strictly limited tasks; the general part of
juridical disciplines, e. g. civil, state and crim
inal law, contains properly legal principles in
the shape of the main legal concepts and regu
lations, so-called constructions, such as family,
property, contract, various types of liabilities,
inheritance, state as a legal entity and as legal
relationship, crime, punishment etc. These le
gal constructions, which formerly referred to
so-called natural law, represent the general the
oretical part of the science of law in which the
ethics of pure will which is considered by Co
hen to be the logic of humanities, the sciences of
spirit, can be oriented. ная, техническая отрасль права (Штаммлер) и
состоит в раскрытии и развитии внутреннего
состава и содержания норм действующего пра
ва согласно правилам формальной логики для
разрешения тесно ограниченных задач; общая
часть юридических дисциплин, напр. граж
данского, государственного, уголовного права,
содержит подлинные юридические начала в
виде основных юридических понятий и положе
ний, т[ак] наз[ываемых] конструкций, как семья,
собственность, договор, различные виды обяза
тельств, наследование, государство как право
вое лицо и правоотношение, преступление, на
казание и т. д. Эти юридические конструкции,
в прежнее время относимые к области т[ак] на
з[ываемого] естественного права, представляют
собою общую теоретическую часть науки пра
ва, в которой может быть ориентирована этика
чистой воли, эта, по мысли Когена, логика гума
нитарных наук, наук о духе. 4 4 The first two parts of the system of philos
ophy, logic and ethics, reveal the idea: the for
mer in a particular limitation and the latter in
boundless generality and perfect purity. The
idea is revealed in the reality of nature not ful
ly, not entirely, but partially and gradually,
step by step limited by conditions and nega
tions; in short, nature is coordinate with and
represents “otherness of the idea”, Anderssein
der Idee, to use Hegel’s term. In contrast, the
ought is the idea in its isolated purity and whole
ness, Allheit, free from particular conditions and
restrictions. 40 Вставка. 41 Вставка вместо зачеркнутого «целостности». 40 Вставка.
41 Вставка вместо зачеркнутого «целостности».
42 Вставка двух слов: «в образе». р
у
42 Вставка двух слов: «в образе». 41 Вставка вместо зачеркнутого «целостности».
42 Вставка двух слов: «в образе». 40 Вставка.
41 Вставка вместо зачеркнутого «целостности». А. Л. Саккетти Степень воплощения идеала в действитель
ности удостоверяется в человеке несравнимым
и незаменимым художественным чувством,
бескорыстным эстетическим наслаждением. Третья часть системы философии, «Эстетика
чистого чувства», ведает принципы прекрасно
го в природе и в искусстве, исследует проявле
ния художественного творчества и наслажде
ния. В трех частях системы, «Логике», «Этике»,
«Эстетике», раскрываются отдельные стороны
всеединого, идеи: условная ограниченность
особенного, безусловная всеобщность всееди
ного, воплощение безусловного и всеобщего в
условном и особенном. Но ведь индивидуаль
ный человек, единичная личность раскрывает
в себе единство и внутреннюю связь всех пои
менованных моментов: частного, всеобщего и
особенного. В человеке связаны и сплетены и
познание, интеллект, и воля, и чувство, каждое
его «переживание», а не только некоторые из
них, многосторонни и многогранны, актив
но-пассивны43, представляют единство позна
ния, воли и чувства. Наконец, всеобщая идея
находит себя самое в единичном индивиде, в
живой человеческой личности. parable and irreplaceable feeling, by selfless
aesthetic pleasure. The third part of the philo
sophical system, Aesthetics of Pure Feeling, deals
with the principles of the beautiful in nature
and in art and studies the manifestations of ar
tistic creativity and pleasure. The three parts of
the system, Logic, Ethics and Aesthetics, look at
different aspects of the all-unity, the idea: the
relative limitation of the particular, the uncon
ditional oneness of the all-unity, the embod
iment of the unconditional and the general in
the conditional and particular. Yet the human
individual, the single person reveals in him
self the unity and inner connection of all the
above-mentioned elements: the singular, the
general and the particular. Interconnected and
interwoven in human being are cognition, in
tellect, will and feeling; every “experience”,
and not only some of them, is many-sided and
multi-faceted, active-passive,20 representing
unity of cognition, will and feeling. Finally, the
general idea finds itself in the singular individu
al, in the living human personality. The individual person, distinguishing and
opposing his own individuality to others,
finds in himself and in others the single com
mon essence, “finds himself in the other” and
the other in himself, becomes conscious of himself,
and attains self-consciousness. The one universal
spirit abides in the infinite variety of individu
al personalities. 43 Вычеркнуто: «(Петражицкий)». 20 Struck out here: (Petrazhitsky). 2 However, the pure idea of all-uni
ty (Allheit) taken as such in its isolation is not
guaranteed to be embodied in the particular
and the singular. On the contrary, the beautiful
in nature and in art demonstrates precisely the
embodiment of the ideal in reality, the whole in
the particular and the special, the idea in its vi
sual representation, in intuition, in image. The
degree to which the ideal is embodied in real
ity is attested in human being by the incom Две первые части системы философии, логи
ка и этика, раскрывают идею: первая в частном
ограничении, вторая — в безграничной все
общности и совершенной чистоте. Идея в дей
ствительности природы раскрывается не цели
ком, не вполне, но постепенно, отчасти, шаг за
шагом, ограниченная условиями и отрицани
ями; словом — природа есть рядоположность,
«инобытие идеи», Anderssein der Idee, говоря сло
вами Гегеля. Напротив, должное есть идея в ее
отрешенной40 чистоте и целостности, Allheit,
свободная от частных условий и ограничений. Но взятая сама по себе в ее отрешенности41 чи
стая идея всеединства (Allheit) не содержит ру
чательства своего воплощения в частном и еди
ничном. Напротив, прекрасное в природе и в
искусстве свидетельствует как раз о воплоще
нии идеального в действительности, целост
ного в частном и особенном, идеи в наглядном
представлении, в созерцании, в образе42. 121 А. Л. Саккетти А. Л. Саккетти The teaching on how the One
Universal Spirit is reflected and refracted in in
dividuals, in these monads, microcosms cre
ated in the image and likeness of the all-unity
constitutes the fourth part of the philosophical
system, Psychology, the teaching on the unity of
cultural self-consciousness, or the human soul. However, Cohen’s psychology has not been
printed yet, leaving us at the mercy of more or
less probable guesses and assumptions. In any
case, psychology in Cohen’s system is called
upon to unite, complete and crown the entire
edifice of philosophy. Отдельный человек, отличая и противопо
лагая свою собственную индивидуальность
другим, находит в себе и в других единую об
щую сущность, «находит себя в другом» и дру
гого в себе, сознает себя, приходит к самосозна
нию. В бесконечном разнообразии индивиду
альных личностей живет единый всеобщий
дух. Учение об отражении и преломлении Еди
ного Всеобщего Духа в индивидах, в этих мона
дах, микрокосмах, созданных по образу и по
добию всеединого, есть четвертая часть систе
мы философии — Психология, учение о един
стве культурного самосознания, или о душе че
ловека. Впрочем, психология Когена доныне не
напечатана, и здесь мы предоставлены на про
извол более или менее вероятных догадок и
предположений. Во всяком случае, психология
в системе Когена призвана объединять, завер
шать и увенчивать все здание философии. 122 A. L. Sakketti Таким образом, система философии, соглас
но замыслу и отчасти выполнению Когена, есть
последовательный ряд отражений и преломле
ний Всеединой и Чистой Истины, Идеи: сна
чала идея «является», «полагается» в ограни
чении и в условном отрицании, в «инобытии»
природы, затем идея раскрывается в отрешен
ной безусловной всеобщности и чистоте как
должное бытие, потом как частное и особенное
в наглядном представлении и художественном
созерцании; наконец, целое находит, обретает
себя в единичном, индивидуальном, приходит
к самосознанию в человеческой личности, в
душе человека. Так последовательно развивает
ся внутренняя жизнь идеи, развертывается по
степенно Система Чистой Философии. 44 Зачеркнута приставка «нео-». А. Л. Саккетти Thus, the system of philosophy, in ac
cordance with the design and partially the
fulfillment, is a sequence of reflections and re
fractions of the One and Pure Truth, the Idea:
first the idea “appears itself” and is “posited”
in restriction and relative negation, in the “oth
erness” of nature, then the idea reveals itself
in isolated unconditional universality and pu
rity as being what ought to be, then as singu
lar and particular in visual representation and
artistic intuition; and finally the whole finds
itself in the singular, in the individual, attains
self-consciousness in the human personality, in
the human soul. Thus the inner life of the idea
progressively develops and the System of Pure
Philosophy gradually unfolds. 5 To Descartes, the founder of the newest
mathematics (analytical geometry) and philo
sophical rationalism (Discourse on the Method,
Metaphysical Reflections on First Philosophy21) Co
hen is brought closer by the scientific spirit of
his philosophy, the link of his philosophising
to mathematics and the resulting rationalism. From Leibniz Cohen borrows the analysis of
the idea of mathematical infinity which is a fea
ture of the newest mathematics. The influence
of Kant’s transcendental philosophy on Cohen
has been mentioned above. Cohen made a sub
stantial step forward and transformed Kant’s
ideas thanks to the analysis of mathematical
infinity which Kant merely approached in his
Antinomies in the “Transcendental Dialectic”. Less stressed by Cohen is the link between his
philosophy and the ideas of Fichte the Elder. Finally, judging from his external manifesta
tions and express statements Cohen was defi
nitely hostile to Hegel’s Absolute Idealism. In
Hegel, as well as Fichte the Elder, Cohen finds
a lack of systematic dismemberment of the
idea, philosophical “romanticism”, manifested
by Fichte in the predominance of the practical
over the theoretical, of what ought to be over
what is, and by Hegel, in contrast, in the iden
tification of concept and idea, in the absorp
tion of the ethical ought by the logical existing. Cohen is equally negative and hostile towards
Fichte’s panmoralism and Hegel’s panlogism. He is particularly averse to the teachings of
Aristotle and Spinoza. Aristotle, according to
Cohen, is the founder of eclectic duality and re
alism in philosophy; “two souls” live in Aristo его учению характер этического идеализма по
преимуществу. Впрочем, эти же черты учение
Когена разделяет с учением Канта о примате,
или первенстве, практического разума над те
оретическим и с этическим идеализмом Фих
те Старшего. С Декартом, этим основателем
новейшей математики (аналитической геоме
трии) и философского рационализма («Рассуж
дение о методе», «Метафизические размышле
ния о первой философии»45), Когена роднит
свойственный его философии научный дух,
связь его философствования с математикою и
проистекающий отсюда рационализм. У Лейб
ница Коген заимствует анализ идеи математи
ческой бесконечности, характеризующий осо
бенность новейшей математики. Влияние тран
сцендентальной философии Канта на Когена
было отмечено выше. При этом существенный
шаг вперед и преобразование идей Канта у Ко
гена явилось как следствие анализа математи
ческой бесконечности, к которой Кант лишь
вплотную подошел в Антиномиях «Трансцен
дентальной диалектики». Менее подчеркивает
Коген связь своей философии с идеями Фихте
Старшего. 5 5 5 The reader familiar with the history of phi
losophy will easily find the features which
bring Cohen’s teaching closer to the systems
of the great creators of the Western Europe
an philosophy: Plato, Descartes, Leibniz, Kant,
Fichte and Hegel. Cohen himself never miss
es an opportunity or an occasion to stress the
historical continuity between his doctrines
and the ideas of Plato, Descartes, Leibniz and
Kant. With Plato, he shares the doctrine of pure
knowledge and being, the doctrine of the Idea;
that is why Cohen’s teaching may be character
ised as a new revival of Plato’s idealism, as con
temporary Platonism. Of particular importance
for understanding the historical continuity of
Cohen’s ideas is the link between his doctrine
and Plato’s doctrine of the idea as a condition
al supposition, hypothesis, ὑπόθεσις, in book VI
of The State, and the supreme idea of the good,
ἀγαθόν, as the unconditional, ἀνυπόθετον. The link
with these teachings of Plato on the suprema
cy of the ethical principle, the idea of the good,
seems to be largely responsible for the pre
dominance of the ethical idea of the ought in Читатель, знакомый с историей филосо
фии, легко найдет черты, сближающие учение
Г. Когена с системами великих творцов запад
ноевропейской философии: Платоном, Декар
том, Лейбницем, Кантом, Фихте и Гегелем. Ко
ген сам не упускает случая и повода указать на
исторически-преемственную связь своих уче
ний с идеями Платона, Декарта, Лейбница и
Канта. С Платоном его роднит учение о чистом
знании и бытии, учение об Идее; поэтому и са
мое учение Когена может быть охарактеризо
вано как новое возрождение Платонова идеа
лизма, как современный платонизм44. Особен
но существенна для понимания исторической
преемственности идей Когена связь его учения
с учением Платона об идее как условном пред
положении, гипотезе, ὑπόθεσις в VI книге «Госу
дарства» и о верховной идее добра, ἀγαθόν как
безусловном, ἀνυπόθετον. Связью с этими учени
ями Платона о верховенстве этического нача
ла, идеи добра, по-видимому, в значительной
степени обусловливается господство этической
идеи должного в системе Когена, придающее 123 А. Л. Саккетти Cohen’s system which lends his doctrine the
character of ethical idealism par excellence. As a
matter of fact, Cohen’s doctrine shares the same
features with Kant’s doctrine of the suprem
acy of practical reason over theoretical reason
and with the ethical idealism of Fichte the El
der. 21 Precisely, Meditations on First Philosophy. 45 Точнее, «Размышления о первой философии». ,
46 Вставка. 45 Точнее, «Размышления о первой философии».
46 Вставка 45 Точнее, «Размышления о первой философии».
46 Вставка. 5 Наконец, если судить по внешним
выражениям и прямым заявлениям Когена,
отношение его к Абсолютному Идеализму Ге
геля прямо враждебно. У Гегеля, как и у Фих
те Старшего, Коген находит отсутствие систе
матического расчленения идеи, философский
«романтизм», выразившийся у Фихте в заси
лии практического начала над теоретическим,
должного над сущим, у Гегеля же, напротив,
в отождествлении понятия и идеи, в поглоще
нии этически46 должного логически сущим. И
панморализм Фихте, и панлогизм Гегеля встре
чают одинаково отрицательное и враждебное
отношение со стороны Когена. Особливую же
неприязнь Коген питает к учениям Аристотеля
и Спинозы. Аристотель, по отзыву Когена, есть
родоначальник эклектического раздвоения и
реализма в философии; «две души» живут в
45 Т
Р
ф
ф 124 A. L. Sakketti Аристотеле: одна обращена к единичным яв
лениям, вещам, другая — к абсолютным «сущ
ностям» вещей, субстанциям. Поэтому Аристо
тель в глазах Когена, в прямую противополож
ность Платону, есть родоначальник обоих ви
дов философского реализма: эмпиризма и мета
физического онтологизма. Спиноза в глазах Ко
гена есть родоначальник философии, противя
щейся систематическому расчленению знания
и сливающей все области его в единой безраз
личной среде, абсолютной субстанции: панте
изм Спинозы, т[аким] обр[азом], есть родона
чальник всяческого «романтизма», философии
тождества Шеллинга и панлогизма Гегеля. tle: one faces singular phenomena, things, and
the other faces absolute “essences” of things,
substances. Thus Aristotle, Cohen believes, en
tirely in contrast to Plato, is the progenitor of
both types of philosophical realism: empiricism
and metaphysical ontologism. Spinoza, in Co
hen’s opinion, is the founder of a philosophy
which opposes systematic dismemberment of
knowledge, blending all its branches in a single
indifferent medium, an absolute substance: Spi
noza’s pantheism is thus the source of all kinds
of “romanticism”, Schelling’s philosophy of iden
tity and Hegel’s panlogism. 6 6 6 One of the features of Cohen’s philosophy,
as has been noted, is orientation in the fact of
science, specifically in mathematical natural sci
ence, i. e. mathematics and pure mechanics. This circumstance lends Cohen’s philosophy its
characteristic one-sidedness, abstract rational
ism and intellectualism. Одну из особенностей философии Когена
составляет, как было отмечено, ориентирова
ние в факте науки и притом — в математиче
ском естествознании, т. е. в математике и чистой
механике. Это обстоятельство придает филосо
фии Когена характерную односторонность, от
влеченный рационализм и интеллектуализм. 22 Görland, a disciple and follower of Cohen, orients
ethics in history: Albert Görland. Ethik als Kritik der
Weltgeschichte. [Reihe:] Wissenschaft und Hypothese.
XIX. Leipzig und Berlin, 1914 (author’s note). 47 Ученик и последователь Когена Гёрланд ориенти
рует этику в истории: Albert Görland. Ethik als Kritik
der Weltgeschichte. [Reihe:] Wissenschaft und Hypoth
ese. XIX. Leipzig und Berlin. 1914 (примеч. авт.). 5 р
у
Гегель, напротив, ориентировал свою фи
лософию в истории и религии: уже его «Фено
менология духа», это предначертание его бу
дущей системы, учение о развитии индивиду
ального и универсального Духа, по существу
есть философия истории. Его система филосо
фии открывается Логикою, т. е. учением об аб
страктном Бытии, о Боге, и завершается учени
ем об Абсолютном Духе, о Самой себя мысля
щей мысли, в которой Дух приходит к самосо
знанию. Коген, в противоположность Гегелю,
ориентирует «Логику чистого познания» в аб
страктных точных науках, математике и чи
стой механике, «Этику чистой воли» — в праве
и науке права47, «Эстетику чистого чувства» —
преимущественно в классическом искусстве. Такое ориентирование философии преимуще Hegel, on the contrary, oriented his philos
ophy in history and religion: already his Phe
nomenology of Spirit, a harbinger of his future
system, the doctrine of the development of the
individual and universal Spirit, is essentially a
philosophy of history. His system of philoso
phy opens with Logic, i. e. a doctrine of abstract
Being, of God and ends with the doctrine of the
Absolute Spirit, of thought that thinks itself,
in which the Spirit attains self-consciousness. Cohen, unlike Hegel, orients The Logic of Pure
Cognition in abstract exact sciences, mathemat
ics and pure mechanics, the Ethics of Pure Will
in law and the science of law22, and the Aesthet
ics of Pure Feeling mainly in classical art. The
orientation of philosophy primarily in abstract, 125 А. Л. Саккетти constructive elements of consciousness lends
Cohen’s philosophy the character of rational
ism, or abstract idealism, as Hegel described the
philosophy of Kant and Kantianism. ственно в абстрактных, конструктивных эле
ментах сознания придает философской мыс
ли Когена характер рационализма, или отвле
ченного идеализма — так Гегель характеризовал
философию Канта и кантианство. y
The philosophies of Hegel and Cohen are
essentially homogeneous systems of idealism:
Hegel’s speculative thinking and Cohen’s pure
cognition reveal essential kinship. However,
there is an important difference of shades be
tween their systems. Cohen’s “pure knowl
edge” is based on mathematics and mechanics
and seeks ultimately to reduce the variety of
qualitative differences to uniform mechanical,
quantitative relations. Accordingly, Cohen pre
fers explanatory building and analysis, paying
comparatively less attention to the classifica
tion of concepts, which unfold a wealth of qual
itative diversity; he clearly prefers Plato the
mathematician to Aristotle the biologist. 7 The features of the philosophical current of
thought represented by Hermann Cohen are
intertwined in a single knot: in his attempt to
solve the religious problem following the path
charted by Kant. Kant, as is well-known, tried
to solve the religious problem by remaining
“exclusively within the limits of reason”, inner
halb der Grenzen der blossen Vernunft. Особенности философского течения мыс
ли, представленного Г. Когеном, сплетаются в
едином узле: в его попытке разрешить религи
озную проблему по пути, проложенному Кан
том. Как известно, Кант попытался дать ре
шение религиозной проблемы, оставаясь «ис
ключительно в пределах разума», innerhalb der
Grenzen der blossen Vernunft. f
Теоретический разум, наука изучает связь
явлений сущего согласно закону причинности,
т. е. необходимой последовательности во време
ни. Практический разум, или нравственный
закон свободы, категорический императив,
предписывает то, что должно быть, дает норму
должного поведения. Согласно «критической»
раздельности, расчлененности теоретическо
го и практического разума, невозможно умоза
ключать прямо и непосредственно от должно
го к сущему, от требований нравственного за
кона к реальной действительности. Решение
религиозной проблемы Кантом в двух словах
сводится к «постулату практического разума»:
для того, чтобы действовать в согласии с требо
ваниями нравственного закона свободы, необ
ходимо иметь уверенность, что нравственное
усилие не останется втуне, не пропадет пона
прасну, но воплотится в действительности. f
Theoretical reason, science studies the con
nection between appearances of the existing in
accordance with the law of causality, i. e. neces
sary sequence in time. Practical reason, or the
moral law of freedom, the categorical impera
tive prescribes what ought to be, sets the norm
of due behavior. In accordance with “critical”
separateness, disconnection between theoreti
cal and practical reason, it is impossible to di
rectly conclude from what ought to be to what
exists, from the demands of the moral law to re
ality. In a nutshell, Kant’s solution of the reli
gious problem boils down to the “postulate of
practical reason”: to act in accordance with the
demands of the moral law of freedom, it is nec
essary to be sure that the moral effort will not
be in vain, will not be wasted, but will be im
plemented in reality. Отсутствие высшей инстанции, ведающей
единство сущего и должного, теоретического и
практического Разума, восполняется у Канта48
верою в существование нравственного миропо
рядка, чем создается ручательство осуществи
мости в действительности нравственно-долж
ного. Постулат нравственной веры гласит: ты
должен, следовательно, ты можешь. 48 Вставка: «у Канта». 5 By
contrast, there is a touch of contempt in He
gel’s attitude to abstract mathematical knowl
edge which reduced the diversity of the world,
reality to the uniformity of quantitative rela
tions, impersonal and indifferent to the vast
array of essential qualitative differences in re
ality. However, Hegel repeatedly stresses the
inherent kinship between his ideas and Aristo
tle’s doctrines. Философия Гегеля и Когена, по существу,
однородные системы идеализма: спекулятив
ное мышление Гегеля и чистое познание Коге
на существенно родственны. Но между систе
мами того и другого есть важная разница в от
тенках. «Чистое знание» у Когена основано на
математике и механике, стремится в конечном
пределе свести разнообразие качественных раз
личий к единообразию механических, количе
ственных отношений. Коген обнаруживает со
ответственное предпочтение объяснительному
построению, анализу, и придает сравнитель
но меньшее значение классификации поня
тий, развертывающих богатство качественных
многообразий, оказывает явное предпочтение
математику Платону перед биологом Аристо
телем. Напротив, Гегель относится несколько
презрительно к абстрактному математическо
му знанию, сводящему разнообразие мира —
действительности к единообразию количе
ственный отношений, безразличному и равно
душному к богатству существенных качествен
ных различий действительности. Вместе с тем
Гегель неоднократно указывает на внутреннее
сродство своих идей с учениями Аристотеля. Thus, Hegel’s philosophy and that of Co
hen have one-sided orientations, one in math
ematics and the other in human history. So
they seem to mutually complement and correct
one another, with the full truth perhaps lying
in their systemic combination. To dig deeper,
the full truth should be sought in a synthesis of
Plato and Aristotle, of mathematical building
and classification of concepts, abstract ideal
ism and concrete realism. An attempt to syn
thesise Plato and Aristotle is known to have
been made in the philosophy of Plotinus and
Neo-Platonism. Итак, как философия Гегеля, так и фило
софия Когена односторонне ориентированы,
одна в математике, другая в истории человече
ства. Поэтому они как бы взаимно пополняют
и исправляют одна другую, и полная истина,
быть может, — в их систематическом соедине
нии. Если восходить глубже, то полноту исти
ны следует искать в синтезе Платона и Аристо
теля, математического построения и класси
фикации понятий, абстрактного идеализма и
конкретного реализма. Попытка синтеза Пла
тона и Аристотеля, как известно, дана в фило
софии Плотина и неоплатонизма. 126 A. L. Sakketti A. L. Sakketti 7 7 7 Коген, со
храняя в общем решение Канта, в системе фи
лософии делает дальнейший шаг: он форму
лирует верховный «закон правды-истины»,
утверждающий единство сущего и должного бы
тия, всеединство чистой идеи. Однако как «за
кон правды-истины», так и развитие идеи Бо Kant makes up for the absence of a higher
authority in charge of the unity of what exists
and what ought to be, of theoretical and practi
cal Reason, by faith in the existence of the mor
al world order, which guarantees that what
morally ought to be can be realised. The pos
tulate of moral faith says: you must, therefore
you can. Cohen, while generally adhering to
Kant’s solution, takes a further step in his sys
tem of philosophy: he formulates the supreme
“law of truth-verity” which asserts the unity of
what is and what ought to be, the all-unity of the
pure idea. However, Cohen, following Kant, re
fers both the “law of truth-verity” and the de
velopment of the idea of Deity to the Ethics of 127 А. Л. Саккетти жества Коген вслед за Кантом относит к «Этике
чистой воли». Такая односторонность равноцен
на односторонности Гегеля, относившего поня
тие Бога к логике, или учению о бытии. Прав
да, такая односторонность Гегеля возмещалась
тем, что раскрытие абсолютного духа он видит
в истории человеческой мысли. Наконец, П. На
торп склонен отнести решение той же пробле
мы к эстетике. Тем не менее отнесение религи
озной проблемы как в область логики, так равно
в область49 этики либо эстетики, т. е. отдельной
части системы философии, не может быть при
знано состоятельным решением вопроса. Pure Will. Such one-sidedness is equal to the
one-sidedness of Hegel who referred the con
cept of God to logic, or the doctrine of being. True, Hegel’s one-sidedness was compensat
ed for because he believed that the absolute
spirit revealed itself through the history of hu
man thought. Finally, Natorp tends to tackle
the solution of this problem through aesthet
ics. Even so, referring the religious problem to
the realm of logic, or ethics, or aesthetics, i. e. to one part of the system of philosophy, is not a
satisfactory solution of the issue. y
As we have seen, Cohen’s system of philos
ophy is, as it were, a series of reflections and
refractions of the single ray of the Pure Idea. 23 [The entire text of the following footnote handwritten
at the bottom of sheets 29-30 of the typewritten copy,
including obverse sides, has been crossed out.] In his
declining years in the work: Der Begriff der Religion im
System der Philosophie. Philosophische Arbeiten. X. Buch,
1. Heft. Giessen, 1915, Cohen recognises that in The
Ethics of Pure Will he did not stop short of dissolving
religion in morality: «Ich habe die methodische Konsequenz
nicht gescheut, daß die Religion in Ethik sich auflösen müsse»
(ibid., p. 42). However, even in this new work Cohen,
while admitting the originality, Eigenart, of religious
content, nevertheless refuses to see it as an independent
area of consciousness, Bewusstseinsart, along with pure
cognition, pure will and pure feeling and reduces
the originality of religious consciousness to a special у
р
52 [Весь следующий текст сноски, вписанный от руки
внизу страниц 29—30 машинописи, включая оборот
ные стороны, зачеркнут:] На склоне лет, в сочине
нии: Der Begriff der Religion im System der Philosophie.
Philosophische Arbeiten. X. B[uch,] 1. Heft. Giessen. 1915,
Коген признает, что в «Этике чистой воли» он не по
боялся растворить религию в нравственности: «Ich habe
die methodische Konsequenz nicht gescheut, daß die Religion
in Ethik sich auflösen müsse» (назв[анное] соч[инение,]
стр. 42). Однако и в этом новом произведении Коген,
признавая своеобразие, Eigenart, религиозного содер
жания, тем не менее отказывается видеть в нем само
стоятельное направление сознания, Bewusstseinsart на
ряду с чистым познанием, чистой волею и чистым
чувством, и своеобразие религиозного сознания сводит р
50 Вставка вместо зачеркнутого «Истины». 49 Вставка: «равно в область». р
у
51 Вставка: «в государстве и человечестве». 49 Вставка: «равно в область». 7 Logic revealed the refraction of the all-one idea
in the individual and the particular, in the sci
entific law and the concept, ethics revealed the
pure and detached wholeness of the idea, All
heit in the state and in humanity, aesthetics
finds the embodiment and manifestation of the
same whole idea, harmony in visual represen
tation and artistic intiution. Finally, the all-uni
ty acquires its image and likeness, becomes
conscious of itself in the unity of the individu
al personality. According to Cohen, psychol
ogy, i. e. the doctrine of the unity of human
being’s consciousness, should crown and com
plete the whole edifice of the system of philos
ophy in which God has a dedicated annexe, or
rather a modest chapel in the huge building of
ethics… 23 We know that Hegel’s system, on the р
р
Мы уже видели, что система философии
Когена представляет собою как бы ряд отра
жений и преломлений единого луча Чистой
Идеи50. Логика вскрыла преломление всееди
ной идеи в частном и особенном, в научном за
коне и в понятии, этика раскрыла чистую от
решенную целостность идеи, Allheit, в государ
стве и человечестве51, эстетика находит в на
глядном представлении и художественном со
зерцании воплощение и проявление той же це
лостной идеи, гармонии. Наконец, в единстве
индивидуальной личности всеединство обре
тает свой образ и подобие, сознает самое себя. По мысли Когена, психология, т. е. учение о
единстве сознания человека, должна увенчи
вать и завершать все здание системы филосо
фии, в которой Богу отведен особый флигель,
вернее — скромная домовая часовня в обшир
ном корпусе этики…52 Мы знаем, что, наобо 128 A. L. Sakketti рот, система Гегеля увенчивалась не психоло
гиею, не учением о микрокосме, о душе челове
ка: учение об абсолютном Духе, о чистой себя
самое мыслящей и все из себя порождающей
мысли, «философия самой философии» завер
шает все здание системы Гегеля. И нам думает
ся, что превыше и над совокупностью прелом
ленных и отраженных моментов чистой идеи,
образующих части системы Когена, разверза
ется подлинная бездонная глубина Вечности contrary, was crowned not by psychology, not
by the doctrine of the microcosm, of the human
soul: the teaching on the absolute Spirit, pure
thought that thinks itself and generates every
thing from within itself, “philosophy of philos
ophy itself” accomplishes the whole building of
Hegel’s system. 7 We believe that over and above
the totality of refracted and reflected elements
of the pure idea that form parts of Cohen’s
system there opens up the genuine fathom к особому видоизменению, модификации содержания чи
стого сознания, «eine neue Modifikation desjenigen Inhalts,
dessen Erzeugung durch eine reine Bewusstseinsart bereits
gesichert ist» (там же, стр. 44). С другой стороны, в том
же новейшем сочинении Коген, опирясь на ветхоза
ветные тексты, подчеркивает тождество идеи Бога и бы
тия и Его единичность, Einzigkeit (точнее — единствен
ность) в иудейском монотеизме. Таким образом, Бог, со
гласно истолкованию Когена, есть самое Единственное
Бытие. В связи с этим получают новое освещение так
же учение Канта о «постулате практического разума»
и мысли самого Когена в «Этике чистой воли» о «зако
не правды-истины». Бог как Самое единственное и не
сравненное Бытие отличен от бывания, Dasein, приро
ды; Он есть основание бывания природы, Daseinsgrund. За
кон «правды-истины», т. е. единство сущего и должно
го, превращается в идею сохранения мироздания, вселен
ной, а Бог — в виновника ее непреходящей действительно
сти: «Gott wird daher in der Idee der Erhaltung der Welt zum
Urheber ihrer unaufhörlichen Wirklichkeit». Идея Бога озна
чает ручательство за непрестанное пребывание при
роды ради бесконечного развития нравственности,
«die Idee Gottes bedeutet die Gewähr, daß immerdar Dasein
sein werde für die unendliche Fortführung der Sittlichkeit». Поэтому идея Бога остается основным понятием, но от
нюдь не исходной точкой обоснования, а его завер
шением: «Er bleibt vielmehr ein Grundbegriff, allerdings
nicht für den Beginn der Fudamentierung, sondern allein für
ihre Vollendung; aber diese Vollendung könnte ohne ihn nicht
erfolgen. So bringt der Gott der Religion eine Bereicherung in
die Ethik, ohne die sie ein Torso bleibe» (назв[анное] соч[и
нение,] стр. 51). Согласно основному методу «ориен
тирования» философии в положительных данных,
фактах сознания, приведенные мысли Когена полу
чают подтверждение и обоснование в ветхозаветных
библейских текстах, заимствованных им преимуще
ственно из книг пророков. Таким образом, и для обо
снования философии религии (Коген с недоверием
относится к этому названию) сохраняет обязательное
значение идея единства метода философии. Вообще в
приведенных мыслях Когена мы не нашли чего-либо
совершенно нового; скорее всего, это — дальнейшее
развитие прежних идей. При всем том представляет
ся непонятным, каким образом религиозное сознание
остается в глазах Когена лишь «новою модификаци
ею чистого содержания сознания». Ведь даже если со
гласиться с Когеном, что идея Бога завершает и увен
чивает здание философии, то это завершение и венец
есть самостоятельная идея (примеч. авт.). 7 к особому видоизменению, модификации содержания чи
стого сознания, «eine neue Modifikation desjenigen Inhalts,
dessen Erzeugung durch eine reine Bewusstseinsart bereits
gesichert ist» (там же, стр. 44). С другой стороны, в том
же новейшем сочинении Коген, опирясь на ветхоза
ветные тексты, подчеркивает тождество идеи Бога и бы
тия и Его единичность, Einzigkeit (точнее — единствен
ность) в иудейском монотеизме. Таким образом, Бог, со
гласно истолкованию Когена, есть самое Единственное
Бытие. В связи с этим получают новое освещение так
же учение Канта о «постулате практического разума»
и мысли самого Когена в «Этике чистой воли» о «зако
не правды-истины». Бог как Самое единственное и не
сравненное Бытие отличен от бывания, Dasein, приро
ды; Он есть основание бывания природы, Daseinsgrund. За
кон «правды-истины», т. е. единство сущего и должно
го, превращается в идею сохранения мироздания, вселен
ной, а Бог — в виновника ее непреходящей действительно
сти: «Gott wird daher in der Idee der Erhaltung der Welt zum
Urheber ihrer unaufhörlichen Wirklichkeit». Идея Бога озна
чает ручательство за непрестанное пребывание при
роды ради бесконечного развития нравственности,
«die Idee Gottes bedeutet die Gewähr, daß immerdar Dasein
sein werde für die unendliche Fortführung der Sittlichkeit». Поэтому идея Бога остается основным понятием, но от
нюдь не исходной точкой обоснования, а его завер
шением: «Er bleibt vielmehr ein Grundbegriff, allerdings
nicht für den Beginn der Fudamentierung, sondern allein für
ihre Vollendung; aber diese Vollendung könnte ohne ihn nicht
erfolgen. So bringt der Gott der Religion eine Bereicherung in
die Ethik, ohne die sie ein Torso bleibe» (назв[анное] соч[и
нение,] стр. 51). Согласно основному методу «ориен
тирования» философии в положительных данных,
фактах сознания, приведенные мысли Когена полу
чают подтверждение и обоснование в ветхозаветных
библейских текстах, заимствованных им преимуще
ственно из книг пророков. Таким образом, и для обо
снования философии религии (Коген с недоверием
относится к этому названию) сохраняет обязательное
значение идея единства метода философии. Вообще в
приведенных мыслях Когена мы не нашли чего-либо
совершенно нового; скорее всего, это — дальнейшее
развитие прежних идей. При всем том представляет
ся непонятным, каким образом религиозное сознание
остается в глазах Когена лишь «новою модификаци
ею чистого содержания сознания». Ведь даже если со
гласиться с Когеном, что идея Бога завершает и увен
чивает здание философии, то это завершение и венец
есть самостоятельная идея (примеч. авт.). modification of the content of pure consciousness, “eine neue
Modifikation desjenigen Inhalts, dessen Erzeugung durch
eine reine Bewusstseinsart bereits gesichert ist” (ibid., p. 44). 7 On the other hand, in the same latest work Cohen,
proceeding from Old Testament texts, stresses the
identity of the idea of God and being and His uniquenessy,
Einzigkeit (precisely, singularity) in Judaist monotheism. Thus God, as interpreted by Cohen, is the most Unique
Being. This throws new light on Kant’s teaching on
“the postulate of practical reason” and Cohen’s own
thoughts on “the law of truth-verity” in Ethics of Pure
Will. God as the most Unique and incomparable Being
is different from the existence, Dasein, of nature. He is
the foundation of the existence of nature, Daseinsgrund. The law of “truth-verity”, i. e. the unity of what is and
what ought to be, turns into the idea of the preservation
of the universe, and God into the cause of its intransient
reality: “Gott wird daher in der Idee der Erhaltung der Welt
zum Urheber ihrer unaufhörlichen Wirklichkeit.” The idea of
God is the guarantee of the continuous existence of nature
for the sake of infinite development of morality, “die Idee
Gottes bedeutet die Gewähr, daß immerdar Dasein sein werde
für die unendliche Fortführung der Sittlichkeit.” Therefore
the idea of God remains a main concept, not, however,
for the starting point of the grounding but only for the
completion: “Er bleibt vielmehr ein Grundbegriff, allerdings
nicht für den Beginn der Fudamentierung, sondern allein für
ihre Vollendung; aber diese Vollendung könnte ohne ihn nicht
erfolgen. So bringt der Gott der Religion eine Bereicherung
in die Ethik, ohne die sie ein Torso bleibe” (ibid., p. 51). In
accordance with the main method of the “orientation” of
philosophy in positive data, facts of consciousness, the
above ideas of Cohen receive confirmation and grounding
in Old Testament biblical texts which he took mainly from
the books of prophets. Thus, to ground the philosophy
of religion (Cohen mistrusts this novel appellation) the
idea of the unity of the method of philosophy remains
obligatory. In general, we have not found anything
totally new in the above-cited thoughts of Cohen; rather,
this is an elaboration of his former ideas. For all that, it
remains unclear how religious consciousness remains in
Cohen’s eyes only a “modification of the pure content of
consciousness”. If one agrees with Cohen that the idea
of God completes and crowns the edifice of philosophy
then this completion and crowning is a self-contained
idea (author’s note). 7 modification of the content of pure consciousness, “eine neue
Modifikation desjenigen Inhalts, dessen Erzeugung durch
eine reine Bewusstseinsart bereits gesichert ist” (ibid., p. 44). On the other hand, in the same latest work Cohen,
proceeding from Old Testament texts, stresses the
identity of the idea of God and being and His uniquenessy,
Einzigkeit (precisely, singularity) in Judaist monotheism. Thus God, as interpreted by Cohen, is the most Unique
Being. This throws new light on Kant’s teaching on
“the postulate of practical reason” and Cohen’s own
thoughts on “the law of truth-verity” in Ethics of Pure
Will. God as the most Unique and incomparable Being
is different from the existence, Dasein, of nature. He is
the foundation of the existence of nature, Daseinsgrund. The law of “truth-verity”, i. e. the unity of what is and
what ought to be, turns into the idea of the preservation
of the universe, and God into the cause of its intransient
reality: “Gott wird daher in der Idee der Erhaltung der Welt
zum Urheber ihrer unaufhörlichen Wirklichkeit.” The idea of
God is the guarantee of the continuous existence of nature
for the sake of infinite development of morality, “die Idee
Gottes bedeutet die Gewähr, daß immerdar Dasein sein werde
für die unendliche Fortführung der Sittlichkeit.” Therefore
the idea of God remains a main concept, not, however,
for the starting point of the grounding but only for the
completion: “Er bleibt vielmehr ein Grundbegriff, allerdings
nicht für den Beginn der Fudamentierung, sondern allein für
ihre Vollendung; aber diese Vollendung könnte ohne ihn nicht
erfolgen. So bringt der Gott der Religion eine Bereicherung
in die Ethik, ohne die sie ein Torso bleibe” (ibid., p. 51). In
accordance with the main method of the “orientation” of
philosophy in positive data, facts of consciousness, the
above ideas of Cohen receive confirmation and grounding
in Old Testament biblical texts which he took mainly from
the books of prophets. Thus, to ground the philosophy
of religion (Cohen mistrusts this novel appellation) the
idea of the unity of the method of philosophy remains
obligatory. In general, we have not found anything
totally new in the above-cited thoughts of Cohen; rather,
this is an elaboration of his former ideas. For all that, it
remains unclear how religious consciousness remains in
Cohen’s eyes only a “modification of the pure content of
consciousness”. 7 If one agrees with Cohen that the idea
of God completes and crowns the edifice of philosophy
then this completion and crowning is a self-contained
idea (author’s note). 129 А. Л. Саккетти less depth of the Eternity of the All-One Being
and Life Itself. The latest philosophical thought
(Emil Lask, Benedetto Croce, Lanz) is imbued
with a vague awareness of the highest stage of
philosophy in the shape of the logic of philoso
phy. With Hegel, we find the philosophy of ab
solute spirit, the philosophy of philosophy; in
the olden days philosophers looked for the first
philosophy, prima philosophia. Cohen’s pure
philosophy, firmly oriented in scientific knowl
edge, should in any case methodically, step by
step, lead to the sphere of the Absolute Truth
and Eternal Life. Otherwise it may fail to per
form its Platonic mission… Всеединого Бытия и Самой Жизни. Новейшая
философская мысль (Эмиль Ласк, Бенедетто
Кроче, Ланц) преисполнена неясного сознания
наивысшей ступени философии в виде логики
философии. У Гегеля мы находим философию
абсолютного духа, философию философии,
в старину философы искали первой филосо
фии, prima philosophia. Чистая философия Ко
гена, твердо ориентированная в научном зна
нии, должна во всяком случае методически,
шаг за шагом привести к сфере Абсолютной
Истины и Вечной Жизни. Иначе она грозит не
выполнить своей платоновской53 миссии… To cite this article: To cite this article:
Sakketti, A. L., 2021. Philosophy of Hermann Co
hen (Publication, Foreword and Commentary by
N. A. Dmitrieva). Kantian Journal, 40(2), pp. 95-130. http://dx.doi.org/10.5922/0207-6918-2021-2-4 To cite this article:
Sakketti, A. L., 2021. Philosophy of Hermann Co
hen (Publication, Foreword and Commentary by
N. A. Dmitrieva). Kantian Journal, 40(2), pp. 95-130. http://dx.doi.org/10.5922/0207-6918-2021-2-4 О публикаторе Нина Анатольевна Дмитриева, доктор философ
ских наук, профессор, Московский педагогический
государственный университет, Москва; Балтий
ский федеральный университет им. И. Канта, Ка
лининград, Россия; Междисциплинарный центр
изучения европейского Просвещения, Галле-Вит
тенбергский университет им. М. Лютера, Галле,
Германия. Translated from the Russian by Evgeni N. Filippov Translated from the Russian by Evgeni N. Filippov Translated from the Russian by Evgeni N. Filippov 53 Вставка вместо зачеркнутого слова «нео-платониче
ской». ПРЕДСТАВЛЕНО ДЛЯ ВОЗМОЖНОЙ ПУБЛИКАЦИИ В ОТКРЫТОМ ДОСТУ
ПЕ В СООТВЕТСТВИИ С УСЛОВИЯМИ ЛИЦЕНЗИИ CREATIVE COMMONS
ATTRIBUTION (CC BY) (HTTP://CREATIVECOMMONS.ORG/LICENSES/BY/4.0/) The publisher Prof. Dr. Nina A. Dmitrieva, Moscow Pedagogical
State University, Moscow, Russia; Immanuel Kant
Baltic Federal University, Kaliningrad, Russia. E-mail: na.dmitrieva@mpgu.su;
NDmitrieva@kantiana.ru
ORCID: https://orcid.org/0000-0003-1092-0321 Prof. Dr. Nina A. Dmitrieva, Moscow Pedagogical
State University, Moscow, Russia; Immanuel Kant
Baltic Federal University, Kaliningrad, Russia. E-mail: na.dmitrieva@mpgu.su;
NDmitrieva@kantiana.ru
ORCID: https://orcid.org/0000-0003-1092-0321 Prof. Dr. Nina A. Dmitrieva, Moscow Pedagogical
State University, Moscow, Russia; Immanuel Kant
Baltic Federal University, Kaliningrad, Russia. E-mail: na.dmitrieva@mpgu.su;
NDmitrieva@kantiana.ru
ORCID: https://orcid.org/0000-0003-1092-0321 E-mail: na.dmitrieva@mpgu.su;
NDmitrieva@kantiana.ru E-mail: na.dmitrieva@mpgu.su;
NDmitrieva@kantiana.ru ORCID: https://orcid.org/0000-0003-1092-0321 © Дмитриева Н. А., 2021. Для цитирования: Саккетти А. Л. Философия Германа Когена / публ.,
предисл., коммент. Н. А. Дмитриевой // Кантов
ский сборник. 2021. Т. 40, № 2. С. 95—130. doi: 10.5922/0207-6918-2021-2-4 Саккетти А. Л. Философия Германа Когена / публ.,
предисл., коммент. Н. А. Дмитриевой // Кантов
ский сборник. 2021. Т. 40, № 2. С. 95—130. doi: 10.5922/0207-6918-2021-2-4 © Dmitrieva N. A., 2021. © Дмитриева Н. А., 2021. 53 Вставка вместо зачеркнутого слова «нео-платониче
ской». 130
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https://openalex.org/W2611662026
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https://inria.hal.science/hal-01424929v3/file/paper_agmk_rem_sens_ver2.pdf
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English
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Object-Based Classification of Grasslands from High Resolution Satellite Image Time Series Using Gaussian Mean Map Kernels
|
Remote sensing
| 2,017
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cc-by
| 18,911
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Object-based classification of grasslands from high
resolution satellite image time series using Gaussian
mean map kernels
Maïlys Lopes Mathieu M Fauvel Stéphane Girard David Sheeren Maïlys Lopes, Mathieu M. Fauvel, Stéphane Girard, David Sheeren To cite this version: Maïlys Lopes, Mathieu M. Fauvel, Stéphane Girard, David Sheeren. Object-based classification of
grasslands from high resolution satellite image time series using Gaussian mean map kernels. Remote
Sensing, 2017, 9 (7), pp.Article 688. 10.3390/rs9070688. hal-01424929v3 Distributed under a Creative Commons Attribution 4.0 International License Article
Object-based classification of grasslands from high
resolution satellite image time series using Gaussian
mean map kernels Mailys Lopes1*, Mathieu Fauvel1, Stéphane Girard2 and David Sheeren1 Mailys Lopes1*, Mathieu Fauvel1, Stéphane Girard2 and David Sheeren1
1
Dynafor, University of Toulouse, INRA, INPT, INPT-EI PURPAN, 31326 Castanet Tolosan, France;
mailys.lopes@inra.fr, mathieu.fauvel@ensat.fr, david.sheeren@ensat.fr. 2
Team Mistis, INRIA Rhône-Alpes, LJK, 38334 Montbonnot, France; stephane.girard@inria.fr. *
Correspondence: mailys.lopes@inra.fr; Tel.: +33-534-32-39-27 Mailys Lopes1*, Mathieu Fauvel1, Stéphane Girard2 and David Sheeren1
1
Dynafor, University of Toulouse, INRA, INPT, INPT-EI PURPAN, 31326 Castanet Tolosan, France
mailys.lopes@inra.fr, mathieu.fauvel@ensat.fr, david.sheeren@ensat.fr. 2
Team Mistis, INRIA Rhône-Alpes, LJK, 38334 Montbonnot, France; stephane.girard@inria.fr. *
Correspondence: mailys.lopes@inra.fr; Tel.: +33-534-32-39-27 Abstract: This paper deals with the classification of grasslands using high resolution satellite image
1
time series. Grasslands considered in this work are semi-natural elements in fragmented landscapes,
2
i.e., they are heterogeneous and small elements. The first contribution of this study is to account for
3
grassland heterogeneity while working at the object level by modeling its pixels distributions by a
4
Gaussian distribution. To measure the similarity between two grasslands, a new kernel is proposed
5
as a second contribution: the α-Gaussian mean kernel. It allows to weight the influence of the
6
covariance matrix when comparing two Gaussian distributions. This kernel is introduced in Support
7
Vector Machine for the supervised classification of grasslands from south-west France. A dense
8
intra-annual multispectral time series of Formosat-2 satellite is used for the classification of grasslands
9
management practices, while an inter-annual NDVI time series of Formosat-2 is used for old and
10
young grasslands discrimination. Results are compared to other existing pixel- and object-based
11
approaches in terms of classification accuracy and processing time. The proposed method shows
12
to be a good compromise between processing speed and classification accuracy. It can adapt to the
13
classification constraints and it encompasses several similarity measures known in the literature. It is
14
appropriate for the classification of small and heterogeneous objects such as grasslands. 15 Keywords: Supervised classification; SVM; Gaussian mean map kernels; kernel methods; object
16
analysis; grasslands. 17 HAL Id: hal-01424929
https://inria.hal.science/hal-01424929v3
Submitted on 13 Jun 2017 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Submitted to Remote Sens., pages 1 – 25 Keywords: Supervised classification; SVM; Gaussian mean map kernels; kernel methods; object
16
analysis; grasslands.
17 www.mdpi.com/journal/remotesensing www.mdpi.com/journal/remotesensing 1. Introduction
18 Most of studies focusing on grasslands have agronomic
45
applications, such as estimating biomass productivity and growth rate [17–19] or derivating biophysical
46
parameters like Leaf Ara Index (LAI), Fraction of Photosynthetically Active Radiation (fPAR) and
47
chlorophyll content [20–24]. Studies having biodiversity conservation schemes such as assessing plant
48
diversity and plant community composition in a grassland are usually based on ground spectral
49
measurements or airborne acquisitions at a very high spatial resolution [25–31]. However, such
50
acquisitions are time-consuming and expensive and thus they do not allow for continuous monitoring
51
of grasslands over the years. 52 Using satellite remote sensing images, grasslands have been studied a lot at a regional scale with
53
medium spatial resolution sensors (i.e., MODIS, 250m/pixel [17,18,32]), where the Minimum Mapping
54
Unit (MMU) is at least of hundreds of meters. This scale is suitable for large, extensive, homogeneous
55
and contiguous regions like steppes [33], but not for fragmented landscapes which are usually found
56
in Europe and in France particularly [34,35]. These fragmented landscapes are made of a patchwork
57
of different land covers which have a small area [35]. In these types of landscapes, grasslands can
58
be smaller (less than 10,000m2) than the pixel resolution [36] (see Figure 1 for a graphical example). 59
As a consequence, pixels containing grasslands are usually a mixture of other contributions, which
60
can limit the analysis [37,38]. As examples, Poças et al. [39] had to select large contiguous areas of
61
semi-natural grasslands in a mountain region of Portugal to be able to use SPOT-VEGETATION data
62
(1-km resolution). Halabuk et al. [40] also had to select only one MODIS pixel per homogeneous
63
sample site in Slovakia to detect cutting in hay meadows. A 30-m pixel resolution is still not sufficient
64
for grassland characterization. Indeed, Lucas et al. [41] and Toivonen and Luoto [42] showed that it
65
was more difficult to classify fragmented and complex elements [43], like semi-natural grasslands, than
66
homogeneous habitats, using Landsat imagery. Price et al. [44] classified six grassland management
67
types in Kansas using six Landsat images, but the accuracy of the classification was not satisfying (less
68
than 70%). Therefore, to detect small grasslands in fragmented landscapes, high spatial resolution
69
images are required [36,45,46]. 70 (a)
(b)
Figure 1. 1. Introduction
18 Grasslands are semi-natural elements that represent a significant source of biodiversity in farmed
19
landscapes [1–4]. They provide many ecosystem services such as carbon storage, erosion regulation,
20
food production, crop pollination, biological regulation of pests [5], which are linked to their plant and
21
animal composition. 22 Different factors impact on grassland biodiversity conservation. Among them, the age of a
23
grassland (i.e, the time since last ploughing/sowing) is directly related to its plant and animal
24
composition. Old "permanent" grasslands, often called semi-natural grasslands, hold a richer
25
biodiversity than temporary grasslands [2,6–8]. Indeed, they had time to establish and stabilize their
26
vegetation cover, contrarily to temporary grasslands which are part of a crop rotation. Additionally,
27
agricultural management of grasslands (i.e, mowing, grazing, fertilizing, reseeding...) influences their
28
structure and composition [9–12]. Management is essential for their biodiversity conservation because
29
its prevents from the woody establishment. Conversely, an intensive use constitutes a threat for this
30
biodiversity [12,13]. Therefore, it is important to know the age of a grassland and to identify the
31 www.mdpi.com/journal/remotesensing 2 of 25 Version June 12, 2017 submitted to Remote Sens. management practices in order to monitor their effect on biodiversity and related services. But these
32
factors are defined at different temporal scales: over the years for the age of a grassland, and during a
33
vegetation season (i.e., a year) for the management practice. 34 Usually, ecologists and agronomists characterize grasslands at the parcel scale through field
35
surveys. However, these surveys require important human and material resources, the knowledge of
36
the assessor and a sampling strategy, which make them expensive and time-consuming [14]. They are
37
thus limited in spatial extent and in temporal frequency, limiting grassland characterization to a local
38
scale and over a short period of time. 39 Conversely, remote sensing offers the possibility to provide information on landscapes over large
40
extents, thanks to the broad spatial coverage and regular revisit frequency of satellite sensors [15]. In
41
this context, satellite images have already appeared to be an appropriate tool to monitor vegetation
42
over large areas with a high temporal resolution. 43 In the remote sensing literature, grasslands have relatively not been studied much compared to
44
other land covers like crops or forest [16]. 1. Introduction
18 However, new missions like Sentinel-2 [55], with very high revisit frequency (5 days)
90
and high spatial resolution (10 meters in four spectral channels, 20 meters in six channels) provide
91
new opportunities for grasslands monitoring over the years in fragmented landscapes [54] at no cost,
92
thanks to the ESA free data access policy. For instance, the high spatial resolution is assumed to make
93
possible the identification of grassland-only pixels in the image and several pixels can belong to the
94
same grassland plot. Hence, the analysis can be done at the object level, not at the pixel level, which is
95
suitable for landscape ecologists and agronomists who usually study grasslands at the parcel scale [56]. 96
Thus, object-oriented approaches are more likely to characterize grasslands ecologically [57,58]. Yet, a
97
lot of works consider pixel-based approaches without any spatial constraints [17,42,44,48,49,52,59]. 98 p
pp
y p
At the object level, grasslands are commonly represented by their mean NDVI [18]. But such
99
representation might be too simple since it does not account for the heterogeneity in a grassland. 100
Sometimes, distributions of pixels as individual observations are still better than the mean value
101
to represent grasslands, as in [54]. Lucas et al. [41] used a rule-based method on segmented areas
102
for habitat mapping but it did not work well on complex and heterogeneous land covers. Esch et
103
al. [60] also used an object-oriented method on segmented elements then represented by their mean
104
NDVI. These methods based on mean modeling do not capture well grasslands heterogeneity. Other
105
representations can be found in the literature, taking standard deviation and object texture features as
106
variables [61], but they were not applied to time series. In our knowledge, these methods do not use
107
the high spatial and the high temporal resolutions jointly. Moreover, all these studies used vegetation
108
indices as a variable, although it has been shown that classification results are better when using more
109
spectral information [35,62]. 110 To deal with the high spatio-spectro-temporal resolutions new satellite sensors are now offering,
111
dimension reduction is usually performed through the use of a vegetation index such as NDVI [50,52,63,
112
64], PCA [65] or spectro-temporal metrics [35,66]. But a large amount of spectro-temporal information
113
is lost with these solutions. Franke at al. 1. Introduction
18 Digitalized grasslands (in red) from the dataset used in this study on (a) a Sentinel-2 image
(10m pixel resolution) and (b) a MODIS image (250m). (b) (a) (b) (a) Figure 1. Digitalized grasslands (in red) from the dataset used in this study on (a) a Sentinel-2 image
(10m pixel resolution) and (b) a MODIS image (250m). 3 of 25 Version June 12, 2017 submitted to Remote Sens. For high spatial resolution images (about 10m/pixel), few intra-annual images are usually
71
available for a given location [47]. However, Buck et al. [48] concluded that three RapidEye images per
72
year were not enough to detect the mowing practices in grasslands. It was confirmed by Franke et al. 73
[49] who classified grassland use intensity into four categories: Semi-natural Grassland, Extensively
74
Used Grassland, Intensively Used Grassland and Tilled Grassland. They increased the classification
75
accuracy when increasing the number of RapidEye images from three to five scenes. Additionally,
76
Schmidt et al. [50] concluded that about seven to ten images, depending on the vegetation index used,
77
are a good tradeoff between the amount of satellite data and classification accuracy of grassland use
78
intensity. Some works report results with few images per year, such as Dusseux et al. [51], but they
79
worked on LAI. In their study for mapping grassland habitat using intra-annual RapidEye imagery,
80
Schuster et al. [52] concluded the more acquisition dates used, the better the mapping quality. 81 [
]
q
,
pp
g q
y
Given the heterogeneity of grasslands in fragmented landscapes, their phenological cycle and the
82
punctuality of the anthropogenic events (e.g., mowing), dense high spatial resolution intra-annual time
83
series are necessary to identify the grassland management types [36,52–54]. Moreover, to discriminate
84
semi-natural grasslands from temporary grasslands, inter-annual time series are necessary. Until
85
recently, satellite missions offering high revisit frequency (1-16 days) had coarse spatial resolution
86
(i.e., NOAA AVHRR - 1km, MODIS - 250/500m). Conversely, high spatial resolution missions did
87
not provide dense time series and/or were costly (i.e., QuickBird, RapidEye). For these reasons
88
and compared to crops, grasslands differentiation through Earth observations is still considered as a
89
challenge [52]. 1. Introduction
18 [49] developed an indicator of the spectral variability of a pixel
114
over the time series, the Mean Absolute Spectral Dynamics, but its efficiency was assessed using a
115
decision tree algorithm. Decision trees are usually not recommended because they tend to over-fit the
116
data [67]. Therefore, the high spatio-spectro-temporal resolutions have not really been addressed in
117
the literature of remote sensing classification. Indeed, such time series bring new methodological and
118
statistical constraints given the high dimension of data (i.e., number of pixels and number of spectral
119
and temporal measurements). Dealing with more variables increases the number of parameters to
120 Version June 12, 2017 submitted to Remote Sens. 4 of 25 estimate, increasing the computation time and making the computation unstable (i.e., ill-conditioned
121
covariance matrices...) [68,69]. Hence, conventional models are not appropriate if one wants to use
122
all the spectro-temporal information of time series with high spatial and temporal resolutions. Thus,
123
classifying grasslands with this type of data is still considered as a challenge [52]. 124 estimate, increasing the computation time and making the computation unstable (i.e., ill-conditioned
121
covariance matrices...) [68,69]. Hence, conventional models are not appropriate if one wants to use
122
all the spectro-temporal information of time series with high spatial and temporal resolutions. Thus,
123
classifying grasslands with this type of data is still considered as a challenge [52]. 124 In the present study, we introduce a model suitable for the classification of grasslands using
125
satellite image time series (SITS) with a high number of spectro-temporal variables (e.g., Sentinel-2
126
data). Two temporal scales are considered in this work: (i) an inter-annual time series of three years to
127
discriminate old grasslands from young grasslands and (ii) an intra-annual time series to identify the
128
management practices. Note that in this work, the objects are not found from segmentation [38] but
129
from existing dataset in a polygon form. 130 The first contribution of this study is to model a grassland at the object level while accounting
131
for the spectral variability within a grassland. We consider that the distribution of the pixel spectral
132
reflectance in a given grassland can be modeled by a Gaussian distribution. The second contribution
133
is to propose a measure of similarity between two Gaussian distributions that is robust to the high
134
dimension of the data. 1. Introduction
18 This method is based on the use of covariance through mean maps. The
135
last contribution is the application of the method to old and young grasslands discrimination and of
136
management practices classification, which are non common applications in remote sensing. Moreover,
137
in our knowledge, mean maps have not yet been used on Gaussian distributions for supervised
138
classification of SITS at the object level. 139 In the next section, the materials used for the experimental part of this study are presented. 140
Then the methods, including the different types of grassland modeling and the measures of similarity
141
between distributions are introduced in section 3. Following that, we experiment the proposed
142
methods on the classification of a real dataset in section 4. Finally, conclusions and prospects are given
143
in section 5. 144 2.2. Satellite data
156 The Whittaker filter is a non-parametric filter which has a smoothing parameter
171
that controls the roughness of the reconstructed curve. It has been successfully applied to smooth
172
NDVI time series in the literature [72–75]. The smoother was adapted for unequally spaced intervals
173
and accounted for missing data (see [62] for a detailed description of the method). The smoothing
174
parameter was the same for all the pixels. It was equal to 105 for year 2013 and to 104 for 2012 and
175
2014, after an ordinary cross-validation done on a subset of the pixels for each year. An example of
176
smoothing on a grassland pixel is provided in Figure 4. This pixel is hidden by a light cloud during
177
one image acquisition (red cross). Notice that the smoothing is done at the cost of under-estimating
178
the local maxima of the temporal profile. 179 03
05
07
09
11
03
05
07
09
11
03
05
07
09
11
2012
2013
2014
Figure 3. Formosat-2 acquisition dates in 2012 (green dots), 2013 (blue dots) and 2014 (red dots) used
in this experiment. Figure 3. Formosat-2 acquisition dates in 2012 (green dots), 2013 (blue dots) and 2014 (red dots) used
in this experiment. 01 02 03 04 05 06 07 08 09 10 11 12
0.02
0.04
0.06
0.08
0.10
Blue
01 02 03 04 05 06 07 08 09 10 11 12
0.02
0.04
0.06
0.08
0.10
Green
01 02 03 04 05 06 07 08 09 10 11 12
0.02
0.04
0.06
0.08
0.10
Red
01 02 03 04 05 06 07 08 09 10 11 12
0.20
0.30
NIR
Figure 4. Example of time series reconstruction (blue dots) with Whittaker smoother for a pixel of a
grassland in the four spectral bands. The black crosses correspond to the original 2013 Formosat-2 time
series and the red ones correspond to missing/noisy data due to the clouds. The x-axis represents the
month of year 2013 and the y-axis is the reflectance. 2.1. Study site
146 The study site is located in south-west France, near the city of Toulouse (about 30km), in a
147
semi-rural area (center coordinates: 43◦27′36′′N 1◦8′24′′E, Figure 2). This region is characterized by a
148
temperate climate with oceanic and Mediterranean influences. The average annual precipitation is
149
656mm and the average temperature is 13◦C. The north of the site, closer to the urban area of Toulouse,
150
is flat, whereas the south-west of the site is hilly. The eastern part corresponds to the Garonne river
151
floodplain and this location is dominated by crop production. Within this study site, livestock farming
152
is declining in favor of annual crop production. Grasslands are mostly used for forage or silage
153
production. Some grasslands, located in the south-western part of the area, are pastures for cattle or
154
sheep. The extent of the area is included in the satellite image extent (Figure 2) and is about 24 × 24km2. 155 Figure 2. Study site location in south-west France. It is included in the satellite image extent. Figure 2. Study site location in south-west France. It is included in the satellite image extent. 5 of 25 Version June 12, 2017 submitted to Remote Sens. 2.2. Satellite data
156 Time series of Formosat-2 were used is this experiment. Formosat-2 has four spectral bands with
157
an 8-meter spatial resolution: B1 "Blue" (0.45 - 0.52µm), B2 "Green" (0.52 - 0.6µm), B3 "Red" (0.63 -
158
0.69µm), B4 "Near Infra-Red (NIR)" (0.76 - 0.9µm). The extent of an acquisition is 24km × 24km. The
159
images were all acquired with the same viewing angle. They were orthorectified, radiometrically and
160
atmospherically corrected by the French Spatial Agency (CNES). They were provided by the Center
161
for the Study of the Biosphere from Space (CESBIO) in reflectance with a mask of clouds and shadows
162
issued from the MACCS (Multi-sensor Atmospheric Correction and Cloud Screening) processor [70],
163
in the frame of the Kalideos project. 164 For the inter-annual analysis, we used all the acquisitions of the consecutive years 2012 (13
165
observations), 2013 (17 observations) and 2014 (15 observations) (Figure 3 and Figure S1 in the
166
supplementary materials). The acquisitions of year 2013 and of year 2014 were used separately for the
167
classification of management practices. 168 To reconstruct the time series due to missing data (clouds and their shadows), the Whittaker
169
filter [71] was applied pixel-by-pixel on the reflectances in each spectral band for each year
170
independently. The Whittaker filter is a non-parametric filter which has a smoothing parameter
171
that controls the roughness of the reconstructed curve. It has been successfully applied to smooth
172
NDVI time series in the literature [72–75]. The smoother was adapted for unequally spaced intervals
173
and accounted for missing data (see [62] for a detailed description of the method). The smoothing
174
parameter was the same for all the pixels. It was equal to 105 for year 2013 and to 104 for 2012 and
175
2014, after an ordinary cross-validation done on a subset of the pixels for each year. An example of
176
smoothing on a grassland pixel is provided in Figure 4. This pixel is hidden by a light cloud during
177
one image acquisition (red cross). Notice that the smoothing is done at the cost of under-estimating
178
the local maxima of the temporal profile. 179 To reconstruct the time series due to missing data (clouds and their shadows), the Whittaker
169
filter [71] was applied pixel-by-pixel on the reflectances in each spectral band for each year
170
independently. 2.3.1. Old and young grasslands
185 In this study, "old" grasslands are 14 years old or more, whereas "young" grasslands are less
186
than five years old. The French agricultural land use database (Registre Parcellaire Graphique) was
187
used to extract the grasslands depending on their age. It registers on an annual basis the cultivated
188
areas declared by the farmers in a GIS. Grasslands are declared as "permanent" or "temporary". 189
Permanent grasslands are at least five years old, whereas temporary grasslands are less than five
190
years old (Commission Regulation EU No 796/2004). For every plot declared as a grassland in 2014,
191
its age was computed from the previous years declarations. We kept only the grasslands which
192
were at least 14 years old in 2014 ("old"), and the grasslands which were less than 5 years old in
193
2014 ("young"). A negative buffer of 8 meters was then applied to all the polygons to eliminate
194
the edge effects (Figure 5). Then they were rasterized using the GDAL command gdal_rasterize
195
(http://www.gdal.org/gdal_rasterize.html) to obtain the pixels inside each grassland. Only the
196
grasslands having an area of at least 1,000m2 were kept to ensure a minimum number of 16 pixels to
197
represent each grassland. In the end, there were 59 old grasslands (at least 14 years old) and 416 young
198
grasslands (Table 1), for an average area of about 26,600m2. 199 (a)
(b)
(c)
(d)
Figure 5. False color Formosat-2 images of the same grassland on two close dates (June and October)
in 2013 and 2014 with the same color scale. (a) 2013-06-06, (b) 2013-10-27, (c) 2014-06-05, (d) 2014-10-23. The blue line represents the polygon limits of the grassland. (b) (a) (b) (a) (c) (d) (d) (c) Figure 5. False color Formosat-2 images of the same grassland on two close dates (June and October)
in 2013 and 2014 with the same color scale. (a) 2013-06-06, (b) 2013-10-27, (c) 2014-06-05, (d) 2014-10-23. The blue line represents the polygon limits of the grassland. 2.2. Satellite data
156 01 02 03 04 05 06 07 08 09 10 11 12
0.02
0.04
0.06
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Blue
Red 01 02 03 04 05 06 07 08 09 10 11 12
0.02
0.04
0.06
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0.10
Green
NIR 01 02 03 04 05 06 07 08 09 10 11 12
0.20
0.30 01 02 03 04 05 06 07 08 09 10 11 12
0.02
0.04
0.06
0.08
0.10 Figure 4. Example of time series reconstruction (blue dots) with Whittaker smoother for a pixel of a
grassland in the four spectral bands. The black crosses correspond to the original 2013 Formosat-2 time
series and the red ones correspond to missing/noisy data due to the clouds. The x-axis represents the
month of year 2013 and the y-axis is the reflectance. Figure 4. Example of time series reconstruction (blue dots) with Whittaker smoother for a pixel of a
grassland in the four spectral bands. The black crosses correspond to the original 2013 Formosat-2 time
series and the red ones correspond to missing/noisy data due to the clouds. The x-axis represents the
month of year 2013 and the y-axis is the reflectance. 6 of 25 Version June 12, 2017 submitted to Remote Sens. For the intra-annual time series, we used all the spectral information. Therefore, the smoothed
180
time series associated with each of the four spectral bands were concatenated to get a unique time
181
series per pixel. For the inter-annual time series, as using all the spectral bands would result in a too
182
large number of variables to process, we worked on the NDVI, computed from the red and NIR bands. 183 For the intra-annual time series, we used all the spectral information. Therefore, the smoothed
180
time series associated with each of the four spectral bands were concatenated to get a unique time
181
series per pixel. For the inter-annual time series, as using all the spectral bands would result in a too
182
large number of variables to process, we worked on the NDVI, computed from the red and NIR bands. 183 Table 1. Composition of the old and young grasslands dataset. Class
Nb of grasslands
Nb of pixels
Old
59
31,166
Young
416
129,348
Total
475
160,514 Class
Nb of grasslands
Nb of pixels
Old
59
31,166
Young
416
129,348
Total
475
160,514 The information of the agricultural practices performed in the crops are not featured in the
201
land use database. Therefore, this dataset comes exclusively from field data. As mentioned in the
202 The information of the agricultural practices performed in the crops are not featured in the
201
land use database. Therefore, this dataset comes exclusively from field data. As mentioned in the
202 7 of 25 Version June 12, 2017 submitted to Remote Sens. introduction, ground data is difficult to obtain in ecology since field work is fastidious. A field survey
203
was conducted in May 2015 to determine the past and current management practices of 52 grasslands
204
by interviewing the farmers or grasslands’ owners. The practices remained stable for the years 2013
205
and 2014. Four management types during a vegetation cycle were identified: one mowing, two
206
mowings, grazing and mixed (mowing then grazing). We eliminated the type "two mowings" of the
207
dataset because of its under-representation (only three grasslands). 208 The management types were used as classes for the classification (Table 2). The grasslands were
209
digitalized manually after field work. A negative buffer of 8 meters was then applied to eliminate the
210
edge effects, before rasterizing the polygons. The average grasslands surface area is about 10,000m2. 211
The smallest grassland is 1,632m2 (which represents 25 Formosat-2 pixels) and the largest is 47,111m2
212
(735 pixels) (Figure 6). 213 Table 2. Grassland management types and composition of the dataset. Class
Nb of grasslands
Nb of pixels
Mowing
34
6,265
Grazing
10
1,193
Mixed
8
1,170
Total
52
8,628
0
200
400
600
800
0
5
10
15
ni
Number of grasslands
Figure 6. Histogram of grasslands size in number of pixels ni. The red line corresponds to the number
of spectro-variables d = 68 in 2013. Table 2. Grassland management types and composition of the dataset. Figure 6. Histogram of grasslands size in number of pixels ni. The red line corresponds to the number
of spectro-variables d = 68 in 2013. 3. Methods
214 3.1. Grassland modeling
215 3.1. Grassland modeling
215 3.1. Grassland modeling
215 In this work, each grassland gi is composed of a given number ni of pixels xik ∈Rd, where k is the
216
pixel index such as k ∈{1, ..., ni}, i ∈{1, . . . , G}, G is the total number of grasslands, N = ∑G
i=1 ni is
217
the total number of pixels, d = nBnT is the number of spectro-temporal variables, nB is the number of
218
spectral bands and nT is the number of temporal acquisitions. In the experimental part, when working
219
on the intra-annual time series of 2013 using the four spectral bands, d = 4 × 17 = 68. In 2014, d =
220
4 × 15 = 60. When working on the inter-annual times series using NDVI, d = 1 × (13 + 17 + 15) = 45. 221
With each grassland gi are associated a matrix Xi of size (ni × d) and a response variable yi ∈R which
222
corresponds to its class label. 223 In this work, each grassland gi is composed of a given number ni of pixels xik ∈Rd, where k is the
216
pixel index such as k ∈{1, ..., ni}, i ∈{1, . . . , G}, G is the total number of grasslands, N = ∑G
i=1 ni is
217
the total number of pixels, d = nBnT is the number of spectro-temporal variables, nB is the number of
218
spectral bands and nT is the number of temporal acquisitions. In the experimental part, when working
219
on the intra-annual time series of 2013 using the four spectral bands, d = 4 × 17 = 68. In 2014, d =
220
4 × 15 = 60. When working on the inter-annual times series using NDVI, d = 1 × (13 + 17 + 15) = 45. 221
With each grassland gi are associated a matrix Xi of size (ni × d) and a response variable yi ∈R which
222
corresponds to its class label. 223 p
In the following, two types of grassland modeling are discussed, at the pixel level and at the
224
object level. A more informative object level modeling is then proposed. Then, similarity measures are
225
discussed. 226 In the following, two types of grassland modeling are discussed, at the pixel level and at the
224
object level. 3.1. Grassland modeling
215 A more informative object level modeling is then proposed. Then, similarity measures are
225
discussed
226 Version June 12, 2017 submitted to Remote Sens. 8 of 25 3.2. Similarity measure
255 3.2. Similarity measure
255 3.1.2. Object level
235 At the object level, the mean vector µi of the pixels belonging to gi is generally used to represent
236
gi. It is estimated empirically by:
237 At the object level, the mean vector µi of the pixels belonging to gi is generally used to represent
236
gi. It is estimated empirically by:
237 ˆµi = 1
ni
ni
∑
l=1
xil. (1) (1) In this case, a vector ˆµi ∈Rd and a response variable yi ∈R are associated with each grassland. 238
This representation might be limiting for heterogeneous objects such as grasslands since the
239
spectro-temporal variability is not encoded. To illustrate this bias, Figure 7 shows on the left the
240
set of pixels values in the NIR band for two grasslands (a and b). From this figure, it can be seen that if
241
the mean vector captures the average behavior, higher variability can be captured by including the
242
variance/covariance (middle and right plots). The figure shows that the first and second eigenvectors
243
of the covariance matrix capture well the general trend in the grassland and the main variations due to
244
different phenologic behaviors in the grassland. This information cannot be recovered by considering
245
the variance feature only: covariance must also be included. 246 In this study, to account for the spectro-temporal variability, we assume that the distribution of
247
pixels xi is, conditionally to grassland gi, a Gaussian distribution N (µi, Σi), where Σi is the covariance
248
matrix estimated empirically by:
249 ˆΣi =
1
ni −1
ni
∑
l=1
(xil −ˆµi)(xil −ˆµi)⊤. (2) (2) In this case, we associate with each gi its estimated distribution N (ˆµi, ˆΣi) and a response variable
250
yi ∈R. The Gaussian modeling encodes first and second order information on the grassland by
251
exploiting the variance-covariance information. It is worth noting that if we constraint ˆΣi = Id, the
252
identity matrix of size d, for i ∈[1, . . . , G], the Gaussian modeling is reduced to the mean vector. In the
253
following, N (ˆµi, ˆΣi) is denoted by Ni. 254 In this case, we associate with each gi its estimated distribution N (ˆµi, ˆΣi) and a response variable
250
yi ∈R. The Gaussian modeling encodes first and second order information on the grassland by
251
exploiting the variance-covariance information. 3.1.2. Object level
235 It is worth noting that if we constraint ˆΣi = Id, the
252
identity matrix of size d, for i ∈[1, . . . , G], the Gaussian modeling is reduced to the mean vector. In the
253
following, N (ˆµi, ˆΣi) is denoted by Ni. 254 In this case, we associate with each gi its estimated distribution N (ˆµi, ˆΣi) and a response variable
250
yi ∈R. The Gaussian modeling encodes first and second order information on the grassland by
251
exploiting the variance-covariance information. It is worth noting that if we constraint ˆΣi = Id, the
252
identity matrix of size d, for i ∈[1, . . . , G], the Gaussian modeling is reduced to the mean vector. In the
253
following, N (ˆµi, ˆΣi) is denoted by Ni. 254 3.1.1. Pixel level
227 The representation of a grassland at the pixel level has been used a lot in the remote sensing
228
literature [17,42,44,48,49,52,59]. The grassland can either be represented by all its pixels or by one pixel
229
when the spatial resolution of the pixel is too coarse, see for instance [39,40]. In this representation,
230
a sample is a pixel. Therefore, with each xik is associated the response variable yi of gi, but xik is
231
processed independently of all other xik′ of gi. However, this representation usually leads to aberrant
232
classification results (e.g., salt and pepper effect) [38], which are not expected when working at the
233
grassland level. 234 3.1.2. Object level
235 3.1.2. Object level
235 3.2.1. Similarity measure between distributions
256 3.2.1. Similarity measure between distributions
256 3.2.1. Similarity measure between distributions
256 For classification purposes, a similarity measure between each pair of grasslands is required. 257
With pixel-based or mean modeling approaches, conventional kernel methods such as Support Vector
258
Machine (SVM) with a Radial Basis Function (RBF) kernel can be used since the explanatory variable
259
is a vector. However for a Gaussian modeling, i.e., when the explanatory variable is a distribution,
260
specific derivations are required to handle the probability distribution as an explanatory variable. 261 For classification purposes, a similarity measure between each pair of grasslands is required. 257
With pixel-based or mean modeling approaches, conventional kernel methods such as Support Vector
258
Machine (SVM) with a Radial Basis Function (RBF) kernel can be used since the explanatory variable
259
is a vector. However for a Gaussian modeling, i.e., when the explanatory variable is a distribution,
260
specific derivations are required to handle the probability distribution as an explanatory variable. 261 Many similarity functions generally used to compare two Gaussian distributions (e.g.,
262
Kullback-Leibler divergence [76], Jeffries-Matusita distance which is based on Bhattacharyya
263
distance [77]) require the inversion of the covariance matrices and the computation of their
264
determinants. For a conventional multivariate Gaussian model, the number of parameters to estimate
265 Version June 12, 2017 submitted to Remote Sens. 9 of 25 9 of 25 02 03 04 05 06 07 08 09 10 11 12
0.2
0.4
02 03 04 05 06 07 08 09 10 11 12
0.2
0.4
02 03 04 05 06 07 08 09 10 11 12
0.2
0.4
(a)
02 03 04 05 06 07 08 09 10 11 12
0.2
0.4
02 03 04 05 06 07 08 09 10 11 12
0.2
0.4
02 03 04 05 06 07 08 09 10 11 12
0.2
0.4
(b)
Figure 7. Examples of 2013 time series evolution in the NIR reflectance band of Formosat-2 for a
grassland of management practice (a) "mowing" and (b) "grazing". The x-axis is the month of the year. The y-axis is the NIR reflectance value. The plot on the left shows the evolution of all the pixels in the
grassland and the temporal mean of these pixels in red. each grassland is d(d + 3)/2 (d parameters for the mean vector and d(d + 1)/2 parameters for the symmetric covariance matrix). In the case where d is large, the number of parameters to estimate can
267
be much larger than the number of samples, making the inverse problem ill-posed. This issue is faced
268
in this study because grasslands are small elements of the landscape. They are characterized by a
269
number of spectro-temporal variables which is about of the same order as the number of pixels ni (see
270
Figure 6). Therefore, most of the estimated covariance matrices are singular and their determinants are
271
null. Hence, conventional similarity measures used for moderate dimensional Gaussian distributions
272
are not suitable for high dimensional Gaussian distributions. In the following, we propose to use
273
mean map kernels and we introduce a derivation of mean map kernels to weight the influence of the
274
covariance matrix. 275 3.2.1. Similarity measure between distributions
256 The plot in the middle shows the temporal
mean in red, the temporal mean +0.2× the first eigenvector in blue and the temporal mean −0.2× the
first eigenvector in black. The plot on the right shows the temporal mean in red, the temporal mean
+0.2× the second eigenvector in blue and the temporal mean −0.2× the second eigenvector in black. 02 03 04 05 06 07 08 09 10 11 12
0.2
0.4
(a) 02 03 04 05 06 07 08 09 10 11 12
0.2
0.4 02 03 04 05 06 07 08 09 10 11 12
0.2
0.4 (a) (a)
02 03 04 05 06 07 08 09 10 11 12
0.2
0.4
(b) 02 03 04 05 06 07 08 09 10 11 12
0.2
0.4 02 03 04 05 06 07 08 09 10 11 12
0.2
0.4 (b) Figure 7. Examples of 2013 time series evolution in the NIR reflectance band of Formosat-2 for a
grassland of management practice (a) "mowing" and (b) "grazing". The x-axis is the month of the year. The y-axis is the NIR reflectance value. The plot on the left shows the evolution of all the pixels in the
grassland and the temporal mean of these pixels in red. The plot in the middle shows the temporal
mean in red, the temporal mean +0.2× the first eigenvector in blue and the temporal mean −0.2× the
first eigenvector in black. The plot on the right shows the temporal mean in red, the temporal mean
+0.2× the second eigenvector in blue and the temporal mean −0.2× the second eigenvector in black. 3.2.2. Mean map kernels between distributions
276 Mean map kernels are similarity measures which operate on distributions [78]. They have
277
been used in remote sensing for semi-supervised pixel-based learning in [79]. In their work, the
278
authors define the similarity between two distributions pi and pj as the average of all pairwise kernel
279
evaluations over the available realizations of pi and pj (i.e., pixels that belong to grasslands gi or gj). It
280
corresponds to the empirical mean kernel [79, eq.(8)]:
281 Ke(pi, pj) =
1
ninj
ni,nj
∑
l,m=1
k(xil, xjm),
(3) (3) where ni and nj are the number of pixels associated with pi and pj respectively, xil is the lth realization
282
of pi, xjm is the mth realization of pj and k is a semi-definite positive kernel function. 283 where ni and nj are the number of pixels associated with pi and pj respectively, xil is the lth realization
282
of pi, xjm is the mth realization of pj and k is a semi-definite positive kernel function. 283 Version June 12, 2017 submitted to Remote Sens. on June 12, 2017 submitted to Remote Sens. 10 of 2 10 of 25 It is possible to include prior knowledge on the distributions by considering the generative mean
284
kernel [78]:
285 Kg(pi, pj)
=
Z
Rd
Z
Rd k(x, x′) ˆpi(x) ˆpj(x′)dxdx′. (4) (4) Note that eq. (3) acts on the realizations of pi while eq. (4) acts on its estimation. When deal
286 Note that eq. (3) acts on the realizations of pi while eq. (4) acts on its estimation. When dealing with a
286
large number of samples, the latter can drastically reduce the computational load with respect to the
287
former. 288 Note that eq. (3) acts on the realizations of pi while eq. (4) acts on its estimation. When dealing with a
286
large number of samples, the latter can drastically reduce the computational load with respect to the
287
former. 288 Note that eq. (3) acts on the realizations of pi while eq. (4) acts on its estimation. When dealing with a
286
large number of samples, the latter can drastically reduce the computational load with respect to the
287
former. 288 In our grassland modeling, pi and pj are assumed to be Gaussian distributions. In that case, if k is
289
a Gaussian kernel such as k(x, x′) = exp(−γ
2 ∥x −x′∥2), eq. 3.2.2. Mean map kernels between distributions
276 (4) reduces to the so-called Gaussian mean
290
kernel [80]:
291 KG(Ni, Nj) =
exp
−0.5(ˆµi −ˆµj)T
ˆΣi + ˆΣj + γ−1Id
−1
(ˆµi −ˆµj)
| ˆΣi + ˆΣj + γ−1Id|0.5
,
(5) (5) where γ is a positive regularization parameter coming from the Gaussian kernel k and | · | stands for
292
the determinant. 293 This kernel is not normalized, i.e., KG(Ni, Ni) ̸= 1, but the normalization can be achiev
294 This kernel is not normalized, i.e., KG(Ni, Ni) ̸= 1, but the normalization can be achieved easily ˜KG(Ni, Nj)
=
KG(Ni, Nj)
KG(Ni, Ni)0.5KG(Nj, Nj)0.5
=
KG(Ni, Nj)|2 ˆΣi + γ−1Id|0.25|2 ˆΣj + γ−1Id|0.25. (6) (6) With respect to the Kullback-Leibler divergence (KLD) and the Jeffries-Matusita distance (JMD),
295
the Gaussian mean kernel introduces a ridge regularization term γ−1Id in the computation of the inverse
296
and of the determinant [81]. Thus, the Gaussian mean kernel is more suitable to measure the similarity
297
in a high dimensional space than KLD and JMD. The value of γ tunes the level of regularization. It is
298
tuned during the training process as a conventional kernel parameter. 299 However, in the case of very small grasslands, two problems remain. The first lies in the ridge
300
regularization: in this case, so low γ values are selected that it becomes too much regularized and it
301
deteriorates the information. The second problem is that the estimation of the covariance matrix has
302
a large variance when the number of samples used for the estimation is lower than the number of
303
variables. Therefore, the covariance matrix becomes a poorly informative feature. In the following, we
304
propose a new kernel function that allows to weight the covariance features with respect to the mean
305
features. 306 3.2.3. α-Gaussian Mean Kernel
307 318 p
q
3. α →+∞: We get a distance which works only on the covariance matrices. It is therefore equivalent
319
to an object modeling where only the covariance is considered. 320 3. α →+∞: We get a distance which works only on the covariance matrices. It is therefore equivalent
319
to an object modeling where only the covariance is considered. 320 4. γ →+∞and α = 2: The α-Gaussian mean kernel simplifies to a RBF kernel built with the
321
Bhattacharyya distance computed between Ni and Nj. 322 4. γ →+∞and α = 2: The α-Gaussian mean kernel simplifies to a RBF kernel built with the
321
Bhattacharyya distance computed between Ni and Nj. 322 This proposed kernel thus includes several similarity measures known in the literature. 323
Furthermore, new similarity measures can be defined by choosing different parameters configuration. 324
The α-Gaussian mean kernel (αGMK) is therefore more flexible since it can adapt to the classification
325
constraints:
326 •
Whether the heterogeneity of the object is relevant or not,
327
Wh
h
h
b
h
b
f
l
d h
b
f
bl
h
h
l g
y
j
•
Whether the ratio between the number of pixels and the number of variables is high or low. 3.2.3. α-Gaussian Mean Kernel
307 Depending on the level of heterogeneity and the size of the grassland, the covariance matrix could
308
be more or less important for the classification process. We propose a kernel including an additional
309
positive parameter α which allows to weight the influence of the covariance matrix, the α-generative
310
mean kernel:
311 Kα(pi, pj)
=
Z
Rd
Z
Rd k(x, x′) ˆpi(x)(α−1) ˆpj(x′)(α−1)dxdx′. (7) (7) When pi and pj are Gaussian distributions, k is a Gaussian kernel and the normalization is applied,
312
the expression gives rise to the α-Gaussian mean kernel:
313 When pi and pj are Gaussian distributions, k is a Gaussian kernel and the normalization is applied,
312
the expression gives rise to the α-Gaussian mean kernel:
313 11 of 25 Version June 12, 2017 submitted to Remote Sens. ˜Kα(Ni, Nj) =
exp
−0.5(ˆµi −ˆµj)T
α( ˆΣi + ˆΣj) + γ−1Id
−1
(ˆµi −ˆµj)
|α( ˆΣi + ˆΣj) + γ−1Id|0.5
|2α ˆΣi + γ−1Id|0.25|2α ˆΣj + γ−1Id|0.25. (8) (8) The proof is given in the appendix. It is interesting to note that particular values of α and γ lead to
314
known results:
315 The proof is given in the appendix. It is interesting to note that particular values of α and γ lead to
314
known results:
315 1. α = 0: In this case, eq. (8) reduces to the Gaussian kernel between the mean vectors. It becomes
316
therefore equivalent to an object modeling where only the mean is considered. 317 1. α = 0: In this case, eq. (8) reduces to the Gaussian kernel between the mean vectors. It becomes
316
therefore equivalent to an object modeling where only the mean is considered. 317 therefore equivalent to an object modeling where only the mean is considered. 317
2. α = 1: It corresponds to the Gaussian mean kernel defined in eq. (6). 318
3. α →+∞: We get a distance which works only on the covariance matrices. It is therefore equivalent
319
to an object modeling where only the covariance is considered. 320
4. γ →+∞and α = 2: The α-Gaussian mean kernel simplifies to a RBF kernel built with the
321 q
j
g
y
α = 1: It corresponds to the Gaussian mean kernel defined in eq. (6). q
j
g
y
2. α = 1: It corresponds to the Gaussian mean kernel defined in eq. (6). 4. Experiments on grasslands classification
329 In this section, the experiments for grassland classification are detailed. We first introduce the
330
seven competitive methods, then the classification protocol is described and we finally present and
331
discuss the results. 332 In this section, the experiments for grassland classification are detailed. We first introduce the
330
seven competitive methods, then the classification protocol is described and we finally present and
331
discuss the results. 332 4.1. Competitive methods
333
Several existing pixel-based and object-based classification methods using SVM are presented
334
below. They are compared to assess the effectiveness of the proposed object-based method which relies
335
on the weighted use of the covariance matrix, αGMK, for the classification of grasslands. 336
4.1.1. Pixel-based and mean modeling
337
These conventional methods use a RBF kernel. 338
•
PMV (Pixel Majority Vote): The pixel-based method was described in section 3.1.1. It classifies
339
each pixel with no a priori information on the object which the pixel belongs to. In order to
340
compare to other object level methods, one class label is extracted per grassland by a majority
341
vote done among the pixels belonging to the same grassland. 342
•
µ (mean): The distribution of the pixels reflectance of gi is modeled by its mean vector µi (see
343
section 3.1.2). 344
4.1.2. Divergence methods
345
These methods are based on a distance D between two Gaussian distributions. They are used in a
346
Gaussian kernel such as KD(Ni, Nj) = exp(−
D2
ij
σ ), with σ > 0:
347
•
HDKLD (High Dimensional Kullback-Leibler Divergence): This method uses the Kullback-Leibler
348
divergence for Gaussian distributions with a regularization on covariance matrices such as
349
described in [82]. 350 Several existing pixel-based and object-based classification methods using SVM are presented
334
below. They are compared to assess the effectiveness of the proposed object-based method which relies
335
on the weighted use of the covariance matrix, αGMK, for the classification of grasslands. 336 Several existing pixel-based and object-based classification methods using SVM are presented
334
below. They are compared to assess the effectiveness of the proposed object-based method which relies
335
on the weighted use of the covariance matrix, αGMK, for the classification of grasslands. 336 •
PMV (Pixel Majority Vote): The pixel-based method was described in section 3.1.1. It classifies
339
each pixel with no a priori information on the object which the pixel belongs to. 4. Experiments on grasslands classification
329 360 •
EMK (Empirical Mean Kernel): This method uses the empirical mean map kernel of eq. (3) and it
357
is pixel-based. 358
•
GMK (Gaussian Mean Kernel): This method is based on the normalized Gaussian mean kernel
359
(eq. 6). 360
•
αGMK (α-Gaussian Mean Kernel): This method is based on the proposed normalized α-Gaussian
361
mean kernel (eq. 8). 362 •
GMK (Gaussian Mean Kernel): This method is based on the normalized Gaussian mean kernel
359
(eq. 6). 360
•
αGMK (α-Gaussian Mean Kernel): This method is based on the proposed normalized α-Gaussian
361
mean kernel (eq. 8). 362 Figure 8 illustrates the relationships between the different methods. The characteristics of each
363
method are synthesized in Table 3. 364 Figure 8 illustrates the relationships between the different methods. The characteristics of each
363
method are synthesized in Table 3. 364 For memory issues during the SVM process, the number of pixels processed for the old and young
365
grasslands classification was divided by 10 for the two methods based on pixels (PMV and EMK). 366
Only 1 pixel out of 10 was kept per grassland. 367 g
Pixel
PMV
Object
Realizations
EMK
Moments
µ
Σ
N
BD
GMK
HDKLD
αGMK
Figure 8. Contribution of the proposed method in grassland analysis for supervised classification. αGMK consists in a general modeling of the grassland at the object level and it encompasses several
known modelings. The underlined methods are tested in this study. PMV, EMK and µ are not based on
Gaussian modeling while the others are. g
Pixel
PMV
Object
Realizations
EMK
Moments
µ
Σ
N
BD
GMK
HDKLD
αGMK Object Moments N Realizations GMK
HDKLD Figure 8. Contribution of the proposed method in grassland analysis for supervised classification. αGMK consists in a general modeling of the grassland at the object level and it encompasses several
known modelings. The underlined methods are tested in this study. PMV, EMK and µ are not based on
Gaussian modeling while the others are. Table 3. Characteristics of the methods used in this study. Table 3. Characteristics of the methods used in this study. Table 3. Characteristics of the methods used in this study. Method
PMV
EMK
µ
HDKLD
BD
GMK
αGMK
Level
Pixel
Object
Object
Object
Object
Object
Object
Expl. 4. Experiments on grasslands classification
329 In order to
340
compare to other object level methods, one class label is extracted per grassland by a majority
341
vote done among the pixels belonging to the same grassland. 342 g
p
g
g
g
•
µ (mean): The distribution of the pixels reflectance of gi is modeled by its mean vector µi (see
343
section 3.1.2). 344 These methods are based on a distance D between two Gaussian distributions. They are used in a
346
Gaussian kernel such as KD(Ni, Nj) = exp(−
D2
ij
σ ), with σ > 0:
347 •
HDKLD (High Dimensional Kullback-Leibler Divergence): This method uses the Kullback-Leibler
348
divergence for Gaussian distributions with a regularization on covariance matrices such as
349
described in [82]. 350 Version June 12, 2017 submitted to Remote Sens. 12 of 25 on June 12, 2017 submitted to Remote Sens. 12 of 25 •
BD (Bhattacharyya Distance): This method uses the Bhattacharyya distance in the case of Gaussian
351
distributions:
352 •
BD (Bhattacharyya Distance): This method uses the Bhattacharyya distance in the case of Gaussian
351
distributions:
352 •
BD (Bhattacharyya Distance): This method uses the Bhattacharyya distance in the case of Gaussian
351
distributions:
352 B(Ni, Nj) = 1
8(ˆµi −ˆµj)⊤ ˆΣi + ˆΣj
2
−1
(ˆµi −ˆµj) + 1
2 ln
|
ˆΣi+ ˆΣj
2
|
| ˆΣi|0.5| ˆΣj|0.5
. Small eigenvalues of the covariance matrices are shrinked to the value 10−5 to make the
353
computation tractable [83]. 354 Small eigenvalues of the covariance matrices are shrinked to the value 10−5 to make the
353
computation tractable [83]. 354 Small eigenvalues of the covariance matrices are shrinked to the value 10−5 to make the
353
computation tractable [83]. 354 3. Mean map kernel based methods 4.1.3. Mean map kernel based methods
355 These methods are based on mean map kernels presented in section 3.2:
356 •
EMK (Empirical Mean Kernel): This method uses the empirical mean map kernel of eq. (3) and it
357
is pixel-based. 358
GMK (G
i
M
K
l) Thi
th d i b
d
th
li
d G
i
k
l •
EMK (Empirical Mean Kernel): This method uses the empirical mean map kernel of eq. (3) and it
357
is pixel-based. 358
•
GMK (Gaussian Mean Kernel): This method is based on the normalized Gaussian mean kernel
359
(eq. 6). 4. Experiments on grasslands classification
329 variable
xik
xik
µi
Ni
Ni
Ni
Ni
Kernel
RBF
RBF
RBF
KHDKLD
KB
˜KG
˜Kα
Parameters
σ, C
σ, C
σ, C
σ, C
σ, C
γ, C
γ, α, C
Nb of samples
16,250 / 8,628
16,250 / 8,628
475 / 52
475 / 52
475 / 52
475 / 52
475 / 52 Version June 12, 2017 submitted to Remote Sens. 13 of 25 13 of 25 4.2. Classification protocol
368 We compared the efficiency in terms of classification accuracy and processing time of all the
369
presented methods by classifying the two grassland datasets on inter-annual and intra-annual time
370
series (section 2). 371 For each method, a Monte Carlo procedure was performed on 100 runs. For each run, the dataset
372
was split randomly into training and testing datasets (75% for training and 25% for testing), preserving
373
the initial proportions of each class. The same grasslands were selected for a given Monte Carlo
374
repetition regardless of the method. 375 During each repetition, the optimal parameters were tuned by cross-validation based on the best
376
F1 score. Table 4 contains the parameters grid search for all the methods. Note that a wide grid was
377
searched for the parameter α of αGMK to further analyze the distribution of selected values. The
378
penalty parameter C of the SVM process was chosen empirically and fixed to C = 10, after running
379
several simulations. The classification accuracy for each repetition was assessed by the F1 score
380
computed from the confusion matrix. The overall accuracy (OA) was computed but it is not presented
381
here, because it does not reflect well the accuracy of the classification since unbalanced datasets were
382
used. 383 In order to compare each pair of methods, a Wilcoxon rank-sum test was processed on the pair of
384
distributions of the 100 F1 scores. This nonparametric test is designed for two independent samples
385
that are not assumed to be normally distributed [84]. It tests if the two samples are drawn from
386
populations having the same distribution. 387 In order to compare each pair of methods, a Wilcoxon rank-sum test was processed on the pair of
384
distributions of the 100 F1 scores. This nonparametric test is designed for two independent samples
385
that are not assumed to be normally distributed [84]. It tests if the two samples are drawn from
386
populations having the same distribution. 387 The kernels and the SVM were implemented in Python through the Scikit library [85]. 388 Table 4. Parameters tested for each method during cross-validation. Method
Parameters values
Inter-annual analysis
Intra-annual analysis
PMV
σ ∈{20, 21, . . . , 210}
σ ∈{2−17, 2−16, . . . , 2−10}
EMK
σ ∈{20, 21, . . . , 210}
σ ∈{2−18, 2−17, . . . 4.2. Classification protocol
368 , 2−10}
µ
σ ∈{20, 21, . . . , 210}
σ ∈{2−18, 2−17, . . . , 2−10}
HDKLD
σ ∈{210, 211, . . . , 220}
σ ∈{215, 216, . . . , 225}
BD
σ ∈{20, 21, . . . , 210}
σ ∈{210, 211, . . . , 218}
GMK
γ ∈{20, 21, . . . , 210}
γ ∈{2−17, 2−18, . . . , 2−10}
αGMK
γ ∈{20, 21, . . . , 210}
γ ∈{2−18, 2−17, . . . , 2−13}
α ∈{0, 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 50}
α ∈{0, 10−3, 10−2, 10−1, 0.3, 0.5, 0.7, 0.9, 1, 2, 5, 10, 15, 20, 25} Table 4. Parameters tested for each method during cross-validation. 4.3. Results
389 4.3. Results
389 4.3.1. Old and young grasslands: inter-annual time series
390
Figure 9 sums up the old and young grasslands classification results for each method over the
391
100 repetitions as a boxplot of F1 scores. The Kappa coefficients can be found in Figure S2 in the
392
supplementary materials. Since the cross-validation was not based on the Kappa coefficient, the results
393
are discussed in terms of F1 scores. The method reaching the best scores is αGMK with a F1 average of
394
0.71 followed by PMV and GMK with an average of 0.69. 395
Table 5 contains the Wilcoxon rank-sum test statistics between each pair of methods. It tests
396
the null hypothesis that the two sets of observations are drawn from the same distribution. The
397
null hypothesis is rejected if the test statistics is greater than 1.96 with a confidence level of 5%
398
(p-value < 0.05). In this case, it accepts the alternative hypothesis that values in one population are
399
more likely to be larger than the values from the other. The two best methods, αGMK and PMV are not
400
significantly different. But αGMK is significantly better than all the other methods, whereas PMV is
401
not significantly different than the mean map methods (EMK and GMK). The worst method is HDKLD
402
with a mean F1 of 0.59. 403 4.3.1. Old and young grasslands: inter-annual time series
390 14 of 25 Version June 12, 2017 submitted to Remote Sens. PMV
EMK µ
HDKLDBD
GMK
αGMK
0.4
0.6
0.8
F1
Figure 9. 4.2. Classification protocol
368 Boxplot of F1 score repartitions for the classification of the old and young grasslands. The
line in the box stands for the median whereas the dot stands for the mean. PMV
EMK µ
HDKLDBD
GMK
αGMK
0.4
0.6
0.8
F1 F1 Figure 9. Boxplot of F1 score repartitions for the classification of the old and young grasslands. The
line in the box stands for the median whereas the dot stands for the mean. Table 5. Absolute value of Wilcoxon rank-sum test statistics on F1 score for the old and young
grasslands classification. ** indicates the results are significantly different, i.e., p-value < 0.05. Method
PMV
µ
HDKLD
BD
EMK
GMK
αGMK
PMV
-
3.52**
8.66**
4.83**
1.93
0.98
1.32
µ
-
7.48**
1.76
1.55
2.28**
4.80**
HDKLD
-
5.68**
8.26**
8.65**
9.77**
BD
-
3.23**
3.95**
6.09**
EMK
-
0.94
3.35**
GMK
-
2.42**
αGMK
- In terms of processing load and time, the pixel-based methods are clearly the most demanding. 404
Indeed, processing the 160,514 pixels was not possible with SVM, so we had to reduce the number
405
of samples. These issues are not faced with object-oriented methods. The fastest methods are µ and
406
HDKLD, but they did not reach acceptable classification accuracies. The best method in terms of ratio
407
accuracy/processing time is αGMK. It is appropriate for processing a large number of grasslands. 408 2. Management practices: intra-annual time series 4.3.2. Management practices: intra-annual time series
409 The classifications accuracies for management practices are shown in Figure 10 (F1 score) and in
410
Figure S3 in the supplementary materials (Kappa coefficient) for year 2013 and for year 2014. 411 The classifications accuracies for management practices are shown in Figure 10 (F1 score) and in
410
Figure S3 in the supplementary materials (Kappa coefficient) for year 2013 and for year 2014. 411 The classifications accuracies for management practices are shown in Figure 10 (F1 score) and in
410
Figure S3 in the supplementary materials (Kappa coefficient) for year 2013 and for year 2014. 411 The classifications accuracies for management practices are shown in Figure 10 (F1 score) and in
ure S3 in the supplementary materials (Kappa coefficient) for year 2013 and for year 2014. In terms of classification accuracy, methods based on divergences (BD and HDKLD) provided
412
the worst results. Pixel-based methods, mean modeling method and mean generative kernel methods
413
provided similar results in terms of F1 score, except for PMV which was significantly worse than the
414
others for the year 2013. αGMK provided the highest values in 2013 (average F1 of 0.65) but it was not
415
significantly better than the others for this dataset. Indeed, due to the very low number of grasslands
416
composing this dataset, confusion matrices were quite similar whatever the method. It is therefore
417
difficult to compare the methods efficiency in this configuration. 418 Nevertheless, this dataset makes possible the comparison in terms of processing times, because
419
the same spectral information was used for all the methods. Figure 11 illustrates the training processing
420
time relative to the one of PMV versus the average F1 score for each method. In terms of computational
421
time, the pixel-based methods required the largest processing times. BD was also very long, mainly
422
because of the shrinkage procedure. Mean modeling was the fastest, followed closely by HDKLD. 423
αGMK and GMK were equivalent in terms of computational times. For this configuration with a low
424
number of grasslands, the mean modeling was the most efficient in terms of accuracy/processing time
425
ratio. 426 Nevertheless, this dataset makes possible the comparison in terms of processing times, because
419
the same spectral information was used for all the methods. Figure 11 illustrates the training processing
420
time relative to the one of PMV versus the average F1 score for each method. In terms of computational
421
time, the pixel-based methods required the largest processing times. BD was also very long, mainly
422
because of the shrinkage procedure. Mean modeling was the fastest, followed closely by HDKLD. 423
αGMK and GMK were equivalent in terms of computational times. For this configuration with a low
424
number of grasslands, the mean modeling was the most efficient in terms of accuracy/processing time
425
ratio. 426 Version June 12, 2017 submitted to Remote Sens. 15 of 25 15 of 25 PMV
EMK
µ
HDKLD
BD
GMK
αGMK
0.2
0.4
0.6
0.8
1
F1
PMV
EMP
µ
HDKLD
BD
GMK
αGMK
0.2
0.4
0.6
0.8
1
(a)
(b)
Figure 10. 4.4. Discussion
429 The purpose of this work was to develop a model suitable for the classification of grasslands from
430
dense inter- or intra-annual SITS and robust to the dimension of the data. The proposed method based
431
on a weighted use of the covariance, namely αGMK, was compared to several competitive methods. 432 It is worth noting that the times series of 2014 produced higher classification accuracies (maximum
427
F1 average of 0.73 for GMK) than the time series of 2013 (maximum F1 average of 0.65 for αGMK).
428 It is worth noting that the times series of 2014 produced higher classification accuracies (maximum
427
F1 average of 0.73 for GMK) than the time series of 2013 (maximum F1 average of 0.65 for αGMK). 428 4.4. Discussion
429 The classifications accuracies for management practices are shown in Figure 10 (F1 score) and in
ure S3 in the supplementary materials (Kappa coefficient) for year 2013 and for year 2014. Boxplot of F1 score repartitions for classification of management practices using time series
of year (a) 2013 and (b) 2014. The line in the box stands for the median whereas the dot stands for the
mean. PMV
EMK
µ
HDKLD
BD
GMK
αGMK
0.2
0.4
0.6
0.8
1
F1
(a) PMV
EMP
µ
HDKLD
BD
GMK
αGMK
0.2
0.4
0.6
0.8
1
(b) 1 (b) (a) Figure 10. Boxplot of F1 score repartitions for classification of management practices using time series
of year (a) 2013 and (b) 2014. The line in the box stands for the median whereas the dot stands for the
mean. 0
0.2
0.4
0.6
0.8
1
0.5
0.55
0.6
0.65
µ
αGMK
PMV
HDKLD
BD
EMK
GMK
Relative time to PMV
F1
Figure 11. Relative training processing times to PMV and average F1 of each method for intra-annual
time series of 2013. Figure 11. Relative training processing times to PMV and average F1 of each method for intra-annual
time series of 2013. It is worth noting that the times series of 2014 produced higher classification accuracies (maximum
427
F1 average of 0.73 for GMK) than the time series of 2013 (maximum F1 average of 0.65 for αGMK). 428 4.4.1. Methods efficiency
433 The methods efficiency are discussed for the old and young grasslands classification, since the
434
results provided with the other dataset are not significantly different, mostly because of the small
435
dataset size. 436 The methods efficiency are discussed for the old and young grasslands classification, since the
434
results provided with the other dataset are not significantly different, mostly because of the small
435
dataset size. 436 The divergence methods (BD and HDKLD) provided the worst results, showing that they are not
437
robust enough to a high dimensional space. 438 The divergence methods (BD and HDKLD) provided the worst results, showing that they are no
ust enough to a high dimensional space. Although they provided results close to the best results, pixel-based methods (PMV and EMK)
439
are the most demanding in terms of computational time and they do not scale well with the number of
440
pixels. Indeed, they have to process N pixels instead of G grasslands with G ≪N. Therefore, we had
441 Although they provided results close to the best results, pixel-based methods (PMV and EMK)
439
are the most demanding in terms of computational time and they do not scale well with the number of
440
pixels. Indeed, they have to process N pixels instead of G grasslands with G ≪N. Therefore, we had
441 Version June 12, 2017 submitted to Remote Sens. 16 of 25 to reduce the number of pixels used for the classification. Using them on a large area might be difficult,
442
as the old and young grasslands dataset showed. 443 Representing grasslands by the estimated distribution of their set of pixels decreases the
444
complexity during the SVM process. Therefore, object level methods offer a lower computational load
445
when compared to empirical mean kernels and pixel-based methods. 446 The mean generative kernel methods performed significantly better than the mean-only method
Among them, αGMK performed better than GMK. It was also one of the most stable methods. In this context, including the covariance information helps to discriminate grasslands. However,
449
if the dimensionality is not properly handled, it deteriorates the process (e.g., BD and HDKLD). In this
450
case, it is preferable to use the mean values only. αGMK offers the possibility to weight the influence
451
of the covariance information compared to the mean. 4.4.1. Methods efficiency
433 As a result, it provided better results than the
452
mean modeling and than GMK, since it encompasses both. 453 It is furthermore interesting to analyze the optimal values of the weightening parameter α found
454
during the cross-validation and the average of associated F1 scores (Figure 12). The highest F1 scores
455
were reached for high values of ˆα. The worst F1 scores were obtained with ˆα < 2 and the value ˆα = 0
456
was never selected. It shows the importance of the covariance information in grasslands modeling: the
457
heterogeneity in a grassland must be accounted for and it is not entirely well represented by the mean
458
only. 459 0.1
0.5
1
2
5
10
15
20
25
50
0
5
10
15
20
25
ˆα
ˆα count
0.6
0.65
0.7
Average F1
Figure 12. Bar plot of ˆα values chosen by cross-validation and the average of associated F1 scores (red
dots) for the classification of old and young grasslands using αGMK. NB: The value ˆα = 0 was never
selected. 0.1
0.5
1
2
5
10
15
20
25
50
0
5
10
15
20
25
ˆα
0.6
0.65
0.7
Average F1 ˆα Figure 12. Bar plot of ˆα values chosen by cross-validation and the average of associated F1 scores (red
dots) for the classification of old and young grasslands using αGMK. NB: The value ˆα = 0 was never
selected. 4.4.4. Grassland typology
498 On the whole, the classification did not reach high accuracies (F1 maximum average of 0.73 for
499
management practices and of 0.71 for old and young grasslands classification). This can be explained
500
by the unbalanced dataset with under-representation of grazing and mixed grasslands in the first
501
application and under-representation of old grasslands in the second one. These classes obtained the
502
lowest producer and user accuracies (cf. Tables S1 and S2 in the supplementary materials) because
503
of their limited number of samples for training the models. The methods should be tested on a more
504
balanced dataset of grasslands classes. 505 Moreover, as many times emphasized, semi-natural grasslands (which are present in these
506
datasets) are characterized by their high level of heterogeneity. Therefore, there might be a large amount
507
of intra-class variability because of grasslands diversity. The discrimination might be improved by
508
using more distinct classes: intensively used grasslands against extensively used grasslands, artificial
509
(monospecific) grasslands against semi-natural grasslands for instance. 510 4.4.2. Grassland modeling
460 Therefore, the classification process takes more time than GMK fo
476 It also includes several object level methods known in the literature. However, this is at the cost of one
475
more parameter to tune. Therefore, the classification process takes more time than GMK for instance. 476
Above all, although it is the first application of generative mean kernels in remote sensing
477
classification, the α-Gaussian mean kernel proved its efficiency and stability in these experiments. The
478
results suggest it is appropriate for grasslands classification. 479 It also includes several object level methods known in the literature. However, this is at the cost of one
475
more parameter to tune. Therefore, the classification process takes more time than GMK for instance. 476
Above all, although it is the first application of generative mean kernels in remote sensing
477
classification, the α-Gaussian mean kernel proved its efficiency and stability in these experiments. The
478
results suggest it is appropriate for grasslands classification. 479 4.4.3. Acquisition dates
480 However, the scope of this study was to
496
develop a method which is able to use a given time series, without having to process a date selection. 497 In general, the results could also be enhanced by removing some winter images which can have
495
a negative influence on the entire annual time series [40]. However, the scope of this study was to
496
develop a method which is able to use a given time series, without having to process a date selection. 497 4.4.3. Acquisition dates
480 For the management practice classification, using time series of 2014 produced better results
481
than using 2013. It might be explained by the acquisition dates in the time series. Although 2014
482
has less images, more clear images were acquired during Spring compared to 2013 which has a
483
lack of acquisitions in April and May (Figure 3). Indeed, many studies showed that the best season
484
to discriminate grasslands is during the growing season [36,49,53,54]. Spring is the period of the
485
vegetation cycle where the management practices begin. Therefore, it is easier to differentiate the
486
practices during this period. It might thus affect the accuracy of the classification of year 2013. 487 It is not shown in this experiment, but using only one or two years of acquisitions to discriminate
488
old from young grasslands did not produce sufficient classification accuracies. This is the reason
489
why three years of data were used. Old "permanent" grasslands are supposed to have a more stable
490
phenology over the years than the young "temporary" grasslands which have been recently sown
491
(less than five years) [6]. The young grasslands phenology is closer to crops in their very first years. 492
We suppose this makes possible their discrimination with inter-annual SITS. However, the optimal
493
number of years needed to discriminate these types of grasslands could constitute a research topic. 494 It is not shown in this experiment, but using only one or two years of acquisitions to discriminate
488
old from young grasslands did not produce sufficient classification accuracies. This is the reason
489
why three years of data were used. Old "permanent" grasslands are supposed to have a more stable
490
phenology over the years than the young "temporary" grasslands which have been recently sown
491
(less than five years) [6]. The young grasslands phenology is closer to crops in their very first years. 492
We suppose this makes possible their discrimination with inter-annual SITS. However, the optimal
493
number of years needed to discriminate these types of grasslands could constitute a research topic. 494 In general, the results could also be enhanced by removing some winter images which can have
495
a negative influence on the entire annual time series [40]. 4.4.2. Grassland modeling
460 Following on from the methods discussion, the choice of modeling grasslands pixels distribution
461
by a Gaussian distribution makes sense in this context. It is particularly well appropriate for
462
semi-natural grasslands, which are very heterogeneous, contrary to crops or annual "artificial"
463
grasslands which can be assimilated to crops. 464 However, modeling grasslands by the mean only produced equivalent results to the methods
465
based on Gaussian modeling for the classification of management practices, contrary to the old and
466
young grassland discrimination. Indeed, management practices are supposed to be uniform at the
467
grassland scale. Therefore the mean appears to be sufficient for this application, contrary to the old and
468
young grasslands discrimination, which requires capturing more variations between the grasslands. 469
The best modeling might be different depending on the application. Moreover, some grasslands are so
470
small that the covariance matrix is too badly estimated. 471 However, modeling grasslands by the mean only produced equivalent results to the methods
465
based on Gaussian modeling for the classification of management practices, contrary to the old and
466
young grassland discrimination. Indeed, management practices are supposed to be uniform at the
467
grassland scale. Therefore the mean appears to be sufficient for this application, contrary to the old and
468
young grasslands discrimination, which requires capturing more variations between the grasslands. 469
The best modeling might be different depending on the application. Moreover, some grasslands are so
470
small that the covariance matrix is too badly estimated. 471 In the proposed kernel, this modeling was made flexible by regularizing the weight given to the
472
covariance matrix. αGMK benefits from its high level of adaptability in front of the object configuration:
473
no choice has to be made between a Gaussian or a mean modeling since the method encompasses both. 474 In the proposed kernel, this modeling was made flexible by regularizing the weight given to the
472
covariance matrix. αGMK benefits from its high level of adaptability in front of the object configuration:
473
no choice has to be made between a Gaussian or a mean modeling since the method encompasses both. 474 17 of 25 Version June 12, 2017 submitted to Remote Sens. 17 of 25 It also includes several object level methods known in the literature. However, this is at the c
475
more parameter to tune. 4.5. Prediction of management practices on the land use database grasslands
532 To show the efficiency of αGMK, we classified all the grasslands from the French agricultural land
533
use database (RPG) covered by the Formosat-2 time series to predict their management practice in 2014. 534
All the plots declared as grasslands in 2014, i.e., "permanent grassland" and "temporary grassland"
535
regardless of their age, were selected. After applying a negative buffer of 8m and rasterizing the
536
polygons, we removed the plots representing less than 10 Formosat-2 pixels. In the end, there were 797
537
grassland plots covered by the extent of Formosat-2 for a total of 252,472 pixels. 538 The multispectral SITS of 2014 was used. The SVM was trained on the whole field data
539
(section 2.3.2) using the same grid search as in the experiments. The parameters chosen after
540
cross-validation based on F1 score were ˆα = 5 and ˆγ = 2−15. Then, the model was used to predict the
541
management practices of the 797 grasslands of the land use database. 542 The classification accuracy could not be assessed since the true labels of the grasslands are not
543
known. However, as described in the study site, a spatial distribution of the classes could be expected. 544
Indeed, grazed and mixed grasslands should be found in the south-west of the site, whereas more
545
mown grasslands should be in the north. 546 An extract of the classification result is shown in Figure 13. It represents the classified grasslands in
547
their raster format. As expected, most of the grazed and mixed grasslands are located in the south-west
548
of the image, whereas the north of the image is mostly composed of mown grasslands. Therefore,
549
αGMK was very likely able to classify with an acceptable accuracy the grasslands management
550
practices without any a priori geographic information. However, specific care should be considered, as
551
not all the possible management practices were predicted. For instance, grasslands mown twice or
552
unused grasslands were not in the training dataset, but it does not mean these managements do not
553
exist in the rest of the data. The method deserves to be tested with an exhaustive grassland typology
554
to produce more detailed grasslands maps. 4.5. Prediction of management practices on the land use database grasslands
532 555 In terms of processing times, the proposed method is able to classify 800 grasslands, representing
556
more than 250,000 pixels, at the object level from a high spatial resolution SITS within a few seconds
557
on a conventional personal computer. 558 4.4.5. Comparison with existing works
511 In our knowledge, only the work of Möckel et al. [86] relates to the classification of grasslands age
512
using remote sensing data. They reached a Kappa value of 0.77 in classifying three different grassland
513
age-classes. However, they used airborne hyperspectral data from a single date. Their recommendation
514
was to use multitemporal data to improve the classification or to use satellite hyperspectral data to
515
monitor grasslands over wider areas. Our study was based on using multispectro-temporal satellite
516
data, but our proposed method would also work with hyperspectral data. 517 As described in the introduction, few studies have been carried out on the analysis of semi-natural
518
grasslands using high spatio-spectro-temporal resolutions SITS. Usually, methods were pixel-based
519
and they were applied on a few images or on a precise date selection to avoid dealing with the high
520 Version June 12, 2017 submitted to Remote Sens. 18 of 25 dimension of data [42,44,49]. Schuster et al. [52] successfully classified grassland habitat using 21
521
RapidEye images on a pixel basis, but there was no mention of the processing times. 522 At the object level using a time series, grasslands were often represented by their mean NDVI,
523
such as in [60], who noticed the difficulty to discriminate grasslands from crops because of mean
524
seasonal NDVI similarities. The closest configuration might be the work of Zillman et al. [35], who
525
used an object-based analysis and spectral reflectances combined with seasonal statistics of vegetation
526
indices for mapping grasslands across Europe. The seasonal statistics were particularly relevant in the
527
classification, because they captured well the spectral diversity of the grassland phenology. The use of
528
these metrics could be considered for discriminating grassland management practices which impact on
529
the phenology. The authors also concluded that the object-based analysis improves the classification
530
compared to a pixel-based classification. However, the objects were determined by segmentation. 531 4.5. Prediction of management practices on the land use database grasslands
532 5. Conclusion
559 This study aimed at developing a model for the classification of grasslands using satellite image
560
time series with a high number of spectro-temporal variables. A grassland modeling at the object
561
level was proposed. To deal with grasslands heterogeneity, their pixels distribution was modeled by
562
a Gaussian distribution. Then, to measure the similarity between two grasslands, i.e, two Gaussian
563
distributions, a kernel function based on mean maps was introduced, namely the α-Gaussian Mean
564
Kernel. The proposed method was compared to existing pixel-based and object-based classification
565
methods for the supervised classification of grassland using inter- and intra-annual SITS. The Gaussian
566
mean kernels provided the highest classification accuracies, showing that the covariance information
567 19 of 25 Version June 12, 2017 submitted to Remote Sens. Figure 13. Extract of the management practices classification of the grasslands from the French
agricultural land use database (RPG) in 2014. The background is a May, 2014 Formosat-2 image in the
NIR channel. Figure 13. Extract of the management practices classification of the grasslands from the French
agricultural land use database (RPG) in 2014. The background is a May, 2014 Formosat-2 image in the
NIR channel. must be accounted for. In terms of processing times, the object-based methods were much faster than
568
pixel-based methods. 569 Several contributions have been made in this work. The first lies in the grasslands pixels
570
distribution modeling at the object level. A flexible kernel was proposed to encompass both Gaussian
571
and mean modeling of grasslands, so no choice has to be made between these two modelings. It can
572
therefore be used on homogeneous objects such as artificial grasslands, or on very small objects, as well
573
as on heterogeneous semi-natural grasslands. The second contribution is that this kernel is suitable for
574
high dimensional data in a small ground sample size context. It enables the use of all the multispectral
575
data instead of a single vegetation index or the use of a long time series. Also, it can be used on a
576
whole time series without dates selection. Indeed, this new kernel offers very low computational load. 577
It can therefore be applied on a large dataset. With this kernel, we were able to process and to classify
578
more than 250,000 pixels on a conventional personal computer within a few seconds. 5. Conclusion
559 Even if it is the
579
first application of generative mean kernels in remote sensing classification, the αGMK proved its
580
efficiency and stability in these experiments. It is a good compromise between processing speed and
581
accuracy for the classification of grasslands. 582 The αGMK deserves to be tested on a larger dataset with more balanced classes. Seasonal statistics
583
could be used to improve the representation of grassland phenology. These ideas will be considered in
584
the future. This method was designed to deal with the dense SITS which will be provided by Sentinel-2
585
and to efficiently produce maps from this type of data. Other applications of the method are still
586
possible (e.g., small and heterogeneous objects such as peatlands, urban areas...). 587 20 of 25 20 of 25 Version June 12, 2017 submitted to Remote Sens. Supplementary Materials: The following are available online at www.mdpi.com/link, Figure S1: True color
588
composite images of the Formosat-2 time series of 2014; Figure S2: Boxplot of Kappa coefficient repartitions for the
589
classification of the old and young grasslands; Table S1: Average user accuracy (UA) and producer accuracy (PA)
590
(%) over the 100 repetitions for each class, 1: Old, 2: Young; Figure S3: Boxplot of Kappa coefficient repartitions
591
for classification of management practices using time series of year (a) 2013 and (b) 2014; Table S2: Average user
592
accuracy (UA) and producer accuracy (PA) (%) over the 100 repetitions for each class, 1: Mowing, 2: Mixed, 3:
593
Grazing. 594 Acknowledgments: This work was partially supported by a CJS INRA-INRIA contract and by the grant Défi
595
Mastodons-CNRS. The authors would like to thank CNES and CESBIO for providing the pre-processed Formosat-2
596
data. Special thanks to M. Lang for playing a major role in the field work, to D. Dallery for designing the processing
597
chain to compute the grasslands age from the RPG, and to R. Carrié for his careful reviewing of the introduction. 598
We would like to thank the anonymous reviewers for their constructive comments. 599 Acknowledgments: This work was partially supported by a CJS INRA-INRIA contract and by the grant Défi
595
Mastodons-CNRS. The authors would like to thank CNES and CESBIO for providing the pre-processed Formosat-2
596
data. Special thanks to M. Lang for playing a major role in the field work, to D. 5. Conclusion
559 Dallery for designing the processing
597
chain to compute the grasslands age from the RPG, and to R. Carrié for his careful reviewing of the introduction. 598
We would like to thank the anonymous reviewers for their constructive comments. 599 Author Contributions: M.L., M.F. and S.G. conceived the model, M.L., M.F. and S.G. conceived and designed the
600
experiments; M.L. performed the experiments; M.L. and M.F. analyzed the data; M.L. and M.F. wrote the paper
601
with feedbacks from S.G. and D.S. 602 Author Contributions: M.L., M.F. and S.G. conceived the model, M.L., M.F. and S.G. conceived and designed the
600
experiments; M.L. performed the experiments; M.L. and M.F. analyzed the data; M.L. and M.F. wrote the paper
601
with feedbacks from S.G. and D.S. 602 Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
603
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
604
decision to publish the results. 605 Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
603
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
604
decision to publish the results. 605 Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
603
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
604
decision to publish the results. 605 References
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Pastures. Ecography 1995, 18, 310–317. 617 2. Cousins, S.A.; Eriksson, O. The influence of management history and habitat on plant species richness in a
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636
grassland management treatments after 25 years. Abbreviations
606 The following abbreviations are used in this manuscript:
607
608
BD
Bhattacharyya Distance
EMK
Empirical Mean Kernel
GIS
Geographic Information System
GMK
Gaussian Mean Kernel
HDKLD
High Dimensional Kullback-Leibler Divergence
JMD
Jeffries-Matusita Distance
KLD
Kullback-Leibler Divergence
LAI
Leaf Area Index
NDVI
Normalized Difference Vegetation Index
NIR
Near Infrared
PMV
Pixel Majority Vote
RBF
Radial Basis Function
SITS
Satellite Image Time Series
SVM
Support Vector Machine
αGMK
α-Gaussian Mean Kernel
609 The following abbreviations are used in this manuscript:
607
608
BD
Bhattacharyya Distance
EMK
Empirical Mean Kernel
GIS
Geographic Information System
GMK
Gaussian Mean Kernel
HDKLD
High Dimensional Kullback-Leibler Divergence
JMD
Jeffries-Matusita Distance
KLD
Kullback-Leibler Divergence
LAI
Leaf Area Index
NDVI
Normalized Difference Vegetation Index
NIR
Near Infrared
PMV
Pixel Majority Vote
RBF
Radial Basis Function
SITS
Satellite Image Time Series
SVM
Support Vector Machine
αGMK
α-Gaussian Mean Kernel
609 Appendix
610 Appendix
610 Proof of eq. (8). First, let us write the Gaussian distribution pi to the power of α−1:
611 pi(x|µi, Σi)α−1
=
1
(2π)d/2α ×
1
|Σi|1/2α × exp
n
−0.5(x −µi)⊤(αΣi)−1 (x −µi)
o
=
(2π)
d
2 (1−1
α )
(2π)d/2
× α1/2 × |Σi|
1
2 (1−1
α )
|αΣi|1/2
× exp
n
−0.5(x −µi)⊤(αΣi)−1 (x −µi)
o
=
α1/2(2π)
d
2 (1−1
α )|Σi|
1
2 (1−1
α ) × p(x|µi, αΣi)
=
C(Σi, α)p(x|µi, αΣi). (A1 (A1) Then, plugging eq. (A1) in eq. (7), we get:
612 Then, plugging eq. (A1) in eq. (7), we get:
612 Then, plugging eq. (A1) in eq. (7), we get:
612 Kα(Ni, Nj)
=
C(Σi, α)C(Σj, α)
exp
−0.5(ˆµi −ˆµj)T
α ˆΣi + α ˆΣj + γ−1Id
−1
(ˆµi −ˆµj)
|α ˆΣi + α ˆΣj + γ−1Id|0.5
, which is eq. (5) with the covariance matrix of the Gaussian distribution scaled with α. The constants
613
C(Σi, α) and C(Σj, α) are removed when normalizing the kernel and we get eq. (8). 614 which is eq. (5) with the covariance matrix of the Gaussian distribution scaled with α. The constants
613
C(Σi, α) and C(Σj, α) are removed when normalizing the kernel and we get eq. (8). 614 21 of 25 21 of 25 Version June 12, 2017 submitted to Remote Sens. References
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of Grassland Successional Stages Using Airborne Hyperspectral Imagery. Remote Sensing 2014, 6, 7732.
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f Grassland Successional Stages Using Airborne Hyperspectral Imagery. Remote Sensing 2014, 6, 7732. Pedregosa, F.; Varoquaux, G.; Gramfort, A.; Michel, V.; Thirion, B.; Grisel, O.; Blondel, M.; Prettenhofer, P.;
Weiss, R.; Dubourg, V.; Vanderplas, J.; Passos, A.; Cournapeau, D.; Brucher, M.; Perrot, M.; Duchesnay, E.
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Oxaliplatin resistance is enhanced by saracatinib via upregulation Wnt-ABCG1 signaling in hepatocellular carcinoma
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Oxaliplatin resistance is enhanced by saracatinib
via upregulation Wnt-ABCG1 signaling in
hepatocellular carcinoma Page 1/21
a
ao
Xi'an Jiaotong University
Ge Song
Xi'an Jiaotong University
Zihan Xu
Shaanxi Provincial People's Hospital
Yang Bu
Ningxia Medical University
Fan Chang
Shaanxi Provincial Institute of Microbiology
Fengan Jia
Shaanxi Provincial Institute of Microbiology
Xuelian Xiao
Xi'an Jiaotong University
Xuejiao Ren
Yan'an University
Mei Zhang
Xi'an Jiaotong University
Qingan Jia
(
qajia66@163.com
)
Xi'an Jiaotong University Medical College First Affiliated Hospital
https://orcid.org/0000-0002-3256-
7197
Research article
Keywords: Hepatocellular carcinoma，Oxaliplatin resistance，Saracatinib，Wnt signaling，ABCG1
Posted Date: January 3rd, 2020
DOI: https://doi.org/10.21203/rs.2.10292/v5
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Version of Record: A version of this preprint was published on January 13th, 2020. See the published
version at https://doi.org/10.1186/s12885-019-6480-9. Research article Page 1/21 Page 1/21 Version of Record: A version of this preprint was published on January 13th, 2020. See the published
version at https://doi.org/10.1186/s12885-019-6480-9. Page 2/21 Abstract Background: Chemo-resistance in hepatocellular carcinoma (HCC) is a major problem, and acquired drug
resistance prevents cancer therapies from achieving complete responses. Molecular targeting therapy
presents an opportunity to impede tumor through combination or sequential therapy, while the accurate
effect is vague. Methods: The efficacy of combinations between oxaliplatin and anti-cancer molecular
targeting drugs was screened. Strangely, the combined chemotherapy with oxaliplatin and saracatinib
induced significantly antagonistic effects. Then the antitumor effects of combined treatment with
saracatinib and oxaliplatin were confirmed in wide type HCC as well as in saracatinib- and oxaliplatin-
resistant HCC. RNA sequencing was used to explore the resistance mechanism, and the roles of ABCG1
and Wnt signaling in oxaliplatin resistance were confirmed. Results: Chemotherapy with oxaliplatin and
saracatinib individually induced strong anti-HCC effects, while combined or sequential treatment of HCC
cells with these two drugs exhibited reduced efficacy compared to treatment with the single drugs. And it
was saracatinib treatment caused oxaliplatin resistance. RNA sequencing revealed 458 genes that were
altered by treatment with saracatinib and oxaliplatin. Of these, the gene encoding the ATP-binding
cassette transporter G1 ABCG1 and Wnt-associated genes were significantly upregulated. Upregulation of
ABCG1 and oxaliplatin resistance were associated with activation of Wnt signaling. Interference with
ABCG1 expression or inhibition of Wnt signaling resulted in reversal of the saracatinib-induced oxaliplatin
resistance in HCC. Conclusions: These studies demonstrated that combined or sequential chemotherapy
with oxaliplatin and saracatinib reduced antitumor efficacy, and this antagonism was attributed to the
activation of Wnt signaling and upregulation of ABCG1 by saracatinib. Reagents and antibodies Oxaliplatin, and Src inhibitor saracatinib (AZD0530) were used for the construction of drug-resistant cell
lines, and other anti-cancer molecular targeting drugs were purchased from ApexBio (Houston, TX, USA)
and Selleck (Houston, TX, USA). Monoclonal antibodies to the following proteins were used in western
blot: E-cadherin, vimentin, PCNA, FZD8, DKK1, AXIN2, WNT6, and β-catenin (purchased from Abcam,
Cambridge, MA, USA) and p-LRP6, GSK-3β, AXIN2, cyclin D1, SRC, OCT4, ABCG1, and BCL-2 (purchased
from Proteintech, Chicago, IL, USA). Background In clinical practice, more than 70% of patients with HCC are diagnosed at an advanced stage and are
treated with a non-radical surgical regimen, including transcatheter arterial chemoembolization (TACE)
and systemic chemotherapy[1]. Oxaliplatin has commonly been used, although the efficacy of oxaliplatin
for HCC is poor, due to the presence of both intrinsic and acquired resistance. Oxaliplatin resistance in
HCC is a major medical problem, and methods for improvement of the response to this chemotherapeutic
are urgently needed[2]. Molecular targeting therapy presents a therapeutic opportunity to impede tumor
relapse and reverse drug resistance, while the accurate combined effect is not yet clear in HCC. In this study, the efficacy of combinations between oxaliplatin and anti-cancer molecular targeting drugs
was screened, and saracatinib treatment actually induced resistance to oxaliplatin treatment was proved. Then we evaluated the response of HCC to oxaliplatin and saracatinib in vitro and in vivo, and RNA
sequencing revealed that the antagonistic relationship between saracatinib and oxaliplatin stemmed
from activation of the Wnt signaling pathway, resulting in increased expression of the ATP-binding
cassette transporter G1 ABCG1. Finally, we proved interference with ABCG1 expression or inhibition of
Wnt signaling resulted in reversal of the saracatinib-induced oxaliplatin resistance in HCC. These findings
indicate that combination or sequential therapy with oxaliplatin and saracatinib have negative effects on
HCC via upregulation Wnt-ABCG1 signaling. In this study, the efficacy of combinations between oxaliplatin and anti-cancer molecular targeting drugs
was screened, and saracatinib treatment actually induced resistance to oxaliplatin treatment was proved. Then we evaluated the response of HCC to oxaliplatin and saracatinib in vitro and in vivo, and RNA
sequencing revealed that the antagonistic relationship between saracatinib and oxaliplatin stemmed
from activation of the Wnt signaling pathway, resulting in increased expression of the ATP-binding
cassette transporter G1 ABCG1. Finally, we proved interference with ABCG1 expression or inhibition of
Wnt signaling resulted in reversal of the saracatinib-induced oxaliplatin resistance in HCC. These findings
indicate that combination or sequential therapy with oxaliplatin and saracatinib have negative effects on
HCC via upregulation Wnt-ABCG1 signaling. Page 3/21 Page 3/21 Cell lines and Animals Human HCC cell lines MHCC97L, which has high metastatic potential (established at Fudan University,
Shanghai, China; RRID: CVCL_4973), and Hep3B, which has low metastatic potential (American Type
Culture Collection, Rockville, MD, USA; RRID: CVCL_0326), were obtained from the Liver Cancer Institute of
Fudan University (Shanghai, China). All cells were maintained in Dulbecco’s Modified Eagle’s Medium
(DMEM; GIBCO, Grand Island, NY, USA) and supplemented with 10% fetal bovine serum (FBS; GIBCO) at
37°C in a humidified incubator with 5% CO2. Cells were routinely screened for the presence of
mycoplasma (Mycoplasma Detection Kit, Roche Diagnostics, Indianapolis, IN, USA). Male BALB/c nu/nu mice (aged 4–6 weeks and weighing approximately 20 g) were obtained from the
Chinese Academy of Science (SLRC, Shanghai, China) and raised in a controlled environment with 25°C
under standard pathogen-free conditions and a natural light/dark cycle (morning 8:00; afternoon 8:00),
and were provided with water and standard diet. Animal protocols were approved by the ethics committee
on Experimental Animals of Xi’an Jiaotong University. In vitro drug sensitivity assay MHCC97L cells were seeded in 96-well plates at 2500 cells per well. Twelve hours after plating, cells were
treated with anti-cancer molecular targeting drugs library (including 29 inhibitors in PI3K, MAPK signaling
et al). After 72h of incubation at 37°C in a 5% CO2 humidified incubator, cell viability was analyzed using
Cell Counting Kit 8 (CCK8; Dojindo, Gaithersburg, MD, USA). The drugs were stored and diluted according
to the manufacturers' instructions. Cell viability assay Wild-type MHCC97L and Hep3B cells were grown in 96-well plates in medium containing 2 μmol/L
oxaliplatin and increasing concentrations of saracatinib for 24, 48, 72, and 96 h. Additionally, wild-type
MHCC97L and Hep3B cells were grown in medium containing 2 μmol/L saracatinib and increasing
concentrations of oxaliplatin for 24, 48, 72, and 96 h. Cell proliferation assays were performed with CCK8. Results were expressed as absorbance of each well at 450 nm (OD450). RNA interference The siRNA duplexes for ABCG1 were chemically synthesized by Qiagen, Inc. (Valencia, CA, USA). The
following ABCG1 siRNA sequences were constructed: 5’-CGTGGATGAGGTTGAGACA-3’(forward) and 5’-
GGTGGACAACAACTTCACA-3’ (reverse). Chemically synthesized mock siRNA (fluorescein-labeled, non-
silencing) was also purchased from Qiagen, Inc. The human full-length cDNA of ABCG1 were obtained
from Genesent (shanghai China) and then cloned into the pCDH lentiviral expression vector (System
Biosciences). Using the In-Fusion HD Cloning Kit (Takara), the amplified fragment was inserted into the
plasmid pCDH (between XbaI and EcoRI sites). Flag-tagged ABCG1 in pCDH vector was from Genesent
(shanghai China). Generation of oxaliplatin- and saracatinib-resistant HCC cell lines Page 4/21 MHCC97L and Hep3B cells were grown in T25 flasks and treated with saracatinib (2 μmol/L and 1
μmol/L) followed by the addition of increasingly higher concentrations of saracatinib until the MHCC97L
cells became stably resistant to 4 μmol/L saracatinib and the Hep3B cells became stably resistant to 2
μmol/L saracatinib. These resistant cells were re-named MHCC97L-Src and Hep3B-Src. Oxaliplatin-
resistant HCC cell lines were generated as previously described [3]. MHCC97L cells that were stably
resistant to 2 μmol/L oxaliplatin were re-named MHCC97L-Oxa, and Hep3B cells that were stably
resistant to 1 μmol/L oxaliplatin were re-named Hep3B-Oxa. MHCC97L and Hep3B cells were grown in T25 flasks and treated with saracatinib (2 μmol/L and 1
μmol/L) followed by the addition of increasingly higher concentrations of saracatinib until the MHCC97L
cells became stably resistant to 4 μmol/L saracatinib and the Hep3B cells became stably resistant to 2
μmol/L saracatinib. These resistant cells were re-named MHCC97L-Src and Hep3B-Src. Oxaliplatin-
resistant HCC cell lines were generated as previously described [3]. MHCC97L cells that were stably
resistant to 2 μmol/L oxaliplatin were re-named MHCC97L-Oxa, and Hep3B cells that were stably
resistant to 1 μmol/L oxaliplatin were re-named Hep3B-Oxa. Colony formation To investigate the effect of combination treatment with saracatinib and oxaliplatin on HCC cells,
MHCC97L and Hep3B (1×103 cells/well) were plated in 6-well plates and cultured with DMEM containing
5% FBS with saracatinib (2 μmol/L) and/or oxaliplatin (2 μmol/L). Culture medium was replaced every 3
d, and the colonies were fixed with ice-cold 4% paraformaldehyde after 14 d. Cells were stained with
Giemsa (Sigma, St. Louis, MO, USA) and photographed at ×5 magnification. Animal Model and Treatment Procedures MHCC97L (5×106) were implanted subcutaneously into the upper left flank region of mice to establish
subcutaneous xenografts. The synergistic effects of the combination therapy of oxaliplatin (10mg/kg)
and saracatinib (20mg/kg) were evaluated. Twenty nude mice bearing subcutaneous xenografts were
randomly divided into the control, oxaliplatin, saracatinib, and oxaliplatin + sorafenib groups (n = 5 per
group). Tumor weights were evaluated in 4 weeks after the treatments. Another eighteen nude mice
bearing subcutaneous xenografts were randomly divided into the oxaliplatin, oxaliplatin + safacatinib,
oxaliplatin + saracatinib + siABCG1 groups (n = 6 per group). Small interfering RNA ABCG1 was used for
local injection every 10 days. Tumor weights were also evaluated in 4 weeks after the treatments. Page 5/21 Intraperitoneal injection of pentobarbital (5mg/kg) combined with cervical spondylolisthesis was used for
the sacrifice of mice after the study. Intraperitoneal injection of pentobarbital (5mg/kg) combined with cervical spondylolisthesis was used for
the sacrifice of mice after the study. RNA sequencing and bioinformatics analysis RNA sequencing (Shanghai OE Biotech Co. Ltd., China) was used to compared MHCC97L with MHCC97L-
Src and MHCC97L with MHCC97L-Oxa. Principal components analysis (PCA) and hierarchical clustering
were performed using the R program. PCA was used to visualize differences between groups. Hierarchical
cluster analysis was used to evaluate a set of dissimilarities, serving as a “complete” method for
analyzing different genes based on the same gene ontology (GO). The GO seq R package was used to
perform GO enrichment analysis of different gene clusters. KEGG enrichment analysis of different gene
clusters were implemented using the cluster Profiler R package, and the cutoff for significance was set as
p=0.05. Raw sequencing data is publicly available at NCBI (GEO accession number GSE129071). Cell cycle assays MHCC97L cells were starved in serum-free medium for 24 h and then grown in oxaliplatin (2 μmol/L)
and/or saracatinib (2 μmol/L) for 48 h. Cell cycle analyses and quantification of genomic DNA
fragmentation were performed using the Cell Cycle Detection Kit (KeyGen, Nanjing, China) according to
the manufacturer’s protocol. Cell cycle distributions were analyzed by flow cytometry using a Becton
Dickinson FACS Calibur (Franklin Lakes, NJ, USA). Combination chemotherapy with oxaliplatin and saracatinib exhibited antagonistic effects The efficacy of combinations between oxaliplatin and anti-cancer molecular targeting drugs was
screened. Strangely, the combined chemotherapy with oxaliplatin and saracatinib induced significantly
antagonistic effects (Figure 1A). The HCC cell lines MHCC97L and Hep3B exhibited significant anti-tumor
effects with lower proliferation rates than wild-type cells following treatment with oxaliplatin or
saracatinib individually; however, combination treatment with the two drugs resulted in a higher
proliferation rate, indicating impaired antitumor efficacy with combined treatment (Figure 1B). To confirm
this phenomenon, plate colony formation assays were performed. Compared to untreated cells, colony
diameters were significantly decreased following treatment with oxaliplatin (257.51±55.60 μm vs. 705.16±170.81 μm; p=0.041) or saracatinib (287.57±71.36 μm vs. 705.16±170.81 μm; p=0.0025)
individually. Combination therapy, on the other hand, resulted in larger colony diameters than oxaliplatin
(455.16±86.12 μm vs. 257.51±55.60 μm; p=0.0086) or saracatinib (455.16±86.1 μm vs. 287.25±71.36
μm; p=0.0245) treatment alone (Figure 1C). Next, we analyzed the cell cycle distributions to further
evaluate the observed changes in cell proliferation. The percentages of cells in S phase decreased
following treatment with either oxaliplatin (22.321±0.67% vs. 29.48±1.06%; p＜0.0001) or saracatinib
(23.59±1.76% vs. 29.48±1.06%; p=0.003). Combination therapy with oxaliplatin and saracatinib resulted
in significantly increased percentages of cells in S phase compared to treatment with oxaliplatin
(26.55±0.39% vs. 22.321±0.67%; p＜0.0001) or saracatinib (26.55±0.39% vs. 23.59±1.76%; p=0.0026)
individually (Figure 1D). Furthermore, the tumor weight of the subcutaneous xenografts were larger in the
combination treatment group than oxaliplatin (3.06±0.16 g vs. 1.51±0.39 g; p=0.0005) or saracatinib
(3.06±0.16 g vs. 1.97±0.32 μm; p=0.0008) treatment alone (Figure 1E). We next revalidated the effects of the combined therapy on the response of HCC cells. MHCC97L
(130.6±16.62 μmol/L vs. 20.85±4.86 μmol/L; p=0.0063) and Hep3B (28.67±5.59 μmol/L vs. 5.29±1.29
μmol/L; p=0.0247) cells exhibited significantly increased IC50 values in response to oxaliplatin when
treated with saracatinib. Interestingly, however, MHCC97L (0.79±0.11 μmol/L vs. 4.81±0.57 μmol/L;
p=0.0056) and Hep3B (2.05±0.32 μmol/L vs. 2.62±0.47 μmol/L; p=0.0631) cells exhibited reduced IC50
values in response to saracatinib when treated with oxaliplatin as well (Figure 2A). These findings
suggest that saracatinib treatment in combination with oxaliplatin increases oxaliplatin resistance in
HCC, and it was the treatment with saracatinib caused oxaliplatin resistance. Additionally, we
investigated the effects of the two chemotherapy drugs on protein expression in the two HCC cell lines
MHCC97L and Hep3B. Statistical analysis A two-sided Student’s t-test was performed to evaluate the statistical significance of differences in
means. Experiments were performed at least three times, and p<0.05 was considered statistically
significant. Statistical analyses were performed using SPSS 15.0 software for Windows (SPSS Inc. Chicago, IL, USA). Page 6/21 Page 6/21 Page 6/21 Sequential chemotherapy reduced the antitumor efficacy of oxaliplatin on saracatinib-resistant HCC In order to simulate the clinical sequential chemotherapy, HCC cell lines were treated continually with
oxaliplatin to generate oxaliplatin-resistant cell lines (MHCC97L-Oxa and Hep3B-Oxa) that exhibited
decreased intercellular adhesion and spindle-shaped cell morphology (Figure 3A). Compared to wild-type
HCC cells, MHCC97L-Oxa (66.67±9.01 μmol/L vs. 31.67±4.04 μmol/L; p=0.0254; Figure 3B, a) and
Hep3B-Oxa (19.21±2.69 μmol/L vs. 5.45±1.23 μmol/L; p=0.0212; Figure 3B, c) exhibited increased
oxaliplatin IC50 values. The oxaliplatin-resistant HCC cell lines were next treated with increasing
concentrations of saracatinib, resulting in a decrease of IC50 values in MHCC97L-Oxa (1.23±0.31 μmol/L
vs. 4.30±0.97 μmol/L; p=0.0141; Figure 3C, a) and Hep3B-Oxa (1.14±0.11 μmol/L vs. 2.62± 0.47 μmol/L;
p=0.0333; Figure 3C, b) compared to the parental wild-type HCC cells, still exhibiting more sensitive to
saracatinib. Saracatinib-resistant MHCC97L and Hep3B cells (MHCC97L-Src and Hep3B-Src) were generated similarly
via continuous treatment with saracatinib, and these lines exhibited enhanced intercellular adhesion and
appeared as agglomerated cell clumps (Figure 3A). Compared to wild-type cells, MHCC97L-Src
(40.07±2.88 μmol/L vs. 4.81±0.57 μmol/L; p=0.0024; Figure 3B, b) and Hep3B-Src (38.36±3.17 μmol/L
vs. 2.62±0.47 μmol/L; p=0.009; Figure 3B, d) exhibited increased saracatinib IC50 values. The saracatinib-
resistant HCC cell lines were then treated with oxaliplatin at increasing concentrations, yielding
significantly enhanced resistance to oxaliplatin with increased IC50 in both MHCC97L-Src (108.71±11.24
μmol/L vs. 20.85±4.86 μmol/L; p=0.0092; Figure 3D, a) and Hep3B-Src (27.01±4.59 μmol/L vs. 5.29±1.29
μmol/L; p=0.0106; Figure 3D, b) compared to wild-type cells. Additionally, we investigated protein expression in oxaliplatin- and saracatinib-resistant cell lines. MHCC97L-Oxa and Hep3B-Oxa exhibited downregulation of PCNA and E-cadherin and upregulation of
vimentin and OCT4, while MHCC97L-Src and Hep3B-Src exhibited downregulation of PCNA, upregulation
OCT4, and reversion of EMT compared to the parental cell lines (Figure 3E). Together, these results
indicate that sequential chemotherapy reduced the antitumor efficacy of oxaliplatin on saracatinib-
resistant HCC. Combination chemotherapy with oxaliplatin and saracatinib exhibited antagonistic effects Oxaliplatin treatment led to the downregulation of PCNA and the occurrence of
EMT, which was associated with the upregulation of vimentin and the downregulation of E-cadherin. Conversely, saracatinib not only downregulated PCNA expression but also reversed the EMT. MHCC97L
and Hep3B treated with both oxaliplatin and saracatinib exhibited partial upregulation of PCNA and
reversion of EMT (Figure 2B). These findings suggest that saracatinib treatment in combination with
oxaliplatin reduces the antitumor efficacy of these drugs on HCC cells, but reverses the negative effect of
EMT induced by oxaliplatin. Page 7/21 Page 7/21 ABCG1 upregulation and Wnt signaling pathway activation are integral mechanisms involved in the
antagonism between saracatinib and oxaliplatin in HCC The expression of 20,030 genes was compared between wild-type MHCC97L and MHCC97L-Src cells in
three independent experiments (Figure 4A). Gene expression profiles for 1172 genes exhibited differences
(p<0.05) between MHCC97L and MHCC97L-Src, implicating these genes in saracatinib resistance. Furthermore, analysis of these associated “resistance” genes revealed 526 upregulated and 645
downregulated genes. The expression of these 20,300 genes in wild-type MHCC97L and MHCC97L-Oxa Page 8/21 was also compared (Figure 4B). Expression profiles for 720 genes exhibited differences (p<0.05) between
MHCC97L and MHCC97L-Oxa, implicating these genes in oxaliplatin resistance. Of these, 455 were
upregulated, and 265 were downregulated in the two drug-resistant cell lines. A total of 458 altered genes overlapped between the two drug-resistant cell lines, and all of these were
closely related to cell division, growth, angiogenesis, adhesion, and metabolic processes (Figure 4C). KEGG pathway analysis revealed that 20 of the altered genes were related to drug resistance: ABCG1,
ATM, BBC3, BIK, BIRC3, CDKN1A, DLL4, ERBB3, FGF2, FOS, GPER1, IL6, JAG1, MMP2, NRG2, PDGFRB,
PIK3CA, SHC4, TOP2A, TOP2B, and VEGFA. Another 16 genes were related to Wnt signaling: ROCK2,
TCF7L1, WNT6, WNT5A, NKD2, FZD3, FZD8, DKK1, WNT5B, NFATC4, PLCB2, SERPINF1, AXIN2, NFATC1,
PLCB4, and RAC2. Immunoblotting confirmed the upregulation of ABCG1 and Wnt-associated proteins,
including FZD8, DKK1, Axin2, and WNT6 (Figure 4D, E, F and G). Immunohisochemotherapy verified that
the expression of ABCG1 was significantly upregulated after the treatment with oxaliplatin (16.25±4.03
vs. 7.50±3.42 μmol/L; p=0.0162) or saracatinib (20.50±4.51 vs. 7.50±3.42 μmol/L; p=0.0037) in
subcutaneous xenografts tissues. And the combination treatment exhibited higher expression of ABCG1
than oxaliplatin single use (30.50±5.01 vs. 16.25±4.03; p=0.0044; Figure 4H). Therefore, we speculate
that ABCG1 upregulation and Wnt signaling pathway activation are integral mechanisms involved in the
antagonism between saracatinib and oxaliplatin in HCC. Interference with ABCG1 expression or inhibition of Wnt signaling resulted in reversal of the saracatinib-
induced oxaliplatin resistance in HCC Immunoblotting verified that the expression of ABCG1 was significantly downregulated by Wnt/β-catenin
signaling pathway inhibition with KYA1797K in wild-type HCC cell lines, MHCC97L-Src, and Hep3B-Src
(Figure 5A). Following ABCG1 downregulation and, the key cell membrane receptors for Wnt signaling
LRP6 and p-LRP6 were not significantly altered; however, β-catenin was slightly downregulated, and the
expression of PCNA was significantly decreased. ABCG1 restoration could reverse this alteration in
protein levels (Figure 5B). Next, we confirmed the role of ABCG1 and Wnt signaling in oxaliplatin resistance. ABCG1 was silenced
using specific siRNA in MHCC97L cells, resulting in a decreased IC50 to oxaliplatin compared to mock-
treated MHCC97L cells (MHCC97L-Mock; 8.41±2.09 μmol/L vs. 25.59 ± 5.82 μmol/L; p=0.0085; Figure 5C,
a). Following silencing of ABCG1 using siRNA in saracatinib-resistant MHCC97L (MHCC97L-Src-ABCG1-
Sh1), we observed decreased resistance to oxaliplatin (40.43±8.12 μmol/L vs. 103.71±8.74 μmol/L;
p=0.0008; Figure 5C, b) compared to mock-treated cells. Furthermore, silencing of ABCG1 combined with
saracatinib in MHCC97L, there was no significantly increased resistance to oxaliplatin (11.84 ± 2.11 vs. 9.73±1.26 μmol/L; p=0.0874; Figure 5C, c). Additionally, HCC cells treated with the Wnt signaling inhibitor
KYA1797K exhibited decreased resistance to oxaliplatin with reduced IC50 values (11.07±2.02μmol/L vs. 31.67±4.04μmol/L; p=0.0082; Figure 5D, a). Treatment of MHCC97L-Src cells with KYA1797K resulted in Page 9/21 Page 9/21 decreased resistance to oxaliplatin (40.83±8.12 μmol/L vs. 108.71±11.24 μmol/L; p=0.0008; Figure 5D,
b). KYA1797K combined with saracatinib in MHCC97L, there was no significantly increased resistance to
oxaliplatin (11.51 ± 1.44 vs. 15.73±2.95 μmol/L; p=0.0920; Figure 5D, c). decreased resistance to oxaliplatin (40.83±8.12 μmol/L vs. 108.71±11.24 μmol/L; p=0.0008; Figure 5D,
b). KYA1797K combined with saracatinib in MHCC97L, there was no significantly increased resistance to
oxaliplatin (11.51 ± 1.44 vs. 15.73±2.95 μmol/L; p=0.0920; Figure 5D, c). Furthermore, it was confirmed once again that the tumor weight of the subcutaneous xenografts was
larger in combination treatment group using oxaliplatin and saracatinib than oxaliplatin single use
(1.82±0.30 g vs. 1.26±0.23 g; p=0.0050; Figure 5E). And the subcutaneous xenografts was smaller in the
combination treatment group using oxaliplatin, saracatinib, and ABCG1 siRNA local injection (0.87±0.24
g vs. 1.82±0.30 g; p=0.0001; Figure 5E) than combination group only using oxaliplatin and saracatinib. Together, these findings suggest that ABCG1 and Wnt signaling contribute to oxaliplatin resistance in
saracatinib-treated HCC cells. And interference with ABCG1 expression or inhibition of Wnt signaling
resulted in reversal of the saracatinib-induced oxaliplatin resistance in HCC Discussion Liver cancer, most commonly seen as hepatocellular carcinoma (HCC), has high prevalence and
incidence rates in China, which accounts for more than 50% of the total number of liver cancer cases and
deaths in the world [4]. One of the chemotherapeutic drugs for patients with advanced HCC is oxaliplatin,
which initiates apoptosis by inhibiting the replication and transcription of DNA in HCC cells [5]. However,
the efficacy of oxaliplatin on HCC is poor, exhibiting intrinsic and acquired resistance. Therefore, methods
to enhance oxaliplatin treatment responses are urgently needed. In this study, the efficacy of
combinations between oxaliplatin and anti-cancer molecular targeting drugs was screened, and
saracatinib treatment actually induced resistance to oxaliplatin treatment was proved. In many solid tumor cells, including HCC, Src expression level or activity is increased[6-8], promoting
metastasis[9, 10]. Activation of the tyrosine kinase Src is responsible for tumor progression promoted by
insulin-like growth factor 1 receptor (IGF-1R)[11, 12]. Previously, we demonstrated that high expression of
IGF1 was closely associated with the maintenance of stemness in oxaliplatin-resistant HCC cells and that
IGF1-IGF1R signaling blockade effectively increased oxaliplatin sensitivity[13]. Tyrosine kinase Src was
discovered more than 30 years ago as a kinase that is involved in the crosstalk between many signaling
pathways, including the integrin/FAK, Ras/Raf/MEK, PI3K/AKT, and IGF1/IGF1R pathways, and Src
activation promotes cell proliferation, adhesion, invasion, migration, metastasis, and tumorigenesis[14]. Recently, Liu et al.[15] reported that increased expression of Src potentiates ERK activation and reverses
sorafenib resistance in HCC. Thus, inhibition of Src may provide a new strategy for drug combination
studies for HCC treatment[16]. Based on these findings, we expect a synergistic relationship between
oxaliplatin and Src kinase inhibition. Saracatinib (AZD0530) is a potent, orally administered small
molecule that inhibits Src by blocking the ATP binding site of the kinase[17]. However, the combined
chemotherapy with oxaliplatin and saracatinib induced significantly antagonistic effects. Recent research
proved saracatinib failed to demonstrate monotherapeutic efficacy, with undesirable stem cell-promoting
functions in patients with head and neck squamous cell carcinoma[18]. In the present study, we proved
the combined treatment of HCC with oxaliplatin and saracatinib impaired the efficacy of either drug Page 10/21 Page 10/21 individually. And it was mainly saracatinib treatment increased oxaliplatin resistance in HCC. We also
tested the effects of the sequential treatment of HCC with oxaliplatin and saracatinib, and sequential
chemotherapy also reduced the antitumor efficacy of oxaliplatin on saracatinib-resistant HCC. Discussion Based on previous and our current findings, oxaliplatin-resistant HCC cells exhibited decreased
intercellular adhesion and spindle-shaped cell morphology that are characteristic of EMT[19], while
saracatinib-resistant HCC cells exhibited enhanced intercellular adhesion and cell clumping[20]. Immunoblotting further confirmed that oxaliplatin treatment led to the occurrence of EMT as vimentin
was upregulated and E-cadherin was downregulated. Saracatinib significantly inhibited the expression of
PCNA and reversed the EMT. Gene expression analysis revealed 458 genes that were altered in both
saracatinib- and oxaliplatin-resistant HCC, and these genes were related to processes of cell division,
growth, angiogenesis, adhesion, and metabolism. Based on KEGG pathway classification, 20 of these
genes were related to drug resistance, while 16 were related to Wnt signaling activation. Furthermore,
immunoblotting revealed that ABCG1 and the Wnt/β-catenin signaling pathway were both upregulated in
HCC cell lines in the presence of continuous treatment with saracatinib or oxaliplatin. ABCG1 is a cholesterol lipid efflux pump that plays a well-known role in tumor growth, conferring
chemoresistance to various malignant tumors[21]. Several ABC transporters, including ABCG1, are
associated with multidrug resistance (MDR), which is a major obstacle to the effective clinical treatment
of cancer[22]. In the present study, ABCG1 is a downstream protein in Wnt/β-catenin signaling and can be
significantly downregulated by Wnt/β-catenin signaling pathway. Interference with the expression of
ABCG1 or inhibition of Wnt/β-catenin results in decreased oxaliplatin resistance, supporting a role for
these proteins in the acquired drug resistance of HCC. Understanding the molecular pathogenesis of HCC
chemoresistance is key to improving patients' prognosis. Limitation In the present study, we demonstrated that combined or sequential chemotherapy with oxaliplatin and
saracatinib induced antagonistic effects, while our study was only limited to liver cancer, and lack of
deeper mechanistic knowledge of the actions of saracatinib. Therefore, several fundamental questions
remain to be answered concerning the combined or sequential chemotherapy with the two drugs in the
further study. (1) Whether antagonisms are limited to a few specific cancer species? (2) Deeper
mechanisms involved in antagonistic effects of the two drugs are also needed be explored? (3) Can
saracatinib be used in other diseases is also need to be answered? Still, our results do provide some
important clues that may help guide drug selection and therapeutic strategy used in clinical treatments of
cancer. Conclusions Page 11/21 From our experimental results and our review of the literature, we propose the following conclusions. (1)
The combined and sequential chemotherapy with oxaliplatin and saracatinib induces significantly
antagonistic effects. (2) ABCG1 upregulation and Wnt signaling pathway activation are integral
mechanisms involved in the antagonism between saracatinib and oxaliplatin in HCC. (3) Interference with
ABCG1 expression or inhibition of Wnt signaling resulted in reversal of the saracatinib-induced oxaliplatin
resistance in HCC. From our experimental results and our review of the literature, we propose the following conclusions. (1)
The combined and sequential chemotherapy with oxaliplatin and saracatinib induces significantly
antagonistic effects. (2) ABCG1 upregulation and Wnt signaling pathway activation are integral
mechanisms involved in the antagonism between saracatinib and oxaliplatin in HCC. (3) Interference with
ABCG1 expression or inhibition of Wnt signaling resulted in reversal of the saracatinib-induced oxaliplatin
resistance in HCC. Consent for publication Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article. The datasets used
and/or analyzed and materials developed during the current study are available from the corresponding
author by reasonable request. List Of Abbreviations HCC: hepatocellular carcinoma, TACE: transcatheter arterial chemoembolization, DMEM: Dulbecco’s
Modified Eagle’s Medium, FBS: fetal bovine serum, CCK8: Cell Counting Kit 8, PCA: Principal components
analysis, GO: gene ontology, IGF-1R: insulin-like growth factor 1 receptor , MDR: multidrug resistance Ethics approval and consent to participate None of the cell lines used in the present study required ethics approval for their use. Ethical approval
was obtained from the Research Ethics Committee of Xi’an Jiaotong University. (Permit Number:2016-
439). Competing interests The authors declare that they have no competing interests. Acknowledgement Not applicable References 1. Tsurusaki M, Murakami T: Surgical and Locoregional Therapy of HCC: TACE. Liver Cancer 2015,
4(3):165-175 1. Tsurusaki M, Murakami T: Surgical and Locoregional Therapy of HCC: TACE. Liver Cancer 2015,
4(3):165-175. 2. Zhang Y, Huang L, Shi H, Chen H, Tao J, Shen R, Wang T: Ursolic acid enhances the therapeutic
effects of oxaliplatin in colorectal cancer by inhibition of drug resistance. Cancer Sci 2018,
109(1):94-102. 3. Bu Y, Jia QA, Ren ZG, Zhang JB, Jiang XM, Liang L, Xue TC, Zhang QB, Wang YH, Zhang L et al:
Maintenance of Stemness in Oxaliplatin-Resistant Hepatocellular Carcinoma Is Associated with
Increased Autocrine of IGF1. PLoS One 2014, 9(3). 4. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A: Global cancer statistics, 2012. CA
Cancer J Clin 2015, 65(2):87-108. 5. Qin S, Bai Y, Lim HY, Thongprasert S, Chao Y, Fan J, Yang TS, Bhudhisawasdi V, Kang WK, Zhou Y et
al: Randomized, multicenter, open-label study of oxaliplatin plus fluorouracil/leucovorin versus
doxorubicin as palliative chemotherapy in patients with advanced hepatocellular carcinoma from
Asia. J Clin Oncol 2013, 31(28):3501-3508. 6. Sievers E, Trautmann M, Kindler D, Huss S, Gruenewald I, Dirksen U, Renner M, Mechtersheimer G,
Pedeutour F, Aman P et al: SRC inhibition represents a potential therapeutic strategy in liposarcoma. Int J Cancer 2015, 137(11):2578-2588. 7. Roseweir AK, Qayyum T, Lim Z, Hammond R, MacDonald AI, Fraser S, Oades GM, Aitchison M, Jones
RJ, Edwards J: Nuclear expression of Lyn, a Src family kinase member, is associated with poor
prognosis in renal cancer patients. BMC Cancer 2016, 16:229. 7. Roseweir AK, Qayyum T, Lim Z, Hammond R, MacDonald AI, Fraser S, Oades GM, Aitchison M, Jones
RJ, Edwards J: Nuclear expression of Lyn, a Src family kinase member, is associated with poor
prognosis in renal cancer patients. BMC Cancer 2016, 16:229. 8. Leroy C, Shen Q, Strande V, Meyer R, McLaughlin ME, Lezan E, Bentires-Alj M, Voshol H, Bonenfant D,
Alex Gaither L: CUB-domain-containing protein 1 overexpression in solid cancers promotes cancer
cell growth by activating Src family kinases. Oncogene 2015, 34(44):5593-5598. 8. Leroy C, Shen Q, Strande V, Meyer R, McLaughlin ME, Lezan E, Bentires-Alj M, Voshol H, Bonenfant D,
Alex Gaither L: CUB-domain-containing protein 1 overexpression in solid cancers promotes cancer
cell growth by activating Src family kinases. Oncogene 2015, 34(44):5593-5598. 9. Funding This research project was mainly supported by the National Natural Science Foundation of China
(81502694). This research project was also partly supported by the Fundamental Research Funds for the
Central Universities (1191329835), Postdoctoral Science Foundation of China (2015M570330), and Key
projects of Ningxia Natural Science Foundation (NZ15130). The funders had no role in the study design,
data collection and interpretation, or decision to submit the work for publication. Page 12/21 Page 12/21 Authors’ contributions: XL, GS, ZHX, YB, FC, FAJ, XLX, XJR, MZ and QAJ contributed to the study design,
analysis, and interpretation of data. QAJ conceived the study, XL, GS, ZHX and YB performed the majority
of the experiments. FC, FAJ and XLX participated in statistical analysis. XJR and MZ participated in the
establishment of the mice model. QAJ drafted and prepared the manuscript. All authors approved the
final manuscript. Authors’ contributions: XL, GS, ZHX, YB, FC, FAJ, XLX, XJR, MZ and QAJ contributed to the study design,
analysis, and interpretation of data. QAJ conceived the study, XL, GS, ZHX and YB performed the majority
of the experiments. FC, FAJ and XLX participated in statistical analysis. XJR and MZ participated in the
establishment of the mice model. QAJ drafted and prepared the manuscript. All authors approved the
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2017, 96:371-377. Figures g Figure 1 Page 15/21
Figure 1 Figure 1 Page 15/21 Combination chemotherapy using oxaliplatin and saracatinib induced antagonistic effects on HCC cells. (A) The efficacy of combinations between oxaliplatin and anti-cancer molecular targeting drugs was
screened, and the combined chemotherapy with oxaliplatin and saracatinib induced significantly
antagonistic effects. (B) Cell viability assays of MHCC97L and Hep3B cells treated with oxaliplatin or
saracatinib individually or in combination. (C) Colony formation assays were performed on HCC cells
following treatment with either oxaliplatin or saracatinib individually or with both drugs combined. Data
are shown as cell colony diameters. (D) The cell cycle distributions were analyzed, and the proportions of
cells in S phase were determined for HCC cell lines treated with oxaliplatin or saracatinib individually or in
combination. (E) The tumor weight of the subcutaneous xenografts were larger in the combination
treatment group than treatment with the single drugs. Page 16/21 Figure 2
Reconfirmation of the antagonistic effects using oxaliplatin and saracatinib on HCC cells. (A) The effects
of the combined therapy on the response of HCC cells to different concentrations of chemotherapy drugs
were assessed in HCC cell lines via measurement of IC50 values. (B) The expression of PCNA and EMT-
associated biomarkers were detected in HCC cells treated with saracatinib and oxaliplatin individually or
in combination. Figure 2 Reconfirmation of the antagonistic effects using oxaliplatin and saracatinib on HCC cells. (A) The effects
of the combined therapy on the response of HCC cells to different concentrations of chemotherapy drugs
were assessed in HCC cell lines via measurement of IC50 values. (B) The expression of PCNA and EMT-
associated biomarkers were detected in HCC cells treated with saracatinib and oxaliplatin individually or
in combination. Reconfirmation of the antagonistic effects using oxaliplatin and saracatinib on HCC cells. (A) The effects
of the combined therapy on the response of HCC cells to different concentrations of chemotherapy drugs
were assessed in HCC cell lines via measurement of IC50 values. (B) The expression of PCNA and EMT-
associated biomarkers were detected in HCC cells treated with saracatinib and oxaliplatin individually or
in combination. Page 17/21 Page 17/21 Figure 3
Sequential chemotherapy induced decreased antitumor efficacy of oxaliplatin on saracatinib-resistant
HCC
(A) Cell morphology was observed in oxaliplatin and saracatinib-resistant HCC cell lines. (B)
oxaliplatin- and saracatinib resistant HCC cell lines exhibited increased IC50 values to oxaliplatin and
saracatinib separately. (C) Chemoresistance of oxaliplatin-resistant HCC cell lines to saracatinib was
determined with decreased IC50 values compared with wild-type HCC cells. (D) Saracatinib-resistant HCC
cell lines exhibited significantly enhanced resistance to oxaliplatin with an increased IC50 to oxaliplatin. (E) Expression of proteins related to proliferation and EMT in oxaliplatin- and saracatinib-resistant cell
lines were examined by immunoblotting. Figure 3 Sequential chemotherapy induced decreased antitumor efficacy of oxaliplatin on saracatinib-resistant
HCC
(A) Cell morphology was observed in oxaliplatin and saracatinib-resistant HCC cell lines. (B)
oxaliplatin- and saracatinib resistant HCC cell lines exhibited increased IC50 values to oxaliplatin and
saracatinib separately. (C) Chemoresistance of oxaliplatin-resistant HCC cell lines to saracatinib was
determined with decreased IC50 values compared with wild-type HCC cells. (D) Saracatinib-resistant HCC
cell lines exhibited significantly enhanced resistance to oxaliplatin with an increased IC50 to oxaliplatin. (E) Expression of proteins related to proliferation and EMT in oxaliplatin- and saracatinib-resistant cell
lines were examined by immunoblotting. Sequential chemotherapy induced decreased antitumor efficacy of oxaliplatin on saracatinib-resistant
HCC
(A) Cell morphology was observed in oxaliplatin and saracatinib-resistant HCC cell lines. (B)
oxaliplatin- and saracatinib resistant HCC cell lines exhibited increased IC50 values to oxaliplatin and
saracatinib separately. (C) Chemoresistance of oxaliplatin-resistant HCC cell lines to saracatinib was
determined with decreased IC50 values compared with wild-type HCC cells. (D) Saracatinib-resistant HCC
cell lines exhibited significantly enhanced resistance to oxaliplatin with an increased IC50 to oxaliplatin. (E) Expression of proteins related to proliferation and EMT in oxaliplatin- and saracatinib-resistant cell
lines were examined by immunoblotting. Page 18/21 Page 18/21 igure 4 Figure 4
Gene expression profiles in saracatinib- and oxaliplatin-resistant HCC revealed upregulation of A
and activation of Wnt signaling-associated proteins
Gene expression profiles were compared bet
wild-type MHCC97L and MHCC97L-Src (A) and between MHCC97L and MHCC97L-Src (B). (C) A
458 genes were found to be altered in both sets of cells. (D) These genes were related to cell divi
growth, angiogenesis, adhesion, and metabolic processes, and KEGG pathway analysis was perf Figure 4 Gene expression profiles in saracatinib- and oxaliplatin-resistant HCC revealed upregulation of ABCG1
and activation of Wnt signaling-associated proteins
Gene expression profiles were compared between
wild-type MHCC97L and MHCC97L-Src (A) and between MHCC97L and MHCC97L-Src (B). (C) A total of
458 genes were found to be altered in both sets of cells. (D) These genes were related to cell division,
growth, angiogenesis, adhesion, and metabolic processes, and KEGG pathway analysis was performed Page 19/21 Page 19/21 on these altered genes. (E) The altered genes related to drug resistance were partly selected. (F)
Immunoblotting was used to determine the protein expression of the genes that were altered most
dramatically between the wild-type and drug-resistant cell lines. y
yp
g Page 20/21
Figure 5
Upregulation of ABCG1 via alterations in Wnt/β-catenin signaling contributes to the effects of saracatinib
on oxaliplatin resistance of HCC cells
(A) Immunoblotting was used to examine the expression of ABCG1 Figure 5 Upregulation of ABCG1 via alterations in Wnt/β-catenin signaling contributes to the effects of saracatinib
on oxaliplatin resistance of HCC cells
(A) Immunoblotting was used to examine the expression of ABCG1 Upregulation of ABCG1 via alterations in Wnt/β-catenin signaling contributes to the effects of saracatinib
on oxaliplatin resistance of HCC cells
(A) Immunoblotting was used to examine the expression of ABCG1 Page 20/21 Page 20/21 following Wnt/β-catenin signaling pathway inhibition by KYA1797K. (B) Wnt/β-catenin signaling pathway
and PCNA expression was determined following silencing of ABCG1 by siRNA and restoration treatment
in HCC cells. (C). The sensitivity of HCC cells to oxaliplatin was measured as IC50 following silencing of
ABCG1 expression with siRNA or mock siRNA treatment. (D) The sensitivity of cells to oxaliplatin
following treatment with the Wnt signaling inhibitor KYA1797K was determined as IC50 values. (E)
Interference with ABCG1 expression resulted in reversal of the saracatinib-induced oxaliplatin resistance
with smaller subcutaneous xenografts. NC3RsARRIVEGuidelinesChecklistfillable.pdf Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 21/21
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The Effect of Immunosuppressive Adjuvant Kynurenine on Type 1 Diabetes Vaccine
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Frontiers in immunology
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cc-by
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The Effect of Immunosuppressive
Adjuvant Kynurenine on Type 1
Diabetes Vaccine
Jing Sun 1, Jiandong Shi 1, Jianfang Li 1, Meini Wu 1, Yanhan Li 1, Sengquan Jia 1,
Chunli Ma 1,2, Xinyi Wang 1, Zhiyuan Li 1,2, Ningzhu Hu 1 and Yunzhang Hu 1*
1 Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, 1 Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China,
2 Kunming Medical University, Kunming, China 1 Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China,
2 Kunming Medical University, Kunming, China Inducing antigen-specific tolerance is a promising treatment for preventing or reversing
Type 1 diabetes (T1D). In contrast to a vaccine that induces immune responses against
pathogens, a tolerogenic vaccine can suppress immunity against antigens causing
diseases by administrating a mixture of self-antigens with an adjuvant that decreases
the strength of antigen-specific response. Kynurenine (Kyn) is an endogenous substance
that can inhibit the natural killer cell and T cell proliferation and promote the differentiation
of naïve T cells into regulatory T cells (Tregs). In this study, we evaluated the efficacy of Kyn
as a novel suppressive adjuvant. Kyn was co-immunized with GAD65 phage vaccine to
induce Treg cells and tolerogenic responses for the prevention of T1D in NOD mouse
model. Mice were subcutaneously immunized two times with 1011 Pfu (100mL,1012 Pfu/ml)
GAD65 phage vaccine doses mixed with 200 mg of Kyn. Serum antibodies and cytokines
were detected by ELISA and electrochemiluminescence, respectively. Flow cytometry
assay was used to analyze DC and Treg. MTS was used for the analysis of spleen
lymphocyte proliferation. RNA sequencing was used to investigate mRNA and miRNA
expression profiles in spleen lymphocytes. Compared to GAD65 phage vaccine alone,
co-immunization of Kyn and GAD65 phage vaccine resulted in the prevention of
hyperglycemia in 60% of mice for at least one month. Further, Kyn enhances
GAD65-specific Th2-mediated immune responses; regulates the Th1/Th2 imbalance
and increases the secretion of Th2 cytokines and the number of CD4+CD25+Foxp3+T
cells; suppresses DC maturation and GAD65-specific T lymphocyte proliferation. Moreover, we integrated Kyn related miRNA and mRNA expression profiles obtained
from the spleen lymphocyte RNA-sequencing which was stimulated by Kyn in vitro. These
data provide an important basis for understanding the mechanisms underlying Kyn as an
immunosuppressive adjuvant which regulated the immune response. These findings
suggest that Kyn can serve as an effective suppressive adjuvant candidate for Type 1
diabetes vaccines. ORIGINAL RESEARCH
published: 07 July 2021
doi: 10.3389/fimmu.2021.681328 Keywords: immunosuppressive, adjuvant, kynurenine, vaccine, Type 1 diabetes vaccine Edited by:
Sophie Tourdot,
Pfizer, United States Reviewed by:
Morten Agertoug Nielsen,
University of Copenhagen, Denmark
Enayat Nikoopour,
University of Michigan, United States *Correspondence:
Yunzhang Hu
huyunzhangym@126.com Specialty section:
This article was submitted to
Vaccines and
Molecular Therapeutics,
a section of the journal
Frontiers in Immunology Specialty section:
This article was submitted to
Vaccines and
Molecular Therapeutics,
a section of the journal
Frontiers in Immunology
Received: 16 March 2021
Accepted: 14 June 2021
Published: 07 July 2021 Received: 16 March 2021
Accepted: 14 June 2021
Published: 07 July 2021 Citation:
Sun J, Shi J, Li J, Wu M,
Li Y, Jia S, Ma C, Wang X,
Li Z, Hu N and Hu Y (2021)
The Effect of Immunosuppressive
Adjuvant Kynurenine on Type 1
Diabetes Vaccine. Front. Immunol. 12:681328. doi: 10.3389/fimmu.2021.681328 INTRODUCTION Type 1 diabetes (T1D) is an autoimmune disease attributed to
the immune-mediated progressive destruction of b cells in the
pancreatic islets, which results in hyperglycemia. Autoantibodies
against insulin, including 65 kDa glutamic acid decarboxylase
(GAD65), insulinoma-associated protein 2 (IA-2), and zinc
transporter 8 (ZnT8), are proteins associated with secretory
b-cell granules. It can be used as biomarkers of T1D-associated
autoimmunity. These proteins can be identified months to years
before the onset of T1D and served as developing risk markers
(1, 2). Nowadays, many kinds of autoantigen-specific T1D trials
involving oral and nasal insulin or recombinant human
GAD65 formulated with alum have been continuously applied
(17). However, no suitable immunosuppressive drug or
immunosuppressive adjuvant can be used in combination with
it. Therefore, finding new immunosuppressive drugs or
immunosuppressive adjuvants is a key factor for the success of
this treatment strategy. Infiltration of islet antigen-specific T cells, activation of pro-
inflammatory antigen-presenting cells, and loss of Foxp3+
regulatory T cells (Tregs) are three of the most T1D
characteristic immunopathological processes (3). gy
Kynurenine (Kyn) is a tryptophan metabolite produced
through tryptophan-2, 3-dioxygenase (TDO) degradation in
the liver under physiological conditions (18), and through the
indoleamine 2, 3 dioxygenase (IDO) in the extrahepatic tissues
including blood and lymph tissue during infection,
inflammation, or oxidative stress (19, 20). It has been
demonstrated that Kyn activates the cytosolic aryl
hydrocarbon receptor (AHR) in a ligand-receptor manner
(21), and endogenously regulates systemic inflammation and
tolerance. Additionally, AHR has been implicated in various
immune functions, including reduce the activity of natural
killer (NK) cells (22), inhibit the NK cell and T cell
proliferation (23), and promote the differentiation of naïve T
cells into Treg instead of into Th17 cells through preventing
dendritic cell maturation (24), which supports the role of AHR
as an important player in determining the T cell differentiation
(25). Because the Kyn-AHR axis has an effect on the
proliferation of Tregs, it has been considered a potential
therapeutic target for the treatment of autoimmune disorder. In our previous studies, we found that Kyn can serve as an
effective suppressive adjuvant for vaccines. Otherwise, Kyn is
an endogenous substance that is safer than exogenous
substances when considered as a vaccine adjuvant. Nearly 20 million people suffer from T1D worldwide. Although life-long insulin treatment can alleviate symptoms
and delay organ damage, it does not reverse the antigen-
specific T cell responses toward b cells. Abbreviations: T1D, Type 1 diabetes; Kyn, kynurenine; GAD65, 65 kDa glutamic
acid decarboxylase; Tregs, regulatory T cells; NOD, non-obese diabetic; AHR, aryl
hydrocarbon receptor. HIGHLIGHTS nearly 80% of NOD mice, long-term normoglycemia was
restored by repeated administration of GAD65-alum. Moreover, the injection of aluminum salts of GAD65 (GAD65-
Alum) in mice has been shown to reduce GAD-specific Th1 Teff
cells (6, 7). Also, GAD65-Alum (Diamyd®) has been recently
tested in phase II and III clinical trials (8–11), showing drug
safety. In addition, Diamyd® appeared to be superior to placebo
in preserving residual b-cell function at 12–15 months. Moreover, an expansion of IL-10+CD4+ T cells was observed,
indicating the regulatory compartment’s induction (12). Despite
these vaccines’ success in mouse models, these vaccines could not
induce CD4+Foxp3+ Tregs that can balance between beneficial
and harmful effects of inflammation. • Co-immunization of Kyn and GAD65 phage vaccine resulted
in prevention of hyperglycemia in 60% of mice for at least one
month. • Co-immunization of Kyn and GAD65 phage vaccine enhances
GAD65-specific Th2-mediated immune responses; regulates
the Th1/Th2 imbalance and increases the secretion of Th2
cytokines and the number of CD4+CD25+Foxp3+ T cells;
suppresses DC maturation and GAD65-specific T
lymphocyte proliferation. • RNA sequencing provides an important basis for
understanding the mechanisms underlying Kyn as an
immunosuppressive adjuvant which regulated the immune
response. Regulatory T (Treg) cell is believed to have a key role in
preventing autoimmunity. Animal studies have illustrated that
CD4+Foxp3+ Tregs can induce tolerance by suppressing the
functions of Th1 cells and DCs or by releasing inhibitory
cytokines such as TGFb or IL-10 (13), and form the primary
mechanism of peripheral tolerance (14). The most promising
immunotherapy for autoimmune disease treatment in humans is
autologous Treg cell therapy (15). Some studies (16) have shown
that dexamethasone and rapamycin (rapa) could significantly
increase cell numbers and function CD4+CD25+ Treg cells in
animal models. However, these immunosuppressive drugs have
side effects, such as induction of infection and tumor formation. Citation: July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 1 The Effect of Kynurenine on T1D Sun et al. Animal Immunization Female NOD mice of 4–6 weeks of age were purchased from
GemPharmatech Co, Ltd (Nanjing, China). All the animals were
housed in a light- and temperature-controlled environment. All
animal studies (including the mice euthanasia procedure) were
done in compliance with the regulations and guidelines of the
Institute of Medical Biology, Chinese Academy institutional
animal care and conducted according to the AAALAC and the
IACUC guidelines. The mice were randomly divided into four groups (12
animals per group). They were subcutaneously immunized
(subcutaneous injection at different sites on the back of mice)
with phage vaccine alone or co-immunized with Kyn (Sigma,
K8625) in 100 ml final volume listed in Table 1. The animal
immunization schedule and detection program are shown in
Figure 1. Briefly, mice were sacrificed, and splenocytes were
harvested at different time points. Blood samples were collected,
and sera were stored at −20°C until tested. INTRODUCTION Therefore, a novel
treatment strategy is required to improve therapeutic effects. Some scholars (4) believe that b cell autoantigens presented in
non-inflammatory contexts can regulate auto-reactive T cells
and generate b cell protection. Recovering antigen-specific
tolerance or down-regulating the immune response to non-
harmful antigens is a promising way to treat T1D. GAD65 is a major autoantigen in T1D. T-cell reactivity and
autoantibodies against GAD65 are early markers of this
autoimmune disease process. GAD antibodies have been found
in nearly 70–80% of T1D patients at the time of diagnosis (5). Preclinical studies have demonstrated that the administration of
the isoform GAD65 in non-obese diabetic (NOD) mouse model
can prevent autoimmune destruction of pancreatic b-cells. In July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 2 The Effect of Kynurenine on T1D Sun et al. In this study, Kyn was co-immunized with GAD65 phage
vaccine to induce Treg cells and tolerogenic responses for
prevention of T1D in the NOD mouse model. We provided
direct evidence that Kyn, as a novel suppressive adjuvant,
promotes Foxp3+ Treg induction, suppresses dendritic cell
maturation and GAD65-specific T cell proliferation, and
significantly increases IL-10, IL-4 and TGF-b1, decreases of IFN-
g and IL-2 in the NOD mouse model. We also analyzed the
molecular information provided by transcriptome sequencing of
mRNA and miRNA in an in vitro Kyn assay, providing a new
understanding of the underlying immune response mechanism
and a new idea for the development of suppressive adjuvants. formaldehyde was added to phage particles at a volume ratio of
1:4,000 to inactivate the phage to obtain the GAD65
phage vaccine. GAD65 Phage Vaccine Preparation GAD65 Phage Vaccine Preparation
The recombinant GAD65 phage vaccine expressing the 190–320
amino acid sequence of huGAD65 (GenBank: M81882.1) was
constructed in the T7 phage display system by our laboratory. The huGAD65 gene shares 95% amino-acid identity and 98%
conservation with mGAD65 (26), respectively. Briefly, we
inoculated 50 ml 1011 pfu/ml GAD65 phage into 5 ml fresh
Escherichia coli strain BLT5403 with OD600 = 0.6–0.8, cultured
at 37°C and 150 rpm for 3 h. The cultures were diluted 50-fold
into 1,000 ml of Escherichia coli strain BLT5403 with OD600 =
0.6–0.8, cultured at 37°C and 150 rpm for 3–6 h. Bacteria were
collected by centrifugation (30 min at 5,000 rpm.) The
supernatant was mixed in 1:5 volume solution containing 20%
polyethylene glycol 8000 (PEG-8000) and 2.5 M NaCl, then kept
at 4°C overnight to precipitate the phage particles. After that, the
precipitate was collected by centrifugation (30 min at 10,000
rpm.), dissolved in 4 ml of phosphate-buffered saline (PBS), and
centrifuged at 10,000 rpm for 30 min. The phage particles were
then purified by sequential centrifugation of the PBS phage
suspension at 10,000 rpm for 30 min. The precipitate was then
dissolved in 1 ml of 50 mM Tris-HCl buffer, pH 7.5. Then, an
ultrafiltration membrane with a molecular weight of 100 kD was
used to remove endotoxin. After ultrafiltration, the content of
endotoxin in phage preparation was less than 100 EU/ml. Then
the phage concentration was adjusted to 1012 PFU/ml, and the Antibodies and Synthetic Peptide y
p
Fluorescently labeled anti-mouse monoclonal antibodies
including anti-mouse CD4-FITC (RM4-5), anti-mouse CD25-
APC (PC61.5), anti-mouse/Rat-Foxp3 PE (FJK-16s), anti-
CD11c-PE (N418), anti-CD80-APC (16-10A1), anti-IL10-FITC
(JESS-16E3), and isotype controls were purchased
from eBioscience (San Diego, CA, USA). GAD65 specific
peptide acid sequences were TYEIAPVFVLLEYVT,
EYVTLKKMREIIGWPGGSGD, KKGAAALGIGTDSVI,
ALGIGTDSVILIKCDERGK. TABLE 1 | Animal groups and dosage. Groups
Dose
Adjuvant
Injection Times
No. Control
100 ml (1012Pfu/ml)
2
12
Control + KYN
100 ml (1012Pfu/ml)
200 mg KYN
2
12
GAD65
100 ml (1012Pfu/ml)
2
12
GAD65 + KYN
100 ml (1012Pfu/ml)
200 mg KYN
2
12 FIGURE 1 | Schematic Map of immunization schedule. Frontiers in Immunology | www.frontiersin.org
July 2021 | Volume 12 | Article 681328
3 FIGURE 1 | Schematic Map of immunization schedule. FIGURE 1 | Schematic Map of immunization schedule. FIGURE 1 | Schematic Map of immunization schedule. July 2021 | Volume 12 | Article 681328 3 The Effect of Kynurenine on T1D Sun et al. RNA Sequencing We also provided insight into the mechanisms of Kyn and its
influence on the immune response to immune cells. Mouse
splenocytes were performed to assess the immune suppressive
properties of kynurenine and to determine that the adjuvants
used were indeed biologically active. Balb/C mice spleen
lymphocytes (three mice in each group) were isolated as
described above, after that they were stimulated for 12 h by
Kyn (40 mg/ml), which performed at least three independent
experiments. Splenocyte samples were collected after treatment,
after which the total mRNA and microRNA libraries were
prepared and sequenced. Total RNA was extracted using the
RNAfast200 kit (Fastagen Biotech, Hefei, China) according to
the manufacturer’s protocol. The quality control of the isolated
RNA (concentration, RIN, 28S/18S, and size) was performed
with Agilent 4200 Bioanalyzer (Agilent Technologies, Santa
Clara, USA). The strand-specific RNA-seq libraries were
prepared using a NEBNext® Ultra™I RNA Library Prep Kit
for Illumina (NEB, MA, USA) following the manufacturer’s
instructions. The libraries were assessed on the Agilent
Bioanalyzer 4200 system and sequenced on the Illumina Xten
platform. RNA sequencing and reads alignment were performed
by GMINIX Biotechnology Corporation (Shanghai, China). Reads were aligned to mouse genome version mm9. MicroRNA sequencing and RNA sequencing data are
deposited at the Gene Expression Omnibus (GEO; accession
number: GSE164304; GSE165737). Differentially expressed
miRNAs/mRNAs were selected based on the following criteria:
|log2 fold change| > = 1.2 and P-value <0.05. Spleen Lymphocyte Proliferation Analysis
Mice splenocytes were harvested after 4 weeks of the last
treatment. Splenocytes were recovered as a single cell
suspension in RPMI 1640 medium (Hyclone). Erythrocytes
were removed from splenic suspensions by lysis in ACK Lysis
Buffer (Gibco™) for 2 min at 37°C. After that, the 1,640 complete
culture medium containing 10% fetal bovine serum was added,
and the cells were inoculated into 96-well plates at the
concentration of 1 × 105/ml. GAD65 specific peptides were
added as antigen stimulation (final concentration 5 mg/ml);
PMA (positive control, final concentration 5 mg/ml) was added
as positive antigen control. After 48 h of stimulation, the 20 ml
MTS (Promega) was added to each well at the last 4 h of
incubation. The wavelength of 490 nm was used to measure
light absorption value of each well by ELISA plate reader (UVP,
California, USA). Analysis of Treg Mice spleen lymphocytes were harvested as previously described,
and then adjusted to the concentration of 1 × 106/ml and
incubated for 48 h at 37°C and 5% CO2 with 100 ml GAD65
peptide pool containing 5 mg/ml of each individual peptide as
described previously. After that, cells were stained with anti-
mouse CD4-FITC (RM4-5), anti-mouse CD25-APC (PC61.5)
antibodies. Then, the cells were fixed with Fix/Perm buffer
(eBioscience, America), incubated in permeabilization buffer
(eBioscience, America), and stained with anti-mouse/Rat-
Foxp3 PE (FJK-16s) antibody. After washing two times,
samples were re-suspended with PBS and immediately
analyzed on CytoFLEX S Flow Cytometer (BECKMAN
COULTER, USA) as previously described. Determination of Serum Antibodies by ELISA The 96-well flat-bottom plates were coated with purified
recombinant GAD65 protein (purified by our lab) at a
concentration of 0.1mg/ml in coating buffer at 4°C overnight. Plates were washed five times with washing buffer and blocked
with blocking solution at 37°C for 1 h. After that, mice sera were
serially diluted two-fold in blocking solution (starting at 1:100–
1:3,200), and 100ml was added to each well. After incubation for
1 h, the plates were washed five times and incubated with 1:2,000
diluted HRP-labeled goat anti-mouse IgG1 and IgG2a antibodies
(Invitrogen, USA) at 37°C for 1 h. After the final wash, 100ml of
ABTS HRP substrate was added per well and plates were
incubated for 5 min; the reaction was stopped by adding 25 ml
of ABTS peroxidase stop solution. The optical density (OD) of
the plate was measured at 450 nm by ELISA plate reader (UVP,
California, USA). The ELISA test reagents were from the ELISA
Kit Anti-Mouse ABTS® System (KPL Protein Detector™). Determination of Serum Antibodies and Cytokines
by Electrochemiluminescence The concentrations of the IL-2, IL-4, IL-10, IFN-g and TgF-b1 in
the serum samples were examined using the MSD ECLIA
according to the manufacturer’s instructions (MSD, Rockville,
MD, USA). For quality control, a standard curve was prepared. The highest point and background point were selected for the
standard point to confirm that the sample concentration was
within the range of the standard curve. In the preliminary
experiment, the sample was diluted two and eight times; eight
times had undetectable factors, which indicated that dilution of
at least two times was recommended. Three repeated tests were
performed on each sample. Determination of Serum Antibodies
and Cytokines then adjusted to the concentration of 1 × 106/ml and incubated
for 48 h at 37°C and 5% CO2 with 100 ml GAD65 peptides pool
containing 5 mg/ml of each individual peptide as described
previously. Cells were processed as described previously and
stained with anti-CD11c-PE (N418) and anti-CD80-APC (16-
10A1). After permeabilization, samples were stained with
intracellular anti-IL10-FITC (JESS-16E3) fluorescent-labeled
monoclonal antibody. After washing two times, samples were
re-suspended with PBS and immediately analyzed on CytoFLEX
S Flow Cytometer (BECKMAN COULTER, USA). The flow data
was processed using FlowJo10.4 software. Determination of Serum Antibodies by ELISA Flow Cytometry Assay
Analysis of DC Mice splenocytes were harvested after 4 weeks of the last
treatment. Mice spleen lymphocytes were harvested and
washed two times with PBS containing 2% BSA. Cells were July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org The Effect of Kynurenine on T1D Sun et al. Validation of Differentially Expressed
miRNA and mRNA computer SPSS software. A p value of <0.05 was considered
statistically significant. The expression of the selected miRNAs was detected by the stem-
loop qRT-PCR method. Total RNA was extracted to obtain
cDNA by reverse transcription-PCR using GoScript Reverse
Transcription System (Promega, USA) according to the
manufacturer’s protocol. The qPCR assay was performed in
CFX96 Touch Real-Time PCR Detection System (BioRad,
Berkeley, USA). The cycling parameters of qPCR reaction were
as follows: 95°C for 5 min, then 95°C 10 s, and 60°C 30 s for 40
cycles (miRNAs); 95°C for 5 min, then 95°C 10 s, and 50°C 30 s
for 35 cycles (mRNAs), followed by a melting curve to record the
specific PCR product. In the qPCR experiment, the relative
expressions were calculated using the 2–DDCt method with
GAPDH as an endogenous control for mRNA and U6 as an
internal control for miRNA. Each reaction was conducted in
triplicate. The corresponding primers were shown in
Supplementary Table 1. The Effect of Kyn on Suppressed
Hyperglycemia and Diabetes To study the potential immunosuppressive adjuvant effects of
Kyn, we immunized mice with the GAD65 phage vaccine
premixed with the Kyn adjuvant. The immunization and
sample collection schedule is shown in Figure 1. Blood
glucose levels and body weight of NOD mice were detected
at 6, 8, 10, 12, 14, 16, 18, and 20 weeks. Our results showed that
(Figures 2A, B) subcutaneous administration of GAD65
vaccine, or GAD65 vaccine + Kyn, prevented the
development of hyperglycemia in 50% (3/6) and 67% (4/6) of
NOD mice at least one-month (from the 14th week to 18th
week), respectively. Clinical diabetes was defined by
hyperglycemia (blood glucose levels >10.3 mmol/l) in fasted
animals (27); the protective effect began after the 14th week of
initial hyperglycemia in our study. In contrast, no significant Statistical Analyses We also found that the Tgfb1 was
increased when immunized with the adjuvant Kyn + GAD65
vaccine compared with other groups in NOD mice. remission of hyperglycemia was observed in the control group
and control + Kyn group. Weight loss is a significant symptom
of T1D, and we found no changes in weight loss in GAD65 and
GAD65 + Kyn groups. During the 144 days of observation
period (Figures 2C, D), mice in control and control + Kyn
groups had a mortality rate of 33.3% (2/6), while no death was
observed in GAD65 and GAD65 + Kyn groups (mice were in
good condition, weighing between 25 and 30 g). Thus, these
results indicated that co-immunization of Kyn and GAD65
phage vaccine could significantly temporarily reverse diabetes
in NOD mouse model. type immune response, and mainly IgG2a antibodies in mouse
models (28). Compared to the group immunized with GAD65
phage alone, a significantly enhanced level of IgG1 was obtained
in the group immunized with GAD65 phage plus Kyn as an
adjuvant. IgG1 anti-GAD65 antibodies resulted in ratios of IgG1
to IgG2a >1 that was indicative of Th2 polarization (Figure 3). To further determine whether the immunosuppressive effect
of Kyn on diabetes was mediated by Th2 cells,
electrochemiluminescence assay was performed to examine the
effects of Kyn on the generation of Th1 and Th2 cytokines. We
detected the production of IFN-g, IL2, IL-4, IL-10 cytokines
associated with Th1 and Th2 responses in the serum of
immunized NOD mice. Our results revealed that IL-4 and IL-
10 (Th2 cytokines) were significantly up-regulated (Figure 3),
while production of IL-2 and IFN-g (Th1 cytokines) was
significantly down-regulated in Kyn co-immunized mice with
GAD65 phage vaccine compared with other groups, which is
consistent with IgG isotype data. These results indicated that
Kyn could enhance the GAD65 vaccination by shifting the Th1/
Th2 balance toward Th2. We also found that the Tgfb1 was
increased when immunized with the adjuvant Kyn + GAD65
vaccine compared with other groups in NOD mice. Statistical Analyses Data are presented as means ± standard deviations (SDs), and
statistical analyses were performed with professional statistical A
B
C
D
FIGURE 2 | NOD mice were immunized with phage vaccine with or without KYN on weeks 10 and 12. (A, C) Blood glucose/body weight was measured on weeks
6, 8, 10, 12, 14, 16, 18, and 20. The body weight of mice grew when treated with GAD65 phage vaccine with KYN. (B) Mice immunization of GAD65 phage vaccine
with KYN were significantly remission from hyperglycemia in 60% at least one month. (D) The survival curves. B A B D D FIGURE 2 | NOD mice were immunized with phage vaccine with or without KYN on weeks 10 and 12. (A, C) Blood glucose/body weight was measured on weeks
6, 8, 10, 12, 14, 16, 18, and 20. The body weight of mice grew when treated with GAD65 phage vaccine with KYN. (B) Mice immunization of GAD65 phage vaccine
with KYN were significantly remission from hyperglycemia in 60% at least one month. (D) The survival curves. July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 5 The Effect of Kynurenine on T1D Sun et al. type immune response, and mainly IgG2a antibodies in mouse
models (28). Compared to the group immunized with GAD65
phage alone, a significantly enhanced level of IgG1 was obtained
in the group immunized with GAD65 phage plus Kyn as an
adjuvant. IgG1 anti-GAD65 antibodies resulted in ratios of IgG1
to IgG2a >1 that was indicative of Th2 polarization (Figure 3). To further determine whether the immunosuppressive effect
of Kyn on diabetes was mediated by Th2 cells,
electrochemiluminescence assay was performed to examine the
effects of Kyn on the generation of Th1 and Th2 cytokines. We
detected the production of IFN-g, IL2, IL-4, IL-10 cytokines
associated with Th1 and Th2 responses in the serum of
immunized NOD mice. Our results revealed that IL-4 and IL-
10 (Th2 cytokines) were significantly up-regulated (Figure 3),
while production of IL-2 and IFN-g (Th1 cytokines) was
significantly down-regulated in Kyn co-immunized mice with
GAD65 phage vaccine compared with other groups, which is
consistent with IgG isotype data. These results indicated that
Kyn could enhance the GAD65 vaccination by shifting the Th1/
Th2 balance toward Th2. GAD65 Phage Vaccine + Kyn Decreases
GAD65-Specific T Cell Proliferation indicating that Kyn could enhance the ability of DCs to
express IL-10. At the same time, we also observed that Con +
Kyn or GAD65 phage vaccine group could induce more secretion
of IL10 in immature DC cells compared with the control group
(P < 0.05). This result confirmed that GAD65, as an autoimmune
antigen of T1D, can induce certain immune tolerance, and Kyn
could enhance this tolerance effect. In the case of T1D, auto-reactive T cells attack islet cells, most of
which are insulin-specific cytotoxic CD8+ T cells. To explore the
potential underlying mechanisms of GAD65 phage vaccine +
Kyn on diabetes, we measured the autoantigen-specific T cell
activation and proliferation in vivo using T cell proliferation
assay in response to GAD65. As shown in Figure 4, the
proliferative response to GAD65 was significantly lower
(P < 0.05) in spleen lymphocytes isolated from GAD65 phage
vaccine + Kyn immunized mice than those from the GAD65
phage vaccine immunized mice. The proliferation was specific to
GAD65 peptide since challenge with the control BSA peptide in
vitro had no effect (P < 0.05). Our data demonstrated that
GAD65 phage vaccine + Kyn vaccination could inhibit the
proliferation of GAD65 auto-reactive T cells in vivo. GAD65 Phage Vaccine + Kyn Increases
CD4+CD25+ Treg Cells in the NOD
Mouse Model Treg is a subset of CD4+ T cells which are characterized by the
expression of transcription factor Foxp3 and helps to keep
inflammation under control and lower the autoimmune disease
risk in healthy individuals (30). They have been proved to
maintain their regulatory functions for a long-term even in the
absence of antigens that induced their generation and are stable
and transferable (31), thereby permitting the expected induction
of these cells to suppress unwanted immunity (13). In this study,
we evaluated the effect of the Kyn co-immunized with GAD65
phage vaccine on the regulation of Treg cells in spleen
lymphocytes. Spleen lymphocytes were isolated and re-
stimulated in culture with the GAD65 peptides. The cells were
then stained with anti-CD4-FITC, anti-CD25-APC, anti-FoxP3-
PE and analyzed by fluorescence-activated cell sorting (FACS). As shown in Figure 6, with the administration of Kyn, the Kyn +
GAD65 group produced a higher proportion of
CD4+CD25+Foxp3+ T cells in CD4+ T cell populations
compared with the GAD65 group (P < 0.05). A similar result
was observed in the GAD65 group; the proportion of
CD4+CD25+Foxp3+ T cells in CD4+ T cell populations was
significantly improved compared with control or control +
Kyn group, respectively (P < 0.05). These results indicated that
GAD65, as an autoimmune antigen of T1D, could increase the
proportions of CD4 +CD25 +Foxp3 + T cells, and
immunosuppressive adjuvant Kyn could specifically increase
this enhanced effect, which may be associated with the
suppression of T1D progression in the spleen lymphocytes. The Effect of Kyn Shifted the Th1/Th2
Balance Toward Th2 Since the proportion of Th1/Th2 cells is not balanced and the
immune response is skewed to Th1 in diabetic NOD mice and
diabetic patients, it is important to examine whether Kyn can
decrease GAD65-specific inflammatory profiles of T cells. Comparing the antibody responses generated by GAD65 phage
immunizations with or without Kyn adjuvant, the IgG1 and
IgG2a isotypes produced by both immunization ways were
detected. DNA injection into the muscle can produce a Th1 FIGURE 3 | Sera samples were collected to detect cytokines with MSD electochemiluminescence. GAD65 phage vaccine co-immunized with KYN generated
significantly lower secretion of IL-2, IFN-g than the GAD65 phage group did, and higher secretion of IL-10, IL-4, TGF-b1, than any other groups did (*P < 0.05). FIGURE 3 | Sera samples were collected to detect cytokines with MSD electochemiluminescence. GAD65 phage vaccine co-immunized with KYN generated
significantly lower secretion of IL-2, IFN-g than the GAD65 phage group did, and higher secretion of IL-10, IL-4, TGF-b1, than any other groups did (*P < 0.05). July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 6 The Effect of Kynurenine on T1D Sun et al. Kyn Can Suppress Dendritic
Cell Maturation DC promotes immunity and mediates T cell tolerance by direct
elimination, Treg induction, or co-adjustment. Immune
tolerance can be induced by adoptive transfer of immature or
semi-mature DCs, or by self-antigen-presenting DCs under
steady-state conditions (29). Twenty-eight days after the last
immunization, single cells of the spleen lymphocyte were
collected, stained with anti-CD11c-FITC, anti-CD80-APC, and
anti-IL10-PE, and analyzed by fluorescence-activated cell sorting
(FACS) to detect the mature state of DCs. As shown in
Figures 5A, B, the proportion of mature DC cells
(CD11c+CD80+) in the group immunized by Kyn + GAD65
was significantly lower than that in the group immunized by
GAD65 alone, indicating that Kyn can inhibit the maturation of
DC cells when stimulated by GAD65 peptides (P < 0.05). We also detected the changes in IL-10 expression in DC cells
(Figures 5C, D). The highest percentage of IL-10 expression in
DC cells immunized by Kyn + GAD65 phage vaccine was 14.2%,
which was significantly higher compared to other groups, FIGURE 4 | Effects of suppressive adjuvant Kyn on the GAD65 specific T cell
proliferation. The proliferative response to GAD65 was significantly lower in
spleen lymphocytes isolated from GAD65 phage vaccine + Kyn immunized
mice than those from the GAD65 phage vaccine immunized mice (*P < 0.05). Gene Expression Profile of Spleen
Lymphocytes Stimulated by Kyn In Vitro
Analysis of miRNA Expression Profiles Analysis of miRNA Expression Profiles
RNA-sequencing technology uses ultra sequencing technologies
to determine vaccine adjuvant transcriptomic profiles in vivo and
in vitro (32–34). Modigliani Y et al., confirmed that miRNA
expression has an important role in vaccination with aluminum
adjuvant (32). In our study, 91 distinctly expressed miRNAs were
identified between the Kyn stimulated group and the NC group
(>1.2-fold change and p < 0.05 as screening criteria). Among
them, 46 (50.5%) miRNAs were up-regulated, and 45 (49.5%)
were down-regulated (Supplementary Figure 2A). The detailed
information of differentially expressed miRNAs can be seen in
Supplementary Table 2. Additionally, hierarchical clustering
analysis showed the expression profile of 91 differentially
expressed miRNAs in different treatment groups. The results
indicated that these miRNAs were divided into two major FIGURE 4 | Effects of suppressive adjuvant Kyn on the GAD65 specific T cell
proliferation. The proliferative response to GAD65 was significantly lower in
spleen lymphocytes isolated from GAD65 phage vaccine + Kyn immunized
mice than those from the GAD65 phage vaccine immunized mice (*P < 0.05). July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 7 The Effect of Kynurenine on T1D Sun et al. A
B
D
C
FIGURE 5 | Kyn suppressed mouse dendritic cell maturation. At the 16th week, mice spleen lymphocytes were stained and immediately analyzed on Flow
Cytometer. Compared with GAD65 phage vaccine immunization alone, significantly lower percentages of CD80+CD11c+ DC cells were observed in GAD65 phage
vaccine co-immunized with KYN, which suggested that KYN may suppress dendritic cell maturation (A, B). At the same time, immatured-dendritic cells in the
co-immunized group secreted more IL10 than the control group did (C, D) (*P < 0.05). A A C B
D D FIGURE 5 | Kyn suppressed mouse dendritic cell maturation. At the 16th week, mice spleen lymphocytes were stained and immediately analyzed on Flow
Cytometer. Compared with GAD65 phage vaccine immunization alone, significantly lower percentages of CD80+CD11c+ DC cells were observed in GAD65 phage
vaccine co-immunized with KYN, which suggested that KYN may suppress dendritic cell maturation (A, B). At the same time, immatured-dendritic cells in the
co-immunized group secreted more IL10 than the control group did (C, D) (*P < 0.05). the expression of differentially expressed mRNAs was significantly
different between the Kyn-stimulated group and the NC group
(Supplementary Figure 2D). Gene Expression Profile of Spleen
Lymphocytes Stimulated by Kyn In Vitro
Analysis of miRNA Expression Profiles Validation of these trends was
performed on randomly selected mRNAs and was demonstrated
using qRT-PCR (Supplementary Figure 1B). clusters, one representing the negative controls and one
representing Kyn stimulated group. The data illustrated that
these common expression patterns of miRNAs were significantly
different from NC groups (Supplementary Figure 2B). Validation of expression levels for randomly selected miRNAs
was analyzed using qRT-PCR (Supplementary Figure 1A); the
results were consistent with the expression trends shown in the
miRNA sequencing results for the selected miRNAs. Functional Analysis of Common Differentially
Expressed miRNAs Functional Analysis of Common Differentially
Expressed miRNAs y
Transcriptome sequencing was used to analyze the mRNA
expression profiles of spleen lymphocytes stimulated by Kyn
(>1.2-fold change and p<0.05 as screening criteria). After 12 h
of stimulation, differentially expressed mRNAs matching these
criteria were depicted in volcano maps (Supplementary
Figure 2C). A total of 1,436 differentially expressed genes were
significantly different between the Kyn stimulated group and the
NC group (Supplementary Table 3). Among these differentially
expressed mRNAs, 713(49.7%) were up-regulated and 723 (50.3%)
were down-regulated. Additionally, cluster analysis indicated that To further study the role of the identified differentially expressed
miRNAs, we first predicted the miRNA target genes via targetscan
(http://www.targetscan.org/, mirnada (http://www.microrna.org/
and miRWalk (http://129.206.7.150/) databases. A total of 1,073
target mRNAs were predicted from the intersection of the three
databases. These target mRNAs, which were predicted by the
bioinformatics software and confirmed by transcriptome
sequencing, were identified as the preliminarily putative target
genes. Then, the preliminarily putative target genes that present a July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 8 The Effect of Kynurenine on T1D Sun et al. FIGURE 6 | Kyn induced CD4+CD25+Foxp3+ Treg cells. At the 16th week, mice spleen lymphocytes were stained and immediately analyzed on Flow Cytometer. Notably, Kyn still strongly enhanced the percentages of CD4+CD25+Foxp3+ Treg cells when co-immunized with GAD65 phage vaccine (*P < 0.05). FIGURE 6 | Kyn induced CD4+CD25+Foxp3+ Treg cells. At the 16th week, mice spleen lymphocytes were stained and immediately analyzed on Flow Cytometer. Notably, Kyn still strongly enhanced the percentages of CD4+CD25+Foxp3+ Treg cells when co-immunized with GAD65 phage vaccine (*P < 0.05). cell differentiation, Th1 and Th2 cell differentiation, PI3K–Akt
signaling pathways were significantly enriched in down
pathways. In general, these pathways mostly focused on
metabolic regulation and immune regulation. These 11
pathways were selected, and the miRNA–mRNA–pathway
regulatory network was generated based on the previously
selected miRNA-target mRNAs pairs (Figure 9). There were
39 differentially expressed miRNAs including 25 up-regulated
miRNAs and 14 down-regulated miRNAs, and 58 differentially
expressed mRNAs included 17 up-regulated mRNAs and 41
down-regulated mRNAs which were involved in these 11
pathways. In the miRNA–mRNA-pathway-net, we observed
that some genes have an important regulatory effect in these
pathways that up-regulated gene Gadd45a, Il12b, IL10 and
down-regulated gene Igf1, Il1r1, PIK3R1 which were located in
the center nodes (Supplementary Figure 3). Functional Analysis of Common Differentially
Expressed miRNAs negative regulatory relationship between miRNA-target mRNAs
were included. Finally, the differentially expressed target mRNAs
that passed through these screen processes were subject to analysis
using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and
Genomes (KEGG). All Gene Ontology can be seen in
Supplementary Table 4, the significant top 25 GO terms were
described in Figure 7. Up gene GO analysis (Figure 7A) results
showed that the five most-enriched terms are translation,
cytoplasmic translation, negative regulation of transcription by
RNA polymerase II, negative regulation of transcription, DNA-
templated and positive regulation of transcription by RNA
polymerase II. Down gene GO analysis (Figure 7B) results
showed that the five most-enriched terms are regulation of
transcription by RNA polymerase II, immune system process,
defense response to virus, innate immune response and cellular
response to interferon-beta. These data could provide evidence that
miRNA potentially up-regulated GO is mostly involved in gene
transcription, but down-regulated GO is mostly involved in
immune response. Frontiers in Immunology | www.frontiersin.org DISCUSSION administration of recombinant GAD65 protein or peptide in
NOD mice can induce immune tolerance against pancreatic b
cells and thus prevents or delays the development of insulitis and
diabetes. Based on human clinical trials and animal studies (47),
the proposed mechanism includes induction and proliferation of
GAD65-specific Tregs, which down-regulate antigen-specific
auto-reactive T cells and prevent them from attacking the
pancreatic b-cells. Therefore, inducing the proliferation of
antigen-specific regulatory T cells may be one of the most
effective ways to prevent or treat autoimmune diseases. Although animal model studies have demonstrated that GAD65
vaccination can be used to prevent autoimmune diabetes (35–
39), the preventive efficacy needs to be further improved. Successful clinical treatment may be hindered by the
uncertainty of major auto-antigens, an insufficient dosage of
antigen and lack of an ideal adjuvant or low Treg induction to
confer long time tolerance effects (40). In order to address these
issues, we designed an immunosuppressant-kynurenine (Kyn),
as adjuvant co-immunized with a phage-displayed vaccine
containing the 190–320 amino acid sequences of GAD65 to
induce Treg cells and tolerogenic responses and to prevent
diabetes I disease in the NOD mouse model. The use of Tregs as a method to treat inflammation and
autoimmune diseases has been proposed and well-received by
scientists (30). Many clinical trials have proved that the use of
vaccines to treat autoimmune diseases cannot effectively induce
the production of antigen-specific Treg cells, thus inhibiting the
role of pathological T cells. Consequently, it is necessary to add
inhibitory adjuvants in the process of vaccine use. Our previous
studies have shown that Kyn may be used as a suppressive
adjuvant to reduce the immunogenicity of HAV, a TD antigen, in
vivo, and LPS, a TI antigen, in vitro (48). As an endogenous
ligand of AHR, Kyn can activate the AHR signaling pathway and
may control immunity and autoimmunity while providing us
with a new opportunity for autoimmune disease therapeutic. In
this study, GAD65-specific CD4+CD25+FoxP3+Treg cells were
significantly increased in the GAD65 vaccine + Kyn group
compared with the vaccine single immunization group. Our study indicated that subcutaneous administration of
GAD65 phage vaccine + Kyn could prevent the development
of hyperglycemia in 60% (4/6) of NOD mice for at least one
month, which was highly effective for the suppression of T1D in
NOD mice. Weight loss is a significant symptom of T1D. Functional Analysis of Specific Differentially
Expressed miRNAs After KEGG signaling pathway enrichment analysis, the
miRNA–mRNA regulatory network map was generated in
Figure 10. Bioinformatics analysis demonstrated that some
specific miRNAs play a significant role in regulating the
lymphocyte function when stimulated by Kyn. Among the up-
regulated miRNAs with more predicted targets, mmu-miR6916-
5p had 40 predicted targets, followed by mmu-miR674-5p and
mmu-miR34a-5p with 33 and 22 predicted targets, respectively. There were few predictive targets for down-regulated miRNAs,
although mmu-miR155-3p had 34 predictive targets. Consequently, our data could provide evidence that there is a
close relationship between miRNA changes, host gene
expression, and suppressive adjuvant effects. KEGG analysis of these target mRNAs illustrated that the
putative target genes of the selected common miRNAs were
enriched into 186 KEGG signaling pathways. Of these signaling
pathways, 91 KEGG signaling pathways were up-regulated, and
95 were down-regulated. The top 25 signal pathways with the
most enriched genes were depicted by bubbles (Figure 8). Among those signaling pathways (Supplementary Table 5), all
involved in the modulation of Kyn stimulated signal pathways,
including metabolic pathways, FoxO signaling pathway,
cytokine–cytokine receptor interaction, Toll-like receptor
signaling pathway, NF-kappa B signaling pathway, TNF
signaling pathway, MAPK signaling pathway, IL-17 signaling
pathway, were enriched in both up and down pathways. Th17 July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 9 The Effect of Kynurenine on T1D Sun et al. A
B
FIGURE 7 | The top 25 significantly enriched GO terms of the overlapping target genes of differentially expressed miRNAs in spleen lymphocytes of Balb/C mice
(Kyn stimulated vs negative control). (A) Up gene Go analysis. (B) Down gene Go analysis. A B FIGURE 7 | The top 25 significantly enriched GO terms of the overlapping target genes of differentially expressed miRNAs in spleen lymphocytes of Balb/C mice
(Kyn stimulated vs negative control). (A) Up gene Go analysis. (B) Down gene Go analysis. Frontiers in Immunology | www.frontiersin.org DISCUSSION In our
study, no significant changes in weight loss were observed in the
GAD65 phage vaccine and GAD65 phage vaccine + Kyn groups. In addition, no death was observed in these two groups, and mice
were in good condition (weighing between 25 and 30 g). This
greater efficacy may be due to the combination of GAD65
antigen with the immunosuppressant-kynurenine, thus
providing effective immunotherapy against T1D. We also discovered that the proportion of mature DC cells
(CD11c+CD80+) was significantly lower in mice co-immunized In T1D, GAD65 has been identified as a primary auto-antigen
in the pancreas. Some studies (41–46) indicated that the July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 10 The Effect of Kynurenine on T1D Sun et al. A
B
FIGURE 8 | The top 25 significantly enriched KEGG-pathway analyses of overlapped target genes of differentially expressed miRNAs in spleen lymphocytes of
Balb/C mice (Kyn stimulated vs negative control). (A) Up gene KEGG analysis. (B) Down gene KEGG analysis. A B B FIGURE 8 | The top 25 significantly enriched KEGG-pathway analyses of overlapped target genes of differentially expressed miRNAs in spleen lymphocytes of
Balb/C mice (Kyn stimulated vs negative control). (A) Up gene KEGG analysis. (B) Down gene KEGG analysis. responding to GAD65. Additionally, an increase in the release
of IL-10 and TGFb1 was detected in Kyn + GAD65 phage
vaccinated mice, indicating that Kyn + GAD65 phage
immunization shifted the diabetogenic Th1 response to the
regulatory Th2 response. IL-10 is a strong anti-inflammatory
cytokine, which plays an important role in inhibiting Th1 cells by
inhibiting the production of proinflammatory cytokines such as
IFN-g, IL-1 and TNFa. TGF-b, which induces immune tolerance
and counteracts the immunostimulatory effects of checkpoint
inhibitors, has an immunosuppressive effect (53). The secretion
of IL10 and TGF-b and the production of Treg cells complement
each other, which is the key factor for the feasible treatment of
autoimmune diseases. T1D is related to the immune imbalance
caused by excessive activation of Th1 cells and inhibition of Th2
cells. Therefore, whether T1D can be cured or not depends on
whether the damaged Th1/Th2 balance can be effectively
restored by immune regulation. with Kyn, indicating that Kyn as an adjuvant could inhibit the
maturation of DC cells. Frontiers in Immunology | www.frontiersin.org DISCUSSION The highest proportion of IL-10
expression in DC cells in the GAD65 co-immunization Kyn
group was 14.2%, which was significantly higher than that in
other groups, indicating that Kyn could enhance the ability of
DC cells to express IL-10. The mature state of DC cells
stimulated by antigen directly affected the production of the
immune response. Under normal physiological conditions, the
fully matured DCs secrete the pro-inflammatory cytokines, such
as IL-1b, TNF, IL-12, and IL-6 (49). However, DCs can also
perform the opposite function by making T cells tolerant against
the autoantigen-directed immune response, which is necessary to
reduce the autoimmune reactions. The immunogenic and
tolerogenic functions of DCs depend on the balance between
activating and inhibitory signals during DC maturation (50). In T1D patients and NOD animal models, the skewed Th1/
Th2 balance which leads to autoimmune destruction of the b-
cells in the pancreas is a progressive phenomenon, resulting in
the continual loss of these cells (51, 52). Our results showed
higher IgG1 than IgG2a in both GAD65 vaccine and GAD65
vaccine + Kyn adjuvant groups, indicating that the vaccine
treatment could induce Th2 type immune response in NOD
mice, mainly regulating humoral immunity. In our study,
protection from diabetes by Kyn + GAD65 phage
immunization was also associated with a significant reduction
of T cell proliferation and of IFN-g secretion by T cells Our data suggested that Kyn can be used as an
immunosuppressive adjuvant in autoimmune disease. Understanding how lymphocytes interact with adjuvants is
crucial to understanding the mechanisms of this adjuvant and
will be critical in the rational design of future vaccines against
many diseases. In the present study, RNA and miRNA
sequencing was performed in mouse spleen lymphocytes
stimulated by Kyn. Compared with the negative control group,
91 common differentially expressed miRNAs and 1,436 common July 2021 | Volume 12 | Article 681328 11 The Effect of Kynurenine on T1D Sun et al. FIGURE 9 | miRNA–mRNA-pathway-net. Squares indicate identified miRNAs, while circles represent the corresponding target genes. Blue indicates down-regulated
miRNAs or mRNAs while red indicates up-regulated miRNAs or mRNAs. The relationship between miRNAs and genes is shown connected by gray lines. Eleven
significantly enriched pathways associated with splenocytes stimulated by Kyn were depicted using the gray triangles. More details about immune related miRNA–
mRNA-pathway are shown in Supplementary Figure 3. FIGURE 9 | miRNA–mRNA-pathway-net. Frontiers in Immunology | www.frontiersin.org DISCUSSION MicroRNA-329-3p (miR-329-3p)
has been studied in many types of human cancer (60). MiR-155
is one of the most studied miRNAs for its multiple roles in the
control of the innate and adaptive immune processes. Several
studies (61) demonstrated that miR-155 controls differentiation
of CD4+ T cells into the T helper cell subsets (Th1, Th2 and
Th17) (62–64) and that it affects the development of Tregs (65,
66). MiR-155 also regulates CD8+ T cells (67, 68) and is vital for
normal B cell differentiation and antibody production (63, 64,
69). MiR-155 over-expression can enhance the anti-viral, as well
as anti-tumor CD8+ T cell responses in vivo (61). On the other
hand, reducing the expression of miR-155 may cause
downstream cascades and increase the tendency to generate
Th2 cells which secrete type 2 (IL-4, IL-5 and IL-10) cytokines
(70). Therefore, fully understanding vaccine factors that
influence immune response has important implications. It
helps direct and rationally design new and more efficacious
vaccines or adjuvants with better immunogenicity and safety
profiles (32). the maintenance function of hepatocytes, adipocytes, and
epithelial cells. Beyond that, the AhR plays a crucial role in the
control of the adaptive immune response. It controls the
differentiation and activity of specific T-cell subsets and
influences adaptive immune responses by affecting both T cells
and antigen presenting cells (APCs). Kyn is the first metabolite of
tryptophan. As an endogenous substance, it activates the AHR in
a ligand-receptor manner. In our study we found that Kyn can
work as immunosuppressive adjuvant and mainly has a negative
regulatory role in the immune related signaling pathway, offering
plausible molecular mechanisms that may control immunity and
autoimmunity. These findings provide us with new opportunities
for targeted, therapeutic modulation of the immune response. Comparing the most significant up- or down-regulated genes, we
observed that most of the genes are related to MAPK, PI3K-Akt,
FoxO, and NF-kappa B signaling pathway (Gadd45a, Il12b, IL10,
Igf1, Il1r1, PIK3R1). In these genes, PIK3R1 is located at the
central node of miRNA–mRNA-pathway-net which indicated
that it had a strong regulatory effect in the process of
lymphocytes stimulated by Kyn. The immune system is highly
sensitive to manipulation of PI3K signaling pathway. Some
researchers (56) have observed that just a two-fold change in
PI3K signal activity through Akt is sufficient to regulate
lymphocyte homeostasis and induce autoimmunity in mice. DISCUSSION Squares indicate identified miRNAs, while circles represent the corresponding target genes. Blue indicates down-regulated
miRNAs or mRNAs while red indicates up-regulated miRNAs or mRNAs. The relationship between miRNAs and genes is shown connected by gray lines. Eleven
significantly enriched pathways associated with splenocytes stimulated by Kyn were depicted using the gray triangles. More details about immune related miRNA–
mRNA-pathway are shown in Supplementary Figure 3. differentially expressed mRNAs were found in the Kyn
stimulated group. The results of the GO analysis revealed that
the up-regulated genes were mainly enriched in gene expression
processes, such as mRNA processing, RNA splicing and
translation. The down-regulated genes were primarily involved
in immune-related biological processes, such as positive
regulation of tumor necrosis factor production, interferon-
alpha production, and IL-6 production. According to the
KEGG‐pathway analysis, both up-regulated and down- regulated genes play a crucial role in metabolic pathways and
pathways related to immune regulatory function. This is in
agreement with other studies in which a relationship between
metabolic state and the differentiation status of innate and
lymphoid cells (54). Numerous studies (55) have demonstrated that AhR plays an
important role in several normal physiological processes,
including development of the vasculature, construction of the
central nervous system, differentiation of blood cell subsets, and July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 12 The Effect of Kynurenine on T1D Sun et al. FIGURE 10 | miRNA–mRNA interaction network. The size of the point represents the regulatory capacity of a given miRNA. Squares indicate identified miRNAs,
while circles represent the corresponding target genes. Blue indicates down-regulated miRNAs or mRNAs while red indicates up-regulated miRNAs or mRNAs. The
relationship between miRNAs and genes is shown connected by gray lines. FIGURE 10 | miRNA–mRNA interaction network. The size of the point represents the regulatory capacity of a given miRNA. Squares indicate identified miRNAs,
while circles represent the corresponding target genes. Blue indicates down-regulated miRNAs or mRNAs while red indicates up-regulated miRNAs or mRNAs. The
relationship between miRNAs and genes is shown connected by gray lines. 3066-3p (down-regulated) were most variable after stimulation
with Kyn. The integrated analysis of miRNA and mRNA
expression revealed that one miRNA targeting several mRNAs,
mmu-miR6916-5p, mmu-miR674-5p, mmu-miR34a-5p and
mmu-miR155-3p were shown to have more targets. There are
no reports on the function of mmu-miR6916-5p, mmu-miR674-
5p, and mmu-miR-3066-3p. Frontiers in Immunology | www.frontiersin.org REFERENCES 10. Johnny L, Maria F, Maria H, Stina A, Mikael C, Mikael P, et al. GAD
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in Maintaining Self-Tolerance. Science (2015) 348(6234):589–94. doi: 10.1126/science.aaa7017 7. Diane KW, Brian B, Dorothy JB, Linda AD, Stephen EG, Robin G, et al. DISCUSSION MicroRNAs (miRNAs) are increasingly being identified as
key factors in the immune system regulating immune responses. Although miRNA regulation of each target results in only minor
changes in gene expression, these small changes can be amplified
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can be easily observed in the immune system, where miRNAs
modulate many cells’ ultimate fate by developing mature
lymphocytes (57–59). Among the differentially expressed
miRNAs, mmu-miR-329-3p (up-regulated) and mmu-miR- To sum up, our data demonstrated that kynurenine, as an
immunosuppressive adjuvant, can successfully help the phage
vaccine to induce immune tolerance in NOD mice, thus reducing
the symptoms of diabetes. As a physiological substance in vivo,
kynurenine has superior safety as an adjuvant than other
exogenous substances in theory. We believe that kynurenine
may be used as a novel immunosuppressive adjuvant in
autoimmune disease. July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 13 The Effect of Kynurenine on T1D Sun et al. DATA AVAILABILITY STATEMENT Basic Research Projects (2017FB040), the Yunnan Technology
Innovation Talent Projects (2017HB096), and Health Reserve
talents in Yunnan Province (H-2017009). The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and
accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/, GSE164304 https://www.ncbi.nlm.nih.gov/, GSE165737. FUNDING Supplementary Table 4 | Gene Ontology analysis for the target gene of
differentially expressed miRNA. This study was supported by the CAMS Innovation Fund for
Medical Sciences (2017-I2M-3-022), the National Natural
Science Foundation of China (31600741), the Yunnan Applied This study was supported by the CAMS Innovation Fund for
Medical Sciences (2017-I2M-3-022), the National Natural
Science Foundation of China (31600741), the Yunnan Applied Supplementary Table 5 | KEGG pathway analysis for the target gene of
differentially expressed miRNA. ETHICS STATEMENT The animal study was reviewed and approved by Institute of
Medical Biology, Chinese Academy institutional animal care
and conducted. Supplementary Figure 1 | qRT-PCR results. (A) MircoRNAs (B) mRNAs. Supplementary Figure 2 | Analysis of microRNA/mRNA expression profiles in
spleen lymphocytes of Balb/C mice stimulated with Kyn in vitro compared with the
negative control group (NC). (A, C) Volcano plots show the differentially expressed
miRNAs/mRNAs. (B, D) Hierarchical clustering presents the correlation of different
samples. AUTHOR CONTRIBUTIONS Supplementary Figure 3 | Immune related miRNA–mRNA-pathway. Global
analysis of immune related miRNA and mRNA which were enriched in both up and
down pathways. JS and YH designed the experiments. JS, JDS, JL, MW, SJ, XW,
and ZL performed the experiments. JS and JDS analyzed data. JS
wrote the manuscript. YL, CM, and NH provided important
analysis tools. All authors contributed to the article and approved
the submitted version. JS and YH designed the experiments. JS, JDS, JL, MW, SJ, XW,
and ZL performed the experiments. JS and JDS analyzed data. JS
wrote the manuscript. YL, CM, and NH provided important
analysis tools. All authors contributed to the article and approved
the submitted version. Supplementary Table 1 | The primers sequence used for q-PCR. Supplementary Table 2 | Differentially expressed miRNA result. Supplementary Table 3 | Differentially expressed mRNA result. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. 681328/full#supplementary-material REFERENCES Diabetes Metab Diabetes
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open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
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j.immuni.2008.11.010 July 2021 | Volume 12 | Article 681328 Frontiers in Immunology | www.frontiersin.org 16
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Site da revista em redalyc.org ISSN 2178-1036 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 https://doi.org/10.31977/grirfi.v19i3.1296
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Recebido: 20/06/2019 | Aprovado: 21/09/2019
Received: 06/20/2019 | Approved: 09/21/2019 RESUMO: Em Ser e Tempo (1927), Heidegger argumenta que o conhecimento primordial do Dasein se dá no manuseio de entes
mundanos. Merleau-Ponty, por sua vez, descreve na Fenomenologia da Percepção (1945) que o conhecimento é, antes de
tudo, uma intencionalidade corporal intraduzível em termos proposicionais. Mais tarde, em What Computers Can’t Do
(1972), Hubert Dreyfus usa a obra de ambos para apontar os equívocos do paradigma cognitivista, isto é, a abordagem
dominante do então nascente campo da inteligência artificial. Finalmente, em The Embodied Mind: Cognitive Science
and Human Experience (1991), a noção de enativismo - elaborada por Francisco Varela, Evan Thompson e Eleanor
Rosch - surge como uma tentativa de tomar do cognitivismo e do conexionismo a hegemonia nas ciências cognitivas. Dito isso, o objetivo do artigo consiste em descrever o enativismo e apontar sua herança fenomenológica. ENATIVISMO E CONHECIMENTO PRÁTICO Rodrigo Benevides Barbosa Gomes1
Universidade Federal de São Carlos (UFSCar)
https://orcid.org/0000-0002-0220-0503
E-mail: rodrigobenevides23@gmail.com Rodrigo Benevides Barbosa Gomes1
Universidade Federal de São Carlos (UFSCar)
https://orcid.org/0000-0002-0220-0503
E-mail: rodrigobenevides23@gmail.com KEYWORDS: Enactivism; Phenomenology; Heidegger; Merleau-Ponty; Francisco Varela. 1 Doutorando em Filosofia na Universidade Federal de São Carlos (UFScar), São Carlos – SP, Brasil. Artigo publicado em acesso aberto sob a licença Creative Commons Attribution 4.0 International License GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019 GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019 ABSTRACT: In Being and Time (1927), Heidegger claims the primordial knowledge of Dasein comes from coping with mundane
beings. Merleau-Ponty, in turn, argues on Phenomenology of Perception (1945) for knowledge as a bodily activity non
translatable in propositional terms. Later, on What Computers Can’t Do (1972), Hubert Dreyfus use the work of both to
point out the flaws of cognitivism, the dominant paradigm in early days of artificial intelligence. Finally, The Embodied
Mind: Cognitive Science and Human Experience (1991) by Francisco Varela, Evan Thompson and Eleanor Rosch brings
enactivism as an attempt to take from cognitivism and connectionism the hegemony in cognitive sciences. That being
said, the paper describes enactivism while showing its phenomenological heritage. KEYWORDS: Enactivism; Phenomenology; Heidegger; Merleau-Ponty; Francisco Varela. nevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, Artigo publicado em acesso aberto sob a licença Creative Commons Attribution 4.0 International License Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 ISSN 2178-1036 I Em Ser e Tempo (1927), Heidegger afirma que “O tipo de prática mais próxima a nós é,
como indicamos, não a mera cognição perceptiva e sim aquele tipo de ocupação onde se manipula
coisas e as põe em uso; e isso possui o seu próprio tipo de ‘conhecimento’.” (HEIDEGGER, 1962, p. 95, tradução nossa). Com isso, percebe-se que as raízes daquilo que mais tarde viria a ser chamado
de enativismo já residem em Heidegger. No entanto, apesar de Varela, Thompson & Rosch (1991)
admitirem certa influência heideggeriana, o principal interlocutor usado em sua obra é Merleau-
Ponty por conta de um motivo crucial: mesmo ao oferecer uma fenomenologia da inteligência
prática do Dasein que serviu a Hubert Dreyfus como principal argumento contra o cognitivismo,
Heidegger pecou ao não oferecer uma detalhada descrição em termos corporais desse envolvimento
não-representacional entre organismo e ambiente2. Em outras palavras, ao focar na exposição de
uma intencionalidade corporal não-proposicional, Merleau-Ponty serve melhor aos propósitos de
Varela et al., a saber, a compreensão da mente não como algo espacialmente localizável e
decomponível, mas como a dinâmica entre ação corporal e ambiente. Como diria Merleau-Ponty, o
processo de cognição é nada mais que “a dialética do meio e da ação” (MERLEAU-PONTY, 1999,
p. 262) Essa dinâmica, na perspectiva enativista, significa a enação [enactment] de um ambiente,
isto é, o trazer-à-tona de um meio pela ação corporal contínua de um organismo vivo. 2 “Ora, não encontramos em Ser e Tempo nem trinta linhas sobre o problema da percepção; nem dez sobre o problema do corpo.” (DE
WAELHENS, 2006, p. XII).
3 Cf. VARELA, F.; MATURANA, H. De Máquinas e Seres vivos: Autopoiese - A organização do Vivo. Porto Alegre: Ed. Artes
Médicas, 1997. GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
13 GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
13 ABSTRACT: Merleau-
Ponty torna isso claro quando descreve em A Estrutura do Comportamento (1942) que o equilíbrio
vital visado pelo organismo para agir no mundo significa a circunscrição, por assim dizer, de um
meio no qual suas ações ganham sentido em termos de otimização de resultado. [...] cada organismo possui suas condições mais eficazes de atividade e sua própria maneira
de atingir equilíbrio; e os determinantes internos desse equilíbrio se dão por uma atitude
geral em relação ao mundo. [...] o próprio organismo mede a ação das coisas e ele próprio
delimita seu meio por um processo circular que não possui análogo no mundo físico. (MERLEAU-PONTY, 1963, p. 148) Quando Merleau-Ponty afirma que “O senso comum põe o lugar do pensamento na cabeça”
(MERLEAU-PONTY, 1999, p. 202), o fenomenólogo resume a tese enativista por excelência, a
saber, a rejeição da mera equivalência entre mente e cérebro, algo possível para o francês “porque
rejeitamos o formalismo da consciência e fizemos do corpo o sujeito da percepção” (MERLEAU-
PONTY, 1999, p. 303). Antes do surgimento de qualquer sistema nervoso, há uma corporeidade ou
sistema autopoiético que mantém seu equilíbrio vital por meio da enação ou circunscrição não-
representacional de um meio3. Dessa forma, torna-se claro o porquê da explícita adoção da
fenomenologia merleau-pontiana como inspiração na formulação enativista. Gostaríamos de considerar o trabalho desenvolvido ao longo deste livro [The Embodied
Mind] como uma continuação moderna de um programa de investigação iniciado há mais de
uma geração pelo filósofo francês Maurice Merleau-Ponty. Por continuação não queremos
dizer uma consideração erudita do pensamento de Merleau-Ponty no contexto da ciência
cognitiva contemporânea. Pelo contrário, queremos apenas afirmar que os trabalhos de 13 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 ISSN 2178-1036 Merleau-Ponty inspiraram e guiaram o desenvolvimento desta obra. (VARELA et al., 1991,
pp. 15-16) Merleau-Ponty inspiraram e guiaram o desenvolvimento desta obra. (VARELA et al., 1991,
pp. 15-16) Com isso, podemos agora apresentar a abordagem desenvolvida por Varela et al. (1991),
opondo-a aos paradigmas cognitivista e conexionista, além de apontar suas influências
fenomenológicas. II A melhor maneira de entender a abordagem enativista é demonstrar como ela difere do
cognitivismo e do conexionismo4. O cognitivismo confunde-se com o surgimento do campo da
inteligência artificial. 4 A separação entre as abordagens citadas servem a título de simplificação explicativa, pois, como Thompson (2007, p. 4) observa,
não há uma demarcação definitiva que impossibilite abordagens heterogêneas: “Em pesquisas contemporâneas, todas as três
abordagens coexistem, tanto de maneira separada como em várias formas híbridas.” O próprio enativismo pode ser considerado
como uma espécie de síntese entre cognitivismo e conexionismo, como veremos adiante. Além disso, o paradigma do processamento
preditivo também consiste em uma recente abordagem (CLARK, 2013) que possibilita tanto interpretações representacionistas como
enativistas; no entanto, trataremos do enativismo no presente artigo somente em relação ao cognitivismo e ao conexionismo. GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
14 GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
12 22
b
2019
14 ABSTRACT: [...] Essa mesma noção de forma permitirá descrever o modo de 14 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 ISSN 2178-1036 existência dos objetos primitivos da percepção. Estes são, como dizíamos, mais do que
conhecidos como objetos verdadeiros, são vividos como realidades. Certos estados da
consciência adulta permitem entender essa distinção. O campo de futebol não é, para o
jogador, um “objeto”, ou seja, a palavra ideal que pode dar lugar a uma multiplicidade
indefinida de perspectivas e permanecer equivalente sob essas transformações aparentes [...]
O campo não lhe é dado, mas está presente para ele como o termo imanente de suas
intenções práticas; ele e o jogador são um só corpo e o jogador sente, por exemplo, a direção
do gol tão imediatamente quanto a vertical e a horizontal de seu próprio corpo. Não
bastaria dizer que a consciência habita esse meio. Ela nada mais é, nesse momento, que a
dialética do meio e da ação (MERLEAU-PONTY, 2006, pp. 262-263). O corpo não é instrumento da inteligência, o corpo é onde a própria inteligência irrompe. Como Evan Thompson (2007, p. 243) ressalta, “a mente humana está corporificada em todo o nosso
organismo”. A ação corporal inteligente não exige um modelo representacional interno de regras
sequenciais para agir efetivamente em uma dada situação. Mesmo quando há uma intenção
explícita anterior à ação (como no caso do jogador de futebol), as ações propriamente ditas
continuam não passíveis de descrição proposicional. Trata-se, então, do que Dreyfus chama de
absorbed coping, isto é, uma ação irrefletida, o modo de agir transparente que Heidegger chama de
ser-no-mundo e Merleau-Ponty de corpo próprio ou, por vezes, intencionalidade operante ou motriz e
também arco intencional.5 Esse aspecto não-representacional da inteligência prática funciona, em
seus termos mais gerais, como o modo de manutenção do equilíbrio vital do organismo em seu meio,
fazendo do ambiente um sistema percebido pelo corpo não como um amontoado de predicados
fixos, mas como solicitações que possibilitam respostas diversas: “De acordo com Merleau-Ponty, o
que é percebido não são estruturas proposicionais unificadas, mas sim solicitações para agir que
estão mais ou menos indeterminadas.” (DREYFUS, 2013, p. 292). Ou ainda: “o equipamento é
uma solicitação para agir, e não uma entidade com uma caracterização funcional.” (DREYFUS,
2007, p.252). 5 O escopo do presente artigo limita-se a descrever a intencionalidade individual do organismo e sua devida relação com objetos
exteriores. A relação com outrem, por outro lado, é igualmente crucial na descrição da inteligência prática e merece atenção
específica. Porém, apenas apontamos aqui que já em Heidegger e Merleau-Ponty há ricas descrições fenomenológicas acerca do
Mitsein (ser-com), isto é, a relação entre diferentes intencionalidades, acarretando em diversas formulações sobre reconhecimento de
si, moralidade, construção da noção de verdade, sexualidade, etc. Para uma compreensão contemporânea do caráter intrinsecamente
coletivo do Dasein baseada nos recentes dados da antropologia evolutiva, etologia e ciências cognitivas, Cf. TOMASELLO, M. A
Natural History of Human Thinking (2014). ABSTRACT: À época, o argumento dominante defendia que a inteligência humana - desde
correr, jogar futebol, dirigir um carro ou resolver equações - poderia ser traduzível em algoritmos
proposicionais. Em outras palavras, o comportamento humano, segundo o cognitivista, deve ser
entendido em termos de regras heurísticas que, dependendo da ação em questão, poderiam ser
efetivadas tanto de modo consciente quanto inconsciente ao se seguir regras ou passos. A raíz de tal
abordagem provém de Alan Turing, aquele que “sugeriu que um computador digital de alta
velocidade, programado com regras e fatos, poderia exibir comportamento inteligente”
(DREYFUS, 1992, p. IX). Quando Dreyfus fez sua sucedida crítica ao cognitivismo, foi
demonstrado que ao descrever a inteligência puramente em termos proposicionais a posição
cognitivista defende implicitamente a ideia de uma mente transcendental na qual há um “mundo-
lá-fora” que seria representado “aqui-dentro” por modelos internos de regras gerais aplicáveis a
diferentes contextos vividos pelo organismo que, ulteriormente, se traduziriam em ações. Por outro
lado, Dreyfus - apoiado na tradição fenomenológica - argumentou que, na verdade, a ação corporal
não-proposicional já é a chamada “atividade inteligente” do organismo: “todo modo de lidar com o
mundo acontece em um pano de fundo que Heidegger chama de ser-no-mundo, o qual não envolve
nenhum tipo de representação.” (DREYFUS, 2007, p. 254). Apesar de não colocar em termos explícitos, a crítica de Dreyfus - ao incorporar as obras de
Heidegger e Merleau-Ponty - já expressa o mote enativista: aquilo que entendemos por mental já é
o corporal, ou seja, a forma originária de inteligência é nada mais que a inteligência prática, seja o
movimento corpóreo de uma ameba ou de um ser humano em formação. Como Dreyfus relembra:
“Eu comecei a suspeitar que os insights formulados em poltronas existencialistas, especialmente as
de Heidegger e Merleau-Ponty, eram más notícias para aqueles trabalhando nos laboratórios de
inteligência artificial.” (DREYFUS, 2007, p. 247). Dito de outro modo, a mente, na descrição
enativista, significa o looping ou o feedback contínuo da cadeia ininterrupta de ações do organismo
em resposta às solicitações do contexto. .A forma [Gestalt] é uma configuração visual, sonora, ou mesmo anterior à distinção dos
sentidos, em que o valor sensorial de cada elemento é determinado por sua função no
conjunto e varia com ela. g
pp
p
7 O valor do cognitivismo, no entanto, não é questionado por Varela et al. Como dito anteriormente, o enativismo não descarta o
cognitivismo, pois o último é mais um passo no avanço da compreensão da cognição e, acima de tudo, é o modelo que não só GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
15 GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
15
cognitivismo, pois o último é mais um passo no avanço da compreensão da cognição e, acima de tudo, é o modelo que não só GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
12 22
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15
g
, p
p
ç
p
g
ç
,
,
q f
g (
)
J. J., The Ecological Approach to Visual Perception (1986). Natural History of Human Thinking (2014).
6 Cf. GIBSON, J. J., The Ecological Approach to Visual Perception (1986). ABSTRACT: Tais solicitações ou affordances6 se dão por meio de “três modos permanentes e
entrelaçados de atividade corporal - auto-regulação, acoplamento sensório motor e interação
intersubjetiva.” (THOMPSON, 2007, p. 243). O entrelaçamento de tais modos - indicados pelo
enativismo como exigências para o desvelamento ou enactment significativo não-representacional do
meio - não são contemplados pela perspectiva cognitivista, pois nela o funcionamento algorítmico
computacional serve como a base explicativa da cognição, fazendo com que a interface organismo-
meio ou sistema-meio seja essencialmente a reprodução de modelos internos: “O típico modelo
cognitivista toma a forma de um programa para a solução de um problema em determinado
domínio.” (THOMPSON, 2007, p. 5). Dessa forma, o foco cognitivista reside na formalização
proposicional da inteligência. No entanto, tal rigidez formalista dos modelos cognitivistas é
exatamente o empecilho na sua validade explicativa quando lidamos com o tipo de variação
organizacional observada no cérebro7. Com o avanço da neurociência, tornou-se cada vez mais 15 15 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 ISSN 2178-1036 evidente o fato de que algoritmos sequenciais talvez não sejam o melhor modelo para compreender
o cérebro e é aqui que entra o modelo conexionista, o qual, segundo Varela et al., representa um
avanço no entendimento da cognição pelo caráter emergentista de sua explicação mas, como
veremos, ainda compartilha com o cognitivismo o erro de não estabelecer um entrelaçamento não-
representacional entre organismo e ambiente. De qualquer maneira, para encerrarmos a exposição
do paradigma cognitivista, vejamos a afirmação de Varela et al. que, assim como em Dreyfus, nos
lembra que “sem se darem conta, os pesquisadores de inteligência artificial estavam realmente
empenhando-se em fazer da filosofia racionalista um programa de pesquisa.” (DREYFUS, 2007, p. 247), ou seja, o modelo cognitivista não é estranho à história da filosofia. De certo modo, o cognitivismo é, até agora, a mais forte defesa da visão representacional da
mente inaugurada por Descartes e Locke. De fato, Jerry Fodor, um dos mais importantes e
eloquentes expoentes do cognitivismo, chega a afirmar que o único aspecto no qual o
cognitivismo
pode
ser
tomado
como
um
verdadeiro
avanço
em
relação
ao
representacionismo dos séculos XVIII e XIX é o seu uso do computador como um modelo
da mente. (VARELA et al., 1991, p. possibilitou a construção das primeiras e, diga-se de passagem, bem sucedidas inteligências artificiais (tradução de texto, xadrez,
etc.), como também possui direta influência em empreitadas contemporâneas como o desenvolvimento de carros auto-dirigíveis.
8 “O conexionismo surgiu no início da década de 1980, revisitando e revitalizando as ideias da era pré-cognitivista da cibernética.”
(THOMPSON, 2007, p. 8). GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
16 GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
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16 ABSTRACT: 138) Com isso, podemos agora nos debruçar sobre o conexionismo para entender em que sentido o
enativismo difere da abordagem que sucedeu o paradigma cognitivista. III Dessa forma, torna-se necessário uma
descrição emergente e não-rígida da base material que subjaz o processo cognitivo, ou seja, a
constante reorganização das unidades neuronais apontam para a antiga lição da Gestalttheorie: o
todo é mais do que a soma de suas partes, pois não só o todo condiciona suas partes, mas também as
partes determinam o todo, indicando o caráter circular da causalidade da esfera orgânica9. Uma descrição simbólica estágio-por-estágio para um sistema com este tipo de constituição
parece ir contra a direção natural. Além disso, tornou-se cada vez mais claro para os
neurocientistas que precisamos estudar os neurônios como membros de vastos conjuntos que
estão constantemente a desaparecer e a surgir através das suas interações cooperativas e em
que cada neurônio tem respostas múltiplas e que se alteram de um modo dependente do
contexto [...] O cérebro é portanto um sistema altamente cooperativo: as densas
interligações entre os seus componentes implicam que quase tudo o que decorrer será uma
função de tudo aquilo que os componentes se encontram a fazer. (VARELA et al., 1991, pp. 131-132). Além disso, não só determinados contextos servem para a mudança de padrões
organizacionais, como a historicidade combinada de ações e contextos deve ser levada em conta: “se
dois neurônios tendem a ser ativos em conjunto, a sua ligação é fortalecida; caso contrário, é
diminuída. Consequentemente, a conexidade do sistema torna-se inseparável da sua história de
transformação.” (VARELA et al., 1991, p. 124). Em outras palavras, a interligação neuronal não é
um sistema acabado que opera via regras fixas, mas sim um contínuo processo de auto-organização
proveniente do histórico de ações contextuais do organismo. Com isso, o objetivo do paradigma
conexionista é “construir um sistema cognitivo não a partir de símbolos e regras, mas a partir de
componentes simples que se ligariam dinamicamente uns aos outros.” (VARELA et al., 1991, p. 125). A visão é útil para indicar os limites do cognitivista que defende que ver significa um
processamento sequencial de inputs: “Olhar para os circuitos visuais como constituindo um
processador sequencial parece inteiramente arbitrário [...] o comportamento de todo o sistema
parece-se muito mais com uma conversa em uma festa do que com uma cadeia de comando.”
(VARELA et al., 1991, p. 134). III III Varela et al. definem o conexionismo como “a controversa abordagem à cognição na qual as
qualidades auto-organizantes dos agregados biológicos desempenham um papel central.” (1991, p. 118). Se a metáfora principal do paradigma cognitivista era a da mente como um computador, “a
metáfora central do conexionismo é a mente como uma rede neural” (THOMPSON, 2007, p. 9). O
conexionista argumenta que qualquer teorização sobre a cognição deve partir do pressuposto onde
se afirma que “propriedades emergentes são fundamentais na operação do próprio cérebro.”
(VARELA et al., 1991, p. 93). Ideias sobre o caráter emergente dos padrões neuronais já existiam
nos primórdios das ciências cognitivas8 e, durante as Macy Conferences (1946-1953), já havia uma
“extensa discussão sobre o fato de nos cérebros reais parecer não existir qualquer tipo de regras,
nenhum processador lógico central, nem a informação parece encontrar-se armazenada em
endereços precisos.” (VARELA et al., 1991, p. 121). Em suma, o paradigma conexionista surge no
intuito de romper com o uso de modelos fixos de representação interna do mundo: “os cérebros
podem funcionar à base de interligações em massa e de uma forma distribuída, de tal modo que as
ligações reais entre conjuntos de neurônios se alteram em função da experiência.” (VARELA et al.,
1991, p. 131). Por exemplo, pesquisas acerca da alteração neuronal no córtex visual de animais
apontam que “as respostas neuronais estereotipadas se tornaram altamente sensíveis ao contexto
[...] Mesmo uma alteração de postura, embora preservando o mesmo idêntico estímulo sensorial,
altera as respostas neuronais no córtex visual primário.” (VARELA et al., 1991, p. 131). Com isso, o
comportamento global que emerge das unidades simples (no caso, os neurônios) evidenciam uma 16 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 ISSN 2178-1036 organização estreitamente ligada ao contexto. Em suma, o conexionismo surge como a defesa de
uma “capacidade auto-organizativa que não existe no paradigma da manipulação de símbolos.”
(VARELA et al., 1991, p. 122). No cognitivismo, “o processamento da informação simbólica é baseado em regras
sequenciais, aplicadas uma de cada vez.” (VARELA et al., 1991, p. 122), levando-nos a modelos que
não condizem com a investigação empírica do cérebro. 9 Vale ressaltar o elogio de Varela et al. aos modelos emergentistas na explicação de diferentes esferas da natureza, fazendo com que
este tipo de solução seja útil não só na descrição de sistemas neuronais como em outros arranjos da matéria: “parece tornar-se difícil
para qualquer agregado densamente ligado poder escapar às propriedades emergentes; deste modo, as teorias de tais propriedades
constituem uma ligação natural para níveis de descrições diferentes em termos de fenômenos naturais e cognitivos”. (VARELA et
al., 1991, p. 129). GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
17 GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
17 10 Como também por Freud (THOMPSON, 2007). GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
18 III Logo, o argumento de que cada unidade de um sistema cognitivo
possuiria uma sintaxe pré-estabelecida (como no caso de computadores) perde sua fundamentação,
pois “um neurônio individual participa em muitos desses padrões globais e tem pouco significado
quando tomado individualmente” (VARELA et al., 1991, p. 134), ou seja, uma tarefa cognitiva
(e.g., reconhecimento visual de um objeto) deve ser descrita como a emergência de uma Gestalt ou
organização global de conexões neuronais onde a significação funcional das unidades simples 17 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 ISSN 2178-1036 variam de acordo com o contexto. Na abordagem conexionista, “o significado não está localizado
em símbolos particulares; é uma função do estado global do sistema.” (VARELA et al., 1991, p. 139, grifo nosso). Portanto, a função de cada unidade neuronal provém do arranjo situacional. Em situações em que o universo de itens possíveis a ser representado é constrangido e de
fácil definição (quando, por exemplo, um computador é programado, ou quando uma
experiência é conduzida com base num conjunto de estímulos visuais predefinidos), a
especificação de significado é clara. Cada item físico ou funcional discreto é feito para
corresponder a um item externo (o seu significado referencial), uma operação de
correspondência que o observador facilmente executa. Removam-se estes constrangimento e
a forma dos símbolos é tudo o que resta, e o significado transforma-se num fantasma, como
aconteceria no caso de termos que contemplar os padrões de bits num computador cujo
manual de instruções se tivesse perdido. (VARELA et al., 1991, pp. 138-139). Muito do conexionismo está contido na visão enativista. Varela et al. (1991) reconhecem
tanto o valor do cognitivismo, como o avanço do conexionismo em relação ao último. O enativismo,
portanto, não significa um descarte do conhecimento acumulado, mas uma tentativa de refinar a
ciência cognitiva no intuito de auxiliar na construção de uma descrição mais precisa do ser-no-
mundo do Dasein. Um modo inclusivo ou misto parece, então, uma estratégia natural a ser seguida. Uma
ligação frutuosa entre um cognitivismo menos ortodoxo e a visão da emergência, onde as
regularidades simbólicas emergem de processos paralelos distribuídos, é uma possibilidade
concreta,
especialmente
na
IA,
com
a
sua
orientação
pragmática,
voltada
predominantemente para a engenharia. (VARELA et al., 1991, pp. 142-143). Dessa forma, vejamos agora o caráter da dinâmica corporal não-representacional que o
enativismo propõe como elemento crucial para a compreensão da cognição. GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
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18 IV No entanto, o segundo ponto - sistemas onde os inputs e outputs são designados
previamente por um designer - continua a ser o resquício representacionista que impede, segundo a
concepção enativista, uma verdadeira compreensão da cognição e indicam o parentesco entre
cognitivismo e conexionismo que o enativismo pretende superar. implausibilidade neurológica do modelo de sistema físico-simbólico e em diversas deficiências
percebidas no processamento de símbolos em comparação com as redes neurais.” (THOMPSON,
2007, p. 8). Um processo cognitivo em um modelo conexionista, portanto, “corresponde a padrões
emergentes de atividades na rede.” (THOMPSON, 2007, p. 9). Tais padrões possuem um caráter
não só de historicidade (aprendizagem do sistema) como ambiental (constante reatualização do
sentido dos dados exteriores), significando um importante passo para além do restrito e limitante
modelo cognitivista: “Enquanto que o cognitivismo firmemente alojava a mente dentro da cabeça,
o conexionismo ofereceu uma concepção mais dinâmica da relação entre processos cognitivos e o
ambiente.” (THOMPSON, 2007, p. 9). Porém, o problema do conexionismo foi não ter ido longe o
suficiente: “Apesar desses avanços, os sistemas conexionistas não envolviam nenhum acoplamento
sensório-motor com o ambiente, e, em vez disso, operavam na base de inputs e outputs (inicialmente
designados pelo programador do sistema).” (THOMPSON, 2007, p. 9). O primeiro ponto - a
ausência de acoplamento sensório-motor - é um problema óbvio que, hoje em dia, não é mais
ignorado pelos cientistas cognitivos. Há um consenso paradigmático hoje que ecoa a fórmula de
Merleau-Ponty: “todo hábito é ao mesmo tempo motor e perceptivo.” (MERLEAU-PONTY, 1999,
p. 210). No entanto, o segundo ponto - sistemas onde os inputs e outputs são designados
previamente por um designer - continua a ser o resquício representacionista que impede, segundo a
concepção enativista, uma verdadeira compreensão da cognição e indicam o parentesco entre
cognitivismo e conexionismo que o enativismo pretende superar. O conexionismo também herdou do cognitivismo a ideia de que a cognição é basicamente a
resolução de problemas pré-definidos (colocados no sistema por um observador ou designer
exterior) e que a mente é essencialmente o inconsciente cognitivo limitado ao crânio, o
domínio sub-pessoal de representação computacional na mente-cérebro [mind-brain]. O
desacordo do conexionismo com o cognitivismo era sobre a natureza da computação e da
representação
(simbólica
para
cognitivistas,
sub-simbólica
para
conexionistas). (THOMPSON, 2007, pp. GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
19 IV O enativismo significa tomar a corporeidade como central na compreensão da mente. O
aspecto inconsciente ou subpessoal destacado pelo cognitivismo10 não é negado, mas sim associado à
totalidade de um sistema autopoiético, isto é, de uma corporeidade dotada de intencionalidade. Para o cognitivista, a cognição “acontece em um módulo cognitivo central do cérebro separado dos
aspectos perceptivos, emocionais e sensório motores. O inconsciente cognitivo não é nem somático,
nem afetivo, e está firmemente alojado dentro da cabeça.” (THOMPSON, 2007, p. 6). Daí o
resquício behaviorista criticado por Thompson sobre a abordagem cognitivista: não só a dinâmica
subpessoal acontece apartada da corporeidade, como o processo cognitivo é privado dos aspectos
emocionais e fenomenológicos da experiência subjetiva: “Em suma, o cognitivismo não oferece
explicação alguma sobre a mentalidade em termos de experiência subjetiva.” (THOMPSON, 2007,
p. 6). Essa lacuna da teoria cognitivista é evidenciada tanto pelo avanço das neurociências, como
pelas ideias conexionistas, as quais apontam que os padrões emergentes dos arranjos neuronais não
são compreensíveis em termos computacionais ou sequenciais: “a crítica conexionista focou-se na 18 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 ISSN 2178-1036 ISSN 2178-1036 implausibilidade neurológica do modelo de sistema físico-simbólico e em diversas deficiências
percebidas no processamento de símbolos em comparação com as redes neurais.” (THOMPSON,
2007, p. 8). Um processo cognitivo em um modelo conexionista, portanto, “corresponde a padrões
emergentes de atividades na rede.” (THOMPSON, 2007, p. 9). Tais padrões possuem um caráter
não só de historicidade (aprendizagem do sistema) como ambiental (constante reatualização do
sentido dos dados exteriores), significando um importante passo para além do restrito e limitante
modelo cognitivista: “Enquanto que o cognitivismo firmemente alojava a mente dentro da cabeça,
o conexionismo ofereceu uma concepção mais dinâmica da relação entre processos cognitivos e o
ambiente.” (THOMPSON, 2007, p. 9). Porém, o problema do conexionismo foi não ter ido longe o
suficiente: “Apesar desses avanços, os sistemas conexionistas não envolviam nenhum acoplamento
sensório-motor com o ambiente, e, em vez disso, operavam na base de inputs e outputs (inicialmente
designados pelo programador do sistema).” (THOMPSON, 2007, p. 9). O primeiro ponto - a
ausência de acoplamento sensório-motor - é um problema óbvio que, hoje em dia, não é mais
ignorado pelos cientistas cognitivos. Há um consenso paradigmático hoje que ecoa a fórmula de
Merleau-Ponty: “todo hábito é ao mesmo tempo motor e perceptivo.” (MERLEAU-PONTY, 1999,
p. 210). GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
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b
2019
19 IV 9-10) Portanto, o enativismo se afasta das abordagens iniciais das ciências cognitivas pela sua
defesa da não-necessidade de representações para explicar o processo de apreensão do meio pelo
organismo. O uso de representações implica em uma visão onde organismo e ambiente são esferas
independentes de uma relação na qual o primeiro reduz o segundo a símbolos (sejam eles fixos e
unitários ou emergentes e variáveis) que definem sequências para a efetivação de uma ação. Em
suma, “seus modelos [do cognitivismo e do conexionismo] eram descorporificados e abstratos.”
(THOMPSON, 2007, p. 10), ao contrário do enativismo, o qual argumenta que “processos
cognitivos emergem da causalidade circular e não-linear das ininterruptas interações envolvendo o
cérebro, o corpo e o ambiente.” (THOMPSON, 2007, p. 11). Em outras palavras, o enativista
argumenta que a melhor imagem para a mente humana não é nem a do computador digital, nem a
da rede conexionista emergente, mas sim a de um sistema dinâmico corporificado no mundo. Além da intransigente defesa da mente como corporificada e situada, a outra principal
característica do enativismo é a adoção da matemática descritiva da teoria de sistemas dinâmicos
(PORT & VAN GELDER, 1995; VAN GELDER, 1998; THOMPSON, 2007): “a cognição é um
fenômeno intrinsecamente temporal e, dessa forma, deve ser entendida da perspectiva da teoria de
sistemas dinâmicos.” (THOMPSON, 2007, p. 11). Grosso modo, a teoria de sistemas dinâmicos é
uma teoria matemática que usa equações diferenciais que, por conta da dinamicidade dos sistemas
estudados, não tentam estabelecer soluções exatas para o comportamento do sistema, mas sim 19 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 ISSN 2178-1036 apenas projeções de médio e longo prazo. Especificamente sobre seu uso nas ciências cognitivas, o
que interessa ao enativismo é que a perspectiva da teoria de sistemas dinâmicos serve para
construir uma imagem mais próxima do que acontece de fato em sistemas autopoiéticos, isto é, os
dados exteriores ao organismo não são representações internalizadas, mas perturbações que levam o
sistema à procura de ações que mantenham seu equilíbrio vital, isto é, em vez de regras a serem
obedecidas, o enativismo usa a teoria dinâmica para compreender os inputs como perturbações a
serem ultrapassadas por meio da combinação entre errância e historicidade comportamental, ou
seja, o caminho surge pelo caminhar. O dinamismo corporificado oferece uma perspectiva diferente do inconsciente cognitivo
computacional. Não mais o inconsciente cognitivo é visto como uma manipulação de
símbolos ou um reconhecimento de padrões descorporificado separado da ação motora e
emocional no mundo. (THOMPSON, 2007, p. 12). GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
20 GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p 12 22 outubro 2019
20 IV Em vez de instruções a serem seguidas, os inputs são descritos como perturbações à
dinâmica intrínseca do sistema; e os estados internos são compreendidos como compensações
auto-organizadas desencadeadas por perturbações e não como representações do estado de
coisas externas.” (THOMPSON, 2007, p. 11). A citação anterior é crucial para compreender a mente como corporificada, pois ao negar o
aspecto representacional da cognição e compreender os inputs como perturbações a um equilíbrio
vital, o enativismo fica livre para descrever a cognição como uma apreensão corporal criativa, isto
é, um “hábil saber-como” [skillful know-how]. Com isso, a dinâmica de apreensão cognitiva do
organismo pode ser descrita a partir da retroatividade entre percepção e ação. Os processos e estruturas cognitivas emergem de padrões sensório-motores recorrentes que
governam a percepção e a ação em agentes autônomos e situados. A cognição como um hábil
saber-como [skilfull know-how] não é redutível à resolução de problemas pré-especificados,
pois o sistema cognitivo não só impõe os problemas como especifica quais ações devem ser
tomadas para sua solução. (THOMPSON, 2007, p. 11). Dessa forma, os processos subpessoais descritos pelo cognitivismo não são negados, mas
reformulados e atrelados à corporeidade. Enquanto que o inconsciente cognitivista é formulado
como um processo descorporificado de manipulação de representações (passível de transposição
para diferentes sistemas), o inconsciente enativista é o processo emocional e sensório-motor de
apreensão direta do meio via corporeidade, inacessível ao eu-representacional. É aqui, então, que a
influência de Merleau-Ponty fica ainda mais explícita, pois a Fenomenologia da Percepção (1945)
tem como intuito descrever exatamente este inconsciente enativista. Quando Merleau-Ponty afirma
que o “corpo é nosso meio geral de ter um mundo” (MERLEAU-PONTY, 1999, p. 203), o
fenomenólogo francês quer, no fundo, definir a motricidade, isto é, a esfera sensório motora, como a
esfera primordial de engendramento significativo: “O que descobrimos pelo estudo da motricidade
é, em suma, um novo sentido da palavra ‘sentido’” (MERLEAU-PONTY, 1999, p. 203). Portanto,
o inconsciente enativista é, como Merleau-Ponty descreveu, o modo originário de apreensão pré-
reflexiva e não-representacional da corporeidade que faz surgir um meio que, posteriormente, pode
vir então a ser representado. O dinamismo corporificado oferece uma perspectiva diferente do inconsciente cognitivo
computacional. Não mais o inconsciente cognitivo é visto como uma manipulação de
símbolos ou um reconhecimento de padrões descorporificado separado da ação motora e
emocional no mundo. (THOMPSON, 2007, p. 12). O dinamismo corporificado oferece uma perspectiva diferente do inconsciente cognitivo
computacional. IV Não mais o inconsciente cognitivo é visto como uma manipulação de
símbolos ou um reconhecimento de padrões descorporificado separado da ação motora e
emocional no mundo. (THOMPSON, 2007, p. 12). 20 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 ISSN 2178-1036 ISSN 2178-1036 Dessa forma, assim como Merleau-Ponty assume parte da teoria psicanalítica em sua obra11,
também o enativismo assume que “muito do que somos como seres psicológicos e biológicos é, em
certo sentido, inconsciente. Logo, a subjetividade não pode ser entendida sem situá-la em relação à
tais estruturas e processos inconscientes.” (THOMPSON, 2007, p. 12). Lembrando que, assim como
Merleau-Ponty defende a noção de intencionalidade operante para indicar o aspecto anônimo ou
subpessoal do corpo, também Thompson ressalta que, no enativismo, os processos inconscientes não
estão alojados em uma parte específica do corpo, mas sim na dinâmica corpo-ação-ambiente. Portanto, esses processos e estruturas inconscientes, incluindo aqueles descritos como
cognitivos e emocionais, se estendem pelo corpo e atuam em loop pelos ambientes materiais,
sociais e culturais nos quais o corpo está situado; eles não estão limitados a processos neurais
dentro do crânio. (THOMPSON, 2007, p. 12) Dessa forma, para concluirmos, a abordagem enativista pode ser resumida em cinco pontos
(THOMPSON, 2007): primeiro, organismos vivos são corporeidades autônomas que se
autoproduzem (autopoiesis) e, com isso, “fazem surgir seus próprios domínios cognitivos.”
(THOMPSON, 2007, p. 13); em segundo lugar, “O sistema nervoso não processa informação no
sentido computacional, e sim cria sentido.” (THOMPSON, 2007, p. 13); terceiro, o processo de
cognição é “o exercício de um saber-como hábil [skillful know-how] em ações situadas e
corporificadas.” (THOMPSON, 2007, p. 13); em quarto lugar, pelo fato da corporeidade do
organismo não processar informação de modo proposicional, o domínio cognitivo “não é uma esfera
externa, pré-especificada e representada internamente, mas sim um domínio relacional”
(THOMPSON, 2007, p. 13); finalmente, o enativismo não vê a experiência fenomênica ou subjetiva
como um epifenômeno secundário na explicação da mente, mas como um tema central a ser
investigado aos moldes da tradição fenomenológica husserliana em consonância com a ciência
contemporânea, ou seja, o paradigma enativista deve ser entendido como um esforço de
naturalização da fenomenologia: “Para o sucesso deste projeto, devemos fazer uso da biologia, da
neurociência, da psicologia, da filosofia e da fenomenologia. IV [...] Uma vez que a ciência voltar sua
atenção à subjetividade e à consciência, à experiência vivida, então ela não poderá seguir sem o
auxílio da fenomenologia.” (THOMPSON, 2007, p. 14). 11 É conhecido o momento da Fenomenologia da Percepção no qual Merleau-Ponty faz sua análise da patologia do membro fantasma e
se utiliza das contribuições teóricas tanto de Heidegger quanto de Freud. GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
21 GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
12 22
b
2019
21 Considerações finais Para além da exigência de modelos internos como pressuposto do processo cognitivo, o
enativismo propõe a imbricação não-representacional do organismo-ambiente. Não há um mundo-
objetivo-representável fora do organismo que seria internalizado via unidades simbólicas fixas, mas
sim uma atividade contínua e não-proposicional onde a historicidade das ações fazem um mundo de
solicitações vir à tona. Em suma, o enativismo significa que “o animal projeta ele mesmo as normas
de seu meio e coloca ele mesmo os termos de seu problema vital.” (MERLEAU-PONTY, 1999, p. 117). 21 ISSN 2178-1036 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 Griot : Revista de Filosofia, Amargosa - BA, v.19, n.3, p.12-22, outubro, 2019 Autor(a) para correspondência: Rodrigo Benevides Barbosa Gomes, Departamento de Pós-Graduação em Filosofia da
Universidade Federal de São Carlos, Rodovia Washington Luis, km 235, 13566-905, São Carlos – SP, Brasil.
rodrigobenevides23@gmail.com GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
p.12-22, outubro, 2019
22 GOMES, Rodrigo Benevides Barbosa. Enativismo e conhecimento prático. Griot : Revista de Filosofia, Amargosa – BA, v.19, n.3,
22 Referências BARBARAS, Renaud. Francisco Varela: A new idea of perception and life. Phenomenology and the
Cognitive Sciences n. 1: pp. 127-132, 2002. ,
f p
p
f
gy
Cognitive Sciences n. 1: pp. 127-132, 2002. CARMAN, T. The Cambridge Companion to Merleau-Ponty. London: Cambridge University Press,
2005. CARMAN, T. The Cambridge Companion to Merleau-Ponty. London: Cambridge University Press,
2005. CLARK, Andy. Whatever next? Predictive brains, situated agents, and the future of cognitive science. Behavioral and Brain Sciences, 2013. DREYFUS, H. L. Being-in-the-World: A Commentary on Heidegger’s Being and Time, Division I. Cambridge: MIT Press, 1991. DREYFUS, H. L. What Computers Still Can't Do: A critique of Artificial Reason. Cambridge: MIT
Press, 1992. DREYFUS, H. L. Why Heideggerian Artificial Intelligence failed and how fixing it would require
making it more Heideggerian. In: Philosophical Psychology. Londres, 20:2, 2007, p.247-268. HEIDEGGER, M. Being and Time. Translated by John Macquarrie & Edward Robinson. London:
Blackwell, 1962. MERLEAU-PONTY, M. A Estrutura do Comportamento. Tradução de M. V. M. Aguiar. São Paulo:
Martins Fontes, 2006. MERLEAU-PONTY, M. Fenomenologia da Percepção. Tradução de C. A. Ribeiro de Moura. São
Paulo: Martins Fontes, 1999. MERLEAU-PONTY, M. The Structure of Behavior. Boston: Beacon Press, 1963. MOUTINHO, L. D. S. Razão e experiência: ensaio sobre Merleau-Ponty. São Paulo: Ed. Unesp. 2006. SACRINI, M. A. F. Fenomenologia e Ontologia em Merleau-Ponty. Tese de Doutorado em Filosofia -
Faculdade de Filosofia, Letras e Ciências Humanas, USP. São Paulo, 2008. THOMPSON, E. Mind
in Life: Biology, Phenomenology and the Sciences of Mind. Cambridge: Harvard University Press,
2007. VARELA, F.; MATURANA, H. A Árvore do Conhecimento: as bases biológicas da compreensão
humana. São Paulo: Ed. Palas Athena, 2001. VARELA, F., THOMPSON, E. & ROSCHE, E. The Embodied Mind: Cognitive Science and Human
Experience. Massachusetts: MIT Press, 1991. VARELA, F.; WEBER, A. Life after Kant: Natural purposes and the autopoietic foundations of
biological individuality. Phenomenology and the Cognitive Sciences, N. 1: pp. 97-125, 2002 22
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Successful treatment of ciliary body medulloepithelioma with intraocular melphalan chemotherapy: a case report
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BMC ophthalmology
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Successful treatment of ciliary body
medulloepithelioma with intraocular
melphalan chemotherapy: a case report Christina Stathopoulos*, Marie-Claire Gaillard, Julie Schneider and Francis L. Munier Stathopoulos et al. BMC Ophthalmology (2020) 20:239
https://doi.org/10.1186/s12886-020-01512-y Stathopoulos et al. BMC Ophthalmology (2020) 20:239
https://doi.org/10.1186/s12886-020-01512-y (2020) 20:239 * Correspondence: christina.stathopoulos@fa2.ch
Department of Ophthalmology, Jules-Gonin Eye Hospital, Fondation Asile
des Aveugles, University of Lausanne, Avenue de France 15, 1000 Lausanne
7, Vaud, Switzerland Abstract Background: Intraocular medulloepithelioma is commonly treated with primary enucleation. Conservative
treatment options include brachytherapy, local resection and/or cryotherapy in selected cases. We report for the
first time the use of targeted chemotherapy to treat a ciliary body medulloepithelioma with aqueous and vitreous
seeding. Case presentation: A 17-month-old boy with a diagnosis of ciliary body medulloepithelioma with concomitant
seeding and neovascular glaucoma in the right eye was seen for a second opinion after parental refusal of
enucleation. Examination under anesthesia showed multiple free-floating cysts in the pupillary area associated with
iris neovascularization and a subluxated and notched lens. Ultrasound biomicroscopy revealed a partially cystic
mass adjacent to the ciliary body between the 5 and 9 o’clock meridians as well as multiple nodules in the
posterior chamber invading the anterior vitreous inferiorly. Fluorescein angiography demonstrated peripheral retinal
ischemia. Left eye was unremarkable. Diagnosis of intraocular medulloepithelioma with no extraocular invasion was
confirmed and conservative treatment initiated with combined intracameral and intravitreal melphalan injections
given according to the previously described safety-enhanced technique. Ciliary tumor and seeding totally regressed
after a total of 3 combined intracameral (total dose 8.1 μg) and intravitreal (total dose 70 μg) melphalan injections
given every 7–10 days. Ischemic retina was treated with cryoablation as necessary. Three years later, ab interno
trabeculotomy followed by 360° gonioscopy-assisted transluminal trabeculotomy 6 months later was performed for
uncontrolled intraocular pressure despite antihypertensive drugs combined to cyclophotocoagulation and 7
intravitreal anti-VEGF injections for recurrent iris neovascularization. Cataract was removed at the same operative
time. The child has remained disease- and metastasis-free at a 5-year follow-up since the last melphalan injection
(25-month follow-up after the combined lensectomy-trabeculotomy) with a controlled intraocular pressure under
topical quadritherapy and a best corrected Snellen visual acuity of 0.08. Conclusions: We report for the first time complete regression of a non-infiltrating ciliary body medulloepithelioma
with seeding achieved with only a small number of intracameral and intravitreal melphalan injections. Concomitant
secondary neovascular glaucoma and cataract needed appropriate management to allow long-term eye and vision
preservation. Keywords: Intraocular medulloepithelioma, Melphalan, Intracameral injection, Intravitreal injection * Correspondence: christina.stathopoulos@fa2.ch
Department of Ophthalmology, Jules-Gonin Eye Hospital, Fondation Asile
des Aveugles, University of Lausanne, Avenue de France 15, 1000 Lausanne
7, Vaud, Switzerland Background child was referred to a specialized oncology centre for
further investigations. Diagnosis of intraocular medulloe-
pithelioma with anterior chamber seeding and neovascu-
lar glaucoma was made and immediate enucleation
advised. Parents refused the intervention and consulted
us for a second opinion. Examination under general
anesthesia showed multiple free-floating cysts in the
pupillary area of the right eye with iris neovasculariza-
tion and a notched lens that was subluxated superona-
sally (Fig. 2a). Intraocular pressure was controlled (17
mmHg) under topical beta-blocker and oral acetazol-
amide. Fundus examination was normal. Fluorescein
angiography revealed anterior segment leakage with in-
ferior peripheral retinal ischemia. Ultrasound biomicro-
scopy
performed
on
the
12
meridians
showed
a
complete iridocorneal angle closure and an irregular-
surfaced, partially cystic mass adjacent to the ciliary
body between the 5 and 9 o’clock meridians. Multiple
nodules were invading the posterior chamber and anter-
ior vitreous invasion was also seen inferiorly (Fig. 2b). Left eye examination was unremarkable. Clinical appear-
ance and ultrasonographic features confirmed the diag-
nosis of ciliary body medulloepithelioma. There being
no signs of extraocular extension nor ciliary infiltration,
conservative treatment was initiated. After obtaining
parents’
informed
consent,
combined
intracameral
(2.25 μg/0.15 ml) and intravitreal melphalan (30 μg/0.15
ml) injections given according to a previously described
safety-enhanced technique to avoid potential extraocular
tumor spread [7, 8]. Injected doses were similar to the
ones used in the treatment of aqueous and vitreous
seeding in retinoblastoma. Cytopathological analysis of
the aqueous tap performed prior to injection showed
small aggregates of malignant cells with high nuclear
cytoplasmic ratio that were negative for synaptophysin
on immunocytochemistry (Fig. 2c). Partial regression of
both ciliary mass and seeding was observed 1 week later,
encouraging treatment to be repeated. In total, 3 com-
bined intracameral (total dose 8.1 μg) and intravitreal
(total dose 70 μg) melphalan injections were given every
7–10 days, resulting in complete response of the main
tumor
(documented
on
UBM
after
2
combined Intraocular medulloepithelioma is a rare, nonhereditary,
neuroepithelial tumor, usually arising from the nonpig-
mented ciliary epithelium of the ciliary body, more rarely
the optic nerve, the retina or the iris [1]. Its exact inci-
dence is not known. Almost all cases are unilateral, with
no gender or racial predilection. Diagnosis is made
within the first decade of life in about 80% of the cases
[2]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 5 Page 2 of 5 Stathopoulos et al. BMC Ophthalmology (2020) 20:239 Stathopoulos et al. BMC Ophthalmology (2020) 20:239 Background Most patients present with loss of vision, pain, leu-
kocoria or a mass appearing in the anterior chamber [1,
3]. In 5% of the cases, a systemic association with pleur-
opulmonary blastoma caused by heterozygous DICER1
mutations has been found [1]. Histopathologically, intra-
ocular medulloepithelioma can be classified into nonter-
atoid and teratoid types and both can be benign or
malignant. Even in the malignant form, these slowly
growing tumors do not tend, however, to metastatize,
unless extraocular extension occurs [1–3]. Characteristic features of ciliary body medulloepithe-
lioma include a grey-white to fleshy pink mass with a
various quantity of cysts adjacent to the ciliary body, a
lens notching with or without subluxation and a neo-
plastic cyclitic membrane [3, 4]. The cysts within the
mass may break away and float freely into the aqueous
or the vitreous cavity. Coexistence of persistent hyper-
plastic primary vitreous has been reported in 20% of the
cases [3, 4]. Around 50% of the cases are complicated
with glaucoma and/or cataract [1–3]. Treatment options
include enucleation [1–4], plaque brachytherapy [1, 2,
5], surgical resection [2, 4, 6] and cryotherapy [2, 4]. Here we report for the first time a case of intraocular
medulloepithelioma with seeding at presentation suc-
cessfully treated with intracameral and intravitreal mel-
phalan chemotherapy. Discussion and conclusions injections) and aqueous/vitreous seeding (observed after 3
combined injections) (Fig. 2d-e). One week after the final
injections, dense anterior chamber hyphema related to
persistent iris neovascularization occurred. After careful
anterior chamber washout under melphalan perfusion
(15 μg/ml), iris neovascularization was managed with
cryoablation of the peripheral retinal ischemia and intra-
vitreal
anti-vascular
endothelial
growth
factor
(anti-
VEGF). Three years later, the patient underwent ab
interno trabeculotomy followed by a 360° gonioscopy-
assisted transluminal trabeculotomy 6 months later to
manage uncontrolled intraocular pressure despite antihy-
pertensive drugs combined with cyclophotocoagulation
and 7 intravitreal anti-VEGF injections for recurrent iris
neovascularization. Cataract surgery with posterior capsu-
lorhexis, anterior vitrectomy and sulcus implantation of a
three-piece intraocular lens was performed during the first
glaucoma surgery. Cytopathologic analysis of the vitrec-
tomy fluid was negative for tumor cells. One year after the
cataract was removed, YAG-laser capsulotomy was per-
formed for posterior capsular lens opacification. The child
has remained disease and metastasis-free at a 5-year
follow-up since the last chemotherapy with a controlled
intraocular pressure under topical quadritherapy and a
best-corrected Snellen visual acuity of 0.08. injections) and aqueous/vitreous seeding (observed after 3
combined injections) (Fig. 2d-e). One week after the final
injections, dense anterior chamber hyphema related to
persistent iris neovascularization occurred. After careful
anterior chamber washout under melphalan perfusion
(15 μg/ml), iris neovascularization was managed with
cryoablation of the peripheral retinal ischemia and intra-
vitreal
anti-vascular
endothelial
growth
factor
(anti-
VEGF). Three years later, the patient underwent ab
interno trabeculotomy followed by a 360° gonioscopy-
assisted transluminal trabeculotomy 6 months later to
manage uncontrolled intraocular pressure despite antihy-
pertensive drugs combined with cyclophotocoagulation
and 7 intravitreal anti-VEGF injections for recurrent iris
neovascularization. Cataract surgery with posterior capsu-
lorhexis, anterior vitrectomy and sulcus implantation of a
three-piece intraocular lens was performed during the first
glaucoma surgery. Cytopathologic analysis of the vitrec-
tomy fluid was negative for tumor cells. One year after the
cataract was removed, YAG-laser capsulotomy was per-
formed for posterior capsular lens opacification. The child
has remained disease and metastasis-free at a 5-year
follow-up since the last chemotherapy with a controlled
intraocular pressure under topical quadritherapy and a
best-corrected Snellen visual acuity of 0.08. Intraocular medulloepithelioma is a rare tumor of un-
known incidence but still represents the most frequent
pediatric primary malignant intraocular tumor after ret-
inoblastoma [9]. Standards of care do not exist. Case presentation A previously healthy 17-month-old boy was given topical
antibiotics for a right eye redness appearing in the con-
text of an ear, nose and throat infection. One week later,
fixed mydriasis of the same eye was observed, and the
child seen elsewhere for evaluation (Fig. 1). Ophthalmo-
logic examination revealed an intraocular mass and the Fig. 1 Mydriasis with sectorial inferotemporal leukocoria in the right eye of the 17-month-old child as presenting sign of intraocular
medulloepithelioma. Note the darker right eye appearance due to iris neovascularization and the inferior transillumination due to the subluxated
colobomatous lens Fig. 1 Mydriasis with sectorial inferotemporal leukocoria in the right eye of the 17-month-old child as presenting sign of intraocular
medulloepithelioma. Note the darker right eye appearance due to iris neovascularization and the inferior transillumination due to the subluxated
colobomatous lens Stathopoulos et al. BMC Ophthalmology (2020) 20:239 Page 3 of 5 Fig. 2 a. Anterior segment chamber photography of the right eye at diagnosis showing iris neovascularization, multiple free-floating cysts in the
pupillary area as well as a subluxated and notched lens. b. Ultrasound biomicroscopy (35 MHz) on the 6 o’clock meridian displaying an
iridocorneal angle closure and an irregular-surfaced mass with intratumoral cysts adjacent to the ciliary body, growing from the ciliary epithelium
into the posterior chamber without invading the ciliary muscle. Clumps of hyperechogenic material are found in the posterior chamber (cysts)
and the anterior vitreous (dust). c. Cytology analysis of the aqueous tap performed before treatment showing aggregates of malignant small cells
with high nuclear/cytoplasmic ratio and finely granular chromatin. d-e. Anterior segment photography and ultrasound biomicroscopy one month
after 3 intracameral and 3 intravitreal melphalan injections showing complete response of the primary tumor and the aqueous/vitreous seeding. Iris neovascularization had transiently regressed after concomitant intravitreal anti-vascular endothelial factor and cryoablation of the peripheral
ischemic retina Fig. 2 a. Anterior segment chamber photography of the right eye at diagnosis showing iris neovascularization, multiple free-floating cysts in the
pupillary area as well as a subluxated and notched lens. b. Ultrasound biomicroscopy (35 MHz) on the 6 o’clock meridian displaying an
iridocorneal angle closure and an irregular-surfaced mass with intratumoral cysts adjacent to the ciliary body, growing from the ciliary epithelium
into the posterior chamber without invading the ciliary muscle. Clumps of hyperechogenic material are found in the posterior chamber (cysts)
and the anterior vitreous (dust). c. Case presentation Cytology analysis of the aqueous tap performed before treatment showing aggregates of malignant small cells
with high nuclear/cytoplasmic ratio and finely granular chromatin. d-e. Anterior segment photography and ultrasound biomicroscopy one month
after 3 intracameral and 3 intravitreal melphalan injections showing complete response of the primary tumor and the aqueous/vitreous seeding. Iris neovascularization had transiently regressed after concomitant intravitreal anti-vascular endothelial factor and cryoablation of the peripheral
ischemic retina References
d
l 1. Tadepalli SH, Shields CL, Shields JA, Honavar SG. Intraocular
medulloepithelioma - a review of clinical features, DICER 1 mutation, and
management. Indian J Ophthalmol. 2019;67(6):755–62. 2. Kaliki S, Shields CL, Eagle RC Jr, Vemuganti GK, Almeida A, Manjandavida FP,
Mulay K, Honavar SG, Shields JA. Ciliary body medulloepithelioma: analysis
of 41 cases. Ophthalmology. 2013;120(12):2552–9. 3. Broughton WL, Zimmerman LE. A clinicopathologic study of 56 cases of
intraocular medulloepitheliomas. Am J Ophthalmol. 1978;85(3):407–18. 4. Shields JA, Eagle RC Jr, Shields CL, Potter PD. Congenital neoplasms of the
nonpigmented ciliary epithelium (medulloepithelioma). Ophthalmology. 1996;103(12):1998–2006. 5. Poon DS, Reich E, Smith VM, Kingston J, Reddy MA, Hungerford JL, Sagoo
MS. Ruthenium-106 plaque brachytherapy in the primary Management of
Ocular Medulloepithelioma. Ophthalmology. 2015;122(9):1949–51. 6. Canning CR, McCartney AC, Hungerford J. Medulloepithelioma (diktyoma). Br J Ophthalmol. 1988;72(10):764–7. 7. Munier FL, Gaillard MC, Balmer A, Soliman S, Podilsky G, Moulin AP, Beck-
Popovic M. Intravitreal chemotherapy for vitreous disease in retinoblastoma
revisited: from prohibition to conditional indications. Br J Ophthalmol. 2012;
96(8):1078–83. In conclusion, we report herein for the first time a
case of non-invasive ciliary body medulloepithelioma
with seeding at presentation successfully treated with
only a small number of intraocular melphalan injections. To our knowledge, this is the first case of intraocular
medulloepthelioma with seeding to be managed conser-
vatively. Concomitant secondary neovascular glaucoma
and cataract needed appropriate management to allow
long-term eye and vision preservation. Further studies
will help to better define the role of targeted chemother-
apy for intraocular medulloepithelioma in the future. 8. Munier FL, Gaillard MC, Decembrini S, Bongiovanni M, Beck-Popovic M. Intracameral chemotherapy (Melphalan) for aqueous seeding in
retinoblastoma: bicameral injection technique and related toxicity in a pilot
case study. Ocul Oncol Pathol. 2017;3(2):149–55. 8. Munier FL, Gaillard MC, Decembrini S, Bongiovanni M, Beck-Popovic M. Intracameral chemotherapy (Melphalan) for aqueous seeding in
retinoblastoma: bicameral injection technique and related toxicity in a pilot
case study. Ocul Oncol Pathol. 2017;3(2):149–55. 9. Saunders T, Margo CE. Intraocular medulloepithelioma. Arch Pathol Lab
Med. 2012;136(2):212–6. 9. Saunders T, Margo CE. Intraocular medulloepithelioma. Arch Pathol Lab
Med. 2012;136(2):212–6. 10. Zhou M, Xu G, Bojanowski CM, Song Y, Chen R, Sun X, Wang W, Chan CC. Differential diagnosis of anterior chamber cysts with ultrasound
biomicroscopy: ciliary body medulloepithelioma. Acta Ophthalmol Scand. 2006;84(1):137–9. 10. Zhou M, Xu G, Bojanowski CM, Song Y, Chen R, Sun X, Wang W, Chan CC. Discussion and conclusions Its cytotoxic activity is
mediated by its DNA alkylating properties, which leads
to cell death [18]. Melphalan injections directly into the
vitreous or the aqueous humor via intravitreal or intra-
cameral injections respectively, allow high intraocular
tumoricidal drug concentrations to be reached, while by-
passing systemic adverse effects and are therefore cur-
rently the treatment of choice for vitreous and aqueous
seeding in retinoblastoma [19, 20]. Recently intravitreal
melphalan has also been used to salvage two cases with
primary vitreoretinal lymphoma [21]. In our case, intra-
ocular
melphalan
alone
was
sufficient
to
achieve
complete regression, not only of the seeding but also of
the primary solid tumor, presumably because of its fri-
able nature and the absence of concomitant ciliary
muscle infiltration. Injected doses were decided based
on our experience with retinoblastoma. The minimal
tumoricidal drug concentration for intraocular medul-
loepithelioma remains to be established. Posterior capsu-
lar cataract needing surgery 3 years after treatment
completion was the only treatment-related adverse effect
observed in our patient. Acknowledgements
We thank Marc Curchod and Yann Leuba for photography editing. 14. Meel R, Chawla B, Mohanti BK, Kashyap S, Bakhshi S. Ocular
medulloepithelioma chemosensitivity. Ophthalmology. 2010;117(12):2440
e2441–2. 14. Meel R, Chawla B, Mohanti BK, Kashyap S, Bakhshi S. Ocular
medulloepithelioma chemosensitivity. Ophthalmology. 2010;117(12):2440
e2441–2. Discussion and conclusions Large
size or invasive medulloepitheliomas are commonly
treated with primary enucleation [3, 4, 10, 11]. Exenter-
ation in combination with irradiation and/or adjuvant
chemotherapy may be necessary in cases with orbital in-
volvement
[3,
12]. Small
or
medium-size
well-
circumscribed tumors can be treated conservatively with
I-125 or Ru-106 plaque radiotherapy [1, 2, 5]. Local re-
section with iridocyclectomy or partial lamellar sclerou-
vectomy
are
usually
insufficient,
displaying
a
high
recurrence rate requiring secondary enucleation [4, 6,
13]. To date, the role of first line chemotherapy for con-
servative management of medulloepithelioma is not
known, as its use has been restricted to adjuvant or neo-
adjuvant therapy of cases with orbital invasion and/or
metastasis [14–17]. In this report, we describe for the first time the con-
servative management of a non-infiltrating ciliary body
medulloepithelioma complicated with seeding at presen-
tation using intraocular melphalan injections. Presence
of seeding was so far considered as an indication for Stathopoulos et al. BMC Ophthalmology (2020) 20:239 Page 4 of 5 Page 4 of 5 enucleation
[4]. Melphalan
is
a
well-known
drug
employed
in
the
treatment
of
various
solid
and
hematopoietic cancers such as ovarian cancer, breast
cancer or multiple myeloma [18]. Its cytotoxic activity is
mediated by its DNA alkylating properties, which leads
to cell death [18]. Melphalan injections directly into the
vitreous or the aqueous humor via intravitreal or intra-
cameral injections respectively, allow high intraocular
tumoricidal drug concentrations to be reached, while by-
passing systemic adverse effects and are therefore cur-
rently the treatment of choice for vitreous and aqueous
seeding in retinoblastoma [19, 20]. Recently intravitreal
melphalan has also been used to salvage two cases with
primary vitreoretinal lymphoma [21]. In our case, intra-
ocular
melphalan
alone
was
sufficient
to
achieve
complete regression, not only of the seeding but also of
the primary solid tumor, presumably because of its fri-
able nature and the absence of concomitant ciliary
muscle infiltration. Injected doses were decided based
on our experience with retinoblastoma. The minimal
tumoricidal drug concentration for intraocular medul-
loepithelioma remains to be established. Posterior capsu-
lar cataract needing surgery 3 years after treatment
completion was the only treatment-related adverse effect
observed in our patient. enucleation
[4]. Melphalan
is
a
well-known
drug
employed
in
the
treatment
of
various
solid
and
hematopoietic cancers such as ovarian cancer, breast
cancer or multiple myeloma [18]. Consent for publication Written informed consent for publication of clinical details and clinical
images was obtained from the parents of the patient. A copy of the consent
form is available for review by the Editor of this journal. Authors’ contributions CS: acquisition, analysis and interpretation of data. Manuscript draft, review
of the literature, MCG: acquisition, analysis and interpretation of data, help to
draft the manuscript, JS: acquisition of data, review of the literature, FLM:
acquisition, analysis and interpretation of data, critical revisions, manuscript
draft. All authors read and approved the final manuscript. 15. Viswanathan S, Mukul D, Qureshi S, Ramadwar M, Arora B, Kane SV. Orbital
medulloepitheliomas -- with extensive local invasion and metastasis: a series
of three cases with review of literature. Int J Pediatr Otorhinolaryngol. 2008;
72(7):971–5. 15. Viswanathan S, Mukul D, Qureshi S, Ramadwar M, Arora B, Kane SV. Orbital
medulloepitheliomas -- with extensive local invasion and metastasis: a series
of three cases with review of literature. Int J Pediatr Otorhinolaryngol. 2008;
72(7):971–5. 16. Eken M, Evren C, Sanli A, Aydin S, Yavuzer D. Malignant non-teratoid
medulloepithelioma with submandibular metastases. Int J Pediatr
Otorhinolaryngol. 2006;70(1):163–5. 16. Eken M, Evren C, Sanli A, Aydin S, Yavuzer D. Malignant non-teratoid
medulloepithelioma with submandibular metastases. Int J Pediatr
Otorhinolaryngol. 2006;70(1):163–5. References
d
l Differential diagnosis of anterior chamber cysts with ultrasound
biomicroscopy: ciliary body medulloepithelioma. Acta Ophthalmol Scand. 2006;84(1):137–9. 11. Owen RI, Tiffin PA, Steel DH. Echographic features of a case of malignant
intraocular medulloepithelioma. Br J Ophthalmol. 2005;89(1):120–1. 11. Owen RI, Tiffin PA, Steel DH. Echographic features of a case of malignant
intraocular medulloepithelioma. Br J Ophthalmol. 2005;89(1):120–1. 12. Sosinska-Mielcarek K, Senkus-Konefka E, Jaskiewicz K, Kordek R, Jassem J. Intraocular malignant teratoid medulloepithelioma in an adult:
clinicopathological case report and review of the literature. Acta
Ophthalmol Scand. 2006;84(2):259–62. 12. Sosinska-Mielcarek K, Senkus-Konefka E, Jaskiewicz K, Kordek R, Jassem J. Intraocular malignant teratoid medulloepithelioma in an adult:
clinicopathological case report and review of the literature. Acta
Ophthalmol Scand. 2006;84(2):259–62. Abbreviations
UBM: Ultrasound biomicroscopy; VEGF: Vascular endothelial growth factor;
YAG: Yttrium aluminium garnet Abbreviations
UBM: Ultrasound biomicroscopy; VEGF: Vascular endothelial growth factor;
YAG: Yttrium aluminium garnet Funding Funding
There was no source of funding for the research. g
There was no source of funding for the research. 17. Hellman JB, Harocopos GJ, Lin LK. Successful treatment of metastatic
congenital intraocular medulloepithelioma with neoadjuvant
chemotherapy, enucleation and superficial parotidectomy. Am J
Ophthalmol Case Rep. 2018;11:124–7. Abbreviations
l
d 13. Holbach L, Volcker HE, Naumann GO. Malignant teratoid
medulloepithelioma of the ciliary body and glial fibrillary acidic protein. Clinical, histochemical and immunohistochemical findings. Klin Monatsbl
Augenheilkd. 1985;187(4):282–6. 13. Holbach L, Volcker HE, Naumann GO. Malignant teratoid
medulloepithelioma of the ciliary body and glial fibrillary acidic protein. Clinical, histochemical and immunohistochemical findings. Klin Monatsbl
Augenheilkd. 1985;187(4):282–6. Acknowledgements
We thank Marc Curchod and Yann Leuba for photography editing. Acknowledgements
We thank Marc Curchod and Yann Leuba for photography editing. Acknowledgements
We thank Marc Curchod and Yann Leuba for photography editing. 9.
Saunders T, Margo CE. Intraocular medulloepithelioma. Arch Pathol Lab
Med. 2012;136(2):212–6. Competing interests
fl
l No conflicting relationship for any author. No conflicting relationship for any author. The authors have no financial interest in any aspect of this article. The authors have no financial interest in any aspect of this article. Received: 8 April 2020 Accepted: 10 June 2020 Received: 8 April 2020 Accepted: 10 June 2020 11.
Owen RI, Tiffin PA, Steel DH. Echographic features of a case of malignant
intraocular medulloepithelioma. Br J Ophthalmol. 2005;89(1):120–1. Availability of data and materials Not applicable. 18. Bayraktar UD, Bashir Q, Qazilbash M, Champlin RE, Ciurea SO. Fifty years of
melphalan use in hematopoietic stem cell transplantation. Biol Blood
Marrow Transplant. 2013;19(3):344–56. 18. Bayraktar UD, Bashir Q, Qazilbash M, Champlin RE, Ciurea SO. Fifty years of
melphalan use in hematopoietic stem cell transplantation. Biol Blood
Marrow Transplant. 2013;19(3):344–56. Ethics approval and consent to participate
Not applicable. Page 5 of 5 Page 5 of 5 Stathopoulos et al. BMC Ophthalmology (2020) 20:239 Stathopoulos et al. BMC Ophthalmology (2020) 20:239 19. Stathopoulos C, Munier FL: Intravitreal chemotherapy in Clinical Ophthalmic
Oncology 3rd Edition 2019, 6(Retinoblasotma, chapter 15):179–192. 19. Stathopoulos C, Munier FL: Intravitreal chemotherapy in Clinical Ophthalmic
Oncology 3rd Edition 2019, 6(Retinoblasotma, chapter 15):179–192. 20. Munier FL, Moulin A, Gaillard MC, Bongiovanni M, Decembrini S, Houghton
S, Beck-Popovic M, Stathopoulos C. Intracameral chemotherapy for globe
salvage in retinoblastoma with secondary anterior chamber invasion. Ophthalmology. 2018;125(4):615–7. 20. Munier FL, Moulin A, Gaillard MC, Bongiovanni M, Decembrini S, Houghton
S, Beck-Popovic M, Stathopoulos C. Intracameral chemotherapy for globe
salvage in retinoblastoma with secondary anterior chamber invasion. Ophthalmology. 2018;125(4):615–7. 21. Shields CL, Sioufi K, Mashayekhi A, Shields JA. Intravitreal Melphalan for
treatment of primary Vitreoretinal lymphoma: a new indication for an old
drug. JAMA Ophthalmol. 2017;135(7):815–8. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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Abstract. Finally the scheme is applied in an off-line version of the ORCHIDEE LSM over Europe to show that
10
realistic river discharge and temperatures are predicted over the major catchments of the region. The simulated solutions are
largely independent of the atmospheric grid used thanks to the proposed sub-grid approach. Hydrological modelling on atmospheric grids; using graphs of
sub-grid elements to transport energy and water
Jan Polcher1, Anthony Schrapffer2,1, Eliott Dupont3, Lucia Rinchiuso4,5, Xudong Zhou6,
Olivier Boucher3, Emmanuel Mouche4, Catherine Ottlé4, and Jérôme Servonnat4
1LMD-IPSL, CNRS, Ecole Polytechnique, Route de Saclay, 91128 Palaiseau Cedex, France
2Centro de Investigaciones del Mar y la Atmósfera (CIMA), CONICET-UBA, CNRS, Intendente Güiraldes 2160 - Ciudad
Universitaria - Pabellón II - 2do. piso (C1428EGA) C. A. Buenos Aires, Argentina
3Institut Pierre-Simon Laplace (IPSL), CNRS, U. Sorbonne, Paris, France
4LSCE-IPSL, CEA, CNRS, U. Paris-Saclay, Gif-sur-Yvette, France
5METIS-IPSL, CNRS, U. Sorbonne, Paris, France
6Institute of Industrial Science, University of Tokyo, Tokyo, Japan
Correspondence: Jan Polcher (jan.polcher@lmd.ipsl.fr) Correspondence: Jan Polcher (jan.polcher@lmd.ipsl.fr) Hydrological modelling on atmospheric grids; using graphs of
sub-grid elements to transport energy and water
Jan Polcher1, Anthony Schrapffer2,1, Eliott Dupont3, Lucia Rinchiuso4,5, Xudong Zhou6,
Olivier Boucher3, Emmanuel Mouche4, Catherine Ottlé4, and Jérôme Servonnat4
1LMD-IPSL, CNRS, Ecole Polytechnique, Route de Saclay, 91128 Palaiseau Cedex, France
2Centro de Investigaciones del Mar y la Atmósfera (CIMA), CONICET-UBA, CNRS, Intendente Güiraldes 2160 - Ciudad
Universitaria - Pabellón II - 2do. piso (C1428EGA) C. A. Buenos Aires, Argentina
3Institut Pierre-Simon Laplace (IPSL), CNRS, U. Sorbonne, Paris, France
4LSCE-IPSL, CEA, CNRS, U. Paris-Saclay, Gif-sur-Yvette, France
5METIS-IPSL, CNRS, U. Sorbonne, Paris, France
6Institute of Industrial Science, University of Tokyo, Tokyo, Japan
Correspondence: Jan Polcher (jan.polcher@lmd.ipsl.fr)
https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. Abstract. Land Surface Models (LSMs) use the atmospheric grid as their basic spatial decomposition because their main objective is
to provide the lower boundary conditions to the atmosphere. Lateral water flows at the surface on the other hand require a much
higher spatial discretization as they are closely linked to topographic details. We propose here a methodology to automatically higher spatial discretization as they are closely linked to topographic details. We propose here a methodology to automatically
tile the atmospheric grid into hydrological coherent units which are connected through a graph. As water is transported on sub-
5
grids of the LSM, land variables can easily be transferred to the routing network and advected if needed. This is demonstrated
here for temperature. The quality of the river networks generated, as represented by the connected hydrological transfer units,
are compared to the original data in order to quantify the degradation introduced by the discretization method. The conditions
the sub-grid elements impose on the time step of the water transport scheme are evaluated and a methodology is proposed to
find an optimal value. Finally the scheme is applied in an off-line version of the ORCHIDEE LSM over Europe to show that
10
realistic river discharge and temperatures are predicted over the major catchments of the region The simulated solutions are tile the atmospheric grid into hydrological coherent units which are connected through a graph. As water is transported on sub-
5
grids of the LSM, land variables can easily be transferred to the routing network and advected if needed. This is demonstrated
here for temperature. The quality of the river networks generated, as represented by the connected hydrological transfer units,
are compared to the original data in order to quantify the degradation introduced by the discretization method. The conditions
the sub-grid elements impose on the time step of the water transport scheme are evaluated and a methodology is proposed to find an optimal value. Finally the scheme is applied in an off-line version of the ORCHIDEE LSM over Europe to show that
10
realistic river discharge and temperatures are predicted over the major catchments of the region. The simulated solutions are
largely independent of the atmospheric grid used thanks to the proposed sub-grid approach. find an optimal value. 1
Introduction Lateral water transport over continents plays an important role in the Earth system but its implementation in models focusses
on different objectives depending on resolution. In global Earth System Models (ESM), tailored to address climate change
15
issues, the main need is to transport the excess water over land to the oceans so as to close the water cycle. Because of their
coarse resolution the main focus will be on the largest rivers. Regional ESM, as those used for process studies and downscaling
of climate projections, will usually attempt to reproduce more details in the continental water cycle. Lateral water transports
will thus also serve to predict levels of in-land water bodies or inundations and the impact of freshwater flows on coastal
processes Finally for km-scale ESM currently being developed to better represent rainfall and convective processes lateral
20 15 processes. Finally for km-scale ESM currently being developed to better represent rainfall and convective processes, lateral
20 1 1 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. flows allow to redistribute moisture along hill-slopes (Fan et al., 2019). At these resolutions the hypothesis that evaporation is
only fed by local precipitation is not valid any more. Thus rain falling on mountain slopes needs to flow into the valleys where
the vegetation is located which will be able to evaporate it. These are resolutions where hill-slope processes will start to be
important. flows allow to redistribute moisture along hill-slopes (Fan et al., 2019). At these resolutions the hypothesis that evaporation is
only fed by local precipitation is not valid any more. Thus rain falling on mountain slopes needs to flow into the valleys where
the vegetation is located which will be able to evaporate it. These are resolutions where hill-slope processes will start to be
important. Furthermore rivers also transport energy and biogeochemical species (Liu et al., 2020; Lauerwald et al., 2017). Thus their
25
correct representation allows to close the associated global cycles of the Earth system and improve the coupling between
continental and oceanic processes. To enhance the representation of coastal processes in ESMs at all scales, the energy and
nutrient contribution by rivers will be a major topic in the years to come. 25 Land surface models, the components within ESMs dealing with continental processes, have implemented over the last 30
years a very uni-dimensional vision of the water and energy processes at the surface. The main driver of their development
30
was to provide the lower boundary to the atmosphere. As a consequence they have also adopted the spatial discretization of
the atmosphere so as not to introduce any discontinuity in this important coupling. The lateral water transport cuts across this
one dimensional vision and also challenges the use of the atmospheric grid. Indeed, lateral water movements require often
higher resolution than the atmosphere as topographic features are a stronger constraint for the flow of water on land than for the atmosphere. The hydrological community have been free of this constraint of the coupling to the atmosphere and could
35
adopt appropriate spatial discretization, which is often kilometric, for the representation of rivers. ESMs have adopted over the last decades two different and complementary approaches to try and deal with the lateral
water transport on continents. The first and most widespread approach is to abandon the atmospheric grid and interpolate
the fields of water exiting the one dimensional soil moisture (generally surface runoff and deep drainage) towards the grid the atmosphere. The hydrological community have been free of this constraint of the coupling to the atmosphere and could
35
adopt appropriate spatial discretization, which is often kilometric, for the representation of rivers. ESMs have adopted over the last decades two different and complementary approaches to try and deal with the lateral
water transport on continents. The first and most widespread approach is to abandon the atmospheric grid and interpolate
the fields of water exiting the one dimensional soil moisture (generally surface runoff and deep drainage) towards the grid i
g
g
y
p
g
g
which will be used to simulate river flows (Decharme et al., 2012; Branstetter, 2003). From this grid the discharge to the seas
40
is then transmitted to the ocean model. The river routing model becomes an independent component of the ESM meaning
it can be developed and validated separately. Sharing this component with other groups is also facilitated (Kauffeldt et al.,
2016). The main drawback of this approach is that it creates a division in the interactions between the vertical and horizontal
motions of water on continents. It complicates the representation of such important processes as floodplains or irrigation. The
interpolations between the atmospheric and routing grids will need to fulfil the conservation principles and preserve gradients
45
in surface processes which will be used to simulate river flows (Decharme et al., 2012; Branstetter, 2003). From this grid the discharge to the seas
40
is then transmitted to the ocean model. The river routing model becomes an independent component of the ESM meaning
it can be developed and validated separately. Sharing this component with other groups is also facilitated (Kauffeldt et al.,
2016). The main drawback of this approach is that it creates a division in the interactions between the vertical and horizontal
motions of water on continents. It complicates the representation of such important processes as floodplains or irrigation. The The second approach is to use directly the atmospheric grid for the lateral flows (Miguez-Macho et al., 2007). This has been
successfully applied in regional and km-scale ESMs for which the horizontal atmospheric grid are compatible with hydro-
logical processes. 2
The river flow model Before presenting the construction of the graph of hydrological transfer units (HTU), the equations used to transport water and
heat along the network of rivers are presented. They give indications on the properties of the graph which need to be preserved. Finally we will show with ORCHIDEE that the simplification of the digital elevation model introduced by
the transport on a graph of HTUs is small compared to the uncertainty in the atmospheric forcing. The methodology will also
be used to demonstrate the value of a simple implementation of stream temperature
75 The methodology has been particularly successful for flood forecasting where lead time for predictions are short (Yucel et al., 2015). A complementary methodology is to use a hydrological tiling of the atmospheric grid to achieve the
50
effective resolution needed (Ngo-Duc et al., 2007; Clark et al., 2015). The general principle is to keep the vertical movements
of water on the atmospheric grid but distribute the excess moisture over hydrologically consistent and connected tiles so that it
can flow horizontally. In principle this methodology should be able to bridge the gap between coarser atmospheric resolutions
and the level of detail needed for surface flows while keeping a close link between the vertical and horizontal processes. 2 2 It allows to represent floodplains and their Preserving the link between the atmospheric resolution and the sub-grid hydrology facilitates the representation of all the
processes which involve exchanges between vertical and lateral water movements. It allows to represent floodplains and their
impact on evaporation (Companion paper Schrapffer et al. 2022) or water extractions for human activities (de Rosnay et al.,
65
2003; Zhou et al., 2021). In this context the stream temperature can also be more easily simulated as the energy balance
performed on the atmospheric grid can directly interact with open water areas which are part of the lateral flows. impact on evaporation (Companion paper Schrapffer et al. 2022) or water extractions for human activities (de Rosnay et al.,
65
2003; Zhou et al., 2021). In this context the stream temperature can also be more easily simulated as the energy balance
performed on the atmospheric grid can directly interact with open water areas which are part of the lateral flows. The proposed study explores the numerical properties of such a hybrid routing scheme and in particular how it handles
different atmospheric grids. In a first step we will show how the graph of HTU can be built and which properties of the hydrological network need to be preserved. Then we will present criteria which allow to verify the fidelity of the HTU graph and
70
in particular how many sub-grid elements are needed for different resolutions of the atmospheric grid to preserve the original
hydrological information. Once water is transported, criteria are needed to select a time step which ensures that the numerical
solution converges. Finally we will show with ORCHIDEE that the simplification of the digital elevation model introduced by
the transport on a graph of HTUs is small compared to the uncertainty in the atmospheric forcing. The methodology will also
be used to demonstrate the value of a simple implementation of stream temperature. 75 hydrological network need to be preserved. Then we will present criteria which allow to verify the fidelity of the HTU graph and
70
in particular how many sub-grid elements are needed for different resolutions of the atmospheric grid to preserve the original
hydrological information. Once water is transported, criteria are needed to select a time step which ensures that the numerical
solution converges. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. Using the nomenclature proposed by Yamazaki et al. (2013) this last method can be labelled hybrid river models. It uses
55
the vector-based methodology within the atmospheric grid as water is transported between unit-catchments and then uses a
grid-based approach when the flow leaves the mesh. The combination of both yields graphs of hydrological transfer units
(HTU) along which the water will flow within and from grid to grid. In the list of criteria established by Kauffeldt et al. (2016)
to classify large-scale hydrological models, a hybrid routing addresses in particular the two linked to the grid. It should be Using the nomenclature proposed by Yamazaki et al. (2013) this last method can be labelled hybrid river models. It uses
55
the vector-based methodology within the atmospheric grid as water is transported between unit-catchments and then uses a
grid-based approach when the flow leaves the mesh. The combination of both yields graphs of hydrological transfer units
(HTU) along which the water will flow within and from grid to grid. In the list of criteria established by Kauffeldt et al. (2016)
to classify large-scale hydrological models, a hybrid routing addresses in particular the two linked to the grid. It should be able to deal with any atmospheric grid and in particular those based on the more complex icosahedron (Dubos et al., 2015) or
60
cubed-sphere (Kim et al., 2021). The flexibility in resolution is given as the hydrological information which cannot be resolved
with the grid-based flow is treated with the vector-based transport. Preserving the link between the atmospheric resolution and the sub-grid hydrology facilitates the representation of all the
processes which involve exchanges between vertical and lateral water movements. It allows to represent floodplains and their able to deal with any atmospheric grid and in particular those based on the more complex icosahedron (Dubos et al., 2015) or
60
cubed-sphere (Kim et al., 2021). The flexibility in resolution is given as the hydrological information which cannot be resolved
with the grid-based flow is treated with the vector-based transport. Preserving the link between the atmospheric resolution and the sub-grid hydrology facilitates the representation of all the
processes which involve exchanges between vertical and lateral water movements. This characteristic time of each reservoir
is the product of a geometric property, the topographic index symbolized by λ in km , and a constant in s/km which needs to
be determined. We chose to distinguish the topographic index of the stream flow (λi,stream) and the one for the aquifers of the The flow is thus given by the reservoir’s water mass divided by the residence time. This characteristic time of each reservoir
is the product of a geometric property, the topographic index symbolized by λ in km , and a constant in s/km which needs to
be determined. We chose to distinguish the topographic index of the stream flow (λi,stream) and the one for the aquifers of the HTU (λi). The first one can be evaluated based on the geometry of the main river within the HTU. The other one provides a
105
more aggregated view of the geometry of the HTU. The general principle for computing these parameters is : λ =
r
d3
dz
(6) λ =
r
d3
dz (6) (6) where d is the relevant length over which the water flows within the HTU and dz the elevation change along that path. In the
section covering the construction of the HTU graph the computation of the topographic indices is detailed for each case (see
section 3.5). 110 where d is the relevant length over which the water flows within the HTU and dz the elevation change along that path. In the
section covering the construction of the HTU graph the computation of the topographic indices is detailed for each case (see
section 3.5). 110 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. – i + 1 is the downstream store of HTU i. Because we are in a directional graph i + 1 is unique and at one point should be
the ocean or a water body for endorheic basins. – i + 1 is the downstream store of HTU i. Because we are in a directional graph i + 1 is unique and at one point should be
the ocean or a water body for endorheic basins. – For an HTU i there is an ensemble of upstream HTUs which will be denoted with {i−1}. For a basin head the ensemble
will be empty. – For an HTU i there is an ensemble of upstream HTUs which will be denoted with {i−1}. For a basin head the ensemble
will be empty. 90 90 – Fluxes connecting two HTUs are denoted with half indices. The flux leaving HTU i is placed at i + 1/2 Given the above notation, the prognostic equations for water transports are given by : Given the above notation, the prognostic equations for water transports are given by : Given the above notation, the prognostic equations for water transports are given by : ∂Wi,stream
∂t
=
X
j∈{i−1}
(Qj+ 1
2 ,slow + Qj+ 1
2 ,fast + Qj+ 1
2 ,stream) −Qi+ 1
2 ,stream
(1)
∂Wi,fast
∂t
= δi,biRbi −Qi+ 1
2 ,fast
(2)
∂Wi,slow
∂t
= δi,biDbi −Qi+ 1
2 ,slow
(3) 95 95 (4) Here Rbi is the surface runoff (i.e. water which does not infiltrate into the unsaturated zone of the LSM) on the atmospheric grid
and Dbi the drainage exiting the soil moisture reservoir. Both of these fluxes are computed by the land surface model and thus
behave according to the assumptions made there. The fluxes out of the three reservoirs are Qi+ 1
2 ,slow,Qi+ 1
2 ,fast,Qi+ 1
2 ,stream)
and are expressed in kg/s. They are defined as follows :
100 100 Qt
i+ 1
2 ,X =
W t
i,X
λi,X gX
withX ∈{stream,fast,slow} Qt
i+ 1
2 ,X =
W t
i,X
λi,X gX
withX ∈{stream,fast,slow}
(5) (5) The flow is thus given by the reservoir’s water mass divided by the residence time. 2.1
Water transport The flow of water occurs on a directional graph towards the outflow point (Diestel, 2012). As for the moment the focus is on
80
simulating river flows, it is the stream reservoirs which connect the various HTUs in the graph. Each HTU i is contained in
only one atmospheric grid box bi and covers the fraction δi,bi with the sum of all HTU areas equal to the continental surface
provided by the atmospheric model. Thus for the fraction of land in the grid we have : P
i∈bi δi,bi = 1. This means that the HTUs
are a connected supermesh of the land fraction within the atmospheric grid (Farrell et al., 2009) and which represent the river
graph. 85 graph. 85
As the graph is directional we can pose the following indexing convention : graph. 85
As the graph is directional we can pose the following indexing convention : 3 3 where Ai is the area of the HTU and is used to transform the water mass in the stream multiplied by its density (ρ) into an
equivalent height. a is a scaling parameter which allows to adjust the role of the relaxation term and thus explore the relative
importance of initial temperatures and interactions with the environment surrounding the river in the determination of Ti,stream. If a is large, Eq 9 will behave like a pure advection scheme and only adjust the temperature of stream with shallow water levels
130
to the top soil temperatures. As a decreases the interaction of the stream temperature with its environment will increase. This
relaxation will mostly be active on HTUs with low stream reservoir which is physically reasonable. In areas where water flows
in small streams the temperature of the water will be close to the one of the upper soils. This simple relaxation obviously does
not cover the complex interactions of lakes or large rivers with the atmosphere and which are known to impact strongly stream temperature. 135
The numerical solution of these equations is discussed later (Section 5) as it depends on the definition of the HTUs. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. be chosen for the water in the fast and slow reservoirs. The assumption here is that they are determined by the temperature of
the soils of the corresponding atmospheric grid. Our initial assumption is :
115 Ti,fast = Tbi,up
(7)
Ti,slow = Tbi,low
(8) (7)
(8) (7) Ti,fast = Tbi,up Ti,fast = Tbi,up (7)
(8) (8) Ti,slow = Tbi,low where Tbi,up is the soil temperature averaged over the top 0.3m and Tbi,low the mean value over the 3.5−17.5m layers. Currently
the deepest node in the soil temperature diffusion model in ORCHIDEE is 17.5m thus imposing a lower limit on the depth for
the temperature of the slowest aquifer. This situation can evolve in future versions of ORCHIDEE. 20 where Tbi,up is the soil temperature averaged over the top 0.3m and Tbi,low the mean value over the 3.5−17.5m layers. Currently
the deepest node in the soil temperature diffusion model in ORCHIDEE is 17.5m thus imposing a lower limit on the depth for the temperature of the slowest aquifer. This situation can evolve in future versions of OR
120 Evaluating the stream temperature through the advection of heat has to deal with the singularity arising when the reservoir
content goes to zero. A relaxation towards the upper soil temperature was chosen to deal with this indetermination. This allows
to write the following set of equations : Evaluating the stream temperature through the advection of heat has to deal with the singularity arising when the reservoir
content goes to zero. A relaxation towards the upper soil temperature was chosen to deal with this indetermination. This allows
to write the following set of equations : ∂Ti,stream
∂t
=
1
Wi,stream
X
j∈{i−1}
(Qj+ 1
2 ,slowTj,slow + Qj+ 1
2 ,fastTj,fast + Qj+ 1
2 ,streamTj,stream)
−
1
Wi,stream
Qi+ 1
2 ,streamTi,stream + K(Tbi,up −Ti,stream)
(9) (9) (9) (10) where Ai is the area of the HTU and is used to transform the water mass in the stream multiplied by its density (ρ) into an
equivalent height. a is a scaling parameter which allows to adjust the role of the relaxation term and thus explore the relative
importance of initial temperatures and interactions with the environment surrounding the river in the determination of Ti,stream. 2.2
Stream temperature The advection of heat content with lateral water transport is given by the advection of the aquifer temperatures and the interac-
tions with the surrounding landscape and the atmosphere. In order to implement these processes, an initial temperature has to 4 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. Table 1. The atmospheric grids and land/sea masks for which HTU graphs were built to test the methodology. grid name
Source
Resolution
Projection
WFDEI
WFDEI forcing dataset Weedon et al. (2014)
0.5◦
Regular longitude and latitude
E2OFD
MSWEP precipitation product (Beck et al., 2017)
0.25◦
Regular longitude and latitude
MEDCORDEX
MED-CORDEX configuration
20km
Lambert conformal
EuroCORDEX
EuroCORDEX configuration
11km
Cassini projection 1. The atmospheric grids and land/sea masks for which HTU graphs were built to test the methodology. For the present study, the methodology for tiling the atmospheric mesh into graphs of HTUs is exemplified on 4 grids
covering the Euro-Mediterranean region (Table 1) which are either used for off-line or coupled simulations of ORCHIDEE. The domain of computation used here is at least from 20◦East to 60◦West and 20◦to 60◦North. To illustrate the diversity For the present study, the methodology for tiling the atmospheric mesh into graphs of HTUs is exemplified on 4 grids
covering the Euro-Mediterranean region (Table 1) which are either used for off-line or coupled simulations of ORCHIDEE. The domain of computation used here is at least from 20◦East to 60◦West and 20◦to 60◦North. To illustrate the diversity
of grids a small sample over the lower Seine is provided in Figure 1. Only rectangular meshes are considered here but the
5
methodology can be extended to triangular ones. The companion paper (Schrapffer et al. 2022), which presents the floodplains For the present study, the methodology for tiling the atmospheric mesh into graphs of HTUs is exemplified on 4 grids
covering the Euro-Mediterranean region (Table 1) which are either used for off-line or coupled simulations of ORCHIDEE. covering the Euro-Mediterranean region (Table 1) which are either used for off-line or coupled simulations of ORCHIDEE. The domain of computation used here is at least from 20◦East to 60◦West and 20◦to 60◦North. To illustrate the diversity
of grids a small sample over the lower Seine is provided in Figure 1. Only rectangular meshes are considered here but the
145
methodology can be extended to triangular ones. The companion paper (Schrapffer et al. 2022), which presents the floodplains
parametrization, uses HTU graphs built for two atmospheric grids over South America. of grids a small sample over the lower Seine is provided in Figure 1. 3
Building the graph of Hydrological Transfer Units (HTU) This section presents the methodology for constructing Hydrological Transfer Units (HTUs) and connecting them to build a
hydrological network suitable to simulate surface water transport. In contrast to the tiling of the atmospheric grid for vegetation
(Ducoudré et al., 1993; Koster and Suarez, 1992), the connectivity of the sub-grid elements needs to be preserved and form a
140
convergent hydrological graph. It is an evolution of the method discussed by Nguyen-Quang et al. (2018). This section presents the methodology for constructing Hydrological Transfer Units (HTUs) and connecting them to build a
hydrological network suitable to simulate surface water transport. In contrast to the tiling of the atmospheric grid for vegetation 5 3.1
Hydrological digital elevation models
150 In order to bridge this
gap between global coarse and regional high resolution HDEMs we have chosen to also use a 60arcmin version of MERIT
(Yamazaki et al., 2019). This version of MERIT was obtained by upscaling from the high-resolution flow direction data (3 arcsec MERIT Hydro) with the Iterative Hydrography Upscaling (IHU) method (Eilande
165 3.1
Hydrological digital elevation models
150 To perform the HTU decomposition and compute their properties a hydrological digital elevation model (HDEM) is needed. The minimal information required is elevation, flow direction, flow accumulation and distance to the ocean for each pixel. The
elevation should be hydrologically consistent in the sense that no flow should lead water to gain elevation. As we will show
below, this is not a strict requirement. The three data sets listed in Table 2 fulfil these criteria and have been used here to test qi
the methodology. They needed to be standardized as each of these HDEM had a different set of variables required and were
155
not using the same conventions. The original HDEM used in IPSL’s ESM is from Fekete et al. (2000) which is at 0.5◦resolution. It has been enhanced by
flow directions in Antarctica in order to carry meltwater to the ocean and close the global water cycle. Its low resolution was
perfectly suited for the global models used in climate studies such as Ngo-Duc et al. (2007). The advent of higher resolution the methodology. They needed to be standardized as each of these HDEM had a different set of variables required and were
155
not using the same conventions. The original HDEM used in IPSL’s ESM is from Fekete et al. (2000) which is at 0.5◦resolution. It has been enhanced byl atmospheric models means that its usefulness is diminishing. For regional applications the HydroSHEDS data (Lehner and
160
Grill, 2013) was used in ORCHIDEE over the Mediterranean region Nguyen-Quang et al. (2018). The fact that this HDEM
is not global means that it is only applicable for regional studies which do not cover the arctic region. In order to bridge this
gap between global coarse and regional high resolution HDEMs we have chosen to also use a 60arcmin version of MERIT
(Yamazaki et al., 2019). This version of MERIT was obtained by upscaling from the high-resolution flow direction data (3
MERIT H d
)
ith th It
ti
H d
h U
li
(IHU)
th d (Eil
d
t l 2021)
165 atmospheric models means that its usefulness is diminishing. For regional applications the HydroSHEDS data (Lehner and
160
Grill, 2013) was used in ORCHIDEE over the Mediterranean region Nguyen-Quang et al. (2018). The fact that this HDEM
is not global means that it is only applicable for regional studies which do not cover the arctic region. Only rectangular meshes are considered here but the
145
methodology can be extended to triangular ones. The companion paper (Schrapffer et al. 2022), which presents the floodplains
parametrization, uses HTU graphs built for two atmospheric grids over South America. The methodology works on any atmospheric grid as long as the polygons constitutive of the mesh are provided together with
the land/sea mask. Figure 1. Samples over the lower Seine basin of the four atmospheric grids considered here (Table 1). The green colour indicate which
meshes are land for the atmospheric model overlaid over the actual land outline in yellow. Figure 1. Samples over the lower Seine basin of the four atmospheric grids considered here (Table 1). The green colour indicate which
meshes are land for the atmospheric model overlaid over the actual land outline in yellow. 6 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. Table 2. The hydrological data sets used in this study to evaluate the building of routing graph and the simulated river discharge
Data set name
Source
Resolution
Domain covered
Constants used [s/km]
gstream
gfast
gslow
FV
Fekete et al. (2000)
0.5◦
Global including Antarctica
6.0
80.0
600
HydroSHEDS
Lehner and Grill (2013)
30acrsec
South of 60◦N and excluding Antarctica
6.3
80.0
600
MERIT
Yamazaki et al. (2019)
60arcsec
Globe without Antarctica
6.3
80.0
600 ata sets used in this study to evaluate the building of routing graph and the simulated river discharge 3.3
First construction of HTUs A first set of coarse HTUs is build by joining all polygons of the supermesh which flow out of the atmospheric grid at the
same point. Their upstream area is computed according to the HDEM. This provides a first set of HTUs which is quite coarse
180
but ensures that all rivers and flow directions out of the atmospheric grid are preserved. The example in Figure 2c) ( for
nbmax = 18) still carries some elements of this decomposition with the HTU 18 flowing out of the Eastern part of the Northern
edges of the atmospheric grid. These small HTUs will not transport large water amounts but ensure that the total upstream area
of rivers to which they contribute remains correct. At this stage all pixels which contribute to the main outlet of the Rhone in the lower left corner are still in a single and same HTU. This first step will conclude for the case illustrated in Figure 2 with as
185
many HTUs as there are arrows pointing out of the grid. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. This process is performed using the SphericalGeometry library (https://github.com/spacetelescope/spherical_geometry) im-
plemented in Python, which allows to calculate intersections of polygons over a sphere. This supermesh between the atmo-
spheric grid and the HDEM can be saved into a NetCDF file to avoid having to perform this calculation again on the same
175
combination of grids. The advantage of this procedure is that it is applicable to any atmospheric grid even unstructured ones. This is particularly important as atmospheric models evolve towards more complex grids (Dubos et al., 2015). 3.2
Supermesh between atmospheric grids and HDEM The HTUs are built from the supermesh (Farrell et al., 2009) between the atmospheric grid and the hydrological digital elevation
model (HDEM). The initial step is to create this supermesh by calculating the set of polygons of the HDEM grid overlapping
with the coarser mesh. The result is for each atmospheric grid point the list of polygons of intersecting polygones and their
area. At the borders of the atmospheric grid a number of small polygons with their areas and flow direction will be created. 170
This generates some numerical noise but which will be absorbed during the HTU construction process. More importantly it
allows to preserve catchment areas and the diversity of flow directions out of the atmospheric grid. The HTUs are built from the supermesh (Farrell et al., 2009) between the atmospheric grid and the hydrological digital elevation
model (HDEM). The initial step is to create this supermesh by calculating the set of polygons of the HDEM grid overlapping
with the coarser mesh. The result is for each atmospheric grid point the list of polygons of intersecting polygones and their area. At the borders of the atmospheric grid a number of small polygons with their areas and flow direction will be created. 170
This generates some numerical noise but which will be absorbed during the HTU construction process. More importantly it
allows to preserve catchment areas and the diversity of flow directions out of the atmospheric grid. area. At the borders of the atmospheric grid a number of small polygons with their areas and flow direction will be created. 170
This generates some numerical noise but which will be absorbed during the HTU construction process. More importantly it
allows to preserve catchment areas and the diversity of flow directions out of the atmospheric grid. area. At the borders of the atmospheric grid a number of small polygons with their areas and flow direction will be created. 170
This generates some numerical noise but which will be absorbed during the HTU construction process. More importantly it
allows to preserve catchment areas and the diversity of flow directions out of the atmospheric grid. 7 3.4
Sub-division of HTUs An algorithm is needed to sub-divide the larger HTUs to better represent the river network within the atmospheric mesh. The
objective is to divide the HTU at important confluences. There are two types of confluences which need to be considered: – Two large rivers (large global upstream area) meet as illustrate in Figure 2a) around Valence. Figure 2b) shows three
rivers with a large global flow accumulation joining the Rhone river in the grid. – Two large rivers (large global upstream area) meet as illustrate in Figure 2a) around Valence. Figure 2b) shows three
rivers with a large global flow accumulation joining the Rhone river in the grid. 190 – A local river (large local upstream) joins a large river. This is for instance the case for HTU 8 in Figure 2c) which
corresponds to La Barberolle flowing into the Rhone river in Valence. part of the main river upstream of the confluence and with this pixel of the HDEM as it outflow location, (2) the downstream
195
part of the main river which keeps the initial outflow pixel and (3) the tributary which has as outflow pixel the one before the
confluence. If the subdivision (1) or (2) are too small, the HTU is only divided into two parts : the HTU of the main river and
the confluence. The tributary will now flow directly to the outflow point of the original HTU and thus potentially creating a
small topological error. The aim is to avoid generating too small HTUs on the main rivers. This algorithm is iterated until one of two conditions is met :
200 – the tributary has the 4th highest global flow accumulation. – The local upstream area is less than 10% of the grid area. 8 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. Figure 2. A sample case of a regular atmospheric grid decomposed into HTUS over the Rhone valley. a) The geographical context of the
Rhone at Valence, France. b) Flow directions and accumulation for the HDEM pixels overlapping the atmospheric grid. c) Resulting HTU
decomposition using nbmax = 18. d) As previously but for nbmax = 10. The white arrows within each HTU highlight the river segment
used to determine the geometric properties used to compute the residence time of the stream reservoir. 3.4
Sub-division of HTUs The grey dots indicate the location
where one HTU flows into the downstream one. In the two lower panels the red line highlights a sample river segment in the HDEM as is
used to statistically evaluate the river graphs (Section 4). The blue line is the corresponding river segment but now in HTU space. Figure 2. A sample case of a regular atmospheric grid decomposed into HTUS over the Rhone valley. a) The geographical context of the
Rhone at Valence, France. b) Flow directions and accumulation for the HDEM pixels overlapping the atmospheric grid. c) Resulting HTU
decomposition using nbmax = 18. d) As previously but for nbmax = 10. The white arrows within each HTU highlight the river segment
used to determine the geometric properties used to compute the residence time of the stream reservoir. The grey dots indicate the location
where one HTU flows into the downstream one. In the two lower panels the red line highlights a sample river segment in the HDEM as is
used to statistically evaluate the river graphs (Section 4). The blue line is the corresponding river segment but now in HTU space. Figure 2. A sample case of a regular atmospheric grid decomposed into HTUS over the Rhone valley. a) The geographical context of the
Rhone at Valence, France. b) Flow directions and accumulation for the HDEM pixels overlapping the atmospheric grid. c) Resulting HTU
decomposition using nbmax = 18. d) As previously but for nbmax = 10. The white arrows within each HTU highlight the river segment
used to determine the geometric properties used to compute the residence time of the stream reservoir. The grey dots indicate the location
where one HTU flows into the downstream one. In the two lower panels the red line highlights a sample river segment in the HDEM as is
used to statistically evaluate the river graphs (Section 4). The blue line is the corresponding river segment but now in HTU space. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. there is a set of small streams or subterranean flows contributing to the main stream of the HTU. Starting from the topographic
index computed on each pixel of the hydrological grid, sums are computed along all streams up to the outflow point of the
HTU. These sums are then averaged to provide an integrated property for the HTU. This allows to produce a λi which is
representative of the soils and hill-slopes surrounding the river. This was found to work best in the proposed set-up but is only
215
a crude simplification of what is known of hill-slope and riparian processes. If other variables of the LSM, soil moisture for instance, are also simulated at the HTU level then the hill-slope processes
which govern the riparian water exchanges could be parametrized. As suggested by Picourlat et al. (2022) the geometric
characteristics of the HTU and its soil moisture can be used to predict the flow of surface groundwater into the river. there is a set of small streams or subterranean flows contributing to the main stream of the HTU. Starting from the topographic
index computed on each pixel of the hydrological grid, sums are computed along all streams up to the outflow point of the
HTU. These sums are then averaged to provide an integrated property for the HTU. This allows to produce a λi which is
representative of the soils and hill-slopes surrounding the river. This was found to work best in the proposed set-up but is only
215
a crude simplification of what is known of hill-slope and riparian processes. If other variables of the LSM, soil moisture for instance, are also simulated at the HTU level then the hill-slope processes
which govern the riparian water exchanges could be parametrized. As suggested by Picourlat et al. (2022) the geometric
characteristics of the HTU and its soil moisture can be used to predict the flow of surface groundwater into the river. 3.5
Computing topographic indices The residence time of water in the stream reservoirs is given by the topographic index (λi,stream) and a constant (gstream) as
shown in equation 5. λi,stream is determined using equation 6 with the length and elevation change of the river between the
205
inflow pixel with the largest upstream area and the outflow of the HTU. If the HTU is a headwater, then the longest path is used
until an elevation change larger than 30% on the HDEM is reached when moving upstream. This avoids taking into account
the steepest parts of the catchment. The aim is to capture the geometrical feature of the main river flowing through the unit. In
Figure 2d) this would be the elevation change and length along the pixels with a white arrow within each HTU. For the fast and slow reservoirs, the topographic index λi should represent the general characteristics of the whole HTU and
210
its complexity when computing the outflow in equation 5. Therefore, another approach is used based on the hypothesis that For the fast and slow reservoirs, the topographic index λi should represent the general characteristics of the whole HTU and
210
its complexity when computing the outflow in equation 5. Therefore, another approach is used based on the hypothesis that 9 3.6
Reaggregation
220 At this stage, the atmospheric grid can contain more HTUs than requested by the user with the nbmax parameter. The user
may want to choose a simpler river network in order to reduce the memory footprint of the routing scheme. This depends
on the configuration best suited for the user’s need. But we would not recommend to select a values of nbmax below 8 on
a rectangular grid as then the number of outflow directions of the meshes is limited. The reaggregation step of the routing At this stage, the atmospheric grid can contain more HTUs than requested by the user with the nbmax parameter. The user
may want to choose a simpler river network in order to reduce the memory footprint of the routing scheme. This depends
on the configuration best suited for the user’s need. But we would not recommend to select a values of nbmax below 8 on
a rectangular grid as then the number of outflow directions of the meshes is limited. The reaggregation step of the routing pre-processor is to reduce the number of HTU on all grid points of the atmospheric to the nbmax value chosen by the user. The
225
merger of HTUs which will be performed by always favouring the largest HTUs or those with the largest upstream area and try
to preserve the diversity of outflow directions out of the atmospheric grid. The elimination of the smallest HTUs is performed
in 5 steps until the nbmax value is reached : 1. Merge all HTUs of an atmospheric grid which flows into the ocean, 1. Merge all HTUs of an atmospheric grid which flows into the ocean, 2. Merge HTUs which flow to the same neighbouring grid by starting with the smallest. This reduces the border noise by
merging the smallest HTUs which have been generated by the supermesh methodology. 2. Merge HTUs which flow to the same neighbouring grid by starting with the smallest. This reduces the border noise by
merging the smallest HTUs which have been generated by the supermesh methodology. 3. Merge HTUs which belong to the same river and flow out of the mesh. As this is also performed for HTUs flowing out
in different directions it generates topological errors. 4. Merge HTUs which flows into a downstream HTU within the same atmospheric grid, 4. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. 3.7
Positioning of gauging stations Stream gauging stations are a precious tools to validate the simulated water cycle of land system models at catchment scale. 245
For this reason, it is important to be able to localize these stations in the HTU space. Obviously, depending on the fidelity of the
river graph in the HTU space more or less stations can be reliably placed. The position of the stations will be made according
to their geographic position and the error in the upstream area within the model. The user can choose the maximum error in
distance and fraction of upstream area which can be tolerated. After the construction of the HTU graph, the pre-processor will attempt to place each stations within the tolerance selected
250
by the user. The errors will then be minimized to select the HTU which will be considered representative for the given station. This information will then be archived with the HTU network so that the land surface model can monitor the flow out of the
HTU corresponding to the station during the simulation. As the stations are placed in the HTU space, when the characteristics
of the graph change more or less stations can be positioned within the allowed errors. After the construction of the HTU graph, the pre-processor will attempt to place each stations within the tolerance selected
250
by the user. The errors will then be minimized to select the HTU which will be considered representative for the given station. This information will then be archived with the HTU network so that the land surface model can monitor the flow out of the
HTU corresponding to the station during the simulation. As the stations are placed in the HTU space, when the characteristics
of the graph change more or less stations can be positioned within the allowed errors. After the construction of the HTU graph, the pre-processor will attempt to place each stations within the tolerance selected
250
by the user. The errors will then be minimized to select the HTU which will be considered representative for the given station. This information will then be archived with the HTU network so that the land surface model can monitor the flow out of the
HTU corresponding to the station during the simulation. As the stations are placed in the HTU space, when the characteristics
of the graph change more or less stations can be positioned within the allowed errors. 4
Validation of HTU graph
255 A method is needed in order to statistically validate the quality of the HTU graph and identify the deviation from the original
HDEM induced by the reduction of effective resolution operated by the algorithm described above. As HTU graph construction
is designed to work without human supervision and at global scale, a visual inspection is not sufficient. The validation method samples randomly a large number of river segments which are representative of the network. The
length and elevation changes for each of these segments are computed on the HDEM and on the HTU graph to evaluate
260
the errors. The red line in Figures 2c) and d) represent one such segment. Its length and elevation change computed on the
HDEM by summing over all pixels it traverses is our reference. In HTU space, the river segment is represented by the blue
lines and illustrates the large differences with the select truncation (nbmax = 18 and 10 in Figures 2). The properties of the
stream within the HTU is given by the white arrows within the area to which the outflow points belongs (represented by a grey circle). The properties of the segment in HTU space can thus be computed by summing the HTU’s stream length and elevation
265
change along the blue lines. By comparing it to the reference the degradation of the stream properties in the HTU space can be
estimated. The errors of segment properties can be decomposed into a cell and a topological error. Within each HTU we can compare
the sub-segment’s properties to the one of the HTU. This will be called the cellular error. In Figure 2c) the properties of HTU circle). The properties of the segment in HTU space can thus be computed by summing the HTU’s stream length and elevation
265
change along the blue lines. By comparing it to the reference the degradation of the stream properties in the HTU space can be
estimated. The errors of segment properties can be decomposed into a cell and a topological error. Within each HTU we can compare
the sub-segment’s properties to the one of the HTU. This will be called the cellular error. In Figure 2c) the properties of HTU 8 are given by the white arrows and they are different from those of the red line. We will thus have a small cellular error here. 3.6
Reaggregation
220 Merge HTUs which flows into a downstream HTU within the same atmospheric grid, 5. Finally a brute-force method is used to merge the smaller HTUs until nbmax is reached. If this method has to be applied
35
to HTUs with an area higher than 5% of the grid box the user is warned that a higher nbmax should be considered. During the reaggregation step, the HTUs do not need to be connex when merged. This leads to situation like HTU 6 in
Figures 2d) which is composed of areas East and West of the Rhone. As highlighted in the discussion above, in order to simulate a realistic river discharge the length and slope of the main rivers As highlighted in the discussion above, in order to simulate a realistic river discharge the length and slope of the main rivers
need to be well preserved in the HTU decomposition. It is clear that if the truncation steps have to be carried too far because of
240
a poor choice by the user, a poor quality graph will be obtained and a reliable simulation of the stream flow cannot be expected. Below we will present a methodology which allows to verify the quality of the graph and estimate an optimal number of HTU
for a given atmospheric grid resolution. 10 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. This methodology is applied over the largest European rivers (Danube, Rhone, Rhine, Loire and Elbe) for the four atmo-
spheric grids presented above. The relative errors and their statistics are computed over a sample of 8000 segments. To ensure
a good representativeness the length of the segments is chosen between 100 and 1000km. To avoid unrepresentative starting
points, their upstream area should be at least of 10km2. Other values for these parameters of the sampling method were tested
but it did not change the results. 280 280 280 Figure 3. Figure provides the distribution of errors for the elevation change (dz) and length (len) for the 8000 samples over 7 different
truncations. The error bars represent the standard deviation of the error sample. Figure 3. Figure provides the distribution of errors for the elevation change (dz) and length (len) for the 8000 samples over 7 different
truncations. The error bars represent the standard deviation of the error sample. Figure 3 shows the dependence of the average relative errors with the chosen truncation (nbmax). The error bars in the
figure provide the variance of relative errors within the sample of river segments. The variance is driven by the diversity of
segments drawn in terms of length (affecting the relative errors) and the complexity of the hydrological structure. This analysis
is only displayed over 3 basins not to clutter the graphics too much. For the length of the segments the errors are within a Figure 3 shows the dependence of the average relative errors with the chosen truncation (nbmax). The error bars in the
figure provide the variance of relative errors within the sample of river segments. The variance is driven by the diversity of
segments drawn in terms of length (affecting the relative errors) and the complexity of the hydrological structure. This analysis
is only displayed over 3 basins not to clutter the graphics too much. For the length of the segments the errors are within a
range ±10% with the largest standard deviation found for the smallest truncations. For elevation change most relative errors
5
are smaller than 25% with again the largest spread obtained for nbmax = 10. The large variance of errors around the mean
imposes that variations of the mean relative errors need to be tested statistically. 4
Validation of HTU graph
255 270
In the downstream HTU (number 4) on the other hand, the cellular error is zero as the segment overlaps with the main river
(white arrows). The topological error describes differences in the path followed by the rivers. In Figure 2c) at nbmax = 18
the river La Barberolle passes through Valence on its way into the Rhone. For nbmax = 10 (Figure 2d)) this river flows into
another one south of the city before joining the Rhone. This is a topological error which is independent of the properties of 8 are given by the white arrows and they are different from those of the red line. We will thus have a small cellular error here. 270
In the downstream HTU (number 4) on the other hand, the cellular error is zero as the segment overlaps with the main river
(white arrows). The topological error describes differences in the path followed by the rivers. In Figure 2c) at nbmax = 18
the river La Barberolle passes through Valence on its way into the Rhone. For nbmax = 10 (Figure 2d)) this river flows into
another one south of the city before joining the Rhone. This is a topological error which is independent of the properties of
h f th HTU
A thi
i diffi
lt t
tif
it
ill b
ti
t d b
bt
ti
th
ll l
f
th t t l
275 each of the HTUs. As this error is difficult to quantify, it will be estimated by subtracting the cellular error from the total error. 275 11 To this end the mean errors are compared to
the next higher truncation using a simple t-test for two independent samples. This will allow to show if increasing the number
of HTUs used to represent the rivers significantly improves the quality of the graph or not. range ±10% with the largest standard deviation found for the smallest truncations. For elevation change most relative errors
285
are smaller than 25% with again the largest spread obtained for nbmax = 10. The large variance of errors around the mean
imposes that variations of the mean relative errors need to be tested statistically. To this end the mean errors are compared to
the next higher truncation using a simple t-test for two independent samples. This will allow to show if increasing the number
of HTUs used to represent the rivers significantly improves the quality of the graph or not. 285 Let us now analyse how the quality of the HTU graph depends on the resolution of the atmospheric grid and the maximum
290
number of HTUs allowed per grid (nbmax). First we examine in figure 4a) the evolution of the mean relative error with
increasing nbmax on the coarsest grid (WFDEI). For the lowest truncation (nbmax = 10) on the Rhine the mean segment
elevations changes (dz) are lower than those in the HDEM by about 10%. This error is larger at the cell level (dashed lines),
meaning that on average the dz of the HTUs are smaller than the corresponding sub-segment by over 15%. This difference of Let us now analyse how the quality of the HTU graph depends on the resolution of the atmospheric grid and the maximum
290
number of HTUs allowed per grid (nbmax). First we examine in figure 4a) the evolution of the mean relative error with
increasing nbmax on the coarsest grid (WFDEI). For the lowest truncation (nbmax = 10) on the Rhine the mean segment
elevations changes (dz) are lower than those in the HDEM by about 10%. This error is larger at the cell level (dashed lines),
meaning that on average the dz of the HTUs are smaller than the corresponding sub-segment by over 15%. This difference of 12 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. (a) MERIT on WFDEI grid
(b) MERIT on MEDCORDEX grid
(c) MERIT on EuroMED grid
(d) HydroSHEDS on MEDCORDEX grid
Figure 4. It can also be noted that from this truncation onward the mean relative errors in elevation changes and segment length
are stable and small. the optimum is obtained for nbmax = 35. Above this value increasing truncation does not significantly change the mean error
300
any more according to the t-test. At this point we have enough HTUs per 0.5◦grid box that the only error in the selected river
segments are those linked to the fact that the HTU’s properties are slightly different from the elements of the segments crossing
them. It can also be noted that from this truncation onward the mean relative errors in elevation changes and segment length
are stable and small. Figure 4c) shows the same results for the highest resolution considered here (11km). The mean relative errors in dz and len
305
are smaller than the coarser grid considered above, also for high nbmax values. It also demonstrates that the convergence of
the cell and total relative errors occurs already at nbmax = 15. The higher atmospheric resolution facilitates the aggregation
of the hydrological information of the MERIT HDEM which is at 60arcsec resolution. There are about 8 grid cells of the
EuroMED grid in each WFDI grid. But we can obtain similar qualities for the HTU graph in WFDEI with only a little more Figure 4c) shows the same results for the highest resolution considered here (11km). The mean relative errors in dz and len
305
are smaller than the coarser grid considered above, also for high nbmax values. It also demonstrates that the convergence of
the cell and total relative errors occurs already at nbmax = 15. The higher atmospheric resolution facilitates the aggregation
of the hydrological information of the MERIT HDEM which is at 60arcsec resolution. There are about 8 grid cells of the
EuroMED grid in each WFDI grid. But we can obtain similar qualities for the HTU graph in WFDEI with only a little more than twice the number of HTUs. This shows that the algorithm to aggregate the hydrological information at the atmospheric
310
grid captures well the main features which determine the flow of water along the network. Obviously, would the HDEM be of
higher resolution (MERIT is available at 90m) then also for the EuroMED grid more HTUs would be needed to describe the
network. For the MEDCORDEX grid at 20km we have build the HTU network with two different HDEMs. For the MERIT HDEM For the MEDCORDEX grid at 20km we have build the HTU network with two different HDEMs. For the MERIT HDEM
(60arcsec resolution) the convergence of cell and total relative errors occurs at nbmax = 25 and are better than 5% for both
315
the elevation change and length of samples (Figure 4b). When HydroSHEDS (30arcsec resolution) is used the behaviour
changes slightly. Let us first examine the errors for the length. The convergence of errors occurs at nbmax = 35. Because of
the higher resolved hydrological information more HTUs are required for its proper representation. For the segment length,
the mean errors are similar than for the MERIT HDEM. The situation is more complex for the elevation changes of the (60arcsec resolution) the convergence of cell and total relative errors occurs at nbmax = 25 and are better than 5% for both
315
the elevation change and length of samples (Figure 4b). When HydroSHEDS (30arcsec resolution) is used the behaviour
changes slightly. Let us first examine the errors for the length. The convergence of errors occurs at nbmax = 35. Because of
the higher resolved hydrological information more HTUs are required for its proper representation. For the segment length,
the mean errors are similar than for the MERIT HDEM. The situation is more complex for the elevation changes of the segments. Because HydroSHEDS does not provide a hydrologically corrected topography, the errors are much larger than
320
for MERIT (Yamazaki et al., 2012). When following downstream the segments in HydroSHEDS a number of situations are
encountered where the elevation change is negative, i.e. water flow into a pixel with a higher elevation. For shorter segments
this occurs for about 20% of the pixels while for longer segments, which are more likely to traverse flat areas, it can reach
30%. On the HDEM, for the full segment, these errors compensate and the total elevation change can be assumed to be correct. segments. Because HydroSHEDS does not provide a hydrologically corrected topography, the errors are much larger than
320
for MERIT (Yamazaki et al., 2012). When following downstream the segments in HydroSHEDS a number of situations are
encountered where the elevation change is negative, i.e. water flow into a pixel with a higher elevation. Error decomposition for the segment’s elevation changes and length for 5 rivers at 3 different atmospheric resolutions and tw
different HDEMs (b) MERIT on MEDCORDEX grid (b) MERIT on MEDCORDEX grid (a) MERIT on WFDEI grid (a) MERIT on WFDEI grid
(c) MERIT on EuroMED grid (d) HydroSHEDS on MEDCORDEX grid (c) MERIT on EuroMED grid Figure 4. Error decomposition for the segment’s elevation changes and length for 5 rivers at 3 different atmospheric resolutions and two
different HDEMs 13 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. errors is explained by the fact that on average the connections between HTUs are further downstream than on the HDEM, thus
295
compensating partly the cellular error. This large difference between the total and cell mean errors is not an ideal solution as it
means that the actual course of the river is not well respected by the cascade of HTUs. The same behaviour is also found for
the length of the river segments with an even larger compensation by the topology of the river. Figure 4a) also demonstrates
that as nbmax increases the total mean error reduces and is overwhelmingly explained by the cell error. For the WFDEI grid errors is explained by the fact that on average the connections between HTUs are further downstream than on the HDEM, thus
295
compensating partly the cellular error. This large difference between the total and cell mean errors is not an ideal solution as it
means that the actual course of the river is not well respected by the cascade of HTUs. The same behaviour is also found for
the length of the river segments with an even larger compensation by the topology of the river. Figure 4a) also demonstrates
that as nbmax increases the total mean error reduces and is overwhelmingly explained by the cell error. For the WFDEI grid the optimum is obtained for nbmax = 35. Above this value increasing truncation does not significantly change the mean error
300
any more according to the t-test. At this point we have enough HTUs per 0.5◦grid box that the only error in the selected river
segments are those linked to the fact that the HTU’s properties are slightly different from the elements of the segments crossing
them. It can also be noted that from this truncation onward the mean relative errors in elevation changes and segment length
are stable and small. the optimum is obtained for nbmax = 35. Above this value increasing truncation does not significantly change the mean error
300
any more according to the t-test. At this point we have enough HTUs per 0.5◦grid box that the only error in the selected river
segments are those linked to the fact that the HTU’s properties are slightly different from the elements of the segments crossing
them. For shorter segments
this occurs for about 20% of the pixels while for longer segments, which are more likely to traverse flat areas, it can reach
30%. On the HDEM, for the full segment, these errors compensate and the total elevation change can be assumed to be correct. But when decomposing it into pieces the compensations can be interrupted. Furthermore when constructing the HTUs we have
325
imposed that dz cannot be smaller than 0.1m thus creating a large discrepancy with the segment as defined in the HDEM. Figure 4d) shows that for rivers with large flat areas such as the Danube the error is largest while it is relatively small for
catchments dominated by mountainous areas such as the Rhone. But when decomposing it into pieces the compensations can be interrupted. Furthermore when constructing the HTUs we have
325
imposed that dz cannot be smaller than 0.1m thus creating a large discrepancy with the segment as defined in the HDEM. Figure 4d) shows that for rivers with large flat areas such as the Danube the error is largest while it is relatively small for
catchments dominated by mountainous areas such as the Rhone. 14 (a) WFDEI grid and MERIT
(b) EuroMED grid and MERIT
Figure 5. Distribution of errors along the upstream area (or fetch) of the starting point of the segments for the Danube. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. (a) WFDEI grid and MERIT
(b) EuroMED grid and MERIT
Figure 5. Distribution of errors along the upstream area (or fetch) of the starting point of the segments for the Danube. (b) EuroMED grid and MERIT (a) WFDEI grid and MERIT (b) EuroMED grid and MERIT Figure 5. Distribution of errors along the upstream area (or fetch) of the starting point of the segments for the Danube. To determine the main sources of errors in our samples, their distributions relative to th To determine the main sources of errors in our samples, their distributions relative to the upstream area of the initial point
are analysed for the Danube. Figure 5 displays these distributions for the coarsest and finest grids analysed here. First it has to
330
be observed that because of the dominance of small sub-catchments in any river basin, the sample of segments favours starting
points with about 100km2 of fetch. Again we can see for which nbmax the cell and total errors converge but also that the
higher truncations are less needed for larger basins as the convergence there is faster. The mean error in segment length is stable
with changing upstream area. On the coarse grid we can note that for small rivers (fetch < 300m2) the dz error is relatively large. This error is amplified by sampling bias and thus has a major contribution to the mean error. This error in dz for small
335
catchments is consistent with the hypothesis of the decomposition algorithm which favours the properties of the largest river
in a grid box to guide the graph construction. This leads to small catchments having a higher probability to display segment
properties that do not match those of the HTU. large. This error is amplified by sampling bias and thus has a major contribution to the mean error. This error in dz for small
335
catchments is consistent with the hypothesis of the decomposition algorithm which favours the properties of the largest river
in a grid box to guide the graph construction. This leads to small catchments having a higher probability to display segment
properties that do not match those of the HTU. It must be noted that for the EuroMED grid the error in dz increases with the size of the upstream area. This is simply
because segments with large upstream areas are more likely to cross flat areas of the Danube basin. The distribution of segment
340
errors was also computed against the length of the sample (not shown). For both the length and elevation change errors it is
found that they are quite constant except for short segments. As they are likely to have also small upstream areas they display
a relatively large cell error. In conclusion of this section we provide the optimal truncation for each grid used here (Table 3) The values for nbmax are because segments with large upstream areas are more likely to cross flat areas of the Danube basin. The distribution of segment
340
errors was also computed against the length of the sample (not shown). https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. Table 3. The atmospheric grids and land/sea masks for which HTU graphs were built to test the methodology. If not specified otherwise the
MERIT HDEM is used. grid name
Resolution
Optimal truncation for MERIT (nbmax)
Optimal time step [s]
WFDEI
0.5◦
35
1800
E2OFD
0.25◦
15
900
MEDCORDEX
20km
12
900
HydroSHEDS = 25
450
EuroCORDEX
11km
10
450 grid name
Resolution
Optimal truncation for MERIT (nbmax)
Optimal time step [s] 5
Numerical implementation For the constructed HTU graphs the constants gX, which are the inverse of a velocity, need t For the constructed HTU graphs the constants gX, which are the inverse of a velocity, need to be estimated. Tests with OR-
CHIDEE have shown that for the two high resolution HDEMs the same values can be used and for the coarser one the constant CHIDEE have shown that for the two high resolution HDEMs the same values can be used and for the coarser one the constant
for the stream needs to be changed slightly (see Table 2)(Schrapffer, 2022). It is quite likely that these parameters are more
350
dependent on the land surface scheme used here than the HDEM. The repartition of water fluxes between surface runoff (Rbi)
and drainage (Dbi) as well as the inertia of the soil moisture model will play a key role for these constants. Thus the values
given in Table 2 are probably only valid for ORCHIDEE’s current soil moisture model (de Rosnay et al., 2002; d’Orgeval
et al., 2008) in its 2m depth configuration. Using the HTU graphs produced here in any other land surface model will probably
i
t
dj
t th
t
355 for the stream needs to be changed slightly (see Table 2)(Schrapffer, 2022). It is quite likely that these parameters are more
350
dependent on the land surface scheme used here than the HDEM. The repartition of water fluxes between surface runoff (Rbi)
and drainage (Dbi) as well as the inertia of the soil moisture model will play a key role for these constants. Thus the values
given in Table 2 are probably only valid for ORCHIDEE’s current soil moisture model (de Rosnay et al., 2002; d’Orgeval
et al., 2008) in its 2m depth configuration. Using the HTU graphs produced here in any other land surface model will probably require to readjust these parameters. 355
The water continuity (Eq 5) is discretized in time using an explicit numerical scheme. This means that the fluxes Qj,X are
evaluated before the reservoir content (Wi,X) are updated. This choice simplifies the implementation and the interaction with
the parallelisation of the ORCHIDEE code. The heat transport equation 9 is also implemented using an explicit method. In
order for the relaxation term to be able to efficiently avoid the singularity of empty reservoirs it is evaluated implicitly. For both the length and elevation change errors it is
found that they are quite constant except for short segments. As they are likely to have also small upstream areas they display
a relatively large cell error. In conclusion of this section we provide the optimal truncation for each grid used here (Table 3). The values for nbmax are In conclusion of this section we provide the optimal truncation for each grid used here (Table 3). The values for nbmax are
derived from Figure 4 as the point where reductions in errors in the graph are not any more justified by the extra computational
345
cost. derived from Figure 4 as the point where reductions in errors in the graph are not any more justified by the extra computational
345
cost. 15 5
Numerical implementation This q
j
p
The water continuity (Eq 5) is discretized in time using an explicit numerical scheme. This means that the fluxes Qj,X are
evaluated before the reservoir content (Wi,X) are updated. This choice simplifies the implementation and the interaction with
the parallelisation of the ORCHIDEE code. The heat transport equation 9 is also implemented using an explicit method. In
order for the relaxation term to be able to efficiently avoid the singularity of empty reservoirs it is evaluated implicitly. This
flux will thus depend on the current upper soil temperature and the stream temperature at the next time step. 360 flux will thus depend on the current upper soil temperature and the stream temperature at the next time step. 360
The Courant–Friedrichs–Lewy (CFL) condition mandates that for a convergence of the numerical solution the time step
needs to be smaller or equal to the residence time (λi,Xgi,X) of the water in the fastest reservoir of the HTU : the stream. Given the decomposition of the atmospheric grid into HTUs for a finer representation of the hydrological connectivity, a wide
distribution of residence times will be obtained with some values being very short. It is thus not practical to select a time step for the routing scheme based on the smallest residence time on the domain. The scheme thus needs to be able to cope with
365
unstable flux calculations. To this end the numerical solution for the continuity equation (Eq 5) also includes a flux limiter
given by : Qj,X ≤Wi,X∆t. This condition should only activate for reservoirs with low water contents and short residence
times. As this flux limiter also determines the numerical quality of the simulated discharge, the model monitors how often this
condition has to be imposed. A practical solution for choosing the time step of the routing scheme is to select a position within the area weighted distri-
370
bution of residence times of all HTUs within the computational domain. It is proposed to select the time step corresponding A practical solution for choosing the time step of the routing scheme is to select a position within the area weighted distri-
370
bution of residence times of all HTUs within the computational domain. These metrics are computed
with daily values over an 11 year period going from 1983 to 1993. The list of these stations is provided in Appendix A. At each of these points in the graph, the simulated river discharge and
390
temperature are evaluated against a reference configuration. The metrics used to quantify the change of behaviour of the model
are the Nash–Sutcliffe model efficiency (NSE), the correlation and ratio of standard deviation. These metrics are computed
with daily values over an 11 year period going from 1983 to 1993. 5.1
Role of the time step The length of the time step is first tested on the graph produced with MERIT for the WFDEI grid using nbmax = 35 based on
395
the evaluation in section 4. The time step is progressively increased from 225s to 5h and the smallest value is considered to be
our reference. As a first step, we examine the fraction of HTUs where the flux limiter has to be applied to stream flow. As this variable is
quite constant throughout the period analysed, only the annual mean is shown in figure 6 with the selected stations ordered by The length of the time step is first tested on the graph produced with MERIT for the WFDEI grid using nbmax = 35 based on
395
the evaluation in section 4. The time step is progressively increased from 225s to 5h and the smallest value is considered to be
our reference. As a first step, we examine the fraction of HTUs where the flux limiter has to be applied to stream flow. As this variable is
quite constant throughout the period analysed, only the annual mean is shown in figure 6 with the selected stations ordered by The length of the time step is first tested on the graph produced with MERIT for the WFDEI grid using nbmax = 35 based on
395
the evaluation in section 4. The time step is progressively increased from 225s to 5h and the smallest value is considered to be
our reference. As a first step, we examine the fraction of HTUs where the flux limiter has to be applied to stream flow. As this variable is
quite constant throughout the period analysed, only the annual mean is shown in figure 6 with the selected stations ordered by up-stream area along the x-axis. In the y direction the tested time steps are plotted and each dot measures the quality of one
400
simulation compared to the reference. This graphic demonstrates that the flux limiter applies to all catchments independently
of their size but the frequency of its activation is a strong function of the time step used. The horizontal line shows the time step
at which 75% of HTU satisfy the CFL criteria. At this level the flux limiter has to be applied to less than 20% of the HTUs. 5
Numerical implementation It is proposed to select the time step corresponding A practical solution for choosing the time step of the routing scheme is to select a position within the area weighted distri-
370
bution of residence times of all HTUs within the computational domain. It is proposed to select the time step corresponding 16 To focus on the numerical properties of the
routing scheme, a version decoupled from ORCHIDEE is used which is directly forced with daily mean surface runoff, drainage
and temperature profiles extracted from a previous WFDEI-GPCC (Weedon et al., 2014) forced simulation of ORCHIDEE. These fields have been interpolated to the selected time-step for the routing and to the spatial resolution of the atmospheric grid on which the routing scheme is to be evaluated. 385
These simulations are evaluated at a limited number of gauging stations which cover a range of upstream areas and climates. From the over 3800 stations placed on the river graphs of the Euro-Mediterranean region only a few were selected with
upstream areas ranging from 2500 to 8105 km2. The lower limit is given by the spatial resolution of the WFDEI forcing and
ensures that the smallest catchments contain at least one atmospheric grid box. The largest catchment is the one of the Danube. on which the routing scheme is to be evaluated. 385
These simulations are evaluated at a limited number of gauging stations which cover a range of upstream areas and climates. From the over 3800 stations placed on the river graphs of the Euro-Mediterranean region only a few were selected with
upstream areas ranging from 2500 to 8105 km2. The lower limit is given by the spatial resolution of the WFDEI forcing and
ensures that the smallest catchments contain at least one atmospheric grid box. The largest catchment is the one of the Danube. on which the routing scheme is to be evaluated. 385
These simulations are evaluated at a limited number of gauging stations which cover a range of upstream areas and climates. From the over 3800 stations placed on the river graphs of the Euro-Mediterranean region only a few were selected with
upstream areas ranging from 2500 to 8105 km2. The lower limit is given by the spatial resolution of the WFDEI forcing and
ensures that the smallest catchments contain at least one atmospheric grid box. The largest catchment is the one of the Danube. The list of these stations is provided in Appendix A. At each of these points in the graph, the simulated river discharge and
390
temperature are evaluated against a reference configuration. The metrics used to quantify the change of behaviour of the model
are the Nash–Sutcliffe model efficiency (NSE), the correlation and ratio of standard deviation. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. to the 25% quantile of this distribution. This means that 75% of all HTU by area will not violate the CFL criteria while for
the others there is a risk that the solution will not be correct. As a consequence it needs to be verified that this compromise on
the quality of the numerical solution on some HTUs will not affect the simulated discharge at the spatial scales of interest. We
found that the time steps determined with this method are more dependent on the atmospheric resolution than the hydrological
375
truncation. This is quite convenient as with a refining of the atmospheric grid the time step of the processes which have the
land surface scheme as a lower boundary will also decrease. Thus increasing the frequency at which the routing scheme will
need to be called is not a strong constraint. found that the time steps determined with this method are more dependent on the atmospheric resolution than the hydrological
375
truncation. This is quite convenient as with a refining of the atmospheric grid the time step of the processes which have the
land surface scheme as a lower boundary will also decrease. Thus increasing the frequency at which the routing scheme will
need to be called is not a strong constraint. This section explores the convergence of the simulated discharge with the selected time step (∆t), the number of HTUs
(nbmax) and the resolution of the atmospheric grid. The analysis is performed on a number of simulations over the Euro-
380
Mediterranean domain discretized with the atmospheric grids presented in Table 1. To focus on the numerical properties of the
routing scheme, a version decoupled from ORCHIDEE is used which is directly forced with daily mean surface runoff, drainage
and temperature profiles extracted from a previous WFDEI-GPCC (Weedon et al., 2014) forced simulation of ORCHIDEE. These fields have been interpolated to the selected time-step for the routing and to the spatial resolution of the atmospheric grid
on which the routing scheme is to be evaluated
385 (nbmax) and the resolution of the atmospheric grid. The analysis is performed on a number of simulations over the Euro-
380
Mediterranean domain discretized with the atmospheric grids presented in Table 1. 5.1
Role of the time step Thus, when looking at the convergence of the solution with the time step we have to consider that not only the CFL criteria
deteriorates the solution but also the flux limiter
405 17 Figure 6. At each station and for the range of time steps tested, the fraction of HTUs for which the flux limiter has been activated in the
upstream catchment. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. Figure 6. At each station and for the range of time steps tested, the fraction of HTUs for which the flux limiter has been activated in the
upstream catchment. Figure 6. At each station and for the range of time steps tested, the fraction of HTUs for which the flux limiter has been activated in the
upstream catchment. Figure 6. At each station and for the range of time steps tested, the fraction of HTUs for which the flux limiter has been activated in the
upstream catchment. Figure 7 compares for the same set of experiments but now examining the degradation of discharge and stream temperature
with time step when compared to the shortest value (∆t = 225s) using the three metrics discussed above. The Nash–Sutcliffe
model efficiency is close to one for all simulations performed with time steps close to or lower to the recommended value
(horizontal line). The correlation between the reference and the test simulation remains close to one as well as the ratio model efficiency is close to one for all simulations performed with time steps close to or lower to the recommended value
(horizontal line). The correlation between the reference and the test simulation remains close to one as well as the ratio
of variance. In this range of time steps we can consider that the numerical solution has converged for catchments larger than
410
2500km2. Once the 25% quantile of residence times is passed some stations show quick degradation in the simulated discharge
and we can note that generally small catchments are more sensitive than larger ones. On the most downstream stations of the
Danube (to the right of the x-axis) the degradation only becomes apparent for time steps of a few hours. For smaller catchments
this can occur much earlier. 5.1
Role of the time step This degradation is not a linear function of up-stream area as it also depends on the complexity of variance. In this range of time steps we can consider that the numerical solution has converged for catchments larger than
410
2500km2. Once the 25% quantile of residence times is passed some stations show quick degradation in the simulated discharge
and we can note that generally small catchments are more sensitive than larger ones. On the most downstream stations of the
Danube (to the right of the x-axis) the degradation only becomes apparent for time steps of a few hours. For smaller catchments
this can occur much earlier. This degradation is not a linear function of up-stream area as it also depends on the complexity 410 of the river graph and the topography. The degradation of the river discharge is manifested by a reduction of the correlation of
415
daily discharge values with respect to the reference and a decrease of variance. The peak discharges are more strongly affected
by increasing time steps than the base flows. Th
i ht
l
i Fi
7 h
th t t
t
t
i
h l
iti
th
di
h
t i
i
ti
t of the river graph and the topography. The degradation of the river discharge is manifested by a reduction of the correlation of
415
daily discharge values with respect to the reference and a decrease of variance. The peak discharges are more strongly affected
by increasing time steps than the base flows. The right column in Figure 7 shows that stream temperature is much less sensitive than discharge to increasing time steps. This is not surprising given that the rivers have relatively slow fluctuating temperatures. The mass flux is thus the driving by increasing time steps than the base flows. The right column in Figure 7 shows that stream temperature is much less sensitive than discharge to increasing time steps. This is not surprising given that the rivers have relatively slow fluctuating temperatures. The mass flux is thus the driving The right column in Figure 7 shows that stream temperature is much less sensitive than discharge to increasing time steps. This is not surprising given that the rivers have relatively slow fluctuating temperatures. 5.1
Role of the time step The mass flux is thus the driving
constraint for the temporal discretization of the routing scheme
420 The right column in Figure 7 shows that stream temperature is much less sensitive than discharge to increasing time steps. This is not surprising given that the rivers have relatively slow fluctuating temperatures. The mass flux is thus the driving
constraint for the temporal discretization of the routing scheme. 420 constraint for the temporal discretization of the routing scheme. 420
Figure 8 shows only the NSE metric but now for three atmospheric grids and in one case for HTU graphs built with a different
HDEM (HydroSHEDS). The results are very similar to the ones presented above. We can conclude that the 25% quantile of
the residence time is a quite robust method to determine the time step which satisfies the CFL criteria and keeps the flux
limiter to an acceptable level. Comparing the figures in the upper row shows that the criteria recommends a shorter time step constraint for the temporal discretization of the routing scheme. 420
Figure 8 shows only the NSE metric but now for three atmospheric grids and in one case for HTU graphs built with a different
HDEM (HydroSHEDS). The results are very similar to the ones presented above. We can conclude that the 25% quantile of
the residence time is a quite robust method to determine the time step which satisfies the CFL criteria and keeps the flux
limiter to an acceptable level. Comparing the figures in the upper row shows that the criteria recommends a shorter time step for the HydroSHEDS HDEM (See table 3) as it provides hydrological information at 30arcsec resolution. The degradation of
425
the comparison to the reference occurs for longer time steps than for MERIT although we are on the same atmospheric grid
(MEDCORDEX). This can probably be traced back to the quality of the hydrological information provided by both HDEMs. In both cases the methodology for selecting the time step seems to be too conservative on this 20km grid. This results is 18 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. (a) Discharge WFDEI MERIT
(b) Stream temperature MERIT
Figure 7. Comparing simulations on the WFDEI grid (with the MERIT HDEM and nbmax=35) with increasing time step to the reference
at ∆t = 225s. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. 5.2
Role of truncation in HTU space For MERIT on the WFDEI grid, the quality
of the segments starts to decrease for nbmax below 30. This point is not as clearly visible here as the analysed stations all have a relatively large up-stream area. As demonstrated with Figure 5 the errors in the river segments are more pronounced for
450
catchments smaller than 104 km2. To demonstrate the loss of numerical convergence for discharge caused by a coarse HTU
graph stations in small catchments would need to be sampled. But this would not be consistent with a forcing at 0.5◦resolution. have a relatively large up-stream area. As demonstrated with Figure 5 the errors in the river segments are more pronounced for
450
catchments smaller than 104 km2. To demonstrate the loss of numerical convergence for discharge caused by a coarse HTU
graph stations in small catchments would need to be sampled. But this would not be consistent with a forcing at 0.5◦resolution. 5.3
Role of the resolution of the atmospheric grid Based on the analysis of the graphs presented in Section 4 an optimal value for nbmax a Based on the analysis of the graphs presented in Section 4 an optimal value for nbmax and time step has been selected for
each combination of atmospheric grid and HDEM (See Table 3). The objective here is to determine how sensitive the simulated
455
discharge is to the atmospheric grid given the same runoff and discharge fields. As they originate from a WFDEI simulation on
the 0.5◦grid this will be used as our reference here.i Based on the analysis of the graphs presented in Section 4 an optimal value for nbmax and time step has been selected for Based on the analysis of the graphs presented in Section 4 an optimal value for nbmax and time step has been selected for
each combination of atmospheric grid and HDEM (See Table 3). The objective here is to determine how sensitive the simulated
455
discharge is to the atmospheric grid given the same runoff and discharge fields. As they originate from a WFDEI simulation on
the 0.5◦grid this will be used as our reference here. Figure 10 shows for the selected stations the deviation of the three other configurations from the WFDEI-MERIT case. One
can note that the changes in the three metrics used are generally small. There is no systematic dependence of the numerical Figure 10 shows for the selected stations the deviation of the three other configurations from the WFDEI-MERIT case. One
can note that the changes in the three metrics used are generally small. There is no systematic dependence of the numerical i
can note that the changes in the three metrics used are generally small. There is no systematic dependence of the numerical
quality with the grid or catchment size even though the resolutions and projections are quite different. One may note that there
460
is systematic degradation of the NSE efficiency, correlation and ratio of standard deviation when changing the HDME on the
MEDCORDEX grid. The MEDCORDEXHS simulation differ particularly for stations with a catchment larger than 7104km2. This can be expected as it is based on another description of the topography and river flow directions than the reference. Difference accumulate with the size of the basin. When only the resolution changes (E2OFD, MEDCORDEX, EuroMED), the
impact on the simulated variables is low except for a few stations. 5.1
Role of the time step (a) Discharge WFDEI MERIT (b) Stream temperature MERIT ations on the WFDEI grid (with the MERIT HDEM and nbmax=35) with increasing time step to the referen Figure 7. Comparing simulations on the WFDEI grid (with the MERIT HDEM and nbmax=35) with increasing time step to the reference
t ∆t
225 Figure 7. Comparing simulations on the WFDEI grid (with the MERIT HDEM and nbmax=35) with increasing time step to the reference
at ∆t = 225s. (b) HydroSHEDS on MEDCORDEX grid (a) MERIT on the MEDCORDEX grid
(b) HydroSHEDS on MEDCORDEX grid
(c) MERIT on E2OFD grid
(d) MERIT on EuroMED grid
Figure 8. Convergence of simulated discharge with increasing time steps on the four different grids presented in Table 1 and the two HDEM
for the MEDCORDEX grid. Only the Nash-Sutcliffe efficiency is displayed. (a) MERIT on the MEDCORDEX grid (b) HydroSHEDS on MEDCORDEX grid
(d) MERIT on EuroMED grid
ps on the four different grids presented in Table 1 and the two HDEM (a) MERIT on the MEDCORDEX grid (b) HydroSHEDS on MEDCORDEX grid (b) HydroSHEDS on MEDCORDEX grid
(d) MERIT on EuroMED grid (c) MERIT on E2OFD grid Figure 8. Convergence of simulated discharge with increasing time steps on the four different grids presented in Table 1 and the two HDEM
for the MEDCORDEX grid. Only the Nash-Sutcliffe efficiency is displayed. for the MEDCORDEX grid. Only the Nash-Sutcliffe efficiency is displayed. confirmed on the E2OFD and EuroMED grids as the degradation of the numerical solution occurs for time steps larger than the
recommended value. But it has to kept in mind that the runoff forcing used here is an extrapolation of data from a 0.5◦grid and
430
thus it is of lower temporal and spatial variability than one would expect at the higher resolutions presented here. Furthermore
on the higher resolution grids, given the appropriate atmospheric forcing, the user would want to analyse smaller catchments
and more extreme hydrological events. recommended value. But it has to kept in mind that the runoff forcing used here is an extrapolation of data from a 0.5◦grid and
430
thus it is of lower temporal and spatial variability than one would expect at the higher resolutions presented here. Furthermore
on the higher resolution grids, given the appropriate atmospheric forcing, the user would want to analyse smaller catchments
and more extreme hydrological events. 430 19 5.2
Role of truncation in HTU space The same type of analysis can be performed to explore the impact of the number of HTUs used per grid box on the time
435
step. For each case we use the time step proposed by the 25% quantile of the residence time distribution. As the discharge is
evaluated at gauging stations, when nbmax is modified the position of the station within the graph can also be affected. Thus
the results presented here not only evaluate the impact of the truncation on the simulated discharge but also the positioning of
the sampled point. These two factors are not independent and both affect our ability to reproduce observed river flows. The y-axis of Figure 9 now shows the maximum number of HTUs per atmospheric grid ranging from 10 to 45. The config-
440
uration nbmax = 55 is considered as our reference here. The graphics show that the truncation is of less consequence to the
quality of stream levels and temperature, within the range considered here, than the time step. For the smallest value of nbmax
the NSE only falls to 0.75. In none of the three metrics considered here there is an indication that the result depends on catch-
ment size. If one were to consider maximum number of HTUs lower than 8, then not all outflow directions of the rectangular atmospheric grids can be represented and the impact on the simulation would probably be more severe. These extremely low
445
values of nbmax were not considered as they defeat the point of a sub-grid approach to routing. All stations where a significant degradation of the simulation occur, it takes place below nbmax = 35. This result is consis-
tent with the statistical analysis of the river graphs presented above (Section 4). For MERIT on the WFDEI grid, the quality
of the segments starts to decrease for nbmax below 30. This point is not as clearly visible here as the analysed stations all atmospheric grids can be represented and the impact on the simulation would probably be more severe. These extremely low
445
values of nbmax were not considered as they defeat the point of a sub-grid approach to routing. All stations where a significant degradation of the simulation occur, it takes place below nbmax = 35. This result is consis-
tent with the statistical analysis of the river graphs presented above (Section 4). https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. 5.4
Convergence of the numerical solutions It is an important result that for an optimal truncation and time step selected according to the criteria defined above, the
simulated stream flows and temperatures are relatively insensitive to the atmospheric grid on which the climatic forcing is
470
provided. This removes the necessity to adjust the parameters gX of the routing scheme when running at different resolutions. Nevertheless, some caveats have to be kept in mind. The numerical verifications presented above were performed using
output from an ORCHIDEE simulation forced by the WFDEI-GPCC forcing at 0.5◦resolution. Because of its 3 hourly forcing
there is little diurnal variability in the surface runoff and generally the spatial variance is limited by the coarse atmospheric information. The higher resolutions evaluated here were run with variables which do not include all the spatial and temporal
475
variability which would be expected. Thus, when using the routing at high resolution within regional Earth system models the
criteria for selecting the time step might need to be reduced. In particular when moving to convection permitting resolutions
when the intensity of simulated rainfall will modify the behaviour of the land surface model and thus the dynamics of the fluxes
which feed river flows. It is nevertheless reassuring to see in the analysis presented above that for higher resolutions a margin information. The higher resolutions evaluated here were run with variables which do not include all the spatial and temporal
475
variability which would be expected. Thus, when using the routing at high resolution within regional Earth system models the
criteria for selecting the time step might need to be reduced. In particular when moving to convection permitting resolutions
when the intensity of simulated rainfall will modify the behaviour of the land surface model and thus the dynamics of the fluxes
which feed river flows. It is nevertheless reassuring to see in the analysis presented above that for higher resolutions a margin exists for the time step. 480
Once reliable km-scale atmospheric forcing are available the numerical analysis presented here should be revisited with
a particular attention to flood events in small catchments. It will allow to evaluate at which stage a smaller quantile in the
residence time should be selected and how important this choice is for representing the extreme hydrological events we are
interested in. 5.3
Role of the resolution of the atmospheric grid For the E2OFD configuration the station Rekingen on the
465 quality with the grid or catchment size even though the resolutions and projections are quite different. One may note that there
460
is systematic degradation of the NSE efficiency, correlation and ratio of standard deviation when changing the HDME on the
MEDCORDEX grid. The MEDCORDEXHS simulation differ particularly for stations with a catchment larger than 7104km2. This can be expected as it is based on another description of the topography and river flow directions than the reference. Difference accumulate with the size of the basin. When only the resolution changes (E2OFD, MEDCORDEX, EuroMED), the
impact on the simulated variables is low except for a few stations For the E2OFD configuration the station Rekingen on the
465 20 (a) Discharge WFDEI MERIT
(b) Stream temperature MERIT
Figure 9. Simulations on the WFDEI grid (with the MERIT HDEM and ∆t = 900s) with increasing maximum number of HTU are com-
pared to the reference which is chosen to have nbmax = 55. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. (a) Discharge WFDEI MERIT
(b) Stream temperature MERIT
Figure 9. Simulations on the WFDEI grid (with the MERIT HDEM and ∆t = 900s) with increasing maximum number of HTU are com-
pared to the reference which is chosen to have nbmax = 55. (a) Discharge WFDEI MERIT (b) Stream temperature MERIT Figure 9. Simulations on the WFDEI grid (with the MERIT HDEM and ∆t = 900s) with increasing maximum number of HTU are com-
pared to the reference which is chosen to have nbmax = 55. Figure 10. The impact of the atmospheric grid is evaluated by comparing the WFDEI simulation to the three other grids (E2OFD, EuroMED
and MEDCORDEX) and for one grid also another HDEM : MEDCORDEXHS (See Table 3). Figure 10. The impact of the atmospheric grid is evaluated by comparing the WFDEI simulation to the three other grids (E2OFD, EuroMED
and MEDCORDEX) and for one grid also another HDEM : MEDCORDEXHS (See Table 3). Rhine (catchment area of 1.4104 km2) stands out. As it is mostly the correlation and the NSE which are affected it has to be
assumed that it is positioned differently in the river graph. 21 6
Validation of the routing scheme in ORCHIDEE
485 In order to appreciate the importance of the numerical choices presented above, the routing scheme is evaluated jointly with
ORCHIDEE at two different resolutions but only using the MERIT HDEM. WFDEI and E2OFD (Marthews et al., 2020) are
two state of the art atmospheric forcing for land surface models both based on the ERA-I re-analysis (Weedon et al., 2014). With the advent of higher resolution precipitation products it was decided to interpolate WFDEI to the MSWEP (Beck et al., 2017) grid (0.25◦) and use it to bias correct rainfall from the re-analysis. Thus the two driving data are not only available on
490
different atmospheric grids but also differ in the precipitation intensity and spatial distribution. The WFDEI version which was
bias corrected with GPCC (Becker et al., 2013; Schneider et al., 2014) is used here. Figure 11 shows on a Taylor diagram the quality of the simulated discharge when compared to observations at the 35
stations presented in appendix A. For discharge, the database of GRDC (Fekete et al., 2000) (http://grdc.bafg.de) is used. bias corrected with GPCC (Becker et al., 2013; Schneider et al., 2014) is used here. Figure 11 shows on a Taylor diagram the quality of the simulated discharge when compared to observations at the 35
stations presented in appendix A. For discharge, the database of GRDC (Fekete et al., 2000) (http://grdc.bafg.de) is used. It has been enhanced with national sources of information for a number of European countries. The stream temperatures
495
were obtained through the GESM database (Programme, 2017) (https://gemstat.org/). The diagnostics are computed with the
available observations over the period 1983-2002. For discharge these results are well in-line with previous validations of ORCHIDEE (Ngo-Duc et al., 2005) and the experi-
i
d d
i
th d t
i il ti
i
t
f
d (W
t l 2018) Th di
i
b t
th
d l ence gained during the data assimilation experiments performed (Wang et al., 2018). The discrepancies between the models
in the Euro-Mediterranean region can be attributed to missing processes in the land surface model. In this region in particular,
500 in the Euro-Mediterranean region can be attributed to missing processes in the land surface model. In this region in particular,
500 22 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. Figure 11. 6
Validation of the routing scheme in ORCHIDEE
485 Based on the analysis above, we know that if
the forcing are the same, the correlation of discharge is better than 0.9 (except for stations 10 and 11) and the ratio of standard
deviation is close to 1. These metrics can be transferred to the Taylor diagram to see that the difference in disagreement of both simulations with observations cannot be explained by the numerical scheme. This confirms that the difference of imposed
505
atmospheric conditions is the dominant cause of differences in behaviour of the model (Marthews et al., 2020). More original for ORCHIDEE is to verify the quality of the simulated stream temperature. The Taylor diagram might give
the reassuring impression that the relatively simplistic model used here for temperature is satisfactory. First and foremost we
have to remember that stream temperature is less affected by water management than water levels. The temperature in streams is driven by the seasonal cycle of its values over land surfaces and thus the temporal correlations can be expected to be high. 510
On the ratio of standard deviation it can already be noted that the amplitude of the stream temperature is overestimated with
the simple model proposed here. The stations of Porte du Scex and Diepoldsau (Stations 5 and 6 respectively) are outliers and
will be considered in more detail in the next section. To better understand the qualities and limitations of the simple temperature scheme proposed here let us examine two stations To better understand the qualities and limitations of the simple temperature scheme proposed here let us examine two stations
with a large upstream area and an excellent observational record : Lobith on the Rhine and Nagymaros on the Danube (Stations
515
31 and 32 respectively). These stations where also used as validation points by models which attempt to incorporate a much
wider set of processes governing stream temperature (van Vliet et al., 2012; Tokuda et al., 2019; Liu et al., 2020). One notes
that on the Rhine the model has a systematic cold bias (Figure 12). Because of the strongly underestimated winter temperatures with a large upstream area and an excellent observational record : Lobith on the Rhine and Nagymaros on the Danube (Stations
515
31 and 32 respectively). 6
Validation of the routing scheme in ORCHIDEE
485 Taylor diagrams comparing the observed monthly discharge and daily stream temperature to observations available at the 35
stations listed in Table A1. The numbers indicated in the figures correspond to those in the table. Figure 11. Taylor diagrams comparing the observed monthly discharge and daily stream temperature to observations available at the 35
stations listed in Table A1. The numbers indicated in the figures correspond to those in the table. human management of surface water is missing as it has not yet been included in the version of the model used here (Zhou
et al., 2021). The differences between both simulations are of more interest here. Based on the analysis above, we know that if
the forcing are the same, the correlation of discharge is better than 0.9 (except for stations 10 and 11) and the ratio of standard
deviation is close to 1. These metrics can be transferred to the Taylor diagram to see that the difference in disagreement of
both simulations with observations cannot be explained by the numerical scheme. This confirms that the difference of imposed human management of surface water is missing as it has not yet been included in the version of the model used here (Zhou
et al., 2021). The differences between both simulations are of more interest here. Based on the analysis above, we know that if
the forcing are the same, the correlation of discharge is better than 0.9 (except for stations 10 and 11) and the ratio of standard
deviation is close to 1. These metrics can be transferred to the Taylor diagram to see that the difference in disagreement of human management of surface water is missing as it has not yet been included in the version of the model used here (Zhou
et al., 2021). The differences between both simulations are of more interest here. Based on the analysis above, we know that if
the forcing are the same, the correlation of discharge is better than 0.9 (except for stations 10 and 11) and the ratio of standard
deviation is close to 1. These metrics can be transferred to the Taylor diagram to see that the difference in disagreement of human management of surface water is missing as it has not yet been included in the version of the model used here (Zhou
et al., 2021). The differences between both simulations are of more interest here. 6
Validation of the routing scheme in ORCHIDEE
485 Previous studies have shown that The two reference simulations show a systematic underestimation of stream temperature in winter and relatively reliable
525
summer values. These biases do not depend on the size of the catchment and are larger than the differences between the
forcing data. Thus there is room for improvement. It has to be remembered that the soil temperatures over the top 0.3m
are essentially driven by the land surface temperature (LST) simulated by ORCHIDEE. This variable is well known to be
affected by modelling assumptions and errors in the driving data (Huntingford et al., 2000). Previous studies have shown that reproducing remote sensed LST in land surface models is challenging (Barella-Ortiz et al., 2017). 530
Given that our approach is relatively simple, it is feasible to better understand the origin of these biases in the simulated
annual cycle of stream temperature. reproducing remote sensed LST in land surface models is challenging (Barella-Ortiz et al., 2017). 530
Given that our approach is relatively simple, it is feasible to better understand the origin of these biases in the simulated
annual cycle of stream temperature. Given that our approach is relatively simple, it is feasible to better understand the origin of these biases in the simulated
annual cycle of stream temperature. 6
Validation of the routing scheme in ORCHIDEE
485 These stations where also used as validation points by models which attempt to incorporate a much
wider set of processes governing stream temperature (van Vliet et al., 2012; Tokuda et al., 2019; Liu et al., 2020). One notes
that on the Rhine the model has a systematic cold bias (Figure 12). Because of the strongly underestimated winter temperatures 23 (a)
(b)
Figure 12. Mean annual cycle of river discharge and temperature at the Lobith station on the Rhine and Nagymaros on the Danube. For
the observations the inter-annual variability of the monthly values is displayed as error bars. The two reference configurations (WFDEI and
E2OFD) are drawn with plain colors while the two sensitivity experiments carried out with WFDEI are given as dotted lines. (a) (b) (b) (a) Figure 12. Mean annual cycle of river discharge and temperature at the Lobith station on the Rhine and Nagymaros on the Danube. For
the observations the inter-annual variability of the monthly values is displayed as error bars. The two reference configurations (WFDEI and
E2OFD) are drawn with plain colors while the two sensitivity experiments carried out with WFDEI are given as dotted lines. (−5K), the amplitude of the annual cycle is too large. On the Danube the annual cycle of temperat But again, the strong cold bias in winter exaggerates the amplitude of the annual cycle. The underestimated winter temperatures
520
is something which was also found in the HEAT-LINK model on the Danube (Tokuda et al., 2019) while the VIC model (van
Vliet et al., 2012) had stream temperatures for the Rhine which were overestimated in winter. Another striking feature is that
all three models predict a too rapid cooling of the rivers in autumn. Figure 13 confirms that this diagnostic is valid over the 25 stations with a temperature record and is general over the region. The two reference simulations show a systematic underestimation of stream temperature in winter and relatively reliable
525
summer values. These biases do not depend on the size of the catchment and are larger than the differences between the
forcing data. Thus there is room for improvement. It has to be remembered that the soil temperatures over the top 0.3m
are essentially driven by the land surface temperature (LST) simulated by ORCHIDEE. This variable is well known to be
affected by modelling assumptions and errors in the driving data (Huntingford et al., 2000). 6.1
Sensitivity of the stream temperature to model assumptions Stream temperature is determined by two fundamental drivers : 24 Figure 13. Seasonal biases at 25 stations where stream temperature are available for the two reference simulations (blue + symbols DEI and
red dots for E2OFD) and for both sensitivity experiments (green downward triangles for the pure advection of both temperatures and purple
upward triangles for top temperature advection). https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. Figure 13. Seasonal biases at 25 stations where stream temperature are available for the two reference simulations (blue + symbols DEI and
red dots for E2OFD) and for both sensitivity experiments (green downward triangles for the pure advection of both temperatures and purple
upward triangles for top temperature advection). – the temperature at which the water leaves the ground and often referred to as headwater temperature. For instance,
535
van Vliet et al. (2012) compared an empirical relation for the headwater temperature with the case when daily soil
temperature is chosen, without specifying at which depth. Tokuda et al. (2019) use the temperature of runoff water
without specifying how it is estimated in MATSIRO (Takata et al., 2003). Here it is proposed to add to this boundary
condition the distinction between temperature of surface runoff and deep drainage temperatures (Section 2.2). 535 – The energy gained by the stream through interaction with the atmosphere and the landscape as it flows to the ocean. 540
These processes are represented with great detail in some models like HEAT-LINK and VIC. Here they are simplified to
a very basic relaxation towards top ground temperature and thus a variable closely related to LST. Separating these two factors in the deficiencies of the models is key to future developments of the scheme so that errors in the
assumptions for the boundary conditions are not compensated by biases in the interactions of the river with the landscape and
atmosphere. 545 atmosphere. 545
To determine the largest source of error in the simulated stream temperature two sensitivity experiments are carried out with
the WFDEI configuration in which the interaction with the atmosphere and landscape are suppressed by setting a = 105. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. The fact that the interaction with the atmosphere and landscape is suppressed in both these experiments leads to a strong
underestimation of the summer stream temperature (Figure 13). The two largest stations on the Danube and Rhine (Figure 12)
show that without the additional energy brought to the stream by the environment the amplitude of their annual cycle is much
lower. When the near surface temperature is used for both runoff and drainage, the amplitude increases slightly but it is still
555
not at the correct level. Figure 13 shows that this result is valid for all catchment sizes. The differences between WFDEI and
WFDEI_Adv do not systematically increase as the stations have a larger catchment. A conclusion which can be drawn at
this stage is that if rivers only obtain their temperature from the headwaters they do not warm-up enough in summer. This
demonstrates the importance of the exchanges with the atmosphere and landscape during this season. The relaxation to the top soil temperature used here is just a simple transfer of the energy balance performed within ORCHIDEE to the streams. In
560
view of the importance of this process a more complete representation of these exchanges should be aimed for. This includes
the simulation of lake thermodynamics (Bernus and Ottlé, 2022). It also explains why simple empirical relations relating near
surface air temperature and stream temperature work so well (Ducharne, 2008). More interesting for the model development is the dependence of the winter temperature bias on the assumption made for the top soil temperature used here is just a simple transfer of the energy balance performed within ORCHIDEE to the streams. In
560
view of the importance of this process a more complete representation of these exchanges should be aimed for. This includes
the simulation of lake thermodynamics (Bernus and Ottlé, 2022). It also explains why simple empirical relations relating near
surface air temperature and stream temperature work so well (Ducharne, 2008). More interesting for the model development is the dependence of the winter temperature bias on the assumption made for the More interesting for the model development is the dependence of the winter temperature bias on the assumption made for the
headwater values. 6.1
Sensitivity of the stream temperature to model assumptions This
means that the relaxation term only serves its numerical purpose as it is different from zero only when the HTU water levels
tend to zero. The two experiments proposed here are : WFDEI_Adv Runoff and drainage have the temperatures as defined in Section 2.2. 550
WFDEI_Top Both water fluxes leaving the soil moisture scheme have the temperature of the top soil layers (0 −0.3m). g
pi
WFDEI_Top Both water fluxes leaving the soil moisture scheme have the temperature of the top soil layers (0 −0.3m). 25 It is expected that in winter the interaction with the landscape and the atmosphere will cool streams as they are warmer It is expected that in winter the interaction with the landscape and the atmosphere will cool streams as they are warmer
than their environment. This is also what is found here when comparing WFDEI and WFDEI_Adv. Thus, in order to have
575
simultaneously the correct interaction with the atmosphere and correct headwater temperature, the drainage temperature should
be warmer than the 3 −17m assumed here. The comparison with WFDEI_Top shows that when cooler near surface soil
temperatures are used the streams are indeed colder, even without atmospheric interactions. This reveals a strong limitation of
the routing scheme proposed here. As we do not represent well ground water, we do not know its depth or its residence time than their environment. This is also what is found here when comparing WFDEI and WFDEI_Adv. Thus, in order to have
575
simultaneously the correct interaction with the atmosphere and correct headwater temperature, the drainage temperature should
be warmer than the 3 −17m assumed here. The comparison with WFDEI_Top shows that when cooler near surface soil
temperatures are used the streams are indeed colder, even without atmospheric interactions. This reveals a strong limitation of
the routing scheme proposed here. As we do not represent well ground water, we do not know its depth or its residence time than their environment. This is also what is found here when comparing WFDEI and WFDEI_Adv. Thus, in order to have
575
simultaneously the correct interaction with the atmosphere and correct headwater temperature, the drainage temperature should
be warmer than the 3 −17m assumed here. The comparison with WFDEI_Top shows that when cooler near surface soil
temperatures are used the streams are indeed colder, even without atmospheric interactions. This reveals a strong limitation of
the routing scheme proposed here. As we do not represent well ground water, we do not know its depth or its residence time at different horizons. Thus its temperature when it emerges at the surface cannot be correctly established. As wintertime cold
580
bias is also present in the mid-latitude basins reported by Takata et al. (2003) and they have assumed initial temperature to be
that of runoff, the cause is probably the same as in our configuration. Figure 13 shows that the case when drainage has the deep soil temperature and no atmospheric interaction
565
is allowed (WFDEI_Adv) the results are more realistic. In this region, winter is the period of low flows which are dominated
by the groundwater contribution to rivers. Attributing to drainage the warmer deep soil temperature in this season is a step in
the right direction. This is corroborated by the fact that the two stations where the winter errors are the largest (Port du Scex
on the Rhone and Diepolsau on the Rhine), and which are upstream of major lakes, are at the outflow of the Alps. In these headwater values. Figure 13 shows that the case when drainage has the deep soil temperature and no atmospheric interaction
565
is allowed (WFDEI_Adv) the results are more realistic. In this region, winter is the period of low flows which are dominated
by the groundwater contribution to rivers. Attributing to drainage the warmer deep soil temperature in this season is a step in
the right direction. This is corroborated by the fact that the two stations where the winter errors are the largest (Port du Scex
on the Rhone and Diepolsau on the Rhine), and which are upstream of major lakes, are at the outflow of the Alps. In these topologically complex regions the contribution of groundwater to the low flows is particularly important (Floriancic et al.,
570
2019). Rain and melt water from the previous season transits through deep aquifers before reaching the rivers at the bottom of
the valley. It has also to be considered that the rivers flowing out of the Alps are affected by hydropower generation. This has
been shown to significantly warm the streams in winter (Fette et al., 2007). It is expected that in winter the interaction with the landscape and the atmosphere will cool streams as they are warmer topologically complex regions the contribution of groundwater to the low flows is particularly important (Floriancic et al.,
570
2019). Rain and melt water from the previous season transits through deep aquifers before reaching the rivers at the bottom of
the valley. It has also to be considered that the rivers flowing out of the Alps are affected by hydropower generation. This has
been shown to significantly warm the streams in winter (Fette et al., 2007). https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. of the aquifer in order to be able to reproduce the observed trends in stream temperature and predict future evolution when
coupled to ESM. of the aquifer in order to be able to reproduce the observed trends in stream temperature and predict future evolution when
coupled to ESM. This highlights the importance of temperature to validate
the groundwater component simulated in land system models. at different horizons. Thus its temperature when it emerges at the surface cannot be correctly established. As wintertime cold
580
bias is also present in the mid-latitude basins reported by Takata et al. (2003) and they have assumed initial temperature to be
that of runoff, the cause is probably the same as in our configuration. This highlights the importance of temperature to validate
the groundwater component simulated in land system models. In the Alpine region, winter stream temperatures have been shown to warm more slowly than in other seasons (Michel et al.,
2020). As it is attributed to groundwater processes, land system models will need to better simulate this slower component
585 26 7
Discussion and conclusion For the four resolutions tested it is equal or larger as the one typically used for ORCHIDEE when coupled to the atmosphere. The time step can probably be increased if the continuity equation on the sub-grid part of the graph is solved
605
implicitly. This would increase the computational efficiency of the routing scheme as it could be called less frequently by the
land surface scheme. Any routing scheme will have adjustable parameters in order to determine the residence time in the aquifers and surface
reservoirs. ORCHIDEE has one parameter per reservoir represented in the transport scheme. We find that changing the reso- the atmosphere. The time step can probably be increased if the continuity equation on the sub-grid part of the graph is solved
605
implicitly. This would increase the computational efficiency of the routing scheme as it could be called less frequently by the
land surface scheme. Any routing scheme will have adjustable parameters in order to determine the residence time in the aquifers and surface
reservoirs. ORCHIDEE has one parameter per reservoir represented in the transport scheme. We find that changing the reso- reservoirs. ORCHIDEE has one parameter per reservoir represented in the transport scheme. We find that changing the reso
lution of the atmospheric grid or the truncation does not require to readjust these parameters. The simulated discharge are not
610
affected in any significant way by the resolution changes. Tests comparing two km-scale HDEMs also show that the parameters
were largely independent of the hydrological information used to build the HTU graph. This is a very important result as it
means that the routing scheme can be used over a large range of configurations of the ESM without having to be adjusted. Thus
expertise can be transferred from one resolution to the other. Our hypothesis is that the repartition between surface runoff and lution of the atmospheric grid or the truncation does not require to readjust these parameters. The simulated discharge are not
610
affected in any significant way by the resolution changes. Tests comparing two km-scale HDEMs also show that the parameters
were largely independent of the hydrological information used to build the HTU graph. This is a very important result as it
means that the routing scheme can be used over a large range of configurations of the ESM without having to be adjusted. 7
Discussion and conclusion This ensures that the graphs
l
d
d
i i i
h
l
i
l
Th
i
l
i
ill d
d
h
l i
f h which combined a vector-based with a grid-based approach (Yamazaki et al., 2013). It combines the water balance simulated
on the atmospheric cells with the higher hydrological resolution needed to predict the horizontal transfers at the surface. The algorithm introduces a truncation parameter which determines the maximum number of HTU as chosen by the user. This allows for applications which do not require all the hydrological details of river flows to reduce the memory consumption of their land surface model by specifying a lower number of sub-grid elements. With a statistical sampling of random river
595
segments the error introduced by the aggregation of the HDEM information at the HTU level is quantified. We recommend
to use a truncation which reduces the total average error of the segments to the cell level error. This ensures that the graphs
are correctly connected and minimizes the topological error. The optimal truncation will depend on the resolution of the
atmospheric grid and the HDEM used. The graphs of HTU would be useless if the water continuity equation cannot be solved with a reasonable time step. We
600
propose to use the 25% quantile of the area weighted residence time as an optimal time step for the routing scheme. This
ensures that 75% of the HTUs by area are numerically stable. It yields a time step which is compatible with the one of the land
surface model. We find that the time step varies more with the spatial resolution of the atmospheric grid than the truncation
of the graph. For the four resolutions tested it is equal or larger as the one typically used for ORCHIDEE when coupled to The graphs of HTU would be useless if the water continuity equation cannot be solved with a reasonable time step. We
600
propose to use the 25% quantile of the area weighted residence time as an optimal time step for the routing scheme. This
ensures that 75% of the HTUs by area are numerically stable. It yields a time step which is compatible with the one of the land
surface model. We find that the time step varies more with the spatial resolution of the atmospheric grid than the truncation
of the graph. 7
Discussion and conclusion This paper proposes a methodology to decompose any atmospheric grid into a graph of hydrological Transfer Units (HTU)
based on a digital elevation model which includes flow directions (HDEM). This network allows to perform a hybrid routing
590
which combined a vector-based with a grid-based approach (Yamazaki et al., 2013). It combines the water balance simulated
on the atmospheric cells with the higher hydrological resolution needed to predict the horizontal transfers at the surface. This paper proposes a methodology to decompose any atmospheric grid into a graph of hydrological Transfer Units (HTU)
based on a digital elevation model which includes flow directions (HDEM). This network allows to perform a hybrid routing
590
which combined a vector-based with a grid-based approach (Yamazaki et al., 2013). It combines the water balance simulated
on the atmospheric cells with the higher hydrological resolution needed to predict the horizontal transfers at the surface. The algorithm introduces a truncation parameter which determines the maximum number of HTU as chosen by the user. This allows for applications which do not require all the hydrological details of river flows to reduce the memory consumption
of their land
rface model b
pecif ing a lo er n mber of
b grid element
With a tati tical ampling of random ri er
595 This paper proposes a methodology to decompose any atmospheric grid into a graph of hydrological Transfer Units (HTU)
based on a digital elevation model which includes flow directions (HDEM). This network allows to perform a hybrid routing
590
which combined a vector-based with a grid-based approach (Yamazaki et al., 2013). It combines the water balance simulated
on the atmospheric cells with the higher hydrological resolution needed to predict the horizontal transfers at the surface. The algorithm introduces a truncation parameter which determines the maximum number of HTU as chosen by the user. This allows for applications which do not require all the hydrological details of river flows to reduce the memory consumption
of their land surface model by specifying a lower number of sub-grid elements. With a statistical sampling of random river
595
segments the error introduced by the aggregation of the HDEM information at the HTU level is quantified. We recommend
to use a truncation which reduces the total average error of the segments to the cell level error. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. that in summer the energy balance over open water is relatively close to the one simulated by ORCHIDEE and thus a simplei that in summer the energy balance over open water is relatively close to the one simulated by ORCHIDEE and thus a simple
relaxation is a good first approximation. More importantly, the simplicity of the scheme allows to reveal the reason for the poor
620
performance in winter which is also found in more complex models. During this season the bias is caused by the boundary con-
dition of the temperature scheme. The water appears not to stay long or deep enough in the ground in order to reach the streamsi that in summer the energy balance over open water is relatively close to the one simulated by ORCHIDEE and thus a simple
relaxation is a good first approximation. More importantly, the simplicity of the scheme allows to reveal the reason for the poor
620
performance in winter which is also found in more complex models. During this season the bias is caused by the boundary con-
dition of the temperature scheme. The water appears not to stay long or deep enough in the ground in order to reach the streams
with a sufficiently high temperature. It points to a limitation of the groundwater representation in the current formulation of the
routing scheme rather than the temperature scheme. This demonstration of the numerical robustness of the hybrid routing scheme of ORCHIDEE is an excellent starting point
625
for future developments. In a companion paper (Schrapffer et al. 2022) it is shown how the elevation information within the
HTU can be used to predict their flooding and overflow into neighbouring HTUs. This allows to represent the functioning of
fluvial floodplains and their contribution to evaporation and vegetation functioning within ORCHIDEE. L k
i
d
b
l
d
t th HDEM D
i
th
t
ti
h
f th HTU thi i f
ti Lakes, reservoirs or dams can be placed onto the HDEM. During the construction phase of the HTUs this information can
then be transferred and aggregated to the level of the hybrid routing scheme. It can then be used to predict water volumes and
630
functioning of these hydrological elements. This can then be combined with the lake energy balance model (Bernus and Ottlé,
2022) in order to represent consistently the lateral transport of water volumes and thermal energy over continents within ESMs. The HTU graph can be enhanced with adduction networks. They link demand points like irrigated areas, power plants (for
hydro power generation or cooling of other plants) or domestic needs to the most appropriate river. Should the demands not be satisfied locally they can be propagated upstream on the adjoint HTU graph so that water is released by the appropriate dam
635
(Zhou et al., 2021). The proposed vision of lateral water transports within Earth system is an extremely power tool which will
enable us to integrate in land surface models the impact of human water management on the hydrological system and predict
its interaction with the climate. satisfied locally they can be propagated upstream on the adjoint HTU graph so that water is released by the appropriate dam
635
(Zhou et al., 2021). The proposed vision of lateral water transports within Earth system is an extremely power tool which will
enable us to integrate in land surface models the impact of human water management on the hydrological system and predict
its interaction with the climate. The representation of deeper groundwater will need more attention in the future developments of ORCHIDEE as recom-
mended by the community (Gleeson et al., 2021). We have noted that probably only a more realistic representation of ground-
640
water transport will enable to correctly simulate temperatures and water quality parameters in areas with complex hydrogeo-
logical structures. It will have to be seen if a specific HTU graph taking into account the hydrogeology needs to be implemented
or if a full three dimensional representation will be required (Maxwell and Miller, 2005). Code and data availability. The code and its evolution are freely available on the GitLab of the French Research organisations : https://gitlab. in2p3.fr/ipsl/lmd/intro/routingpp. The hydrological digital elevation models used will be made available upon request.The code version and
645
data used for this study are available at https://doi.org/10.5281/zenodo.7058895 Code and data availability. The code and its evolution are freely available on the GitLab of the French Research organisations : https://gitlab. in2p3.fr/ipsl/lmd/intro/routingpp. The hydrological digital elevation models used will be made available upon request.The code version and
645
data used for this study are available at https://doi.org/10.5281/zenodo.7058895 Code and data availability. 7
Discussion and conclusion Thus
expertise can be transferred from one resolution to the other. Our hypothesis is that the repartition between surface runoff and lution of the atmospheric grid or the truncation does not require to readjust these parameters. The simulated discharge are not
610
affected in any significant way by the resolution changes. Tests comparing two km-scale HDEMs also show that the parameters
were largely independent of the hydrological information used to build the HTU graph. This is a very important result as it
means that the routing scheme can be used over a large range of configurations of the ESM without having to be adjusted. Thus
expertise can be transferred from one resolution to the other. Our hypothesis is that the repartition between surface runoff and drainage of excess water in the soil moisture reservoir is the main driver for the optimal values of the parameters. Thus as the
615
representation of the unsaturated soils evolves in ORCHIDEE the parameters of the routing scheme will have to be verified. To demonstrate the value of the hybrid routing scheme on a graph of HTU a simple temperature transport scheme is im-
plemented. It is remarkable that it produces results comparable to much more elaborate schemes. We attribute this to the fact drainage of excess water in the soil moisture reservoir is the main driver for the optimal values of the parameters. Thus as the
615
representation of the unsaturated soils evolves in ORCHIDEE the parameters of the routing scheme will have to be verified. To demonstrate the value of the hybrid routing scheme on a graph of HTU a simple temperature transport scheme is im-
plemented. It is remarkable that it produces results comparable to much more elaborate schemes. We attribute this to the fact 27 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
c⃝Author(s) 2022. CC BY 4.0 License. exchanges with the CIMA in Argentina. IPSL-Climate Graduate School-EUR (ANR-11-IDEX-0004-17-EURE-0006) is thanked for their
650
support to the model development presented here. Author contributions JP developped the code, designed and executed the numerical evaluations, AS contributed to the code
development and evaluation, ED and OB contributed the temperature advection development, LR and JS evaluated the scheme
during its development, XZ contirbuted to the code development and provided the MERIT HDEM, all co-authors discussed
the methodology and contributed to the manuscript. 655 exchanges with the CIMA in Argentina. IPSL-Climate Graduate School-EUR (ANR-11-IDEX-0004-17-EURE-0006) is thanked for their
650
support to the model development presented here. The code and its evolution are freely available on the GitLab of the French Research organisations : https://gitlab. in2p3.fr/ipsl/lmd/intro/routingpp. The hydrological digital elevation models used will be made available upon request.The code version and
645
data used for this study are available at https://doi.org/10.5281/zenodo.7058895 Acknowledgements. GRDC and WCRP are thanked for collecting and distributing the discharge and stream temperature data. We are greatful
to Dai Yamazaki for providing the MERIT hydrological digital elevation model. Without UKCEH’s hospitality the lead author of this study
would not have found the time to perform the work. IPSL’s MesoCentre is thanked for the computer time. ECOS-Sud has funded the 28 https://doi.org/10.5194/egusphere-2022-690
Preprint. Discussion started: 15 September 2022
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T
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Number
River
Station name
up-stream area [km2]
Discharge
Temperature
1
Danube River
Hundersingen
2647.01
1983-2002
-
2
La Seine
Troyes
3410.00
1983-2002
-
3
Vechte
Vechterweerd
3600.00
-
1983-1995
4
La Seine
Mery-sur-seine
3880.00
1983-2002
1983-1996
5
Rhone
Porte Du Scex
5244.00
1983-2002
1983-2002
6
Rhine River
Diepoldsau, Rietbruecke
6119.000
1983-2002
1983-2002
7
Trent, River
Colwick
7486.00
1983-2002
1983-2002
8
La Seine
Pont-sur-seine
8760.00
1983-2002
-
9
Thames, River
Kingston
9948.00
1983-2002
1983-2002
10
Rhone
Chancy, Aux Ripes
10323.00
1983-2002
1986-2002
11
Aare
Brugg
11726.00
1983-2002
1983-2002
12
Rhine River
Rekingen
14718.00
1983-2002
1983-2002
13
Danube River
Ingolstadt
20001.00
1983-2002
-
14
La Seine
Montereau-fault-yonne
21178.00
-
1983-1996
15
La Seine
Saint-fargeau-ponthierry
26290.00
1999-2002
1983-1996
16
La Seine
Alfortville
30800.00
1983-2002
-
17
Rhine River
Basel, Schifflaende
35905.00
1983-1995
1983-2002
18
Guadiana
Badajoz
48530.00
1995-2002
-
19
Guadalquivir
Sevilla
48548.00
-
-
20
Rhine River
Maxau
50196.00
1983-2002
1985-1995
21
Duero
Fonfria
63160.00
1983-1995
1990-1995
22
Danube River
Achleiten
76653.00
1983-2002
1985-1995
23
Ebro, Rio
Tortosa
84230.00
1983-2002
1990-1995
24
Vistula
Warsaw
84945.17
1983-1990
1992-2002
25
Rhine River
Mainz
98206.00
1983-2002
1985-1995
26
Danube River
Korneuburg
101536.60
1996-2002
-
27
Rhine River
Kaub
103488.00
1983-2002
1985-1995
28
Oder River
Hohensaaten-Finow Ap
109564.00
1983-2002
1992-2002
29
La Loire
Montjean-sur-loire
109930.00
1983-2002
1983-1996
30
Danube River
Bratislava
131331.00
1983-2002
1995-1995
31
Rhine River
Lobith
160800.00
1983-2002
1983-1995
32
Danube River
Nagymaros
183533.00
1983-1999
1983-1996
33
Vistula
Tczew
193922.91
1983-2002
1992-2002
34
Danube River
Pancevo
525009.00
1983-2002
-
35
Danube River
Ceatal Izmail
807000.00
1983-2002
-
Stations used for the evaluation of the numerical stability and quality of the simulated discharge and stream temperatu 34
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Hedgehog Pathway as a Potential Intervention Target in Esophageal Cancer
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Cancers
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Received: 22 May 2019; Accepted: 11 June 2019; Published: 13 June 2019 Abstract: Esophageal cancer (EC) is an aggressive disease with a poor prognosis. Treatment
resistance is a major challenge in successful anti-cancer therapy. Pathological complete response after
neoadjuvant chemoradiation (nCRT) is low, thus requiring therapy optimization. The Hedgehog
(HH) pathway has been implicated in therapy resistance, as well as in cancer stemness. This article
focusses on the HH pathway as a putative target in the treatment of EC. Immunohistochemistry on
HH members was applied to EC patient material followed by modulation of 3D-EC cell cultures,
fluorescence-activated cell sorting (FACS), and gene expression analysis after HH pathway modulation. Sonic Hedgehog (SHH) and its receptor Patched1 (PTCH1) were significantly enriched in EC resection
material of patients with microresidual disease (mRD) after receiving nCRT, compared to the control
group. Stimulation with SHH resulted in an up-regulation of cancer stemness in EC sphere cultures,
as indicated by increased sphere formation after sorting for CD44+/CD24−EC cancer stem-like cell
(CSC) population. On the contrary, inhibiting this pathway with vismodegib led to a decrease in
cancer stemness and both radiation and carboplatin resistance. Our results strengthen the role of
the HH pathway in chemoradiotherapy resistance. These findings suggest that targeting the HH
pathway could be an attractive approach to control CSCs. Keywords: Esophageal cancer; Hedgehog pathway; cancer stem cells; treatment resistance cancers cancers Hedgehog Pathway as a Potential Intervention Target
in Esophageal Cancer Da Wang 1,2,3, Peter W. Nagle 1,2,3, Helena H. Wang 1,2,3, Justin K. Smit 3, Hette Faber 1,2,†,
Mirjam Baanstra 1,2, Arend Karrenbeld 4, Roland K. Chiu 1,2,‡, John Th.M. Plukker 3
and Robert P. Coppes 1,2,* Da Wang 1,2,3, Peter W. Nagle 1,2,3, Helena H. Wang 1,2,3, Justin K. Smit 3, Hette Faber 1,2,†,
Mirjam Baanstra 1,2, Arend Karrenbeld 4, Roland K. Chiu 1,2,‡, John Th.M. Plukker 3
and Robert P. Coppes 1,2,* 1
Department of Biomedical Sciences of Cells and Systems, Section Molecular Cell Biology, University of
Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands;
daisywangda@hotmail.com (D.W.); p.w.k.nagle@umcg.nl (P.W.N.); h.h.wang@umcg.nl (H.H.W.);
m.baanstra@umcg.nl (M.B.); r.k.chiu@rug.nl (R.K.C.) 1
Department of Biomedical Sciences of Cells and Systems, Section Molecular Cell Biology, University of
Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands;
daisywangda@hotmail.com (D.W.); p.w.k.nagle@umcg.nl (P.W.N.); h.h.wang@umcg.nl (H.H.W.);
m.baanstra@umcg.nl (M.B.); r.k.chiu@rug.nl (R.K.C.) 2
Department of Radiation Oncology, University of Groningen, University Medical Center Groningen,
9700 RB Groningen, The Netherlands 2
Department of Radiation Oncology, University of Groningen, University Medical Center Groningen,
9700 RB Groningen, The Netherlands 3
Department of Surgery, University of Groningen, University Medical Center Groningen,
9700 RB Groningen, The Netherlands; j.k.smit@umcg.nl (J.K.S.); j.t.m.plukker@umcg.nl (J.T.M.P.) g
j
g
j
p
g
4
Department of Pathology, University of Groningen, University Medical Center Groningen,
9700 RB Groningen, The Netherlands; a.karrenbeld@umcg.nl Correspondence: r.p.coppes@umcg.nl; Tel.: +31-50-363-2501; Fax: +31-50-363-2913 *
Correspondence: r.p.coppes@umcg.nl; Tel.: +31-50-363-2501; Fax: +31-50-363-2913
†
Deceased. ‡
Current address, Department of Health and Life Science, University College Groningen, University of
Groningen, Groningen 9718 BG, The Netherlands. Groningen, Groningen 9718 BG, The Netherlands. Groningen, Groningen 9718 BG, The Netherlands. www.mdpi.com/journal/cancers 1. Introduction Despite modern advances in the treatment of esophageal cancer (EC), most patients still face a
poor prognosis primarily due to locoregional advanced disease, metastases at the time of diagnosis
and early recurrences after initial curative treatment [1]. Esophageal adenocarcinoma (EAC) arising
from gland tissue is one of the two main histological EC types and has the fastest growing incidence in
the western world, increasing 300% within the last 30 years [2]. EAC is currently the predominant Cancers 2019, 11, 821; doi:10.3390/cancers11060821 www.mdpi.com/journal/cancers 2 of 13 Cancers 2019, 11, 821 type in the western world beyond esophageal squamous cell carcinoma (ESCC), which arises from the
epithelium [2,3]. If resectable, both types are commonly treated with neoadjuvant chemoradiotherapy
(nCRT) followed by surgery. However, only 29% of these tumors will reach a pathologic complete
response (pCR), while >50% respond partially and around 20% will not respond at all to nCRT [4]. Moreover, most patients will relapse within 2.5 years [5]. These numbers indicate that there is an
urgent need for treatment optimization. Elucidating tumor regulatory mechanisms that are on the
basis of sustained disease might provide new therapeutic targets. Furthermore, it may provide
tools for developing novel ways to predict therapy response preventing patients from undergoing
unnecessary therapy. y
py
The Hedgehog (HH) pathway, which plays a major key role in embryogenesis, is essential for
tissue development and differentiation [6,7]. In adult tissue, the HH pathway is inactive in normal
tissues except for stem and progenitor cells where it plays a role in the maintenance of homeostasis
and tissue repair [6,7]. Three mammalian HH ligands have been described, namely Sonic Hedgehog
(SHH), Desert Hedgehog (DHH) and Indian Hedgehog (IHH). Of these three ligands, SHH is by
far the best characterized [6,7]. In the presence of SHH ligand, SHH binds to the Patched1 (PTCH1)
transmembrane receptor relieving the inhibitory effect on Smoothened (SMO). Consequently, a cascade
of reactions leads to the activation of GLI family zinc finger 1,2 and 3 transcription factors (GLI1, GLI2
and GLI3). Upon binding of the GLI transcription factors to a protein complex including Suppressor of
Fused (SUFU) (an essential component of HH signaling in vertebrates [8]), GLI transcription factors
translocate to the nucleus where they control the transcription of target genes [7,9,10]. PTCH1 is
transcriptionally induced upon activation of SMO along with the other pathway targets. 1. Introduction Target genes,
such as PTCH1 and GLI1, play a role in maintaining the regulation of the pathway itself, while others
promote cell survival. Aberrant activation or mutation in the key members of the HH pathway is
associated with the development of cancer and resistance to conventional anticancer therapy [7,9]. Moreover, the HH pathway has been linked to a population of cells called cancer stem-like cells
(CSCs) which is a major contributor of therapy resistance [11–13], while PTCH1 is often associated
with cancer stemness [14]. It has previously been shown that CSCs possess features of normal stem
cells such as the ability to self-renew and to generate all other cells of the tumor [11–13], and may
play an important role in tumor-initiation, metastases, recurrences and therapy resistance in different
kinds of cancers [15]. Therefore, this population of cells is known for its aggressiveness and should be
eradicated. Previously, Smit et. al. [16] showed that a subpopulation of a commonly used CSC marker
CD44 expressing cells the CD44+/CD24−expressing cells, displayed more potently CSC characteristics. In vitro, CD44+/CD24−cells yield significantly more spheres and are more resistant to irradiation. In vivo, this population forms tumors earlier which also grow larger in size. Serial transplantation
experiments of first, second and third generation tumors demonstrated a strong correlation between
the proportion of CD44+/CD24−cells and the in vivo growth rate [16]. Furthermore, CD44+/CD24−
was found to be present in 50% of pretreatment tumor biopsies of patients with residual EAC after
treatment in surgical specimens but absent in all biopsies of patients who had a pathological complete
response after nCRT [16]. These results suggest that CSCs are more enriched in the CD44+/CD24−
population compared to CD44+/CD24+ and the unsorted population, and could be used as a readout
for esophageal CSCs. Interestingly, the combination CD44+/CD24−is also commonly used to isolate
breast CSCs [17]. Thus, studying the HH pathway in EC may reveal a new way to eradicate CSCs leading to reduced
therapy resistant tumors. Our aim is to explore whether the HH pathway can be considered as a
potential target for future anti-CSC EC therapy. 2. Results To investigate if the HH pathway is involved in regulating EC CSCs, the level of expression of the
HH members PTCH1 and SHH were assessed in EC resection material from patients that underwent
a surgical resection in our center during the period 2006 and 2011. Sixteen tumor tissue specimens 3 of 13
3
f 12 Cancers 2019, 11, 821 from non or marginally responding EC patients to nCRT (microresidual disease, mRD group with
Mandard tumor regression grade 4 and 5) were compared to 32 matched control tumor specimens of
patients who only had undergone radical surgery (S group). The latter group with unknown therapy
responsiveness represents a mix of all potentially responding groups. Indeed, both PTCH1 and SHH
were significantly higher expressed in mRD tissue compared to S tissue (both p = 0.04, Figure 1A,B). These results indicate that the HH pathway may be related to therapy resistance. specimens from non or marginally responding EC patients to nCRT (microresidual disease, mRD
group with Mandard tumor regression grade 4 and 5) were compared to 32 matched control tumor
specimens of patients who only had undergone radical surgery (S group). The latter group with
unknown therapy responsiveness represents a mix of all potentially responding groups. Indeed, both
PTCH1 and SHH were significantly higher expressed in mRD tissue compared to S tissue (both p =
0.04, Figure 1A,B). These results indicate that the HH pathway may be related to therapy resistance. Figure 1. PTCH1 and SHH are up-regulated in mRD patients compared to surgery alone patients (S). (A) Representative samples of low intensity PTCH1 and weakly positive SHH expression
(respectively upper left and lower left), and high intensity PTCH1 and strong positive SHH
expression (respectively upper right and lower right). (B) Comparison of PTCH1 (p = 0.04) and SHH
(p = 0.04) IHC expression between mRD after neoadjuvant CRT resection specimens (N = 16) and S
specimens (N = 32). Error bars represent standard error of the mean (SEM), *p < 0.05. Figure 1. PTCH1 and SHH are up-regulated in mRD patients compared to surgery alone patients (S). (A) Representative samples of low intensity PTCH1 and weakly positive SHH expression (respectively
upper left and lower left), and high intensity PTCH1 and strong positive SHH expression (respectively
upper right and lower right). 2. Results (B) Comparison of PTCH1 (p = 0.04) and SHH (p = 0.04) IHC expression
between mRD after neoadjuvant CRT resection specimens (N = 16) and S specimens (N = 32). Error
bars represent standard error of the mean (SEM), *p < 0.05. Figure 1. PTCH1 and SHH are up-regulated in mRD patients compared to surgery alone patients (S). Figure 1. PTCH1 and SHH are up-regulated in mRD patients compared to surgery alone patients (S). Figure 1. PTCH1 and SHH are up-regulated in mRD patients compared to surgery alone patients (S). (A) Representative samples of low intensity PTCH1 and weakly positive SHH expression
(respectively upper left and lower left), and high intensity PTCH1 and strong positive SHH
expression (respectively upper right and lower right). (B) Comparison of PTCH1 (p = 0.04) and SHH
(p = 0.04) IHC expression between mRD after neoadjuvant CRT resection specimens (N = 16) and S
specimens (N = 32). Error bars represent standard error of the mean (SEM), *p < 0.05. Figure 1. PTCH1 and SHH are up-regulated in mRD patients compared to surgery alone patients (S). (A) Representative samples of low intensity PTCH1 and weakly positive SHH expression (respectively
upper left and lower left), and high intensity PTCH1 and strong positive SHH expression (respectively
upper right and lower right). (B) Comparison of PTCH1 (p = 0.04) and SHH (p = 0.04) IHC expression
between mRD after neoadjuvant CRT resection specimens (N = 16) and S specimens (N = 32). Error
bars represent standard error of the mean (SEM), *p < 0.05. Figure 1. PTCH1 and SHH are up-regulated in mRD patients compared to surgery alone patients (S). (A) Representative samples of low intensity PTCH1 and weakly positive SHH expression
(respectively upper left and lower left), and high intensity PTCH1 and strong positive SHH
expression (respectively upper right and lower right). (B) Comparison of PTCH1 (p = 0.04) and SHH
(p = 0.04) IHC expression between mRD after neoadjuvant CRT resection specimens (N = 16) and S
specimens (N = 32). Error bars represent standard error of the mean (SEM), *p < 0.05. Figure 1. PTCH1 and SHH are up-regulated in mRD patients compared to surgery alone patients (S). (A) Representative samples of low intensity PTCH1 and weakly positive SHH expression (respectively
upper left and lower left), and high intensity PTCH1 and strong positive SHH expression (respectively
upper right and lower right). 2. Results (B) Comparison of PTCH1 (p = 0.04) and SHH (p = 0.04) IHC expression
between mRD after neoadjuvant CRT resection specimens (N = 16) and S specimens (N = 32). Error
bars represent standard error of the mean (SEM), *p < 0.05. To validate the impact of the HH pathway in the regulation of CSCs that might play a
fundamental role in the observed therapy resistance in patient derived tumor resection material,
CD44+/CD24− was taken as a read-out for cancer stemness after first verifying the sphere forming
ability, as a hallmark of CSCs, of this population in vitro. The sphere forming potential of two
subpopulations of cells were isolated by sorting the 3–15% of the outer extreme of CD44+/CD24− CSC
population and CD44+/CD24+ non-CSC population, in OE21 and OE33 EC cell lines isolated at 70%
confluency (Figure 2A). Five percent of the outer extreme of each subpopulation were isolated by
FACS. Indeed, as shown previously [16], the CD44+/CD24− population formed significantly more
spheres when compared to CD44+/CD24+ population in both OE21 and OE33 cell lines (p = 0.01 and
To validate the impact of the HH pathway in the regulation of CSCs that might play a fundamental
role in the observed therapy resistance in patient derived tumor resection material, CD44+/CD24−
was taken as a read-out for cancer stemness after first verifying the sphere forming ability, as a
hallmark of CSCs, of this population in vitro. The sphere forming potential of two subpopulations
of cells were isolated by sorting the 3–15% of the outer extreme of CD44+/CD24−CSC population
and CD44+/CD24+ non-CSC population, in OE21 and OE33 EC cell lines isolated at 70% confluency
(Figure 2A). Five percent of the outer extreme of each subpopulation were isolated by FACS. Indeed,
as shown previously [16], the CD44+/CD24−population formed significantly more spheres when
compared to CD44+/CD24+ population in both OE21 and OE33 cell lines (p = 0.01 and p = 0.02 4 of 13 Cancers 2019, 11, 821 respectively, Figure 2B,C), which is also known to be resistant to radiation and form tumors more
potently as shown previously [16]. Cancers 2019, 11, x
4 of 12 Figure 2. Enhancement of sphere formation capacity by sorting for CD44+/CD24−. (A) Representative
FACS plots of OE21 and OE33 stained with CD24 FITC and CD44 PE. (B) Representative images of
spheres. Bar indicates 100 µm. (C) Quantification of spheres shown in (B). OE21 CD44+/CD24− vs. 2. Results CD44+/CD24+ (p = 0.01) and OE33 CD44+/CD24− vs. CD44+/CD24+ (p = 0.02, N = 3) spheres after five
days of culture. Error bars represent standard deviation, *p < 0.05. Figure 2. Enhancement of sphere formation capacity by sorting for CD44+/CD24−. (A) Representative
FACS plots of OE21 and OE33 stained with CD24 FITC and CD44 PE. (B) Representative images of
spheres. Bar indicates 100 µm. (C) Quantification of spheres shown in (B). OE21 CD44+/CD24−vs. CD44+/CD24+ (p = 0.01) and OE33 CD44+/CD24−vs. CD44+/CD24+ (p = 0.02, N = 3) spheres after
five days of culture. Error bars represent standard deviation, *p < 0.05. Figure 2. Enhancement of sphere formation capacity by sorting for CD44+/CD24−. (A) Representative
FACS plots of OE21 and OE33 stained with CD24 FITC and CD44 PE. (B) Representative images of
spheres. Bar indicates 100 µm. (C) Quantification of spheres shown in (B). OE21 CD44+/CD24− vs. /
d OE
/
/
h
f
f
Figure 2. Enhancement of sphere formation capacity by sorting for CD44+/CD24−. (A) Representative
FACS plots of OE21 and OE33 stained with CD24 FITC and CD44 PE. (B) Representative images of
spheres. Bar indicates 100 µm. (C) Quantification of spheres shown in (B). OE21 CD44+/CD24−vs. CD44 /CD24
(
0 01)
d OE33 CD44 /CD24
CD44 /CD24
(
0 02 N
3)
h
ft Figure 2. Enhancement of sphere formation capacity by sorting for CD44+/CD24−. (A) Representative
FACS plots of OE21 and OE33 stained with CD24 FITC and CD44 PE. (B) Representative images of
spheres. Bar indicates 100 µm. (C) Quantification of spheres shown in (B). OE21 CD44+/CD24− vs. CD44+/CD24+ (p = 0.01) and OE33 CD44+/CD24− vs. CD44+/CD24+ (p = 0.02, N = 3) spheres after five
days of culture. Error bars represent standard deviation, *p < 0.05. Figure 2. Enhancement of sphere formation capacity by sorting for CD44+/CD24−. (A) Representative
FACS plots of OE21 and OE33 stained with CD24 FITC and CD44 PE. (B) Representative images of
spheres. Bar indicates 100 µm. (C) Quantification of spheres shown in (B). OE21 CD44+/CD24−vs. CD44+/CD24+ (p = 0.01) and OE33 CD44+/CD24−vs. CD44+/CD24+ (p = 0.02, N = 3) spheres after
five days of culture. Error bars represent standard deviation, *p < 0.05. 2. Results (B) Relativ
mRNA expression of CSC related genes in OE33 CD44+/CD24− CSC population, CD24+/CD44
population and control. Shown genes express >2 fold in CD44+/CD24− population compared to
control. (C) Validation of relative mRNA expression of PTCH1 in CD44+/CD24− CSC population
CD24+/CD44+ population and control in OE21 and OE33 by qPCR. PTCH1 was 3.8 fold up-regulated
in CD44+/CD24− CSC population compared to control in OE21 (p = 0.04, N = 3) and 2.3 fold in OE3
(p = 0.04, N = 5). Error bars represent standard deviation. Figure 3. PTCH1 is up-regulated in CD44+/CD24−CSC population in both OE21 and OE33 cell lines
according to a qPCR array of 84 genes related to cancer stemness. (A) Relative mRNA expression of
CSC related genes in OE21 CD44+/CD24−CSC population, CD44+/CD24+ population and control
(obtained from more differentiation form the cell lines derived xenograft tumors, [16]). Shown genes
express >2 fold in CD44+/CD24−population compared to control. Control is set on 1. (B) Relative
mRNA expression of CSC related genes in OE33 CD44+/CD24−CSC population, CD24+/CD44+
population and control. Shown genes express >2 fold in CD44+/CD24−population compared to
control. (C) Validation of relative mRNA expression of PTCH1 in CD44+/CD24−CSC population,
CD24+/CD44+ population and control in OE21 and OE33 by qPCR. PTCH1 was 3.8 fold up-regulated
in CD44+/CD24−CSC population compared to control in OE21 (p = 0.04, N = 3) and 2.3 fold in OE33
(p = 0.04, N = 5). Error bars represent standard deviation. To assess the role of the HH pathway in CSC regulation, the HH pathway was modulated b
exogenously adding its ligand SHH (0.4 µg/mL) to CD44+/CD24− and CD44+/CD24+ cells afte
sorting (similar gating strategies as shown in Figure 1A were used, Supplementary Figure S1A) i
both OE21 and OE33. After adding, SHH induced a significant increase in sphere forming potentia
in both CD44+/CD24− and CD44+/CD24+ sorted cells when compared to control in OE21 and OE3
(Figure 4A), whereas the solvent (PBS + 0.1% BSA) did not affect the sphere forming ability of eithe
cell line (data not shown). 2. Results Next, in order to identify a pathway in an unbiased way that may be involved in the regulation
of the CD44+/CD24− CSC population, a qPCR array of 84 genes related to cancer stemness was
performed on the subpopulations of both OE21 and OE33 cells and were compared to previously
harvested OE21- and OE33-derived xenograft tumor controls representing a more differentiated
population [16]. In this way, genes that were up-regulated in CD44+/CD24− expression compared to
the control could be identified (Supplementary Table S1 and Figure 3A). Genes that had >2-fold
upregulation compared to controls were considered to be upregulated. In OE21, multiple genes were
>2-fold upregulated including PTCH1, a member of the HH pathway. In OE33, fewer genes displayed
>2-fold upregulation, among which was found to be again PTCH1 (Supplementary Table S1 and
Figure 3B). To narrow down genes, the differences of expression between the CD44+/CD24−
population and CD44+/CD24+ population were compared to their controls. Interestingly, PTCH1 was
upregulated in CD44+/CD24− and CD44+/CD24+ populations in both cell lines compared to their
controls (OE21 9.31 and 3.67, and OE33 2.37 and 1.71 respectively). These findings were validated by
separate qPCR analyses (both cell lines p = 0.04, Figure 3C). Next, in order to identify a pathway in an unbiased way that may be involved in the regulation of
the CD44+/CD24−CSC population, a qPCR array of 84 genes related to cancer stemness was performed
on the subpopulations of both OE21 and OE33 cells and were compared to previously harvested OE21-
and OE33-derived xenograft tumor controls representing a more differentiated population [16]. In
this way, genes that were up-regulated in CD44+/CD24−expression compared to the control could be
identified (Supplementary Table S1 and Figure 3A). Genes that had >2-fold upregulation compared
to controls were considered to be upregulated. In OE21, multiple genes were >2-fold upregulated
including PTCH1, a member of the HH pathway. In OE33, fewer genes displayed >2-fold upregulation,
among which was found to be again PTCH1 (Supplementary Table S1 and Figure 3B). To narrow
down genes, the differences of expression between the CD44+/CD24−population and CD44+/CD24+
population were compared to their controls. Interestingly, PTCH1 was upregulated in CD44+/CD24−
and CD44+/CD24+ populations in both cell lines compared to their controls (OE21 9.31 and 3.67, and
OE33 2.37 and 1.71 respectively). These findings were validated by separate qPCR analyses (both cell
lines p = 0.04, Figure 3C). 2. Results 5 of 13
5 of 1 Cancers 2019, 11, 821
Cancers 2019, 11, x Figure 3. PTCH1 is up-regulated in CD44+/CD24− CSC population in both OE21 and OE33 cell line
according to a qPCR array of 84 genes related to cancer stemness. (A) Relative mRNA expression o
CSC related genes in OE21 CD44+/CD24− CSC population, CD44+/CD24+ population and contro
(obtained from more differentiation form the cell lines derived xenograft tumors, [16]). Shown gene
express >2 fold in CD44+/CD24− population compared to control. Control is set on 1. (B) Relativ
mRNA expression of CSC related genes in OE33 CD44+/CD24− CSC population, CD24+/CD44
population and control. Shown genes express >2 fold in CD44+/CD24− population compared t
control. (C) Validation of relative mRNA expression of PTCH1 in CD44+/CD24− CSC population
CD24+/CD44+ population and control in OE21 and OE33 by qPCR. PTCH1 was 3.8 fold up-regulate
in CD44+/CD24− CSC population compared to control in OE21 (p = 0.04, N = 3) and 2.3 fold in OE3
(p = 0.04, N = 5). Error bars represent standard deviation. Figure 3. PTCH1 is up-regulated in CD44+/CD24−CSC population in both OE21 and OE33 cell lines
according to a qPCR array of 84 genes related to cancer stemness. (A) Relative mRNA expression of
CSC related genes in OE21 CD44+/CD24−CSC population, CD44+/CD24+ population and control
(obtained from more differentiation form the cell lines derived xenograft tumors, [16]). Shown genes
express >2 fold in CD44+/CD24−population compared to control. Control is set on 1. (B) Relative
mRNA expression of CSC related genes in OE33 CD44+/CD24−CSC population, CD24+/CD44+
population and control. Shown genes express >2 fold in CD44+/CD24−population compared to
control. (C) Validation of relative mRNA expression of PTCH1 in CD44+/CD24−CSC population,
CD24+/CD44+ population and control in OE21 and OE33 by qPCR. PTCH1 was 3.8 fold up-regulated
in CD44+/CD24−CSC population compared to control in OE21 (p = 0.04, N = 3) and 2.3 fold in OE33
(p = 0.04, N = 5). Error bars represent standard deviation. Figure 3. PTCH1 is up-regulated in CD44+/CD24− CSC population in both OE21 and OE33 cell line
according to a qPCR array of 84 genes related to cancer stemness. (A) Relative mRNA expression o
CSC related genes in OE21 CD44+/CD24− CSC population, CD44+/CD24+ population and contro
(obtained from more differentiation form the cell lines derived xenograft tumors, [16]). Shown gene
express >2 fold in CD44+/CD24− population compared to control. Control is set on 1. 2. Results In contrast, inhibition of the HH pathway using vismodegib (5 nM;
concentration chosen based on the viability of cells treated with vismodegib alone (Supplementar
To assess the role of the HH pathway in CSC regulation, the HH pathway was modulated by
exogenously adding its ligand SHH (0.4 µg/mL) to CD44+/CD24−and CD44+/CD24+ cells after sorting
(similar gating strategies as shown in Figure 1A were used, Supplementary Figure S1A) in both OE21 and
OE33. After adding, SHH induced a significant increase in sphere forming potential in both CD44+/CD24−
and CD44+/CD24+ sorted cells when compared to control in OE21 and OE33 (Figure 4A), whereas the
solvent (PBS + 0.1% BSA) did not affect the sphere forming ability of either cell line (data not shown). In
contrast, inhibition of the HH pathway using vismodegib (5 nM; a concentration chosen based on the
viability of cells treated with vismodegib alone (Supplementary Figure S2)) resulted in a reduction of the 6 of 13
6
f 12 Cancers 2019, 11, 821 CD44+/CD24−CSC phenotype in OE21 and OE33 cells when compared to controls (Figure 4B, FACS
gating strategies are shown in supplementary Figure S1B). Moreover, sphere forming ability also decreased
after vismodegib treatment in both OE21 and OE33 cells (Figure 4C). Compared to other cells within a
tumor, it has previously been shown that CSCs are more resistant to many forms of cancer treatments [15]. Therefore, modulation of the CSC population should illicit a change in radioresistance in OE-21 cells [16]. To this end, OE-21 cells were photon irradiated or treated with carboplatin (a common chemotherapeutic
drug used for esophageal cancer treatment) following 48 hours treatment with vismodegib and their
clonogenic capability was assessed. Indeed, vismodegib-treated cells were found to be more sensitive to
both radiation and carboplatin treatment than control cells (Figure 4D,E). These findings indicate that the
HH pathway may be involved in the regulation of the CD44+/CD24−CSC population increasing cancer
stemness and as a consequence may result in an increased therapy resistance. Inhibiting the pathway
with vismodegib may be of therapeutic value and should be explored further. compared to controls (Figure 4B, FACS gating strategies are shown in supplementary Figure S1B). Moreover, sphere forming ability also decreased after vismodegib treatment in both OE21 and OE33
cells (Figure 4C). Compared to other cells within a tumor, it has previously been shown that CSCs
are more resistant to many forms of cancer treatments [15]. 2. Results Therefore, modulation of the CSC
population should illicit a change in radioresistance in OE-21 cells [16]. To this end, OE-21 cells were
photon irradiated or treated with carboplatin (a common chemotherapeutic drug used for esophageal
cancer treatment) following 48 hours treatment with vismodegib and their clonogenic capability was
assessed. Indeed, vismodegib-treated cells were found to be more sensitive to both radiation and
carboplatin treatment than control cells (Figure 4D,E). These findings indicate that the HH pathway
may be involved in the regulation of the CD44+/CD24− CSC population increasing cancer stemness
and as a consequence may result in an increased therapy resistance. Inhibiting the pathway with
vismodegib may be of therapeutic value and should be explored further. Figure 4. Sonic Hedgehog, ligand of the PTCH1 receptor up-regulates sphere formation of both
CD44+/CD24− and CD44+/CD24+ populations in OE21 and OE33 while vismodegib, a HH pathway
inhibitor, deselects for the CD44+/CD24− population, decreases sphere formation and increases
Figure 4. Sonic Hedgehog, ligand of the PTCH1 receptor up-regulates sphere formation of both
CD44+/CD24−and CD44+/CD24+ populations in OE21 and OE33 while vismodegib, a HH pathway Figure 4. Sonic Hedgehog, ligand of the PTCH1 receptor up-regulates sphere formation of both
CD44+/CD24− and CD44+/CD24+ populations in OE21 and OE33 while vismodegib, a HH pathway
i hibito
de ele t
fo
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Figure 4. Sonic Hedgehog, ligand of the PTCH1 receptor up-regulates sphere formation of both
CD44+/CD24−and CD44+/CD24+ populations in OE21 and OE33 while vismodegib, a HH pathway 7 of 13 Cancers 2019, 11, 821 inhibitor, deselects for the CD44+/CD24−population, decreases sphere formation and increases
radiosensitivity. (A) Relative number of spheres formed in OE21 and OE33 after sorting for
CD44+/CD24−and CD44+/CD24+ populations with the controls set on 1. Cells treated with SHH
formed significantly more spheres in CD44+/CD24−and CD44+/CD24+ populations compared to their
controls (DMSO-treated) (OE21: 1.97 (p = 0.009, N = 3) and 2.33 (p = 0.03, N = 3) fold respectively. OE33: 1.68 (p = 0.04, N = 3) and 2.18 (p = 0.04, N = 3) fold respectively. Spheres were counted after five
days. (B) Percentage of CD44+/CD24−expression in OE21 and OE33 (unsorted populations) control
(DMSO-treated) and vismodegib (5 nM) treated cells. 3. Discussion In this study, we demonstrated that the HH pathway is up-regulated in nCRT patients with
Mandard 4 and 5 statuses after reviewing the resection material and moreover it may be a key player
in the regulation of cancer stemness as shown by the change in the amount CD44+/CD24−CSC
population upon HH pathway modulation. The first observation can be explained by the fact that
nCRT may activate the HH pathway and in turn making cells acquiring more CSC features including
the therapy resistance characteristic [18]. Another clarification for this phenomenon could be that
a subset of cancer cells already has an activated HH pathway prior to therapy that renders them to
survive chemo- and radiotherapy [19–22]. Most likely, a combination of both may occur. The S group
contains patients from <2006 when radical surgery was the standard treatment for esophageal cancer
implicating that this group includes non-responders, partial responders and complete responders. This
could be the reason why the results of PTCH1 and SHH expression between this group and the mRD
group are more condensed. Next, we verified these results in an unbiased qPCR array experiment
with sorted CD44+/CD24−OE21 and OE33 cells compared to sorted CD44+/CD24+ OE21 and OE33
cells, and dissociated OE21 and OE33 cells previously harvested from mouse xenograft tumors [16]. Cells from xenografts represent a more differentiated and heterogeneous group of cells, and thus
represent all four populations of CD44 and CD24 following FACS analysis. Therefore, the xenograft
group was considered as our control group, as the original cell lines did not show expression of the
CD44−/CD24+ or CD44−/CD24−. On the other hand, cells obtained from xenografts may be influenced
by tumor microenvironmental factors, such as hypoxia activating, a number of CSC-related pathways
including the HH pathway. However, our qPCR data show that PTCH1 expression is lower in our
control group compared to the other two groups indicating that even with the potential influence of the
tumor microenvironment, PTCH1 expression is less compared to a ‘pure’ CD44+/CD24−population
suggesting the tumor is indeed composed of more differentiated cells. Co-labelling CD44, CD24 and
PTCH1 for flow cytometry purposes was not favorable due to the lack of conjugated antibodies against
PTCH1. 2. Results Vismodegib treated cells showed significantly a
deselection of the CD44+/CD24−phenotype in both cell lines compared to (DMSO-treated) controls
(OE21 p = 0.02, OE33 p = 0.04, N = 4). (C) Amount of spheres formed in OE21 and OE33 control
and vismodegib (5 nM) treated cells. Spheres were significantly lower in vismodegib treated cells
compared to control (DMSO-treated) in OE21 (p = 0.008, N = 3) and OE33 (p = 0.03, N = 4). Survival
of vismodegib-treated cells in response to (D) radiation and (E) Carboplatin. Error bars represent
standard deviations, *p < 0.05, **p < 0.01. 3. Discussion While vismodegib binds directly to PTCH and SMO and thereby inhibits the activation of
GLI [23], the effect of vismodegib treatment on the expression of other genes which were found to
be upregulated in the CSC subpopulation but not directly involved in the HH pathway (Figure 3)
was not investigated. It would be interesting to determine if the upregulation of these genes was also
abrogated upon vismodegib treatment, as this may indeed further confirm that the CSCs in general
have been targeted. RNA-seq analysis may reveal other interesting targets not present on our cancer
stemness dedicated array. To date, we are the first to show the importance of the HH pathway in controlling cells in a
specific CSC population in EC. The activation of the HH pathway in subsets of EC has been shown
previously [19–22] and is in line with our study. In addition, some studies have shown the association
of the HH pathway with therapy resistance but did not address the involvement of specific CSC Cancers 2019, 11, 821 8 of 13 populations [20,22]. We have demonstrated that inhibiting the HH pathway is related to a reduction
of cells with the cancer stem cell related phenotype (CD44+/CD24−), less sphere forming capability
and more radiosensitivity. Although vismodegib is being tested in the clinic, xenograft models in
combination with HH pathway modulation will provide more information on the potential therapeutic
effectiveness. Furthermore, it would be important for future studies to confirm if the effect was indeed
directly due to the actions of vismodegib on the HH pathway using other HH-targeting molecules
which are currently being used in clinical trials, such as LDE225 [24–26]. Vismodegib, also known as GDC-0449, is a small molecule HH inhibitor that blocks the
interaction between the PTCH-receptors and their ligands [27]. The choice of vismodegib is based
on its advantageous characteristics. It is more potent than the better known cyclopamine and
has more favorable pharmaceutical characteristics. Moreover, it has already been FDA approved
for advanced basal cell carcinoma [27]. Vismodegib is currently used in early trials for treatment
of basal cell carcinomas and medulloblastoma, showing promising early indications in enhancing
treatment responses [28–30]. Therefore, vismodegib could be a promising drug to target esophageal
CSC populations. 4.1. Patient Material Sixteen tumor tissue specimens of microscopic residual disease (mRD group) from EC patients
who showed little or no response to nCRT (Mandard tumor regression grade 4 and 5) following
surgical resection at the University Medical Center Groningen, during the period 2006 and 2011 were
included in the primary study group. Only patients with histologically proven ESCC or EAC after
curative resections were included. Some patient samples were excluded due to lack of sufficient
tissue. Furthermore, all patients had to be treated with nCRT according to the CROSS regimen [4,5]. These 16 tumor specimens were matched (1:2) to 32 control tumor specimens of patients who have
only undergone surgery (S group). The S group contains patients from the period before 2006 when
radical surgery alone was considered as standard therapy for EC. When extrapolating this group,
it will represent the whole treatment group of current nCRT, including none, partial and complete
responders. Matching was based on histology (EAC or ESCC) and depth of tumor invasion (T-stage). Tissue microarrays (TMAs) were constructed as previously described [16]. The study was conducted
according to the guidelines of our Ethical Institutional board (www.ccmo.nl). Archival tissue was
handled according to the Dutch Code for proper use of Human Tissue (www.federa.org). This study did not require an ethics approval or a consent to participate. The human material
used in this study was archival tissue. The study was conducted according to the guidelines of our
Ethical Institutional board (www.ccmo.nl). Archival tissue was handled according to the Dutch Code
for proper use of Human Tissue (www.federa.org). This study was performed in accordance with the
Declaration of Helsinki. 4.2. Immunohistochemical Staining and Evaluation 4.2. Immunohistochemical Staining and Evaluation Immunohistochemical staining was performed on 5 µm tissue sections from archival material using
primary antibodies against PTCH1 (Anti-Patched/PTCH1 antibody Abcam 53715 1:100), SHH (anti
SHH 1:100 Abcam 53281) (Abcam, Cambridge, MA, USA), CD44 (anti-mouse/human CD44 Antibody
Biolegend Cat.103002 1:100) (Biolegend, London, United Kingdom) and CD24 (anti CD24 Abcam
31622 1:100). The tissue sections were de-paraffinized and subsequently immersed in PBS 2% hydrogen
peroxidase to block endogenous peroxidase activity. Antigen-retrieval was performed and the sections
were incubated overnight at 4 ◦C with the primary antibodies. Tissue sections were then incubated
with biotinylated secondary antibodies at 1:300 dilutions. The ABC complex was formed using the
Vectastain Elite ABC HRP kit (Vector Laboratories, Peterborough, United Kingdom). This complex
was visualized with SIGMA FAST 3,3′-diaminobenzidine tablets (Merck KGaA, Darmstadt, Germany). 9 of 13 Cancers 2019, 11, 821 In the final step, sections were counterstained with haematoxylin. Immunohistochemical staining was
digitized using Aperio (ASSA ABLOY, Raamsdonksveer, The Netherlands) or Hamamatsu (Almere,
The Netherlands). These systems are able to process immunohistochemical slides with high quality,
speed and reliability for whole slide imaging. These slides were scored by two independent blinded
researchers (D.W. and J.K.S.) using the IRS method without prior knowledge of the clinical outcome. Random samples of each marker were validated by the pathologist A.K. The percentage of positive
cells was scored into four categories: (0) no staining, (1) <10%, (2) 10–50%, (3) 51–80% and (4) 81–100%. Intensity was scored as (0) negative, (1) weak, (2) medium and (3) strong. An Immuno-Reactivity Score
(IRS) was calculated by multiplying the percentage of positive cells with the intensity score resulting in
a score on a scale of 0–12. For evaluation of SHH the IRS was divided into four groups: negative (IRS
0–1, immunoscore 0); weak positive staining (IRS 2–3, immunoscore 1); moderate positive staining
(IRS 4–8, immunoscore 2) and strong positive staining (IRS 9–12, immunoscore 3) [31]. Since almost all
specimens showed PTCH1 expression in 81–100% of the tumor cells, only intensity for PTCH1 was
determined and categorized into negative or low (intensity 0–1, immunoscore 0) and high (intensity
2–3, immunoscore 1). 4.4. Fluorescence Activated Cell Sorting (FACS) CD44+/CD24−cells and CD44+/CD24+ cells were sorted with both OE21 and OE33 using
antibodies directed against CD44 (BD Biosciences, 550989, San Jose, CA, USA) and CD24 (BD
Biosciences, 555427) with MoFlo Asterios cell sorter (Beckman Coulter, Woerden, The Netherlands). Isotype controls were used to compensate for aspecific binding of the antibodies. CD24 antibody
conjugated with FITC and CD44 antibody conjugated with PE along with their corresponding isotype
controls (BD Biosciences, 555748, 555749, respectively) were used. The most left and right (3–15%)
population of CD44+/CD24−, respectively, CD44+/CD24+ populations were sorted. 4.3. Cell Lines and Cell Culture Two esophageal cancer cell lines were used, OE21 derived from ESCC of the upper esophagus
and OE33 derived from a poorly differentiated Barrett’s associated tumor of the distal esophagus. Both
cell lines were donated by Dr. F.A. Kruyt, Department of Medical Oncology, University Medical Center
Groningen and were independently DNA verified as EC cell lines by the Leibniz Institute DSMZ-German
Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) in 2012. These cell lines
are commonly used in esophageal cancer research. OE21 and OE33 were cultured in GIBCO RPMI
1640 medium (Life Technologies) supplemented with 10% FCS and 1% of penicillin/streptomycin in an
incubator of 5% CO2 and 37 ◦C. Both cell lines were passaged in 1:5 at 70% confluency. 4.4. Fluorescence Activated Cell Sorting (FACS) 4.7. RNA Extraction, First Strand Analysis and qPCR Total RNA was extracted from the sorted cell populations using RNAeasy micro extraction kit
(Qiagen, Venlo, The Netherlands). RNA concentration was measured by Nanodrop (Isogen Life Sciences,
De Meern, The Netherlands. To ensure RNA quality, standards according to the manufacturer’s
datasheet were followed. Subsequently, the first cDNA strand was produced using 500 ng/µL RNA
and SuperScript® VILO™cDNA synthesis kit (Life Technologies, Bleiswijk, The Netherlands). A qPCR
array (Qiagen) of 84 genes related to cancer stemness was performed on the CD44+/CD24−and
CD44+/CD24+ sorted cells of OE21 and OE33. Additionally, solid tumors generated from the same cell
lines obtained from xenografts, as reported previously [16], were used as controls as these populations
represent a differentiated group of cells. The xenografts were generated by isolating either sorted
CD44+/CD24−, CD44+/CD24+ or unsorted cells OE21 and OE33 cells before injecting either of these
populations into animals. These cells were not treated with CRT. These arrays were performed on
Biorad CFX connect 96 wells plate according the protocol of the manufacturer. Genes that were >2-fold
up-regulated in the CD44+/CD24−CSC population compared to controls were considered relevant. The gene that appeared to be up-regulated in both cell lines was validated by separate qPCR analyses. YWHAZ was used as an endogenous control for normalization. qPCR was performed using SYBR
green (Biorad, Lunteren, The Netherlands) to measure the expression of the genes. 4.5. Sphere Culture and Sphere Count 100,000 cells of each sorted population, CD44+/CD24−and CD44+/CD24+, were seeded in
triplicate in each well of a six-well plate with 2 mL of mammocult serum-free medium with supplement
(Stemcell Technologies, Vancouver, Canada). Mammocult serum-free medium was used instead
of Neural Basal A medium supplemented with N2, bFGF and FGF-2 as previously described by
Smit et al. [16]. because of its more potent sphere forming ability in both cell lines without distortion of
the results. The wells were coated with agarose to prevent the attachment of cells onto the bottom of
the plate. The concentration agarose was 0.023 g/mL dissolved in distilled water. A total volume of
0.2 mL of mammocult was added each day to nourish the cells. To ensure balance of the total amount
of formed spheres in both cell lines, 2 × 104 sorted OE33 cells and 1 × 105 OE21 cells were seeded for
the modulation of the HH pathway experiments. Pictures of spheres were taken under the microscope
after five days. For the quantification, only sphere-like structure in a homogenized aliquot of 100 µL
were counted. 10 of 13 10 of 13 Cancers 2019, 11, 821 4.6. HH Pathway Modulation OE21 and OE33 cells were sorted for CD44+/CD24−and CD44+/CD24+ populations and seeded
in each well of a six-well plate coated with agarose to prevent the attachment of the cells onto the
bottom of the plate. For OE21 1 × 105 cells were used for each population while 2 × 104 cells were used
for OE33 to achieve similar amount of spheres in both cell lines. Each sorted population of both cell
lines were cultured in Mammocult medium supplemented by 0.4 µg/mL human recombinant protein
Sonic Hedgehog (R and D systems) reconstituted in PBS + 0.1% BSA, according to manufacturer
datasheet, to generate spheres. This treated population was compared to its control, which consisted
of only Mammocult and PBS + 0.1% BSA. The relative amount of spheres compared to control was
calculated after five days. Five nanomoles of vismodegib reconstituted in DMSO was used according
to manufacturer instructions (IC50 of 3 nM) and was also based on the biological effect in both cell
lines according to viability assays (data not shown). vismodegib also blocks ABC transporter, ABCG2,
however only when applied in relatively higher concentrations (IC50 of 1.4 µM) according to the
manufacturer datasheet. FACS analyses on LSRII (BD Biosciences) were performed on the expression
of CD44+/CD24−and CD44+/CD24+ in OE21 and OE33 after 48 h 5 nM vismodegib treatment in RPMI
media and their controls, which consisted of RPMI supplemented with DMSO. Flow cytometry data
were analyzed using Kaluza (Beckman Coulter, Woerden, The Netherlands). This software offers flow
cytometry analysis software solution designed for high content data. The same cells of both cells lines
used in the FACS experiment were seeded in Mammocult medium to generate spheres. This was done
immediately after collecting the cells for the FACS analyses to ensure reliability and consistency. For
OE21 1 × 105 cells were seeded and for OE33 2 × 104 cells were seeded. Spheres were counted after
five days. 4.8. Clonogenic Assay To assess the sensitivity of vismodegib treated cells, a clonogenic assay was performed. Cells were
seeded in a 12-well plate at 1 × 105 cells per well and incubated with/without vismodegib (5 nM). After
48 hours, cells were photon irradiated using a 137Cs source (IBL 637 Cesium-137 γ-ray machine). After
irradiation cells were washed and harvested from the plate following incubation with trypsin-EDTA. The cells were then counted and seeded in 60 mm cell culture dishes at concentrations ranging from
200–1000 cells depending on the radiation dose or carboplatin concentration. To assess the response to
carboplatin following vismodegib treatment, cells were seeded in triplicate at 500 cells per 60 mm cell 11 of 13 Cancers 2019, 11, 821 culture plate at all carboplatin concentrations. For the irradiation experiments, cells were seeded in
triplicate at multiple concentrations of 200, 500 and 1000 cells per plate per irradiation dose. The cells
were incubated at 37 ◦C and 5% CO2 for 10–12 days, after which they were stained with clonogenic
assay stain solution (50% methanol, 10% acetic acid, 1% w/v Coomassie brilliant blue). Colonies (≥50
cells) were counted and the surviving fraction was calculated. 5. Conclusions Conflicts of Interest: The authors declare that there is no conflict of interest. 4.9. Statistical Analysis All experiments were at least performed three times except for the qPCR array experiment
screening for CSC related genes which was performed in duplicate for each sample and each cell
line. However, the gene of interest was validated separately by qPCR of at least three experiments. Shown data are presented as mean and shown error bars represent standard deviation unless otherwise
stated. Groups were compared with Student’s t-test. IHC results of PTCH1 were compared with the
Fishers exact test and of SHH were compared with the chi-square test for trend using SPSS (version 22). A p-value <0.05 was considered significant. 5. Conclusions In this study, the potential of targeting the Hedgehog pathway to enhance the efficacy of esophageal
cancer was investigated. Enhanced levels of Sonic Hedgehog and its receptor Patched1 were observed
in microresidual disease biopsies compared to biopsies from control patients. In vitro modulation of
the HH pathway efficiently altered CSC populations, while inhibition of the pathway can be used to
enhance sensitivity to common esophageal cancer treatments. The HH pathway therefore offers a
potentially attractive target to enhance esophageal cancer treatment. Supplementary Materials: The supplementary materials are available online at http://www.mdpi.com/2072-6694/
11/6/821/s1. Table S1: Fold change of expression in important cancer stemness-related genes. Figure S1: Gating
strategies of the FACS experiments. (A) The left 3–15% proportion of CD44+/CD24- cells and the right 3–15%
proportion of CD44+/CD24+ cells were sorted for the induction of the HH pathway by SHH. (B) CD44 and CD24
expression in OE21 and OE33 cells of vismodegib (5nM) sample compared to control sample. Gates of all samples
were set individually after correcting for aspecific binding by running isotype controls. Figure S2: Viability curves
of OE21 and OE33 after treatment Vismodegib Relative number of viable cells after different concentrations of
vismodegib treatment compared to no vismodegib treatment (control). Viable cells were counted with trypan blue. 5 nM was subsequently chosen as the concentration to use. Author Contributions: D.W. designed and performed most of the experiments, performed data analysis, and
prepared the manuscript. P.W.N. designed and performed the clonogenic experiments and helped during
the preparation of the manuscript. H.H.W. performed a part of the clonogenic experiment. J.K.S. and R.K.C. helped designing experiments. H.F. and M.B. performed the IHC. experiments and a part of the RNA extraction
experiments respectively. A.K. helped to score the pathological slides. J.T.M.P. had a supervisory role and edited
the manuscript. R.P.C. designed experiments, edited the manuscript and had a supervisory role. Funding: This study was partially funded by the MD/PhD program at the University of Groningen and partially
by the Dutch Cancer Society (KWF, grant no. 10417). Acknowledgments: We would like to thank F.A.E. Kruyt (Department of Medical Oncology, UMCG, The
Netherlands) for kindly provide us the esophageal cell lines OE21 and OE33. Additionally, they would like to
thank their colleagues of the FACS facility, H. Moes, G. Mesander and R.J. van der Leij for whose guidance during
our sorting experiments. Conflicts of Interest: The authors declare that there is no conflict of interest. 1.
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A Systematic Review and Meta-Analysis. J. Am. Acad. Dermatol. 2018, 79, 1089–1100.e17. [CrossRef] 28. Xie, P.; Lefrancois, P. Efficacy, Safety, and Comparison of Sonic Hedgehog Inhibitors in Basal Cell Carcinomas:
A Systematic Review and Meta-Analysis. J. Am. Acad. Dermatol. 2018, 79, 1089–1100.e17. [CrossRef] 29. Sekulic, A.; Migden, M.R.; Basset-Seguin, N.; Garbe, C.; Gesierich, A.; Lao, C.D.; Miller, C.; Mortier, L.;
Murrell, D.F.; Hamid, O.; et al. Long-Term Safety and Efficacy of Vismodegib in Patients with Advanced Basal
Cell Carcinoma: Final Update of the Pivotal ERIVANCE BCC Study. BMC Cancer 2017, 17, 332. [CrossRef] 30. Espinosa-Bustos, C.; Mella, J.; Soto-Delgado, J.; Salas, C.O. State of the Art of Smo Antagonists for Cancer
Therapy: Advances in the Target Receptor and New Ligand Structures. Future Med. Chem. 2019, 11, 617–638. [CrossRef] 31. Kaemmerer, D.; Peter, L.; Lupp, A.; Schulz, S.; Sanger, J.; Baum, R.P.; Prasad, V.; Hommann, M. Comparing of
IRS and Her2 as Immunohistochemical Scoring Schemes in Gastroenteropancreatic Neuroendocrine Tumors. Int. J. Clin. Exp. Pathol. 2012, 5, 187–194. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Opponent intracerebral signals for reward and punishment prediction errors in humans
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ARTICLE Results iEEG data were collected from 20 patients with drug-resistant
epilepsy (see demographical details in Supplementary Table 1 and
Methods section) while they performed an instrumental learning
task (Fig. 1a). Electrode implantation was performed according to
routine clinical procedures, and all targeted brain areas for the
presurgical evaluation were selected strictly according to clinical
considerations with no reference to the current study. However, a number of empirical studies have casted doubt on
this anatomical separation between reward and punishment
learning systems. Part of the confusion might come from the use
of behavioral tasks that allow for a change of reference point, such
that not winning becomes punishing and not losing becomes
rewarding13. The issue is aggravated with decoding approaches
that preclude access to the sign of PE signals, i.e., whether they
increase or decrease with reward versus punishment14. Another
reason for inconsistent findings might be related to the recording
technique: fMRI instead of electrophysiology. Indeed, some
electrophysiological studies in monkeys have recorded reward
and punishment PE signals in adjacent brain regions15,16. In
addition, single-unit recordings in monkeys have identified PE
signals in other brain regions: not only small deep brain nuclei
such as the ventral tegmental area17,18 but also large cortical
territories such as the dorsolateral prefrontal cortex (dlPFC,
refs. 19,20). Patients had to choose between two cues to either maximize
monetary gains (for reward cues) or minimize monetary losses
(for punishment cues). The pairs of cues associated to reward and
punishment learning were intermingled within three to six ses-
sions of 96 trials. In each pair, the two cues were associated to the
two possible outcomes (0/1€ in the reward condition and 0/-1€ in
the punishment condition) with reciprocal probabilities (0.75/
0.25 and 0.25/0.75). Reward and punishment conditions were
matched in difficulty, as the same probabilistic contingencies were
to be learned. Patients were instructed to do their best to max-
imize the monetary gains and to minimize the monetary losses
during the task. No further information was given regarding the
exact task structure. One issue with fMRI is that the temporal resolution makes it
difficult to dissociate the two components of PE – observed and
expected outcome value. The issue arises because the same region
might reflect PE at both the times of option and outcome display. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w An important
conclusion of these studies is that functional specificities relate to
the learning domain (reward versus punishment) and not to the
sign of prediction errors (positive versus negative). These two
factors could be dissociated because reward learning also involves
negative PE (when the expected reward is not delivered), while
punishment learning also involves positive PE (when expected
punishment is avoided). Thus, the suggestion is that some brain
regions may signal reward PE (both positive and negative),
informing whether or not a choice should be repeated, while
other brain regions may signal punishment PE, informing whe-
ther or not a choice should be avoided. However, a number of empirical studies have casted doubt on
this anatomical separation between reward and punishment p
p
g
Yet, different lines of evidence point to an anatomic divide
between reward and punishment learning systems, in relation
with opponent approach and avoidance motor behaviors3,4. First,
fMRI studies have located prediction error (PE) signals in dif-
ferent brain regions, such as the ventral striatum and ven-
tromedial prefrontal cortex (vmPFC) for reward versus the
amygdala, anterior insula (aINS), or lateral orbitofrontal cortex
(lOFC) for punishment5–8. Second, reward and punishment
learning can be selectively affected, for instance by dopaminergic
manipulation and anterior insular lesion9–12. An important
conclusion of these studies is that functional specificities relate to
the learning domain (reward versus punishment) and not to the
sign of prediction errors (positive versus negative). These two
factors could be dissociated because reward learning also involves
negative PE (when the expected reward is not delivered), while
punishment learning also involves positive PE (when expected
punishment is avoided). Thus, the suggestion is that some brain
regions may signal reward PE (both positive and negative),
informing whether or not a choice should be repeated, while
other brain regions may signal punishment PE, informing whe-
ther or not a choice should be avoided. In this task (Fig. 1a), patients (n = 20) are required to choose
between two cues to maximize monetary gains (during reward-
learning) or minimize monetary losses (during punishment-
learning). Reward and punishment PE can then be inferred from
the history of tasks events, using a computational model. We first
identifiy from the 1694 cortical recording sites a set of brain
regions encoding PE, which include vmPFC, lOFC, aINS, and
dlPFC. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w We then specify the dynamics of PE signals in both time
and frequency domains, and compare between reward and pun-
ishment conditions. The main purpose of these analyses is to
assess whether differences between brain regions relate to the sign
(positive versus negative) or to the domain (reward versus pun-
ishment) of PE signals driving choice behavior. ARTICLE ARTICLE Anatomical dissociation of intracerebral signals for
reward and punishment prediction errors in
humans Maëlle C. M. Gueguen
1, Alizée Lopez-Persem
2, Pablo Billeke
3, Jean-Philippe Lachaux4,
Sylvain Rheims
5, Philippe Kahane6, Lorella Minotti6, Olivier David1, Mathias Pessiglione
7,8,9 &
Julien Bastin
1,9✉ Maëlle C. M. Gueguen
1, Alizée Lopez-Persem
2, Pablo Billeke
3, Jean-Philippe Lachaux4,
Sylvain Rheims
5, Philippe Kahane6, Lorella Minotti6, Olivier David1, Mathias Pessiglione
7,8,9 &
Julien Bastin
1,9✉ Whether maximizing rewards and minimizing punishments rely on distinct brain systems
remains debated, given inconsistent results coming from human neuroimaging and animal
electrophysiology studies. Bridging the gap across techniques, we recorded intracerebral
activity from twenty participants while they performed an instrumental learning task. We
found that both reward and punishment prediction errors (PE), estimated from computational
modeling of choice behavior, correlate positively with broadband gamma activity (BGA) in
several brain regions. In all cases, BGA scaled positively with the outcome (reward or pun-
ishment versus nothing) and negatively with the expectation (predictability of reward or
punishment). However, reward PE were better signaled in some regions (such as the ven-
tromedial prefrontal and lateral orbitofrontal cortex), and punishment PE in other regions
(such as the anterior insula and dorsolateral prefrontal cortex). These regions might therefore
belong to brain systems that differentially contribute to the repetition of rewarded choices
and the avoidance of punished choices. 1 Univ. Grenoble Alpes, Inserm, U1216, Grenoble Institut Neurosciences, GIN, Grenoble, France. 2 Frontal Function and Pathology team, Institut du Cerveau,
Sorbonne Université, INSERM U 1127, CNRS UMR 7225, Paris, France. 3 División de Neurociencia, Centro de Investigación en Complejidad Social
(neuroCICS), Facultad de Gobierno, Universidad del Desarrollo, Santiago, Chile. 4 Lyon Neuroscience Research Center, Brain Dynamics and Cognition team,
DYCOG INSERM UMRS 1028, CNRS UMR 5292, Université de Lyon, Lyon, France. 5 Department of Functional Neurology and Epileptology, Hospices Civils
de Lyon and University of Lyon, Lyon, France. 6 Univ. Grenoble Alpes, Inserm, U1216, CHU Grenoble Alpes, Grenoble Institut Neurosciences, GIN,
Grenoble, France. 7 Motivation, Brain and Behavior lab, Centre de NeuroImagerie de Recherche, Institut du Cerveau et de la Moelle épinière, Hôpital de la
Pitié-Salpêtrière, Paris, France. 8 Inserm U1127, CNRS U7225, Université Pierre et Marie Curie (UPMC-Paris 6), Paris, France. 9These authors contributed
equally: Mathias Pessiglione, Julien Bastin. ✉email: julien.bastin@univ-grenoble-alpes.fr 1 NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunications TURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w A A
pproaching reward and avoiding punishment are the two
fundamental drives of animal behavior. In principle, both
reward-seeking
and
punishment-avoidance
could
be
learned through the same algorithmic steps. One the most
straight and simple algorithm postulates that the value of chosen
action is updated in proportion to prediction error1,2, defined as
observed minus expected outcome value. In this simple reinfor-
cement learning model, the only difference is outcome valence:
positive for reward (increasing action value) and negative for
punishment (decreasing action value). The same brain machinery
could therefore implement both reward and punishment learning. With the aim of bridging across species and techniques, we
investigate here PE signals in the human brain, using a time-
resolved
recording
technique:
intracerebral
electro-
encephalography (iEEG). The iEEG signals were collected in
patients implanted with electrodes meant to localize epileptic foci,
while they performed an instrumental learning task. The same
approach was used in one previous study that failed to identify
any anatomical specificity in the neural responses to positive and
negative outcomes26. To assess whether this lack of specificity was
related to the recording technique or to the behavioral task, we
used a task that properly dissociates between reward and pun-
ishment learning, as shown by previous fMRI, pharmacological,
and lesion studies6,11. A
pproaching reward and avoiding punishment are the two
fundamental drives of animal behavior. In principle, both
reward-seeking
and
punishment-avoidance
could
be
learned through the same algorithmic steps. One the most
straight and simple algorithm postulates that the value of chosen
action is updated in proportion to prediction error1,2, defined as
observed minus expected outcome value. In this simple reinfor-
cement learning model, the only difference is outcome valence:
positive for reward (increasing action value) and negative for
punishment (decreasing action value). The same brain machinery
could therefore implement both reward and punishment learning. Yet, different lines of evidence point to an anatomic divide
between reward and punishment learning systems, in relation
with opponent approach and avoidance motor behaviors3,4. First,
fMRI studies have located prediction error (PE) signals in dif-
ferent brain regions, such as the ventral striatum and ven-
tromedial prefrontal cortex (vmPFC) for reward versus the
amygdala, anterior insula (aINS), or lateral orbitofrontal cortex
(lOFC) for punishment5–8. Second, reward and punishment
learning can be selectively affected, for instance by dopaminergic
manipulation and anterior insular lesion9–12. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w c Average performance (correct choice rate, n = 20 patients). Modeled performance is indicated by white and gray disks (using Q-learning + repetition bias and basic Q-learning model, QLr and QL, respectively). Dots
represent individual patients. d Difference between conditions (reward minus punishment correct choice rate) in observed and modeled data. Dots
represent individual patients and error-bars represent mean ± SEM across patients (n = 20). e Inter-patient correlations between modeled and observed
correct choice rate for reward (blue) and punishment (red) learning. Each circle represents one patient. Red line represents the linear regression across
patients (n = 20). f Reaction time (RT) learning curves. Median RT are averaged across patients and the mean (±SEM) is plotted as function of trials
separately for the reward (blue) and punishment (red) conditions. Black horizontal bars represent the outcome of two-sided statistical tests of difference,
using paired Student’s t tests in c and d. ns means not significant and asterisk indicates significance in d (QL > QLr, p = 0.0037; QL > data, p = 0.013). Degrees of freedom (DF)
Bayesian information criterion (BIC)
Mean ± SEM
Learning rate (α)
Mean ± SEM
Inverse temperature (β)
Mean ± SEM
Repetition bias (θ)
Mean ± SEM
QL
2
502 ± 31
0.27 ± 0.04
3.80 ± 0.48
–
QLr
3
430 ± 30
0.26 ± 0.04
3.19 ± 0.43
0.44 ± 0.06 Computational modeling. To generate trial-wise expected values
and prediction errors, we fitted a Q-learning model (QL) to
behavioral data. The QL model generates choice likelihood via a
softmax function of cue values, which are updated at the time of
outcome. Fitting the model means adjusting two parameters
(learning rate and choice temperature) to maximize the likelihood
of observed choices (see methods). Because this simple model left
systematic errors in the residuals, we implemented another model
(QLr) with a third parameter that increased the value of the cue
chosen in the previous trial, thereby increasing the likelihood of
repeating the same choice. We found that including a repetition
bias in the softmax function better accounted for the data, as
indicated by a significantly lower Bayesian Information Criterion
(BIC) for QLr model (t19 = 4.05, p < 0.001; Table 1; one-sample two-tailed Student’s t tests on the difference of BIC). On average,
this QLr model accounts for a more symmetrical performance
between reward and punishment learning (Fig. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Fig. 1 Behavioral task and results. a Successive screenshots of a typical trial in the reward (top) and punishment (bottom) conditions. Patients had to
select one abstract visual cue among the two presented on each side of a central visual fixation cross, and subsequently observed the outcome. Duration is
given in milliseconds. b Average learning curves (n = 20 patients). Modeled behavioral choices (solid line) are superimposed on observed choices (shaded
areas represent mean ± SEM across patients). Learning curves show rates of correct choice (75% chance of 1€ gain) in the reward condition (blue curves)
and incorrect choice (75% chance of 1€ loss) in the punishment condition (red curves). c Average performance (correct choice rate, n = 20 patients). Modeled performance is indicated by white and gray disks (using Q-learning + repetition bias and basic Q-learning model, QLr and QL, respectively). Dots
represent individual patients. d Difference between conditions (reward minus punishment correct choice rate) in observed and modeled data. Dots
represent individual patients and error-bars represent mean ± SEM across patients (n = 20). e Inter-patient correlations between modeled and observed
correct choice rate for reward (blue) and punishment (red) learning. Each circle represents one patient. Red line represents the linear regression across
patients (n = 20). f Reaction time (RT) learning curves. Median RT are averaged across patients and the mean (±SEM) is plotted as function of trials
separately for the reward (blue) and punishment (red) conditions. Black horizontal bars represent the outcome of two-sided statistical tests of difference,
using paired Student’s t tests in c and d. ns means not significant and asterisk indicates significance in d (QL > QLr, p = 0.0037; QL > data, p = 0.013). Table 1 Model parameters and comparison criterion. Fig. 1 Behavioral task and results. a Successive screenshots of a typical trial in the reward (top) and punishment (bottom) conditions. Patients had to
select one abstract visual cue among the two presented on each side of a central visual fixation cross, and subsequently observed the outcome. Duration is
given in milliseconds. b Average learning curves (n = 20 patients). Modeled behavioral choices (solid line) are superimposed on observed choices (shaded
areas represent mean ± SEM across patients). Learning curves show rates of correct choice (75% chance of 1€ gain) in the reward condition (blue curves)
and incorrect choice (75% chance of 1€ loss) in the punishment condition (red curves). Results Thus, if option and outcome display are close in time, the
hemodynamic signals reflecting positive and negative expected
outcome value would cancel each other21,22. The issue can be
solved by adequate jittering between cue and outcome events23,24,
but this has not been systematically used in human fMRI studies. In addition to recording techniques and related timing issues,
discrepant results between human and monkey studies could also
arise from differences in the paradigms25, such as the amount of
training or the particular reward and punishment used to con-
dition choice behavior. Indeed, primary reinforcers used in
monkeys like fruit juices and air puffs may not be exact reward
and punishment equivalents, as are the monetary gains and losses
used in humans. Behavioral performance. Patients were able to learn the correct
response over the 24 trials of the learning session: they tended to
choose the most rewarding cue in the reward condition and avoid
the most punishing cue in the punishment condition (Fig. 1b). Average percentage of correct choices (Fig. 1c) in the reward and
punishment conditions was significantly different from chance
(50%) level (reward: 71.4 ± 3.2%, t19 = 6.69, p < 3 × 10−6; pun-
ishment: 71.5 ± 2.1%, t19 = 10.02, p < 6 × 10−9; difference: t19 =
−0.03; p = 0.98; one-sample and paired-sample two-tailed Stu-
dent’s t tests). Reaction times were significantly shorter in the
reward than in the punishment condition (Fig. 1f; reward: 700 ±
60 ms; punishment: 1092 ± 95 ms; difference: t19 = −7.02, p < 2 ×
10−6). Thus, patients learnt similarly from rewards and punish-
ments, but took longer to choose between cues for punishment
avoidance. This pattern of results replicates behavioral data pre-
viously obtained from healthy subjects6,27. NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w TURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w 1d), while the
standard QL model would learn better in the reward condition,
because reinforcement is more frequent than in the punishment
condition (as patients approach the +1€ and avoid the −1€
outcome). With the QLr model, choices in reward and punish-
ment conditions were captured equally well, with an explained
variance across patients of 87 and 83% (Fig. 1e). p
g
In the following analyses, iEEG activity was regressed against PE
estimated for each participant at each trial in each condition from
the QLr model fit. PE therefore represents our key independent
variable – a hidden variable that in principle could have driven
learning, since it is informed by individual choice behavior. 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Fig. 2 Anatomical location of intracerebral electrodes. a Sagittal and axial slices of a brain template over which each dot represents one iEEG recording
site (n = 1694). Color code indicates location within the four main regions of interest (red: vmPFC, n = 54; green: dlPFC, n = 74; blue: lOFC, n = 70; purple:
aINS, n = 83). b MarsAtlas parcellation scheme represented on an inflated cortical surface. Fig. 2 Anatomical location of intracerebral electrodes. a Sagittal and axial slices of a brain template over which each dot represents one iEEG recordin
site (n = 1694). Color code indicates location within the four main regions of interest (red: vmPFC, n = 54; green: dlPFC, n = 74; blue: lOFC, n = 70; purple
aINS, n = 83). b MarsAtlas parcellation scheme represented on an inflated cortical surface. Fig. 2 Anatomical location of intracerebral electrodes. a Sagittal and axial slices of a brain template over which each dot represents one iEEG recording
site (n = 1694). Color code indicates location within the four main regions of interest (red: vmPFC, n = 54; green: dlPFC, n = 74; blue: lOFC, n = 70; purple:
aINS, n = 83). b MarsAtlas parcellation scheme represented on an inflated cortical surface. iEEG: localizing PE using broadband gamma activity. To
identify brain regions signaling PE, we first focused on broadband
gamma activity (BGA, in the 50−150 Hz range) because it is
known to correlate with both spiking and fMRI activity28–31. BGA was extracted from each recording site and time point and
regressed against PE (collapsed across reward and punishment
conditions) which were generated by the QLr model across trials. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w The location of all iEEG recording sites (n = 1694 bipolar deri-
vations) was labeled according to MarsAtlas parcellation32, and to
the atlas of Destrieux (Destrieux et al.67) for the hippocampus
and the distinction between anterior and posterior insula (Fig. 2). In total, we could map 1473 recording sites into 39 brain parcels. In the following, we report statistical results related to PE signals
tested across the recording sites located within a given parcel. Note that an inherent limitation to any iEEG study is that the
number of recorded sites varies across parcels, which impacts the
statistical power of analyses used to detect PE signals in different
brain regions. across parcels. We also estimated the significance of PE signals at
the site level, by using time-varying regression estimates and
associated p-values, while FDR-correcting for multiple compar-
isons in the time domain (across 97 comparisons in the 0–1.5 s
time window following outcome onset), in accordance with
published methods33. We found eight parcels showing significant
PE signals and displaying a proportion of significant contacts
superior to 20% (Supplementary Table 2). This set of significant
brain
parcels
included
the
aINS,
vmPFC,
dlPFC,
lOFC,
hippocampus, lateral, and caudal medial visual cortex (VCcm
and VCl) and the medial inferior temporal cortex (ITcm). Given
this result and the literature reviewed in the introduction, we
focused on the anterior insula and prefrontal ROIs (vmPFC,
lOFC, and dlPFC) in the hereafter analyses, while the same
analyses
performed
in
the
other
ROIs
are
presented
as
supplementary information. iEEG: localizing PE using broadband gamma activity. To
identify brain regions signaling PE, we first focused on broadband
gamma activity (BGA, in the 50−150 Hz range) because it is
known to correlate with both spiking and fMRI activity28–31. BGA was extracted from each recording site and time point and
regressed against PE (collapsed across reward and punishment
conditions) which were generated by the QLr model across trials. The location of all iEEG recording sites (n = 1694 bipolar deri-
vations) was labeled according to MarsAtlas parcellation32, and to
the atlas of Destrieux (Destrieux et al.67) for the hippocampus
and the distinction between anterior and posterior insula (Fig. 2). In total, we could map 1473 recording sites into 39 brain parcels. In the following, we report statistical results related to PE signals
tested across the recording sites located within a given parcel. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w d Regression estimates of power against PE, averaged over early (0–0.5 s) and late (0.5–1 s) post-stimulus windows for
the lower-frequency bands (θ/α: 4–13 Hz) and over the 0.25–1 s window for higher frequency bands (β: 13–33 Hz and broadband γ: 50–150 Hz). Center
lines, box limits, whiskers, and crosses of the box plots, respectively represent median, interquartile range, and outliers of the data distribution from the n
recording sites. Stars indicate significance (all p values < 0.05) of regression estimates (one-sample, two-sided Student’s t test). Error bars correspond to
inter-sites SEM and dots correspond to individual recording sites. (βPE = 0.03 ± 0.006, sum(t(69)) = 116.1, pc < 1 × 10−3; Fig. 3c). We
next focused on a 0.25–1 s post-outcome time window for
subsequent analyses, as it plausibly corresponds to the computa-
tion of PE. To further quantify statistically how the information
about PE was distributed across frequencies, we averaged
regression estimates over the 0.25–1 s time window for the
broadband gamma and beta bands, and over two separate time
windows to distinguish the early (0–0.5 s) and late (0.5–1 s)
components of theta-alpha band activity (Fig. 3d). As expected,
we found significant PE correlates in BGA in the four ROIs (all p
< 0.05). Furthermore, beta-band activity was also positively
associated with PE in two ROIs (aINS: βPE = 0.11 ± 0.012; t(82)
= 9.17; p < 1 × 10−13; vmPFC: βPE = 0.03 ± 0.008; t(53) = 3.34; p
= 0.0015,
one-sample
two-tailed
Student’s
t
test). Finally,
regarding
the
theta/alpha
band,
regression
estimates
were
significantly above (below) zero in the early (late) time window
in all ROIs (all p values < 0.05). regressed each time and frequency point against PE generated by
the QLr model across trials. This time-frequency analysis con-
firmed the presence of PE signals in BGA following outcome
onset in all ROIs (Fig. 3b). Furthermore, PE was also positively
associated with beta-band (13–33 Hz) power in the aINS and
vmPFC. In the theta/alpha bands (4–8 and 8–13 Hz), there was
an initial positive association (during the first 500 ms after out-
come onset), which was followed by a negative association (from
500 to 1000 ms after outcome onset) in all four ROIs. Thus, the
time-frequency analysis pointed to three other frequency bands in
which power could be associated to PE. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Fig. 3 Investigation of PE signals across frequency bands. a Anatomical localization of the aINS (purple), dlPFC (green), vmPFC (red), and lOFC (blue). All recording sites located in these parcels were included in the ROI analyses. The sample size used to derive statistics in panels b–d are displayed for each
ROI (aINS: n = 83; dlPFC: n = 74; vmPFC: n = 54; lOFC: n = 70). b. Time-frequency decomposition of PE signals following outcome onset. Hotter colors
indicate more positive regression estimates. Horizontal dashed lines indicate boundaries between frequency bands that are investigated in panels c and
d. c Time course of regression estimates obtained from linear fit of BGA with PE modeled across reward and punishment conditions. Solid lines (filled
areas) indicate mean (SEM) across recording sites. Horizontal bold black lines indicate significant clusters (pc < 1 × 10−3; one-sample, two-sided Student’s t
test after cluster-wise correction). d Regression estimates of power against PE, averaged over early (0–0.5 s) and late (0.5–1 s) post-stimulus windows for
the lower-frequency bands (θ/α: 4–13 Hz) and over the 0.25–1 s window for higher frequency bands (β: 13–33 Hz and broadband γ: 50–150 Hz). Center
lines, box limits, whiskers, and crosses of the box plots, respectively represent median, interquartile range, and outliers of the data distribution from the n
recording sites. Stars indicate significance (all p values < 0.05) of regression estimates (one-sample, two-sided Student’s t test). Error bars correspond to
inter-sites SEM and dots correspond to individual recording sites. Fig. 3 Investigation of PE signals across frequency bands. a Anatomical localization of the aINS (purple), dlPFC (green), vmPFC (red), and lOFC (blue). All recording sites located in these parcels were included in the ROI analyses. The sample size used to derive statistics in panels b–d are displayed for each
ROI (aINS: n = 83; dlPFC: n = 74; vmPFC: n = 54; lOFC: n = 70). b. Time-frequency decomposition of PE signals following outcome onset. Hotter colors
indicate more positive regression estimates. Horizontal dashed lines indicate boundaries between frequency bands that are investigated in panels c and
d. c Time course of regression estimates obtained from linear fit of BGA with PE modeled across reward and punishment conditions. Solid lines (filled
areas) indicate mean (SEM) across recording sites. Horizontal bold black lines indicate significant clusters (pc < 1 × 10−3; one-sample, two-sided Student’s t
test after cluster-wise correction). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Note that an inherent limitation to any iEEG study is that the
number of recorded sites varies across parcels, which impacts the
statistical power of analyses used to detect PE signals in different
brain regions. iEEG: PE signals across ROIs and frequency bands. In each ROI
(Fig. 3a), we explored whether activity in other frequency bands
could also be related to PE (collapsed across reward and pun-
ishment conditions). We performed a time-frequency decom-
position of the evoked response around outcome onset and g
For each parcel, we first tested the significance of regression
estimates (averaged over the 0.25–1 s time window following
outcome onset) in a fixed-effect analysis (pooling sites across
patients) and Bonferroni-corrected for multiple comparisons NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w (
)
To confirm this dissociation between reward PE and punish-
ment PE signaling regions, we performed a number of control
analyses, at different levels from iEEG data preprocessing to
model-based regressions. First, the functional dissociation was
unchanged when using an alternative procedure for extracting
BGA (see Methods section and Supplementary Fig. 1) or after
removing recording sites with pathological activity and trials with
artifacts (see Methods section and Supplementary Fig. 2). p
(Q
)
Finally, we explored the effects of domains (reward vs. punishment PEs) in the four other brain regions associated with
PE (Supplementary Fig. 8). BGA in the medial Inferior Temporal
Cortex (mITC) was associated with outcomes (reward and
punishments), but not with expected value, so this region would
not qualify as signaling PE. The three other ROIs (hippocampus
(HPC), lateral visual cortex (lVC), and caudal medial visual
cortex: (cmVC)), showed a dissociation in time, with a short
punishment PE and prolonged reward PE. When averaging the
signal over the 0.25–1-s post-outcome time window, there was no
significant difference between reward and punishment PE in
these regions (all p values < 0.05). There was therefore no strong
evidence for these regions to be associated with either reward or
punishment learning. pp
y
g
Second, we examined whether the functional dissociation could
arise from a differential involvement in the different leaning
phases, as patients typically get more punishments at the
beginning and more reward at the end of a learning session. Also, during the course of leaning, patients could figure out the
mapping between the different pairs of cues and reward versus
punishment domains and hence reframe their expectations. We
checked that the difference between RPE and PPE signals
observed across the four regions of interest was still significant
even after controlling for trial index within learning sessions
(Supplementary Fig. 3). We also checked that the magnitude of
PE signals was constant throughout the learning session. For this
we simply used the contrast between possible outcomes (reward
versus no reward and punishment versus no punishment), in a
model-free analysis. In the RL framework, this contrast should be
stable because it does not depend on what is learned (i.e., on
expectations). Indeed, this contrast of BGA activity following zero
and non-zero outcomes was of similar magnitude in early and
late trials of learning sessions (Supplementary Fig. 4). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w To confirm this observation, we regressed trial-wise power
against PE, in the four ROIs and four frequency bands, for each
time point between −0.2 and 1.5 s around outcome onset. In the
broadband gamma (Fig. 3c), we found a significant cluster-
corrected association with PE in the 0.09–1.00 s window for the
aINS (βPE = 0.08 ± 0.007, sum(t(82)) = 462.2, pc < 1 × 10−3, one-
sample, two-tailed Student’s t test after cluster-wise correction),
0.19–0.97 s for the dlPFC (βPE = 0.06 ± 0.008, sum(t(73)) = 273.5,
pc < 1 × 10−3), 0.30–0.86 s for the vmPFC (βPE = 0.03 ± 0.008,
sum(t(53)) = 115.3, pc < 1 × 10−3) and 0.39–0.94 s for the lOFC To compare the contribution of activities in the different
frequency bands to PE signaling across the four ROIs, we
included them as separate regressors in general linear models
meant to explain PE. The general aim of this analysis was to test 5 TURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w whether lower-frequency bands were adding any information
about PE (compared to BGA alone). We thus compared GLMs
including only BGA to all possible GLMs containing broadband
gamma
plus
any
combination
of
low-frequency
activities. Bayesian
model
selection
(see
Methods)
designated
the
broadband-gamma-only GLM as providing the best account of
PE (Ef = 0.997, Xp = 1). Thus, even if low-frequency activity was
significantly related to PE, it carried redundant information
relative to that extracted from BGA. punishment (Supplementary Fig. 5). In the aIns and dlPFC,
recording sites were more likely to represent punishment
prediction errors whereas in the vmPFC and lOFC, recording
sites were more likely to represent reward prediction errors. iEEG: breaking PE into outcome and expectation signals. We
next decomposed PE signals into outcome and expected value, for
both reward (R and Qr) and punishment (P and Qp), to test
whether the two components were reflected in BGA, at the time
of outcome display. We observed a consistent pattern across
ROIs: while the outcome component was positively correlated
with BGA, the expectation component was negatively correlated
with BGA (Fig. 4b). To further quantify this pattern in each ROI,
we tested regression estimates (averaged over a 0.25–1-s post-
outcome time window) of both outcome and expectation for both
reward and punishment PE (Fig. 4c). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w We found that both com-
ponents of PE were significantly expressed in the vmPFC and
lOFC BGA following reward outcomes and in the aIns and dlPFC
BGA following punishment outcomes (all p values < 0.05). Because baseline correction might affect the decrease in BGA
observed with expected reward or punishment, we checked that
the results were robust (Supplementary Fig. 6) to a change in the
time window used to extract baseline activity (500 ms pre-fixation
epoch instead of 6 s around outcome onset). iEEG: comparison between reward and punishment PE. In the
following analyses, we focused on BGA and tested whether pre-
diction errors estimated in the reward (RPE = R-Qr) and pun-
ishment (PPE = P-Qp) conditions could be dissociated between
the four ROIs previously identified (aINS, dlPFC, vmPFC, and
lOFC). We computed the time course of regression estimates
separately for reward and punishment PE. We observed an
increase of the regression estimate at the time of outcome display,
which differed between conditions and ROIs (Fig. 4a). In aINS
and dlPFC, regression estimates were significantly higher for
punishment than for reward PE, in the 0.23–0.70 s window for
the aINS (βRPE−βPPE = −0.06 ± 0.02, sum(t(82)) = −97.01, pc <
1 × 10−3, one-sample two-tailed Student’s t test after cluster-wise
correction) and in the 0.25–1.5 s for the dlPFC (βRPE−βPPE =
−0.06 ± 0.01, sum(t(73)) = −368.6, pc < 1 × 10−3). An opposite
pattern emerged in the vmPFC and lOFC: regression estimates
were significantly higher for reward PE than for punishment PE,
in the 0.48–1.02 s for the vmPFC (βRPE−βPPE = 0.06 ± 0.01, sum
(t(53)) = 116, pc < 1 × 10−3) and in the 0.72–1.45 s for the lOFC
(βRPE−βPPE = 0.04 ± 0.01, sum(t(69)) = 138.7, pc < 1 × 10−3). We next examined whether expectation signals could be
observed during the delay period preceding outcome delivery, by
averaging regression estimates over a −1.2 to −0.2 time window
before outcome onset. We found (Supplementary Fig. 7) that
expectations tended to be positively related to BGA in all regions
but regression estimates were significantly above zero for
expected punishment only (Qp in aIns and dlPFC), and not for
expected reward (Qr in vmPFC and lOFC). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w This was
observed in both reward PE (vmPFC and lOFC) and punishment
PE (aIns and dlPFC) signaling ROIs. Thus, PE were reliably
signaled from the beginning to the end of learning, with a stable
difference between ROIs more sensitive to reward versus
punishment outcomes. NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunicatio Discussion
H Horizontal bold
es indicate significant difference between conditions (blue: RPE > PPE; red: PPE > RPE; pc < 0.05). Shaded areas represent inter-sites SEM. b Time course
regression estimates obtained from a linear model including both outcome (solid lines) and expected value (dotted lines) components for both reward
and Qr) and punishment (P and Qp) PE. c Regression estimates averaged over the 0.25–1 s time window (represented as shaded gray areas in panels b). ars indicate significance (*p < 0.05, one-sample, two-tailed Student’s t test). Error-bars correspond to inter-sites SEM and dots correspond to individual
cording sites. The sample size (n) used to derive statistics in all panels was: aINS: n = 83 sites; dlPFC: n = 74; vmPFC: n = 54; lOFC: n = 70. RPE
PPE
-0.1
0
0.1
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0
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Qp
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Qr
P
Qp
b
c
a
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Qp
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PE components
Time from outcome (s)
Time from outcome (s)
Regression estimate
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-0.5
0
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0
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0
0.5 b c Regression estimate
c Regression estimate Regression estimate Regression estimate Regression estimate Regression estimate Regression estimate 0
0.5
1
1.5
Time from outcome (s) Fig. 4 Dissociation of reward PE (R-Qr) and punishment PE (P-Qp) signals. a Time course of regression estimates obtained from linear fit of BGA with PE
modeled separately for the reward (blue) and punishment (red) conditions (PPE punishment prediction error, RPE reward prediction error). Horizontal bold
lines indicate significant difference between conditions (blue: RPE > PPE; red: PPE > RPE; pc < 0.05). Shaded areas represent inter-sites SEM. Discussion
H Here, we compared the neural correlates of reward and punish-
ment PE during instrumental learning. We identified a set of
brain regions signaling PE in different frequency bands, the most
informative being BGA. All regions signaled outcomes with
increased BGA and expectations with decreased BGA. However,
there was a partial dissociation: the vmPFC and lOFC emitted
stronger signals for reward PE, whereas the aINS and dlPFC
emitted stronger signals for punishment PE. This anatomical
divide relates to the learning domain (reward versus punishment)
and not to the sign of PE, since in all cases outcomes (whether
positive or negative) were signaled with increased BGA. In the
following, we successively discuss the specification of PE signals
in terms of anatomical location and frequency band, and then the
dissociation between reward and punishment PE. Third, we examined whether the differences between ROIs
signaling reward and punishment PE could arise from differences
in the proportion of recording sites sensitive to reward versus When regressing BGA against PE modeled across learning
conditions, we identified significant correlates in a number of
brain regions. Among the significant ROIs, some (e.g., the vmPFC 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Regression estimate
b RPE
PPE
-0.1
0
0.1
0.2
0.3
aINS (n=83)
Regression estimate
0
0.5
1
1.5
Regression estimate
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0
0.1
0.2
0
0.5
1
1.5
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0
0.1
0.2
P
Qp
R
Qr
-0.5
0
0.5
Regression estimate
R
Qr
P
Qp
b
c
a
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0
0.1
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1
1.5
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1
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Qr
P
Qp
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Qr
P
Qp
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0
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1
1.5
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0
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R
Qr
P
Qp
PE components
Time from outcome (s)
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Regression estimate
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-0.5
0
0.5
-0.5
0
0.5
-0.5
0
0.5
g. 4 Dissociation of reward PE (R-Qr) and punishment PE (P-Qp) signals. a Time course of regression estimates obtained from linear fit of BGA with PE
odeled separately for the reward (blue) and punishment (red) conditions (PPE punishment prediction error, RPE reward prediction error). NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w different ROIs, with an increase in the beta to high-gamma band,
and an increase followed by a decrease in the theta to alpha band. The late decrease may reflect the anti-correlation between theta-
band activity and the other signals (broadband gamma, hemo-
dynamic, and spiking activity) that was documented in previous
studies29,30,38. However, the early increase is more surprising and
suggests that PE are initially signaled in low-frequency activity,
before reaching BGA. Yet when the different frequency bands
were put in competition for predicting PE across trials, low-
frequency activity proved to be redundant, with respect to the
information already contained in BGA. This result is in line with
our results regarding subjective valuation for decision making39:
all the information available in neural activity could be found in
BGA, even if activity in lower-frequency bands also showed sig-
nificant value signals. An important conclusion of our analyses is that the dissocia-
tion was made between reward and punishment PE, not between
positive and negative PE. Indeed, some learning models assume
that positive and negative PE are processed differently, yielding
different learning rates (e.g. refs. 41,42). A strict dissociation
between positive and negative PE (across valence) would imply
that regions signaling reward PE with increased activity would
signal punishment PE with decreased activity, and vice-versa. This would induce an ambiguity for the rest of the brain, as an
omitted reward would be coded similarly to an inflicted pun-
ishment, and an avoided punishment similarly to an obtained
reward. This is not the pattern that we observed: on the contrary,
both reward and punishment PE were positively correlated to
BGA in all regions (at least numerically, if not significantly). Yet
reward and punishment PE could be distinguished by a down-
stream region, from the relative activity of regions more sensitive
to reward and those more sensitive to punishment. Thus, rather
than the sign of PE, the dissociation depended on their domain,
i.e., on whether the PE should reinforce the repetition or the
avoidance of last choice. The timing of PE signals, peaking ~0.5 s after outcome onset,
was roughly compatible with that observed in the hemodynamic
response, which is typically delayed by 5–6 s. The (positive)
correlation with the outcome and the (negative) correlation with
the expectation were simultaneously observed after outcome
display. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Within the punishment-sensitive regions, the aINS was
expected, as it was associated with punishment PE in our fMRI
study using the same task6 and because it is systematically cited in
meta-analyses of fMRI studies searching for neural correlates of
punishment outcomes35,36,40. Surprisingly, the link between aINS
activity and punishment PE has seldom been explored in non-
human primates. This exploration was made possible here by the
development of oblique positioning techniques employed to
implant electrodes, which result in a large spatial sampling of the
insular cortex50. This is important because other iEEG approa-
ches, such as subdural recordings (Ecog), could not explore the
role of the insular cortex in instrumental learning26. The present
result echoes a previous finding, using the same technique, that
aINS BGA signals mistakes in a stop-signal task51. By compar-
ison, punishment PE signals in the dlPFC were less expected,
since they were not observed in fMRI results using the same task,
even if it is not uncommon to observe dlPFC activation following
punishment outcomes35,36,40. The dissociation observed here at
the cortical level between reward versus punishment PE signals
might be related to afferences from different neuromodulatory
systems, such as dopaminergic versus serotonergic pathways,
which have been suggested to play opponent roles in approach
versus avoidance behaviors3,52. Within the reward-sensitive regions, the vmPFC was expected,
given the number of fMRI studies reporting a link between
vmPFC and reward outcome, including those using the same task
as here (Pessiglione et al.6 reanalyzed in Palminteri et al.11) and
meta-analyses34–36,43. The expression of reward PE in the vmPFC
might relate to its position as a main efferent output of midbrain
dopamine
neurons,
following
the meso-cortical
pathway44. Indeed, manipulation of dopaminergic transmission was found to
interfere with reward learning, specifically9,10,22, an effect that
was captured by reward sensitivity in a computational model of
learning in this task6.The observation of reward PE signals in the
lOFC was less expected, because it is generally not reported in
meta-analyses of human fMRI studies and because several elec-
trophysiology studies in animals suggested that, even if orbito-
frontal cortex neurons respond to reward outcomes, they might
not
encode
prediction
errors45,46. However,
the
similarity
between lOFC and vmPFC reward PE signals is consistent with
previous iEEG studies showing similar representation of sub-
jective value and reward outcome in the two regions BGA39,47. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Yet the lOFC and vmPFC reward PE signals may serve different
functions, as was suggested by lesion studies in both human and
non-human primates showing that the lOFC (but not the
vmPFC) is critical for solving the credit assignment problem48,49. Wi hi
h
i h
i i
i
h
INS y
p
g
Although all our ROIs exhibited a similar pattern of response,
they differ in the strength of reward versus punishment PE sig-
nals. This anatomical dissociation between learning systems in
the brain may appear at variance with previous studies reporting
that rewards and punishments are ubiquitously represented all
over the brain14,26. However, these non-specific results were
observed during tasks in which the outcome is either reward or
punishment. Thus, it is understandable that both reward and
punishment regions were mobilized by the outcome in these
previous studies, since being rewarded is not being punished and
vice-versa. Here, PE signals were defined by the comparison
between reward or punishment outcomes and their omission, not
with each other, which enabled a dissociation. Besides, the pre-
vious conclusion was based on the finding that the information
about reward versus punishment outcomes could be recovered
from many brain regions. Had we applied the same decoding
analysis here, we would have reached the same conclusion:
information about reward versus punishment PE could be
recovered in all our ROIs, precisely because their response
depended on outcome valence. In other words, the contrast
between reward and punishment PE would be significant in all
ROIs, precisely because they are differentially sensitive to the two
learning domains. The dissociation between regions signaling
reward and punishment PE may also seem at odds with single-
unit recordings showing reward and punishment PE signals can
be found in neighboring neurons15,16. Yet it should be empha-
sized that the dissociation observed here was only partial, com-
patible with the possibility that some regions contained more
reward-sensitive neurons and others more punishment-sensitive
neurons, even if both types can be found in all regions. g
g
p
Within the punishment-sensitive regions, the aINS was
expected, as it was associated with punishment PE in our fMRI
study using the same task6 and because it is systematically cited in
meta-analyses of fMRI studies searching for neural correlates of
punishment outcomes35,36,40. Surprisingly, the link between aINS
activity and punishment PE has seldom been explored in non-
human primates. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Interestingly, expectations tended to be positively asso-
ciated with BGA, the sign of the correlation being reverted when
the outcome was delivered. These observations, made possible
here by the high temporal resolution of iEEG, are rarely reported
in fMRI studies40. One reason is that the hemodynamic response,
because of its low temporal resolution, may confound positive
expectation at cue onset and negative expectation at outcome
onset, unless the two events are separated by a long delay (as in,
e.g. refs. 23,24). Our double observation corroborates a previous
study showing that the differential response to positive and
negative feedbacks, recorded with intracranial electrodes, is
modulated by reward expectation26. As the response to outcome
can be viewed as an indicator of valence, and the modulation by
expectation as an effect of surprise, it shows that valence and
surprise can be represented in the same brain region, in accor-
dance with the very notion of prediction error signal. Within the reward-sensitive regions, the vmPFC was expected,
given the number of fMRI studies reporting a link between
vmPFC and reward outcome, including those using the same task
as here (Pessiglione et al.6 reanalyzed in Palminteri et al.11) and
meta-analyses34–36,43. The expression of reward PE in the vmPFC
might relate to its position as a main efferent output of midbrain
dopamine
neurons,
following
the meso-cortical
pathway44. Indeed, manipulation of dopaminergic transmission was found to
interfere with reward learning, specifically9,10,22, an effect that
was captured by reward sensitivity in a computational model of
learning in this task6.The observation of reward PE signals in the
lOFC was less expected, because it is generally not reported in
meta-analyses of human fMRI studies and because several elec-
trophysiology studies in animals suggested that, even if orbito-
frontal cortex neurons respond to reward outcomes, they might
not
encode
prediction
errors45,46. However,
the
similarity
between lOFC and vmPFC reward PE signals is consistent with
previous iEEG studies showing similar representation of sub-
jective value and reward outcome in the two regions BGA39,47. Yet the lOFC and vmPFC reward PE signals may serve different
functions, as was suggested by lesion studies in both human and
non-human primates showing that the lOFC (but not the
vmPFC) is critical for solving the credit assignment problem48,49. Discussion
H b Time course
of regression estimates obtained from a linear model including both outcome (solid lines) and expected value (dotted lines) components for both reward
(R and Qr) and punishment (P and Qp) PE. c Regression estimates averaged over the 0.25–1 s time window (represented as shaded gray areas in panels b). Stars indicate significance (*p < 0.05, one-sample, two-tailed Student’s t test). Error-bars correspond to inter-sites SEM and dots correspond to individual
recording sites. The sample size (n) used to derive statistics in all panels was: aINS: n = 83 sites; dlPFC: n = 74; vmPFC: n = 54; lOFC: n = 70. explanation would be that regions such as the dlPFC are more
heterogenous across individuals, such that the group-level ran-
dom-effect analyses typically conducted in fMRI studies might be
less sensitive than the fixed-effect analyses performed here or in
animal studies. Conversely, some key regions consistently found to
signal PE in fMRI studies (e.g., the ventral striatum) are absent in
our results, for the simple reason that they were not sampled by
the electrodes implanted for clinical purposes. Even if our results
provide some insights about the location of PE signals, they
cannot be taken as arising from a fair whole-brain analysis, since
some regions were more frequently sampled than others, biasing
the statistical power and hence the sensitivity of PE detection. and aINS) were classic regions related to prediction errors in
meta-analyses of human fMRI studies34–36, whereas others (e.g.,
the dlPFC and lOFC) were regions where single-neuron firing
activity in non-human primates was shown to correlate with
prediction error19,20,37. Our study thus fills a gap across species
and techniques, confirming that intracerebral BGA is a relevant
neurophysiological signal related to both hemodynamic and
spiking activity, as previously suggested28–31. g
y
y
gg
Yet it raises the question of why fMRI studies, including those
using the same task as here6, often failed to detect PE correlates in
regions such as the dlPFC and lOFC. One possible explanation is
the more stringent correction for multiple comparisons across
voxels in fMRI studies, compared to the correction across ROIs
applied here, or the absence of correction in most animal studies
that
typically
investigate
a
single
brain
region. Another p
y
In all the investigated ROIs, we also found significant links with
activity in lower-frequency bands. Time-frequency decomposi-
tion of PE correlates yielded remarkably similar patterns in the 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w We suggest
that artifact removal may be unnecessary in model-based analyses
of iEEG activity, and that results may actually be more robust if
based on the entire dataset without ad-hoc selection based on
visual inspection. Behavioral task. Patients performed a probabilistic instrumental learning task
adapted from previous studies6,11. Patients were provided with written instructions,
which were reformulated orally if necessary, stating that their aim in the task was to
maximize their financial payoff and that to do so, they had to consider reward-
seeking and punishment-avoidance as equally important (Fig. 1). Patients per-
formed short training sessions to familiarize with the timing of events and with
response buttons. Training procedure comprised a very short session, with only
two pairs of cues presented on 16 trials, followed by 2–3 short sessions of 5 min
such that all patients reached a threshold of 70% correct choices during both the
reward and punishment conditions. During iEEG recordings, patients performed
three to six test sessions on a single testing occurrence (with short breaks between
sessions). Each session was an independent task containing four new pairs of cues
to be learned. Cues were abstract visual stimuli taken from the Agathodaimon
alphabet. Each pair of cues was presented 24 times for a total of 96 trials. The four
cue pairs were divided in two conditions (2 pairs of reward and 2 pairs of pun-
ishment cues), associated with different pairs of outcomes (winning 1€ versus
nothing or losing 1€ versus nothing). The reward and punishment conditions were
intermingled in a learning session and the two cues of a pair were always presented
together. Within each pair, the two cues were associated to the two possible out-
comes with reciprocal probabilities (0.75/0.25 and 0.25/0.75). On each trial, one
pair was randomly presented and the two cues were displayed on the left and right
of a central fixation cross, their relative position being counterbalanced across
trials. The subject was required to choose the left or right cue by using their left or
right index to press the corresponding button on a joystick (Logitech Dual Action). Since the position on screen was counterbalanced, response (left versus right) and
value (good versus bad cue) were orthogonal. The chosen cue was colored in red
for 250 ms and then the outcome was displayed on the screen after 1000 ms. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Reward PE signals were also observed in both aINS and dlPFC
regions, albeit with a lesser sensitivity. This may be interpreted as
an effect of saliency rather than PE22,53, as punishments were less
frequent in the task than rewards (because patients learned to
avoid the former and obtain the latter). However, pure saliency
coding would not explain the responses to punishments observed
in the aINS during Pavlovian learning tasks where high punish-
ments were controlled to be more frequent than low punishments
(e.g. Seymour et al.54) or in gambling tasks where punishment
and reward outcomes were matched (e.g. Petrovic et al.55). Also,
saliency coding would not predict the consequence of aINS
damage, which was found to specifically impair punishment
learning in this task, an effect that was captured by a specific
diminution of the sensitivity to punishment outcome in a com-
putational model11. Yet it remains that reward and punishment
learning are not exact symmetrical processes, since positive
reward PE favors repetition of the same choice, whereas positive
punishment PE pushes to the alternative choice, hence involving
an additional switching process. This switching process might
explain the longer choice RT observed in the punishment con-
dition. The switch might relate to the prolonged implication of
the dlPFC following punishment PE, in keeping with the estab-
lished role of this region in cognitive control56–58. The implica-
tion of the aINS might be more related to the aversiveness of
punishment PE, in line with the role attributed to this region in
pain, interoception, and negative feelings59–61. CHUG-12, study 0907) and from a competent authority (ANSM no: 2009-A00239-
48). CHUG-12, study 0907) and from a competent authority (ANSM no: 2009-A00239-
48). iEEG data acquisition and preprocessing. Patients underwent intracerebral
recordings by means of stereotactically implanted semirigid, multilead depth
electrodes (sEEG). In total, 5–17 electrodes were implanted in each patient. Elec-
trodes had a diameter of 0.8 mm and, depending on the target structure, contained
8–18 contact leads 2-mm-wide and 1.5-mm-apart (Dixi, Besançon, France). Anatomical localizations of iEEG contacts were determined on the basis of post-
implant computed tomography scans or postimplant MRI scans coregistered with
preimplant scans62. Electrode implantation was performed according to routine
clinical procedures, and all target structures for the presurgical evaluation were
selected strictly according to clinical considerations with no reference to the
current study. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Neuronal recordings were conducted using an audio–video-EEG monitoring
system (Micromed, Treviso, Italy), which allowed simultaneous recording of
128–256 depth-EEG channels sampled at 256 Hz (1 patient, note that this patient
was removed from analyses based on broadband gamma activity which could not
be computed given the low sampling rate), 512 Hz (6 patients), or 1024 Hz (12
patients) [0.1–200 Hz bandwidth]. One of the contacts located in the white matter
was used as a reference. Each electrode trace was subsequently re-referenced with
respect to its direct neighbor (bipolar derivations with a spatial resolution of 3.5
mm) to achieve high local specificity by canceling out effects of distant sources that
spread equally to both adjacent sites through volume conduction63. In order to take advantage of our large sample set, all recording sites with an
anatomical label were included in the analyses (i.e., without any exclusion of sites
with artifacts or pathological activity). Nevertheless, to check whether electrodes
with artifacts or pathological activity could have biased the results, we applied a
semi-automatic pipeline: first, bad channels detection was conducted with a
machine learning approach64, secondly, epileptic spikes were detected
automatically with Delphos – Detector of ElectroPhysiological Oscillations and
Spikes –65 and all data were then finally visually inspected to check their quality. We also excluded recording sites that were part of the epileptogenic zone by
identifying with the neurologists (PK and SR) all recording sites involved at seizure
onset and/or sites that were located within the cortical resection (if any) performed
after the sEEG; furthermore, trials during which iEEG activity was higher or lower
than four times the standard deviation of the average signal were excluded. The
results were not affected by this procedure. The data have been collected in patients being treated for
epilepsy. It is unlikely that disease or treatment affected learning
performance because the behavior of our patients was comparable
to that of healthy young participants performing a similar task
(e.g. Palminteri et al.27). We made the assumption that epileptic
activity did not distort the brain signals linked to PEs and
interpreted the data as if they were collected in healthy indivi-
duals. Furthermore, epileptic artifacts are unlikely to covary with
the computational variable (PE) against which brain activity was
regressed. We formally verified that removing artifacts did not
affect the results about PE signals observed in BGA. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w In
order to win money, patients had to learn by trial and error the cue–outcome
associations, so as to choose the most rewarding cue in the reward condition and
the less punishing cue in the punishment condition. Visual stimuli were delivered
on a 19 inch TFT monitor with a refresh rate of 60 Hz, controlled by a PC with
Presentation 16.5 (Neurobehavioral Systems, Albany, CA). In summary, we used human intracerebral BGA to test the a
priori theoretical principle that reward and punishment PE could
be processed by the same brain machinery (one being the nega-
tive of the other). On the contrary, we found that both reward
and punishment PE were positively correlated to BGA in all brain
regions. Yet some regions amplified reward PE signals, and others
punishment PE signals. Thus, the dissociation between reward
and punishment brain systems is not about the sign of the cor-
relation with PE, but about the valence domain of outcomes
(better or worse than nothing). These appetitive and aversive
domains correspond to different behaviors that must be learned:
more or less approach for reward PE and more or less avoidance
for punishment PE. Further research is needed to disentangle the
roles of the different reward and punishment regions in these
learning processes. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w This exploration was made possible here by the
development of oblique positioning techniques employed to
implant electrodes, which result in a large spatial sampling of the
insular cortex50. This is important because other iEEG approa-
ches, such as subdural recordings (Ecog), could not explore the
role of the insular cortex in instrumental learning26. The present
result echoes a previous finding, using the same technique, that
aINS BGA signals mistakes in a stop-signal task51. By compar-
ison, punishment PE signals in the dlPFC were less expected,
since they were not observed in fMRI results using the same task,
even if it is not uncommon to observe dlPFC activation following
punishment outcomes35,36,40. The dissociation observed here at
the cortical level between reward versus punishment PE signals
might be related to afferences from different neuromodulatory
systems, such as dopaminergic versus serotonergic pathways,
which have been suggested to play opponent roles in approach
versus avoidance behaviors3,52. NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunicat ð2Þ The dlPFC ROI
(n = 74 sites) was defined as the inferior and superior bilateral dorsal prefrontal
cortex (MarsAtlas labels: PFrdli and PFrdls). The aINS ROI (n = 83 sites) was
defined as the bilateral anterior part of the insula (Destrieux atlas labels: Short
insular gyri, anterior circular insular sulcus and anterior portion of the superior
circular insular sulcus). g
To assess the contribution of the different frequency bands to prediction errors,
we used the following GLM: PE ¼ βγ ´ Y γ
þ ββ ´ Y β
þ βeθα ´ Y eθα
ð
Þ þ βlθα ´ Y lθα
ð
Þ ð7Þ With βλ, ββ, βeθα, and βlθα corresponding to the regression estimates of the
power time series Y in the broadband gamma, beta, early theta-alpha and late
theta-alpha bands. This GLM was compared to the eight possible alternative GLMs
that combine BGA power to a single other frequency band (beta or early theta-
alpha or late theta-alpha), two additional frequency bands (beta and early theta-
alpha or beta and late theta-alpha or early and late theta-alpha) or all possible
frequency bands (beta and early theta-alpha and late theta-alpha). Computation of single-trial broadband gamma envelopes. Broadband gamma
activity (BGA) was extracted with the Hilbert transform of iEEG signals using
custom MATLAB scripts as follows. iEEG signals were first bandpass filtered in
10 successive 10-Hz-wide frequency bands (e.g., 10 bands, beginning with 50–60
Hz up to 140–150 Hz). For each bandpass filtered signal, we computed the
envelope using standard Hilbert transform. The obtained envelope had a time
resolution of 15.625 ms (64 Hz). Again, for each band, this envelope signal (i.e.,
time-varying amplitude) was divided by its mean across the entire recording ses-
sion and multiplied by 100 for normalization purposes. Finally, the envelope sig-
nals computed for each consecutive frequency bands (e.g., 10 bands of 10 Hz
intervals between 50 and 150 Hz) were averaged together, to provide one single
time-series (the BGA) across the entire session, expressed as percentage of the
mean. This procedure was used to counteract a bias toward the lower frequencies of
the frequency interval induced by the 1/f drop-off in amplitude. Finally, these time-
series were smoothed with a 250 ms sliding window to increase statistical power for
inter-trial and inter-individual analyses of BGA dynamics. ð2Þ Bayesian information criterion (BIC) was computed for each subject and model: BIC ¼ log ntrials
ð
Þ ´ n degrees of freedom
þ 2 ´ LLmax
ð5Þ Y ¼ α þ β ´ PE
ð6Þ ð5Þ Y ¼ α þ β ´ PE ð6Þ Outcome prediction errors (estimated with the QLr model) for each patient and
trial were then Z-scored and used as statistical regressors for iEEG data analysis with β corresponding to the regression estimate on which statistical tests are
conducted. PE corresponds to Prediction errors collapsed across reward and punishment conditions in
Fig. 3 (GLM1) Electrophysiological analyses. Collected iEEG signals were analyzed using
Fieldtrip66 and homemade MATLAB codes. Anatomical labeling of bipolar deri-
vation between adjacent contact-pairs was performed with IntrAnat software62. The 3D T1 pre-implantation MRI gray/white matter was segmented and spatially
normalized to obtain a series of cortical parcels using MarsAtlas32 and the Des-
trieux atlas67. 3D models of electrodes were then positioned on post-implantation
images (MRI or CT). Each recording site (i.e., each bipolar derivation) was thus
labeled according to its position in a parcellation scheme in the patients’ native
space. Thus, the analyzed dataset only included electrodes identified to be in the
gray-matter. g
Either reward or punishment PE in Fig. 4. To quantify the number of recorded sites related to prediction errors (PE) for
each brain parcel (Supplementary Table 2), we assessed significance of regression
estimates in the time domain by applying a correction for multiple comparison
(focusing on the in the [0.25 1 s] time window) using the false discovery rate
algorithm33 and by discarding significant effects lasting <100 ms. Because using
alternative methods to define the individual statistical threshold (FDR, computed
as above, uncorrected p < 0.005 or a common threshold such that the average
regression estimates had to be >0.1) yielded to qualitatively similar results, we used
the common threshold for clarity/display purpose to generate Supplementary
Fig. 5. Regions of interest definition. The vmPFC ROI (54 sites) was defined as the ven-
tromedial PFC plus the fronto-medial part of orbitofrontal cortex bilaterally
(MarsAtlas labels: PFCvm plus mesial part of OFCv and OFCvm). The lOFC ROI
(n = 70 sites) was defined as the bilateral central and lateral parts of the orbito-
frontal cortex (MarsAtlas labels: OFCvl plus lateral parts of OFCv). ð2Þ with R(t) the reinforcement value among −1€, 0€, and +1€. Using the expected
values associated with the two possible cues, the probability (or likelihood) of each
choice was estimated using the softmax rule: Time-frequency decomposition. Time-frequency analyses were performed with the
FieldTrip toolbox for MATLAB. A multitapered time-frequency transform allowed
the estimation of spectral powers (Slepian tapers; lower-frequency range: 4–32 Hz,
6 cycles and 3 tapers per window; higher frequency range: 32–200 Hz, fixed time-
windows of 240 ms, 4–31 tapers per window). This approach uses a steady number
of cycles across frequencies up to 32 Hz (time window durations therefore decrease
as frequency increases) whereas for frequencies above 32 Hz, the time window
duration is fixed with an increasing number of tapers to increase the precision of
power estimation by increasing smoothing at higher frequencies. Pat ¼
eQat=β
eQat=β þ eQbt=β
ð3Þ ð3Þ The constant parameters α and β are the learning rate and choice temperature,
respectively. A second Q-Learning model (QLr) was implemented to account for
the tendency to repeat the choice made on the preceding trial, irrespective of the
outcome. A constant (θ) was added in the softmax function to the expected value of
the option chosen on the previous trial presented the same cues. For example, if a
subject chose option A on trial t: q
y
q
duration is fixed with an increasing number of tapers to increase the precision of
power estimation by increasing smoothing at higher frequencies. Patþ1 ¼
eQatþθ=β
eQatþθ=β þ eQbt=β
ð4Þ ð4Þ General linear models. Frequency envelopes of each recording site were epoched on
each trial and time locked to the outcome onset (−3000 to 1500 ms). Each time
series was regressed against the variables of interest to obtain a regression estimate
per time point and recording site. In all GLMs, normalized power (Y) was regressed
across trials against prediction error signal PE (normalized within patients) at every
time point: We optimized model parameters by minimizing the negative log likelihood
(LLmax) of choice data using MATLAB fmincon function, initialized at multiple
starting points of the parameter space, as previously described (Palminteri et al.27). Methods
P ti
t
I Patients. Intracerebral recordings were obtained from 20 patients (33.5 ± 12.4
years old, 10 females, see demographical details in Supplementary Table 1) suf-
fering from pharmaco-resistant focal epilepsy and undergoing presurgical evalua-
tion. They were investigated in two epilepsy departments (Grenoble and Lyon). To
localize epileptic foci that could not be identified through noninvasive methods,
neural activity was monitored in lateral, intermediate, and medial wall structures in
these patients using stereotactically implanted multilead electrodes (stereotactic
intracerebral electroencephalography, iEEG). All patients gave written informed
consent and the study received approval from the ethics committee (CPP 09- Behavioral analysis. Percentage of correct choice (i.e., selection of the most
rewarding or the less punishing cue) and reaction time (between cue onset and
choice) were used as dependent variables. Statistical comparisons between reward
and punishment learning were assessed using two-tailed paired t-tests. All statis-
tical analyses were performed with MATLAB Statistical Toolbox (MATLAB
R2017a, The MathWorks, Inc., USA). 9 TURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w Computational modeling. A standard Q-learning algorithm (QL) was used to frequency band of interest47 instead of filtering across consecutive frequency bands
yielded to a similar pattern of result (Supplementary Fig. 1). For each trial, baseline
correction (Z-score) was applied using the average and standard deviation com-
puted over a 6-s epoch centered around outcome onset (−3 to 3 s). We also verified
that using a 500-ms fixation epoch preceding the outcome to z-score each trial
yielded similar results. p
g
Q
g
g
(Q )
model choice behavior. For each pair of cues, A and B, the model estimates the
expected value of choosing A (Qa) or B (Qb), according to previous choices and
outcomes. The initial expected values of all cues were set at 0, which corresponded
to the average of all possible outcome values. After each trial (t), the expected value
of the chosen stimuli (say A) was updated according to the rule: Qatþ1 ¼ Qat þ α*δt
ð1Þ ð1Þ Computation of envelopes in lower frequencies. The envelopes of theta, alpha and
beta bands were extracted in a similar manner as the broadband gamma frequency
except that steps were 1 Hz for θ and α and 5 Hz for β. The ranges corresponding to
the different frequency bands were as follows: broadband gamma was defined as
50–150 Hz, beta as 13–33 Hz, alpha as 8–13 Hz, and theta as 4–8 Hz. For each trial,
baseline correction (Z-score) was applied using the average and standard deviation
computed over a 6-s epoch centered around outcome onset (−3 to 3 s). The outcome prediction error, δ(t), is the difference between obtained and
expected outcome values: δt ¼ Rt þ Qat
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previously shown to maximize the signal/noise ratio to detect task-related mod-
ulation of BGA by effectively smoothing the signal across frequencies and time to
detect BGA modulations that exhibit across trials and across sites variability in
terms of precise timing and frequency signature28,39,51,68–73. In addition, we also
checked whether estimating BGA using a single bandpass filter across the whole The model comparison was conducted using the VBA toolbox (Variational
Bayesian Analysis toolbox; available at http://mbb-team.github.io). Log-model
evidence obtained in each recording site was taken to a group-level, random-effect,
Bayesian model selection (RFX-BMS) procedure74. RFX-BMS provides an
exceedance probability (Xp) that measures how likely it is that a given model is
more frequently implemented, relative to all the others considered in the model
space, in the population from which samples are drawn. For the separate investigation of prediction error components, two separate
analyses were conducted for reward and punishment PE. For each analysis, power
time-series Y was regressed against both outcome (R or P) and expectation (Qr or
Qp): Y ¼ α þ β1 ´ R þ β2 ´ Q
ð8Þ ð8Þ With β1 and β2 corresponding to the outcome (R or P) and expectation (Qr or
Qp) regression estimates. For all GLMs, significance of regressors was assessed using one-sample two-
tailed t-test. T-values and p-values of those tests are reported in the result section. Once regions of interest were identified, significance was assessed through
permutation tests within each ROI. The pairing between power and regressor 10 NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunicatio ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23704-w 50. Afif, A., Minotti, L., Kahane, P. & Hoffmann, D. Anatomofunctional
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13-TECS-0013) and from the European Union Seventh Framework Program (FP7/2007–
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appropriate credit to the original author(s) and the source, provide a link to the Creative
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pursuit coding system. Neurology 87, 1517–1518 (2016). © The Author(s) 2021 NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunications 12 NATURE COMMUNICATIONS | (2021) 12:3344 | https://doi.org/10.1038/s41467-021-23704-w | www.nature.com/naturecommunicatio
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https://openalex.org/W4313233110
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https://hal.ehesp.fr/hal-03922908/file/irv13091.pdf
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English
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Preparing for the upcoming 2022/23 influenza season: A modelling study of the susceptible population in Australia, France, Germany, Italy, Spain and the United Kingdom
|
Influenza and other respiratory viruses
| 2,022
|
cc-by
| 3,795
|
To cite this version: Bronke Boudewijns, John Paget, Marco del Riccio, Laurent Coudeville, Pascal Crépey. Preparing for
the upcoming 2022/23 influenza season: A modelling study of the susceptible population in Australia,
France, Germany, Italy, Spain and the United Kingdom. Influenza and Other Respiratory Viruses,
2022, 10.1111/irv.13091. hal-03922908 Preparing for the upcoming 2022/23 influenza season: A
modelling study of the susceptible population in
Australia, France, Germany, Italy, Spain and the United
Kingdom Bronke Boudewijns, John Paget, Marco del Riccio, Laurent Coudeville, Pascal
Crépey Bronke Boudewijns, John Paget, Marco del Riccio, Laurent Coudeville, Pascal
Crépey S H O R T C O M M U N I C A T I O N S H O R T C O M M U N I C A T I O N HAL Id: hal-03922908
https://ehesp.hal.science/hal-03922908v1
Submitted on 4 Jan 2023 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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teaching and research institutions in France or
abroad, or from public or private research centers. Received: 9 December 2022
Accepted: 11 December 2022
DOI: 10.1111/irv.13091 Received: 9 December 2022
Accepted: 11 December 2022 Funding information /onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Bronke Boudewijns1
|
John Paget1
|
Marco Del Riccio1,2
|
Laurent Coudeville3
|
Pascal Crépey4 1Netherlands Institute for Health Services
Research (Nivel), Utrecht, The Netherlands
2Department of Health Sciences, University of
Florence, Florence, Italy
3Sanofi, Modeling Epidemiology and Data
Science, Lyon, France
4EHESP, CNRS, Inserm, Arènes - UMR 6051,
RSMS – U 1309, Université de Rennes,
Rennes, France 1Netherlands Institute for Health Services
Research (Nivel), Utrecht, The Netherlands
2Department of Health Sciences, University of
Florence, Florence, Italy
3Sanofi, Modeling Epidemiology and Data
Science, Lyon, France
4EHESP, CNRS, Inserm, Arènes - UMR 6051,
RSMS – U 1309, Université de Rennes,
Rennes, France Preparing for the upcoming 2022/23 influenza season:
A modelling study of the susceptible population in
Australia, France, Germany, Italy, Spain and the
United Kingdom Bronke Boudewijns1
|
John Paget1
|
Marco Del Riccio1,2
|
Laurent Coudeville3
|
Pascal Crépey4 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2022 Th A th
I fl
d Oth
R
i
t
Vi
bli h d b J h
Wil
& S
Ltd Influenza Other Respi Viruses. 2022;1–5. Abstract We analysed the influenza epidemic that occurred in Australia during the 2022 winter
using an age-structured dynamic transmission model, which accounts for past
epidemics to estimate the population susceptibility to an influenza infection. We
applied the same model to five European countries. Our analysis suggests Europe
might experience an early and moderately large influenza epidemic. Also, differences
may arise between countries, with Germany and Spain experiencing larger epidemics,
than France, Italy and the United Kingdom, especially in children. Correspondence
Pascal Crépey, Université de Rennes, EHESP,
CNRS, Inserm, Arènes - UMR 6051, RSMS – U
1309, Rennes, France. Email: pascal.crepey@ehesp.fr K E Y W O R D S
Australia, COVID-19, Europe, Influenza, modelling, susceptibility Funding information
This study was funded by a research grant
(FluCov project) from Sanofi. Funding information
This study was funded by a research grant
(FluCov project) from Sanofi. © 2022 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd. 1
|
INTRODUCTION 2 Before the COVID-19 pandemic, influenza was responsible for
15 000–70 000 deaths and up to 50 million symptomatic cases
every year in the European Union (EU) and European Economic
Association (EEA).1 After April 2020 and during the 2020/21 winter,
influenza circulation was massively reduced by the emergence of
SARS-CoV-2
and
the
related
non-pharmaceutical
interventions
(NPIs).2 NPIs have been largely lifted in the EU/EEA and influenza
co-circulated with SARS-CoV-2 during the 2021/22 winter,3 and it is
expected to make a stronger return during the 2022/23 winter.4
A number of news reports have looked at the influenza season in
Australia
and
predicted
alarming
scenarios
for
the
Northern
Hemisphere.5 We adapted an age-structured SEIR-like transmission model, which
accounts for the impact of past influenza epidemics on the suscepti-
bility for influenza in the population.6 The model was calibrated with
laboratory-confirmed detections7,8 and burden estimates9,10 to repli-
cate the dynamic of strain specific influenza epidemics over the period
2012–2022 in Australia and in the five largest countries in Western
Europe: France, Germany, Italy, Spain and the United Kingdom. Age-
stratified contact matrices in this model were based on the Mistry
et al. model.11 Our model accounts for influenza vaccination and
effectiveness (i.e., some vaccinated may still be susceptible), and this
allows an estimation of the level of immunity acquired after an influ-
enza epidemic and its progressive exponential waning over time wileyonlinelibrary.com/journal/irv Influenza Other Respi Viruses. 2022;1–5. 2 BOUDEWIJNS ET AL. 2 The influenza season in Europe (Northern Hemisphere) typically
lasts from November to April, with the number of cases usually peak-
ing around February.18 In 2020/21, there were hardly any influenza
detections (see Figure 1) in France, Germany, Italy, Spain and the
United Kingdom, and the following winter of 2021/22 was generally
mild, with a late influenza season dominated by influenza A(H3N2). There was some variation in influenza activity between countries; in
the United Kingdom, the season started relatively early, but the peak
was postponed in all five countries. Spain was the only country with
two distinct peaks, and the United Kingdom had the lowest levels of
laboratory detections compared with previous seasons. (average duration of 6 years for influenza A). In all six countries, the
vaccination coverage rates (VCRs) for influenza were collected and
included in the model.12,13 The United Kingdom was the only country
that reached the WHO VCR target of 75% for people aged 65 years
and older, with 81% in 2020/21 and 82% in 2021/22. Spain was the
second country closest to meet the target with 69% in 2021/22. We
also introduced the impact of NPIs on influenza transmission by using
the outcomes of a COVID-19 model14 in which country-specific levels
of NPIs were estimated as part of model calibration to reported cases,
hospitalizations and deaths.14,15 Figure 2 displays the proportion of susceptible persons per coun-
try and age-group from 2017 to 2022 for influenza A. Because vacci-
nated individuals remain partially susceptible, both vaccinated and
unvaccinated persons are presented. Results for Australia showed an
overall increase in the proportion of susceptible persons (+10.1%),
ahead of the 2022 influenza season compared with the reference
(maximum susceptible population between 2017 and January 2020). The estimated susceptible population was also higher in Germany
(+8.0%) and Spain (+6.2%), and lower in the United Kingdom
(1.6%). The percentage increase compared with the reference was
highest in the youngest two age-groups in both Australia (0–4 and
5–14 years) and the five European countries (0–1 and 2–17 years). The susceptibility picture in Germany and Spain looked most like the
one in Australia; in France and Italy, the proportion of susceptible 3
|
RESULTS Figure 1 displays the number of laboratory-confirmed influenza
detections per week from 2017–2022, available through FluNet.8 In
the Southern Hemisphere, the influenza season in Australia usually
starts in May and lasts until October.16 Following the emergence of
SARS-CoV-2 and the implementation of strong NPIs, including heavy
restrictions on travel to and from Australia, there was very little influ-
enza activity until the winter of 2022. After a 2-year absence, this sea-
son was characterised by an early appearance (mid-April 2022) and a
relatively high peak in influenza detections in June, followed by a
steep decrease and the season was dominated by influenza A (mostly
A(H3N2), if subtyped).17 y on [04/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Cr F I G U R E 1
Weekly number of laboratory confirmed influenza detections, reported to FluNet, since the 2017 season in Australia and
2017/18 season in France, Germany, Italy, Spain and the United Kingdom. The period preceding the COVID-19 pandemic is shown with a blue
background, the period with most COVID-19 related non-pharmaceutical interventions (NPIs) and few influenza cases is shown with a yellow
background, the period in 2022 after most NPIs were lifted per country28,29 is shown with a green background. s (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License F I G U R E 1
Weekly number of laboratory confirmed influenza detections, reported to FluNet, since the 2017 season in Australia and
2017/18 season in France, Germany, Italy, Spain and the United Kingdom. The period preceding the COVID-19 pandemic is shown with a blue
background, the period with most COVID-19 related non-pharmaceutical interventions (NPIs) and few influenza cases is shown with a yellow
background, the period in 2022 after most NPIs were lifted per country28,29 is shown with a green background. 17502659, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.13091 by Ecole des hautes etudes en sante publique (EHESP), Wiley Online Lib BOUDEWIJNS ET AL. 3 F I G U R E 2
Evolution of the population susceptibility to influenza A (H1N1 and H3N2) since 2017 by age group in Australia, Germany, Spain,
France, Italy and the United Kingdom. Horizontal red lines show the maximum level of susceptibility before January 2020. 3
|
RESULTS The percentage of
increase (or decrease) displayed on the plots are computed for the highest susceptibility level ahead of the 2022 (Australia) and 2022–2023
(Europe) influenza season compared with the red lines. Transparent areas stand for susceptible who vaccinated. , Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.13091 by Ecole des hautes etudes en sante publique (EHESP), Wiley Online Library on [04/01/2023]. See the Terms and F I G U R E 2
Evolution of the population susceptibility to influenza A (H1N1 and H3N2) since 2017 by age group in Australia, Germany, Spain,
France, Italy and the United Kingdom. Horizontal red lines show the maximum level of susceptibility before January 2020. The percentage of
increase (or decrease) displayed on the plots are computed for the highest susceptibility level ahead of the 2022 (Australia) and 2022–2023
(Europe) influenza season compared with the red lines. Transparent areas stand for susceptible who vaccinated. F I G U R E 3
Influenza and SARS-CoV-2 detections in Australia, between January 2020 and September 2022 //onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License F I G U R E 3
Influenza and SARS-CoV-2 detections in Australia, between January 2020 and September 2022 vaccination levels that are relatively high compared with Europe (68.3%
in persons aged 65 years and over). Vaccination against influenza is
recommended for anyone over 6-months-old and is available free of
charge for certain risk groups.19 In addition, in June and July of 2022,
some states (Queensland and Western Australia) provided the vaccine
free of charge to all inhabitants over 6 months to increase vaccine
uptake.20,21 One difference between Australia and Europe is that
Australia had no real influenza activity since the start of COVID-19 pan-
demic, but Europe experienced some mild influenza activity during the
2021/22 winter, and this will affect the proportion of the population
that is susceptible to influenza due to infections. persons did not increase as much. In the United Kingdom, the esti-
mated susceptible population only exceeded the reference in the two
youngest age-groups. ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS
We would like to thank Willemijn van Waarden for help with acces-
sing the WHO influenza and SARS-CoV-2 datasets. We would like to thank Willemijn van Waarden for help with acces-
sing the WHO influenza and SARS-CoV-2 datasets. ETHICS STATEMENT Ethical approval was not required for this study as all data used within
this work were part of routine surveillance. An early rise in influenza detections is possible, especially in coun-
tries with the highest increase in the susceptible population. Germany
has already reported an increase in influenza detections in late
November 2022, just like the United States, Canada and Mexico.23 A
similar early start was observed in Australia and also described in
Chile,24 where the season was characterised by an early start and
atypical course, but fewer hospitalisations, compared with before the
COVID-19 pandemic. An important factor that will impact the upcom-
ing influenza epidemics in Europe will be the specific viruses that cir-
culate in Europe. Some countries in the southern hemisphere had an
H3N2 dominant season, such as Australia, Chile24 and Argentina,25
while others had an H1N1 dominant season (South Africa). The H1N1
epidemic in South Africa, was then followed by a separate influenza B
epidemic.26 Another important factor would be the emergence of a
new SARS-CoV-2 variant, which could impact influenza activity (see
Figure 3 for how this played out in Australia), for example, via the
implementation of NPIs. 4
|
DISCUSSION The 2022 influenza season in Australia was characterised by an early
onset and a slightly shorter and sharper epidemic than previous seasons
(see Figure 1). The number of laboratory-confirmed detections was very
high compared with previous seasons; however, primary care data
showed low rates and the hospitalisation rate was average.17 Factors
that could have influenced the duration of the influenza season were
the rise of a new SARS-CoV-2 Omicron variant (see Figure 3) and the Predictive modelling can be an important tool to generate possi-
ble influenza activity scenarios. In these models, a crucial role is given
to the level of susceptibility for influenza in the population, which is BOUDEWIJNS ET AL. AUTHOR CONTRIBUTIONS Bronke Boudewijns: Investigation; writing-original draft; writing-
review and editing. John Paget: Conceptualization; investigation;
writing-original draft; writing-review and editing. Marco Del Riccio:
Investigation; writing-review and editing. Laurent Coudeville: Data
curation; formal analysis; investigation; methodology; writing-review
and
editing. Pascal
Crépey:
Conceptualization;
formal
analysis;
investigation; methodology; writing-original draft; writing-review and
editing. ORCID Bronke Boudewijns
https://orcid.org/0000-0002-7214-4586
John Paget
https://orcid.org/0000-0002-1503-2481
Marco Del Riccio
https://orcid.org/0000-0002-2742-0297
Pascal Crépey
https://orcid.org/0000-0003-1852-0752 4 altered by both infection and vaccination. Our approach allows one to
reconstruct the immune status of the population based on past epi-
demics. We then used the susceptible population metric to interpret
influenza epidemics that occurred in Australia and to assess what may
be expected this winter in Europe. Based exclusively on this metric, a
larger than usual influenza outbreak could be expected in the five
European countries. However, given that the largest overall increase
in the susceptible population was seen in Australia and this did not
lead to an extreme caseload, or increased number of hospitalisations
due to influenza in 2022, a scenario with a very large outbreak in
Europe seems unlikely. In addition, the highest levels of susceptibility
increase are found in the young population (0–14 years for Australia
and 0–17 years for the five European countries), a population where
influenza circulation could be high, but probabilities of severe out-
comes remain generally low. However, another study found that a
10–60% increase in the population susceptibility might lead to a maxi-
mum of onefold to fivefold rise in peak magnitude and a onefold to
fourfold rise in the epidemic size for the upcoming 2022–2023
influenza season.22 especially
in
children,
compared
with
France,
Italy
and
the
United Kingdom. These results highlight the importance of monitoring
and modelling the level of immunity against influenza across coun-
tries. They also depend on the European population maintaining their
prevention and control measures, including influenza vaccination. DATA AVAILABILITY STATEMENT Not applicable. One limitation of our study is that we have focussed our analysis
on influenza A. This was a methodological choice as (a) influenza A is
typically the dominant virus circulating in the population (B generally
represents only 23% of detections in a season),27 (b) influenza B did
not play an important role in influenza epidemics in Australia and the
other southern hemisphere countries, and (c) our estimation of sus-
ceptibility is mainly illustrative and should only be used for relative
comparisons between countries. Another limitation is that these are
modelled estimates of susceptibility, which would be important to
compare to serological data. Finally, our analysis was limited by some
of the data inputs (e.g., the VCR data for Germany was limited). CONFLICT OF INTEREST BB and MDR report no conflicts of interest, PC reports consulting
fees from Sanofi non-related to this project, LC is an employee of
Sanofi and JP reports that Nivel has received funding for influenza
research projects from Sanofi, the Fondation de France and WHO. PEER REVIEW The peer review history for this article is available at https://publons. com/publon/10.1111/irv.13091. doi:10.1016/S2214-
109X(22)00358-8 23. Nivel. FluCov-Bulletin – end-November 2022. Utrecht; 2022. Report
No.: 17. 11. Mistry D, Litvinova M, Pastore y Piontti A, et al. Inferring high-
resolution human mixing patterns for disease modeling. Nat Commun. 2021;12(1):323. doi:10.1038/s41467-020-20544-y 24. Barraza MFO. Influenza Incidence and Vaccine Effectiveness During
the Southern Hemisphere Influenza Season—Chile, 2022. MMWR
Morbidity and Mortality Weekly Report 2022;71. 12. Del Riccio M, Lina B, Caini S, et al. Increase of influenza vaccination
coverage rates during the COVID-19 pandemic and implications for
the upcoming influenza season in northern hemisphere countries and
Australia. Eurosurveillance. 2021;26(50):2101143. 25. Nivel. FluCov-Bulletin – July 2022. Utrecht; 2022. Report No.: 12. 26. Nivel. FluCov-Bulletin – September 2022. Utrecht; 2022. Report
No.: 14. 13. VCR datasources. Australia: National Centre for Immunisation
Research and Surveillance. Influenza vaccination coverage data. Sydney: 2022. Available from: https://www.ncirs.org.au/influenza-
vaccination-coverage-data; France: Santé publique France. Bulletin
épidémiologique grippe, semaine 22. Bilan préliminaire de l’épidémie
de grippe 2021–22. Paris: 2022 1-6-2022; Germany: Robert Koch
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Organizational Sustainability and Value Creation in Collaborative Networks
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Lecture notes in computer science
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Organizational Sustainability and Value Creation in
Collaborative Networks
Pierluca Di Cagno, Bianca D’agostinis, Barbara Livieri
To cite this version:
Pierluca Di Cagno, Bianca D’agostinis, Barbara Livieri. Organizational Sustainability and Value
Creation in Collaborative Networks. 15th Working Conference on Virtual Enterprises (PROVE), Oct
2014, Amsterdam, Netherlands. pp.349-356, �10.1007/978-3-662-44745-1_34�. �hal-01392134�
HAL Id: hal-01392134
https://inria.hal.science/hal-01392134
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Distributed under a Creative Commons Attribution 4.0 International License
Organizational Sustainability and Value Creation in
Collaborative Networks
Pierluca Di Cagno, Bianca D’Agostinis and Barbara Livieri
Department of Economic Sciences
University of Salento, Lecce, Italy
{name.surname}@unisalento.it
Abstract. Collaborative Networks are becoming ever more important as a
lunchpad for the achievement of competitive advantages and for the creation of
socio-economic benefits. According to the relational view theory, joint efforts
can indeed generate relational rents. However, task coordination is not a sufficient means for value creation. Indeed, Collaborative Networks have to create a
link between several aspects, such as trust, culture of collaboration, knowledge
sharing, managerial processes, incentive systems, ethical code and so on in order to create cooperation and, thus, value. Aim of this work is to analyze how
these aspects affect each other and how they affect value creation within collaborative networks. In order to do so, we develop a model, based on UML and
e3value, in which the main factors impacting on value creation and value exchanges within CNOs are represented. After the description of the model, we
analyze a case study of a CNO.
Keywords: Collaborative Networks, Value Creation, Sustainability; e-3 value;
Value exchanges; Relational rents
1
Introduction
In the actual economic context, Collaborative Networks (CNOs) represents a
lunchpad for the enhancement of firms’ competitiveness and for the creation of socioeconomic and environmental benefits. CNOs can be seen as a system made by several
“entities that are largely autonomous, geographically distributed, and heterogeneous
in terms of their operating environment, culture, social capital and goals […]”, enabled by new technologies [1]. In this scenario, nodes of a CNO share information,
resources and competencies with the aim of create new value through the broadening
of enterprise boundaries.
However, the sustainability of CNOs, from not only a financial perspective but also
from the organizational one is the premise for value creation. Indeed, collaboration
implies mutual trust and commitment, giving relevance to behavioral and managerial
principles, ethic codes, collaboration culture and incentive systems. Several authors
have studied the role of trust and commitment in inter-organizational settings [2, 3],
however, at the best of our knowledge, no one of them studied these factors with a
systematic approach that led to modeling and requirements. Nonetheless, CNOs are
adfa, p. 335, 2011.
© Springer-Verlag Berlin Heidelberg 2011
336
P. Di Cagno, B. D’Agostinis and B. Livieri
complex entities, whose understanding is not immediate, therefore it becomes necessary to understand and model the determinants of value creation in CNOs [4].
In this work, we propose a model of sustainability and value creation within CNO.
In particular, we represent these aspects by means of UML and e3-value, an ontology
based methodology used to define requirements for software systems taking into account the value exchange among different individuals. In this way, we were able to
model and analyze value exchanges and co-creation in Collaborative Networks.
The works is structured as follows. Section II is for the analysis of related works
concerning the use of e3value. In Section III we pose the basis for our model, which
we present, by means of a visual representation, in Section IV. In Section V we propose a case study. Finally, in Section VI we discuss our model and the application to
the case study and we draw conclusions.
2
Related Works
The advent of e-business has led to the necessity of the development of e-business
models. The e-business model has to address the formulation of requirements through
a rigorous method [5]. In this scenario, Gordijn and Akkermans [5, 6] proposed a
multi-viewpoint approach for the business model development: a) value viewpoint,
that represents the value creation and exchange process; b) process viewpoint, that
represents business process and tasks; c) Information System viewpoint, that describes the Information Systems required for business processes.
From a value point of view, e3-value is commonly used for the modeling of value
exchanges. The e3-Value is in fact an ontology-based methodology for defining business models for business networks [5] incorporating concepts from requirements engineering and conceptual modeling (including a graphical notation). Its main focus is
on identifying and analyzing how value is created, exchanged and consumed within a
multi-actor network. Hence, taking the economic value perspective and visualizing
what is exchanged, i.e. which kind of economic value, and by whom [7]. An economic value exchange, and consequently the e3-value ontology as a whole, is based on the
principle of reciprocity emphasizing the dual character of business transactions. This
“give and take”- approach, described by means of specific “value-exchange diagrams”, denotes that every actor offers something of value, such as money, physical
goods, services, or capabilities, and gets a value in return. In this scenario moneyexchange aspects are included in the value-exchange diagrams, and this semi-formal
description is very useful for business analysis [5] but also for requirement specification on ePayments and for other aspects (decision making, user profiling, market
segmentation, …) directly connected to monetization in eServices. This approach can
bring several benefits, such as better communication among actors, improvement of
the decision making process and a more complete view of business operations and
business perspective. In particular, when applied to CNOs can help in the understanding of value exchanges among partners and external actors, thus extending existing
reference models, such as ARCON.
Organizational Sustainability and Value Creation in Collaborative Networks
3
337
A Collaboration Model: Theoretical Foundation
In literature, several types of partnerships among firms have been identified, characterized by the deepness of the sharing relations and by the integration level of firms.
In particular, Camarinha-Matos and Afsarmanesh [1, 8] identified the following integration levels: (a) networking, which consists of the information exchange for mutual
benefits, with no common goals; (b) coordination, which consists of the coordination
of autonomous tasks, connected to a common goal; (c) cooperation, which consists in
resource and information sharing, with the aim of achieving compatible goals; (d)
collaboration, which is the highest integration level and consists of the sharing of
information, resources and responsibility in order to organize and develop activities
for a common goal.
Only in this last sense we can talk of collaborative network which have been defined as “a network consisting of a variety of entities that are largely autonomous,
geographically distributed, and heterogeneous in terms of their operating environment, culture, social capital and goals, but that collaborate to better achieve common
or compatible goals, and whose interactions are supported by computer network”.
[1]. CNOs enable several benefits, among which the increase in the success rate of
enterprises, also in turbulent markets, and the increased capability to achieve shared
goals [1]. Indeed, through alliances, it is possible to enhance enterprise size, gain
access to new markets and to external know-how, create synergies, share resources
and risks, integrate firms’ resources with the ones of partners and enhance innovation
processes, with a result of increased value creation [9]. Collaboration among enterprises enable the finding of resources not easily accessible on the market or that cannot be easily created. Also, the interactions among partners can be generated the relational capital [10], defined as a set of relations among firms which facilitate the exploitation of economic activities. Resources are exchanged when this is mutually
convenient and there is mutual trust, which exists when: a) partners confide in a reliable and transparent exchange of resources; b) a firm believes that other partners are
pursuing actions that are beneficiary also for the firms itself and c) that these actions
are not prejudicial [11]. Indeed, trust has been seen as “the cornerstone of strategic
partnership” [12], whereas the lack of trust can bring to the failure of the alliance [13]
and whereas if associated with commitment can bring to “outcomes that promote
efficiency, productivity and effectiveness” [14].
In the field of strategic alliances, trust is often a cognition-based trust, which
comes from rational choices based on information, rather than from emotional bonds
between individuals (affect-based trust), that follow in case of recurring interactions
between firms. In particular, cognition-based trust comes from previous interactions,
reputation on the market, ecological and ethical responsibility, organizational context,
similarities in firms’ characteristics. The searching for trust based relations have to
rationales: a) CNOs with a high level of trust are more likely to succeed and to create
value; b) relations among partners with a high level of trust requires much less time
and commitment to be managed [15]. For these reasons, the partner selection is often
based on previous interactions, reputation and potential degree of conflict [15].
338
P. Di Cagno, B. D’Agostinis and B. Livieri
Recognizing the importance of model frameworks for CNO, the ARCON model
was developed by [4, 16]. In the model, four endogenous elements are analyzed,
namely structural dimension, componential dimension, functional dimension, behavioral dimension. The behavioral dimension regards the principles, policies and rules
of governance that guide who operated within the CNO in order to make it more efficient [17]. Indeed, CNO governance is particularly relevant, since it’s a requirement
for the effective monitoring and control of relations. Inter-organizational relations
create a not-planned operative context, which at the same time is not entirely spontaneous [18]. Moreover, another relevant element is risk, which has been analyzed towards three perspectives. In [19] firms choose to collaborate in order to face market
risks; in [17] risk is the result of CNOs, while in [20] trust derives from risk and, at
the same time, is affected by trust. However, cooperation could lead to a reputational
risk, coming from non-socially responsible behavior of partners that could affect the
whole CNO.
In short, the advantages of CNOs are fully exploited only when there is trust
among partners, an adequate governance, ethical codes and a culture of collaboration.
4
SuMCNOs: A CNOs Sustainability Model
4.1
Method
For the development of our model, we selected UML as means to represent general
factors and variable, and e3-value for the analysis of value exchanges in CNOs, since
it’s a business ontology specifically designed for the analysis of value constellations
[5]. After reviewing more than 50 papers related to CNOs, published on international
conferences and journals, we identified three variables that have a great impact on
value creation. We modeled these variables with a UML class diagram. To this purpose, we considered the nodes of the networks, the CNOs and the variables that impact on value creation as classes, since a class is an abstraction of things (or instances): in other words, classes represent a whole set of objects that share methods and
attributes [21]. Moreover, we analyzed the value exchanges within networks by
means of e3value. To this aim, we modeled CNOs as composite actors made by other
business actors. Accordingly to e3value standard notation, we show the value activities that contribute to reaching the CNOs goals as value activities, while the value
exchanges have been depicted as connection between value ports, assigned to each
actor. Finally, we applied this model to a case of study, for which we draw the corresponding e-3 value diagram. All the diagrams are drawn with Microsoft Visio and
e3value Editor.
4.2
Model representation
This section provides a representation of our proposal, in order to exemplify our modeling approach. In the first part of the model, based on UML, we represent the determinants that impact on value creation and exchange within CNOs. As stated in the
theoretical foundation section, the main aspects concern trust, resource sharing and
Organizational Sustainability and Value Creation in Collaborative Networks
339
commitment. For the purpose of this study, we intend value as the result of the activities of the individual firm and of the collaboration with other entities. In this sense,
the value refers not only to financial benefits, but also to socio-environmental ones.
In Fig. 1, we represent the candidate classes and the candidate associations of our
model. In particular, each Node is part of a CNO; both Node and CNO are children of
the parent class BusinessEntity, which is a generalization of their concepts.
Also, nodes share Resource, which is a generalization of TangibleResource,
FinancialResource and IntangibleResource. To the primary association
between Node and Resource, we tied an association class Sharing since the
association is, in turn, associated with other classes. In a similar way, to the primary
association between Node and CNO, we tied an association class Participating
since the association itself has some attributes, namely Trust, Commitment and
RelationalCapital. We choose to represent these attributes as classes, in order
enable the association with other classes. In more detail, Trust is affected by
several factors, whereas the Factor class is a generalization of InternalFactor, which is an attribute of the association class Participating, and ExternalFactor. RelationalCapital, which is an attribute of Participating,
is also associated with the class BusinessEntity. Tying RelationalCapital
both to the class Participating and to the class BusinessEntity enables its
attribution to the level on which the information is available.
Moreover, RelationalCapital is enabled by:
─ Trust and Commitment, which are attributes of the class Participating:
this relation doesn’t need to be represented, since are all attributes of the same association class, therefore the association it’s implicit;
─ The Sharing of resources: if nodes don’t share their resources, it’s difficult to
find relational capital, since it’s strictly linked with how much firms work togheter.
The associate class Sharing, in turn, is enabled by Commitment and Trust,
as explained in Section III.
Fig. 1. UML class diagram representing value creation in CNOs
340
P. Di Cagno, B. D’Agostinis and B. Livieri
The second part of the model is represented by means of e3 value ontology,
through which we depict the value exchanges inside and outsides the collaborative
network. To this purpose, we take into account two types of CNOs and, in particular,
a supply chain and a horizontal strategic alliance.
In Fig. 2, value exchanges in a supply chain are represented. SupplyChain is a
composite actor made by the actors Supplier and InternalCustomer. Supplier has a value activity Production and sell its products and services (value link)
to InternalCustomer, who in exchange pay the price (money) to the supplier.
Supplier also sells its product towards ExternalCustomer: the benefits of the
CNO with respect to a market-driven environment is that products and services within
the alliance can be sold at a lower price or can be more specific for the customer’s
needs. Also, InternalCustomer has a value activity Production and sell its
final products and services to FinalCustomer. The social benefit is that CNO,
under certain circumstances, enhance products quality and reduce prices.
Fig. 2. Representation of value exchanges in CNOs
Moreover, exchanges within a horizontal alliance are represented. As depicted in
Fig. 3, the value exchanges don’t happen between the partners of the CNOs, but between the nodes and the CNO itself, whereas the benefits for nodes are usually intangibles.
Fig. 3. Representation of value exchanges of horizontal strategic alliances
5
Case Study
The Network of Bathing Establishments of Viareggio is a collaborative network born
in 2011 with the aim of increasing the competitiveness of the participants (85 bathing
establishments). The main objectives of the network include (a) the increase of securi-
Organizational Sustainability and Value Creation in Collaborative Networks
341
ty on the beaches, (b) the improvement of environmental sustainability and (c) the
coordination in supplying services.
For the purpose of the analysis of value creation, we will analyze a specific project
realized by the CNO regarding the safety of bathing establishments. The projects
enabled services of medical aid and first support with AED (Automated External
Defibrillator). In order to achieve this result, the network bought 41 defibrillators and
offered courses for about 150 staff operators. As depicted in Fig. 3, Node1 and
Node2 pay a fee in order to access to the project, whilst NetworkOfBathingEstablishments buys from AEDSupplier 41 defibrillators and from CouseErogator a service (course offering), under the payment of a fee. Finally, NetworkOfBathingEstablishments offers this service and the defibrillators to
Node1 and Node2. This enables Node1 and Node2to offer more services to their
clients, which in return will get a safer environment also for elderlies and for people
with health problems.
Fig. 4. Representation of the value exchanges in the Network of Bathing Establishments
In this case, trust mechanisms between nodes have been enabled by the continuous
interaction among partners and by the network manager, who helped in the switch
from a internal-based culture, where every external entity is a competitor, towards a
collaborative culture, where each node is seen as relevant part for value creation for
the CNO and for the creation of social benefits.
6
Discussion and Conclusions
In this paper, we propose a model for collaborative networks that takes into account
both the factors affecting value creation and the value exchanges within the network.
In particular, we described by means of UML the role of commitment and trust in the
participation relationship and the variables that affect these two factors. Also, we
analyzed through e3value the value exchanges within CNOs taking into account two
different scenarios, namely a supply chain and a horizontal strategic alliance. Finally,
we used e3value ontology in order to describe a case study of a horizontal network
between bathing establishments. Whilst previous research only analyzed single ena-
342
P. Di Cagno, B. D’Agostinis and B. Livieri
bling factors or value chains, we jointly examined these perspectives and we developed a model, named SuMCNOs, which can be easily applied to different cases in
order to understand the sustainability of CNOs. Future works will include the extension of the scenarios investigated.
7
References
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o
. УДК 517.12
ТЕОРЕМЫТАУБЕРОВА ТИПА НА СЛУЧАЙ АБСОЛЮТНОЙ
СУММИРУЕЮСТИ В СТЕПЕНИ р
ДВОЙННХ
РЯДОВ
Т.Н. ярковая
преобразуєм двойной ряд
a й
б
ZU
,
Srain =? 2 Ча
|
k,t=f
ка (=7
_
(тп)
с помощью матрицн
С == С,
i
следующим образом:
|
2
(та),
-
ти паба
мо»
(2)
(тп)|
Пусть Скот бл
, где Ахню І, Вайвияв-
дяртся
| Plo~ матрицами
[1]
Для заданных положительных последовзтельностей. En}. {каб
положим
|
4
ft
4
т.
1
чо
(т " Бет
Ст
jen os
=”,
се
г
т
Une =L.Ami?
бі-д б,
НЕ
ПЕ‘
jae
со
8
Lt
Pui -/ би 2 Кі ДА
1=ё
Jal
fee
С. метод, для которого
ЙОсурк
чо ЙРис Ї
являются
[Alр
- матрицами [1]
и, кроме того, имеют место
ot FP ap
=Omt),
2% AO By
=
x. bpme ті,
Oe
=
и! =О(1) ТЕОРЕМЫТАУБЕРОВА ТИПА НА СЛУЧАЙ АБСОЛЮТНОЙ
СУММИРУЕЮСТИ В СТЕПЕНИ р
ДВОЙННХ
РЯДОВ
Т.Н. ярковая
преобразуєм двойной ряд
a й
б
ZU
,
Srain =? 2 Ча
|
k,t=f
ка (=7
_
(тп)
с помощью матрицн
С == С,
i
следующим образом:
|
2
(та),
-
ти паба
мо»
(2)
(тп)|
Пусть Скот бл
, где Ахню І, Вайвияв-
дяртся
| Plo~ матрицами
[1]
Для заданных положительных последовзтельностей. En}. {к
положим
|
4
ft
4
т. 1
чо
(т " Бет
Ст
jen os
=”,
се
г
т
Une =L.Ami? бі-д б,
НЕ
ПЕ‘
jae
со
8
Lt
Pui -/ би 2 Кі ДА
1=ё
Jal
fee
С. метод, для которого
ЙОсурк
чо ЙРис Ї
являются
[Alр
- матрицами [1]
и, кроме того, имеют место б (2) обозначим через
[ (С, 5рою. Говопят, что последовательность
{Sm} eR ,
если
3°
бр бра Фа),
р ep Smp= Omit) ,
9 ради
=:
банк Sui
ml=t2
2ви Su , Пукаїд,,. ОХОДЯТСЯ. я
,
277 Е
. -
(p)
В дальнейшем будем писать
Имп= є""а),
если ЗМиті
|
р
абсолютно сходится в стезени р
к нулю и
упав? (1), если
évmn}
абсолютно сходитія в степени в. Теорема I. ели ложной ряд (1), для когорого ітив,
ICAL) - суммируєм г
OS
, то условия
tmn =OU)©,„то "@)
tran 08) Tmn=O4) взллотся достаточными для его абсолют-
5
ной сходимости в степечи Е
XK
9
. /
Локазательство. Прехле всего заметим,
что
нь
Ия
=<
SR,
Simin =2_ & lenбкддіт
,
(3)
Kat
|
м
ft
г
<
тя = 2. lao br. =
Ж, 2 ai Сті,
У
ГРУ
l=1
27
(4. обозначим через
[ (С, 5рою. Говопят, что последовательность
{Sm} eR ,
если
3°
бр бра Фа),
р ep Smp= Omit) ,
9 ради
=:
банк Sui
ml=t2
2ви Su , Пукаїд,,. ОХОДЯТСЯ. я
,
277 Е
. -
(p)
В дальнейшем будем писать
Имп= є""а),
если ЗМиті
|
р
абсолютно сходится в стезени р
к нулю и
упав? (1), если
évmn}
абсолютно сходитія в степени в. Теорема I. ели ложной ряд (1), для когорого ітив,
ICAL) - суммируєм г
OS
, то условия
tmn =OU)©,„то "@),
tran 08) Tmn=O4) взллотся достаточными для его абсолют-
5
ной сходимости в степечи Е
XK
9
. /
Локазательство. Прехле всего заметим,
что
нь
Ия
=<
SR,
Simin =2_ & lenбкддіт
,
(3)
Kat
|
м
ft
г
<
тя = 2. lao br. УДК 517.12
ТЕОРЕМЫТАУБЕРОВА ТИПА НА СЛУЧАЙ АБСОЛЮТНОЙ
СУММИРУЕЮСТИ В СТЕПЕНИ р
ДВОЙННХ
РЯДОВ
Т.Н. ярковая
преобразуєм двойной ряд
a й
б
ZU
,
Srain =? 2 Ча
|
k,t=f
ка (=7
_
(тп)
с помощью матрицн
С == С,
i
следующим образом:
|
2
(та),
-
ти паба
мо»
(2)
(тп)|
Пусть Скот бл
, где Ахню І, Вайвияв-
дяртся
| Plo~ матрицами
[1]
Для заданных положительных последовзтельностей. En}. {каб
положим
|
4
ft
4
т.
1
чо
(т " Бет
Ст
jen os
=”,
се
г
т
Une =L.Ami?
бі-д б,
НЕ
ПЕ‘
jae
со
8
Lt
Pui -/ би 2 Кі ДА
1=ё
Jal
fee
С. метод, для которого
ЙОсурк
чо ЙРис Ї
являются
[Alр
- матрицами [1]
и, кроме того, имеют место
ot FP ap
=Omt),
2% AO By
=
x. bpme ті,
Oe
=
и! =О(1) =
Ж, 2 ai Сті,
У
ГРУ
l=1
27
(4. (3) В таком случае, применяя emaЗАбеля ,
fa
a
. . =4
і
>и:т_Enttakaт] б
у
гаї
[=1
i=? 4=1
— Кир /7 р,УМ Ai Фиг. Следовательно,
если использовать
(4),
20,
4°, получим
Sinn ат 7 Ent Уна)ду (т) го А, бт:2 fer =
pr-peo (РІ
21
]=
[=
2=. ра
. pt
г
i]
ле
з _
5
Po
rp
. Sm р
= (т [бы Ки
Rn і2-Вітті
Pep кр
утр
р-»со
bt
n
п
_©2
ео
2
2ди = 7 виє 7 аду ті —
(21
bat
Іст
fal
jm
ef
се
І4
aL. (21 08)7("дваС),
КН
откуда и следует наше утверкдение. А тогда, как показчвает-
Senaseuosse
1,
71
72 (та)"див є
we aeyeИ
мм
-
)
Sinn ==‘les
пл). Пусть
@=2,<х,<...<Жи=@,
ху, суз «... «УР. Положим бл= 1(С»,Уя)
пля заданной в области
Да /осу дежхеф, Cs y<6 4
фуккции
(09)
и пусть
суммы
м
м
ПП
р
22(тп)!Абу)
оз
в й»! равномерно ограничены относительно любого внбора
(Жул $ { Y
Тогда будем говорить, что
f(xy}
имеют ограниченную />-
вариацяю в 1)
. |
Ряд (Г)
ЇАЇр
~ comarpyen, если
(ху) =.x “уша
есть функция ограниченной р - вариация в д. Теорема 2. Если двойной ряд (1), для которого ФаЄ м.А,Безверяенно для системы двух лифференихальных уравяєний y= fly2),
7‘ == Ут, 2) яти
he,м
|
https://openalex.org/W4205244886
|
http://old.scielo.br/pdf/rbeaa/v26n2/1807-1929-rbeaa-26-02-0097.pdf
|
English
| null |
Co-inoculation of Anabaena cylindrica and Azospirillum brasilense during initial growth and chloroplast pigments of corn
|
Revista Brasileira de Engenharia Agrícola e Ambiental
| 2,022
|
cc-by
| 4,752
|
Co-inoculation of Anabaena cylindrica and Azospirillum brasilense
during initial growth and chloroplast pigments of corn1 Co-inoculação de Anabaena cylindrica e Azospirillum brasilense
durante o crescimento inicial e pigmentos cloroplásticos do milho Osvaldo Matsuo2* , Claudemir Zucareli2 , Eduardo H. Horácio2 ,
Luiz A. R. Alves2 & Otavio J. G. A. Saab2 1 Research developed at Universidade Estadual de Londrina, Londrina, PR, Brazil
2 U i
id d E
d
l d L
d i
L
d i
PR B
il 1 Research developed at Universidade Estadual de Londrina, Londrina, PR, Brazil
2 Universidade Estadual de Londrina, Londrina, PR, Brazil 2 Universidade Estadual de Londrina, Londrina, PR, Brazil HIGHLIGHTS:
Anabaena cylindrica associated with Azospirillum brasilense favored the development and growth of corn. Plant growth-promoting bacteria promote morphoanatomical alterations in the roots. Anabaena cylindrica and Azospirillum brasilense provide efficient water and nutrients absorption. GHLIGHTS:
Anabaena cylindrica associated with Azospirillum brasilense favored the development and growth of corn. Plant growth-promoting bacteria promote morphoanatomical alterations in the roots. Anabaena cylindrica and Azospirillum brasilense provide efficient water and nutrients absorption. ABSTRACT: Although the isolated effects of the cyanobacteria Anabaena cylindrica and the bacteria Azospirillum
brasilense are well-known, the co-inoculation of both can promote other benefits to corn plants’ biological nitrogen
fixation and growth. The aim was to evaluate the initial growth and chloroplast pigment levels of corn hybrids co-
inoculated with A. brasilense and A. cylindrica. The experiment was conducted in greenhouse conditions using the
completely randomized design in a factorial scheme 2 × 4 with two hybrids, Balu 184 and Balu 280 Pro, and four
inoculation treatments: no inoculation, inoculation of A. cylindrica, inoculation of A. brasilense, and the co-inoculation
of A. cylindrica + A. brasilense. Co-inoculation resulted in higher root growth and dry mass of the aerial part in both
hybrids; however, the hybrid Balu 280 Pro presented higher responsivity. The co-inoculation of A. brasilense and A. cylindrica increased initial corn growth without altering the photosynthetic pigment levels. Key words: Zea mays L., Anabaena cylindrica, diazotrophs bacteria, microalgae, plant-growth-promoting bacteria RESUMO: Embora comprovados os efeitos isolados das cianobactérias Anabaena cylindrica bem como da bactéria
Azospirillum brasilense, na fixação biológica de nitrogênio e na promoção de crescimento de plantas de milho, a co-
inoculação de ambas pode ampliar outros benefícios às plantas. O objetivo foi avaliar o crescimento inicial e teores
de pigmentos cloroplastídicos de híbridos de milho co-inoculados com A. brasilense e A. cylindrica. • Ref. 249250 – Received 01 Mar, 2021
* Corresponding author - E-mail: tga.mats@gmail.com
• Accepted 01 Aug, 2021 • Published 01 Sept, 2021
Edited by: Hans Raj Gheyi Revista Brasileira de Engenharia Agrícola e Ambiental
Campina Grande, PB – http://www.agriambi.com.br – http://www.scielo.br/rbeaa
ISSN 1807-1929
v.26, n.2, p.97-102, 2022
Brazilian Journal of Agricultural and Environmental Engineering Revista Brasileira de Engenharia Agrícola e Ambiental
Campina Grande, PB – http://www.agriambi.com.br – http://www.scielo.br/rbeaa
ISSN 1807-1929
v.26, n.2, p.97-102, 2022
Brazilian Journal of Agricultural and Environmental Engineering ISSN 1807-1929 DOI: http://dx.doi.org/10.1590/1807-1929/agriambi.v26n2p97-102 Material and Methods The experiment was conducted in a greenhouse conditions
in the Agronomy Department of the Universidade Estadual de
Londrina, in the municipality of Londrina, PR, located at the
geographical coordinates: 23º 20’ 23.45” S and 51º 12’ 32.28” W,
between October 2017 and January 2018.h The co-inoculation of plant-growth-promoting bacteria
(PGPB) is an advanced agricultural technology of proven
efficiency that promotes higher root growth (Yegorenkova et
al., 2016). The co-inoculation in corn crops of plant-growth-
promoting bacteria may favor root and aerial parts growth due
to efficient nutrient absorption. It also reduces the need for
synthetic fertilizers, environmental and production costs and
promotes more profitable and sustainable systems (Andrade
et al., 2014a; Prasanna et al., 2015; Muro-Pastor et al., 2017). The experiment was conducted adopting a completely
randomized design in a factorial scheme 2 × 4 with four
replicates using the corn hybrid cultivars Balu 184 22C (H1)
and Balu 280 Pro 20C (H2), and four inoculation treatments:
control (no inoculation) (T1), isolated inoculation of A. cylindrica (T2) and A. brasilense (T3), and co-inoculation of
A. cylindrica + A. brasilense (T4). Azospirillum brasilense is a widely known PGPB,
commercialized and used in corn crops due to its biological
nitrogen fixation (BNF) action, hormone synthesis, phosphate
solubility, and increased tolerance to biotic and abiotic stresses
(Strzelczyk et al., 1994; Gupta et al., 2013; Hashtroudi et al.,
2013). The free-living cyanobacteria A. cylindrica occurs in
many different environments (Shariatmadari et al., 2013) and
fixate nitrogen through their heterocysts (Muro-Pastor et al.,
2017). They also produce hormones and present nutrient-rich
mucilage and soil conditioner bio-compounds (Prasanna et al.,
2015). Nevertheless, studies with inoculated and co-inoculated
cyanobacteria in agriculture are still scarce. The cyanobacteria were obtained from the Laboratory
Collection of the Universidade Federal do Rio Grande -
FURG (Rio Grande do Sul, Brazil) and reproduced in the
Microbiology Laboratory of the Instituto Agronômico do
Paraná, the state agricultural research center. The inoculant was
prepared following Gavilanes et al. (2020) for an approximate
concentration of 108 UFC mL-1. The A. brasilense used was the
commercial inoculant NITROBACTER AZP© containing the
strains AbV5 and AbV6 with a concentration of 2.0 x 108 UFC
mL-1, following the manufacturer’s instructions.h Evidence suggests an associative potential of A. cylindrica
and A. brasilense through BNF enhancing growth and,
consequently, corn development and grain yield (Prasanna
et al., 2015). The seeds were inoculated before sowing. Osvaldo Matsuo et al. 98 Co-inoculation of Anabaena cylindrica and Azospirillum brasilense
during initial growth and chloroplast pigments of corn1 O experimento
foi conduzido em condições de casa de vegetação sob o delineamento inteiramente casualizado, em esquema fatorial
2 × 4, com dois híbridos: Balu 184 e Balu 280 Pro e quatro tratamentos de inoculação: ausência de inoculação,
inoculação de A. cylindrica, inoculação de A. brasilense e a co-inoculação de A. cylindrica + A. brasilense. A co-
inoculação resultou no maior crescimento da raiz e massa seca da parte aérea nos dois híbridos, porém com maior
responsividade para o híbrido Balu 280 Pro. A co-inoculação combinada de A. brasilense e A. cylindrica incrementou
o crescimento inicial do milho, sem alterações nos teores dos pigmentos fotossintéticos. Palavras-chave: Zea mays L., Anabaena cylindrica, bactérias diazotróficas, microalgas, bactérias promotoras de
crescimento vegetal • Ref. 249250 – Received 01 Mar, 2021
* Corresponding author - E-mail: tga.mats@gmail.com
• Accepted 01 Aug, 2021 • Published 01 Sept, 2021
Edited by: Hans Raj Gheyi
This is an open-access article
distributed under the Creative
Commons Attribution 4.0
International License. This is an open-access article
distributed under the Creative
Commons Attribution 4.0
International License. This is an open-access article
distributed under the Creative
Commons Attribution 4.0
International License. Material and Methods In the inoculation
and co-inoculation, the dose of cyanobacteria applied was
5.0 mL kg-1 of seeds (Gavilanes et al., 2020). For the A. brasilense, the dose applied was 4 mL kg-1 of seeds, following
the manufacturer’s instructions.h Exploring the co-inoculation potential of cyanobacteria
such as A. cylindrica combined with A. brasilense can reduce
the use of nitrogen fertilizers, the production costs, and favor
environment conservation (Hungria et al., 2015; Marini et al.,
2015; Fukami et al., 2016; Milléo & Cristófoli, 2016; Gavilanes
et al., 2020). The interaction with other production factors,
such as the corn cultivar, must be considered. Thus, the aim
of the present study was to evaluate the initial growth and
chloroplast pigment levels of corn hybrids co-inoculated with
A. brasilense and A. cylindrica. The inoculation was conducted by adding to the seeds the
bacteria A. brasilense and A. cylindrica, providing an even
distribution of the inoculant in the seed. After the inoculation,
the seeds were shade dried under room conditions with the
lowest temperature of 16.2 °C, highest of 27.6 °C, relative
air humidity of 75.7%, insolation of 2.2 hours of sunlight
then seeded. Sowing was conducted with four seeds per pot
and eight days after sowing (DAS), after the plants emerged,
thinning was conducted to keep one plant per pot. Rev. Bras. Eng. Agríc. Ambiental, v.26, n.2, p.97-102, 2022. Figure 1. Meteorological data of the experimental period 2017-2018 Figure 1. Meteorological data of the experimental period 2017-2018 Rev. Bras. Eng. Agríc. Ambiental, v.26, n.2, p.97-102, 2022. Rev. Bras. Eng. Agríc. Ambiental, v.26, n.2, p.97-102, 2022. Co-inoculation of Anabaena cylindrica and Azospirillum brasilense during initial growth and chloroplast pigments of corn 99 for the levels of chlorophyll a, b, and carotenoids, respectively. The measurements of pigment levels were performed through
the Eqs. 2, 3, and 4 (Barbosa et al., 2017): for the levels of chlorophyll a, b, and carotenoids, respectively. The measurements of pigment levels were performed through
the Eqs. 2, 3, and 4 (Barbosa et al., 2017): The soil, classified as Latossolo Vermelho distróférrico
(Oxisol) (EMBRAPA, 2018), after sieving was used as a
substrate and conditioned in pots with a volume of 18.0 L. (
)
1000 A434 1.90Chl a
63.14Chl b
Car
214
⋅
−
−
=
(4) (4) Through soil analysis, dolomitic limestone was applied at a
dose equivalent to 4.5 t ha-1, corresponding to the application
in each pot with the substrate in a dose equivalent to 40.0 g
in 18 dm3 (31 x 30 x 25 cm). The mixture was incorporated
manually in a container with a higher capacity, and after
homogenization, the substrate returned to the pot. For
fertilization of maintenance, a granular fertilizer was applied
with the formulation NPK (4-30-10) in the dose of 400.0 kg ha-1,
equivalent to 4.0 g per pot. Topdressing nitrogen fertilization
was performed manually on the soil surface of the pot and
the plant’s surroundings in the vegetative stage V4, applying
ammonium sulfate (21-00-00-24S) at the dose of 50.0 kg N ha-1.h The data were submitted to normality (Shapiro Wilk) and
homogeneity (Bartlett) tests, analysis of variance, and means
comparison by Tukey’s test at p ≤ 0.05. The value of RL was
transformed into the potency 2 and the logarithm (x) in the
base 10 for RDP and APDP, following the assumptions of
normality and homogeneity to perform the ANOVA, presenting
a significant F-value with p ≤ 0.01 for the respective tests. Rev. Bras. Eng. Agríc. Ambiental, v.26, n.2, p.97-102, 2022. Results and Discussions The evaluation of growth characteristics was conducted in
the vegetative stage V8, with the completely expanded eighth
leaf. Each plant’s leaf area (LA), expressed in cm2 of leaf, was
measured based on the total length (L) and width in the leaves’
middle third (W). The data were obtained following Eq. 1,
proposed by Francis (1969): Interactions between the hybrids and the inoculations
were verified for the variables LA, RV, RL, RDP, and APDP. In
the variables PH, levels of Chl a, Chl b, and Car, there was a
significant effect for the isolated inoculation (Table 1). if
The leaf area of the hybrid H2 at T2 (69.12 cm2) was higher
than H1 (59.64 cm2) (Table 2). Moreover, there was a significant
difference in the treatments’ mean and higher value for the
isolated treatment T2 with the hybrid H2 in comparison to the
control treatment T1 and the combined inoculation T4. The
difference between the hybrids is probably because of genetic
interaction between cultivars and the inoculation of the PGPB
with topdressing nitrogen fertilization increased nutrient
absorption, resulting in a higher leaf area. Similar results were
found by Gavilanes et al. (2019), in a greenhouse, with superior
responses for LA in the interaction between cyanobacteria
inoculation and nitrogen fertilization in corn crops. Zeffa et
al. (2019) suggest that the positive response for the efficiency
of the inoculation of A. brasilense (Ab-V5) for phytometric
parameters varies according to the genotype. (1) LA
0.75 C L
= (1) LA
0.75 C L
= where: where: L
- leaf length; and, L
- leaf length; and, W - leaf width. The root volume (RV) was determined by measuring
the water column displacement in a graduated cylinder. After cleaning, the roots were individually put in a graded
cylinder with 100 mL of water, and the difference in volume
displacement provided the root volume in cubic centimeters
(cm3). Root length (RL) was determined by a graded scale
measuring from the root apex to its insertion in the stem in
centimeters. The root dry phytomass (RDP) and aerial part dry
phytomass (APDP) were determined after drying in an oven
at a constant temperature of 65.0 °C. The plant height (PH)
was measured from the soil surface until the insertion of the
expanded eighth leaf (V8) using a graded scale. Material and Methods The
soil chemical characteristics were determined before applying
the treatments: pH (CaCl2) 4.5; 3.48 cmolc dm-3 of Ca2+;
0.8 cmolc dm-3 of Mg2+; 4.96 cmolc dm-3 of H+Al; 2.22 mg dm-3
of P; 0.38 cmolc dm-3 of K; 1.73 g dm-3 of Total Nitrogen; 20.37
g dm-3 of OM; and 4.81 cmolc dm-3 of effective CEC. According
to the soil chemical analysis, fertilization was conducted based
on the recommendation for corn crops (IAPAR, 2003).h (
)
Chl a
11.24 A663
2.04 A645
=
⋅
−
⋅
(
)
Chl b
20.13 A645
4.19 A663
=
⋅
−
⋅
(2)
(3) (
)
Chl a
11.24 A663
2.04 A645
=
⋅
−
⋅
(2) (2) (3) (
)
1000 A434 1.90Chl a
63.14Chl b
Car
214
⋅
−
−
=
(4) Results and Discussions When the co-inoculation was used, the difference between
the hybrids was not observed, supporting the results obtained Table 1. Results of the analyses of variance for initial growth
and chloroplast pigment levels of the corn hybrids Balu 184
(H1) and Balu 280 Pro (H2), regarding the co-inoculation of
Azospirillum brasilense and Anabaena cylindrica The level of chlorophyll ‘a’ (Chl ‘a’), chlorophyll ‘b’ (Chl ‘b’),
and carotenoids (Car) were measured using the methodology
of Whitham et al. (1971). Leaf tissues were extracted, and the
mass was determined (0.2 g). After that, they were macerated
in liquid nitrogen and then transferred to a test tube with
10.0 mL of acetone 80%. The tubes were wrapped in aluminum
foil, with the matte side turned to the exterior and conditioned
in a refrigerator at a temperature of 8.0 ± 2 °C for a week. The next procedure was the centrifugation of extracts. The
supernatants were submitted to absorbance (A) readings with
a spectrophotometer in the wavelengths 663, 645, and 434 nm ns - Not significant; * - Significant at p ≤ 0.05 e ** - Significant at p ≤ 0.01; (1)
Transformation into the Potency 2; (2) Transformation into Log (x) Rev. Bras. Eng. Agríc. Ambiental, v.26, n.2, p.97-102, 2022. Osvaldo Matsuo et al. 100 Table 2. Mean values and values of the interaction between
hybrids vs. inoculation (H x I) for initial growth and the
chloroplast pigment levels evaluated in the vegetative phase
of the corn hybrids Balu 184 (H1) and Balu 280 Pro (H2),
regarding the inoculation of A. brasilense and A. cylindrica,
applied in the seed regulator substances. Similar studies indicate that A. cylindrica
stimulated root growth through phytohormones (auxins and
cytokinins) and other substances in different corn hybrids
with different responses for the inoculation of microorganisms
concerning plants’ development and growth (Prasanna et al.,
2015; Marini et al., 2015; Zeffa et al., 2019). pp
T1 - Control; T2 - Inoculation A. brasilense; T3 - Inoculation A. cylindrica; T4 - A. brasilense + A. cylindrica; (1) Transformation Log (x); Means followed by the same letter,
lower case in the row and upper case in the column, do not differ among them according
to the Tukey’s test at p ≤ 0.05 f
)
Calzavara et al. (2018), while studying Bacillus sp. and
A. Results and Discussions cylindrica; (1) Transformation Log (x); Means followed by the same letter,
lower case in the row and upper case in the column, do not differ among them according
to the Tukey’s test at p ≤ 0.05 by Gavilanes et al. (2020) in field research that did not observe
interactions between the inoculation of hybrids with the leaf
area index. The authors suggest a lack of specific responses
for corn genotypes, concluding that it is a facilitator factor for
the application recommendations of A. cylindrica associated
with A. brasilense. Prasanna et al. (2015) attribute a positive effect for
corn growth produced by a biofilm rich in nutrients
and phytohormones, synthesized by cyanobacteria and
rhizobacteria, with different responses between hybrids for the
agronomic characteristics. This fact highlights the necessity
to investigate better the combination between hybrid and
microorganisms in the inoculant. Moreover, Marini et al. (2015), in a study about the
interaction between corn genotype and the bacteria
Azospirillum, concluded that the inoculation of A. brasilense
increased corn APDP and LA, also noting the necessity of
further studies about more responsive genotypes. Calzavara
et al. (2018) related root structural alterations and increased
the aerial part vegetal mass, nutrient absorption, and root
exudation.hif In the variable RV, an interaction effect (Table 2) occurred
in T4, with differences between the hybrids, and the mean of
the hybrid H2 was higher than the hybrid H1. Moreover, T4
was higher than the other treatments with both hybrids. For
the variable RL, an interaction between the hybrids H2 and
H1 occurred in T3, and a higher mean for H2 (Table 2). The
combined inoculation in T4, independently of the hybrids,
produced a higher mean than the other treatments. The
higher root growth in volume and length between the hybrids
and the treatment can be attributed to the PGPB and the
synergistic effect of the combined inoculation of A. cylindrica
and A. brasilense, responsible for the synthesis of plant growth The variable PH demonstrated a significant difference
between the inoculation treatments, with a lower mean for
T2 than the co-inoculation T4 (Table 2). Possibly, the higher
energy consumption of the plant when generating exudates by
the root, which are a food source for the inoculated bacteria,
influenced the lower PH of the isolated treatment T2. Results and Discussions brasilense, attributed to the growth promoter bacteria
a strong influence on root anatomy, widening the area of
the metaxylem vessel elements (MVE), favoring water
and nutrients absorption. According to Li et al. (2018),
the application of auxins in the initial stage of corn plants
stimulated the concentration in the roots, and a higher density
of lateral roots was observed. However, the continual supply of
exogenous auxin is fundamental to provide the morphological
and anatomical changes during the plant growth stages. It is
different from the supply through PGPB microorganisms,
such as the co-inoculation of A. cylindrica (Andrade et al.,
2014b; Prasanna et al., 2015) and A. brasilense (Cassán &
Diaz-Zorita, 2016). The interaction between the hybrids and the inoculation for
the variables RDP and APDP, and the combined inoculation
T4 did not significantly differ between the hybrids H1 and
H2. Nevertheless, T2 and T3 of isolated inoculations resulted
in an H2 higher than H1 (Table 2). In this sense, the increase
of RDP and APDP of the hybrid H2 compared with H1 can
be attributed to the hybrid H2’s higher mass than H1, and its
affinity with PGPB inoculation. Thus, RDP and APDP of H2
and H1 in the combined inoculation T4 were close, and, in the
treatments’ mean, T4 in both hybrids was higher than the other
treatments (Table 2). Possibly, the higher biological activity in
the corn rhizosphere with the co-inoculation of A. cylindrica
and A. brasilense, associated with the transformations of the
plants’ vascular system and the nitrogen supply, promoted
plant growth and the increase of corn phytomass. Calzavara
et al. (2018) suggest that the inoculation of A. brasilense and
Bacillus sp. with topdressing nitrogen fertilization in corn did
not reduce the roots’ biomass compared with the control plants. Still, the transformations in the roots influenced the increase
of vegetal biomass. T1 - Control; T2 - Inoculation A. brasilense; T3 - Inoculation A. cylindrica; T4 - A. brasilense + A. cylindrica; (1) Transformation Log (x); Means followed by the same letter,
lower case in the row and upper case in the column, do not differ among them according
to the Tukey’s test at p ≤ 0.05 T1 - Control; T2 - Inoculation A. brasilense; T3 - Inoculation A. cylindrica; T4 - A. brasilense + A. Conclusions 1. Co-inoculation provided higher root growth and dry
phytomass in the aerial part in both hybrids.h Gupta, V.; Ratha, S. K.; Sood, A.; Chaudhary, V.; Prasanna, R.; New
insights into the biodiversity and applications of cyanobacteria
(blue-green algae)-Prospects and challenges. Algal Research, v.2,
p.79-97, 2013. https://doi.org/10.1016/j.algal.2013.01.006 2. The co-inoculation of A. brasilense and A. cylindrica
increases the initial growth of corn. 2. The co-inoculation of A. brasilense and A. cylindrica
increases the initial growth of corn. Hashtroudi, M. S.; Ghassempour, A.; Riahi, H.; Shariatmadari, Z.;
Khanjir, M. Endogenous auxins in plant growth-promoting
cyanobacteria-Anabaena vaginicola and Nostoc calcicola. Journal
of Applied Phycology, v.25, p.379-386, 2013. https://doi. org/10.1007/s10811-012-9872-7 Acknowledgements The authors would like to thank the Conselho Nacional
de Desenvolvimento Científico e Tecnológico - CNPq for
the scholarship granted and the financial aid to develop the
research. Hungria, M.; Nogueira, M. A.; Araujo, R. S. Soybean seed co-
inoculation with Bradyrhizobium spp. and Azospirillum brasilense:
A new biotechnological tool to improve yield and sustainability. American Journal of Plant Scienses, v.6, p.811-817, 2015. https://
doi.org/10.4236/ajps.2015.66087 Results and Discussions in current agriculture: From
the laboratory to the field. Soil Biology & Biochemistry, v.103,
p.117-130, 2016. https://doi.org/10.1016/j.soilbio.2016.08.020 p.117-130, 2016. https://doi.org/10.1016/j.soilbio.2016.08.0 EMBRAPA - Empresa Brasileira de Pesquisa Agropecuária. Sistema
brasileiro de classificação de solos. 5.ed. Brasília, Embrapa, 2018. 356p. In this study, the co-inoculation of A. cylindrica with
A. brasilense in the seeds promoted a positive effect in the
development and root growth of corn (RV, RL, and MSR) and
the plant (MSA) compared with the absence of inoculation
T1 and the isolated inoculation of plant-growth-promoting
bacteria T2 and T3. The combined bacteria’s synergy probably
stimulated this fact in the production, the action of nutrient-
rich PGPB bio-compounds, the hybrids more responsive to
morphoanatomical alterations in the root, and the widening
of the water and nutrient’s uptake and absorption area. Francis, C. A.; Rutger, J. N.; Palmer, A. F. E. Rapid method for
plant leaf area estimation in maize (Zea mays L.). Crop
Science, v.9, p.537-539, 1969. https://doi.org/10.2135/
cropsci1969.0011183X000900050005x Fukami, J.; Nogueira, M. A.; Araujo, R. S.; Hungria, M. Accesing
inoculation methods of maize and wheat with Azospirillum
brasilense. AMB Express, v.6, p.1-13, 2016. https://doi. org/10.1186/s13568-015-0171-y Gavilanes, F. Z.; Andrade, D. S.; Zucareli, C.; Horácio, E. H.; Yunes,
S. J.; Barbosa. A. P.; Alves, L. A. R.; Cruzatty L. G., Maddela, N. R., Guimarães, M. de F. Co-inoculation of Anabaena cylindrica
with Azospirillum brasilense increases grain yield of maize
hybrids. Rhizosphere, v.15, p.1-8, 2020. https://doi.org/10.1016/j. rhisph.2020.100224 In this study, the association of A. cylindrica and A. brasilense positively influenced root growth, revealing the
great potential for higher development and grain yield in corn
crops. However, more pieces of field research are necessary to
verify the results obtained and the interaction between PGPB
and the hybrids and other cultivation factors such as the crop
environment and management. Gavilanes, F. Z.; Andrade, D. S.; Zucarelli, C.; Yunes, J. S.; Amaral, H.;
Costa, R. M. da; Raia, D. Garcia, M; Guimarães, M de F. Efecto de
la inoculación con cianobactéria y coinoculación con Azospirillum
brasilense sobre características fitométricas em maíz. Bioagro,
v.31, p.193-202, 2019. Results and Discussions In the
combined treatment T4, a positive synergistic effect might have
occurred stimulated by PGPB bio-compounds, although it did
not present a significant difference from the control treatment. In this context, the lower PH of the isolated inoculation T2 may Rev. Bras. Eng. Agríc. Ambiental, v.26, n.2, p.97-102, 2022. Co-inoculation of Anabaena cylindrica and Azospirillum brasilense during initial growth and chloroplast pigments of corn Co-inoculation of Anabaena cylindrica and Azospirillum brasilense during initial growth and chloroplast pigments of corn 101 be related to the plant’s higher energy waste to liberate root
exudates as a food source for diazotrophs (Lima et al., 2019). Andrade, D. S.; Machineski, G. da S.; Lovato, G. M.; Colozzi Filho,
A.; Goes, K. C. G. P. de. Inoculação de cianobactérias em
leguminosas e gramíneas. In: Andrade, D. S. (ed.) Microalgas de
águas continentais. Londrina: IAPAR, 2014b. Cap.12, p.445-476. The photosynthetic pigments (chlorophyll and b) and
photoprotective (carotenoid) may sign the physiological stage
of corn. The mean values obtained in the treatments indicate a
significant difference only between the treatments T1 and T2,
with a lower mean for T2. However, there was no significant
difference for the treatments T3 and T4 compared with the
control treatment (Table 2). Probably, the co-inoculation
results did not present alterations because the evaluated
pigment levels are generally variables of indirect evaluation
regarding plant growth. Barbosa, A. P; Meschede, D. K.; Alves, G. A. C.; Freiria, G. H.; Furlan,
F. F.; Alves, L. A. R.; Junco, M. C. Paspalum notatum growth and
pigment content in response to the application of herbicides. Revista Brasileira de Herbicidas, v.16, p.142-151. 2017. https://
doi.org/10.7824/rbh.v16i2.520 Calzavara, A. K.; Paiva, P. H. G.; Gabriel, L. C.; Oliveira, A. L. M.;
Milani, K; Oliveira, H. C.; Bianchini, E., Pimenta, J. A.; Oliveira,
M. C. N. de; Dias-Pereira, J.; Stolf-Moreira, R. Associative
bactéria influence maize (Zea mays L.) growth, physiology and
root anatomy under diferente nitrogen levels. Plant Biology, v.20,
p.870-878, 2018. https://doi.org/10.1111/plb.12841 Nevertheless, Gavilanes et al. (2019) reported, in a
greenhouse, an increase in chlorophyll b level with the
combined inoculation of A. cylindrica and A. brasilense. In
another field research, Gavilanes et al. (2020) observed higher
chlorophyll levels in corn with co-inoculation than the control
treatment. Cassán F.; Diaz-Zorita, M. Azospirillum sp. in current agriculture: From
the laboratory to the field. Soil Biology & Biochemistry, v.103, Cassán F.; Diaz-Zorita, M. Azospirillum sp. Rev. Bras. Eng. Agríc. Ambiental, v.26, n.2, p.97-102, 2022. Literature Cited IAPAR - Instituto Agronômico do Paraná. Sugestão de adubação
e calagem para culturas de interesse econômico no Estado do
Paraná. Londrina: IAPAR, 2003, 30p. Andrade, D. S.; Colozzi Filho, A.; Souto, A. R.; Scherer, A.; Costa, A.;
Guedes, C. L. B.; Angelo, E. A.; Lima, F. de A. F. de; Machineski, G.;
Barbosa, G. M. de C.; Silva, H. R. da; Gatti, I. C. de A.; Cardoso,
J.; Matos, M. A. de; Miyasawa, M.; Silva, M. R. L. da; Gomes, P. C. Microalgas de águas continentais. 1.ed. Londrina: IAPAR,
2014a, 343p. Li, Z.; Zhang, X; Zhao, Y; Li, Y.; Zhang, G; Peng, Z.; Zhang, J. Enhancing auxin accumulation in maize root tips improves root
growth and dwarfs plant height. Plant Biotechnology Journal, v.16,
p.86-99, 2018. https://doi.org/10.1111/pbi.12751 Rev. Bras. Eng. Agríc. Ambiental, v.26, n.2, p.97-102, 2022. Osvaldo Matsuo et al. 102 Shariatmadari, Z.; Riahi, H.; Hashtroudi, M. S.; Ghassempour, A.;
Aghashariatmadary, Z. Plant growth promoting cyanobacteria
and their distribution in terrestrial habitats of Iran. Soil science
and Plant Nutrition, v.59, p.535-547, 2013. https://doi.org/10.10
80/00380768.2013.782253 Lima, T. M. de; Pascoal, P. R. P.; Carvalho, F. J.; Lana, R. M. Q. Adubação de milho crioulo com resíduos orgânicos em associação
com Azospirillum brasilense. Revista Brasileira de Agropecuária
Sustentável, v.9, p.9-16, 2019. https://doi.org/10.21206/rbas. v9i2.3881 Marini, D.; Guimarães, V. F.; Dartora, J.; Lana, M. do C.; Pinto Júnior,
A. S. Growth and yield of corn hybrids in response to association
with Azospirillum brasilense and nitrogen fertilization. Revista
Ceres, v.62, p.117-123, 2015. https://doi.org/10.1590/0034-
737X201562010015 Strzelczyk, E.; Kampert, M.; Li, C. Y. Cytokinin-like substances and
ethylene production by Azospirillum in media with different
carbon sources. Microbiological Research, v.149, p.55-60, 1994. https://doi.org/10.1016/S0944-5013(11)80136-9 https://doi.org/10.1016/S0944-5013(11)80136-9 737X201562010015 Whitham, F. H.; Blaydes, D. F.; Devlin, R. M. Experiment in plant
physiology. New York: David van Nostrand Company, 1971. p.55-58. Milléo, M. V. R.; Cristófoli, I. Avaliação da eficiência agronômica
da inoculação de Azospirillum sp. na cultura do milho. Revista
Scientia Agraria, v.17, p.14-23, 2016. https://doi.org/10.5380/
rsa.v17i3.44630 Yegorenkova, I. V.; Tregubova, K. V.; Burugin, G. L.; Matora, L. Y.;
Ignatov, V. V. Assessing the efficacy of co-inoculation of wheat
seedling with the associative bacteria Paenibacillus polymyxa
1465 and Azospirillum brasilense Sp245. Canadian Journal of
Microbiology, v.62, p.279-285, 2016. https://doi.org/10.1139/
cjm-2015-0647 Muro-Pastor, A.; Brenes-Álvarez; Vioque, A. A combinatorial
strategy of alternative promoter use during differentiation of a
heterocystous cyanobacterium. Environmental Microbiology
Reports, v.9, p.449-458, 2017. https://doi.org/10.1111/1758-
2229.12555 Zeffa, D. M.; Perini, L. Rev. Bras. Eng. Agríc. Ambiental, v.26, n.2, p.97-102, 2022. Literature Cited J.; Mayara B. S.; Sousa N. V. de; Scapim, C. A.;
Oliveira, A. L. M. de; Amaral Júnior, A. T. do; Gonçalves, L. S. A. Azospirillum brasilense promote increases in growth and nitrogen
use efficiency of maize genotypes. Plos One, v.14, p.1-19, 2019. https://doi.org/10.1371/journal.pone.0215332 Prasanna, R.; Hossain, F.; Babu, S.; Bidyarani, N.; Adak, A.; Verma, S.;
Shivay, Y. S.; Nain, L. Prospecting cyanobacterial formulations as
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10.1080/02571862.2015.1025444
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Swiss LGBTIQ+ Panel - 2021 Summary Report.
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FOREWORD In Switzerland, individuals who identify as lesbian, gay, bisexual, trans, intersex,
queer, and other sexual or gender minorities (LGBTIQ+) still face prejudice,
discrimination, and structural inequalities. The situation, however, is changing. In February 2020, 63.1% of Swiss voters approved the extension of the anti-
discrimination law to protect sexual minorities from discrimination based on their
sexual orientation. In September 2020, the national council approved a project to
simplify the process for trans or gender-variant persons to change their gender
in ofcial records. In addition, further legal changes (e.g., legalization of same-
sex marriage) are currently being discussed in the government. Due to these
rapid changes, it is important to assess (and communicate about) changes in the
situation of LGBTIQ+ individuals over time. In Switzerland, individuals who identify as lesbian, gay, bisexual, trans, intersex,
queer, and other sexual or gender minorities (LGBTIQ+) still face prejudice,
discrimination, and structural inequalities. The situation, however, is changing. In February 2020, 63.1% of Swiss voters approved the extension of the anti-
discrimination law to protect sexual minorities from discrimination based on their
sexual orientation. In September 2020, the national council approved a project to
simplify the process for trans or gender-variant persons to change their gender
in ofcial records. In addition, further legal changes (e.g., legalization of same-
sex marriage) are currently being discussed in the government. Due to these
rapid changes, it is important to assess (and communicate about) changes in the
situation of LGBTIQ+ individuals over time. To achieve this goal, we initiated the Swiss LGBTIQ+ Panel in 2019: An annual
survey about the situation of LGBTIQ+ individuals in Switzerland. The panel covers
personal experiences of LGBTIQ+ individuals (support, discrimination, coming
out), emerging topics in the Swiss context (protection against discrimination,
simplification of administrative procedure to change legal identity documents to
match experienced gender, legalizing same-sex marriage), as well as actions to
advance the legal situation in Switzerland. Each year we will have a core section
assessing a set of fixed questions which allows us to examine potential changes over
time (e.g., support, discrimination, coming out) and additional sections assessing
the current situation (e.g., legal changes) and topics that our participants consider
relevant (e.g., school). Shortly before 2020, we launched the second wave of the Swiss LGBTIQ+ Panel. SWISS LGBTIQ+
PANEL
20 2 0 S U MM ARY REPO RT
Tabea Hässler & Léıla Eisner Tabea Hässler & Léıla Eisner Tabea Hässler & Léıla Eisner Final written report by Dr. Tabea Hässler (University of Zurich) and Dr. Léïla Eisner
(University of Lausanne) with the support of Raja Liechti and Edith Häsler. The
principle authors contributed equally to the work. Acknowledgment: This report was financially supported by the Swiss National Science
Foundation through an AGORA grant (CRARP1_198594) awarded to Dr. Tabea Hässler
and Dr. Léïla Eisner. We thank Soraya Burger for the design and Matteo Antonini,
Fatma Agrebi, Edith Häsler, Olenka Dworakowski, Francesco Pedrazzini, and Lynn
Heydasch for the translation and proofreading. We further thank Emmanuelle Anex and
Leo Theißing for their valuable feedback. Finally, we thank all the LGBTIQ+
associations, magazines, and individuals for sharing and promoting our survey and all
respondents for making this report possible. Please cite the report as: Hässler, T., & Eisner, L. (2020). Swiss LGBTIQ+ Panel - 2020 Summary
Report. https://doi.org/10.31234/osf.io/kdrh4 Please cite the report as: Hässler, T., & Eisner, L. (2020). Swiss LGBTIQ+ Panel - 2020 Summary
Report. https://doi.org/10.31234/osf.io/kdrh4 1 Sexual orientation and gender identity are distinct, so people can be members of both sexual and gender
minorities. In order to keep the survey duration uniform, we have assigned people to one version of
questionnaire. However, there was an option to fill in the version for both sexual and gender minorities. FOREWORD To account for the specific challenges (e.g., legal situation, discrimination) that
diferent subgroups of the LGBTIQ+ community face, we designed diferent
versions of a web questionnaire that were tailored to sexual minorities (i.e.,
individuals with a minority sexual orientation such as gay, lesbian, bisexual, or
pansexual people) and gender minorities (e.g., individuals identifying as trans
or intersex).1 Further, interested cisheterosexual individuals were also invited to III participate in the survey. All versions were translated into French, German, Italian,
and English. Thanks to the help of many LGBTIQ+ organizations, magazines, and
individuals who widely shared our study on diferent media, 1’792 people replied
to our questionnaire from December 2019 to July 2020. This document presents
a summary of the key findings of the survey. participate in the survey. All versions were translated into French, German, Italian,
and English. Thanks to the help of many LGBTIQ+ organizations, magazines, and
individuals who widely shared our study on diferent media, 1’792 people replied
to our questionnaire from December 2019 to July 2020. This document presents
a summary of the key findings of the survey. participate in the survey. All versions were translated into French, German, Italian,
and English. Thanks to the help of many LGBTIQ+ organizations, magazines, and
individuals who widely shared our study on diferent media, 1’792 people replied
to our questionnaire from December 2019 to July 2020. This document presents
a summary of the key findings of the survey. IV GLOSSARY21 GLOSSARY
Asexual
A term used to describe a person who does not experience
sexual attraction. Bisexual
A term used to describe a person who is attracted to more
than one gender. Distinct from pansexual, which includes
attraction to people regardless of gender. Cis female
Someone who was assigned female at birth and identifies and
lives as a woman. Cis-heterosexual
Used in this report to refer to people whose gender identity
matches their sex assigned at birth (i.e., who are not members
of gender minorities) and who are exclusively attracted to
another gender. Cis male
Someone who was assigned male at birth and identifies and
lives as a man. Coming out
When a person first tells someone about their sexual
(public)
orientation or gender identity. Gay man
A man who is attracted to other men. Gender identity
A person’s internal sense of their own gender. Gender identity –
An umbrella category used to describe individuals who choose
Other
‘other’ as the category for their gender identity. In this
category, participants reported, for instance, identifying as
agender, gender fluid, gender questioning, drag queen, queer,
demiboy, or as not identifying with any gender. Gender minority
Individuals with a minority gender identity such as trans
members
or intersex people. 2 Please note that the definitions belong to the community and might change over time. Asexual
A term used to describe a person who does not experience
sexual attraction. Bisexual
A term used to describe a person who is attracted to more
than one gender. Distinct from pansexual, which includes
attraction to people regardless of gender. Cis female
Someone who was assigned female at birth and identifies and
lives as a woman. Cis-heterosexual
Used in this report to refer to people whose gender identity
matches their sex assigned at birth (i.e., who are not members
of gender minorities) and who are exclusively attracted to
another gender. Cis male
Someone who was assigned male at birth and identifies and
lives as a man. Coming out
When a person first tells someone about their sexual
(public)
orientation or gender identity. Gay man
A man who is attracted to other men. Gender identity
A person’s internal sense of their own gender. Gender identity –
An umbrella category used to describe individuals who choose
Other
‘other’ as the category for their gender identity. GLOSSARY21 In this
category, participants reported, for instance, identifying as
agender, gender fluid, gender questioning, drag queen, queer,
demiboy, or as not identifying with any gender. Gender minority
Individuals with a minority gender identity such as trans
members
or intersex people. 2 Please note that the definitions belong to the community and might change over time. A term used to describe a person who does not experience
sexual attraction. A term used to describe a person who is attracted to more
than one gender. Distinct from pansexual, which includes
attraction to people regardless of gender. Someone who was assigned female at birth and identifies and
lives as a woman. Used in this report to refer to people whose gender identity
matches their sex assigned at birth (i.e., who are not members
of gender minorities) and who are exclusively attracted to
another gender. Gender minority
Individuals with a minority gender identity such as trans
members
or intersex people. 2 Please note that the definitions belong to the community and might change over time. 2 Please note that the definitions belong to the community and might change over time. V Heterosexual
A term used to describe a person who is exclusively attracted
to another gender. Also referred to as straight. Homosexual
A term used to describe a person who is emotionally,
romantically, or sexually attracted to members of the same
gender. Intersex
An umbrella term for people with sex characteristics
(hormones, chromosomes, and external/internal reproductive
organs) that difer from those typically expected of a male or
female individual. Joint adoption
A term used to describe adoption by two partners. Contrary to
stepchild adoption, joint adoption by same-sex parents is
currently not legal in Switzerland. Lesbian woman
A woman who is attracted to other women. LGBTIQ+
An abbreviation used to refer to all people who identify as
lesbian, gay, bisexual, trans, intersex, queer, or as having any
other minority sexual orientation or gender identity. Minority sexual
Used in this report to refer to anyone not identifying as
orientation
heterosexual. This includes individuals identifying as gay,
lesbian, bisexual, pansexual, queer, etc. Non-binary
An umbrella term used to describe gender identities where the
individual does not identify exclusively as a man or a woman. There are many categories included within this, such as
agender, genderqueer, and gender fluid. Pansexual
Attraction to people regardless of their gender. GLOSSARY21 Same-sex
A term used to describe the legal union between two people
marriage
of the same gender. Sexual minority
Individuals with a minority sexual orientation such as
members
homosexual (gay, lesbian), bisexual, or pansexual people. A term used to describe a person who is exclusively attracted
to another gender. Also referred to as straight. A term used to describe a person who is emotionally,
romantically, or sexually attracted to members of the same
gender. An umbrella term for people with sex characteristics
(hormones, chromosomes, and external/internal reproductive
organs) that difer from those typically expected of a male or
female individual. A term used to describe adoption by two partners. Contrary to
stepchild adoption, joint adoption by same-sex parents is
currently not legal in Switzerland. An abbreviation used to refer to all people who identify as
lesbian, gay, bisexual, trans, intersex, queer, or as having any
other minority sexual orientation or gender identity. An umbrella term used to describe gender identities where the
individual does not identify exclusively as a man or a woman. There are many categories included within this, such as
agender, genderqueer, and gender fluid. Sexual orientation Describes who a person is emotionally, romantically or
sexually attracted to. xual orientation An umbrella category used to describe individuals who choose
ther
‘other’ as the category for their sexual orientation. In this
category, participants mentioned, for instance, identifying as
asexual (partly with romantic attraction), demisexual, fluid,
polyamorous, polysexual, heteroflexible, homoflexible, queer,
questioning, as well as not liking categories. Umbrella term used to describe individuals who have a gender
identity that is diferent to the gender assigned at birth. Non-
binary people may or may not consider themselves to be
trans. Someone who was assigned male at birth but identifies and
lives as a woman. Someone who was assigned female at birth but identifies and
lives as a man. A term used mainly by people who identify with a minority
sexual orientation and/or gender identity. The process of exploring your own sexual orientation and/or
gender identity. VI VI Sexual orientation Describes who a person is emotionally, romantically or
sexually attracted to. Sexual orientation An umbrella category used to describe individuals who choose
- Other
‘other’ as the category for their sexual orientation. In this
category, participants mentioned, for instance, identifying as
asexual (partly with romantic attraction), demisexual, fluid,
polyamorous, polysexual, heteroflexible, homoflexible, queer,
questioning, as well as not liking categories. Trans
Umbrella term used to describe individuals who have a gender
identity that is diferent to the gender assigned at birth. Non-
binary people may or may not consider themselves to be
trans. Trans women
Someone who was assigned male at birth but identifies and
lives as a woman. Trans men
Someone who was assigned female at birth but identifies and
lives as a man. Queer
A term used mainly by people who identify with a minority
sexual orientation and/or gender identity. Questioning
The process of exploring your own sexual orientation and/or
gender identity. Sexual orientation Describes who a person is emotionally, romantically or
sexually attracted to. Sexual orientation Describes who a person is emotionally, romantically or
sexually attracted to. CONTENTS FOREWORD
III
GLOSSARY
V
IMPORTANT METHODOLOGICAL NOTES
2
KEY FINDINGS
3
THE RESULTS
4
WHO RESPONDED? 5
SECTION 1: COMING OUT
7
SECTION 2: SUPPORT AND EXPERIENCED DISCRIMINATION 10
SECTION 3: SCHOOL CONTEXT
13
SECTION 4: WELL-BEING
16
SECTION 5: SITUATION IN SWITZERLAND
17
SECTION 6: POST-VOTING SURVEY
19
SECTION 7: FUTURE DIRECTIONS
24 THE SWISS LGBTIQ+ PANEL 1 IMPORTANT METHODOLOGICAL NOTES Before interpreting the results of this report, please read these important
methodological notes. The second survey of the Swiss LGBTIQ+ Panel was hosted online for 7 months. An online survey was considered the best way to reach out to a large number of
LGBTIQ+ and cis-heterosexual respondents and allowed respondents to provide
anonymous and confidential responses. Individuals who voluntarily participated in
our study were mostly re-contacted via e-mail or informed by LGBTIQ+ and other
organizations through posts, articles, newsletters, and chats. Though the number
of respondents to the survey was large, we still need to be careful when interpreting
the data and extrapolating from the findings. The sample was self-selected and is
not representative of the entire LGBTIQ+ population in Switzerland. In particular,
LGBTIQ+ individuals who are/were not connected to LGBTIQ+ organizations or not
“out” are probably less represented in our study. These people may have diferent
experiences to those who are connected to the LGBTIQ+ scene. In addition, most
cis-heterosexual individuals who participated in this survey heard about the survey
from LGBTIQ+ individuals (although some university students took part as well)
and might generally be more supportive of LGBTIQ+ issues than the general Swiss
population. This implies that responses by cis-heterosexual individuals displayed
in the present report are particularly NOT representative of the cis-heterosexual
population. Please be aware of this when interpreting the results displayed in this
report. 2 KEY FINDINGS LGBTIQ+ individuals in Switzerland still face structural inequalities, experience
discrimination, and don’t feel fully accepted everywhere. These inequalities are more
pronounced among gender minority members, who report particularly high levels of
structural inequalities. Furthermore, 16% of gender minority members and 8% of sexual
minority members reported having been physically attacked because of their gender
identity/sexual orientation in the year before the survey. Both sexual and gender minority members reported that people reacted more
positively to their coming out than what they expected beforehand. One reason for this
overestimation of negative reactions is that one’s sexual orientation/gender identity
is concealable and that we often lack information about others’ opinions. Hence, it
is essential that allies indicate their support (e.g., inclusive policies and messages)
to foster a climate of inclusion in which LGBTIQ+ individuals feel safe to come out. A valuable source of support for both members of sexual and gender minorities
are friends and other LGBTIQ+ individuals. While most sexual minority members feel
supported by their family members, evidence is more mixed among gender minority
members: some are supported by their family, while others experience discrimination. Even though LGBTIQ+ students are particularly vulnerable, sexual orientation and gender
identity are often not covered in school. Half of the youngest participants (under 21
years of age) report that LGBTIQ+ issues were not at all addressed during their entire
school period. This disregard of LGBTIQ+ topics stands in stark contrast to the needs
of LGBTIQ+ pupils and students, who indicated that greater inclusiveness, greater
visibility, and greater support from teachers and classmates would have helped them. The vote on extending the anti-discrimination law and the discussion surrounding it led to
mixed emotions. While many participants reported feeling relieved and happy, some felt
sad that one-third of people voted against the extension. Further, many gender minorities
reported feeling left out. Diferent subgroups within the LGBTIQ+ spectrum report challenges such as being
medicalized, neglected, or not seen as a valid identity. These challenges stem not only
from the general society but also from within the LGBTIQ+ community. 3 WHO RESPONDED? In total, 1’792 individuals participated in the 2020 survey: 1’276 filled out the
sexual minority version of the questionnaire, 188 the gender minority version,
13 both the sexual and gender minority versions, and 315 the cis-heterosexual
version. A summary of participants’ sexual orientation, gender identity, age group,
geographical area, education, and religion is presented in Table 1 below. For example, 50.3% respondents (902 people) identified as homosexual, 15.9%
(285 people) as bisexual, 9.5% (171 people) as pansexual, 17.8% (319 people) as
heterosexual, 2.1% (38 people) as asexual, and 4.3% (77 people) as another sexual
orientation (demisexual, questioning, queer and other). In total, 1’792 individuals participated in the 2020 survey: 1’276 filled out the
sexual minority version of the questionnaire, 188 the gender minority version,
13 both the sexual and gender minority versions, and 315 the cis-heterosexual
version. A summary of participants’ sexual orientation, gender identity, age group,
geographical area, education, and religion is presented in Table 1 below. In total, 1’792 individuals participated in the 2020 survey: 1’276 filled out the
sexual minority version of the questionnaire, 188 the gender minority version,
13 both the sexual and gender minority versions, and 315 the cis-heterosexual
version. A summary of participants’ sexual orientation, gender identity, age group,
geographical area, education, and religion is presented in Table 1 below. For example, 50.3% respondents (902 people) identified as homosexual, 15.9%
(285 people) as bisexual, 9.5% (171 people) as pansexual, 17.8% (319 people) as
heterosexual, 2.1% (38 people) as asexual, and 4.3% (77 people) as another sexual
orientation (demisexual, questioning, queer and other). Table 1. Who responded? THE RESULTS In this section, we relay some of the main findings from the 2020 survey. Because
diferent versions of the questionnaire were tailored to sexual minorities’ and
gender minorities’ rights, we will, in what follows, present the findings of sexual
minority, gender minority, and cis-heterosexual respondents separately. In this section, we relay some of the main findings from the 2020 survey. Because
diferent versions of the questionnaire were tailored to sexual minorities’ and
gender minorities’ rights, we will, in what follows, present the findings of sexual
minority, gender minority, and cis-heterosexual respondents separately. WHO RESPONDED? Participants by
TOTAL
HOMO-
SEXUAL
BISEXUAL
PANSEXUAL
HETERO-
SEXUAL
ASEXUAL
OTHER
Sexuality %
100%
50.3%
15.9%
9.5%
17.8%
2.1%
4.3%
N
1’792
902
285
171
319
38
77
Participants by
CIS
FEMALE
CIS
MALE
TRANS
FEMALE
TRANS
MALE
NON-
BINARY
OTHER
Gender %
51.5%
36.0%
2.1%
2.4%
6.0%
2.1%
N
923
645
37
43
107
37
Participants by
Under 20
20-29
30-39
40-49
50-59
Over 60
Age group %
9.1%
44.0%
20.0%
12.4%
9.2%
5.5%
N
162
787
357
221
164
98
Participants by
GERMAN
FRENCH
ITALIAN
ROMANSH
BILINGUAL
Geo area %
62.6%
30.9%
2.4%
1.2%
2.9%
N
1’122
553
43
22
52
Participants by
NO UNI
UNI DEGREE
OTHER
Education %
40.2%
53.5 %
6.25%
N
720
959
112
Participants by
ATHEIST
CATHOLIC
PROTES-
TANT
JEWISH
MUSLIM
BUDDHISM
OTHER
Religion %
58.3%
15.2%
15.7%
0.6%
0.7%
1.8%
7.8%
N
1’044
273
281
10
13
32
139
Note. Percentages have been rounded and may not add up to 100%. Note. Percentages have been rounded and may not add up to 100%. Note. Percentages have been rounded and may not add up to 100%. 5 In Table 2, we show the sample composition in greater detail, separating out
respondents by both sexual orientation and gender identity. The numbers in
brackets represent trans participants. For example, the second line can be read as follows: There are 50 bisexual male
participants, 7 of them are trans. There are 213 bisexual female participants, 10 of
them are trans. There are 18 bisexual non-binary participants and 15 of them are
trans. There are 4 bisexual participants who do not identify as either female, male,
or non-binary and 2 of them are trans. Table 2. Sample Composition Sexual Orientation/
Gender identity
Male
Female
Non-binary
Other
Heterosexual
89 (8)
225 (2)
5 (5)
0 (0)
Bisexual
50 (7)
213 (10)
18 (15)
4 (2)
Pansexual
31 (13)
90 (6)
39 (32)
11 (9)
Homosexual
502 (12)
371 (10)
20 (7)
9 (4)
Asexual
3 (2)
25 (4)
7 (6)
3 (3)
Other
12 (0)
32 (1)
20 (18)
13 (9)
Total
687 (42)
956 (33)
109 (83)
40 (27)
Note In brackets: Individuals identifying as trans Note. In brackets: Individuals identifying as trans. Note. In brackets: Individuals identifying as trans. 6 COMING OUT EXPECTATION VS. REALITY Respondents to the survey were asked to indicate how they thought members
of diferent groups would react to their coming out. They were then asked to
indicate how members of these diferent groups actually reacted to their coming
out. Sexual minority and gender minority participants could indicate values
from 1 (Very negative) to 7 (Very positive). Therefore, a higher number indicates
more positive expectations/reactions. Results are displayed in Figure 1 (sexual
minorities) and Figure 2 (gender minorities). Please note that we included only
the people who outed themselves in the relevant context (e.g., 1’113 of 1’289 sexual
minority participants outed themselves among their family). In general, results
indicate that sexual minority and gender minority participants expected that their
friends would react more positively to their coming out than others. This pattern
was also found for actual reactions to the coming out. Notably, however, patterns
indicate that sexual and gender minority participants expected that the reactions
to their outing would be more negative than they actually were across all groups. Coming Out Expectation vs. Reality: Sexual Minorities
Figure 1. Coming Out Among Sexual Minorities: Expected Versus Real Reaction
5.7
5.7
6.3
5.5
5.1
4.8
4.6
5.6
4.6
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Figure 2. Coming Out Among Gender Minorities: Expected Versus Real Reaction
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&YQFDUBUJPO 3FBMJUZ 7 CONTEXT OF COMING OUT Next, respondents in the survey were asked in which contexts they were out and
among how many people. Importantly, one’s sexual orientation/gender identity
(e.g., one’s trans identity) might not always be relevant. To illustrate, once trans
individuals transitioned, some might not feel the need to reveal that they are
trans (since they pass as the gender they identify as). COMING OUT EXPECTATION VS. REALITY However, this measure still
provides a valid estimate for how openly people can talk about their identity and
current relationship/activities. We grouped the answers into three categories: (1)
Being out to none or a few people, (2) approximately half of the people, and
(3) most/all people. The results are shown separately for sexual minority (see
Figure 3) and gender minority members (see Figure 4). Please keep in mind that
respondents could also choose that a context was not applicable for them (e.g., if
they do not visit a church or any other religious setting). Therefore, the valid
number of responses vary widely between contexts. Sexual minority group members (see Figure 3 below) were out among most of their
friends followed by their family. Half of the respondents for whom the categories
acquaintances, university, workplace, and sport were applicable were openly out
to most/all people. However, half of the respondents did not come out in the
school context and more than half among their neighbors. Finally, two-thirds of the
respondents for which the category church/religious organization was applicable
were not out in this context. Context Coming Out: Sexual Minorities
Figure 3. Context of Coming Out Among Sexual Minority Members
67.4%
40.9%
33.2%
34.1%
50.9%
57.2%
7.0%
26.1%
23.6%
10.9%
11.3%
17.6%
16.4%
11.5%
11.2%
10.8%
24.2%
14.9%
21.7%
47.8%
49.2%
49.4%
37.6%
31.7%
82.1%
49.7%
61.5%
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%%*++..%%0 Figure 3. Context of Coming Out Among Sexual Minority Members 8 Members of gender minorities (see Figure 4 below) were on average less likely than
sexual minority members to reveal their gender identity. Two-thirds of respondents
were out to most/all of their friends, while one fifth was not out among their friends
at all. Almost half of the gender minority respondents were out among most/all
family members, while more than one third chose not to out themselves to family
members. In the workplace, university, school, sport, and church contexts as well
as among their neighbors, gender minorities were particularly guarded: between
half and two-thirds of respondents did not reveal their gender identity to (almost)
everyone. Context Coming Out: Gender Minorities
Figure 4. COMING OUT EXPECTATION VS. REALITY Context of Coming Out Among Gender Minority Members
67.2%
64.8%
54.6%
58.9%
62.8%
68.0%
20.9%
42.9%
34.5%
8.6%
9.1%
9.9%
10.5%
11.8%
12.2%
17.3%
27.5%
17.8%
24.1%
26.1%
35.5%
30.5%
25.5%
19.9%
61.7%
29.6%
47.7%
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$POUFYU$PNJOH0VU(FOEFS.JOPSJUJFT Figure 4. Context of Coming Out Among Gender Minority Members 9 SECTION 2:
SUPPORT AND EXPERIENCED DISCRIMINATION SUPPORT BY DIFFERENT GROUPS
In this section, we present findings related to support and experienced
discrimination. First, members of sexual and gender minorities were asked to
indicate how supported they felt in diferent contexts (see Figure 5). Respondents
could choose values from 1 (Not at all) to 7 (Totally) or that a context was not
applicable for them (e.g., if they do not participate in any sports). Thus, higher
numbers correspond to higher perceived support. Please note that valid numbers
of responses vary widely between contexts (from 267 to 1’264 responses for
sexual minorities and 52 to 194 for gender minorities). For instance, the level of
felt support from the church context reported in Figure 5 corresponds to answers
from 267 sexual minority participants for whom the church context was relevant/
applicable. Both members of sexual and gender minorities reported that they felt most
supported by their friends, followed by the LGBTIQ+ scene, and their families. Respondents reported mixed support from their neighbors, school, university,
workplace, and sports team, and felt little support from their church/other religious
settings. Overall, members of gender minorities felt less supported than members
of sexual minorities.
0 10
Supported by
Figure 5. Support by Social Group
2.5
3.2
3.7
3.4
3.1
3.0
3.9
5.9
4.3
5.3
2.7
4.5
4.5
4.3
3.5
3.3
4.6
6.2
5.0
5.7
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!41 (ŏ)%*+.%0%!/ŏ
!* !.ŏ)%*+.%0%!/ 10 EXPERIENCES OF DISCRIMINATION Next, members of sexual and gender minorities were asked to indicate how often
they experienced diferent types of discrimination in the past 12 months. We
grouped the answers into two categories: yes, experienced discrimination in the
past 12 months vs. no, experienced no discrimination in the past 12 months. In
Figure 6, we report the proportion of people who experienced a particular form
of discrimination. Based on previous feedback from our participants, we added
unwanted disclosure and being stared at in public spaces. A large majority of both members of sexual and gender minorities reported that
they have been exposed to jokes, felt that their sexual orientation/gender identity
has been not taken seriously, and that people stare at them in public spaces. SECTION 2:
SUPPORT AND EXPERIENCED DISCRIMINATION A
large majority of members of gender minorities furthermore reported structural
discrimination, unwanted disclosure, and social exclusion. Importantly, 16% of
gender minority members and 8% of sexual minority members reported having
been the target of physical violence within the last year. When comparing individual
subgroups, lesbian, bi-, and pansexual women as well as trans respondents
reported experiencing sexual harassment by men, while this seems to be less of a
problem for gay men. Experienced Discrimination in the Past 12 Months
Figure 6. Types of Experienced Discrimination
55.5%
78.1%
16.0%
77.9%
55.0%
14.0%
40.7%
44.2%
76.5%
86.0%
36.6%
64.3%
8.2%
39.8%
33.4%
9.3%
37.2%
30.1%
50.8%
81.0%
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
UNWANTED DISCLOSURE
STARING
PHYSICAL VIOLENCE
STRUCTURAL DISCRIMINATION
EXCLUSION
SEXUAL HARASSMENT WOMEN
SEXUAL HARASSMENT MEN
BULLYING
NOT TAKEN SERIOUSLY
JOKES
Percentage of participants who experienced each form of discrimination
Sexual minorities
Gender minorities Experienced Discrimination in the Past 12 Months Figure 6. Types of Experienced Discrimination 11 CONTEXTS OF DISCRIMINATION Because we know that discrimination can occur in very diferent situations, we
wanted to get a better understanding of the contexts in which LGBTIQ+ individuals
feel discriminated against. Again, respondents could choose values from 1 (Never)
to 7 (Very often) or that a context was not applicable to them. Please keep in
mind that numbers of valid responses vary widely between contexts (from 338
to 1’207 responses for sexual minorities and 47 to 181 for gender minorities). Members of gender minorities reported experiencing more discrimination in all
contexts assessed (see Figure 7). Gender minority participants reported that they
sometimes experience discrimination by legal institutions, in school, university,
the workplace, church/other religious settings, hospitals, public spaces, and by
their families (all means around 3). Members of sexual minorities reported that
they more often experienced discrimination in public spaces, church, and by legal
institutions (all means above 2) than in other contexts. Context of Discrimination
Figure 7. Context of Discrimination
2.9
3.0
2.9
3.2
2.1
2.5
2.9
3.0
3.0
2.0
2.3
1.6
2.8
1.8
2.6
2.1
1.6
2.0
1.2
1.4
1.8
1.5
1.7
1.4
1.8
1.3
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ŏ!* !.ŏ)%*+.%0%!/ Context of Discrimination !41 (ŏ)%*+.%0%!/ŏ
ŏ!* !.ŏ)%*+.%0%!/ Figure 7. SECTION 2:
SUPPORT AND EXPERIENCED DISCRIMINATION Context of Discrimination 12 EXPERIENCES AT SCHOOL Last year, we asked participants to indicate what topics we should address in the
future. The school context was one of the most named. Therefore, we decided to
add a section on experiences of LGBTIQ+ individuals at school. Participants were
asked to indicate whether the topics of sexual orientation and/or gender identity
were addressed at school. In general, both topics were rarely addressed at school:
A majority of the participants (64.4%, 1’153 persons) indicated that neither sexual
orientation nor gender identity were addressed at school. Only 6.4% (115 persons)
of participants indicated that both sexual orientation and gender identity were
addressed. Another 21.3% (381 persons) indicated that sexual orientation but not
gender identity was discussed at school, while 1.3% (24 persons) indicated that
gender identity but not sexual orientation was discussed at school. Finally, 6.5%
(116 persons) indicated being unsure. BBecause the situation has changed over
time, we looked at the diferences in responses between age groups (under 21,
21-30, 31-40, over 40 years old). Results displayed in Figure 8 indicate that sexual
orientation and to a lesser extent gender identity are increasingly discussed at
school. Yet, 50% of the youngest respondents still report that sexual orientation
and gender identity have not been discussed at all in school. Discussions About LGBTIQ+ Issues at School by Age Group
Figure 8. Discussions at School by Age Groups
15.9
5.8
3.0
3.3
29.5
25.1
19.6
10.2
0.5
0.8
1.8
1.1
50.0
61.6
69.1
80.1
4.1
6.6
6.5
5.3
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20
40
60
80
100
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+*ņ0ŏ'*+3 Figure 8. Discussions at School by Age Groups Figure 8. Discussions at School by Age Groups 13 EXPERIENCES AT SCHOOL EXPERIENCES AT SCHOOL But
none of us talked about it.”
– 26-year-old gay man
“The constant
heteronormative assumptions
by my friends made it very
GLIÀFXOWWRFRPHRXWWRWKHPZLWKRXW
feeling as if I had been lying to them,
even though I hadn’t been. Once, our class
discussed the topic of marriage equality on
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been strong opponents to it.”
– 17-year-old gay man EXPERIENCES AT SCHOOL To gather experiences of LGBTIQ+ individuals at school, we asked respondents
to write down their experiences belonging to a sexual and/or gender minority
at school. Below, you can find some selected answers (we chose answers in the
respective language; thus, the quotes difer across the English, French, German,
and Italian versions of the report). ´9HU\ GLIÀFXOW 14
at school. Below, you can find some selected answers (we chose answers in the
respective language; thus, the quotes difer across the English, French, German,
and Italian versions of the report). “There is no effort
made to include LGBTIQ+ people
in public school. We often are seen as
outsiders; no education is made about our
rights and the discrimination we are victims of. Sex education classes and biology classes forget
us completely. It was quite violent to have powerful
people completely washing away any identity that wasn‘t
heteronormative or binary, especially when they could
have used this power and those subjects to tell other
people about us. I also know it was way more violent
IRUSHRSOHZKRKDGDOUHDG\ÀJXUHGRXWWKH\ZHUHSDUW
RIWKH/*%7,4FRPPXQLW\ ,RQO\LGHQWLÀHGDVDQ
ally at the time of public school).“
– 20-year- old trans nonbinary person
“I felt like I
had to hide it. I was
WHUULÀHGVRPHSHRSOHZRXOG
notice something was different
about me.”
– 24-year-old lesbian
“I came out during
school. Most people were
supportive or didn‘t care,
and the exposure to other
/*%74SHRSOHGHÀQLWHO\KHOSHG
me accept myself.”
– 16-year-old lesbian woman
´9HU\GLIÀFXOW
It was basically
impossible for me to come
out. It would have meant almost
total social exclusion, and many,
many jokes and bullying.”
– 29-year-old bisexual man
“The school uses the gender-star (*)
EHWZHHQJHQGHUHGQRXQVDGGUHVVLQJSHRSOHDQG,ÀQGWKDW
very important because it makes me feel seen and valid.”
– trans non-binary person
“I was not
out - got beaten
up anyways.”
– 54-year-old gay man
“Being the weird one and
kind of looked at as «oh she‘s just like
that». I was excluded from parties and sexually
assaulted at the ones I attended on the grounds that
bisexual people want sexual encounters all the time,
and that we‘re promiscuous.”
– 21-year-old bisexual woman
“Out of hundreds of
pupils, only one was publicly out
and he was heavily bullied for it. It
felt like a very hostile environment and not
safe for coming out, so no one did. In the years
after graduating, so many of my classmates did,
indicating I was not the only one struggling. “I came out during
school. Most people were
supportive or didn‘t care,
and the exposure to other
/*%74SHRSOHGHÀQLWHO\KHOSHG
me accept myself.”
– 16-year-old lesbian woman 14
at school. Below, you can find some selected answers (we chose answers in the
respective language; thus, the quotes difer across the English, French, German,
and Italian versions of the report).
“There is no effort
made to include LGBTIQ+ people
in public school. We often are seen as
outsiders; no education is made about our
rights and the discrimination we are victims of.
Sex education classes and biology classes forget
us completely. It was quite violent to have powerful
people completely washing away any identity that wasn‘t
heteronormative or binary, especially when they could
have used this power and those subjects to tell other
people about us. I also know it was way more violent
IRUSHRSOHZKRKDGDOUHDG\ÀJXUHGRXWWKH\ZHUHSDUW
RIWKH/*%7,4FRPPXQLW\ ,RQO\LGHQWLÀHGDVDQ
ally at the time of public school).“
– 20-year- old trans nonbinary person
“I felt like I
had to hide it. I was
WHUULÀHGVRPHSHRSOHZRXOG
notice something was different
about me.”
– 24-year-old lesbian
“I came out during
school. Most people were
supportive or didn‘t care,
and the exposure to other
/*%74SHRSOHGHÀQLWHO\KHOSHG
me accept myself.”
– 16-year-old lesbian woman
´9HU\GLIÀFXOW
It was basically
impossible for me to come
out. It would have meant almost
total social exclusion, and many,
many jokes and bullying.”
– 29-year-old bisexual man
“The school uses the gender-star (*)
EHWZHHQJHQGHUHGQRXQVDGGUHVVLQJSHRSOHDQG,ÀQGWKDW
very important because it makes me feel seen and valid.”
– trans non-binary person
“I was not
out - got beaten
up anyways.”
– 54-year-old gay man
“Being the weird one and
kind of looked at as «oh she‘s just like
that». I was excluded from parties and sexually
assaulted at the ones I attended on the grounds that
bisexual people want sexual encounters all the time,
and that we‘re promiscuous.”
– 21-year-old bisexual woman
“Out of hundreds of
pupils, only one was publicly out
and he was heavily bullied for it. It
felt like a very hostile environment and not
safe for coming out, so no one did. In the years
after graduating, so many of my classmates did,
indicating I was not the only one struggling. But
none of us talked about it.”
– 26-year-old gay man
“The constant
heteronormative assumptions
by my friends made it very
GLIÀFXOWWRFRPHRXWWRWKHPZLWKRXW
feeling as if I had been lying to them,
even though I hadn’t been. Once, our class
discussed the topic of marriage equality on
DGD\ZKHQ,ZDVDEVHQWDQG,ZDVKRUULÀHG
WRÀQGRXWWKDWVRPHRIP\FODVVPDWHVKDG
been strong opponents to it.”
– 17-year-old gay man ´9HU\GLIÀFXOW
It was basically
impossible for me to come
out. It would have meant almost
total social exclusion, and many,
many jokes and bullying.”
– 29-year-old bisexual man ´9HU\GLIÀFXOW
It was basically
impossible for me to come
out. It would have meant almost
total social exclusion, and many,
many jokes and bullying.”
– 29-year-old bisexual man “I felt like I
had to hide it. I was
WHUULÀHGVRPHSHRSOHZRXOG
notice something was different
about me.”
– 24-year-old lesbian “The school uses the gender-star (*)
EHWZHHQJHQGHUHGQRXQVDGGUHVVLQJSHRSOHDQG,ÀQGWKDW
very important because it makes me feel seen and valid.”
– trans non-binary person “I was not
out - got beaten
up anyways.”
– 54-year-old gay man “Being the weird one and
kind of looked at as «oh she‘s just like
that». I was excluded from parties and sexually
assaulted at the ones I attended on the grounds that
bisexual people want sexual encounters all the time,
and that we‘re promiscuous.”
– 21-year-old bisexual woman “Out of hundreds of
pupils, only one was publicly out
and he was heavily bullied for it. It
felt like a very hostile environment and not
safe for coming out, so no one did. In the years
after graduating, so many of my classmates did,
indicating I was not the only one struggling. But
none of us talked about it.”
– 26-year-old gay man “I came out during
school. Most people were
supportive or didn‘t care,
and the exposure to other
/*%74SHRSOHGHÀQLWHO\KHOSHG
me accept myself.”
– 16-year-old lesbian woman “There is no effort
made to include LGBTIQ+ people
in public school. We often are seen as
outsiders; no education is made about our
rights and the discrimination we are victims of. Sex education classes and biology classes forget
us completely. It was quite violent to have powerful
people completely washing away any identity that wasn‘t
heteronormative or binary, especially when they could
have used this power and those subjects to tell other
people about us. I also know it was way more violent
IRUSHRSOHZKRKDGDOUHDG\ÀJXUHGRXWWKH\ZHUHSDUW
RIWKH/*%7,4FRPPXQLW\ ,RQO\LGHQWLÀHGDVDQ
ally at the time of public school).“
– 20-year- old trans nonbinary person 14 WHAT COULD HELP/COULD HAVE HELPED? Finally, we asked respondents to indicate what helped them or what would have
helped them being an LGBTIQ+ individual in school. 15
elped them being an LGBTIQ+ individual in school. “It would have helped
if teachers would have been more
inclusive (when they were talking in
JHQHUDODQGLQVH[HGXFDWLRQFODVVHV DQGÀUPO\
stood up against homo/trans-phobic bullying. Also, if there would have been more education
around LGBTQ+ issues in general.”
– 22-year-old bisexual woman
“No one was
visible at school: there
were no gay kids, no gay
teachers, no gay parents – it
basically didn‘t exist. I don‘t
want to think how it would have
been to be a trans kid in such an
environment.”
– 33-year-old bisexual
woman
“It helped that there
were others who were openly out. More representation would have been
nice though. Our teachers only talked about
heterosexual relationships (even during lessons
which would not have needed any analogies in that
department) and the topic of gender identity etc. was never breached. It would have been nice to
have another point of view.“
– 21-year-old asexual woman
“Having
more
LGBTIQ+ role
models.”
– 28-year-old
gay man
“Supportive teachers, LGBT
education and awareness in school, in courses. And also LGBT presence in general. In school books, on
posters or advertisement (not always show heterosexual couples)
etc. Basically, VISIBILITY and a strong commitment from the school
management to punish bullying/discrimination in this regard. Also, it
is important for heterosexual people at school to understand what
LGBT people are undergoing. This of course is not only important
for LGBT but for everyone, for the society.” – 29-year-old
bisexual man
“[…]The teachers
need to model acceptance,
the staff as well, they need to
prove that they have zero tolerance
for abuse, mobbing, bullying, assault,
sexism, racism, or other negative
behaviors like that […].”
– 40-year-old queer woman
“That
teachers (+the media)
would talk more about it
in general and especially in
a positive light. 90% of the
e I was hearing about the LGBTQ+
mmunity as a child, it appeared
ke a problem. […] It would have
mendously helped me to see some
ositive coming out stories or
st older members of the LGBTQ+
community living a happy and
IXOÀOOLQJOLIHµ
– 24-year-old lesbian woman
“My friends helped me
a lot, having a community or group
that you can rely on and share with. WHAT COULD HELP/COULD HAVE HELPED? At
university, knowing that there were a few queer
inclusive associations was also helpful. It would
have helped to hear more about us (and other minorities
and oppressed communities). Inclusive and neutral language
ZRXOGKDYHEHHQJUHDWQRQELQDU\EDWKURRPVWRREXWÀUVWO\
being introduced through the class material (bio, sex ed,
philosopy, history, law...) to those issues and getting
out of this white binary heteronormative view of
everything...”
– 20-year-old non-binary trans person “No one was
visible at school: there
were no gay kids, no gay
teachers, no gay parents – it
basically didn‘t exist. I don‘t
want to think how it would have
been to be a trans kid in such an
environment.”
– 33-year-old bisexual
woman “[…]The teachers
need to model acceptance,
the staff as well, they need to
prove that they have zero tolerance
for abuse, mobbing, bullying, assault,
sexism, racism, or other negative
behaviors like that […].”
– 40-year-old queer woman “That
teachers (+the media)
would talk more about it
in general and especially in
a positive light. 90% of the
time I was hearing about the LGBTQ+
community as a child, it appeared
like a problem. […] It would have
tremendously helped me to see some
positive coming out stories or
just older members of the LGBTQ+
community living a happy and
IXOÀOOLQJOLIHµ
24
ld l
bi me
+
“My friends helped me
a lot, having a community or group
that you can rely on and share with. At
university, knowing that there were a few queer
inclusive associations was also helpful. It would
have helped to hear more about us (and other minorities
and oppressed communities). Inclusive and neutral language
ZRXOGKDYHEHHQJUHDWQRQELQDU\EDWKURRPVWRREXWÀUVWO\
being introduced through the class material (bio, sex ed,
philosopy, history, law...) to those issues and getting
out of this white binary heteronormative view of
everything...” me
“My friends helped me
a lot, having a community or group
that you can rely on and share with. At
university, knowing that there were a few queer
inclusive associations was also helpful. It would
have helped to hear more about us (and other minorities
and oppressed communities). Inclusive and neutral language
ZRXOGKDYHEHHQJUHDWQRQELQDU\EDWKURRPVWRREXWÀUVWO\
being introduced through the class material (bio, sex ed,
philosopy, history, law...) to those issues and getting
out of this white binary heteronormative view of
everything...” – 24-year-old lesbian woman 15 SECTION 4:
WELL-BEING In this section we report on respondents’ subjective well-being. We asked sexual
and gender minority members as well as cis-heterosexual respondents about
both their positive afect (i.e., feeling enthusiastic, happy, and satisfied) and their
negative afect (i.e., feeling sad, helpless, and rejected) in the last 12 months (see
Figure 9). This allowed us to compare the well-being between the respondents. Values range from 1 (Very rarely) to 7 (Very frequently), thus higher numbers
indicate both higher positive and negative afect. Cis-heterosexual respondents
and members of sexual minorities do not significantly difer in positive afect and
negative afect, while members of gender minorities report slightly less positive
afect and more negative afect. This indicates that members of gender minorities
feel worse of than both cis-heterosexual individuals and members of sexual
minorities. In this section we report on respondents’ subjective well-being. We asked sexual
and gender minority members as well as cis-heterosexual respondents about
both their positive afect (i.e., feeling enthusiastic, happy, and satisfied) and their
negative afect (i.e., feeling sad, helpless, and rejected) in the last 12 months (see
Figure 9). This allowed us to compare the well-being between the respondents. Values range from 1 (Very rarely) to 7 (Very frequently), thus higher numbers
indicate both higher positive and negative afect. Cis-heterosexual respondents
and members of sexual minorities do not significantly difer in positive afect and
negative afect, while members of gender minorities report slightly less positive
afect and more negative afect. This indicates that members of gender minorities
feel worse of than both cis-heterosexual individuals and members of sexual
minorities. Well-Being
3.2
5.0
4.1
4.7
3.4
5.1
ŏ
ŏ
.+)ŏāŅ!.5ŏ. .!(5Ņŏ0+ŏĈŅ!.5ŏ".!-1!*0(5Ņ
!41 (ŏ)%*+.%0%!/ŏŏ
ŏ!* !.ŏ)%*+.%0%!/ŏ
ŏŏŏ%/ġ$!0!.+/!41 (/ Well-Being
3.2
5.0
4.1
4.7
3.4
5.1
ŏ
ŏ
.+)ŏāŅ!.5ŏ. .!(5Ņŏ0+ŏĈŅ!.5ŏ".!-1!*0(5Ņ
!41 ( )%*+.%0%!/
!* !. )%*+.%0%!/
%/ġ$!0!.+/!41 (/ ŏ !41 (ŏ)%*+.%0%!/ŏŏ
ŏ!* !.ŏ)%*+.%0%!/ŏ
ŏŏŏ%/ġ$!0!.+/!41 (/ Figure 9. Well-Being 16 WISH TO GET MARRIED AND HAVE CHILDREN Currently, LGBTIQ+ individuals in Switzerland are not allowed to marry, adopt
children other than the child of their partner, or use assisted procreation. Because
same-sex marriage and assisted procreation are currently being discussed in the
Swiss government, we asked participants to indicate whether they wish to get
married (see Figure 10) and to have children (see Figure 11). We first report the proportion of people who are not married but wish to do so. Results indicate that more than half of the sexual minority participants (55.1%,
712 people) wish to get married despite not currently being allowed to do so. Furthermore, one quarter of gender minority participants (27.7%, 51 people) wish
to get married. Wish to Get Married
Figure 10. Wish to Get Married
41.4%
27.7%
55.1%
27.9%
34.8%
16.3%
26.3%
29.9%
25.9%
4.4%
7.6%
2.8%
ŏ
ŏ ĨāĂąĉĥāĂĉĊĩŏ
ŏ
ŏ ĨāĉąĥĂĀāĩŏ
ġ ŏĨĂĆāĥăāĆĩ
!/
+
+*ņ0ŏ'*+3
0$!. Next, we report the proportion of people who currently do not have children but
wish to have children. Therefore, participants who already have children were
not included (9.0%, 116 sexual minority participants; 17.9%, 36 gender minority
participants; and 24.8%, 78 cis-heterosexual participants). Of the childless
participants, more than one third of sexual minority (35.6%, 418 people), one fifth
of gender minority (21.8%, 36 people), and half of the cis-heterosexual participants
(56.1%, 133 people) indicated that they want to have children. Wish to Have Children
Figure 11. Wish to Have Children
56.1%
21.8%
35.6%
17.3%
40.0%
33.5%
23.6%
29.1%
26.3%
3.0%
9.1%
4.5%
ŏ
ŏĨāāĈăĥāĂĉĊĩŏ
ŏ
ŏĨāćĆĥĂĀāĩŏ
ġ ŏĨĂăĈĥăāĆĩ
!/ŏ
ŏ+ŏŏŏŏŏ+*ņ0ŏ'*+3ŏŏŏ ŏ0$!. 17 FEBRUARY VOTING: EXTENDING THE DISCRIMINATION LAW Political changes regarding same sex marriage and assisted procreation are
currently discussed in the parliament. Furthermore, 2020 saw a popular vote
on discrimination. On the 9th of February 2020, 63.1% of Swiss citizens voted
in favor of extending the discrimination law to include sexual orientation. Please
note, however, that gender identity is still not included in this law, even though
our data reveal that members of gender minorities face more discrimination than
sexual minority members. ENGAGEMENT The public vote on extending the anti-discrimination law allowed us to investigate
the impact of political changes on both LGBTIQ+ and cis-heterosexual individuals. We re-contacted participants one month after the public vote on anti-discrimination
to answer a few additional questions. In total, 636 individuals participated in both
the post-voting survey and the main survey (473 sexual minority members, 88
gender minority members, and 75 cis-heterosexual individuals). Among all three
groups, over 80% reported having voted (86.5% sexual minority members, 84.1%
gender minority members, and 82.7% of cis-heterosexual individuals). Before the vote, LGBTIQ+ individuals and allies engaged in various behaviors (e.g.,
putting up a rainbow flag) to encourage others to vote in favor of extending
the antidiscrimination law. Below, we report the proportion of participants who
indicated that they engaged in the relevant activity (see Figure 12). In general,
most sexual minority, gender minority, and cis-heterosexual participants reported
having talked to cis-heterosexual individuals to motivate them to vote. Further,
we see diferences between sexual/gender minorities and cis-heterosexual
participants as the latter engaged significantly less than the former. The public vote on extending the anti-discrimination law allowed us to investigate
the impact of political changes on both LGBTIQ+ and cis-heterosexual individuals. We re-contacted participants one month after the public vote on anti-discrimination
to answer a few additional questions. In total, 636 individuals participated in both
the post-voting survey and the main survey (473 sexual minority members, 88
gender minority members, and 75 cis-heterosexual individuals). Among all three
groups, over 80% reported having voted (86.5% sexual minority members, 84.1%
gender minority members, and 82.7% of cis-heterosexual individuals). Before the vote, LGBTIQ+ individuals and allies engaged in various behaviors (e.g.,
putting up a rainbow flag) to encourage others to vote in favor of extending
the antidiscrimination law. Below, we report the proportion of participants who
indicated that they engaged in the relevant activity (see Figure 12). In general,
most sexual minority, gender minority, and cis-heterosexual participants reported
having talked to cis-heterosexual individuals to motivate them to vote. Further,
we see diferences between sexual/gender minorities and cis-heterosexual
participants as the latter engaged significantly less than the former. Actions to Motivate People to Vote
Figure 12. WISH TO GET MARRIED AND HAVE CHILDREN We asked people who participated in our survey before
February 8th to indicate their willingness to participate in the vote, the expected
outcome of the vote, and their awareness of campaigns against and/or in favor of
the law change February 8th to indicate their willingness to participate in the vote, the expected
outcome of the vote, and their awareness of campaigns against and/or in favor of
the law change. We found similar patterns between sexual minority, gender minority, and cis-
heterosexual participants for the expected outcome of the vote and intentions to
vote. On average, participants expected that around 55.2% of Swiss citizens would
vote in favor of the law (compared to 63.1% in the actual voting). Further, the vast
majority (85.6%) of participants were planning to vote. While the campaign awareness did not difer between sexual and gender minority
participants, cis-heterosexual participants were less likely to have seen a campaign
about the voting than LGBTIQ+ individuals. Indeed, many LGBTIQ+ participants
(36.1%) only saw a campaign in favor of the extension of the law, 27.9% saw
campaigns from both sides, 7.0% only saw a campaign against the extension of the
discrimination law, and 29.1% did not see any campaign. Among cis-heterosexual
participants, 23.3% cis-heterosexual participants only saw a campaign in favor of
the extension of the law, 16.0% saw campaigns from both sides, 2.9% only saw a
campaign against the extension of the discrimination law, and a majority of cis-
heterosexual participants (57.8%) did not see any campaign. Thus, the topic was
less salient among those individuals less directly afected by it. 18 ENGAGEMENT Actions to Motivate People to Vote – Post-voting Survey
26.5
15.9
4.4
2.9
36.2
6.0
23.5
66.7
5.9
53.7
25.6
7.3
10.0
62.2
7.5
59.3
80.5
19.5
48.3
18.9
7.2
9.1
57.1
8.3
59.1
81.8
30.1
0
20
40
60
80
100
RAINBOW FLAG
DISTRIBUTING FLYERS
STAND AT PUBLIC PLACE
POSTCARDS
SOCIAL MEDIA POST
POSTERS IN PUBLIC SPACES
TALKING TO LGBTIQ+
INDIVIDUALS
TALKING TO HETEROSEXUALS
DONATING MONEY
Percentages (%) of Yes - I did this action
Sexual minorities
Gender minorities
Cis-heterosexuals Percentages (%) of Yes I did this action
Sexual minorities
Gender minorities
Cis-heterosexuals Figure 12. Actions to Motivate People to Vote – Post-voting Survey 19 IMPACT OF THE VOTE AND CAMPAIGN Next, we asked LGBTIQ+ and cis-heterosexual participants to indicate how the
political campaign around the vote as well as the outcome of the vote afected
them. In general, many sexual minority, gender minority, and cis-heterosexual
participants reported being relieved and/or happy about the outcome of the
vote. Some were disappointed by the fact that only sexual orientation (and not
also gender identity) was included in the law, while many participants reported a
mixed feeling, being happy (especially about the proportion of yes in the French
and Italian speaking parts of Switzerland) but still sad to see that more than 30%
of people voted against the law change. For instance, someone wrote “I was really
happy and relieved that the law passed. However, it is frustrating to see that there
are still 37% of voters who don’t deem protection against discrimination important
enough. Also, I’m angry that trans and intersex persons still won’t be protected”. Below you will find statements on how individuals perceived the impact of the
campaign. 20
“Having a public
discussion about LGBT thematics
is often a moment where the people who
are strongly homophobic (and LGBT-phobic) become
public about their opinion, which can be very hurtful. Personally, I have the chance to have an accepting
surrounding thus I had the chance to experience a lot of
support. ENGAGEMENT But I also experienced hate and I was once again
reminded that basic human rights are sadly still neither basic
nor accepted by everyone and even if it‘s not the majority
that is against the extension of the law, it‘s enough to
have a minority of homophobic people that will cause
an incredible amount of damage.”
– 25-year-old lesbian woman
“I appreciated
the fact that people
discussed the situation
of LGBTIQIA+ people in
Switzerland, I felt that we were
more visible than usual.”
– 27-year-old bisexual woman 20 “Some of my friends
became more aware of the
shockingly few rights that the LGBT
community has in Switzerland (many
were under the impression that anti-
discrimination laws existed, and same sex
marriage was legal).”
– 19-year-old lesbian woman
“It sucks to
hear people (who don’t
know I’m queer) talk about
how they planned either on
voting against it or not voting
at all.”
– 27-year-old pansexual woman
“People who
are not in favor of
LGBTIQ+ rights tend to be
relatively quiet about it most of
the time, so it was very disheartening
to see a political campaign against
LGBTIQ+ rights in public life, however,
it was also very supportive to see so
many people and organizations outright
stating their support for extending
the discrimination law. Overall, a
FRQÁLFWHGSHULRGµ
– 27-year-old gay man
“I was incredibly
pleased to see my sexual
orientation represented in a national
campaign, the word «bisexuals» on posters
at every corner <3 I was sad to see that
the lack of protection for trans people was
hardly mentioned.”
– 30-year-old bisexual woman
“As a trans
and aromantic asexual
individual, the results of it
all did not concern me personally,
since we seem to have been excluded
from it early on (yes, I‘m still bitter
about that). But the discussions showed
me once again how hateful and stupid
some people are
and how much I live in a bubble.”
– 22-year-old asexual non-
binary person
“It
gave further
insights into the
Swiss society‘s take
on LGB rights.”
– 31-year-old
homosexual woman “Some of my friends
became more aware of the
shockingly few rights that the LGBT
community has in Switzerland (many
were under the impression that anti-
discrimination laws existed, and same sex
marriage was legal).”
– 19-year-old lesbian woman
“It sucks to
hear people (who don’t
know I’m queer) talk about
how they planned either on
voting against it or not voting
at all.”
– 27-year-old pansexual woman
“People who
are not in favor of
LGBTIQ+ rights tend to be
relatively quiet about it most of
the time, so it was very disheartening
to see a political campaign against
LGBTIQ+ rights in public life, however,
it was also very supportive to see so
many people and organizations outright
stating their support for extending
the discrimination law. Overall, a
FRQÁLFWHGSHULRGµ
– 27-year-old gay man
“I was incredibly
pleased to see my sexual
orientation represented in a national
campaign, the word «bisexuals» on posters
at every corner <3 I was sad to see that
the lack of protection for trans people was
hardly mentioned.”
– 30-year-old bisexual woman
“As a trans
and aromantic asexual
individual, the results of it
all did not concern me personally,
since we seem to have been excluded
from it early on (yes, I‘m still bitter
about that). But the discussions showed
me once again how hateful and stupid
some people are
and how much I live in a bubble.”
– 22-year-old asexual non-
binary person
“It
gave further
insights into the
Swiss society‘s take
on LGB rights.”
– 31-year-old
homosexual woman “Some of my friends
became more aware of the
shockingly few rights that the LGBT
community has in Switzerland (many
were under the impression that anti-
discrimination laws existed, and same sex
marriage was legal).”
– 19-year-old lesbian woman “People who
are not in favor of
LGBTIQ+ rights tend to be
relatively quiet about it most of
the time, so it was very disheartening
to see a political campaign against
LGBTIQ+ rights in public life, however,
it was also very supportive to see so
many people and organizations outright
stating their support for extending
the discrimination law. Overall, a
FRQÁLFWHGSHULRGµ
– 27-year-old gay man “As a trans
and aromantic asexual
individual, the results of it
all did not concern me personally,
since we seem to have been excluded
from it early on (yes, I‘m still bitter
about that). But the discussions showed
me once again how hateful and stupid
some people are
and how much I live in a bubble.”
– 22-year-old asexual non-
binary person 21 IMPACT OF THE VOTE ON WELL-BEING IMPACT OF THE VOTE ON WELL-BEING We also assessed how the outcome of the vote impacted participants’ well-being. Many challenges in 2020, such as the outbreak of Covid-19 and the debates around
climate change, might have impacted people’s well-being. We, therefore, asked
participants to which degree their well-being was negatively or positively afected
by diferent factors (see Figures 13, 14, 15). In general, the large majority of sexual minority, gender minority, and cis-
heterosexual participants reported being negatively afected by the outbreak of
the virus and climate change. Please note that the survey was initiated in early
March 2020 (right before the lockdown in Switzerland). Further, a majority of
sexual minority participants (53.1%) indicated being positively afected by political
changes such as voting on extending the anti-discrimination law to cover protection
on the basis of one’s sexual orientation. Fewer gender minority participants
(42.9%) – who were not included in the antidiscrimination law– and even fewer cis-
heterosexual participants (26.8%) reported being positively afected by political
changes. Well-being Changes: Sexual Minorities
Figure 13. Changes in Well-being for Sexual Minorities – Post-voting Survey
29.4%
11.1%
56.8%
63.5%
29.9%
29.4%
35.9%
34.2%
28.2%
63.9%
41.2%
53.1%
9.0%
8.3%
6.2%
ŏ
ŏ
ŏ
ŏ
!# 0%2!(5ŏ ""! 0! +0ŏ ""! 0! +/%0%2!(5ŏ ""! 0! Well-being Changes: Sexual Minorities ure 13. Changes in Well-being for Sexual Minorities – Post-voting Survey 22 Well-being Changes: Gender Minorities
Figure 14. Changes in Well-being for Gender Minorities – Post-voting Survey
33.3%
14.3%
64.3%
54.8%
38.6%
23.8%
42.9%
32.1%
34.5%
55.4%
42.9%
42.9%
3.6%
10.7%
6.0%
ŏ
ŏ
ŏ
ŏ
!# 0%2!(5ŏ ""! 0! +0ŏ ""! 0! +/%0%2!(5ŏ ""! 0! ure 14. Changes in Well-being for Gender Minorities – Post-voting Survey Well-being Changes: Cis-heterosexuals
Figure 15. Changes in Well-being for Cis-heterosexual Participants – Post-voting
Survey
25.4%
18.3%
52.1%
67.1%
31.4%
29.6%
54.9%
36.6%
24.3%
60.0%
45.1%
26.8%
11.3%
8.6%
8.6%
ŏ
ŏ
ŏ
ŏ
!# 0%2!(5ŏ ""! 0! +0ŏ ""! 0! +/%0%2!(5ŏ ""! 0! Well-being Changes: Cis-heterosexuals
Figure 15. Changes in Well-being for Cis-heterosexual Participants – Post-voting
Survey
25.4%
18.3%
52.1%
67.1%
31.4%
29.6%
54.9%
36.6%
24.3%
60.0%
45.1%
26.8%
11.3%
8.6%
8.6%
ŏ
ŏ
ŏ
ŏ
!# 0%2!(5ŏ ""! 0! +0ŏ ""! 0! +/%0%2!(5ŏ ""! 0! Figure 15. IMPACT OF THE VOTE ON WELL-BEING Changes in Well-being for Cis-heterosexual Participants – Post-voting
Survey 23 SECTION 7:
FUTURE DIRECTIONS Unfortunately, even in LGBT spaces
there are sometimes guys who don’t
respect women’s boundaries.”
– 22-year-old lesbian woman “Bisexual people
often aren‘t taken seriously,
or there‘s a number of harmful
stereotypes (being greedy, unable to
decide, etc.) that can come from members of
the LGBTIQ+ community or from outside that
community.”
– 25-year-old bisexual woman “As a pansexual
person some people
expect of me to think of
everyone as attractive or feel
an attraction to everyone since it
doesn‘t matter who and only their
character.”
– 20-year-old pansexual woman “As a pansexual
person some people
expect of me to think of
everyone as attractive or feel
an attraction to everyone since it
doesn‘t matter who and only their
character.”
– 20-year-old pansexual woman “The biggest issue for
asexuals is the medicalisation of our
sexual identity. [...] Medical and
PHQWDOKHDOWKSHUVRQQHORIWHQWU\WRÀ[WKHZURQJ
It is noteworthy to say that the asexual person in
question is likely to experience those things even if
they are sex positive and enjoy having sex. [...] Society
in general often either assumes something is wrong with
us for not feeling sexual attraction or accuse us
of making it all up to be special. […].”
– 22-year-old asexual woman
t “Homosexuality in
the binary experience is slowly
VWDUWLQJWRÀQGLWVSODFHLQVRFLHW\
<HWWKHÁXLGLW\LQVH[XDOLW\DVZHOODQG
gender identity and all the individuals that
do not associate with a clear letter (L-G-
B-T) are neglected by society. Regarding the
discrimination in the community, race and
gender raise walls.” ´'LIÀFXOW\OLYLQJDVRXU
identity in «serious» social
groups (workplace, university) if
we haven’t fully
transitioned to the point where it is
XQQRWLFHDEOH9HU\KDUGWRÀQGURPDQWLF
sexual partners. The process of transitioning
is still very blurry in terms of where
to go/what is required/support groups or
organizations etc. In the LGBTQ community:
some gay/bi people don’t see trans people as
real (rare but I’ve seen it). Usually being
trans and homosexual can be viewed badly
especially lesbian trans women.”
– 19-year-old trans man – 20-year-old non-binary queer person “I think in
Switzerland, people
don‘t see nonbinary people as
trans or even as a valid gender
identity […]. I have to educate my
friends and family about the LGBTQ+
community and explain my sexuality and
gender identity. Furthermore, I think
that cishets /society doesn‘t understand
why certain words they use are offensive
DQGLQVHQVLWLYHZKLFKLVVRPHWKLQJ,ÀQG
sad. SECTION 7:
FUTURE DIRECTIONS The second wave of the Swiss LGBTIQ+ Panel has been a great success thanks
to the help of various LGBTIQ+ and other organizations, LGBTIQ+ magazines,
student assistants, and individual eforts. In this 2020 summary report, we have
provided an overview of the second wave of data collection. As in the previous
wave, the data reveals that members of sexual and gender minorities in
Switzerland still face structural inequalities, experience discrimination, and do
not feel fully accepted everywhere. As in 2019, we find that these inequalities
are more pronounced among gender minority members – such as trans and
non-binary people – making them a particularly vulnerable group within
the LGBTIQ+ community. This year’s survey placed a specific focus on the
school context, where LGBTIQ+ individuals seem particularly vulnerable, and
greater inclusiveness, visibility, and support from teachers and students are
needed. Another focus of this year’s survey was the voting on extending the
anti-discrimination law, which led to creative eforts to mobilize individuals but
also mixed emotions. Many LGBTIQ+ individuals and cis-heterosexual allies
reported feeling relieved, while some gender minority members reported feeling
left out. In the future, we would like to examine how the situation of LGBTIQ+ individuals
is changing over time. We aim to report how experiences of discrimination,
support, and contexts of coming out have changed since our first survey. Moreover, based on participants’ answers to this year’s survey about their
personal experience in the LGBTIQ+ community (see selected answers on the
next page), we aim to better understand how LGBTIQ+ individuals with diferent
backgrounds feel integrated into the LGBTIQ+ community and what can be
done to make all people feel welcomed. Finally, your help is vital to drawing
valid conclusions. The Swiss LGBTIQ+ Panel is only possible due to the support
of many LGBTIQ+ and cis-heterosexual individuals. Therefore, we want to thank
you for your contribution and hope that many people will continue to participate
in our panel in the future. 24 “I think as
women we experience a
mixture of homophobia and sexism
where lesbians are often very
sexualized / not taken seriously by
men. SECTION 7:
FUTURE DIRECTIONS However, living […] in Switzerland in
general isn‘t the worst place for queer
people and there is a friendly, warm
and loving community here, even for
people my age.”
– non-binary trans person 25 CONTACT DETAILS
Please cite the report as:
Hässler, T., & Eisner, L. (2020). Swiss LGBTIQ+ Panel - 2020
Summary Report. https://doi.org/10.31234/osf.io/kdrh4
To reach the study coordinators
Dr. Tabea Hässler
University of Zurich
email: tabea.haessler@uzh.ch
Dr. Léïla Eisner
University of Lausanne
email: leila.eisner@unil.ch
Soraya Burger
Design & Illustration,
email: info@sorayaburger.de
https://sorayaburger.de www.swiss-lgbtiq-panel.ch
Facebook: Swiss Lgbtiq+ Panel
Twitter: @lgbtiqpanel
Instagram: @swisslgbtiqpanel
|
https://openalex.org/W4390668489
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https://www.cambridge.org/core/services/aop-cambridge-core/content/view/7746B2E632072F877587AFD0AFD0820C/S2040174423000375a.pdf/div-class-title-involuntary-tobacco-smoke-exposures-from-conception-to-18-years-increase-midlife-cardiometabolic-disease-risk-a-40-year-longitudinal-study-div.pdf
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English
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Involuntary tobacco smoke exposures from conception to 18 years increase midlife cardiometabolic disease risk: a 40-year longitudinal study
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Journal of developmental origins of health and disease
| 2,023
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cc-by
| 9,197
|
Keywords: Maternal smoking; involuntary smoke;
environmental tobacco smoke;
cardiometabolic disease; hypertension;
diabetes; Collaborative Perinatal Project;
DOHaD Corresponding author:
Xiaozhong Wen;
Email: xiaozhongwen@hotmail.com Corresponding author:
Xiaozhong Wen;
Email: xiaozhongwen@hotmail.com Corresponding author:
Xiaozhong Wen; www.cambridge.org/doh www.cambridge.org/doh www.cambridge.org/doh Journal of Developmental
Origins of Health and Disease Journal of Developmental
Origins of Health and Disease Original Article Zhongzheng Niu1
, Lina Mu1, Stephen L. Buka2, Eric B. Loucks2, Meng Wang1,3,4,
Lili Tian5 and Xiaozhong Wen6 Cite this article: Niu Z, Mu L, Buka SL,
Loucks EB, Wang M, Tian L, and Wen X. (2023)
Involuntary tobacco smoke exposures from
conception to 18 years increase midlife
cardiometabolic disease risk: a 40-year
longitudinal study. Journal of Developmental
Origins of Health and Disease 14: 689–698. doi: 10.1017/S2040174423000375 1Department of Epidemiology and Environmental Health, School of Public Health and Health Professions, The State
University of New York at Buffalo, Buffalo, NY, USA; 2Department of Epidemiology, Brown University School of Public
Health, Providence, RI, USA; 3RENEW Institute, The State University of New York at Buffalo, Buffalo, NY, USA;
4Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington,
Seattle, WA, USA; 5Department of Biostatistics, School of Public Health and Health Professions, The State University of
New York at Buffalo, Buffalo, NY, USA and 6Division of Behavioral Medicine, Department of Pediatrics, Jacobs School of
Medicine and Biomedical Sciences, The State University of New York at Buffalo, Buffalo, NY, USA Received: 23 June 2023
Revised: 29 October 2023
Accepted: 29 November 2023
First published online: 8 January 2024 Received: 23 June 2023
Revised: 29 October 2023
Accepted: 29 November 2023
First published online: 8 January 2024 Abstract Few population studies have sufficient follow-up period to examine early-life exposures with later
life diseases. A critical question is whether involuntary exposure to tobacco smoke from
conception to adulthood increases the risk of cardiometabolic diseases (CMD) in midlife. In the
Collaborative Perinatal Project, serum-validated maternal smoking during pregnancy (MSP) was
assessed in the 1960s. At a mean age of 39 years, 1623 offspring were followed-up for the age at
first physician-diagnoses of any CMDs, including diabetes, heart disease, hypertension, or
hyperlipidemia. Detailed information on their exposure to environmental tobacco smoke (ETS)
in childhood and adolescence was collected with a validated questionnaire. Cox regression was
used to examine associations of in utero exposure to MSP and exposure to ETS from birth to 18
years with lifetime incidence of CMD, adjusting for potential confounders. We calculated midlife
cumulative incidences of hyperlipidemia (25.2%), hypertension (14.9%), diabetes (3.9%), and
heart disease (1.5%). Lifetime risk of hypertension increased by the 2nd -trimester exposure to
MSP (adjusted hazard ratio: 1.29, 95% confidence interval: 1.01–1.65), ETS in childhood (1.11,
0.99–1.23) and adolescence (1.22, 1.04–1.44). Lifetime risk of diabetes increased by joint
exposures to MSP and ETS in childhood (1.23, 1.01–1.50) or adolescence (1.47, 1.02–2.10). These
associations were stronger in males than females, in never-daily smokers than lifetime ever
smokers. In conclusion, early-life involuntary exposure to tobacco smoke increases midlife risk of
hypertension and diabetes in midlife. Introduction Heart disease, stroke, and diabetes, together known as cardiometabolic disease (CMD), cause
18 million deaths in the globe each year, more than any other causes.1 Although CMD is mainly
diagnosed in a later life, cumulating evidence has suggested that its disease process initiates in
early life.2,3 This early initiation has also been associated with various early-life environmental
exposures.4 Of particular interests are involuntary exposures to tobacco smoke, including in
utero exposure to maternal smoking during pregnancy (MSP) and environmental tobacco
smoke (ETS) exposure after birth.5 In utero exposure to MSP has been associated with low birth
weight, childhood obesity, and decreased kidney volume, while ETS exposure after birth may
further potentiate the risk of diabetes and hypertension.6–10 However, there is little research
directly linking early-life involuntary exposure to tobacco smoke with later-life CMD, mostly
due to the challenge of long follow-up.5,11 In addition, MSP and ETS are correlated, because
mothers who smoked during pregnancy are likely to continue smoking after delivery and thus
expose the offspring to ETS, generating a “double-hit”.12 However, whether early-life exposure
to the “double-hit” of both MSP and ETS may have additive effect on CMD remains unknown. We a prior selected sex and offspring’s own smoking as potential effect modifiers. Introduction Previous
studies have suggested sex differences in the susceptibility of exposure to maternal stressors
during pregnancy where females were more likely to be protected by estrogens and earlier linear
growth compared to males.13 In addition, cardiovascular responses to maternal stressors during
pregnancy (e.g., undernutrition) and early-life stressors (emotional and physical stimuli) differ
by sex, where males had higher resting vascular resistance while females had higher cardiac
sympathetic activation.14 Moreover, risk of midlife CMD is also higher in males than females.15 Heart disease, stroke, and diabetes, together known as cardiometabolic disease (CMD), cause
18 million deaths in the globe each year, more than any other causes.1 Although CMD is mainly
diagnosed in a later life, cumulating evidence has suggested that its disease process initiates in
early life.2,3 This early initiation has also been associated with various early-life environmental
exposures.4 Of particular interests are involuntary exposures to tobacco smoke, including in
utero exposure to maternal smoking during pregnancy (MSP) and environmental tobacco
smoke (ETS) exposure after birth.5 In utero exposure to MSP has been associated with low birth
weight, childhood obesity, and decreased kidney volume, while ETS exposure after birth may
further potentiate the risk of diabetes and hypertension.6–10 However, there is little research
directly linking early-life involuntary exposure to tobacco smoke with later-life CMD, mostly
due to the challenge of long follow-up.5,11 In addition, MSP and ETS are correlated, because
mothers who smoked during pregnancy are likely to continue smoking after delivery and thus
expose the offspring to ETS, generating a “double-hit”.12 However, whether early-life exposure
to the “double-hit” of both MSP and ETS may have additive effect on CMD remains unknown. © The Author(s), 2024. Published by Cambridge
University Press in association with The
International Society for Developmental
Origins of Health and Disease (DOHaD). This is
an Open Access article, distributed under the
terms of the Creative Commons Attribution
licence (http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted re-use,
distribution and reproduction, provided the
original article is properly cited. © The Author(s), 2024. Published by Cambridge
University Press in association with The
International Society for Developmental
Origins of Health and Disease (DOHaD). This is
an Open Access article, distributed under the
terms of the Creative Commons Attribution
licence (http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted re-use,
distribution and reproduction, provided the
original article is properly cited. We a prior selected sex and offspring’s own smoking as potential effect modifiers. Introduction Previous
studies have suggested sex differences in the susceptibility of exposure to maternal stressors
during pregnancy where females were more likely to be protected by estrogens and earlier linear
growth compared to males.13 In addition, cardiovascular responses to maternal stressors during
pregnancy (e.g., undernutrition) and early-life stressors (emotional and physical stimuli) differ
by sex, where males had higher resting vascular resistance while females had higher cardiac
sympathetic activation.14 Moreover, risk of midlife CMD is also higher in males than females.15 Involuntary tobacco smoke exposures from
conception to 18 years increase midlife
cardiometabolic disease risk: a 40-year
longitudinal study Involuntary tobacco smoke exposures from
conception to 18 years increase midlife
cardiometabolic disease risk: a 40-year
longitudinal study Environmental tobacco smoke exposure Similarly, one’s own smoking has been an established risk factor for
CMD.15,16 Thus, we also stratified the analyses by adult smoking
status to explore potentially different pathways with or without
adult smoking. At the adult follow-up telephone interview, trained staff used a
questionnaire to assess each participant’s exposure to ETS in early life. The questionnaire had similar questions for two periods: birth to 10
years (childhood) and 11–18 years (adolescence). Participants
answered if they lived together with the following potential caretakers:
the biological mother, biological father, other female, and other male
caregivers, in each year from birth to 18 years. If they lived together,
participants further answered ‘Did your (fill in type of caretaker) ever
smoke (cigarettes, pipes, cigars) in your home for a year or longer? If
YES, how old were you then. (circle all ages that apply, from birth to 1,
then each year from 1 to 18).’ Participants also answered ‘About how
many CIGARETTES did your (fill in type of caretaker) smoke per
day, on average? (1 pack = 20 cigarettes). (Selection from None, 0.5, 1,
1.5, 2 or more packs); During these years that your (fill in type of
caretaker) smoked in the home, about how many hours per day were
you exposed to his/her smoke, on average? (Fill in exact hours).’. The
questionnaire also included other questions to help the participant to
recall each smoker’s behavior: if family photos from childhood/
adolescence often showed the smoker’s smoking, and in which
situations the smoker smoked when the participant was around (e.g.,
during meals and while driving). In addition to the four possible
caregivers listed above, a fifth smoker’s smoking behavior and the
relationship with the participant were asked in a similar manner, but
the information was not included in the analyses because very few
participants (<1%) reported a fifth smoker. Because the pack of
smoked cigarettes and the hour of exposure to each smoker were
asked for the whole periods in childhood and adolescence, we
calculated the average ETS exposure quantity (hour-pack) from each
caregiver in each of these two periods using the following formula:
ETS quantity = (number of years smoked in the home * average pack
per day smoked * hours per day exposed)/year in the period. We then
combined the ETS quantity in hour-packs/day across all caregivers as
the final ETS exposure in childhood and adolescence, respectively. Cardiometabolic disease At the adult follow-up, participants filled a questionnaire on their
health status and medical history. Participants were asked if a
doctor had ever told them that they had any of the following
CMDs: high blood pressure (hypertension), high cholesterol
(hypercholesterolemia), stroke, heart attack, angina, congestive
heart failure, or diabetes. Considering the severity of the diseases
and the small number of cases, we combined stroke, heart attack,
angina, coronary heart disease, and congestive heart failure into
one group named “heart disease”. For hypertension and diabetes, a
second question was asked about whether the condition was only
diagnosed during pregnancy. Pregnancy-induced hypertension
and gestational diabetes were not considered as CMD diagnoses in
current analyses. If a CMD was reported, further questions were
asked on the age at the first diagnosis and current medication use. Study population Participants were offspring born to mothers who were enrolled at
two New England sites (Boston, MA and Providence, RI, total
N = 15,721) of the Collaborative Perinatal Project (CPP) from
1959 to 1966. The CPP was a multicenter, national-wide birth
cohort study (N = 55,908) on prenatal and perinatal factors of
children’s health.18 Detailed information on mothers’ health and
behaviors was collected, including their smoking habits in each
trimester of pregnancy. In 2001–2004, a follow-up project, the New
England Family Study (NEFS), randomly selected 4579 adult
offspring born to mothers of the CPP to fill a screening
questionnaire, among whom 3121 mailed back the questionnaire. Eventually, 1674 finished the follow-up questionnaire and 1623
with complete information made to the final analytical sample. Compared to the remaining unselected 11,142 who were also born
to mothers of CPP at New England sites, the final analytical sample
had similar distributions of maternal smoking and other key
covariates, such as maternal age at delivery.17,20 There is a higher
proportion of female offspring in the NEFS than in the original
cohort, possibly due to a lower response rate in males than in
females, as has been seen in other studies.21 All participants
provided informed consent at enrollment. The study was reviewed
by Institutional Review Boards at Harvard School of Public Health,
Brown University, and University at Buffalo. Maternal smoking during pregnancy At each prenatal visit, mothers reported whether they smoked
cigarettes since the last visit and, if so, the average number of
cigarettes smoked per day. For women enrolled in the second
(49.4%) or third (18.6%) trimester, their smoking behaviors before
enrollment were imputed using the information collected from the
earliest prenatal visits. A validation study demonstrated strong
agreement (Kappa = 0.83) between serum cotinine and self-
reported smoking in a subsample of the CPP participants.22 We
converted the number of cigarettes to packs (1 pack = 20
cigarettes) in each trimester as the trimester-specific quantity of
in utero exposure to MSP and then averaged over the three
trimesters as the whole pregnancy exposure in further analyses. https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press 690 Z. Niu et al. Z. Niu et al. Environmental tobacco smoke exposure Retrospective recalling of childhood and adolescence exposure to ETS
has been widely used in life course studies, with high agreement in the
status, duration, and severity of ETS exposure between participants’
recall and the responses from their surrogates (e.g., parents).23 In
addition, the questions that we used have been validated and
recommended to assess ETS exposure in this field.24 g
We analyzed data from a 40-year longitudinal study, the New
England Family Study (NEFS), that collected detailed information
on MSP in the 1960s and then followed the adult offspring in the
2000s for CMD occurrence and a detailed history of ETS exposure
from birth to 18 years.17,18 Although the prevalence of smoking was
much higher in the 1960s (over 50%) compared to the present
(about 10%),19 the biological mechanism underlying the association
of early-life involuntary exposure to smoking with midlife CMD risk
should not change with time substantively, and therefore findings
based on this historical cohort remain relevant, especially to test the
developmental origins of health and disease (DOHaD) hypotheses. We hypothesized that (1) the risk of CMD increased with in utero
exposure to MSP and early-life exposure to ETS, and (2) the
increased risk could be more striking when exposed both in utero
and in early life, among males or active smokers. https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press Results As shown in Table 1, mothers were enrolled at an average age of
25 years (SD 5.8). Most of the mothers were White (86.4%), had
10th–12th-grade education (63.7%), were married (89.9%), and
had normal BMI (18.5–25 Kg/m2, 73.4%). The median daily
cigarette consumption was 3.0 cigarettes in the first trimester and
increased to 3.5 and 3.8 cigarettes in the second and the third
trimester, respectively. The prevalence of low birth weight
(<2500 g) and preterm birth (<37 weeks) were 8.1% and 7.9%,
respectively. The offspring were exposed to ETS for a median of 4.0
(Q1, Q3: 0.3, 10.0) hour-packs/day in childhood and 2.0 (Q1, Q3: 0,
7.5) hour-packs/day in adolescence. Offspring were followed-up at
an average age of 39.1 years (SD: 1.9), 40.7% of them were males,
and 50.2% were daily smokers. Detailed descriptions and histo-
grams of MSP/ETS exposures are shown in Supplemental
Figure S3. As shown in Table 2, risk for hypertension significantly
increased by 1.29 times (adjusted HR [aHR], 95% CI: 1.01–1.65)
for each pack increment of in utero exposure to MSP in the second
trimester. The increased risk was similar in the first (aHR: 1.27,
95% CI: 0.99–1.63) and the third (aHR: 1.23, 95% CI: 0.96–1.28)
trimester, although not significant. In utero exposure to MSP in
each trimester was also associated with increased risk of diabetes,
although not significantly. There is no significant association of in
utero exposure to MSP in each trimester with hypercholester-
olemia or heart disease. The analyses included a set of potential confounders based on
the literature review and the hypothesized causal structure analyses
using directed acyclic graphs (Supplemental Figures S1–S2).26
Different sets of confounders have been identified according to the
exposure period. A general set of confounders was adjusted for all
exposure periods, including study site, maternal age, race,
education, marital status, offspring’s sex and race. For in utero
MSP exposure, pre-pregnancy BMI and parity were further
adjusted. For ETS exposures in childhood or adolescence, maternal
smoking during the whole pregnancy (average pack/day) and birth
weight were further adjusted; and for ETS exposure in adolescence,
both in utero MSP exposure over the whole pregnancy and ETS
exposure in childhood were further adjusted. We deemed offspring
current smoking as a potential mediator on the path from exposure
to outcome, therefore we did not adjust for adult current smoking,
which could underestimate the total effect. Statistical analyses We used frequencies and proportions to describe the distributions of
categorical
variables and Chi-squared tests to compare
the
distribution of categorical variables by maternal smoking status
during pregnancy (yes/no). We used the exact probability test to
compare the lifetime incidence rates of CMD by maternal smoking
status during pregnancy.25 The potentially non-linear relationship
between MSP or ETS exposure and CMD risk was tested by
incorporating a spline term of exposure for the risk of CMD in a
generalized additive model. Given none of the spline terms reached
statistical significance (Supplemental Table S1), we treated MSP or
ETS as a continuous variable. We used Cox proportional hazards
regression models to estimate the hazard ratios (HRs) and 95%
confidence interval (CI) for incident hypertension, hypercholester-
olemia, diabetes, and heart disease by each pack/day increment of in
utero exposure to MSP and by each hour-pack/day increment in ETS
exposure in childhood and adolescence. Survival time was based on
the duration from birth to the age at the first health-professional
diagnosis or age at the adult follow-up if none of these conditions was
diagnosed. For ETS exposure, the reported HRs were scaled for an
interquartile-range increase in hour-pack/day among those with
exposure to facilitate interpretation, i.e., 9.8 hour-pack/day for
childhood ETS exposure and 8.0 hour-pack/day for adolescence
ETS exposure. To avoid reverse causality, CMD cases that were
diagnosed before 18 years were excluded from the analyses (N = 24
for hypercholesterolemia, 17 for hypertension, and 7 for diabetes). No
cases of heart disease occurred before 18 years. We checked the
proportional hazards assumption by testing the statistical significance
of the exposures’ interactions with the survival time. p
p
All analyses were conducted in SAS 9.4 (Cary, NC). Covariates In the CPP, mothers reported their race and perinatal character-
istics, including age at prenatal enrollment (year), education (<9th, https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press 691 Journal of Developmental Origins of Health and Disease 10th–12th, and >12th grades), marital status (married/other), and
parity. We calculated their pre-pregnancy body mass index (BMI,
Kg/m2) using self-reported pre-pregnancy weight and height at
prenatal visits in the original CPP. Birth weight (grams) and
gestational duration (weeks) were obtained from birth records. At
the adult follow-up, the offspring participants reported their age,
race, and current active smoking status (yes/no). We a priori selected two potential effect modifiers, i.e.,
offspring’s sex and own active smoking behavior, giving their
important roles in the CMD risk.27 Specifically, we calculated the
P-value for interaction between sex and in utero, childhood, or
adolescence exposure; and estimated the stratum-specific hazard
ratio by offspring sex (male/female). Similarly, we calculated the
stratum-specific hazard ratio by offspring’s smoking status (ever/
never-daily smoking). For the sensitivity analyses to assess the potential dose-response
relationship, we restricted the analytic sample by excluding those
without MSP/ETS exposure in each period. https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press Results (Continued) study
Characteristics
Overall
N (%)
Overall
1,623
Maternal characteristics
Site
Boston
858 (52.9)
Providence
765 (47.1)
Maternal age at delivery (year), Mean (SD)
25.0 (5.8)
Maternal race/ethnicity
White
1,403 (86.4)
African American
208 (12.8)
Others
12 (0.7)
Maternal education
2–9 grades
392 (24.2)
10–12 grades
1,033 (63.6)
>12 grades
198 (12.2)
Maternal marital status – married
1,459 (89.9)
Maternal pre-pregnancy BMI groups
Undernutrition
125 (7.7)
Normal
1,191 (73.4)
Overweight/obese
307 (18.9)
Parity
0
357 (22.0)
1
381 (23.5)
≥2
885 (54.5)
Maternal smoking during pregnancy (average cig/day), median (Q1, Q3)
Whole pregnancy
3.6 (0.0, 17.4)
1st trimester
3.0 (0.0, 17.0)
2nd trimester
3.5 (0.0, 17.5)
3rd trimester
3.8 (0.0, 18.6)
Offspring characteristics
Birth weight categories
<2500 g
131 (8.1)
2500–4000 g
1,377 (84.8)
>4000 g
115 (7.1)
Gestational duration categories
<37 weeks
128 (7.9)
37–42 weeks
1,354 (83.4)
>42 weeks
141 (8.7)
Offspring sex – male
661 (40.7)
Offspring race – white
1,352 (83.3)
ETS exposure (average hour-pack/day), median (Q1, Q3)
0–18 years
3.5 (0.5, 9.2) significant sex differences in the associations were observed for
other CMDs. There is a significant interaction between adult offspring’s own
smoking status (ever/never-daily smoking) and both in utero
exposures to MSP and ETS exposure with the risk of diabetes
(Table 5). Among never-daily smokers, both in utero exposure to
MSP in the first (aHR: 2.23, 95% CI: 1.18–4.22) or the second
(aHR: 2.35, 95% CI: 1.24–4.47) trimester and ETS exposure in
childhood (aHR: 1.38, 95% CI: 1.12–1.71) or adolescence (aHR:
1.45, 95% CI: 1.01–2.08) were associated with increased risk of
diabetes. The associations with CMDs were all non-significant
among ever-daily smokers. Results Interquartile range increment of one hour-pack/day in
exposure to ETS in childhood (aHR: 1.11, 95% CI: 0.99–1.23)
and adolescence (aHR: 1.22, 95% CI: 1.04–1.44) were both
associated with hypertension, even after adjusting for in utero
exposure to MSP and earlier ETS exposure (for adolescence)
(Table 3). When stratified by in utero exposure to MSP, risk of
hypertension by both childhood and adolescent ETS exposure
remained significant only in those with in utero MSP exposure,
although the P-value for interaction was >0.05. Risk of diabetes
significantly increased with both childhood (aHR: 1.23, 95% CI:
1.01–1.50) and adolescence (aHR: 1.47, 95% CI: 1.02–2.10) ETS
exposure among those with joint in utero exposure to MSP. In sensitivity analyses in a subsample with any exposure to MSP
and ETS (excluding unexposed participants), there was no
meaningful changes in the main effect estimation for the
association
of
MSP
or
ETS
exposure
with
CMD
risk
(Supplemental Table S2). In addition to adjusting for in utero exposure in the analyses of
childhood and adolescent exposure, we also estimated the potential
effect modification of in utero exposure. Specifically, we calculated
the P-value for the product term of in utero exposure during the
whole pregnancy (pack/day) and ETS exposure (hour-pack/day) in
childhood and adolescence. We further calculated the stratum-
specific hazard ratio of childhood and adolescent ETS exposure by
maternal smoking status during pregnancy (yes/no). There is a significant interaction between sex and both in utero
exposure to MSP and childhood ETS exposure with the risk of
hypertension (Table 4). For sex-specific analysis, associations of in
utero exposure to MSP, childhood and adolescent ETS with
hypertension were more striking in males than females. No 692 Z. Niu et al. Table 1. (Continued)
Characteristics
Overall
N (%)
Childhood (0–10 years)
4.0 (0.3, 10.0)
Adolescence (11–18 years)
2.0 (0.0, 7.5)
Age at adult follow-up, Mean (SD)
39.1 (1.9)
Ever daily smoker
815 (50.2) Table 1. Maternal and offspring characteristics in 1623 participants of the NEFS
study Table 1. Maternal and offspring characteristics in 1623 participants of the NEFS
study Table 1. Discussion In a 40-year longitudinal cohort, we found involuntary exposures
to tobacco smoke from conception to adolescence were associated
with increased risk of hypertension in midlife. The risk of
hypertension was even higher, and the risk of diabetes was also
increased with joint exposure to both in utero MSP and childhood/
adolescent ETS. Such associations were overall stronger among
males than females and among never-daily smokers than ever-
daily smokers. We did not find any significant association of early-
life
involuntary
exposure
to
tobacco
smoke
with
midlife
hyperlipidemia or heart disease. Our findings on the association of in utero exposure to MSP
with increased risk of hypertension in adulthood were consistent
with some previous studies,6,28 but not all of them.29,30 A meta-
analysis that combined four previous studies found high study
heterogeneity
(I2 > 95%,
P < 0.0001)
and
a
non-significant
combined association, suggesting critical roles of study design
(prospective vs. retrospective) and exposure assessment accuracy
in elucidating the association of in utero exposure to MSP with
adult hypertension.30 Our study provides important contribution
in this aspect given our prospective collection of MSP, which was
not likely to be biased by offspring’s health outcomes decades later. We also found the point estimates were generally comparable
across the three trimesters of pregnancy, suggesting the whole
pregnancy could be important for the association of maternal
smoking with future risk of hypertension in offspring. Note in our
1960s pregnancy cohort, only a small proportion (<5%) of mothers
changed smoking behavior over pregnancy. Research using recent
cohorts in which more women quit smoking at various gestational
weeks is needed to further examine whether sensitive win-
dows exist. The risk of hypertension also increased with exposure to ETS
postnatally from birth to 18 years. In previous studies with
postnatal ETS exposure,10,31 in utero exposure status was often
unknown. Because
pregnant
women
who
smoked
during
pregnancy were likely to continue smoking after giving birth, https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press Journal of Developmental Origins of Health and Disease 693 Table 2. Discussion Cox regression model for associations of in utero exposure to maternal smoking with adult cardiometabolic disease
Exposure periods
Hyperlipidemia
Hypertension
Diabetes
Heart disease
N case
409
241
49
24
Person year
60,661
61,458
62,931
63,362
Lifetime incidence rate (per 1000 person-year)
6.74
3.92
0.78
0.38
1st-trimester exposure to maternal smoking, per pack/day
Unadjusted HR (95% CI)
1.06 (0.88–1.28)
1.21 (0.95–1.53)
1.31 (0.78–2.20)
0.97 (0.45–2.10)
Adjusted HR (95% CI)
1.03 (0.85–1.24)
1.27 (0.99–1.63)
1.25 (0.74–2.13)
0.92 (0.42–2.05)
2nd-trimester exposure to maternal smoking, per pack/day
Unadjusted model
1.10 (0.92–1.32)
1.22 (0.97–1.55)
1.34 (0.80–2.24)
0.95 (0.44–2.05)
Adjusted model
1.07 (0.88–1.29)
1.29 (1.01–1.65)
1.30 (0.77–2.20)
0.91 (0.41–2.01)
3rd-trimester exposure to maternal smoking, per pack/day
Unadjusted model
1.10 (0.92–1.33)
1.16 (0.91–1.48)
1.13 (0.65–1.97)
0.90 (0.41–1.98)
Adjusted model
1.08 (0.89–1.30)
1.23 (0.96–1.58)
1.10 (0.62–1.94)
0.88 (0.38–2.02)
Whole pregnancy exposure to maternal smoking, per pack/day
Unadjusted model
1.09 (0.90–1.32)
1.20 (0.94–1.53)
1.27 (0.74–2.18)
0.94 (0.43–2.06)
Adjusted model
1.06 (0.87–1.28)
1.27 (0.99–1.64)
1.23 (0.71–2.13)
0.90 (0.40–2.05)
HRs measure the effect of a single pack/day increase in maternal smoking during pregnancy on the hazard of adult cardiometabolic disease. Adjusted HR were controlled for study site, maternal
age, race, education, marital status, pre-pregnancy BMI, parity, offspring’s sex and race. del for associations of in utero exposure to maternal smoking with adult cardiometabolic disease HRs measure the effect of a single pack/day increase in maternal smoking during pregnancy on the hazard of adult cardiometabolic disease. Adjusted HR were controlled for study site, maternal
age, race, education, marital status, pre-pregnancy BMI, parity, offspring’s sex and race. HRs measure the effect of a single pack/day increase in maternal smoking during pregnancy on the hazard of adult cardiometabolic disease. Adjusted HR were controlled for study site, maternal
age, race, education, marital status, pre-pregnancy BMI, parity, offspring’s sex and race. offspring’s own lifetime daily smoking status modified the
association of in utero and postnatal ETS exposure with midlife
risk of hypertension and diabetes. Although adult smoking could
be a mediator on the path from MSP/ETS exposure to midlife
CMD, our findings on the significant associations among never-
daily smokers suggest the underlying mechanisms may be
independent of adult smoking. This is also supported by the
insignificant associations among ever daily smokers. Another
possible explanation for such effect modification is that smokers
were less likely to participate research studies,37 especially those
with CMD. Therefore, interpretation of our findings among
smokers should take extra caution. Discussion children exposed to in utero maternal smoking were also more
likely to be exposed to postnatal ETS. In utero exposure could
potentially confound the association of postnatal ETS exposure
with adult hypertension risk. In the present study, we first adjusted
for in utero exposure to maternal smoking when postnatal ETS was
the main exposure and found no meaningful changes in the point
estimate of the association between ETS and CMD. Further, when
the analyses on postnatal ETS were stratified by in utero exposure,
the increased risk of hypertension mostly remained among
offspring with in utero exposure but not among those without. These findings suggested the importance of accounting for earlier
exposure when assessing the effect of postnatal ETS exposure. g
In addition to hypertension, the risk of diabetes also
increased when the offspring were jointly exposed to in utero
maternal smoking and postnatal ETS. To the best of our
knowledge, we are the first to report such a possible synergetic
effect, although the individual effect has been reported
previously.29,32 Two previous studies reported association of
in utero exposure to maternal smoking with the increased risk of
diabetes (including type 2 and gestational diabetes).29,32
Another two studies showed early-life ETS exposure was
associated with risk factors of diabetes, including higher BMI
and increased risk of obesity.11,33 Other than hypertension and
diabetes, we also found a non-significantly increased risk of
heart disease with ETS exposure in male adolescents, likely
because the number of heart disease was small. https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press Discussion Nevertheless, our
findings were consistent with previous retrospective studies that
reported
associations
of
childhood
or
adolescence
ETS
exposures with increased carotid artery intima-media thick-
ness,34 brachial artery flow-mediated dilatation,32 carotid
atherosclerotic plaque,10 and risk of CHD and stroke.35 A few plausible mechanisms may explain the association of in
utero exposure to maternal smoking with adult hypertension, such
as upregulated maternal blood pressure tone, nicotine and other
harmful substances, placental dysfunction that induce oxidative
stress, inflammation, arterial endothelium injury, and epigenetic
modification.38–40 Similarly, early-life exposure to ETS may
potentiate the vulnerability of children because children have a
higher respiration rate relative to body size than adults, partially
developed detoxification mechanisms, and sensitive cardiovascular
and endocrine system development during the rapid growth
period.5,41 Cumulative evidence has suggested that a small disturbance of
the cardiovascular system in early life could be amplified over time
and subsequently accelerate the development of cardiovascular
disease in later life.42,43 Therefore, protecting children from
involuntary tobacco smoke is an important primordial prevention
strategy of cardiovascular disease. Banning smoking in public
places mitigates ETS in the general population,44 but studies also
reported an increasing trend of ETS exposure in children, possibly
because their parents switched to smoking more at home.45,46
Other intervention strategies such as parental smoking cessation,
smoke-free
home,
and
indoor
air
filtration
warrant
consideration.47,48 p q
We found offspring’s sex could modify the association of in
utero and postnatal ETS exposure with CMD. One possible
explanation for this sex difference could be the protective effects of
hormones and the healthier lifestyle in the female.36 We also found 694 Z. Niu et al. Table 3. Discussion Cox regression model for associations of childhood and adolescence exposure to ETS with adult cardiometabolic disease
Exposure periods
Hyperlipidemia
Hypertension
Diabetes
Heart disease
Total sample (N = 1623)
N case
409
241
49
24
Person year
60,661
61,458
62,931
63,362
Lifetime incidence rate (per 1000 person-year)
6.74
3.92
0.78
0.38
Childhood (0–10 years) exposure to ETSa
Unadjusted HR (95% CI)
1.08 (0.99–1.16)
1.10 (0.99–1.21)
1.23 (1.03–1.47)
1.19 (0.91–1.54)
Adjusted HR (95% CI)
1.07 (0.98–1.17)
1.11 (0.99–1.23)
1.18 (0.97–1.45)
1.16 (0.88–1.52)
Adolescence (11–18 years) exposure to ETSa
Unadjusted HR (95% CI)
1.08 (1.00–1.18)
1.16 (1.05–1.28)
1.31 (1.12–1.53)
1.28 (1.03–1.60)
Adjusted HR (95% CI)
1.05 (0.92–1.20)
1.22 (1.04–1.44)
1.35 (0.98–1.86)
1.44 (0.94–2.20)
Offspring without in utero exposure (N = 660)
N case
158
83
13
9
Person year
24,694
25,043
25,613
25,783
Lifetime incidence rate (per 1000 person-year)
6.40
3.31
0.51
0.35
Childhood (0–10 years) exposure to ETSa
Unadjusted HR (95% CI)
1.18 (0.88–1.58)
1.07 (0.70–1.62)
0.19 (0.02–2.00)
1.84 (0.78–4.34)
Adjusted HR (95% CI)
1.18 (0.88–1.59)
1.16 (0.77–1.74)
0.16 (0.02–1.81)
1.65 (0.69–3.98)
Adolescence (11–18 years) exposure to ETSa
Unadjusted HR (95% CI)
1.11 (0.90–1.36)
1.08 (0.80–1.45)
0.01 (0.00–6.05)
1.63 (1.06–2.52)
Adjusted HR (95% CI)
1.10 (0.88–1.37)
1.17 (0.86–1.58)
0.01 (0.00–5.96)
1.59 (0.96–2.64)
Offspring with in utero exposure (N = 963)
N case
235
141
30
15
Person year
35,967
36,415
37,318
37,579
Lifetime incidence rate (per 1000 person-year)
6.53
3.87
0.80
0.40
Childhood (0–10 years) exposure to ETSa
Unadjusted HR (95% CI)
1.07 (0.98–1.18)
1.10 (0.98–1.23)
1.24 (1.03–1.49)
1.10 (0.79–1.54)
Adjusted HR (95% CI)
1.05 (0.95–1.16)
1.12 (1.00–1.25)
1.23 (1.01–1.50)
1.10 (0.78–1.56)
Adolescence (11–18 years) exposure to ETSa
Unadjusted HR (95% CI)
1.07 (0.97–1.19)
1.17 (1.05–1.30)
1.33 (1.14–1.55)
1.10 (0.77–1.58)
Adjusted HR (95% CI)
1.02 (0.87–1.19)
1.24 (1.03–1.49)
1.47 (1.02–2.10)
1.08 (0.64–1.84)
P-interaction*
Childhood (0–10 years)
0.21
0.91
0.07
0.08
Adolescence (11–18 years)
0.71
0.55
0.12
0.27
Adjusted HR were controlled for study site, maternal age, race, education, marital status, maternal smoking during pregnancy (average pack/day), offspring’s sex, race and birth weight; For ETS
exposure in adolescence, ETS exposure in childhood was additionally adjusted. *P-interaction tests the significance of the product term of ETS with maternal smoking during the whole pregnancy (yes/no). Discussion aHRs measure the effect of an IQR increase in daily exposure to ETS on the hazard of adult cardiometabolic disease (IQR = 9 8 hour-packs/day for ETS exposure in childhood 8 0 in adolescence) This study was strengthened by a comprehensive measure of
involuntary tobacco smoke exposure from conception to 18 years
and the longitudinal follow-up. This study had several potential
limitations. As reported previously, female offspring were over-
represented in the adult follow-up, thus could introduce a potential
collider bias.20 However, controlling sex as a confounder and
stratification analyses by sex could potentially reduce this bias. Information on maternal smoking during pregnancy was self-
reported, although measurement error was unlikely to differ by
midlife CMDs substantially. Information on ETS exposure was
recalled by the adult offspring, thus recall bias might exist given
they were aware of their CMD diagnoses. This is a common
limitation among life course studies. Prior publications indicated
that use of recommended questions and survey methods could https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press Journal of Developmental Origins of Health and Disease 695 Table 4. Discussion ons of early-life exposure to in utero maternal smoking or ETS with adult cardiometabolic disease All models were adjusted for study site, maternal age, race, education, marital status, offspring’s race; for intrauterine exposures, pre-pregnancy BMI, parity were also adjusted; for ETS, maternal
smoking during pregnancy (average pack/day) and birthweight, were adjusted; and for ETS exposure in adolescence, ETS exposure in childhood was additionally adjusted. aHRs measure the effect of a single pack increase in maternal smoking during pregnancy or an IQR increase in daily exposure to ETS on the hazard of adult cardiometabolic disease (IQR = 9.8
hour-packs/day for ETS exposure in childhood, 8.0 in adolescence). that our analyses were hypothesis-driven.51 Thus, we could not rule
out the possibility of false positive results. Although the overall
sample size was fairly large, the number of CMD cases was modest,
given the relatively young age (~40s) of adult offspring. yield reasonably accurate information on ETS exposure years and
the number of exposure pack-years.24 Our questionnaire utilized
these recommended questions and there were additional efforts
such as using family photos to facilitate participants to recall early-
life exposure. CMD status and age at first diagnosis were self-
reported and thus subject to measurement errors. Previous
validation studies suggested an acceptable level of reliability of
self-reported CMD outcomes in the US population.49,50 Residual
confounding might exist given potential measurement errors in
some controlled confounders such as time-varying socioeconomic
status. Shared environment could affect both MSP/EST and
offspring CMD risk. Although we have adjusted key factors that
could reflect some aspects of home and neighborhood environ-
ment (e.g., study site, education, age, marital status, race), residual
confounding by other factors such as physical activity and diet was
not adjusted. We did not adjust for multiple comparisons given yield reasonably accurate information on ETS exposure years and
the number of exposure pack-years.24 Our questionnaire utilized
these recommended questions and there were additional efforts
such as using family photos to facilitate participants to recall early-
life exposure. CMD status and age at first diagnosis were self-
reported and thus subject to measurement errors. Previous
validation studies suggested an acceptable level of reliability of
self-reported CMD outcomes in the US population.49,50 Residual
confounding might exist given potential measurement errors in
some controlled confounders such as time-varying socioeconomic
status. Shared environment could affect both MSP/EST and
offspring CMD risk. Discussion Although we have adjusted key factors that
could reflect some aspects of home and neighborhood environ-
ment (e.g., study site, education, age, marital status, race), residual
confounding by other factors such as physical activity and diet was
not adjusted. We did not adjust for multiple comparisons given https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press Discussion Sex-stratified associations of early-life exposure to in utero maternal smoking or ETS with adult cardiometabolic disease
Exposure periods
Hyperlipidemia
Hypertension
Diabetes
Heart disease
Among female offspring (N = 962)
N case
210
98
26
14
Person year
35,982
36,705
37,185
37,456
Lifetime incidence rate (per 1000 person-year)
5.84
2.67
0.70
0.37
1st-trimester exposure to maternal smokinga
1.17 (0.89–1.54)
1.03 (0.66–1.60)
1.19 (0.56–2.53)
0.61 (0.17–2.23)
2nd-trimester exposure to maternal smokinga
1.22 (0.93–1.59)
1.03 (0.66–1.60)
1.22 (0.58–2.58)
0.61 (0.17–2.20)
3rd-trimester exposure to maternal smokinga
1.18 (0.89–1.56)
0.99 (0.63–1.54)
0.97 (0.43–2.18)
0.68 (0.20–2.38)
Whole pregnancy exposure to maternal smokinga
1.20 (0.90–1.58)
1.01 (0.65–1.59)
1.13 (0.52–2.48)
0.62 (0.17–2.27)
Childhood (0–10 years) exposure to ETSa
1.10 (0.99–1.22)
1.00 (0.84–1.19)
1.10 (0.82–1.46)
1.19 (0.85–1.65)
Adolescence (11–18 years) exposure to ETSa
1.08 (0.92–1.26)
1.17 (0.95–1.44)
1.25 (0.85–1.86)
1.17 (0.68–2.02)
Among male offspring (N = 661)
N case
183
126
17
10
Person year
24,679
24,753
25,746
25,906
Lifetime incidence rate (per 1000 person-year)
7.42
5.09
0.66
0.39
1st-trimester exposure to maternal smokinga
0.93 (0.69–1.26)
1.57 (1.12–2.20)
1.50 (0.67–3.37)
1.59 (0.50–5.11)
2nd-trimester exposure to maternal smokinga
0.95 (0.71–1.29)
1.55 (1.11–2.18)
1.60 (0.71–3.60)
1.55 (0.48–5.01)
3rd-trimester exposure to maternal smokinga
1.00 (0.74–1.36)
1.48 (1.04–2.10)
1.40 (0.60–3.27)
1.29 (0.37–4.45)
Whole pregnancy exposure to maternal smokinga
0.96 (0.71–1.30)
1.56 (1.10–2.20)
1.52 (0.66–3.51)
1.49 (0.44–5.04)
Childhood (0–10 years) exposure to ETSa
0.97 (0.82–1.14)
1.24 (1.08–1.41)
1.30 (0.99–1.72)
1.05 (0.58–1.92)
Adolescence (11–18 years) exposure to ETSa
0.95 (0.78–1.15)
1.27 (1.04–1.54)
1.45 (0.98–2.14)
1.39 (0.79–2.44)
P-interaction
1st-trimester exposure to maternal smokinga
0.22
0.06
0.52
0.15
2nd-trimester exposure to maternal smokinga
0.16
0.09
0.48
0.15
3rd-trimester exposure to maternal smokinga
0.33
0.12
0.42
0.26
Whole pregnancy exposure to maternal smokinga
0.22
0.08
0.47
0.17
Childhood (0–10 years) exposure to ETSa
0.10
0.03
0.36
0.94
Adolescence (11–18 years) exposure to ETSa
0.14
0.28
0.39
0.29
All models were adjusted for study site, maternal age, race, education, marital status, offspring’s race; for intrauterine exposures, pre-pregnancy BMI, parity were also adjusted; for ETS, maternal
smoking during pregnancy (average pack/day) and birthweight, were adjusted; and for ETS exposure in adolescence, ETS exposure in childhood was additionally adjusted. aHRs measure the effect of a single pack increase in maternal smoking during pregnancy or an IQR increase in daily exposure to ETS on the hazard of adult cardiometabolic disease (IQR = 9.8
hour-packs/day for ETS exposure in childhood, 8.0 in adolescence). Conclusion In summary, we found in utero exposure to maternal smoking
during pregnancy as well as exposure to ETS in childhood and
adolescence might increase midlife risk of hypertension. In addition,
joint exposure to both maternal smoking during pregnancy and ETS
in childhood and adolescence might increase the risk of diabetes. Males and never-daily smokers seemed to have even higher risk of
midlife CMD if they were exposed to tobacco smoke in early life. Future studies are needed to replicate our findings. https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press 696 Z. Niu et al. Table 5. Offspring smoking-stratified associations of early-life exposure to in utero maternal smoking or childhood/adolescence ETS with adult cardiometabolic
disease Table 5. Conclusion aHRs measure the effect of a single pack increase in maternal smoking during pregnancy or an IQR increase in daily exposure to ETS on the hazard of adult cardiometabolic disease adolescence, ETS exposure in childhood was additionally adjusted. aHRs measure the effect of a single pack increase in maternal smoking during pregnancy or an IQR increase in daily exposure to ETS on the hazard of adult cardiometabolic disease
(IQR = 9.8 hour-packs/day for ETS exposure in childhood, 8.0 in adolescence). adolescence, ETS exposure in childhood was additionally adjusted. aHRs measure the effect of a single pack increase in maternal smoking during pregnancy or an IQR increase in daily exposure to ETS on the hazard of adult cardiometabolic disease
(IQR = 9.8 hour-packs/day for ETS exposure in childhood, 8.0 in adolescence). should any endorsements be inferred by AHA, NIH, HRSA, HHS or the US
Government. The sponsors had no role in writing the manuscript or the
decision to submit it for publication. Conclusion Offspring smoking-stratified associations of early-life exposure to in utero maternal smoking or childhood/adolescence ETS with adult cardiometabolic
disease
Exposure periods
Hyperlipidemia
Hypertension
Diabetes
Heart disease
Never-daily smoker (N = 808)
N case
171
118
21
12
Person year
30,345
30,696
31,431
31,549
Lifetime incidence rate (per 1000 person-year)
5.64
3.84
0.67
0.38
1st-trimester exposure to maternal smokinga
1.04 (0.77–1.41)
1.37 (0.96–1.95)
2.23 (1.18–4.22)
0.72 (0.19–2.63)
2nd-trimester exposure to maternal smokinga
1.11 (0.82–1.50)
1.35 (0.95–1.92)
2.35 (1.24–4.47)
0.71 (0.19–2.64)
3rd-trimester exposure to maternal smokinga
1.06 (0.77–1.44)
1.41 (0.97–2.04)
1.92 (0.92–4.02)
0.90 (0.25–3.19)
Whole pregnancy exposure to maternal smokinga
1.07 (0.78–1.46)
1.39 (0.96–2.00)
2.27 (1.13–4.56)
0.77 (0.21–2.82)
Childhood (0–10 years) exposure to ETSa
1.00 (0.86–1.15)
1.16 (1.00–1.33)
1.38 (1.12–1.71)
1.26 (0.86–1.86)
Adolescence (11–18 years) exposure to ETSa
1.00 (0.84–1.19)
1.21 (1.01–1.46)
1.45 (1.01–2.08)
1.26 (0.77–2.08)
Ever daily smokers (N = 815)
N case
222
106
22
12
Person year
30,316
30,762
31,500
31,813
Lifetime incidence rate (per 1000 person-year)
7.32
3.45
0.70
0.38
1st-trimester exposure to maternal smokinga
1.09 (0.83–1.44)
1.28 (0.85–1.92)
0.46 (0.16–1.37)
1.29 (0.41–4.10)
2nd-trimester exposure to maternal smokinga
1.09 (0.83–1.43)
1.28 (0.85–1.92)
0.50 (0.18–1.43)
1.25 (0.40–3.94)
3rd-trimester exposure to maternal smokinga
1.14 (0.87–1.50)
1.10 (0.73–1.66)
0.53 (0.19–1.48)
0.94 (0.27–3.19)
Whole pregnancy exposure to maternal smokinga
1.11 (0.84–1.47)
1.22 (0.81–1.85)
0.49 (0.17–1.42)
1.16 (0.35–3.84)
Childhood (0–10 years) exposure to ETSa
1.09 (0.97–1.23)
1.09 (0.93–1.27)
0.74 (0.42–1.30)
1.05 (0.65–1.68)
Adolescence (11–18 years) exposure to ETSa
1.06 (0.90–1.25)
1.24 (0.99–1.55)
0.86 (0.47–1.60)
1.20 (0.65–2.23)
P-interaction
1st-trimester exposure to maternal smokinga
0.76
0.65
0.01
0.34
2nd-trimester exposure to maternal smokinga
0.48
0.53
0.01
0.35
3rd-trimester exposure to maternal smokinga
0.72
0.20
0.02
0.70
Whole pregnancy exposure to maternal smokinga
0.65
0.42
0.01
0.43
Childhood (0–10 years) exposure to ETSa
0.63
0.64
0.03
0.56
Adolescence (11–18 years) exposure to ETSa
0.96
0.64
0.07
0.69
All models were adjusted for study site, maternal age, race, education, marital status, offspring’s sex, race, and age at adult follow-up; for intrauterine exposures, pre-pregnancy BMI, parity, birth
weight category, preterm, or post-term were also adjusted; for ETS, maternal smoking during pregnancy (average pack/day) and birth weight were also adjusted; and for ETS exposure in All models were adjusted for study site, maternal age, race, education, marital status, offspring’s sex, race, and age at adult follow-up; for intrauterine exposures, pre-pregnancy BMI, parity, birth
weight category, preterm, or post-term were also adjusted; for ETS, maternal smoking during pregnancy (average pack/day) and birth weight were also adjusted; and for ETS exposure in
adolescence, ETS exposure in childhood was additionally adjusted. https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press References de Jonge LL, Harris HR, Rich-Edwards JW, et al. Parental smoking in
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found at https://doi.org/10.1017/S2040174423000375 Supplementary material. The supplementary material for this article can be
found at https://doi.org/10.1017/S2040174423000375 Acknowledgments. This work was supported by the American Heart
Association (AHA) pre-doctoral fellowship awarded to Zhongzheng Niu
(Grant number: 20PRE35120245). We acknowledge the participants and other
collaborators from the Early Determinant of Health Study and the New England
Family Study. Dr. Wen’s time effort was supported through the Health
Resources and Services Administration (HRSA) of the US Department of
Health
and
Human
Services
(HHS)
under
R40MC31880
entitled
“Socioeconomic disparities in early origins of childhood obesity and body
mass index trajectories”; Clinical and Translational Science Award Pilot Study
support from National Center for Advancing Translational Sciences, National
Institutes of Health (NIH) grant UL1TR001412; and R21 exploratory research
support from the Eunice Kennedy Shriver National Institute of Child Health
and Human Development, NIH grant R21HD091515 (all awarded to
Xiaozhong Wen). The information, content and/or conclusions are those of
the author and should not be construed as the official position or policy of, nor Author contribution. Zhongzheng Niu: Conceptualization, Methodology,
Software, Formal analysis, Data Curation, Writing – Original Draft, Review &
Edit; Lina Mu: Conceptualization, Methodology, Writing – Review & Edit,
Supervision; Stephen L. Buka: Conceptualization, Methodology, Writing –
Review & Edit; Eric B. Loucks: Conceptualization, Methodology, Writing –
Review & Edit; Meng Wang: Conceptualization, Methodology, Writing – Review
& Edit; Lili Tian: Conceptualization, Methodology, Writing – Review & Edit;
Xiaozhong Wen: Conceptualization, Methodology, Writing – Review & Edit,
Supervision. Financial support. All authors have no financial relationships relevant to this
article to disclose. Competing interests. All authors have no conflicts of interest to disclose. https://doi.org/10.1017/S2040174423000375 Published online by Cambridge University Press 697 Journal of Developmental Origins of Health and Disease Ethical standard. The authors assert that all procedures contributing to this
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Outcome modelling strategies in epidemiology: traditional methods and basic alternatives
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International journal of epidemiology
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Outcome modelling strategies in epidemiology:
traditional methods and basic alternatives
Sander Greenland,1 Rhian Daniel2 and Neil Pearce2,3* 1Department of Epidemiology and Department of Statistics, University of California, Los Angeles, CA,
USA, 2Department of Medical Statistics, London School of Hygiene and Tropical Medicine, London, UK
and 3Centre for Public Health Research, Massey University, Wellington, New Zealand *Corresponding author. Department of Medical Statistics, Faculty of Epidemiology and Population Health, London School
of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK. E-mail: neil.pearce@lshtm.ac.uk *Corresponding author. Department of Medical Statistics, Faculty of Epidemiology and Population Health, London S
of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK. E-mail: neil.pearce@lshtm.ac.uk Accepted 5 February 2016 Accepted 5 February 2016 Education Corner Abstract Controlling for too many potential confounders can lead to or aggravate problems of
data sparsity or multicollinearity, particularly when the number of covariates is large in
relation to the study size. As a result, methods to reduce the number of modelled covari-
ates are often deployed. We review several traditional modelling strategies, including
stepwise regression and the ‘change-in-estimate’ (CIE) approach to deciding which
potential confounders to include in an outcome-regression model for estimating effects
of a targeted exposure. We discuss their shortcomings, and then provide some basic al-
ternatives and refinements that do not require special macros or programming. Throughout, we assume the main goal is to derive the most accurate effect estimates
obtainable from the data and commercial software. Allowing that most users must stay
within standard software packages, this goal can be roughly approximated using basic
methods to assess, and thereby minimize, mean squared error (MSE). • The main goal of a statistical analysis of effects should be the production of the most accurate (valid and precise) ef-
fect estimates obtainable from the data and available software. • The main goal of a statistical analysis of effects should be the production of the most accurate (valid and precise) ef-
fect estimates obtainable from the data and available software. • This goal is quite different from that of variable selection, which is to obtain a model that predicts observed outcomes
well with the minimal number of variables; this prediction goal is only indirectly related to the goal of change-in-esti-
mate approaches, which is to obtain a model that controls most or all confounding with a minimal number of
variables. • This goal is quite different from that of variable selection, which is to obtain a model that predicts observed outcomes
well with the minimal number of variables; this prediction goal is only indirectly related to the goal of change-in-esti-
mate approaches, which is to obtain a model that controls most or all confounding with a minimal number of
variables. • We illustrate some basic alternative modelling strategies that focus more closely on accurate effect estimation as
measured by mean squared error (MSE) and which can be implemented by practitioners with limited programming
and consulting resources. Downloaded from https://academic.oup.com/ije/article-abstract/45/2/565/2572664
by Cardiff University user
on 13 November 2017 V
C The Author 2016. Published by Oxford University Press on behalf of the International Epidemiological Association.
565
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
reuse, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Epidemiology, 2016, 565–575
doi: 10.1093/ije/dyw040
Advance Access Publication Date: 20 April 2016
Education Corner International Journal of Epidemiology, 2016, 565–575
doi: 10.1093/ije/dyw040
Advance Access Publication Date: 20 April 2016
Education Corner stributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
ion in any medium, provided the original work is properly cited. Multicollinearity and mean squared error:
modified CIE approaches One issue that is not explicitly considered or discussed in
most epidemiological strategies is that of multicollinearity
of covariates with exposure, i.e. when exposure is nearly a
linear combination of other variables in the model. This
problem becomes most obvious in propensity-score ana-
lyses when the exposure is so well predicted that there is
little overlap in the exposed and unexposed scores. With
multicollinearity, exposure effect estimates become un-
stable, as reflected by large standard errors. To combine bias and variance considerations when
dealing with genuine confounders, consider estimation of
an exposure effect measure represented by a single coeffi-
cient b, such as a rate difference or log risk ratio. The bias
B in an estimator of b is the difference between the ex-
pected value (mean) l of the estimator and the ‘true’ popu-
lation value b, so B ¼ l – b. The standard error (SE) of the
estimator is just its standard deviation around that mean l;
SE2 is thus the estimator’s variance. The mean squared
error (MSE) of the estimator of b combines these proper-
ties via the equation MSE ¼ B2 þ SE2.24–27 Reducing mul-
ticollinearity by dropping variables can decrease the
variance (SE2) component of the MSE, but may also in-
crease the bias B in the estimator of b if the dropped vari-
ables are indeed necessary to adjust for, given the retained
variables. Thus we seek ways of reducing the SE of the esti-
mator (e.g. by removing a source of multicollinearity)
without seriously increasing its bias B, so that the MSE is
reduced.24,25,27 We also assume that data checking, description and
summarization have been done carefully.2 Finally, we as-
sume that all quantitative variables have been: re-centreed
to ensure that zero is a meaningful reference value present
in the data; and rescaled so that their units are meaningful
differences within the range of the data;3 and that univari-
ate distributions and background (contextual) information
have been used to select categories or an appropriately
flexible form (e.g. splines) for detailed modelling.3 Elsewhere we have discussed the issues involved in sim-
ply adjusting for all measured potential confounders.1 This
approach can be valid when the number of covariates is
not too large in relation to the study size and the included
covariates
are
not
highly
predictive
of
exposure. Scope, aims and assumptions As with our initial review,1 our coverage is not intended for
highly skilled practitioners; rather, we target teachers, stu-
dents and working epidemiologists who would like to do
better with data analysis, but who lack resources such as R
programming skills or a bona fide modelling expert commit-
ted to their project. Throughout, we assume that we are
applying a conventional risk or rate regression model (e.g. logistic, Cox or Poisson regression) to estimate the effects of
an exposure variable X on the distribution of a disease vari-
able Y while controlling for other variables, and that the
outcome is uncommon enough so that distinctions among
risk, rate and odds ratios can be ignored. The other variables
include forced variables, such as age and sex, which we may
always want to control, and may also include unforced vari-
ables about which we are unsure whether to control. Abstract • We illustrate some basic alternative modelling strategies that focus more closely on accurate effect estimation as
measured by mean squared error (MSE) and which can be implemented by practitioners with limited programming
and consulting resources. V
C The Author 2016. Published by Oxford University Press on behalf of the International Epidemiological Association. 565
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
reuse, distribution, and reproduction in any medium, provided the original work is properly cited. V
C The Author 2016. Published by Oxford University Press on behalf of the International Epidemiological Association. V
C The Author 2016. Published by Oxford University Press on behalf of the International Epidemiological Association. 565
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 565 y
y
p
g
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
reuse, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Epidemiology, 2016, Vol. 45, No. 2 566 These include intermediates (variables on the causal path-
way between exposure and diseases) and their descend-
ants4 and any other variable influenced by the exposure or
outcome.5–7 These also include variables that are not part
of minimal sufficient adjustment sets, whose control may
increase bias.4–11 We assume that these variables have
been identified and eliminated e.g. using causal dia-
grams4,6,8 to display contextual theory,12 leaving us with a
set of potential adjustment covariates (often called ‘poten-
tial confounders’), including those variables that we are
reasonably confident would reduce bias if controlled and
our study size were unlimited. We focus only on basic se-
lection from these variables, leaving aside many difficult
issues about model specification and diagnostics,3,13–19
time-varying exposures and confounders, interactions and
mediation.20–23 Introduction We have recently reviewed traditional approaches to con-
founder selection for outcome (risk) and treatment (propen-
sity) models, including significance-testing and ‘change-in-
estimate’ (CIE) approaches.1 We argued that the main goal
of a statistical analysis of effects should be the production of
the most accurate (valid and precise) effect estimates obtain-
able from the data and available software. Allowing that
most users must stay within standard software packages,
this goal can be roughly approximated using basic methods
to minimize estimated mean squared error (MSE). We here
provide an illustrated overview of this approach. Downloaded from https://academic.oup.com/ije/article-abstract/45/2/565/2572664
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on 13 November 2017 Multicollinearity and mean squared error:
modified CIE approaches Nonetheless, controlling too many variables can lead to or
aggravate problems arising from data sparsity or from high
multiple correlation of exposure with the controlled con-
founders (which we term multicollinearity), in which case
one may seek to reduce the number of modelled covariates. Several formal methods seek to minimize MSE in effect
estimation with uncertain confounders, but require special
programming.19,28,29 We will describe a more crude ap-
proach that extends ordinary CIE approaches1 to consider There are of course variables for which control may be
inappropriate based on preliminary causal considerations. International Journal of Epidemiology, 2016, Vol. 45, No. 2 567 estimated MSE minimization using ordinary software out-
puts. Suppose we selectively delete confounders from a full
model and see what happens to the exposure coefficient es-
timate and its standard error. Assuming the full-model esti-
mate is unbiased, we can then estimate the bias Breduced
from the deletion by the difference between the reduced-
model estimate ^breduced and full-model estimate ^bfull. This
step leads to the following equations for estimating the
change in MSE (DMSE) from reducing the model by delet-
ing the confounder: with potassium excluded vs 0.290 with potassium
included). Thus, controlling for potassium appears to
be no worse in accuracy, in addition to having smaller
approximate bias. Next, consider potassium as the main exposure: we
obtain a lower RMSE (0.095) for the potassium coeffi-
cient when including sodium compared with excluding
sodium (0.130); thus controlling for sodium appears to
be preferable. DMSE ¼ MSEreduced– MSEfull
¼ Breduced2 þ SEreduced2– SEfull2
¼ ð^breduced–^bfullÞ2–
SEfull2– SEreduced2
¼
DB
ð
Þ2– D SE2
As with CIE, the exposure-coefficient change resulting
from covariate deletion can be assessed by examining the
estimated change directly, and also with a collapsibility
test, i.e. a test of the hypothesis that the deletion does not
change the exposure coefficients.31–33 One caution to these
approaches is that an accurate assessment of confounding
may require examining changes from moving groups of
variables. Regardless of the number of covariates being
deleted, however, if there is one exposure term X, then a
one degree of freedom chi-squared statistic for this hypoth-
esis is vc
2 ¼ (DB)2/D(SE2).33 Deleting a variable when
DMSE > 0 is equivalent to deleting the variable when vc
2
< 1, which corresponds to P > 0.32 for collapsibility. Downloaded from https://academic.oup.com/ije/article-abstract/45/2/565/2572664
by Cardiff University user
on 13 November 2017 Multicollinearity and mean squared error:
modified CIE approaches Appendix 1 (available as Supplementary data at IJE on-
line) gives further details, describes a generalization of this
test to exposures represented by multiple terms and sug-
gests avenues for improvement. where (DB)2 estimates the squared-bias increase from the
deletion and D(SE2) estimates the variance decrease from
the deletion. A positive difference, i.e. (DB)2 > D(SE2), in-
dicates that the deletion increased the MSE; a negative dif-
ference indicates that the deletion reduced the MSE. We
say ‘indicates’ because, of course, we have only rough esti-
mates of B, SE and MSE, and ^bfull, which will be approxi-
mately unbiased only when the model, the set of measured
confounders and the sample size are all sufficient for ap-
proximate validity. This approach is illustrated in Box 1,
with an example involving two correlated variables, so-
dium and potassium intake. To
illustrate
the
general
algorithms,
denote
by
W1,. . .,WJ those variables (such as age and sex) that we
want forced into all our models along with exposure X be-
cause they are expected to be important confounders or
modifiers of the exposure effect measure, or because they
are known strong risk factors that everyone wants to see in
adjustment; this list could include age splines, sex and eth-
nicity indicators etc. Our chief concern will be with the re-
maining
variables
U1,. . .,UH,
whose
importance
for
adjustment is highly uncertain. Box 1 Associations of sodium and potassium intake at age 4 months with blood pressure (BP) at age 7 years29
Model
Exposure
variables*
Coefficient
estimate
SE for coefficient
Coefficient bias
estimate
Indicates
bias
Indicates large
collinear
Root MSE
estimate*
1
Sodium
0.518
0.290
Referent
0.290
Potassium
0.099
0.095
Referent
0.095
2a
Sodium
0.708
0.225
0.190
Yes
Yes
0.294
2b
Potassium
0.206
0.074
0.107
Yes
Yes
0.130
*All analyses are adjusted for energy intake at 4 or 8 months, age at BP measurement, sex, socioeconomic position (maternal and paternal education), family
social class, maternal age at childbirth, parity, birthweight, gestational age, breastfeeding, smoking during pregnancy, sodium intake at 7 years. ssociations of sodium and potassium intake at age 4 months with blood pressure (BP) at age 7 years29 Table 2. Hypothetical results from rate regressions in which a covariate is or is not a confounder or a source of multicollinearity
Model Model variables
Exposure
coefficient
estimate
Rate ratio
estimate
SE for
coeff. 95% CL
Coefficient
bias estimate*
Indicates
bias? Indicates
strongly
collinear? Root MSE
estimate*
Collapsibility
v2 and P-value33
1
X,W1. . .WJ, U1. . .UH 0.693
2.00
0.24
1.25,3.20
Referent
0.24
Some mutually exclusive alternative possibilities under model 2 (minimal model in which all unforced variables U1. . .UH are dropped)
2a
X,W1. . .WJ
0.693
2.00
0.24
1.25, 3.20 0
No
No
0.24
0, P ¼ 1
2b
X,W1. . .WJ
1.099
3.00
0.20
2.03, 4.44 0.405
Yes
No
0.45
9.34, P ¼ 0.002
2c
X,W1. . .WJ
0.693
2.00
0.14
1.52, 2.63 0
No
Yes
0.14
0, P ¼ 1
2d
X,W1. . .WJ
1.099
3.00
0.14
2.28, 3.95 0.405
Yes
Yes
0.43
4.03, P ¼ 0.04
*Taking model 1 as the referent (‘gold standard’). ults from rate regressions in which a covariate is or is not a confounder or a source of multicollinearity *Taking model 1 as the referent (‘gold standard’). model (0.24), because the minimal model involves a large
increase in confounding and a relatively smaller decrease
in multicollinearity. The task is then to identify a com-
promise model (including some but not all the variables in
question) in which multicollinearity is reduced, but there is
negligible increase in confounding. This could occur, for
example, if the variables most responsible for confounding
were distinct from the variables most responsible for multi-
collinearity. Candidate variables can be assessed by drop-
ping each variable in turn from the full model. Box 1 We consider an example from a study of sodium in-
take in infancy (age 4 months) and blood pressure at 7
years.30 The analysis involved adjusting for a relatively
large number of potential confounders (see Table 1). A
potentially important confounder was potassium intake
at the same age, which was strongly correlated with
sodium intake (r ¼ 0.81). This was reflected in an in-
crease in the standard error for the sodium coefficient
when potassium was also included in the model.30
The authors therefore note that ‘due to high sodium-
potassium correlations, effect of sodium independent
of potassium could not be estimated with reasonable
precision’, and they therefore did not control for potas-
sium in the analyses. Some hypothetical modelling results are shown in
Table 2. We suppose result 1 is from a full model for the dis-
ease rate with exposure, the forced variables and all poten-
tial confounders. Results 2a–d then illustrate the four
mutually exclusive possible outcomes of comparing a full
(maximal)
model
including
the
potential
confounders
(forced and unforced variables) with a minimal model
including only the main exposure and the forced variables. Result 2a suggests little or no confounding or multicolli-
nearity problems, since there is little difference between the
basic and full models; we might therefore prefer the simpli-
city of reporting estimates from the minimal model. In contrast, result 2b suggests there is confounding by the We did RMSE analyses (Table 1), which showed that
although there was an increase in the SE of the so-
dium coefficient when potassium is included in the
model (compare model 1 with model 2a), the reduc-
tion in SE from deleting potassium from the model is
offset by the increase in bias (sodium RMSE ¼ 0.294 by Cardiff Universit
on 13 November 2017 International Journal of Epidemiology, 2016, Vol. 45, No. 2 568 Table 1. Box 1 Of course,
this process may fail to identify any acceptable model re-
duction, in which case the options are to stay with the full
model or else turn to more sophisticated methods such as
penalized estimation or hierarchical (multilevel or mixed)
models to improve accuracy.13,34–37 unforced variables, as seen by contrasting the exposure rate
ratios from model 1 and model 2b, indicating that it is ne-
cessary to control at least some of the unforced variables. Results 2c and 2d involve large multicollinearity, as
indicated by the difference (0.14 compared with 0.24) in
the standard error for the main exposure coefficient. The
more favourable situation is when the factors causing mul-
ticollinearity are very weak confounders, so they can be
deleted from the model without increasing the MSE of the
exposure-effect estimate. This situation is indicated when
deleting these factors leaves the exposure-effect estimate
virtually unchanged, but greatly reduces its standard error
(as in result 2c), suggesting that the minimal model pro-
vides more accurate estimates of the exposure effect (i.e. it
has a smaller MSE). Again, we caution that this smaller
standard error does not account for the preliminary testing
and is thus too small by an unknown amount. Table 1 gives effect estimates without and with adjust-
ment for the Uh, which provides a basis for discussing the
plausibility of residual confounding. For example, if ad-
justment using imperfectly measured Uh removes more
than one-half of the excess rate associated with a particu-
lar main exposure, then it is reasonable to speculate that
adjustment
with better Uh information would have
removed most of the excess rate. Thus it can be worth-
while to present estimates from different degrees of
adjustment. It is more difficult to proceed when multicollinearity
arises from a strong confounder (result 2d), since the in-
crease in precision due to deleting such a confounder may
be more than offset by an increase in confounding.26 We
thus must consider the net impact of reducing the SE of the
exposure-effect estimate while increasing its bias, and we
do so by directly comparing square roots of estimated MSE
(RMSE); we use the square roots to put the results back on
the scale of the effects and biases. Based on the above considerations, Box 2 outlines one
backward-deletion strategy for screening out potential con-
founders. Downloaded from https://academic.oup.com/ije/article-abstract/45/2/565/2572664
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on 13 November 2017 1.
Baseline specification 1. Baseline specification b)
If any candidate in the model has DMSE < 0 (indicating
its deletion reduces MSE), drop the one with the small-
est (most negative) DMSE and go to step (a) if there is
any candidate left in the model. Otherwise (if there is
no candidate Uh left in the model, or none left have
DMSE < 0), stop and use the current model. 1.1 Select the variables that are appropriate to in-
clude, using a causal directed acyclic graph
(DAG)
to
exhibit
theorized
causal
relations
among variables identified a priori as potentially
important for estimating the effects of interest. 1.1 Select the variables that are appropriate to in-
clude, using a causal directed acyclic graph
(DAG)
to
exhibit
theorized
causal
relations
among variables identified a priori as potentially
important for estimating the effects of interest. 1.2 Divide the variables into three classes: (i) the
main exposure X; (ii) forced-in variables (e.g. age, sex) which are always included in the model
(W1. . .WJ); and (iii) the non-forced variables
which will be candidates for deletion (U1. . .UH). We can also derive a parallel forward-selection strategy
starting with the basic model when there are more poten-
tial confounders to consider than can reasonably fit at once
(e.g. when using too many of them results in sparse-data
bias, thus spuriously inflating (DB)2): 1.3 Run a ‘full’ model including all main exposure
terms, forced-in variables and non-forced vari-
ables from 1.3, with no exposure-covariate prod-
ucts. [If full model does not converge or the
results indicate sparse-data bias, change to a for-
ward-selection
strategy,
or
use
hierarchical
(multilevel or mixed) or penalized modelling
methods.] F1)
Fit the basic model, with no exposure-covariate
products. F2)
Enter the following expansion loop, starting with the
basic model as the ‘current model’: a)
For each candidate variable that is not in the current
model, re-run the model expanded with its terms Uh
and compute the DMSE from adding those terms. 2. Variable selection
Enter the following reduction loop, starting with
the full model as the ‘current model’: b)
If any candidate Uh not in the model has DMSE > 0
(indicating its addition reduces MSE), enter the one
with the largest DMSE and go to step (a) if any
candidate remains left out. Box 2 Variable selection based on backward deletion
using estimated MSE reduction Box 2 Variable selection based on backward deletion
using estimated MSE reduction DMSE ¼ ð^breduced–^bcurrentÞ2–
SEcurrent
2– SEreduced
2
DMSE ¼ ð^breduced–^bcurrentÞ2–
SEcurrent
2– SEreduced
2
Box 1 This strategy is intended as a set of options, In result 2d, the estimated RMSE from the minimal
model is substantially larger (0.43) than from the full International Journal of Epidemiology, 2016, Vol. 45, No. 2 569 rather than a prescription; it would be applicable in set-
tings in which a full model can be fit without problems,
there is not an inordinate number of potential confounders
to consider and there is no clear and strong heterogeneity. One implementation is as follows: across the included covariates, even if heterogeneity
is present.38–40 B2)
Enter the following reduction loop, starting with the
full model as the ‘current model’: B2)
Enter the following reduction loop, starting with the
full model as the ‘current model’: a)
For each candidate variable that remains in the current
model, re-run the model without its terms (the Uh that
represent it) and compute the resulting DMSE relative
to the current model from dropping those terms; again, a)
For each candidate variable that remains in the current
model, re-run the model without its terms (the Uh that
represent it) and compute the resulting DMSE relative
to the current model from dropping those terms; again, a) B1)
Fit the full model, with no exposure-covariate prod-
ucts. This model provides an average regression Full model Model 2a in Table 3 shows the results of the full max-
imum likelihood (ML) model, adjusting for all potential
confounders; there is a substantial change in the odds
ratio for milk (from 2.46 to 1.50), but there is also an in-
crease in the SE for the coefficient estimate (from 0.225
to 0.257). Model 2b is the full model fit using the Firth
adjustment for coefficient-estimate
bias.42,43 This is
used as the ‘standard’ to estimate the bias of the other
models, and is combined with the bootstrap SEs to esti-
mate the RMSE. Overall, the milk coefficients from the
full models have a much lower RMSE (0.262, 0.251)
than in the basic model (0.567) because the increase in Box 3 We consider an example from a study of the preva-
lence of atopy in a small town and neighbouring vil-
lages in Poland in 2003.41 In the current analysis, we
estimate the association between ‘no current unpas-
teurized milk consumption’ and current atopy status. It
was plausible that lack of unpasteurized milk con-
sumption could increase the risk of atopy. Because
drinking unpasteurized milk happens mostly in rural
settings, however, there are a number of other expos-
ures which may be related to both unpasteurized milk
consumption and the prevalence of atopy. Model 3a in Table 1 shows the results of a forwards
stepwise logistic regression (using P < 0.20 as the cri-
terion for inclusion) with milk, age group and sex as
forced variables; Town, Firstborn, Current smoker,
Town as a child, Parents farmers, Parents kept poultry
and Parents kept horses were also selected. Model 3b
is again a forwards stepwise logistic regression but
uses P < 0.05 as the criterion for inclusion. Model 3c
and d are the backwards stepwise procedures with
P < 0.20 and P < 0.05, respectively. AIC Main exposure: never drinking unpasteurized milk (1:
never vs 0: regularly/sometimes). Model 4a in Table 1 shows the results of using the Akaike
Information Criterion (AIC)14 where variables were forward
selected to achieve the largest increase in AIC at each
step. Model 4b is from using AIC for backwards deletion. Forced variables: age-group (seven categories), sex. Live in town (yes/no) or village
Live on a farm (yes/no)
Contact (regular/occasional) with cows, pigs, poultry,
sheep or goats, horses
Work (regular/occasional) milking cows, cleaning barns,
collecting eggs
Firstborn (yes/no)
Number of siblings (1, 2, 3þ)
Current smoker (yes/no)
Lived in town (yes/no) or village as a child
Lived on a farm (yes/no) as a child
Parents were farmers (yes/no)
Family kept cows, pigs, poultry, sheep or goats, horses. Model 5a and b was selected in parallel to 4a and b
but using the Bayesian Information Criterion.14 Relative change-in-estimate approach Relative change-in-estimate approach Only town residence (in addition to the forced vari-
ables of age group and sex) produced a substantial
change in the estimate for milk; once this was in the
model, no other variable changed the milk odds ratio
estimate by more than 10%, leading to model 6a. Model 6b is from the analogous backwards procedure
and resulted in the same model. Basic model Model 1 in Table 3 shows the results of the basic ana-
lysis for milk, adjusted for the forced variables (age-
group and sex). Downloaded from https://academic.oup.com/ije/article-abstract/45/2/565/2572664
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on 13 November 2017 1.
Baseline specification Otherwise (if there are no
more unselected candidates, or if none left out have
DMSE > 0), stop and use the current model. 2.1 For each candidate variable that remains in the
current model, re-run the model without its terms
(the Uh that represent it)and compute the result-
ing DMSE relative to the current model from
dropping those terms: 2.1 For each candidate variable that remains in the
current model, re-run the model without its terms
(the Uh that represent it)and compute the result-
ing DMSE relative to the current model from
dropping those terms: Both the above approaches can be viewed as a modifica-
tion of conventional testing strategies in one major way:
the test of the confounder coefficient is replaced by a test
of collapsibility of the exposure coefficient over the con-
founder. This test is easily constructed from ordinary out-
puts (see Appendix 1, available as Supplementary data at
IJE online) and is appropriately sensitive to the confounder
relation to exposure as well as to its relation to disease. It
can also be viewed as a modification of CIE strategy that
allows for random error in the observed change and for the
possible variance reduction from deletion. ð^breduced–^bcurrentÞ2–
SEcurrent
2– SEreduced
2
2.2 If any candidate has DMSE < 0, drop the one
with the smallest (most negative) DMSE and go
to step 4.2 if there are any candidates left in the
model. Otherwise (if there is no candidate Uh left
in the model, or none left have DMSE < 0), stop
and use the current model. 2.2 If any candidate has DMSE < 0, drop the one
with the smallest (most negative) DMSE and go
to step 4.2 if there are any candidates left in the
model. Otherwise (if there is no candidate Uh left
in the model, or none left have DMSE < 0), stop
and use the current model. 3. Assessment
of
heterogeneity
(effect-measure
modification) In Box 3, these approaches are applied to a study of
atopy in Poland, and their results are compared with other
common approaches. 3.1 Assess heterogeneity in a series of supplementary
analyses, focusing on covariates of a priori interest Downloaded from https://academic.oup.com/ije/article-abstract/45/2/565/2572664
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on 13 November 2017 International Journal of Epidemiology, 2016, Vol. 45, No. 2 570 SE from including all potential confounders is small in
comparison with the change in the coefficient estimate. 1.
Baseline specification Traditional stepwise regression SE from including all potential confounders is small in
comparison with the change in the coefficient estimate. Box 3 RMSE Model 7a in Table 1 shows the results of using RMSE
reduction for forward selection in two different ways. Model 7a1 used (at each step) the larger of the two
models being compared as the reference for estimat-
ing RMSE reduction, and is thus analogous to the
other procedures, whereas model 7a2 used the full
model as the reference for each step. Model 7b is the
backwards version of the same procedure. Model 7b1
used (at each step) the larger of the two models being
compared as the reference (for estimating the RMSE),
whereas model 7b2 used the full model as the refer-
ence for each step. Discussion In this example, all of the modelling approaches yielded
reasonably similar findings—the full model (Firth bias-
adjusted) yielded an OR of 1.47, and all of the other
approaches produced ORs in the range of 1.42 to 1.51. The RMSEs were also similar, smaller than that of the
full model and substantially smaller than that for the
basic model. The fact that there exist models with lower
estimated RMSE than the models selected by the RMSE
procedures 7ab (using the larger of the two models as
the reference) illustrates how a procedure that selects
or rejects variables one at a time (forwards or back-
wards) does not always find the model with the overall
optimal value of the criterion being used. Another problem however is that logistic coefficients
are in general not collapsible, in that there will be differ-
ences between the actual (underlying) coefficients with and
without a given covariate if the covariate predicts the out-
come, even if that covariate is not a confounder by virtue
of being independent of exposure.6 This difference will be
negligible unless the outcome is common, in which case it
will be advisable to switch to estimation of collapsible ef-
fect measures (such as risk ratios and differences), e.g. by
regression standardization.13 In this example, Town is the only variable whose inclu-
sion/exclusion in the model has much impact on the
exposure effect estimate. Town is also highly predict-
ive of the outcome. Thus, all methods select it, and
whatever else they happen to select makes very little
difference for any of the measures considered. For the
same reasons, the bootstrap 95% CIs (which take vari-
able selection into account) were in general only
slightly larger than the ‘standard’ 95% CIs. We there-
fore see little apparent advantage of one method over
another in this example. Nonetheless, in a setting with
strong confounding by intercorrelated groups of mul-
tiple confounders, we might find more stark differ-
ences among the results from different methods. Discussion Like more sophisticated but computationally intensive
methods,19 the strategies we describe differ from stepwise
regression and other purely predictive approaches, in that
their goal is to improve accuracy of exposure effect esti-
mates rather than to simply predict outcomes. At the same
time, recognizing that the gap between state-of-the-art
methods and what is done in most publications has only
grown over time, they are intended to fall within the scope
of the limits on software and effort that constrain typical
researchers. Thus, parsimony is replaced by the goal of
minimizing error in effect estimation. A related point is that, as with parsimony, pursuit of
goodness-of-fit may lead to inappropriate decisions about
confounder control; in particular, some variables may not
be included in the model because they do not significantly
improve the fit, even though they are important confound-
ers. ‘Global’ tests of fit are especially inadequate for con-
founder selection13 since there can be many ‘good-fitting’ Downloaded from https://academic.oup.com/ije/article-abstract/45/2/565/2572664
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on 13 November 2017 Penalization Following previous recommendations,37,44 we included
two analyses with weakly informative shrinkage priors
for each coefficient. The first analysis used a log-F(1,1)
(Haldane) prior distribution for each coefficient, which is
equivalent to using an F(1,1) prior distribution for the by Cardiff Universit
on 13 November 2017 International Journal of Epidemiology, 2016, Vol. 45, No. 2 571 odds ratio (antilog) from each coefficient, and assigns
95% probability to the odds ratio falling between 1/648
and 648. The second analysis used a log-F(2,2) (standard
logistic) distribution for each coefficient, which is equiva-
lent to using an F(2,2) prior distribution for the odds
ratio from each coefficient, and assigns 95% probability
to the odds ratio falling between 1/39 and 39. The priors
were imposed by adding two pseudo-observations for
each coefficient to the actual data file, with weights of
1=2 for the F(1,1) prior and weights of 1 for the F(2,2)
prior, then fitting the full model to the augmented data
set by maximum likelihood, with the constant term
replaced by an indicator for ‘actual-data record’ and
weights of 1 for all actual-data records.36,45,46 to the high significance level and therefore liberal inclusion
implicit in using DMSE ¼ 0 as the decision point. Nonetheless, the strategy could be improved by using boot-
strapping or cross-validation to estimate DMSE and set
confidence intervals. A further problem with using CIE strategies for logistic
regression is that it is possible the change in estimate is
largely due to more sparse-data bias (i.e. too few subjects
at crucial combinations of variables) in the full-model esti-
mate ^bfull rather than increased confounding in the
reduced-model estimate ^breduced. For a binary exposure X
and disease Y, this problem becomes noticeable when there
are much fewer than about 4 subjects per confounder coef-
ficient at each exposure-disease combination; for example,
with 7 confounder terms we would want at least 4(7) ¼ 28
subjects in each cell of the two-way XY table for some as-
surance that sparse-data bias in ^bfull is small. One way to
avoid this problem is to switch to penalized estimation;
it is also possible to apply the above reduction algo-
rithms after minimal penalization to reduce sparse-data
bias.44–48 Downloaded from https://academic.oup.com/ije/article-abstract/45/2/565/2572664
by Cardiff University user
on 13 November 2017 Some limitations As with most variable-selection procedures including step-
wise and CIE, confidence intervals obtained by combining
the final point estimate and SE from the above strategy are
not theoretically valid. Simulation studies24,25 so far sug-
gest that this invalidity is negligible in typical settings, due International Journal of Epidemiology, 2016, Vol. 45, No. 2 572 Table 3. Model-adjusted associations of current unpasteurized milk consumption with current atopy status40
Model
Model variables*
Exposure
coefficient
estimate
SE for
coefficient
OR
95% CL
for OR
Estimated
bias and
RMSE
Bootstrap SE†
for coefficient
Bootstrap 95%
CL‡ for OR
1 (basic)
Milk
0.899
0.225
2.46
1.58, 3.82
0.516
0.567
0.236
1.59, 3.97
2a (ML full)
Milk
All other variables#
0.406
0.257
1.50
0.91, 2.48
0.023
0.262
0.261
0.89, 2.46
2b (Firth)42,43
Milk
All other variables#
0.383
0.252
1.47
0.91, 2.40
0.000
0.251
0.251
0.89, 2.37
3a (forwards stepwise,
P < 0.20)
Milk
Town
Firstborn
Current smoker
Town as a child
Parents farmers
Parents kept poultry
Parents kept horses
0.390
0.244
1.48
0.91, 2.38
0.007,
0.261
0.261
0.87, 2.43
3b (forwards stepwise,
P < 0.05)
Milk
Town
Current smoker
Town as a child
Parents kept poultry
0.383
0.243
1.47
0.91, 2.36
<0.001
0.261
0.261
0.88, 2.44
3c (backward stepwise,
P < 0.20)
Milk
Town
Firstborn
Current smoker
Parents farmers
Parents kept poultry
Parents kept horses
0.398
0.244
1.49
0.92, 2.40
0.015
0.261
0.261
0.88, 2.47
3d (backward stepwise,
P < 0.05)
Milk
Town
Current smoker
Parents farmers
Parents kept poultry
Parents kept horses
0.414
0.244
1.51
0.94, 2.44
0.031
0.265
0.263
0.93, 2.61
4a (forwards AIC)
Milk
Town
Horses
Firstborn
Current smoker
Parents kept poultry
0.381
0.243
1.46
0.91, 2.36
0.002
0.260
0.260
0.86, 2.39
4b (backward AIC)
Milk
Town
Horses
Firstborn
Current smoker
Parents kept poultry
Parents kept horses
Parents farmers
0.398
0.244
1.49
0.92, 2.40
0.015
0.262
0.262
0.88, 2.48
5a (forwards BIC)
Milk
Town
Current smoker
Parents kept poultry
0.393
0.243
1.48
0.92, 2.39
0.010
0.264
0.264
0.88, 2.45
(Continued) 3. Model-adjusted associations of current unpasteurized milk consumption with current atopy status40 (Continued) International Journal of Epidemiology, 2016, Vol. 45, No. 2 573 Table 3. Downloaded from https://academic.oup.com/ije/article-abstract/45/2/565/2572664
by Cardiff University user
on 13 November 2017 Some limitations Continued
Model
Model variables*
Exposure
coefficient
estimate
SE for
coefficient
OR
95% CL
for OR
Estimated
bias and
RMSE
Bootstrap SE†
for coefficient
Bootstrap 95%
CL‡ for OR
5b (backward BIC)
Milk
Town
Current smoker
Parents kept poultry
0.393
0.243
1.48
0.92, 2.39
0.010
0.264
0.264
0.87, 2.45
6a (forwards CIE)
Milk
Town
0.400
0.242
1.49
0.93, 2.39
0.017
0.255
0.254
0.93, 2.56
6b (backward CIE)
Milk
Town
0.400
0.242
1.49
0.93, 2.39
0.017
0.255
0.254
0.92, 2.52
7a (forwards RMSE,
larger model as
referent)
Milk
Town
Poultry
Collecting eggs
Number of siblings
Parents kept cows
Parents kept poultry
0.363
0.245
1.44
0.89, 2.32
0.020
0.258
0.257
0.86, 2.35
7b (backward RMSE,
larger model as
referent)
Milk
Town
Poultry
Collecting eggs
Firstborn
0.350
0.243
1.42
0.88, 2.29
0.017
0.257
0.256
0.84, 2.28
8a (forwards RMSE,
full model as
referent)
Milk
Town
0.400
0.242
1.49
0.93, 2.39
0.033
0.263
0.261
0.88, 2.45
8b (backward RMSE,
full model as
referent)
Milk
Town
Parents kept cows
Parents kept poultry
0.407
0.242
1.50
0.94, 2.42
0.024
0.264
0.263
0.89, 2.51
9a penalization by log-
F(1,1) priors§45
Milk
All other variables#
0.396
0.253
1.49
0.90, 2.44
0.013
0.253
0.253
0.90, 2.42
9b penalization by log-
F(2,2) priors¶45
Milk
All other variables#
0.389
0.250
1.47
0.90, 2.41
0.006
0.246
0.246
0.90, 2.36 *All analyses are adjusted for age group and sex. †Based on 4000 bootstrap samples. ‡Bias-corrected and accelerated (BCa) with 4000 resamples.56
#Town, farm, cows, pigs, poultry, sheep/goats, horses, milking cows, cleaning barns, collecting eggs, firstborn, number of siblings, current smoker, lived in
town or village as a child, parents were farmers, family kept cows, family kept pigs, family kept poultry, family kept sheep or goats, family kept horses. §Equivalent to F(1,1) prior for odds ratio; 95% prior limits are 1/648, 648. ¶Equivalent to F(2,2) prior for odds ratio; 95% prior limits are 1/39, 39. But the harsh reality is that even databases of studies with
hundreds of thousands of patients often face severe limits
in crucial categories, such as the number of exposed cases. *All analyses are adjusted for age group and sex. References Second, as we have emphasized, simple selection meth-
ods (such as stepwise, CIE and apparent MSE change) do
not take account of random variability introduced by data-
based model selection. Thus, without cross-validation or
some other adjustment, the standard error of the resulting
effect estimate is not correctly estimated by taking the
standard error computed from the final model.15 With
methods that focus on the effect estimate, however, the
eliminated variables are generally those that have only
weak relations to exposure or disease, the resulting prob-
lem is limited.25 Where such problems are of concern, they
can be mitigated by the use of shrinkage, penalization and
related
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be possible, but some accuracy improvement may be ex-
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sion is of uncertain benefit. models that correspond to very different confounder effects
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then why wouldn’t we? If indeed we have numbers so large
that there is no problem from controlling too many vari-
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grant agreement no 668954. Union’s Seventh Framework Programme (FP7/2007-2013) / ERC
grant agreement no 668954. Some limitations based model reduction will produce biased estimates be-
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on 13 November 2017 Acknowledgements We thank Deborah Lawlor for suggesting the example used in Box
1, and for the analysis summarized in Table 3. We thank Barbara
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on 13 November 2017
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https://openalex.org/W4311869187
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http://jotis.triatmamulya.ac.id/index.php/jotis/article/download/79/68
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Indonesian
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STRATEGI PENGEMBANGAN DESA WISATA AAN KECAMATAN BANJARANGKAN KLUNGKUNG
|
Journal of Tourism and Interdiciplinary Studies
| 2,022
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cc-by
| 5,290
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Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 ISSN 2807-5129 (media online) Abstract A tourism village is a community consisting of residents of a limited area who can interact
directly with each other under management and have awareness and awareness to play a
role together by adapting different individual skills. Tourism villages are formed to realize
the community so that they can act as direct actors in an effort to increase readiness and
awareness in responding to tourism potential or tourist attractions in the area of each
village. This study aims to formulate a development strategy for the tourism village of Aan
as a tourism destination in Klungkung. Aan Village has enormous potential but faces a
number of obstacles in its development. This is indicated by the diversity of tourism
potentials originating from nature, culture and man-made tourist attractions. The research
was conducted through a qualitative descriptive analysis using SWOT analysis to find
strategies for developing the potential of the Aan tourism village and tourism area life cycle
analysis. Data collection techniques through observation, in-depth interviews,
documentation and literature study. Retrieval of informants was done by purposive
sampling. The results of the study show that to maximize the development of the
attractiveness of Aan Village as a tourist destination, it is necessary to develop strategies
and programs such as community empowerment, development of supporting infrastructure,
and collaboration with stakeholders and the private sector in creating sustainable
programs. Desak Made Purnama Dewi¹*, I Made Gede Darma Susila², I Made Suwitra Wirya³
¹²³Fakultas Bisnis dan Pariwisata Universitas Triatma Mulya
Jalan Kubu Gunung Tegal Jaya Dalung
*e-mail: purnama.dewi@triatmamulya.ac.id Desak Made Purnama Dewi¹*, I Made Gede Darma Susila², I Made Suwitra Wirya³
¹²³Fakultas Bisnis dan Pariwisata Universitas Triatma Mulya
Jalan Kubu Gunung Tegal Jaya Dalung
*e-mail: purnama.dewi@triatmamulya.ac.id Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 ISSN 2807-5129 (media online) kepariwisataan, pemerintah mengembangkan desa wisata yang bertujuan untuk
meningkatkan pertumbuhan ekonomi, kesejahteraan rakyat, menghapus kemiskinan,
mengatasi pengangguran, melestarikan alam, lingkungan dan sumber daya, serta
memajukan kebudayaan. Namun, jika dalam pengembangannya itu tidak dipersiapkan dan
dikelola dengan sangat baik maka dapat juga menimbulkan berbagai permasalahan yang
merugikan wisatawan ataupun masyarakat. Maka dari itu untuk menjamin supaya
pariwisata dapat berkembang secara baik dan berkelanjutan serta mendatangkan manfaat
bagi wisatawan maupun masyarakat maka perlu pengkajian secara mendalam terhadap
semua sumber dan daya pendukungnya. Pengembangan desa wisata juga merupakan salah
satu bentuk percepatan pembangunan desa secara terpadu untuk mendorong transformasi
sosial, budaya, dan ekonomi desa. Karena itu, tiap daerah dan desa perlu mencermati
potensi yang dimilikinya untuk diangkat dan dikembangkan agar memberikan nilai tambah
manfaat serta menghasilkan produktivitas yang tinggi untuk meningkatkan kesejahteraan
masyarakat setempat. Desa wisata menjadi salah satu destinasi pariwisata tanah air dengan
potensi yang besar bagi perkembangan sektor pariwisata dan ekonomi kreatif di Indonesia. Desa wisata merupakan pengembangan suatu wilayah desa yang pada dasarnya tidak
merubah apa yang sudah ada akan tetapi lebih cenderung kepada pengembangan potensi
desa yang ada dengan melakukan pemanfaatan kemampuan unsur- unsur yang ada di dalam
desa yang berfungsi sebagai atribut produk wisata dalam skala yang kecil menjadi
rangkaian aktivitas atau kegiatan pariwisata dan mampu menyediakan serta memenuhi
serangkaian kebutuhan perjalanan wisata baik dari aspek daya tarik maupun sebagai
fasilitas pendukung (Muljadi, 2012). Desa Wisata adalah suatu kawasan pedesaan yang
menawarkan keseluruhan dari suasana yang mencerminkan keaslian dari pedesaaan itu
sendiri mulai dari sosial budaya, adat istiadat, keseharian, memiliki arsitektur bangunan
dan struktur tata ruang desa yang khas dan dari kehidupan sosial ekonomi atau kegiatan
perekonomian yang unik dan menarik serta mempunyai potensi untuk dikembangkanya
berbagai komponen kepariwisataan, misalnya atraksi, akomodasi, makanan-minuman,
cinderamata, dan kebutuhan wisata lainnya (Soetarso dan Mulyadin, 2013) y (
y
)
Di Bali jumlah desa wisata yang dimiliki terhitung hingga tahun 2021 sebanyak 294
desa wisata yang tersebar diseluruh pulau dewata. Menurut Kadis Pariwisata Provinsi Bali
Tjok Bagus Pemayun adanya tren tatanan era baru bahwa pariwisata dewasa ini lebih
banyak melihat ke ruang terbuka di desa-desa (CNN, 2022). Klungkung merupakan salah
satu kabupaten terkecil yang ada di Bali. Jumlah desa wisata yang dimiliki hingga tahun
2021 sebanyak 19 desa wisata. Pendahuluan Pengembangan pariwisata merupakan peran penting bagi pembangunan suatu daerah. Dengan adanya kegiatan pariwisata di suatu daerah maka daerah-daerah yang memiliki
potensi pariwisata akan dapat lebih mudah berkembang dan maju. Pengembangan
pariwisata adalah suatu usaha untuk mengembangkan atau memajukan objek wisata agar
lebih baik dan menarik ditinjau dari segi tempat dan segala yang ada didalamnya untuk
dapat menarik minat wisatawan untuk mengunjunginya (Anindita,2015). Perkembangan
pariwisata sangat tergantung pada jumlah kunjungan wisatawan, baik wisatawan domestik
maupun wisatawan mancanegara (Damanik dan Teguh, 2012). Makin banyak kunjungan
semakin besar peluang destinasi untuk mendapatkan devisa dari sektor pariwisata sebagai
sumber pendapatan pajak dan lapangan kerja. Pemerintah memacu pengembangan desa
wisata untuk memeratakan pembangunan melalui manfaat ekonomi pariwisata (Putra dan
Pitana,
2010). Pengembangan
pariwisata
menurut
Swarbrooke
(dalam
http:
//www.scribd.com) merupakan suatu rangkaian upaya untuk mewujudkan keterpaduan
dalam penggunaan berbagai sumber daya pariwisata dan mengintegrasikan segala bentuk
aspek di luar pariwisata yang berkaitan secara langsung maupun tidak langsung akan
kelangsungan pengembangan pariwisata. Pengembangan pariwisata adalah salah satu cara
untuk membuat suatu obyek wisata menjadi menarik dan dapat membuat para pengunjung
tertarik untuk mengunjunginya (Yoeti, 1987). Sejalan dengan tujuan pembangunan 90 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Desa Aan ditetapkan sebagai desa wisata ke-19 berdasarkan
Peraturan Bupati Klungkung Nomor 8 Tahun 2021 tentang Perubahan atas Peraturan
Bupati Nomor 2 tahun 2017 Tentang Penetapan Desa Wisata. Desa Aan Kecamatan
Banjarangkan memiliki beragam potensi alam yang bila dikelola dengan benar maka akan
dapat memberikan manfaat yang luar biasa bagi warganya. Sejumlah potensi tersebut
diantaranya sumber air yang melimpah, perbukitan dengan pemandangannya yang
menakjubkan, hamparan persawahan serta berbagai potensi lainnya. Beberapa potensi
tersebut, ada yang sudah digarap namun ada pula yang belum digarap akibat keterbatasan
pendanaan (Pemdes Aan,2021). Pengembangan pariwisata di Desa Aan sudah
mendapatkan perhatian yang serius dari Pemerintahan Kabupaten Klungkung. Bupati
Suwirtha selaku Bupati Klungkung telah beberapa kali melakukan kunjungan ke Desa Aan
untuk melihat potensi yang dimiliki. Dalam pengembangan sebuah daerah wisata tidaklah
mudah karena terdapat beberapa kendala yang mesti dihadapi seperti kendala geografis
wilayah, kondisi masyarakat, biaya dan kendala lain yang bisa saja hanya ditemui di suatu
daerah baru berkembang. Sebagai desa wisata yang baru dikukuhkan, desa Aan
memerlukan strategi yang tepat dalam pengembangannya, yang mampu memberikan
kontribusi positif tidak hanya bagi masyarakat sekitar namun juga bagi keberlangsungan
ekosistem sekaligus memelihara berbagai potensi alam dan budaya yang hidup di desa Aan. 91 ISSN 2807-5129 (media online) Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Berdasarkan beberapa permasalah tersebut, maka perlu dilakukan penelitian lebih lanjut
untuk dapat merumuskan dan memfomulasikan sebuah pengembangan yang relevan. Berdasarkan beberapa permasalah tersebut, maka perlu dilakukan penelitian lebih lanjut
untuk dapat merumuskan dan memfomulasikan sebuah pengembangan yang relevan. Landasan Teori Untuk menjawab pengembangan pariwisata yang terdapat di desa Aan, maka landasan
teori yang digunakan dalam penelitian ini yaitu analisis siklus hidup destinasi wisata
(Tourism area life cycle) dan analisis SWOT. Tourism area life cycle merupakan suatu
konsep yang di terapkan atau digunakan dalam pengembangan suatu daerah wisata. Kerangka ini merupakan sebuah alur natural akan tetapi baru diteliti oleh Butler (1980),
sebagai sarana untuk menjelaskan seberapa banyak sebuah destinasi wisata bergerak
melalui beberapa siklus. Siklus dimulai dengan tahap penemuan (exploration) yang
menunjukkan potensi pariwisata berada pada tahapan identifikasi dan menunjukkan
destinasi memiliki potensi untuk dikembangkan menjadi daya tarik atau destinasi wisata. Tahapan selanjutnya yaitu pelibatan (involvement) masyarakat lokal mengambil inisiatif
dengan menyediakan berbagai pelayanan jasa untuk para wisatawan yang mulai
menunjukkan tanda-tanda peningkatan dalam beberapa periode. Pada tahap pengembangan
(development) telah terjadi kunjungan wisatawan dalam jumlah besar dan pemerintah
sudah berani mengundang investor nasional atau internasional untuk menanamkan modal
di kawasan wisata yang akan dikembangkan. Tahap konsolidasi (consolidation) sektor
pariwisata menunjukkan dominasi dalam struktur ekonomi pada suatu kawasan dan ada
kecenderungan dominasi jaringan international semakin kuat memegang peranannya pada
kawasan wisata atau destinasi tersebut. Pada tahap stagnasi (stagnation) angka kunjungan
tertinggi telah tercapai dan beberapa periode menunjukkan angka yang cenderung stagnan. Setelah stagnasi ada dua kemungkinan yang dapat terjadi terhadap kelangsungan destinasi
wisata yaitu tahap decline atau tahap rejuvenation. Terjadinya penurunan atau terjadinya peremajaan tergantung dari kebijakan yang
diambil oleh pengelola destinasi wisata bersangkutan. Analisis SWOT digunakan untuk
menghasilkan strategi pengelolaan dan program pengembangan daya tarik wisata dengan
mengidentifikasi faktor kekuatan, kelemahan, peluang dan ancaman yang menjadi
landasan untuk menetapkan strategi pengelolaan dan program pengembangan daya tarik
wisata di desa wisata Aan. Analisis SWOT adalah identifikasi berbagai faktor secara
sistematis untuk merumuskan strategi sebuah destinasi wisata. Model perencanaan yang
digunakan untuk mengevaluasi kekuatan (strengths), kelemahan (weaknesses), peluang
(opportunities), dan ancaman (threats). Keempat faktor itulah yang membentuk akronim
SWOT (strengths, weaknesses, opportunities, threats). SWOT memuat kondisi lingkungan
internal dan eksternal. Lingkungan Internal terdiri atas Strengths dan Weaknesses
sedangkan lingkungan eksternal mencakup Opportunities dan Threats yang dihadapi dunia
bisnis (Rangkuti, 2013). Analisis SWOT (SWOT analysis) yakni mencakup upaya-upaya
untuk mengenali kekuatan, kelemahan, peluang, dan ancaman yang menentukan
pengembangan sebuah daya tarik wisata. Metode Penelitian Rancangan penelitian menggunakan pendekatan kualitatif, yaitu penelitian yang
bertujuan mengungkapkan gejala secara holistik dan konstektual melalui pengumpulan
data dari latar alami dengan memanfaatkan diri peneliti sebagai instrument kunci. Proses
dan makna berdasarkan perfektif subyek lebih ditonjolkan dalam penelitian ini. Teknik
pengumpulan data melalui observasi, daftar pertanyaan, wawancara mendalam (in-depth
interview), studi kepustakaan, dan studi dokumentasi. Pengambilan sampel menggunakan
teknik purposive sampling, yaitu teknik pengambilan sumber data dengan pertimbangan
tertentu. Analisis data yang digunakan adalah teknik analisis data kualitatif, dengan 92 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 ISSN 2807-5129 (media online) Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 langkah-langkah; pengumpulan data, reduksi data, penyajian data dan penarikan
kesimpulan. Hasil dan Pembahasan 1. Potensi Pengembangan Desa Wisata Aan
Berdasarkan sejarahnya, kata Aan berasal dari kata Ea, yaitu nama dari sebuah
pohon karena wilayah tersebut banyak terdapat pohon Ea. Diawali dengan kedatangan
Jero Gede Pasek beserta pengiringnya yang menemukan wilayah tersebut dan
merambas pohon Ea untuk dijadikan tempat tinggal. Setelah membangun perumahan
penduduk, lalu I Gede Pasek merencanakan pembangunan desa. Perangkat desa mulai
diadakan seperti banjar patus, tempat-tempat ibadah sangat diutamakan seperti
Kahyangan desa, Penataran Gunung Kawi dan lain-lain. Wilayah Desanya terbagi
menjadi 3 banjar: 1) Banjar Kelodan (Banjar Peken sekarang) 2) Banjar Tengah (Banjar
Pasek sekarang) 3) Banjar Kaleran (Banjar Carik Dalem/Swelegiri sekarang). Demikian pula banjar adatnya dibagi menjadi 2 bagian yaitu Adat Banjar Patus dan
Adat Banjar Pura. Setelah desa beserta perlengkapanya selesai dibangun maka
pembangunan selanjutnya diarahkan kepada sektor pertanian. Bendungan/empelan
segera dibangun, sehngga pengairan menjadi teratur dan lancar. Pondok abasan yang
dulunya menjadi tempat tinggal, dirubah dijadikan persawahan yang sangat subur. Semenjak itulah seluruh areal dari hutan Ea diubah namanya menjadi Desa Aan
(Pemdes Aan, 2022). Gambar 1. Kantor Pemerintahan Desa Aan Gambar 1. Kantor Pemerintahan Desa Aan Secara administratif desa Aan terbagi atas 4 (empat) Dusun meliputi Dusun
Sengkiding, Dusun Swelagiri, Dusun Pasek dan Dusun Peken. Luas wilayah Desa
Aan adalah 398 Ha, yang terdiri dari lahan sawah seluas 200,960 Ha, Tanah Tegalan
seluas 162,280 Ha, sedangkan tanah Pekarangan mencapai 25,330 Ha. Desa Aan
memiliki 1,250 Ha tanah Kuburan serta tanah lainnya 9,630 Ha. Desa Aan adalah satu
dari tigabelas desa di wilayah Kecamatan Banjarangkan, dengan jarak + 8 km arah kota
dari Semarapura. Desa Aan memiliki berbatasan dengan Desa Timuhun di sebelah
utara, Tukad Jinah di sebelah timur, di sebelah selatan adalah Desa Tihingan dan Desa
Getakan di sebelah barat. Berdasarkan letak, daerah, dan klasifikasi desa maka Desa
Aan merupakan wilayah bukan pantai, katagori daerah pedesaan, dan klasifikasi
swasembada (Pemdes Aan, 2022). Desa Aan memiliki beragam potensi alam, diantaranya sumber air yang
melimpah, perbukitan dengan pemandangannya yang menakjubkan, hamparan
persawahan serta berbagai potensi lainnya. Selain itu ada sejumlah daerah tujuan
wisata dan sarana penunjang seperti; Museum Sukanta Wahyu, Air Terjun Celek Celek,
Air Terjun Gebyug, Aan Secret Waterfall, pelukatan/pesiraman, Monumen Batu 93 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
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Vol. 2 No. 2 Desember 2022, 90-100 Kembar, jalur tracking di tengah persawahan, Petapan candi selamat datang, pasar
UMKM, program wisata menginap di rumah warga, serta potensi wisata lainnya. Daya
tarik wisata yang dimiliki untuk dikembangkan di desa Aan seperti air terjun,
persawahan, hingga perbukitan dengan view point-nya. Wisatawan yang berkunjung
dapat melihat keistimewaan masing-masing air terjunnya seperti air terjun Celek-celek,
air terjun Gebyug dan Aan Secret Waterfall. Dari ketiga air terjun yang ada, untuk saat
ini Aan Secret Waterfall telah dikelola dan dikenal dikalangan wisatawan. Jalur menuju
Aan Secret Waterfall melewati hamparan sawah yang asri, sehingga tidak
membosankan ketika menuruni tanjakan menuju air terjun. Selain itu pengunjung juga
disediakan area peristirahatan sejenak berupa kedai tradisional yang menyajikan
makanan tradional khas desa Aan dengan harga yang sangat terjangkau. Menurut Perbekel Desa Aan I Wayan Wira Adnyana, keberadaan Air Terjun
Celek-celek pada zaman dahulu dijadikan sebagai jalur penyeberangan dari Desa Aan
menuju Desa Nyalian dengan cara menaiki tebing air terjun. Ketika memanjat tebing
setinggi 30 meter itu, warga memasukkan tangan pada celah-celah tebing yang dibuat
sedemikian rupa agar bisa naik. Saat memasukkan tangan mencari celah diantara tebing
dalam bahasa Bali istilahnya nyelek tebing, sehingga dimasyarakat terkenal dengan
istilah air terjun Celek-celek. Pengembangan Air Terjun Celek-celek dimulai sejak
akhir 2021, yang terintegrasi dengan Museum Sukanta Wahyu, di Banjar Sengkiding. Untuk mencapai lokasi air terjun dari Museum Sukanta Wahyu hanya berjarak 200
meter, dan menuruni sekitar 50 anak tangga, selain itu juga bisa dijangkau dari akses
jalan sebelah barat Balai Banjar Sengkiding. Para wisatawan bisa menikmati air terjun dan melanjutkan ke Petapan park. Wisatawan dapat bersantai dan menikmati pemandangan hamparan persawahan nan
hijau yang berpadu sempurna dengan barisan perbukitan. Di Petapan Park, wisatawan
juga dapat menikmati berbagai kudapan khas daerah setempat dan melakukan berbagai
aktivitas menarik seperti; bermain river tubing, memancing ikan di air deras,
menikmati pemandangan persawahan, bermain sling serta berbagai jenis aktivitas
permainan lainnya. Searah dengan jalur menuju Petapan Park terdapat pula UMKM
lebah madu trigona atau madu Kelle. Kelompok Sari Amertha merupakan UMKM
lebah trigona (lebah hitam) yang memproduksi produk-produk sabun berbahan madu
dan propolisnya menjadi produk bernilai tinggi. Gambar 2. Petapan Park dan UMKM Madu Kelle di Desa Aan Gambar 2. Petapan Park dan UMKM Madu Kelle di Desa Aan Tujuan wisata selanjutnya yang ada di desa Aan yaitu Puncak Pengukur-ukuran. Di puncak bukit ini wisatawan dapat melihat wilayah desa Aan dengan bebas, mulai
dari sunrise hingga sunset terlihat sangat apik dari Puncak Pengukur-ukuran. Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 94 ISSN 2807-5129 (media online) Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Pemandangan gunung, sawah hingga laut menjadi perpaduan sempurna untuk
dijadikan konten media sosial. j
Dari segi aksesibilitas, desa Aan memiliki infastruktur jalan yang sangat
memadai dan dapat dilewati dengan berbagai macam kendaraan bermotor seperti
kendaraan roda empat jenis minibus, mobil atau kendaraan roda dua. Fasilitas
pendukung lainnya, terdapat aneka warung makanan sehingga wisatawan lebih mudah
mencari kebutuhan yang diperlukan ketika berada di desa Aan, juga fasilitas telepon
dan jaringan internet yang terjangkau. Posisi Desa Wisata Aan
Menurut Teori Tourism Area Life Cycle
Tahapan
Ciri-ciri
Hasil
Observasi
Sesuai
Tidak
Sesuai
1. Tahap Ekplorasi
(Exploration)
a) Suatu tempat sebagai potensi wisata baru
ditemukan
b) Lokasinya sulit dicapai namun diminati oleh
sebagian kecil wisatawan
c) Wisatawan tertarik dengan daerah yang
belum tercemar
2. Tahap
Keterlibatan
(Involvement)
a) Adanya keterlibatan masyarakat lokal
b) Suatu daerah menjadi destinasi wisata yang
ditandai dengan adanya promosi
c) Adanya inisiatif masyarakat lokal dalam
membangun daerahnya
d) Adanya kunjungan wisatawan meskipun
masih minim
3. Tahap
Pengembangan
(Development)
a) Adanya investasi dari luar
b) Daerah semakin terbuka secara fisik
c) Fasilitas lokal mulai tersisih dan digantikan
fasilitas dengan standar Internasional
d) Mulai
muncul
atraksi
buatan
untuk
menambah atraksi wisata yang asli
4. Tahap
Konsolidasi
(Consolidation)
a) Daerah dan dominasi perekonomian dipegang
oleh jaringan Internasional
b) Jumlah kunjungan wisatawan masih naik
c) Fasilitas lama mulai ditinggalkan
5. Tahap Stagnasi
(Stagnation)
a) Kapasitas berbagai faktor melebihi daya
dukung yang dimiliki sehingga menimbulkan
dampak tersendiri
b) Kalangan industri sudah mulai bekerja berat
untuk memenuhi kapasitas
c) Atraksi buatan sudah mendominasi
d) Citra awal mulai meredup dan destinasi sudah
tidak popular lagi
6. Tahap
Penurunan
(Decline)
a) Wisatawan mulai beralih ke destinasi baru
b) Banyak
fasilitas
pariwisata
yang
dialihfungsikan
untuk
kegiatan
non-
Posisi Desa Wisata Aan 95 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100
SSN 2807-5129 (media online)
pariwisata,
sehingga
destinasi
menjadi
semakin tidak menarik bagi wisatawan
c) Partisipasi lokal mulai meningkat lagi
d) Destinasi wisata bisa berkembang menjadi
destinasi
kelas
rendah
atau
secara
keseluruhan kehilangan jati diri
7. Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Tahap
Peremajaan
(Rejuvenation)
a) Perubahan bisa terjadi setelah adanya peran
serta berbagai pihak, menuju perbaikan atau
peremajaan
b) Adanya inovasi dalam pengembangan produk
baru dengan memanfaatkan sumber daya
yang sebelumnya tidak dimanfaatkan
Sumber: Hasil Observasi Penulis, 2022. Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 ISSN 2807-5129 (media online) Berdasarkan data-data yang diperoleh dilapangan, hasil penelitian menyimpulkan
bahwa desa wisata Aan saat ini masih dalam tahapan involvement atau pelibatan
masyarakat. Pada tahap involvement memiliki ciri-ciri; kunjungan wisatawan masih
sedikit, komunikasi antara masyarakat dan wisatawan masih tinggi, fasilitas mulai
dibangun, masyarakat bersama dengan pemangku kebijakan mulai menyediakan
fasilitas pendukung, pola hidup sosial budaya masyarakat mulai berubah merespon
terhadap perubahan ekonomi yang terjadi, promosi mulai dilakukan. Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Adapun faktor eksternal yang menghambat (ancaman) pengembangan desa
wisata Aan sebagai daya tarik yaitu; adanya persaingan yang sama dengan beberapa
desa wisata lainnya yang ada di Klungkung seperti Nusa Penida yang memang telah
dikenal lebih dahulu, daya tarik yang dimiliki seperti wisata alam, pengunungan, air
terjun juga dimiliki oleh desa wisata lainnya, alih fungsi lahan yang terjadi seiring
perkembangan sehingga wisata alam yang dimiliki mengalami penurunan, terjadinya
pergeseran budaya lokal akibat perkembangan pariwisata, komersialisasi budaya lokal
yang berorientasi pada keuntungan. Tabel 1. Analisis SWOT Pengembangan Desa Wisata Aan
Faktor Internal
Faktor Eksternal
Strength/Kekuatan (S)
1. Tiga (3) obyek air terjun
2. Petapan Park
3. Museum Sukanta Wahyu
4. Monumen Batu Kembar
5. UMKM budidaya madu
Trigona
6. Jalur tracking untuk
menikmati suasana alam
7. Pemandangan alam masih
alami
8. Warga masyarakat yang
ramah
9. Akses yang mudah dicapai
10. Akses jariangan internet dan
telepon terjangkau
Weakness/ Kelemahan (W)
1. Masyarakat belum
kompeten dibidang
pariwisata
2. Kurangnya sarana dan
prasarana pendukung di
obyek wisata seperti toilet
3. Jalur tracking menuju ke
obyek wisata pada musim
hujan kurang mendukung
dan sedikit curam
4. Belum adanya gapura
sebagai pertanda memasuk
desa wisata Tabel 1. Analisis SWOT Pengembangan Desa Wisata Aan Faktor Internal 1. Masyarakat belum
kompeten dibidang
pariwisata 2. Kurangnya sarana dan
prasarana pendukung di
obyek wisata seperti toilet 5. UMKM budidaya madu
Trigona 3. Jalur tracking menuju ke
obyek wisata pada musim
hujan kurang mendukung
dan sedikit curam 7. Pemandangan alam masih
alami 8. Warga masyarakat yang
ramah 4. Belum adanya gapura
sebagai pertanda memasuki
desa wisata Faktor Eksternal 9. Akses yang mudah dicapai 10. Akses jariangan internet dan
telepon terjangkau Strategi S-T (Strength Threat)
Memanfaatkan kekuatan untuk
menghadapi ancaman
1. Meningkatkan kualitas
pengelolaan , pelayanan agar Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100
ISSN 2807-5129 (media online) Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100
ISSN 2807-5129 (media online) Strategi W-T (Weakness
Threat)
Mengatasi kelemahan dalam
rangka memanfaatkan
peluang . Faktor Internal dan Eksternal yang Mempengaruhi Pengembangan Desa Wisata
Aan 2. Faktor Internal dan Eksternal yang Mempengaruhi Pengembangan Desa Wisata
Aan
Faktor Internal yang Mempengaruhi Pengembangan Desa Wisata Aan
Analisis faktor internal bertujuan untuk mengidentifikasi potensi-potensi wisata,
aset yang dimiliki atau segala sesuatu yang terdapat di daerah tujuan wisata
(Yoeti,2000). Berdasarkan hasil pengumpulkan data yang dilakukan, faktor internal
yang dimiliki mendukung (kekuatan ) pengembangan desa wisata Aan yakni Museum
Sukanta Wahyu, Air Terjun Celek Celek, Air Terjun Gebyug, Aan Secret Waterfall,
pelukatan/pesiraman, Monumen Batu Kembar, jalur tracking di tengah persawahan,
Petapan Park, UMKM lebah madu hitam Trigona (kelle), program wisata menginap di
rumah warga, serta potensi wisata lainnya. Selain potensi wisata tersebut, akses menuju
obyek wisata tersebut juga mudah dijangkau. Sesungguhnya, faktor internal bersifat
menghambat pengembangan desa wisata Aan antara lain; kurangnya sumber daya
manusia yang berkompeten di bidang pariwisata, kurangnya sarana dan prasarana
pendukung pada masing-masing obyek wisata, seperti toilet (WC), jalur tracking
menuju air terjun sedikit curam dan apabila musim hujan susah untuk dijangkau, dan
belum adanya gapura Selamat Datang / Desa Wisata yang dibuat pada perbatasan desa. Faktor Eksternal yang Mempengaruhi Pengembangan Desa Wisata Aan. Faktor Eksternal yang Mempengaruhi Pengembangan Desa Wisata Aan. Analisis faktor eksternal mencakup peluang dan ancaman yang berasal dari luar
lingkungan daya tarik wisata. Analisis faktor eksternal bertujuan untuk
mengidentifikasi peluang yang mendorong pengembangan daya tarik wisata serta
ancaman yang mempengaruhi pengembangan daya tarik wisata. Berdasarkan hasil
pengumpulan data, faktor eksternal (peluang) yang mendukung pengembangan desa
wisata Aan antara lain; adanya kecenderungan desa wisata Aan mengarah kepada
wisata alam, peran aktif perangkat desa serta pemerintahan kabupaten Klungkung
dalam mendukung pengembangan pariwisata di desa Aan melalui bantuan dan
kebijakan, adanya kemajuan teknologi yang mempermudah dalam promosi pariwisata,
serta citra Bali sebagai daerah tujuan wisata favorit di Indonesia bahkan dunia. 96 ISSN 2807-5129 (media online) Opportunities/ Peluang Strategi
W-O
(Weakness
Opportunities) Strategi
S-O
(Strength
Opportunities) Strategi
S-O
(Strength
Opportunities) 1. Kecenderungan
mengarah pada
wisata alam Strategi
yang
menggunakan
kekuatan
dan
memanfaatkan
peluang Strategi yang meminimalkan
kelemahan
untuk memanfaatkan peluang 2. Peran serta
masyarakat untuk
ikut melestarikan
lingkungan 1. Meningkatkan pengelolaan
DTW dan melengkapi
fasilitas pariwisata 1. Meningkatkan pelatihan
SDM yang profesional 2. Melengkapi sarana dan
prasarana baik jalur
menuju DTW, toilet
maupun gapura batas desa
melalui kebijakan dana
desa 2. Meningkatkan kemampuan
manajemen Pokdarwis dalam
mengelola bantuan
pemerintah g
g
3. Peranan masyarakat
melalui karang
taruna p
3. Memanfaatkan letak strategis
untuk sarana promosi desa
wisata 4. Peranan
pemerintahan desa
dalam membangun
pariwisata Meningkatkan anggaran,
koordinasi antara
stakeholder, dukungan
pemerintah untuk
membangun fasilitas
penunjang Strategi S-T (Strength Threat)
Memanfaatkan kekuatan untuk
menghadapi ancaman
1. Meningkatkan kualitas
pengelolaan , pelayanan agar Strategi W-T (Weakness
Threat)
Mengatasi kelemahan dalam
rangka memanfaatkan
peluang 97 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 ISSN 2807-5129 (media online) akibat
perkembangan
pariwisata
3. Pergeseran budaya
lokal akibat
perkembangan
pariwisata
4. Komersialisasi
budaya lokal
menjadi budaya
yang berorientasi
pada uang akibat
perkembangan
pariwisata
3. Pergeseran budaya
lokal akibat
perkembangan
pariwisata
4. Komersialisasi
budaya lokal
menjadi budaya
yang berorientasi
pada uang
dapat bersaing dengan desa
wisata lainnya
2. Memanfaatkan konsep CBT
sebagai pedoman mengatasi
pergeseran dan komersialisasi
budaya
3. Memperkuat lembaga adat
untuk meningkatkan kesadaran
masyarakat dalam mengelola
desa wisata berbasis
masyarakat
1. Meningkatkan kesadaran
masyarakat mengantisipasi
persaingan desa wisata
2. Meningkatkan pelatihan dan
dana untuk mengelola desa
wisata
3. Meningkatkan kemampuan
manajemen pengurus dan
Pokdarwis, dukungan
pemerintah dan stakeholder
sehingga pariwisata berbasis
msyarakat masih tetap ada dapat bersaing dengan desa
wisata lainnya dapat bersaing dengan desa
wisata lainnya 1. Meningkatkan kesadaran
masyarakat mengantisipasi
persaingan desa wisata
2. Meningkatkan pelatihan dan
dana untuk mengelola desa
wisata
3. Meningkatkan kemampuan
manajemen pengurus dan
Pokdarwis, dukungan
pemerintah dan stakeholder
sehingga pariwisata berbasis
msyarakat masih tetap ada 2. Memanfaatkan konsep CBT
sebagai pedoman mengatasi
pergeseran dan komersialisasi
budaya 3. Pergeseran budaya
lokal akibat
perkembangan
pariwisata 3. Memperkuat lembaga adat
untuk meningkatkan kesadaran
masyarakat dalam mengelola
desa wisata berbasis
masyarakat 3. Meningkatkan kemampuan
manajemen pengurus dan
Pokdarwis, dukungan
pemerintah dan stakeholder
sehingga pariwisata berbasis
msyarakat masih tetap ada Berdasarkan hasil matriks analisis SWOT diatas berikut diuraikan 4 strategi yang
digunakan Berdasarkan hasil matriks analisis SWOT diatas berikut diuraikan 4 strategi yang
digunakan. Strategi
S-O
(Strength
Opportunities) g
a) Strategi S-O (Strength-Opportunities), menginventarisasi daya tarik wisata,
membuat pemetaan kawasan wisata, memilih dan menentukan daya tarik wisata
utama yang akan dijadikan brand mark, dan memaksimalkan peranan / partisipasi
masyarakat untuk memanfaatkan letak wilayah strategis desa Aan. b) Strategi W-O (Weakness – Opportunities), melakukan pertemuan, pelatihan dan
pengembangan SDM, membuka usaha pariwisata seperti homestay, peningkatan
UMKM melalui produk-produknya, kuliner khas daerah, membangun fasilitas
wisata yang masih terbatas dan pengembangan sesuai dengan rencana dan
pemetaan kawasan dengan menjalin komunikasi antara pemangku kebijakan. c) Strategi ST (Strenght-Threat), mengembangkan potensi wisata dan meningkatkan
pelayanan terhadap wisatawan melalui usaha pariwisata sehingga meminimalisir
ancaman terjadinya alih fungsi lahan. d) Strategi WT (Weakness-Threat), melakukan pertemuan, seminar, diskusi,
workshop untuk meningkatkan kemampuan pengurus Pokdarwis dalam mengelola
bantuan dari pemerintah atau pihak lainnya. 1. Meningkatkan kesadaran
masyarakat mengantisipasi
persaingan desa wisata
2. Meningkatkan pelatihan dan
dana untuk mengelola desa
wisata
3. Meningkatkan kemampuan
manajemen pengurus dan
Pokdarwis, dukungan
pemerintah dan stakeholder
sehingga pariwisata berbasis
msyarakat masih tetap ada Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Simpulan Berdasarkan analisis potensi dan faktor internal eksternal diatas dapat ditarik tiga
simpulan berikut. 1. Desa wisata Aan memiliki banyak potensi menarik seperti air terjun Aan Secret
Waterfall, air terjun Celek-celek, air terjun Gebyug, pemandangan bukit dan
persawahan yang masih alami dengan jalur tracking-nya, Petapan park yang
menyajikan berbagai aktivitas menarik seperti memancing di air deras, aneka
permainan, sling (ayunan) serta jenis kegiatan menarik lainnya yang dapat dilakukan
di Petapan Park. Selain wisata alam, terdapat pula museum serta UMKM yang
mengelola budidaya madu kelle, beserta produk-produk kesehatan yang dihasilkan
dari budidaya tersebut y
2. Berdasarkan analisis siklus hidup destinasi wisata, desa wisata Aan berada pada
tahapan involvement atau pelibatan masyarakat. Pada tahap involvement memiliki ciri-
ciri; kunjungan wisatawan masih sedikit, komunikasi antara masyarakat dan
wisatawan masih tinggi, fasilitas mulai dibangun, masyarakat bersama dengan
pemangku kebijakan mulai menyediakan fasilitas pendukung, pola hidup sosial
budaya masyarakat mulai berubah merespon terhadap perubahan ekonomi yang
terjadi, promosi mulai dilakukan. j
p
3. Strategi yang perlu dikembangkan untuk menyukseskan pengembangan desa Aan
sebagai desa wisata yaitu (a) Program pengembangan dengan Strength-
Opportunities; menginventarisasi daya tarik wisata dan memilih salah satu daya tarik
wisata utama yang akan dijadikan brand mark, pengembangan pariwisata berbasis
masyarakat, peningkatan kualitas atraksi wisata, perbaikan aksesibilitas, amenities
dan kelembagaan. (b) Program Pengembangan dengan Weakness-Opportunities;
penataan kawasan wisata, pembuatan jalan setapak yang lebih baik lagi menuju obyek
wisata, membangun gapura desa wisata pada pintu masuk desa, membuat papan nama
pada obyek wisata, menentukan kebijakan penyelenggaraan pariwisata, pelatihan dan
pengembangan kemampuan masyarakat. (c) Program pengembangan dengan
Strenght-Threat; mengoptimalkan seluruh fasilitas, sarana dan prasarana, koordinasi
seluruh pemangku kebijakan dan melakukan promosi melalui digital marketing. (d)
Program pengembangan dengan Weakness-Threat: membuat produk wisata dan
melakukan promosi produk wisata. 3. Strategi yang perlu dikembangkan untuk menyukseskan pengembangan desa Aan
sebagai desa wisata yaitu (a) Program pengembangan dengan Strength-
Opportunities; menginventarisasi daya tarik wisata dan memilih salah satu daya tarik
wisata utama yang akan dijadikan brand mark, pengembangan pariwisata berbasis
masyarakat, peningkatan kualitas atraksi wisata, perbaikan aksesibilitas, amenities
dan kelembagaan. (b) Program Pengembangan dengan Weakness-Opportunities;
penataan kawasan wisata, pembuatan jalan setapak yang lebih baik lagi menuju obyek
wisata, membangun gapura desa wisata pada pintu masuk desa, membuat papan nama
pada obyek wisata, menentukan kebijakan penyelenggaraan pariwisata, pelatihan dan
pengembangan kemampuan masyarakat. (c) Program pengembangan dengan
Strenght-Threat; mengoptimalkan seluruh fasilitas, sarana dan prasarana, koordinasi
seluruh pemangku kebijakan dan melakukan promosi melalui digital marketing. Simpulan (d)
Program pengembangan dengan Weakness-Threat: membuat produk wisata dan
melakukan promosi produk wisata. Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100
ISSN 2807-5129 (media online) ISSN 2807-5129 (media online) Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 3. Program Pengembangan 3. Program Pengembangan
Program pengembangan daya tarik wisata desa Aan diuraikan berdasarkan strategi
Strength-Opportunities, Weakness-Opportunities, Strength-Threat dan Weakness-
Threat. Program pengembangan daya tarik wisata desa Aan diuraikan berdasarkan strategi
Strength-Opportunities, Weakness-Opportunities, Strength-Threat dan Weakness-
Threat. a) Program pengembangan dengan Strength-Opportunities; menginventarisasi daya
tarik wisata dan memilih salah satu daya tarik wisata utama yang akan dijadikan
brand mark, pengembangan pariwisata berbasis masyarakat, peningkatan kualitas
atraksi wisata, perbaikan aksesibilitas, amenities dan kelembagaan b) Program Pengembangan dengan Weakness-Opportunities; penataan kawasan
wisata, pembuatan jalan setapak yang lebih baik lagi menuju obyek wisata,
membangun gapura desa wisata pada pintu masuk desa, membuat papan nama
pada obyek wisata, menentukan kebijakan penyelenggaraan pariwisata, pelatihan
dan pengembangan kemampuan masyarakat. c) Program pengembangan dengan Strenght-Threat; mengoptimalkan seluruh
fasilitas, sarana dan prasarana, koordinasi seluruh pemangku kebijakan dan
melakukan promosi melalui digital marketing. d) Program pengembangan dengan Weakness-Threat: membuat produk wisata dan
melakukan promosi produk wisata. 98 Daftar Pustaka A.J, Muljadi, 2012, Kepariwisataan dan Perjalanan. Jakarta: Raja Grafindo Persada. p 12
Butler, R.W. 1980. “The Concept of a Tourist Area Cycle of Evolution: Implications for
Management of Resources”, The Canadian Geographer, 24(1), pp 5-12 CNN Indonesia. 2022. “Banyak Dikunjungi Turis Asing Jumlah Desa Wisata di Bali
Melonjak” https://www.cnnindonesia.com/gaya-hidup/20220815192253-269-
834969/banyak-dikunjungi-turis-asing-jumlah-desa-wisata-di-bali-melonjak y
j
g
g j
j
Gunn C. 1994, Tourism Planning; Basic, Concepts and Cases, Taylor and Francis, USA. Marceilla Hidayat, 2011, Strategi Perencanaan Dan Pengembangan Objek Wisata (Studi
Kasus Pantai Pangandaran Kabupaten Ciamis Jawa Barat), Tourism and Hospitality
Essentials (THE) Journal, Vol. I, No. 1, 2011 – 34 Marceilla Hidayat, 2011, Strategi Perencanaan Dan Pengembangan Objek Wisata (Studi
Kasus Pantai Pangandaran Kabupaten Ciamis Jawa Barat), Tourism and Hospitality
Essentials (THE) Journal, Vol. I, No. 1, 2011 – 34 Parsons. 1990. Tourism area life cycle of Parsons dan Pemikirannya. Sebuah Pengantar. Alih Bahasa Hartono Hadikusumo. Bandung: PT.Tiara Parsons. 1990. Tourism area life cycle of Parsons dan Pemikirannya. Sebuah Pengantar. Alih Bahasa Hartono Hadikusumo. Bandung: PT.Tiara Priasukmana Soetarso dan R. Mohamad Mulyadin, 2013, Pembangunan Desa Wisata:
Pelaksanaan Undang-undang Otonomi Daerah, jurnal, hlm 38 Priasukmana Soetarso dan R. Mohamad Mulyadin, 2013, Pembangunan Desa Wisata:
Pelaksanaan Undang-undang Otonomi Daerah, jurnal, hlm 38
Rangkuti, F. 2005. Analisis SWOT Teknik Membedah Kasus Bisnis. Jakarta: Gramedia
Rangkuti, F. 2013. Analisis SWOT. Jakarta: Gramedia
Sugiyono 2005 Memahami Penelitian Kualitatif Bandung: CV Alfabeta Priasukmana Soetarso dan R. Mohamad Mulyadin, 2013, Pembangunan Desa Wisata:
Pelaksanaan Undang-undang Otonomi Daerah, jurnal, hlm 38 Priasukmana Soetarso dan R. Mohamad Mulyadin, 2013, Pembangunan Desa Wisata:
Pelaksanaan Undang-undang Otonomi Daerah, jurnal, hlm 38
Rangkuti, F. 2005. Analisis SWOT Teknik Membedah Kasus Bisnis. Jakarta: Gramedia
k i
2013 A
li i S
O
k
G
di Rangkuti, F. 2005. Analisis SWOT Teknik Membedah Kasus Bisnis. Jakarta: Gramedia Rangkuti, F. 2013. Analisis SWOT. Jakarta: Gramedia Sugiyono. 2005. Memahami Penelitian Kualitatif. Bandung: CV Alfabeta 99 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100
SSN 2807-5129 (media online)
Sugiyono. 2014. Metode Penelitian Manajemen. Bandun: Alfabeta
Sukmadinata. 2006. Metode Penelitian Kualitatif. Bandung: Graha Akasara
Website Resmi Pemerintahan Desa Aan. 2022. https://aan.desa.id/
Website
Resmi
Pemerintahan
Kabupaten
Klungkung. 2021. https://klungkungkab.go.id/berita/detail/bupati-suwirta-terima-audiensi-pokdarwis-
desa-aan-bahas-rencana-pengembangan-desa-wisata Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100 Jounal of Tourism and Interdisciplinary Studies (JoTIS)
Vol. 2 No. 2 Desember 2022, 90-100
ISSN 2807-5129 (media online)
Sugiyono. 2014. Metode Penelitian Manajemen. Bandun: Alfabeta
Sukmadinata. 2006. Metode Penelitian Kualitatif. Daftar Pustaka Bandung: Graha Akasara
Website Resmi Pemerintahan Desa Aan. 2022. https://aan.desa.id/
Website
Resmi
Pemerintahan
Kabupaten
Klungkung. 2021. https://klungkungkab.go.id/berita/detail/bupati-suwirta-terima-audiensi-pokdarwis-
desa-aan-bahas-rencana-pengembangan-desa-wisata ISSN 2807-5129 (media online) 100
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English
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Seborrheic Pemphigoid
|
Case reports in dermatological medicine
| 2,014
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cc-by
| 2,864
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Enzo Errichetti,1 Giuseppe Stinco,1 Enrico Pegolo,2 Nicola di Meo,3
Giusto Trevisan,3 and Pasquale Patrone1 1 Institute of Dermatology, Department of Experimental and Clinical Medicine, University of Udine,
San Michele Hospital, Piazza Rodolone 1, Gemona del Friuli, 33013 Udine, Italy p
y
Institute of Anatomic Pathology, Department of Medical and Biological Sciences, University of Udine, 2 Institute of Anatomic Pathology, Department of Medical and Biological Sciences, University of Udine,
University Hospital of Santa Maria della Misericordia, Piazzale Santa Maria della Misericordia 15, 33100 Udine, Italy
3 Institute of Dermatology and Venereology, University of Trieste, Maggiore Hospital, Piazza Ospedale 1, 34100 Trieste, Italy University Hospital of Santa Maria della Misericordia, Piazzale Santa Maria della Misericordia 15, 33100 Udine,
3 Institute of Dermatology and Venereology, University of Trieste, Maggiore Hospital, Piazza Ospedale 1, 34100 Tri Correspondence should be addressed to Enzo Errichetti; enzoerri@yahoo.it Received 13 January 2014; Revised 13 July 2014; Accepted 4 August 2014; Published 18 August 2014 Academic Editor: Thomas Berger Copyright © 2014 Enzo Errichetti et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Seborrheic pemphigoid (SP), first described in 1969 by Schnyder, is a peculiar variant of BP which clinically resembles pemphigus
erythematosus, since it is characterized by ruptured bullae and erosions covered with crusts involving the seborrheic areas. To the
best of our knowledge, from the first description only four other cases of SP have been reported, of which two are in the English
literature. We report an additional case of SP in a 56-year-old man with cervical spondylogenic myelopathy with very impaired
mobility. Hindawi Publishing Corporation
Case Reports in Dermatological Medicine
Volume 2014, Article ID 768217, 4 pages
http://dx.doi.org/10.1155/2014/768217 Hindawi Publishing Corporation
Case Reports in Dermatological Medicine
Volume 2014, Article ID 768217, 4 pages
http://dx.doi.org/10.1155/2014/768217 Hindawi Publishing Corporation
Case Reports in Dermatological Medicine
Volume 2014, Article ID 768217, 4 pages
http://dx.doi.org/10.1155/2014/768217 1. Introduction A 56-year-old man presented to our clinic with a three-
month history of recurrent and mild itchy bullae quickly
evolving in erosive lesions covered by crusts located on
his scalp, forehead, auricular and periauricular regions, and
interscapular area (seborrheic sites). The patient had been
previously diagnosed as having impetigo and seborrheic
dermatitis; however, specific therapies for these disorders
were found to be completely ineffective. His medical history
included an untreated progressive (during previous 4 years)
slight bilateral age-related sensorineural hearing loss (pres-
bycusis); bipolar disorder (since he was 35 years old) con-
trolled from about 5 years with quetiapine, duloxetine, and
lorazepam; and cervical spondylogenic myelopathy with very
limited mobility (from about 2 years) which had worsened
considerably in the last six months. The cervical problem has
been treated only with physical therapies (but the patient is
currently waiting for a surgical treatment). The man denied
other drugs intake and health issues. Physical examination
revealed very few flaccid blisters, several erosions (some of
which with a peripheral epithelial collarette), and hematic
and serous crusts on a slightly erythematous background Bullous pemphigoid (BP) is a chronic, autoimmune, often
pruritic, subepidermal, blistering dermatosis occurring
mainly in elderly individuals aged 70 years and older. Classi-
cally, patients present with large tense bullae on apparently
normal or erythematous skin, located at the sides of the neck,
axillae, groins, upper inner aspects of the thighs, and abdo-
men. Not rarely, excoriated, eczematous, papular, or urticarial
lesions may precede the onset of the blisters [1, 2]. In addition
to the more classic findings, several atypical presentations
of BP have been described, including forms confined to a
particular cutaneous district (paralyzed extremity, pretibial
area, umbilicus, vulva, and irradiated or peristomal sites) and
variants presenting with vesicular, erythroderma, vegetating,
dyshidrotic dermatitis-like, prurigo nodularis-like, toxic
epidermal necrolysis-like, ecthyma-like, or pemphigus ery-
thematosus-like lesions [2, 3].h The latter variant is known as “seborrheic pemphigoid”
and is a very rare form of BP, since there are only a few
cases reported in the literature. We report an additional case
of seborrheic pemphigoid in a patient with cervical spon-
dylogenic myelopathy with very impaired mobility. 1. Introduction Case Reports in Dermatological Medicine 2 (a)
(b)
(c)
Figure 1: Several erosions (some of which with a peripheral epithelial collarette) and hematic and serous crusts on a slightly erythematous
background on forehead (a), right periauricular region (b), and interscapular area (c); a flaccid blister (arrow) is visible under the right ear
lobe (b). (a) (b) (c) (a) (c) (b) Figure 1: Several erosions (some of which with a peripheral epithelial collarette) and hematic and serous crusts on a slightly erythematous
background on forehead (a), right periauricular region (b), and interscapular area (c); a flaccid blister (arrow) is visible under the right ear
lobe (b) (a)
(b)
(c)
(d)
Figure 2: Subepidermal bulla containing eosinophils with an eosinophilic inflammatory cell infiltrate in the superficial dermis (H and
E staining, magnification ×100) (a); detail of eosinophils in the subepidermal blister (H and E staining, magnification ×400) (b). Direct
immunofluorescence tests show deposition of IgG (c) and C3 (d) at the basement membrane zone (200x). (b) (a) (b) (a) (d) (c) (c) (d) Figure 2: Subepidermal bulla containing eosinophils with an eosinophilic inflammatory cell infiltrate in the superficial dermis (H and
E staining, magnification ×100) (a); detail of eosinophils in the subepidermal blister (H and E staining, magnification ×400) (b). Direct
immunofluorescence tests show deposition of IgG (c) and C3 (d) at the basement membrane zone (200x). in epidermal extracts; furthermore, the BP180 antibodies
were also detected (value of 31.7 U/mL; cutoff value for
positivity: 15.0 U/mL) by an enzyme-linked immunosorbent
assay (ELISA) BP180-NC16a diagnosis kit. The detection of
antinuclear and antiextractable nuclear antigens antibodies
was negative. On the basis of clinical, histological, and
laboratory findings, a diagnosis of seborrheic pemphigoid
was made. Treatment with oral methylprednisolone at the
dosage of 0.5 mg/kg/die mg produced a rapid improvement. After 5 weeks the patient achieved a complete remission and
the steroid was gradually tapered during the subsequent 6 (Figures 1(a), 1(b), and 1(c)); Nikolsky’s sign was absent. No other significant skin or mucosal lesions were seen. A
6 mm punch biopsy specimen was taken from the edge of
a blister of right periauricular region and submitted for
histological examination, which showed subepidermal bulla
containing eosinophils with an eosinophilic inflammatory
cell infiltrate in the superficial dermis (Figures 2(a) and
2(b)). Direct immunofluorescence (IF) of perilesional skin
detected IgG and C3 deposition at the basement membrane
zone (Figures 2(c) and 2(d)). References [1] E. Ruocco, A. Aurilia, G. Simonetti, E. Cozzani, A. Baroni, and
G. Argenziano, “Bullous pemphigoid: three atypical cases,” Acta
Dermato—Venereologica, vol. 82, no. 3, pp. 222–223, 2002. [2] G. Di Zenzo, R. della Torre, G. Zambruno, and L. Borradori,
“Bullous pemphigoid: from the clinic to the bench,” Clinics in
Dermatology, vol. 30, no. 1, pp. 3–16, 2012. y
g
The reasons underlying the peculiar location of the
lesions in seborrheic areas in SP are not clear. It is well-known
that the regional variability in the BP antigens skin expression
may play a role in the distribution of the lesions of BP, given
that the greatest concentration of BP antigens is in the skin of
flexor surfaces of the arm, leg, and thigh, the most common
sites involved in BP [19]. On the basis of this finding, it is
possible to speculate that subjects with SP could present a
higher expression of BP antigens in the seborrheic areas than
the rest of the skin surface. Unfortunately, our patient denied
further skin biopsies and therefore we have not been able
to assess this hypothesis. We hope that future studies may
evaluate this assumption. Another possible explanation for
the peculiar localization of the lesion in SP could be that
unknown factors typical of seborrheic areas may trigger or
aggravate the disease in susceptible individuals. In this view,
it is important to underline the ability of Malassezia yeast,
which is notoriously localized to seborrheic sites, to activate
the complement system, via either the alternative pathway
or the classical pathway, with the possibility to amplify the
complement-mediated inflammation which is characteristic
of BP [20]. Considering that an increase of the static pool [3] “Blistering diseases,” in Dermatology, O. Braun-Falco, G. Plewig, H. H. Wolff, and W. H. C. Burgdorf, Eds., pp. 676–680,
Springer, Berlin, Germany, 2nd edition, 2000. [4] S. Welke, “Seborrheic pemphigoid (Schnyder),” Hautarzt, vol. 31, no. 1, pp. 18–20, 1980. [5] M. U. Schnyder, “Seborrheic pemphigoid. A new nosologic
entity?” Bulletin de la Societe Francaise de Dermatologie et de
Syphiligraphie, vol. 76, no. 3, article 320, 1969. [6] P. Pailheret, Courtin, and Testard, “Seborrheic pemphigoid,”
Bulletin de la Soci´et´e Franc¸aise de Dermatologie et de Syphiligra-
phie, vol. 5, pp. 772–773, 1959. [7] S. Maissa and A. Relias, “Seborrheic pemphigoid developing
into foliaceous pemphigus,” Bulletin de la Soci´et´e Franc¸aise de
Dermatologie et de Syphiligraphie, vol. 63, pp. 417–418, 1956. [8] P. J. Conflict of Interests The authors declare that there is no conflict of interests
regarding the publication of this paper. When a blister is available, its histological examination
shows a picture comparable to the classic form of BP, with a
subepidermal cleavage [1]. Direct IF test of perilesional skin,
which generally reveals linear deposition of IgG and C3 at
the dermoepidermal junction [1], and detection of circulating
IgG antibodies against basement membrane zone (indirect
IF), BP230, and/or BP180 may be of aid in the diagnosis [1, 18].h 1. Introduction The result of immunoblotting
showed IgG autoantibodies which reacted against BP230 Case Reports in Dermatological Medicine 3 months up to dose of 0.1 mg/kg/die. The value of circulating
anti-BP180-NC16a antibodies (ELISA test) was also progres-
sively decreased (26.4 U/mL after 5 weeks, 21.6 U/mL after
3 months, and 18.1 U/mL after 6 months). Currently, after 9
months from the start of steroid therapy, the patient is free of
disease and presents a value of circulating anti-BP180-NC16a
antibodies under the cutoff value for positivity (13.2 U/mL)
with a dose of methylprednisolone of 0.1 mg/kg/die. of already secreted sebum due to immobility and muscular
paralysis plays a permissive role for growth of Malassezia
yeast [21], the possible involvement of this microorganism
could also explain a possible correlation between SP and cer-
vical spondylogenic myelopathy with very limited mobility
in our patients. In fact, albeit we can not exclude a coin-
cidental association, there are three points that support a
such link: the development of the disease after a relatively
brief period from the significant worsening of the motor
abilities, the age of onset lower than the average [3], and the
well-known correlation between classical pemphigoid and
neurodegenerative processes [22, 23]. 3. Discussion Seborrheic pemphigoid (SP) is a peculiar variant of BP which
clinically resembles pemphigus erythematosus (known also
as seborrheic pemphigus), since it is characterized by rup-
tured bullae and erosions covered with crusts involving the
seborrheic areas [4]. The first instance of SP reported in the
literature dates back to 1969, when Schnyder described a case
in an elderly female [5]. It is important to underline that all
cases [6–16] described as “SP” before Schnyder’s report were
actually instances of pemphigus erythematosus [5]. Such
confusion was probably caused by the lack of availability
of reliable serological tests and direct IF test of perilesional
skin, which are very helpful particularly in the cases without
detectable bullae [1, 17, 18]. To the best of our knowledge,
since the first description four other instances of SP, similar to
our case and original report of Schnyder, have been reported
[1, 4, 17, 18], of which two are in English language in 1991
[18] and 2002 [1], respectively. Regarding the latter report, the
authors emphasized a possible association between losartan
intake and unleashing of the lesions in their patient [1]. Although SP may sometimes be confused with seborrheic
dermatitis or impetigo, the main differential diagnosis is
pemphigus erythematosus. In our opinion, the negativity of
Nikolsky’s sign, as described in the present case, may help to
suspect a SP rather than pemphigus erythematosus, since in
the latter it is almost always present [24]. Anyhow, only the
histology, direct immunofluorescence examination, and sero-
logical studies allow us to definitely distinguish SP from pem-
phigus erythematosus and the other conditions mentioned
above. With regard to the therapy, according to other authors
[3], our case confirms that relatively low dosages of systemic
corticosteroids are effective in SP. References Michel and M. Blanchon, “Results obtained with cortisone
in a case of generalized seborrheic pemphigoid,” Bulletin de la
Soci´et´e Franc¸aise de Dermatologie et de Syphiligraphie, vol. 59,
no. 1, pp. 93–96, 1952. [9] J. Watrin, P. Michon, C. Michon, J. Beurey, and R. Leduc,
“Humoral study of a case of seborrheic pemphigoid and favo-
rable therapy with ACTH and blood transfusions,” Bulletin de 4 Case Reports in Dermatological Medicine la Soci´et´e Franc¸aise de Dermatologie et de Syphiligraphie, vol. 59,
no. 1, pp. 39–41, 1952. [10] C. Fassotte, “A case of seborrheic pemphigoid (Senear-Usher
syndrome),” Archives Belges de Dermatologie et de Syphiligra-
phie, vol. 7, no. 3, pp. 208–213, 1951. [11] R. Weille, “Case of seborrheic pemphigoid; monosymptomatic
and benign evolution,” Bulletin de la Soci´et´e Franc¸aise de Der-
matologie et de Syphiligraphie, vol. 58, no. 4, pp. 353–356, 1951. [12] P. Michel, M. Blanchon, and J. S. Paul, “Case of seborrheic
pemphigoid with an implacable and fatal evolution in spite of
quinacrine and cortisone,” Bulletin de la Soci´et´e Franc¸aise de
Dermatologie et de Syphiligraphie, vol. 58, no. 4, pp. 350–355,
1951. [13] J. Watrin and P. Jeandidier, “Duhring’s disease or seborrheic
pemphigoid,” Bulletin de la Soci´et´e Franc¸aise de Dermatologie
et de Syphiligraphie, vol. 58, no. 4, pp. 347–349, 1951. [14] A. Touraine, “Seborrheic pemphigoid (Senear-Usher syndrome,
pemphigus erythematosus); general study; nosological situa-
tion,” Bulletin de la Soci´et´e Franc¸aise de Dermatologie et de
Syphiligraphie, vol. 58, no. 2, pp. 113–124, 1951. [15] E. Griveaud and J. Duverne, “Seborrheic pemphigoid (Senear-
Usher syndrome); evolution and treatment; histological and
biological study,” Bulletin de la Soci´et´e Franc¸aise de Dermatolo-
gie et de Syphiligraphie, vol. 58, no. 2, pp. 90–112, 1951. [16] J. Margarot, P. Rimbaud, and P. Izarn, “Seborrheic pemphigoid;
evolution toward pemphigus foliaceus,” Bulletin de la Soci´et´e
Franc¸aise de Dermatologie et de Syphiligraphie, vol. 57, no. 4, pp. 382–384, 1950. [17] I. Schneider and S. Husz, “Seborrhoic pemphigoid,” Hautarzt,
vol. 37, no. 3, pp. 149–151, 1986. [18] K. Tamaki, T. Furuya, Y. Kubota, A. Uno, and S. Shimada, “Seb-
orrheic pemphigoid and polymorphic pemphigoid,” Journal of
the American Academy of Dermatology, vol. 25, no. 3, pp. 568–
570, 1991. [19] D. J. Goldberg, M. Sabolinski, and J. C. Bystryn, “Regional varia-
tion in the expression of bullous pemphigoid antigen and loca-
tion of lesions in bullous pemphigoid,” Journal of Investigative
Dermatology, vol. 82, no. 4, pp. 326–328, 1984. [20] H. R. Ashbee and E. References G. V. Evans, “Immunology of diseases asso-
ciated with Malassezia species,” Clinical Microbiology Reviews,
vol. 15, no. 1, pp. 21–57, 2002. [21] M. Mastrolonardo, A. Diaferio, and G. Logroscino, “Seborrheic
dermatitis, increased sebum excretion, and parkinson’s disease:
a survey of (im)possible links,” Medical Hypotheses, vol. 60, no. 6, pp. 907–911, 2003. [22] G. Stinco, P. Mattighello, M. Zanchi, and P. Patrone, “Multiple
sclerosis and bullous pemphigoid: a casual association or a
pathogenetic correlation?” European Journal of Dermatology,
vol. 12, no. 2, pp. 186–188, 2002. [23] G. Stinco, R. Codutti, M. Scarbolo, F. Valent, and P. Patrone,
“A retrospective epidemiological study on the association of
bullous pemphigoid and neurological diseases,” Acta Dermato-
Venereologica, vol. 85, no. 2, pp. 136–139, 2005. [24] “Pemphigus,” in Dermatology, J. L. Bolognia, J. L. Iorizzo, and
R. P. Rapini, Eds., pp. 422–423, Mosby Elsevier, St. Louis, Mo,
USA, 2nd edition, 2008. Submit your manuscripts at
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https://www.frontiersin.org/articles/10.3389/fmedt.2023.1241368/pdf?isPublishedV2=False
|
English
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Feasibility of the preparation of cochleate suspensions from naturally derived phosphatidylserines
|
Frontiers in medical technology
| 2,023
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cc-by
| 8,487
|
Feasibility of the preparation
of cochleate suspensions
from naturally derived
phosphatidylserines
Søren Kristensen
1, Khadeija Hassan
1, Nadia Skarnager Andersen
1,
Frank Steiniger
2 and Judith Kuntsche
1*
1Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark,
2Center for Electron Microscopy, Jena University Hospital, Jena, Germany EDITED BY
Irina Le-Deygen,
Lomonosov Moscow State University, Russia
REVIEWED BY
Wei Shao,
Westlake University, China
Angelina Angelova,
UMR8612 Institut Galien Paris Sud (IGPS),
France
*CORRESPONDENCE
Judith Kuntsche
kuntsche@sdu.dk
RECEIVED 16 June 2023
ACCEPTED 17 August 2023
PUBLISHED 06 September 2023
CITATION
Kristensen S, Hassan K, Andersen NS, Steiniger F
and Kuntsche J (2023) Feasibility of the
preparation of cochleate suspensions from
naturally derived phosphatidylserines. Front. Med. Technol. 5:1241368. doi: 10.3389/fmedt.2023.1241368 EDITED BY
Irina Le-Deygen,
Lomonosov Moscow State University, Russia
REVIEWED BY
Wei Shao,
Westlake University, China
Angelina Angelova,
UMR8612 Institut Galien Paris Sud (IGPS),
France
*CORRESPONDENCE
Judith Kuntsche
kuntsche@sdu.dk
RECEIVED 16 June 2023
ACCEPTED 17 August 2023
PUBLISHED 06 September 2023
CITATION
Kristensen S, Hassan K, Andersen NS, Steiniger F
and Kuntsche J (2023) Feasibility of the
preparation of cochleate suspensions from
naturally derived phosphatidylserines. Front. Med. Technol. 5:1241368. doi: 10.3389/fmedt.2023.1241368 EDITED BY
Irina Le-Deygen,
Lomonosov Moscow State University, Russia
REVIEWED BY
Wei Shao,
Westlake University, China
Angelina Angelova,
UMR8612 Institut Galien Paris Sud (IGPS),
France EDITED BY
Irina Le-Deygen,
Lomonosov Moscow State University, Russia Søren Kristensen
1, Khadeija Hassan
1, Nadia Skarnager Andersen
1,
Frank Steiniger
2 and Judith Kuntsche
1* 1Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark,
2Center for Electron Microscopy, Jena University Hospital, Jena, Germany Introduction: Cochleates are cylindrical particles composed of dehydrated
phospholipid bilayers. They are typically prepared by addition of calcium ions to
vesicles
composed
of
negatively
charged
phospholipids
such
as
phosphatidylserines (PS). Due to their high physical and chemical stability, they
provide an interesting alternative over other lipid-based drug formulations for
example to improve oral bioavailability or to obtain a parenteral sustained-
release formulation. Kristensen S, Hassan K, Andersen NS, Steiniger F
and Kuntsche J (2023) Feasibility of the
preparation of cochleate suspensions from
naturally derived phosphatidylserines. Front. Med. Technol. 5:1241368. doi: 10.3389/fmedt.2023.1241368 Methods: In the present study, the feasibility to prepare cochleate suspensions
from
soy
lecithin-derived
phosphatidylserines
(SPS)
was
investigated
and
compared to the “gold standard” dioleoyl-phosphatidylserine (DOPS) cochleates. The SPS lipids covered a large range of purities between 53 and >96% and
computer-controlled mixing was evaluated for the preparation of the cochleate
suspensions. Electron microscopic investigations were combined with small-
angle x-ray diffraction (SAXD) and Laurdan generalized polarization (GP) analysis
to characterize particle structure and lipid organization. © 2023 Kristensen, Hassan, Andersen, Steiniger
and Kuntsche. cochleates,
phosphatidylserine,
drug
delivery,
lipid
formulation,
physicochemical
characterization, electron microscopy TYPE Original Research
PUBLISHED 06 September 2023
DOI 10.3389/fmedt.2023.1241368 1. Introduction in patent applications (16, 17). In addition, some recent studies
used soy-phosphatidylserine to prepare cochleates incorporating
lipid-A as an oral vaccine adjuvant system (18) and amphotericin
B for oral administration (9), respectively. However, there is still
very
limited
information
about
the
morphology
and
lipid
structure
in
cochleate
suspensions
prepared
from
natural
phosphatidylserines in the scientific literature. Cochleates, which have first been described by Papahadjopoulos
in 1975 (1) are formed by specific binding of multivalent cations
such as calcium ions to negatively charged phospholipids such as
phosphatidylserine (PS). The binding of calcium ions results in
dehydration of the phospholipid headgroup, collapse and fusion of
the vesicles followed by formation of lamellar sheets which roll up
to form cochleate cylinders (1–3). By addition of EDTA, a calcium
chelator, the cochleates lose their structure and large vesicles are
obtained again (1). Due to their solid structure, cochleate particles
possess a considerably high chemical and physical stability, what
makes
them
interesting
for
drug
formulation
(4,
5). The
predominant application for cochleate suspensions is oral drug
delivery and the prospect of this formulation strategy could clearly
be illustrated for amphotericin B-loaded cochleate suspensions
(6–11). However, cochleate formulations have generally been
suggested for a broad range of drug administration and the
interested reader is referred a recent comprehensive review article (5). Cochleates, which have first been described by Papahadjopoulos
in 1975 (1) are formed by specific binding of multivalent cations
such as calcium ions to negatively charged phospholipids such as
phosphatidylserine (PS). The binding of calcium ions results in
dehydration of the phospholipid headgroup, collapse and fusion of
the vesicles followed by formation of lamellar sheets which roll up
to form cochleate cylinders (1–3). By addition of EDTA, a calcium
chelator, the cochleates lose their structure and large vesicles are
obtained again (1). Due to their solid structure, cochleate particles
possess a considerably high chemical and physical stability, what
makes
them
interesting
for
drug
formulation
(4,
5). The
predominant application for cochleate suspensions is oral drug
delivery and the prospect of this formulation strategy could clearly
be illustrated for amphotericin B-loaded cochleate suspensions
(6–11). However, cochleate formulations have generally been
suggested for a broad range of drug administration and the
interested reader is referred a recent comprehensive review article (5). Feasibility of the preparation
of cochleate suspensions
from naturally derived
phosphatidylserines
Søren Kristensen
1, Khadeija Hassan
1, Nadia Skarnager Andersen
1,
Frank Steiniger
2 and Judith Kuntsche
1*
1Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark,
2Center for Electron Microscopy, Jena University Hospital, Jena, Germany This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is
permitted, provided the original author(s) and
the copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic practice. No use, distribution or reproduction is
permitted which does not comply with these
terms. Results: Despite some differences in particle morphology, cochleate suspensions
with similar internal lipid structure as DOPS cochleates could be prepared from
SPS with high headgroup purity (≥96%). Suspensions prepared from SPS with
lower purity still revealed a remarkably high degree of lipid dehydration and
well-organized lamellar structure. However, the particle shape was less defined,
and the typical cochleate cylinders could only be detected in suspensions
prepared with higher amount of calcium ions. Finally, the study proves the
feasibility to prepare suspensions of cochleates or cochleate-like particles
directly from a calcium salt of soy-PS by dialysis. 01 Frontiers in Medical Technology frontiersin.org Kristensen et al. 10.3389/fmedt.2023.1241368 GRAPHICAL ABSTRACT GRAPHICAL ABSTRACT GRAPHICAL ABSTRACT Frontiers in Medical Technology 1. Introduction Information
about
membrane rigidity and lipid dehydration can be obtained by
analyzing the Laurdan GP (22). The energy of Laurdan’s
emission depends on the polarity of the environment of the
headgroup of the Laurdan molecule. In a polar environment, the
fluorophore loses some energy due to dipole-dipole interactions
and emission is shifted to higher wavelengths. This effect can be
used to investigate membrane rigidity and structure (23, 24). In a
fluid membrane, some Laurdan molecules will be deep inside the
membrane (non-polar environment, emission wavelength around
430 nm), whereas others are close to the hydrated headgroups of
the phospholipids and thus in contact with water molecules
(emission wavelength around 490 nm). By setting the intensities
at both wavelength in relation to each other, the general
polarization (GP) can be calculated on a scale from −1 (only
polar) to 1 (only non-polar). 1. Introduction In
the
first
studies
on
cochleate
formation,
isolated
phosphatidylserine from bovine brain with high purity has been
used (1, 2), but synthetic phospholipids, especially DOPS, have
then mostly been applied to prepare cochleates (6–8, 12) and
especially for structural analysis (3, 13, 14). From an industrial
perspective of drug development, costs and availability of the
excipients are highly relevant and cheaper alternatives such as
naturally derived phosphatidylserines are of interest especially for
oral
drug
delivery
(15). Accordingly,
the
use
of
phosphatidylserines derived from soy lecithin has been described The aim of the present study was thus to explore particle
structure
and
lipid
organization
in
cochleate
suspensions
prepared by phosphatidylserines derived from soy lecithin in
comparison to the gold standard DOPS (Scheme 1) and applying
different experimental methods for comprehensive structural
analysis. Phosphatidylserine can be obtained from lecithin by
enzymatic headgroup modification (19) or by precipitation of the
fraction of negatively charged lipids from raw lecithin followed
by purification and eventually recrystallization into the water-
soluble
sodium
salt. In
the
present
work,
seven
phosphatidylserines
derived
from
soy
lecithin
differing
in
headgroup purity and salt form (sodium vs. calcium salt) have
been studied (Table 1). In addition, the feasibility of direct
processing the calcium salt into a cochleate formulation has been
evaluated. This is of special practical and economical interest, as
this could eliminate additional processing steps upon PS isolation
and purification from lecithin. In
the
first
studies
on
cochleate
formation,
isolated
phosphatidylserine from bovine brain with high purity has been
used (1, 2), but synthetic phospholipids, especially DOPS, have
then mostly been applied to prepare cochleates (6–8, 12) and
especially for structural analysis (3, 13, 14). From an industrial
perspective of drug development, costs and availability of the
excipients are highly relevant and cheaper alternatives such as
naturally derived phosphatidylserines are of interest especially for
oral
drug
delivery
(15). Accordingly,
the
use
of
phosphatidylserines derived from soy lecithin has been described For comprehensive characterization of lipid organization in the
cochleate cylinders, electron microscopy was combined with small-
angle
x-ray
diffraction
(SAXD)
and
Laurdan
generalized
polarization
(GP)
analysis. Whereas
scanning
electron
microscopy (SEM) provides information about the particle shape 02 frontiersin.org Kristensen et al. 10.3389/fmedt.2023.1241368 SCHEME 1
Graphical presentation of the study design. Graphical presentation of the study design. TABLE 1 Specifications of the soybean lecithin-derived lipids according to the specifications from the manufacturer (lipoid). The sample codes derived
from the batch numbers (numbers in bold). PS, phosphatidylserine; PA, phosphatidic acid; PC, phosphatidylcholine. 1. Introduction and surface, the particle inner structure and lipid organization can
be visualized by cryo-transmission electron microscopy (cryo-
TEM) as well as TEM of thin cross sections of the sample
(3, 20). Information about the lipid organization can also be
determined by SAXD. The highly ordered, dehydrated lamellar
arrangement of the lipids in the cochleate structure results in a
very sharp 1st order reflection in the small-angle range indicating
the thickness of the lipid lamellae [5.1 and 5.2 nm for DSPS and
DOPS
cochleates,
respectively
(14,
21)]. Information
about
membrane rigidity and lipid dehydration can be obtained by
analyzing the Laurdan GP (22). The energy of Laurdan’s
emission depends on the polarity of the environment of the
headgroup of the Laurdan molecule. In a polar environment, the
fluorophore loses some energy due to dipole-dipole interactions
and emission is shifted to higher wavelengths. This effect can be
used to investigate membrane rigidity and structure (23, 24). In a
fluid membrane, some Laurdan molecules will be deep inside the
membrane (non-polar environment, emission wavelength around
430 nm), whereas others are close to the hydrated headgroups of
the phospholipids and thus in contact with water molecules
(emission wavelength around 490 nm). By setting the intensities
at both wavelength in relation to each other, the general
polarization (GP) can be calculated on a scale from −1 (only
polar) to 1 (only non-polar). Different
methods
for
the
preparation
of
cochleate
suspensions have been described in the literature (3, 12, 13, 25)
where the simple addition of calcium chloride solution to the
PS-vesicle suspension (“trapping method”) was selected as a
starting point in this study. However, as standardization of
manual addition of the calcium chloride solution is difficult to
achieve,
preparation
of
cochleate
suspensions
by
using
a
computer-controlled
mixing
device
and
by
dialysis
was
evaluated (Scheme 2). and surface, the particle inner structure and lipid organization can
be visualized by cryo-transmission electron microscopy (cryo-
TEM) as well as TEM of thin cross sections of the sample
(3, 20). Information about the lipid organization can also be
determined by SAXD. The highly ordered, dehydrated lamellar
arrangement of the lipids in the cochleate structure results in a
very sharp 1st order reflection in the small-angle range indicating
the thickness of the lipid lamellae [5.1 and 5.2 nm for DSPS and
DOPS
cochleates,
respectively
(14,
21)]. Frontiers in Medical Technology PS, phosphatidylserine; PA, phosphatidic acid; PC, phosphatidylcholine. 2.1. Materials Dioleoyl phosphatidylserine (DOPS, ≥99%) was obtained from
Avanti Polar Lipids Inc. (U.S.) and soybean phosphatidylserines
(SPS) of varying purities (Table 1) were provided by the Lipoid
GmbH (Germany). TRIZMA preset-crystals pH 7.4, calcium
chloride dihydrate (Ph.Eur.), sodium azide (≥99.5%), EDTA
tetrasodium dihydrate (>99%) and chloroform (≥99.0%, Ph.Eur.,
stabilized with ∼1% ethanol) were obtained from Sigma, sodium
chloride (Ph.Eur.) from VWR and Laurdan (6-dodecanoyl-2-
dimethylaminonaphtalene)
from
Molecular
Probes
(Thermo- Code
Batch number
PS (%)
PA (%)
PC (%)
Cation
Description
SPS-10
599,990–2160010/001
≥96
4
0.4
Na+
Enzymatic modification from purified soybean PC
SPS-13
599,990–2160013/001
65
23
2
Na+/Ca2+
Enzymatic modification from soybean PC
SPS-14
599,990–2160014/001
55
20
5
Na+/Ca2+
SPS13 mixed with soybean lecithin
SPS-16
599,990–2170016/001
73
12
N/A
Na+
SPS-15 converted to Na+ salt
SPS-15
599,990–2170015/001
65
12
N/A
Ca2+
Calcium salt of PS from soybean lecithin
SPS-19
599,990–2180019/914
74
N/A
N/A
Ca2+
Calcium salt of PS from soybean lecithin
SPS-34
538,500–2160034/911
53
N/A
N/A
Ca2+
Calcium salt of PS from soybean lecithin 03 Frontiers in Medical Technology frontiersin.org frontiersin.org 10.3389/fmedt.2023.1241368 Kristensen et al. SCHEME 2
Schematic presentation of the methods used for the preparation of the cochleate suspensions. SCHEME 2
Schematic presentation of the methods used for the preparation of the cochleate suspensions. 8°C until use. Specifications of the liposomes used in this study
are provided in Table 2. Fisher Scientific). Purified water was obtained from a Milli-Q
Advantage A10 system (Millipore). 2.3.1. Trapping method 100 µl 140 mM calcium chloride solution in Tris buffer was
added dropwise to 1 ml diluted liposome suspension (10 mg/ml
lipid) under magnetic stirring (250 rpm) to reach a lipid/calcium
ion molar ratio of about 1:1. 2.3.2. Controlled mixing Equal volumes of liposome suspension (20 mg/ml) and
calcium chloride solution in Tris buffer were mixed at a
controlled mixing speed of 80 µl/s. The self-constructed device
consisted of two disposable 1-ml syringes with the syringe
plungers connected to each a motor and the syringe outlets to a
Y-junction (inner diameter 0.5 mm). If not stated otherwise, the
calcium chloride concentration was adjusted to reach a lipid/
calcium ion molar ratio of 1:1. If necessary, the pH was adjusted
to 7.4. TABLE 2 Specifications of the liposomes used for cochleate preparation. TABLE 2 Specifications of the liposomes used for cochleate preparation. Lipid
EDTA
(mM)
DLS
Method for
cochleate
preparation
Diameter
(nm)
PdI
DOPS
0
65 ± 3
0.216 ± 0.018
TM, CM
SPS-10
0
103 ± 4
>0.5
TM, CM
SPS-10
10
37 ± 1
0.238 ± 0.001
CM
SPS-13
10
70 ± 1
0.238 ± 0.001
CM
SPS-14
10
71 ± 1
0.230 ± 0.005
CM
SPS-15
30
36 ± 1
0.222 ± 0.004
CM
SPS-16
10
36 ± 1
0.224 ± 0.007
CM
SPS-19
25–30
30 ± 1
0.223 ± 0.009
CM, D
SPS-34
30
64 ± 1
0.234 ± 0.004
CM
PdI, polydispersity index; TM, tapping method; CM, controlled mixing (80 µl/s); D,
dialysis. 2.2. Preparation of liposomes The different methods used for cochleate preparation in this
study are schematically presented in Scheme 2. Liposomes (20 mg/ml lipid) were prepared either by the lipid-
film method or by directly dispersing the lipid in buffer under
mechanical
agitation
(Heidolph
Multi-reax
set
to
500 rpm;
Heidolph Instr., Germany) overnight. If not stated otherwise,
10 mM Tris buffer pH 7.4 preserved with 0.02% (w/v) sodium
azide (Tris buffer) was used. To facilitate dispersion of the SPS
lipids containing calcium ions, an adequate amount of EDTA
was
added
to
the
buffer
(Table
2). The
crude
liposome
dispersions were submitted to bath sonication (35 kHz, Bandelin
Sonorex Digitech, 2–6 cycles à 15 min, Bandelin electronic
GmbH & Co. KG, Germany) to obtain suspensions of small
unilamellar vesicles. The liposome suspensions were stored at 4– Frontiers in Medical Technology PdI, polydispersity index; TM, tapping method; CM, controlled mixing (80 µl/s); D,
dialysis. 2.6.2. Cryo-TEM A drop of diluted (1:1 in Tris buffer) sample was placed on a
holey copper grid (Quantifoil R 1.2/1.3, 400 mesh) and rapidly
frozen in liquid ethane (about −180°C). A cryo-transfer unit
(Gatan 626, Gatan Inc., U.S.) was used to transfer the frozen
specimen
into
the
pre-cooled
cryo-transmission
electron
microscope (CM 120, Philipps, Netherlands). The specimen was
viewed under low dose conditions (120 kV), and images were
recorded with a CCD camera (FastScan F114, TVIPS GmbH,
Germany). 2.6.1. Conventional TEM A small droplet of non-diluted formulation was placed on a
holy carbon grid (Quantifoil R2/1, Quantifoil Micro Tools
GmbH, Germany) placed on filter paper and air dried for five
minutes. The samples were viewed in a transmission electron
microscope (CM120, Philipps, Netherlands) at 120 kV. Images
were recorded with a 2k CMOS Camera (F216, TVIPS GmbH,
Germany). 2.5.2. High-resolution SEM The diluted (1:10 or 1:5) sample was sonicated for 4 min at room
temperature (Elmasonic P, Elma Schmiedhauer GmbH, Germany,
37 kHz, 50% power) and 5 µl sample was then placed on a copper
grid
(Quantifoil
R,
1.2/1.3,
Quantifoil
Micro
Tools
GmbH,
Germany). The sample was rinsed with a small amount of Tris
buffer before being air-dried on top of a filter paper. The copper
grid was then placed on an aluminum SEM specimen stub and
coated with 7-nm platinum layer (CCU-010, Safematic GmbH,
Switzerland). The samples were examined in a LEO 1,530 Gemini
SEM (Carl Zeiss GmbH Jena, Germany) operated at 4 kV
acceleration voltage. Images were acquired by the InLens detector. 2.3.3. Dialysis 1 ml liposome suspension (20 mg/ml lipid) was filled in a
dialysis cell (Pur-A-Lyzer, MWCO 12.5 kDa, Sigma Aldrich) and
placed in a 1,000 ml beaker containing 700 ml 250 mM calcium
chloride solution in Tris buffer. Dialysis was carried out under
magnetic stirring and light protection for 24 h with a buffer
change
after
2
and
4 h. Samples
were
withdrawn
at
predetermined time points from the dialysis cell to monitor the
formation of cochleates. Frontiers in Medical Technology 04 frontiersin.org Kristensen et al. Kristensen et al. 10.3389/fmedt.2023.1241368 2.6.3. Resin-embedded cross sections Samples were prepared as described previously (3). Briefly, the
sample was pelleted by centrifugation and stained in 100 mM
cacodylate buffer pH 7.4 with 1% osmium tetroxide for 2 h prior
dehydration
in
50%
ethanol. The
dehydrated
sample
was
embedded in epoxy resin Araldite CY 212 (Agar Scientic Ltd.,
UK). After polymerization, the block was cut into thin sections
(70–100 nm) using an Ultracut E ultramicrotome (Reichert-Jung,
Germany) at room temperature. Sections were placed on copper
grids and examined in the TEM at 120 kV (CM 120, Philipps,
Netherlands). Repeat distances of the bilayer structure were
determined directly from the TEM images of by analyzing 100 ×
100 nm section of the image by Fourier Transform (FFT) in
ImageJ 1.52p. 2.6. Transmission electron microscopy
(TEM) Vesicle size of the liposome suspensions was determined by
DLS (DelsaMax Pro, Beckman Coulter Life Science, U.S.). The
diluted (1:1,000 in Tris buffer) liposomes were measured 6 times
over
10
s
at
20°C
in
backscattering
mode
(163.5°). The
hydrodynamic diameter (z-average) and polydispersity index
(PdI) were calculated by the instrument’s cumulant analysis
(DelsaMax version 1.0.1.6. Beckman Coulter). Results given as
average and standard deviation of the six acquisitions. 2.5.1. Conventional SEM The diluted (1:10 or 1:5) sample was placed on a polycarbonate
filter (0.5 × 0.5 cm, 0.4 µm pore size, Whatman Nuclepore Track-
etched membrane, Sigma) and air-dried on top of a filter paper. The dried sample was then placed on an aluminum SEM
specimen stub and coated with 15-nm gold layer (JFC-1100, Jeol
Ltd., Japan). The samples were examined in a LEO-435VP SEM
(accelerating voltage 10 kV) or a Quanta 2,000 SEM (FEI, U.S.,
acceleration voltage 15–20 kV) and images were acquired with
Everhart-Thornley detectors. Frontiers in Medical Technology frontiersin.org 2.8. Laurdan fluorescence spectroscopy 10 µl 0.1 mM Laurdan solution in anhydrous ethanol was
mixed
with
500 µl
diluted
sample
(0.5 mg/ml
lipid)
and
equilibrated under light protection on a shaker (IKA Vibrax IKA
GmbH & Co. KG, Germany, 250 rpm). Fluorescence emission
spectra were recorded with a Cary Eclipse instrument (Varian,
Agilent, U.S.) at room temperature from 380 nm to 600 nm and
an excitation wavelength of 370 nm. Generalized polarization
(GP) values were calculated taking the fluorescence intensities at
430 nm and 490 nm into account: GP ¼ I430 I490
I430 þ I490 (2) 2.7. Small-angle x-ray diffraction (SAXD) As
expected, the Laurdan emission spectra of DOPS and SPS-10
liposomes were bimodal (Figure 3A, dotted lines) resulting in
slightly negative GP values between −0.1 and −0.2, as typically
for membranes in the fluid state. Binding of calcium ions results
in dehydration of the phospholipid headgroups and formation of
an anhydrous multilamellar structure. Accordingly, a distinct
change in the Laurdan emission spectra with an emission
maximum around 430 nm was observed (Figure 3A, closed
lines). Similar GP values around 0.5 were measured for both 2.7. Small-angle x-ray diffraction (SAXD) The
highly organized lamellar internal structure of the cochleate
particles could be visualized for both DOPS (Figures 2A–C) and
SPS-10 (Figures 2D–F) cochleates. The lamellar repeat distances
of the lipid lamellae were 5.1 ± 0.1 nm (n = 11) and 4.8 ± 0.1 nm
(n = 13)
for
DOPS
and
SPS-10
cochleates,
respectively. Importantly, the tightly packed lamellar structure could also be
visualized in the more spherically shaped particles (marked in
Figures 2C,F). To get more quantitative information about the
lipid dehydration, Laurdan GP analysis was carried out. As
expected, the Laurdan emission spectra of DOPS and SPS-10
liposomes were bimodal (Figure 3A, dotted lines) resulting in
slightly negative GP values between −0.1 and −0.2, as typically
for membranes in the fluid state. Binding of calcium ions results
in dehydration of the phospholipid headgroups and formation of
an anhydrous multilamellar structure. Accordingly, a distinct
change in the Laurdan emission spectra with an emission
maximum around 430 nm was observed (Figure 3A, closed
lines). Similar GP values around 0.5 were measured for both the typical cochleate structures with high aspect ratio were
observed
in
SEM
in
addition
to
more
spherical
particles
(Figures 1A–C). In good agreement with literature (3, 13), some
of the cochleate cylinders had an inner water channel (marked
with arrows in Figure 1C). In the SPS-10 suspensions, the
particles were considerably smaller, but the elongated particles
indicate the formation of cochleate cylinders (Figures 1D–F). As
in the DOPS suspension, the cochleate cylinders were coexisting
with more spherical particles. Due to the solid nature of the
particles, the formulations could directly be visualized in TEM
(e.g., direct analysis of the dried sample) where they had a rather
similar appearance as observed in cryo-TEM (Figure 2). The
highly organized lamellar internal structure of the cochleate
particles could be visualized for both DOPS (Figures 2A–C) and
SPS-10 (Figures 2D–F) cochleates. The lamellar repeat distances
of the lipid lamellae were 5.1 ± 0.1 nm (n = 11) and 4.8 ± 0.1 nm
(n = 13)
for
DOPS
and
SPS-10
cochleates,
respectively. Importantly, the tightly packed lamellar structure could also be
visualized in the more spherically shaped particles (marked in
Figures 2C,F). To get more quantitative information about the
lipid dehydration, Laurdan GP analysis was carried out. 2.7. Small-angle x-ray diffraction (SAXD) y
One droplet of the sample was placed on a gold sample carrier
BU012 129-T (BAL-TEC AG, Lichtenstein), and rapidly frozen by
plunge-freezing in liquid nitrogen-cooled propane/ethane (50:50). The samples were then transferred into a VCT 100 cryo-transfer
system (BAL-TEC AG, Lichtenstein), which was continuously
cooled with liquid nitrogen. After connecting the VCT 100
system to a MED 020 high-vacuum coating system (BAL-TEC
AG, Lichtenstein), samples were fractured and deep-etched at
−95°C for 5 min and finally sputter-coated with 2–3 nm gold. Using the VCT 100 cryo-transfer system, samples were finally
transferred under vacuum and cryo conditions into a scanning
electron microscope Leo 1,530 Gemini (Carl Zeiss GmbH Jena,
Germany) onto a liquid nitrogen cooled (at −140°C) sample
holder. Images were recorded digitally with an InLens SE
detector (Carl Zeiss GmbH Jena, Germany) at 4 kV acceleration
voltage. Small-angle x-ray diffraction patterns were recorded at room
temperature with a SAXSess mc2 instrument
(Anton Paar
GmbH, Austria, x-ray wavelength: 0.154 nm, CCD-SCX 4,300
detector) using a flow-through capillary. Each sample was
measured 50 times over 30 s (1,500 s in total) and desmearing of
the raw data was performed with SAXSquant software. The
lamellar repeat distance (d-spacing) was calculated from the first
order reflection according to Bragg’s equation with λ the
wavelength of the x-rays (0.154 nm, Cu Kα) and θ the scattering
angle: d ¼
l
2 sinu
(1) (1) In the figures, the scattering intensities are plotted against the
scattering vector s with s = 2sinθ/λ = 1/d. In the figures, the scattering intensities are plotted against the
scattering vector s with s = 2sinθ/λ = 1/d. Frontiers in Medical Technology 05 frontiersin.org frontiersin.org 10.3389/fmedt.2023.1241368 10.3389/fmedt.2023.1241368 Kristensen et al. the typical cochleate structures with high aspect ratio were
observed
in
SEM
in
addition
to
more
spherical
particles
(Figures 1A–C). In good agreement with literature (3, 13), some
of the cochleate cylinders had an inner water channel (marked
with arrows in Figure 1C). In the SPS-10 suspensions, the
particles were considerably smaller, but the elongated particles
indicate the formation of cochleate cylinders (Figures 1D–F). As
in the DOPS suspension, the cochleate cylinders were coexisting
with more spherical particles. Due to the solid nature of the
particles, the formulations could directly be visualized in TEM
(e.g., direct analysis of the dried sample) where they had a rather
similar appearance as observed in cryo-TEM (Figure 2). 3.1. Comparison of DOPS and SPS ≥96%
(SPS-10) To evaluate the general feasibility to prepare cochleate
suspensions from naturally derived lipids with similar well-
organized lipid organization as found in DOPS cochleates, the
soy-PS with highest head-group purity (≥96%; SPS-10) was
applied. Cochleate suspensions were prepared by the standard
trapping method (10 mg/ml lipid, lipid/calcium ion molar ratio
of 1:1). Addition of calcium ions to the liposome suspensions resulted
in immediate flocculation for both lipids. In the DOPS suspension, FIGURE 1
Representative high-resolution SEM (A,B,D–F) and cryo SEM (C) images of DOPS (A–C) and SPS-10 (D–F) cochleate suspensions. Cochleate suspensions
were prepared by the trapping method at a lipid/calcium ion molar ratio of 1:1. Selected cochleate cylinders with an inner water channel are marked with
an arrow. Additional SEM images are presented in the supplementary material (Supplementary Figures S1 and S2). 3. Results
3.1. Comparison of DOPS and SPS ≥96%
(SPS-10) 3. Results FIGURE 1 FIGURE 1
Representative high-resolution SEM (A,B,D–F) and cryo SEM (C) images of DOPS (A–C) and SPS-10 (D–F) cochleate suspensions. Cochleate suspensions
were prepared by the trapping method at a lipid/calcium ion molar ratio of 1:1. Selected cochleate cylinders with an inner water channel are marked with
an arrow. Additional SEM images are presented in the supplementary material (Supplementary Figures S1 and S2). 06 Frontiers in Medical Technology frontiersin.org Kristensen et al. 10.3389/fmedt.2023.1241368 FIGURE 2
Representative TEM (A,B,D) and cryo-TEM (C,E,F) images of DOPS (A–C) and SPS-10 (D–F) cochleate suspensions. Cochleate suspensions were prepared by
the trapping method at a lipid/calcium ion molar ratio of 1:1. A spherical particle with well-organized lamellar structure i marked with an arrow in c and f. FIGURE 2
Representative TEM (A,B,D) and cryo-TEM (C,E,F) images of DOPS (A–C) and SPS-10 (D–F) cochleate suspensions. Cochleate suspensions were prepared by
the trapping method at a lipid/calcium ion molar ratio of 1:1. A spherical particle with well-organized lamellar structure i marked with an arrow in c and f. FIGURE 3
Laurdan emission spectra (A) and small-angle x-ray diffraction (SAXD) patterns (B) from DOPS (red) and SPS-10 (blue) liposome (dashed lines) and
cochleate (closed lines) suspensions. Note the presence of second and third order reflections in (B) Cochleate suspensions were prepared by the
trapping method at a lipid/calcium ion molar ratio of 1:1. FIGURE 3
Laurdan emission spectra (A) and small-angle x-ray diffraction (SAXD) patterns (B) from DOPS (red) and SPS-10 (blue) liposome (dashed lines) and
cochleate (closed lines) suspensions. Note the presence of second and third order reflections in (B) Cochleate suspensions were prepared by the
trapping method at a lipid/calcium ion molar ratio of 1:1. FIGURE 3
Laurdan emission spectra (A) and small-angle x-ray diffraction (SAXD) patterns (B) from DOPS (red) and SPS-10 (blue) liposome (dashed lines) and
cochleate (closed lines) suspensions. Note the presence of second and third order reflections in (B) Cochleate suspensions were prepared by the
trapping method at a lipid/calcium ion molar ratio of 1:1. 07 07 Frontiers in Medical Technology Frontiers in Medical Technology frontiersin.org 10.3389/fmedt.2023.1241368 Kristensen et al. DOPS and SPS-10 cochleates indicating a similar degree of
dehydration of the lipid bilayers. SAXD results confirmed the
well-organized
lamellar
structure
of
the
lipid
particles
(Figure 3B, closed lines) by the presence of a very sharp 1st
order reflection. FIGURE 1 Even the much weaker reflections of 2nd and
3rd order (marked with arrows in Figure 3B) could be detected. Based on the position of the reflections, the thickness of the lipid
lamella (d-spacing) was determined to be of 5.1 and 4.9 nm for
the DOPS and SPS-10 cochleates, respectively. In contrast, only
weak and broad reflections were measured for the corresponding
liposome suspensions (Figure 3B, dotted lines). Interestingly, the
reflection was more distinct for SPS-10 than DOPS liposomes
which may be explained by the presence of residual calcium ions
in the starting lipid. As the calcium salt of PS cannot be
dispersed in an aqueous medium, some crystalline lipid material
will be present in the liposome suspension. This is likely also the
reason for the larger diameter and PdI in DLS measurements of
these liposomes (Table 2). This explanation is supported by the
observation that addition of small amounts of EDTA upon
liposome preparation resulted in a distinctly smaller size and
similar PdI values as measured for the other vesicle suspensions
(Table 2). resin and thin sections were then viewed in the TEM (Figure 4). The overall appearance (images on the left) was similar as
observed for DOPS cochleates in a previous study (3). Well-
organized lamellar structures could clearly be visualized at higher
magnification allowing an estimation of the lamellar repeat
distance (see also Supplementary Figure S5). The average
lamellar repeat distances were between 4.2 and 4.4 nm (n = 4),
e.g., somewhat smaller than those determined by TEM original
samples and SAXD (4.9 nm). In good agreement with this
results, considerably high Laurdan GP values were determined
(Figure 5A) with a trend of decreasing GP with decreasing
amount of negatively charged lipids. This trend was very clear
for the suspensions prepared from the sodium salts (100%, 88%,
85% and 75% of total negatively charged lipids for SPS-10, SPS-
13, SPS-16 and SPS-14, Figure 5A). High GP values were also
obtained
for
the
SPS-19
and
SPS-34
suspensions,
which,
however, contained a higher amount of calcium ions (lipid/
calcium ion molar ratio about 1:2). Moreover, these lipids likely
will also contain other (not specified) negatively charged lipids,
which may contribute to the high degree of lipid dehydration
and organization. In all suspensions, the characteristic SAXD
reflection was detected, and the lamellar repeat distance (d =
4.9 nm) was similar for all suspensions. 3.3. Direct preparation of cochleate
suspensions from calcium-SPS DOPS and SPS-10 cochleate suspension were also prepared by
controlled mixing of equal volumes of liposomes and calcium
chloride solution at a mixing speed of 80 µl/s where suspensions
with
similar
properties
were
obtained
(Supplementary
Figure S3). All further samples were thus prepared by controlled
mixing at 80 µl/s if not stated otherwise. From a practical and economic point of view, direct processing
of the water-insoluble calcium salt of PS to prepare the cochleate
suspensions is of interest and has been evaluated in this study. Cochleate suspensions were prepared from the most promising
SPS calcium salt from the screening experiments (SPS-19). To
disperse the water-insoluble calcium PS in the aqueous buffer for
liposome preparation, EDTA was added to the buffer (Table 2). Cochleate formulations with different lipid/calcium ion molar
ratios (1:1, 1:2, 1:5 and 1:10) were then prepared to determine
the optimal composition with respect to homogeneity (aggregate
size) and degree of dehydration (high GP values). In addition, a
suspension was also prepared by dialyzing liposomes directly
against a calcium chloride solution to remove the EDTA, which
initially was added for liposome preparation. All cochleate
suspensions were prepared in triplicate. FIGURE 1 The reflections were,
however, broader and less intensive as shown for selected
formulations in Figure 5B. Despite the differences in particle shape, the results clearly
indicate that cochleates with similar structural lipid organization
as in DOPS cochleates can be prepared from naturally derived
phosphatidylserines with high headgroup purity. Similarly as
DOPS
cochleates,
the
SPS-10
cochleate
suspensions
were
physically stable (particle structure and redispersibility) for at
least half a year. Frontiers in Medical Technology frontiersin.org 3.2. Screening of naturally derived
phosphatidylserines with different purities In
the
next
step,
a
range
of
naturally
derived
phosphatidylserines of varying purities (PS between 53 and 96%)
and salt forms (sodium or calcium salt or a mixture of both,
Table 1) were screened for their ability to form cochleates. To
facilitate dispersion of the lipid, EDTA was added in an adequate
amount (Table 2) and the concentration of calcium chloride
solution used for cochleate preparation was adjusted accordingly
to reach lipid/calcium ion molar ratios of about 1:1 or 1:2 for the
sodium (SPS-10, SPS-13, SPS-14) and calcium salts (SPS-15, SPS-
19, SPS-34), respectively (Table 2). Independently on the lipid/calcium ion ratio, flocculated
suspensions were obtained and the characteristic SAXD reflection
(d-spacing 4.9 nm) was detected in all suspensions shortly after
preparation (not shown). As expected, the aggregation tendency
(aggregate size) increased with increasing amounts of added
calcium chloride. There was also an increase in GP with
increasing lipid/calcium molar ratio (Figure 6A) up to a ratio of
1:5 reaching then GP values between 0.4 and 0.5 (Figure 6A). However, GP values declined distinctly during storage in all
samples prepared by direct mixing (Figure 6A). It can be
speculated that the EDTA in the suspension interferes with the All samples showed immediate flocculation upon mixing with
calcium ions, but the characteristic cochleate structures (e.g.,
cochleate cylinders) could not be seen in SEM (Supplementary
Figure S4). To get more information about the inner structure of
the particles, selected suspensions (SPS-10, SPS-13, SPS-15 and
SPS-19) were stained with osmium tetroxide, embedded in epoxy 08 frontiersin.org Kristensen et al. 10.3389/fmedt.2023.1241368 FIGURE 4
TEM images of resin-embedded samples of cochleate suspensions prepared from SPS of varying purities (SPS-10, SPS-13, SPS-15, SPS-19, controlled
mixing, 80 µl/s). The buffer for liposome preparation contained 100 mM sodium chloride and EDTA (Table 2). The calcium chloride concentration
was adjusted according to the added EDTA to reach a lipid/calcium ion molar ratio of about 1:1 (sodium salts) or 1:2 (calcium salts). See
Supplementary Figure S5 for details on determination of the lamellar thickness. FIGURE 4
TEM images of resin-embedded samples of cochleate suspensions prepared from SPS of varying purities (SPS-10, SPS-13, SPS-15, SPS-19, controlled
mixing, 80 µl/s). The buffer for liposome preparation contained 100 mM sodium chloride and EDTA (Table 2). The calcium chloride concentration
was adjusted according to the added EDTA to reach a lipid/calcium ion molar ratio of about 1:1 (sodium salts) or 1:2 (calcium salts). Frontiers in Medical Technology 3.2. Screening of naturally derived
phosphatidylserines with different purities See
Supplementary Figure S5 for details on determination of the lamellar thickness. particle structure over time. To circumvent this problem, cochleate
suspensions were prepared by dialysis. The process of lipid
dehydration during dialysis could clearly be followed by the
increasing Laurdan GP values and a plateau was reached after
about 1 h (Figure 6B). Most importantly, the GP values did not decrease distinctly upon storage (Figure 6A) indicating improved
stability compared to the samples prepared by direct mixing. Remarkably, the SPS-19 cochleate suspensions prepared with
lipid/calcium chloride molar ratio of 1:5 and by dialysis had a
rather similar morphology as those prepared from the purer SPS- Frontiers in Medical Technology 09 frontiersin.org Kristensen et al. 10.3389/fmedt.2023.1241368 FIGURE 5
Laurdan GP (A, n = 3) and SAXD pattern (B) of cochleate suspensions prepared from SPS of varying purities (table 1, controlled mixing, 80 µl/s). The mass
percent of negatively charged lipids (PS + PA) is indicated in the figure. The buffer for liposome preparation contained 100 mM sodium chloride and EDTA
(Table 2). The calcium chloride concentration was adjusted according to the added EDTA to reach a lipid/calcium ion molar ratio of about 1:1 (sodium
salts) or 1:2 (calcium salts). FIGURE 6
Laurdan GP (n = 3) of SPS-19 formulations measured after preparation and after storage (A) and during dialysis (B) cochleate suspensions were prepared
by controlled mixing (80 µl/s) or dialysis. 25 mM EDTA was added to prepare liposomes and the calcium chloride concentration was adjusted accordingly
to reach the desired lipid/calcium ion molar ration between 1:1 and 1:10. Frontiers in Medical Technology
10
frontiersin.org FIGURE 5
Laurdan GP (A, n = 3) and SAXD pattern (B) of cochleate suspensions prepared from SPS of varying purities (table 1, controlled mixing, 80 µl/s). The mass
percent of negatively charged lipids (PS + PA) is indicated in the figure. The buffer for liposome preparation contained 100 mM sodium chloride and EDTA
(Table 2). The calcium chloride concentration was adjusted according to the added EDTA to reach a lipid/calcium ion molar ratio of about 1:1 (sodium
salts) or 1:2 (calcium salts). FIGURE 5
Laurdan GP (A, n = 3) and SAXD pattern (B) of cochleate suspensions prepared from SPS of varying purities (table 1, controlled mixing, 80 µl/s). The mass
percent of negatively charged lipids (PS + PA) is indicated in the figure. 4. Discussion Overall, particles with a tightly packed and dehydrated lamellar
structure
could
be
prepared
from
soybean
lecithin-derived
phosphatidylserines. The formulation with a resemblance closest
to the cylindrical DOPS cochleates was obtained from the soy-PS
with highest purity (SPS-10, PS ≥96%). In both formulations,
cochleate cylinders were co-existing with more compact particles. The similar GP-values and the presence of the characteristic
sharp SAXD reflection indicate a highly ordered lipid structure. The shorter lamellar repeat distance (d-spacing, 4.9 nm) of the
soy-PS particles is in good agreement with results obtained in a
recent study (9) and can be explained by the mixed fatty acid
chains
in
this
naturally
derived
lipid. For
dipalmitoyl
phosphatidylserine (DPPS)-calcium, for example, the lamellar
repeat distance is about 4.5 nm (26) and thus distinctly smaller
than that of DOPS-calcium [5.1 nm (14)]. Suspensions which were prepared from lipids with lower purity
and lipid/calcium ion molar ratios of 1:1 (sodium salts) or 1:2
(calcium
salts)
resulted
in
particles
with
less
well-ordered
structures and dehydration (broader and less intensive SAXD
reflection, lower GP values, no formation of cylindrical particles). However, the well-defined lamellar arrangement seen in electron
microscopy and similar d-spacing as determined for SPS-10
cochleates indicate that at least some regions in the particles had
a lipid organization and headgroup dehydration similarly to
particles obtained from the purer PS. The content of neutral
lipids such as phosphatidylcholine (PC) is likely the determining
factor for the overall less well-ordered particle structure, and one
may expect at least a partial segregation of the lipids in the
lamellar structure. Addition of calcium ions to vesicles composed
of an equimolar mixture of DOPS and DOPC, for example,
resulted in segregation of DOPS in the tightly packed lipid
organization of cochleates, while DOPC remained in fluid-phase
(27). Considering the lipids used in the present study, the PC
content specified by the manufacturer ranged from < 1% in the
purified phosphatidylserine to above 5% but might be higher in
the lipids with no specified PC content. Therefore, PC may
introduce less ordered domains in the lipid particles resulting in
lower Laurdan GP values and a broader SAXD reflections. For As the lecithin-derived lipids may contain residual calcium ions
from the processing stage, addition of EDTA was needed to
facilitate complete lipid dispersion in buffer and subsequent vesicle
formation. However, there are some concerns with respect to an
interference with the cochleate structure. 3.2. Screening of naturally derived
phosphatidylserines with different purities The buffer for liposome preparation contained 100 mM sodium chloride and EDTA
(Table 2). The calcium chloride concentration was adjusted according to the added EDTA to reach a lipid/calcium ion molar ratio of about 1:1 (sodium
salts) or 1:2 (calcium salts). FIGURE 6
Laurdan GP (n = 3) of SPS-19 formulations measured after preparation and after storage (A) and during dialysis (B) cochleate suspensions were prepared
by controlled mixing (80 µl/s) or dialysis. 25 mM EDTA was added to prepare liposomes and the calcium chloride concentration was adjusted accordingly
to reach the desired lipid/calcium ion molar ration between 1:1 and 1:10. FIGURE 6
Laurdan GP (n = 3) of SPS-19 formulations measured after preparation and after storage (A) and during dialysis (B) cochleate suspensions were prepared
by controlled mixing (80 µl/s) or dialysis. 25 mM EDTA was added to prepare liposomes and the calcium chloride concentration was adjusted accordingly
to reach the desired lipid/calcium ion molar ration between 1:1 and 1:10. 10 Frontiers in Medical Technology frontiersin.org Kristensen et al. 10.3389/fmedt.2023.1241368 FIGURE 7
Representative SEM images of SPS-10 (A,B) and SPS-19 cochleate suspensions prepared by dialysis (C,D) and by controlled mixing (80 µl/s, lipid/calcium
ion molar ratio of 1:5, E,F). Note the cylindrical cochleate particles marked with an arrow. FIGURE 7
Representative SEM images of SPS-10 (A,B) and SPS-19 cochleate suspensions prepared by dialysis (C,D) and by controlled mixing (80 µl/s, lipid/calcium
ion molar ratio of 1:5, E,F). Note the cylindrical cochleate particles marked with an arrow. 10 (Figure 7). Importantly, some cochleates with the typical
cylindrical particle shape could be detected (marked with arrows
in Figures 7B,D). affinity towards calcium ions has been suggested to disperse
cochleate aggregates (21) and may present an alternative to facilitate
the dispersion of calcium-SPS. However, low water solubility of the
formed
calcium
citrate
is
the
limiting
factor
in
this
case. Preparation of cochleate suspension by dialysis, where the EDTA is
removed simultaneously to the addition of calcium, presents an
elegant alternative as stable suspensions were obtained this way. Frontiers in Medical Technology Publisher’s note All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed
or endorsed by the publisher. Author contributions The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmedt.2023. 1241368/full#supplementary-material SK and JK: Conceptualization; SK, FS, and JK: Methodology; SK,
KH, NSA, and FS: Investigation; SK and JK: Writing - original draft Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Acknowledgments The authors thank Lipoid GmbH for providing lecithin-
derived phosphatidylserines, Heike Bunjes for providing the
possibility to carry out SAXD measurements at the University
of Braunschweig and Ursula Jahn for her support during SAXD
analysis. 4. Discussion Indeed, decreasing GP
values measured during storage for the SPS-19 formulations with
remaining EDTA has been observed in this study despite the rather
large amount of added calcium ions. Sodium citrate with lower 11 frontiersin.org Kristensen et al. 10.3389/fmedt.2023.1241368 the investigated SPS-calcium salt (SPS-19), increasing amounts of
calcium ions resulted in a higher degree of dehydration (higher
Laurdan GP value). A similar effect has been described for
mixtures of phosphatidic acid and phosphatidylcholine (28). preparation; SK, KH, NSA, FS, and JK: Writing - review and editing;
JK: Project administration and funding acquisition. All authors
contributed to the article and approved the submitted version. preparation; SK, KH, NSA, FS, and JK: Writing - review and editing;
JK: Project administration and funding acquisition. All authors
contributed to the article and approved the submitted version. In conclusion, cochleate particles with similar structure and
thus functionality as DOPS cochleates can be prepared from
lecithin-derived phosphatidylserine with high head group purity
(>96%) despite some differences in particle morphology (aspect
ratio). Particles
with
a
well-organized
lamellar
dehydrated
structure could also be obtained from less pure SPS lipids,
however, with a lower degree of structural order and dehydration
depending on the lipid/calcium ion ratio. Importantly, the results
of the study indicate the feasibility of direct processing of a
calcium SPS salt by adding EDTA for liposome formation
followed by dialysis to remove EDTA and add calcium ions for
the formation of cochleates. Considering drug incorporation,
small
domains
with
less
ordered
structure
may
even
be
advantageous, as the loading capacity in the highly ordered, rigid
lipid structure of cochleates can be expected to be limited for
most drugs. Altogether, naturally derived phosphatidylserines
present
an
interesting
and
promising
option
for
further
development of cochleate formulations for drug delivery. Ethics statement Ethical review and approval was not required for this study in
accordance with the national legislation and the institutional
requirements. Funding This research was funded by the Phospholipid Research Center
Heidelberg, grant number JKU-2016-048/1-1. Data availability statement The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author. 8. Batista-Duharte A, Lastre M, Romeu B, Portuondo DL, Tellez-Martinez D,
Manente FA, et al. Antifungal and immunomodularory activity of a novel cochleate 5. Lipa-Castro A, Legrand FX, Barratt G. Cochelate drug delivery systems: an
approach to their characterization. Int J Pharm. (2021) 610:121225. doi: 10.1016/j.
ijpharm.2021.121225 7. Santangelo R, Paderu P, Delmas G, Chen Z, Mannino R, Zarif L, et al. Efficiency of
oral cochleate-amphotericin B in a mouse model of systemic candidiasis. Antimicrob
Agents Chemother. (2000) 44:2356–60. doi: 10.1128/AAC.44.9.2356-2360.2000 6. Zarif L, Graybill JR, Perlin D, Najvar L, Bocanegra R, Mannino RJ. Antifungal
activity of amphotericin B cochleates against Candida albicans infection in a mouse
model. Antimicrob Agents Chemother. (2000) 44:1463–9. doi: 10.1128/AAC.44.6.
1463-1469.2000 Frontiers in Medical Technology 5. Lipa-Castro A, Legrand FX, Barratt G. Cochelate drug delivery systems: an
approach to their characterization. Int J Pharm. (2021) 610:121225. doi: 10.1016/j.
ijpharm.2021.121225
6. Zarif L, Graybill JR, Perlin D, Najvar L, Bocanegra R, Mannino RJ. Antifungal
activity of amphotericin B cochleates against Candida albicans infection in a mouse
model. Antimicrob Agents Chemother. (2000) 44:1463–9. doi: 10.1128/AAC.44.6.
1463-1469.2000
7. Santangelo R, Paderu P, Delmas G, Chen Z, Mannino R, Zarif L, et al. Efficiency of
oral cochleate-amphotericin B in a mouse model of systemic candidiasis. Antimicrob
Agents Chemother. (2000) 44:2356–60. doi: 10.1128/AAC.44.9.2356-2360.2000
8. Batista-Duharte A, Lastre M, Romeu B, Portuondo DL, Tellez-Martinez D,
Manente FA, et al. Antifungal and immunomodularory activity of a novel cochleate References 1. Papahadjopoulos D, Vail WJ, Jacobson K, Poste G. Cochleate lipid cylinders:
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on phosphatidylserine membranes: experiments and all-atomic simulations. Biophys. J. (2012) 102:2095–103. doi: 10.1016/j.bpj.2012.03.009 3. Nagarsekar K, Ashtikar M, Thamm J, Steiniger F, Schacher F, Fahr A, et al. Electron microscopy and theoretical modeling of cochleates. Langmuir. (2014)
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08982100009031116 8. Batista-Duharte A, Lastre M, Romeu B, Portuondo DL, Tellez-Martinez D,
Manente FA, et al. Antifungal and immunomodularory activity of a novel cochleate 12 Frontiers in Medical Technology frontiersin.org Kristensen et al. Kristensen et al. 10.3389/fmedt.2023.1241368 for amphotericin B delivery against Sporothrix schenckii. Int Immunopharmacol. (2016) 40:277–87. doi: 10.1016/j.intimp.2016.09.008 19. Schneider M. Major sources, composition and processing. In: Gundstone
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US 2015/0140074 A1, 2015. 18. Wang N, Wang T, Zhang M, Chen R, Deng Y. Using procedure of
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16:1662–7. doi: 10.1021/la990922j 13 13 frontiersin.org Frontiers in Medical Technology
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Can Social Protection Improve Sustainable Development Goals for Adolescent Health?
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cc-by
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RESEARCH ARTICLE Background The first policy action outlined in the Sustainable Development Goals (SDGs) is the imple-
mentation of national social protection systems. This study assesses whether social protec-
tion provision can impact 17 indicators of five key health-related SDG goals amongst
adolescents in South Africa. Editor: David O. Carpenter, Institute for Health &
the Environment, UNITED STATES
Received: April 11, 2016
Accepted: October 1, 2016
Published: October 17, 2016 Editor: David O. Carpenter, Institute for Health &
the Environment, UNITED STATES * Lucie.Cluver@spi.ox.ac.uk OPEN ACCESS OPEN ACCESS
Citation: Cluver LD, Orkin FM, Meinck F, Boyes
ME, Yakubovich AR, Sherr L (2016) Can Social
Protection Improve Sustainable Development
Goals for Adolescent Health? PLoS ONE 11(10):
e0164808. doi:10.1371/journal.pone.0164808
Editor: David O. Carpenter, Institute for Health &
the Environment, UNITED STATES
Received: April 11, 2016
Accepted: October 1, 2016
Published: October 17, 2016
Copyright: © 2016 Cluver et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Citation: Cluver LD, Orkin FM, Meinck F, Boyes
ME, Yakubovich AR, Sherr L (2016) Can Social
Protection Improve Sustainable Development
Goals for Adolescent Health? PLoS ONE 11(10):
e0164808. doi:10.1371/journal.pone.0164808 Lucie D. Cluver1,2*, F. Mark Orkin3, Franziska Meinck1, Mark E. Boyes1,4, Alexa
R. Yakubovich1, Lorraine Sherr5 Lucie D. Cluver1,2*, F. Mark Orkin3, Franziska Meinck1, Mark E. Boyes1,4, Alexa
R. Yakubovich1, Lorraine Sherr5 1 Centre for Evidence-Based Intervention, Department of Social Policy & Social Intervention, University of
Oxford, Oxford, United Kingdom, 2 Department of Psychiatry and Mental Health, University of Cape Town,
Cape Town, South Africa, 3 DPHRU, School of Clinical Medicine, and DST-NRF Centre of Excellence in
Human Development, University of the Witwatersrand, Johannesburg, South Africa, 4 Health Psychology
and Behavioural Medicine Research Group, School of Psychology and Speech Pathology, Curtin University,
Perth, Australia, 5 Department of Infection & Population Health, University College London, London, United
Kingdom a1111 Methods We conducted a longitudinal survey of adolescents (10–18 years) between 2009 and 2012. Census areas were randomly selected in two urban and two rural health districts in two
South African provinces, including all homes with a resident adolescent. Household receipt
of social protection in the form of ‘cash’ (economic provision) and ‘care’ (psychosocial sup-
port) social protection, and health-related indicators within five SDG goals were assessed. Gender-disaggregated analyses included multivariate logistic regression, testing for inter-
actions between social protection and socio-demographic covariates, and marginal effects
models. Copyright: © 2016 Cluver et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: Data are available
from the UK Data Archive https://discover. ukdataservice.ac.uk/catalogue/?sn=851277&type=
Data%20catalogue, Identifier 10.5255/UKDA-SN-
851277. Introduction Last year, the Millennium Development Goals expired. Replacing them are the Sustainable
Development Goals (SDGs): an ambitious 17 overarching goals with targets and indicators that
UN member states will use to guide human development policy over the next fifteen years. If
the SDGs are to move from aspirations to an implementable strategy, evidence-basedinterven-
tions need to be rapidly decided upon and scaled up [1]. The SDGs identify poverty and
inequality as major barriers to health for the world’s most structurally deprived populations. They combine ambitious human development outcomes, such as eliminating hunger and gen-
der disparities, with ‘policy actions’ designed to achieve those outcomes. Competing Interests: The authors have declared
that no competing interests exist. In doing so, the SDGs offer both great challenges and potential solutions. The first identified
policy action, SDG 1.3, is to ‘implement nationally appropriate social protection systems and
measures for all and by 2030 achieve substantial coverage of the poor and vulnerable’. Defini-
tions of social protection vary, but often follow Devereux and Sabates-Wheeler: ‘Social protec-
tion describes all public and private initiatives that provide income or consumption transfers
to the poor, protect the vulnerable against livelihood risks, and enhance the social status and
rights of the marginalised; with the overall objective of reducing the economic and social vul-
nerability of poor, vulnerable and marginalised groups’. Importantly, they clarify that social
protection can be provided in both formal ‘public and private’, and by informal ‘collective or
community-level’ sources [2]. A key vulnerable group are adolescents in Africa. Despite many advances, Sub-Saharan
Africa remains the region with the lowest Human Development Index, lowest life expectancy,
and greatest health and gender inequalities.[3] In the past decade, South Africa has emerged as
the world’s least economically equal society,[4] with persisting racial divides.[3] Adolescents
and youth are hard-hit: HIV-infection rates amongst young African females are rising,[5] as
are violent deaths amongst young males,[6] and sexual violence remains amongst the highest
in the world.[7] For these adolescents, social protection may offer both scaleability and potential. Existing
research focuses on government-provided cash transfers, which have been shown to improve
mental health, education and sexual health [8], and whilst debate remains around conditional-
ity of such transfers, most governments in the region have favoured unconditional or ‘soft’ con-
ditioned provision. However, cash transfers may not be enough. Interpretation National social protection systems are not a panacea, but findings suggest that they have
multiple and synergistic positive associations with adolescent health outcomes. Such sys-
tems may help us rise to the challenges of health and sustainable development. Social Protection and the Sustainable Development Goals violence perpetration, no effects were found and more targeted or creative means will be
needed to reach adolescents on these challenging burdens. Foundation (UK, www.nuffieldfoundation.org)
[grant number OPD/31598], the Health Economics
and HIV/AIDS Research Division at the University
of KwaZulu-Natal (South Africa; www.heard.org.za)
[grant number R14304/AA002], the John Fell Fund
(UK, www.admin.ox.ac.uk/pras/jff) [grant number
103/757], the Leverhulme Trust (UK, www. leverhulme.ac.uk) [grant number PLP-2014-095],
the University of Oxford’s ESRC Impact
Acceleration Account, the Community Care study
funded by Sweden-Norad, Helpage and UNICEF,
and the European Research Council under the
European Union’s Seventh Framework Programme
[FP7/2007-2013, grant agreement 313421]. The
funders had no role in study design, data collection
and analysis, decision to publish or preparation of
the manuscript. Findings Social protection was associated with significant adolescent risk reductions in 12 of 17 gen-
der-disaggregated SDG indicators, spanning SDG 2 (hunger); SDG 3 (AIDS, tuberculosis,
mental health and substance abuse); SDG 4 (educational access); SDG 5 (sexual exploita-
tion, sexual and reproductive health); and SDG 16 (violence perpetration). For six of 17
indicators, combined cash plus care showed enhanced risk reduction effects. Two interac-
tions showed that effects of care varied by poverty level for boys’ hunger and girls’ school
dropout. For tuberculosis, and for boys’ sexual exploitation and girls’ mental health and Funding: This work was supported by the
Economic and Social Research Council (UK; www. esrc.ac.uk) and the National Research Foundation
(South Africa; www.nrf.ac.za) [grant number RES-
062-23-2068], UNICEF, the National Department of
Social Development (South Africa; www.dsd.gov. za), the Claude Leon Foundation (South Africa;
www.leonfoundation.co.za), the Nuffield 1 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 NE | DOI:10.1371/journal.pone.0164808
October 17, 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Social Protection and the Sustainable Development Goals risk behaviors and additionally reached boys. [11] For education outcomes, cash transfers have
also been shown to benefit girls more than boys, but cash and care combinations have not been
investigated [12]. Cash transfers and care programmes are being implemented in many low-income settings,
particularly in Africa and South America, but many programmes remain small-scale. ‘Cash’
and ‘care’ can take many forms, are focused at different beneficiarygroups, and differ in extent
between and within countries–for example Kenya’s main cash transfer is targeted at house-
holds of orphaned and vulnerable children, whilst South Africa’s is for all low-income families
[10]. Countries such as Malawi, Tanzania and Uganda provide free primary schooling, but con-
cerns remain about continuing fees, costly textbooks and lack of secondaryeducation provision
[13]. Psychosocial care for children and adolescents is often received in the home or school
setting, following evidence that family-based support is the most sustainable and effective
approach for child development [14], but this often requires external support for strugglingor
vulnerable caregivers–primarily from NGOs and including a range from general social support
to structured parenting programmes [15]. State adoption of broader social protection policies would thus represent a considerable
political and fiscal undertaking.[16].A cost-benefit analysis of a cash transfer delivered as part
of a randomised trial in Malawi showed HIV, mental health and education benefits for adoles-
cent girls. If governments considered co-financing between departmental budgets, benefits out-
weigh costs for each sector [17]. With the advent of the SDGs, comprising 17 new goals with a
wide range of targets and suggested indicators, it is opportune to examine whether cash alone,
care alone or cash plus care has traction with more than one of the health-relevant SDG out-
comes. This would help identify pathways for structural intervention whereby multiple benefits
could be anticipated. It is also important to understand whether social protection shows differ-
ent patterns of effect for boys and girls: Gender equality is itself an SDG 5, but a step in achiev-
ing this requires gender-disaggregated examination of potential programming. This South African study presents an opportunity to test these potential impacts.[18]
Between 2009–2012, the government extended the receipt of child-focusedcash transfers to
adolescents from an age-limit of 12 to 18. Additionally, roll-out of free schooling and school
meals was underway for highest-deprivation districts. Methods Participants and procedures Simultaneously, international organisa-
tions were expanding provision of psychosocial ‘care’ support to adolescents, through NGO
and parenting support. During this period, access and take-up of all of these interventions was
uneven, as typifies large-scale programming and family-based care in any resource-constrained
context. This uneven scale-up of both ‘cash’ and ‘care’ social protection allowed rigorous test-
ing of associations with health outcomes in a real-world African context. Given that ‘natural
experiments’ are quasi-experimental rather than randomised designs, it is also desirable to
check whether social protection effects are modifiedby interactions with each other or with
any socio-demographicfactors that also predict adolescent health outcomes. This study thus has three aims: 1) to test associations of social protection and indicators for
health-relevant targets of five SDG goals, amongst highly deprived African adolescent boys and
girls; 2) to test for potential interactions between social protection and socio-demographicco-
predictors of adolescent health; 3) to test where ‘cash’ and/or ‘care’ forms of social protection
are effective, or where combined ‘cash plus care’ can provide additive benefits. Introduction For example in South Africa,
cash showed protective effects against HIV-risk behaviors for girls, but less or no impacts on
boys [9, 10]. Subsequent HIV-focused studies then tested whether the addition of other types
of social protection provisions may increase the impact of cash transfers. In South Africa, cash
and other types of economic support such as school feeding were tested in combination with
psychosocial care provisions such as positive parenting, good supervision from a primary care-
giver or teacher support. The combination of ‘cash plus care’ further protected girls from HIV- 2 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Participants and procedures 3515 adolescents aged 10–18 (56.7% female) were interviewedat baseline (2009–10) and fol-
lowed up at one year (2011–12). Baseline refusal rate was <2.5% and retention rate 96.8%. 3 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Social Protection and the Sustainable Development Goals Two urban and two rural health districts, all low-income, high HIV-prevalence and majority
black African, were selected within two South African provinces: Mpumalanga and the West-
ern Cape. Within each health district, census enumeration areas were randomly sampled until
sample size was reached. In each area, every household was visited and included in the study if
they had a resident adolescent. One randomly-selected adolescent per household was inter-
viewed face-to-face for 60–70 minutes. Questionnaires and consent forms were translated and
checked with back-translation into Xhosa, Zulu, Sotho, Swati and Tsonga, and adolescents
chose their language of participation. Two urban and two rural health districts, all low-income, high HIV-prevalence and majority
black African, were selected within two South African provinces: Mpumalanga and the West-
ern Cape. Within each health district, census enumeration areas were randomly sampled until
sample size was reached. In each area, every household was visited and included in the study if
they had a resident adolescent. One randomly-selected adolescent per household was inter-
viewed face-to-face for 60–70 minutes. Questionnaires and consent forms were translated and
checked with back-translation into Xhosa, Zulu, Sotho, Swati and Tsonga, and adolescents
chose their language of participation. Ethical protocols were approved by university Institutional Review Boards of Oxford, Cape
Town and KwaZulu-Natal, and by provincial Health, Education and Social Development
Departments. Voluntary written informed consent was obtained from adolescents and primary
caregivers, and to ensure full understanding of the study, information and consent processes
were additionally read aloud to all participants. No incentives were given apart from refresh-
ments and certificates of participation. Interviewers were trained in working with vulnerable
youth and confidentiality was maintained except when participants were at risk of significant
harm or requested assistance. Where adolescents reported recent abuse, rape, or risk of signifi-
cant harm, referrals were made to child protection, HIV/AIDS, and health services,with fol-
low-up support. Social Protection and the Sustainable Development Goals protection scales),[28] transactional sexual exploitation (sex in exchange for food, shelter,
school fees, transport, or money) or age-disparate sex (sexual partner more than five years
older than the adolescent).[21] Lack of access to sexual and reproductive health (SDG 5.6) was
measured for adolescent girls only, as pregnancy or childbirth prior to age 17. SDG 16: ‘Promote peacefuland inclusive societies’. Adolescent violence perpetration
(SDG 16.1) was measured using violence items from the delinquency and aggression subscales
of the Child Behaviour Checklist,[29, 30] and included past-month robbery, vandalism, carry-
ing of a knife or a gun. SDG 1.3: Social Protection. Due to strong evidence that both cash provision and psycho-
social care require sustained and predictable duration in order to maintain effects on child
development,[31] each type of cash and care was coded positively only if received at both base-
line and one-year follow-up. Variables measured receipt of ‘cash only’, ‘care only’, and ‘cash
plus care’. ‘Cash’ was grouped as either direct cash transfers or ‘in kind’ transfers of free education and
food, following evidence that families use cash primarily for food and school expenses.[32]
Thus, ‘cash’ social protection was measured as accessing one or more of child-focusedcash
transfer (household access to either a government Child Support or Foster Child grant),[33] or
free schooling (free school and textbooks) and school feeding (daily, free school-provided
meals). Access to ‘care’ social protection was sustained receipt of 1 of positive parenting (e.g. pri-
mary caregiver praise and warmth) and good parental monitoring (e.g. household rules and
consistent supervision),measured using the Alabama Parenting Questionnaire,[34] and
teacher social support (social, practical and emotional) using a standardized scale [35] and
dichotomized as ‘high support’. Covariates Covariates included both baseline socio-demographics and potential confounders of social pro-
tection access or SDG outcomes, including predictors for grant receipt. These included adoles-
cent age (>13), urban/rural site (using census definitions), and informal housing. Poverty was
measured using the SA Social Attitudes survey’s basic necessities for children and coded as
missing more than two necessities [36]. Household employment whether anyone in the house-
hold had a job (full or part-time). Number of children (>2) in the household used a ‘household
map’. Female primary caregiver was measured, with primary caregiver identified as ‘the person
who lives with you and looks after you most’. Possession of a birth certificate was measured as
a potential predictor of cash and school access. Outcome measures Five SDGs were identified as highly relevant to adolescent health: hunger (SDG 2), health
(SDG 3), education (SDG 4), gender equality (SDG 5), and peaceful societies (SDG 16). Within
each goal, indicators of negative health outcomes were selected that were measurable and had
potential to be impacted by social protection. All measures were taken at baseline and follow-
up. SDG 2 ‘End hunger, achieve food security’. Adolescent hunger (SDG 2.1) was measured
using SA National Food Consumption Survey items[19] and defined as experiencingmore
than one day of insufficient food in the home during the past week. SDG 3 ‘Ensure healthy lives’. HIV-risk behaviour (SDG 3.3) was assessed as one or more
of four high-risk behaviours from the National Survey of HIV and Sexual Behaviour amongst
Young South Africans.[20, 21] Unprotected sex was inconsistent/no condom use during past
year sex; multiple sexual partners was three or more past-year partners,[22] sex whilst using
substances included inebriation or any drug use; early debut was initiation of sex below age 15. Tuberculosis (SDG 3.3) was measured as pulmonary tuberculosis disease using an 8-item
WHO symptom checklist.[23] A conservative threshold was set of four or more symptoms of
fever, discoloured sputum, fatigue, weight loss, and night sweats in addition to two or more
symptoms of coughing blood, chest pains, and cough for more than three weeks.[24] Mental
health risk (SDG 3.4) used standardized psychometric tools, measuring one or more clinical-
level disorder of depression (Child Depression Inventory Short Form),[25] anxiety (Revised
Children’s Manifest Anxiety Scale),[26] or suicidality (MINI International Psychiatric Inter-
view for Children and Adolescents).[27] Substance and alcohol misuse (SDG 3.5) used 15 items
from the National Survey of HIV and Risk Behaviour [21] and included past-year weekly or
more frequent alcohol use, inebriation, and any drug use including marijuana, mandrax or
crystal methamphetamine. SDG 4 ‘Inclusive and equitable education’. School non-enrolment (SDG 4.1) was school
dropout (prior to completion of senior school) due to any cause. SDG 5 ‘Achieve gender equality and empower women and girls’. Sexual violence and
exploitation of girls (SDG 5.2) was any of past-year sexual abuse, rape (using UNICEF child 4 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Socio-demographics, SDG indicators and social protection As shown in Table 1, the sample was 56.7% female and food insecurity was 34.7% (girls) and
29.1% (boys). Rates of past-year HIV-risk behaviour and mental health disorder ranged from
11.0–20.5%. School dropout was 4.9% (girls) and 3.5% (boys) and past-year sexual violence
was 10.1% (girls) and 5.9% (boys). Self-reportedviolence perpetration was 9.3% (girls) and
13.9% (boys). Receipt of social protection was equal for girls and boys: ‘cash’ alone 43.0/43.3%,
‘care’ alone 10.3/10.8%, and ‘cash plus care’ 34.9/31.5%. Adolescents deceased or lost to follow-up Adolescents deceasedor lost to follow-up (n = 114, 3.3%) did not differ at baseline from those
retained on rates of TB, school dropout, sexual violence/exploitation, pregnancy or violence
perpetration,receipt of ‘cash’ or ‘care’. However, they were more food insecure (χ2 = 8.99,
p = 0.003) and reported more HIV-risks (χ2 = 7.26, p = 0.007), substance use (χ2 = 8.18,
p = 0.004), and mental health disorder (χ2 = 5.15, p = 0.023). Although a one-year follow-up of
96.8% is extremely high, some of the most vulnerable participants were deceasedor untraceable
and thus findings may slightly under-estimate risks. Social Protection and the Sustainable Development Goals only those covariates and social protection factors significant at p < .05. Fourth, we checked
for potential effect modifications, arising from interactions between cash and care social pro-
tections, and between them and covariates. Using the third, final model for each SDG indicator
(, we ran multivariable regressions with interaction terms for cash and care, for cash and any
significant covariate, and for care and any significant covariate. Fifth, significant instances of
social protection predictors and covariates were entered into marginal effects analyses in
STATA. This demonstrated how the predicted probability of the outcome may change when
cash or care were accessed, as well as showing potential additive effects of cash plus care, whilst
holding covariates at mean values. For SDG 5.6 (pregnancy or childbirth), results are shown
only for girls. Analyses Analyses were conducted in five stages disaggregated by gender using SPSS 22 and Stata 13. First, we tested for differences between youth lost and retained at follow-up on baseline socio-
demographic characteristics, SDG health indicators, and social protection access. Second,
using the sample of adolescents retained across both time points (n = 3401, 97%), we examined
socio-demographics,negative SDG health outcomes, and social protection access. Third, we
tested associations between each SDG indicator and dummy variables for ‘cash’ and ‘care’
social protection, following the sequential approach recommended by Hosmer and Lemeshow
[37]. For each SDG indicator at follow-up as the outcome, we ran three logistic regression
models, each controlling for that SDG indicator at baseline [38]. The first model included all
potential covariates alongside the two social protection factors. The second model retained
covariates and social protection factors significant at p < .10, and the third model included 5 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Associations of social protection and SDG health indicators Logistic regression models, disaggregated by gender, showed that social protection was associ-
ated with reduced risks in 12 of 17 measured health-related indicators pertaining to five SDG
goals. For pulmonary tuberculosis, no associations were shown for either boys or girls. Regres-
sions controlled for covariates and baseline SDG risk. For five SDG indicators (HIV-risk
behaviour, tuberculosis, substance use, school non-enrolment and hunger) patterns of associa-
tion with social protection showed similar risk-reduction effects amongst boys and girls. For
sexual violence, risk-reduction associations were only shown for girls, and for violence perpe-
tration and mental health, risk-reduction associations were only shown for boys. There were
no associations showing social protection increasing risk for either gender. For boys (see Table 2), cash social protection was significantly associated with five SDG
indicators: reduced HIV-risk behaviour (OR 0.69 CI 0.50–0.95); reduced mental health disor-
der (OR 0.67 CI 0.47–0.96); reduced substance use (OR 0.61 CI 0.42–0.89); reduced school
dropout (OR 0.10 CI 0.05–0.19) and reduced violence perpetration (OR 0.67 CI 0.48–0.93). Care social protection was significantly associated with four SDG indicators: reduced hunger
(OR 0.50 CI 0.34–0.73); reduced HIV-risk behaviour (OR 0.56 CI 0.41–0.77); reduced sub-
stance use (OR 0.36 CI 0.23–0.57) and reduced violence perpetration (OR 0.59 CI 0.43–0.81)
(Table 2). 6 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Social Protection and the Sustainable Development Goals Table 1. Socio-demographic characteristics of the sample. PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Social Protection and the Sustainable Development Goals Table 1. (Continued)
Girls (n = 1926)
Boys (n = 1475)
Sustainable Development Goals
2.1 Hunger (1 or more day/week)
668 (34.7%)
429 (29.1%)***
3.3 HIV risk behaviour
262 (13.6%)
302 (20.5%)***
3.4 Tuberculosis
60 (3.1%)
31 (2.1%)
3.4 Mental health risk
281 (14.6%)
162 (11.0%)**
3.5 Substance Abuse
106 (5.5%)
143 (9.7%)***
4.1 School non-enrolment
95 (4.9%)
52 (3.5%)*
5.2 Sexual violence or exploitation
195 (10.1%)
87 (5.9%)***
5.6 Pregnancy
71 (3.7%)
-
16.1 Violence perpetration
180 (9.3%)
205 (13.9%)***
Social protection
Cash only
829 (43.0%)
639 (43.3%)
Care only
199 (10.3%)
159 (10.8%)
Cash plus care
672 (34.9%)
464 (31.5%)*
Covariates
Adolescent aged over 13
936 (48.6%)
705 (47.8%)
Rural location
969 (50.3%)
712 (48.3%)
3 or more children in the home
948 (49.2%)
650 (44.1%)**
2 or more basic necessities missing
1137 (59.0%)
845 (57.3%)
Informal housing
624 (32.4%)
444 (30.1%)
Job in household
1454 (75.5%)
1136 (71.1%)
Child has birth certificate
1829 (95.0%)
1414 (95.9%)
Female primary caregiver
1759 (91.3%)
1297 (87.9%)**
*** p < .001
** p < .01
* p < .05. p values associated with Chi Square tests. doi:10.1371/journal.pone.0164808.t001 No interactive effects between cash and care were shown for boys, but additive effects of
cash and care were associated with greater risk reductions in three SDG indicators (see Fig 1). Among boys, substance use incidence in the past year was 13.6% without cash and care provi-
sion, 8.7% with cash provision, 5.4% with care, and 3.3% with cash plus care. For violence per-
petration, incidence was 20.1% without cash and care provision, 14.4% with cash provision,
12.9% with care alone, and 9.0% with cash plus care,. For HIV-risk behaviour, incidence was
23% without cash and care, 17.5% with cash alone, 14.7% with care alone, and 10.6% with cash
plus care. There was only one statistically significant interaction of cash or care with a socio-demo-
graphic covariate, among boys: poverty and care on the SDG indicator of hunger. Among boys
who were less poor, care had a markedly greater effect on reducing hunger (36.1% reduced to
21.8%) than among boys who were poorer (46.6% reduced to 44.2%) (see Fig 2A). Associations of social protection and SDG health indicators Girls (n = 1926)
Boys (n = 1475)
Sustainable Development Goals
2.1 Hunger (1 or more day/week)
668 (34.7%)
429 (29.1%)***
3.3 HIV risk behaviour
262 (13.6%)
302 (20.5%)***
3.4 Tuberculosis
60 (3.1%)
31 (2.1%)
3.4 Mental health risk
281 (14.6%)
162 (11.0%)**
3.5 Substance Abuse
106 (5.5%)
143 (9.7%)***
4.1 School non-enrolment
95 (4.9%)
52 (3.5%)*
5.2 Sexual violence or exploitation
195 (10.1%)
87 (5.9%)***
5.6 Pregnancy
71 (3.7%)
-
16.1 Violence perpetration
180 (9.3%)
205 (13.9%)***
Social protection
Cash only
829 (43.0%)
639 (43.3%)
Care only
199 (10.3%)
159 (10.8%)
Cash plus care
672 (34.9%)
464 (31.5%)*
Covariates
Adolescent aged over 13
936 (48.6%)
705 (47.8%)
Rural location
969 (50.3%)
712 (48.3%)
3 or more children in the home
948 (49.2%)
650 (44.1%)**
2 or more basic necessities missing
1137 (59.0%)
845 (57.3%)
Informal housing
624 (32.4%)
444 (30.1%)
Job in household
1454 (75.5%)
1136 (71.1%)
Child has birth certificate
1829 (95.0%)
1414 (95.9%)
Female primary caregiver
1759 (91.3%)
1297 (87.9%)**
Girls (n = 1926)
Boys (n = 1475)
Sustainable Development Goals
2.1 Hunger (1 or more day/week)
668 (34.7%)
429 (29.1%)***
3.3 HIV risk behaviour
262 (13.6%)
302 (20.5%)***
3.4 Tuberculosis
60 (3.1%)
31 (2.1%)
3.4 Mental health risk
281 (14.6%)
162 (11.0%)**
3.5 Substance Abuse
106 (5.5%)
143 (9.7%)***
4.1 School non-enrolment
95 (4.9%)
52 (3.5%)*
5.2 Sexual violence or exploitation
195 (10.1%)
87 (5.9%)***
5.6 Pregnancy
71 (3.7%)
-
16.1 Violence perpetration
180 (9.3%)
205 (13.9%)***
Social protection
Cash only
829 (43.0%)
639 (43.3%)
Care only
199 (10.3%)
159 (10.8%)
Cash plus care
672 (34.9%)
464 (31.5%)*
Covariates
Adolescent aged over 13
936 (48.6%)
705 (47.8%)
Rural location
969 (50.3%)
712 (48.3%)
3 or more children in the home
948 (49.2%)
650 (44.1%)**
2 or more basic necessities missing
1137 (59.0%)
845 (57.3%)
Informal housing
624 (32.4%)
444 (30.1%)
Job in household
1454 (75.5%)
1136 (71.1%)
Child has birth certificate
1829 (95.0%)
1414 (95.9%)
Female primary caregiver
1759 (91.3%)
1297 (87.9%)**
(Continued) Table 1. Socio-demographic characteristics of the sample. PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 7 / 20 Social Protection and the Sustainable Development Goals Table 2. Logistic regression models showing associations between social protection receipt and SDG indicators among boys (N = 1475). As shown in Table 3, amongst girls, cash social protection was significantly associated with
five SDG indicators: reduced HIV-risk behaviour (OR 0.64 CI 0.46–0.87), reduced substance
abuse (OR 0.46 CI 0.30–0.70), reduced school dropout (OR 0.14 CI 0.09–0.24), reduced sexual
exploitation (OR 0.67 CI 0.48–0.93), and reduced pregnancy (OR 0.46 CI .27–0.78). Care social
protection was significantly associated with four SDG indicators: reduced hunger (p < .001 OR 8 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Social Protection and the Sustainable Development Goals Table 2. Step 1
SDG 2.1
Hunger
SDG 3.3 AIDS
SDG 3.3 TB
SDG 3.4
Mental Health
SDG 3.5
Substance
Abuse
4.1 School non-
enrolment
5.2 Sexual
violence or
exploitation
16.1 Violence
perpetration
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
(95%
CI)
(95%
CI)
(95%
CI)
(95%
CI)
(95%
CI)
(95% CI)
(95%
CI)
(95%
CI)
Cash
1.03
.841
0.67
.017
0.78
.56
0.71
0.074
0.57
.006
0.11
< .001
0.85
.535
0.67
.019
(0.78–
1.36)
(0.48–
0.93)
(0.34–
1.81)
(0.48–
1.04)
(0.39–
0.85)
(0.10–
0.21)
(0.52–
1.41)
(0.47–
0.94)
Care
0.74
.011
0.58
.001
0.66
.661
0.76
0.138
0.37
< .001
0.48
.063
0.67
.107
0.61
.004
(0.58–
0.93)
(0.42–
0.79)
(0.31–
1.43)
(0.53–
1.09)
(0.23–
0.57)
(0.23–
1.04)
(0.42–
1.09)
(0.44–
0.86)
Baseline
outcome
1.98
< .001
5.5
< .001
6.38
< .001
2.59
< .001
2.12
< .001
50.18
< .001
3.28
.002
1.59
.009
(1.56–
2.51)
(3.86–
7.85)
(2.56–
15.90)
(1.73–
3.90)
(1.45–
2.09)
(13.77–
182.94)
(1.54–
6.96)
(1.13–
2.24)
Older than 13
years
1.17
.172
4.19
< .001
0.69
.340
1.22
0.258
3.67
< .001
1.57
.192
2.63
< .001
1.44
.021
(0.93–
1.48)
(3.03–
5.80)
(0.32–
1.48)
(0.86–
1.72)
(2.41–
5.59)
(0.80–
3.10)
(1.60–
4.32)
(1.06–
1.97)
Urban
location
1.6
< .001
1.13
.414
0.73
.731
0.81
0.252
1.29
.190
1.35
.368
0.96
.867
1.09
.579
(1.27–
2.02)
(0.84–
1.53)
(0.34–
1.58)
(0.57–
1.15)
(0.88–
1.89)
(0.70–
2.58)
(0.60–
1.53)
(0.80–
1.50)
>3 children in
home
0.98
.856
0.78
.100
2.05
.060
0.94
0.735
0.64
.027
1.12
.746
0.87
.547
0.82
.202
(0.78–
1.23)
(0.58–
1.05)
(0.97–
4.33)
(0.67–
1.33)
(0.43–
0.95)
(0.57–
2.17)
(0.55–
1.37)
(0.60–
1.12)
Has birth
certificate
0.85
.562
1.07
.853
0.33
.094
0.83
0.64
0.68
.346
0.69
.527
1.28
.696
0.51
.035
(0.49–
1.48)
(0.52–
2.21)
(0.09–
1.21)
(0.39–
1.80)
(0.31–
1.52)
(0.22–
2.19)
(0.37–
4.37)
(0.27–
0.95)
Female
caregiver
0.87
.411
0.94
.787
1.16
.819
0.86
0.541
1.33
.309
0.59
.192
0.68
.203
1.18
.488
(0.62–
1.22)
(0.62–
1.43)
(0.34–
3.99)
(0.54–
1.39)
(0.77–
2.33)
(0.26–
1.31)
(0.38–
1.23)
(0.74–
1.88)
Missing >2
necessities
1.96
< .001
1.12
.473
1.87
.140
1.3
0.173
0.95
.810
3.53
.002
0.78
.295
0.99
.942
(1.52–
2.53)
(0.82–
1.54)
(0.82–
4.28)
(0.89–
1.88)
(0.64–
1.41)
(1.58–
7.86)
(0.49–
1.25)
(0.71–
1.37)
Informal
housing
1.58
< .001
1.43
.026
0.67
.384
1.4
0.07
1.18
.414
1.76
.092
0.69
.176
1.18
.342
(1.23–
2.03)
(1.05–
1.97)
(0.27–
1.66)
(0.97–
2.01)
(0.79–
1.75)
(0.91–
3.41)
(0.41–
1.18)
(0.84–
1.64)
Job in
household
0.83
.841
0.94
.528
1.44
.104
0.98
0.882
1.01
.912
0.74
.162
1.11
.440
1.15
.139
(0.72–
0.95)
(0.78–
1.13)
(0.93–
2.23)
(0.80–
1.22)
(0.80–
1.28)
(0.49–
1.13)
(0.85–
1.47)
(0.96–
1.39)
Constant
0.23
< .001
0.1
< .001
0.04
0.004
0.2
0.004
0.06
< .001
0.06
0.003
0.06
< .001
0.25
0.005
Step 2
SDG 2.1
Hunger
SDG 3.3 AIDS
SDG 3.3 TB
SDG 3.4
Mental Health
SDG 3.5
Substance
Abuse
4.1 School non-
enrolment
5.2 Sexual
violence or
exploitation
16.1 Violence
perpetration
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
(95%
CI)
(95%
CI)
(95%
CI)
(95%
CI)
(95%
CI)
(95% CI)
(95%
CI)
(95%
CI)
Cash
-
-
0.71
.033
-
-
0.67
.027
0.61
0.011
0.1
< .001
-
-
0.67
.016
(Continued)
PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016
9 / 20 (Continued) PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 9 / 20 For HIV-risk
behaviour, incidence was 14.5% without cash and care, 10.1% with cash provision, 9.7% with
care, and 6.7% with cash plus care. For sexual exploitation, incidence was 14.3%, without cash
or care, 10.9% with cash provision, 10.3% with care and 7.7% with cash plus care. Note. For each outcome, interactions between (a) cash and care and (b) each of cash and care and the covariates in the model were tested. None were
statistically significant except for the interaction between care and missing necessities for the outcome hunger, as shown in step 3, and thus only significant
effects are illustrated. doi:10.1371/journal.pone.0164808.t002
PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016
11 / 20 Note. For each outcome, interactions between (a) cash and care and (b) each of cash and care and the covariates in the model were tested. None were
statistically significant except for the interaction between care and missing necessities for the outcome hunger, as shown in step 3, and thus only significant
effects are illustrated. 0.63 CI 0.51–0.77); reduced HIV-risk behaviour (OR 0.67 CI 0.49–0.91); reduced substance use
(OR 0.32 CI 0.19–0.53); and reduced sexual exploitation (OR 0.71 CI 0.52–0.98). No interactive effects between cash and care were shown for girls, but additive effects of
cash and care were associated with maximised risk reductions in three SDG indicators (see Fig
1). Among girls, substance use incidence in the past year was 10.5% without cash or care provi-
sion, 5.1% with cash provision, 3.6% with care, and 1.7% with cash plus care. For HIV-risk
behaviour, incidence was 14.5% without cash and care, 10.1% with cash provision, 9.7% with
care, and 6.7% with cash plus care. For sexual exploitation, incidence was 14.3%, without cash
or care, 10.9% with cash provision, 10.3% with care and 7.7% with cash plus care. 0.63 CI 0.51–0.77); reduced HIV-risk behaviour (OR 0.67 CI 0.49–0.91); reduced substance use
(OR 0.32 CI 0.19–0.53); and reduced sexual exploitation (OR 0.71 CI 0.52–0.98). (
)
p
(
)
No interactive effects between cash and care were shown for girls, but additive effects of
cash and care were associated with maximised risk reductions in three SDG indicators (see Fig
1). Among girls, substance use incidence in the past year was 10.5% without cash or care provi-
sion, 5.1% with cash provision, 3.6% with care, and 1.7% with cash plus care. (Continued)
(.52-
.97)
(0.47–
0.96)
(0.42–
0.89)
(0.05–
0.19)
(0.48–
0.93)
Care
0.73
.008
0.57
< .001
-
-
-
-
0.36
< .001
0.5
.072
-
-
0.59
.001
(0.58–
0.92)
(.41-
.77)
(0.23–
0.57)
(0.24–
1.06)
(0.43–
0.81)
Baseline
outcome
1.98
< .001
5.46
< .001
-
-
2.78
< .001
2.22
< .001
53.1
< .001
-
-
1.58
.009
(1.57–
2.51)
(3.83–
7.77)
(1.87–
4.15)
(1.53–
3.22)
(15.12–
186.51)
(1.12–
2.22)
Older than 13
years
-
-
4.23
< .001
-
-
-
-
3.63
< .001
-
-
-
-
1.45
.017
(3.07–
5.84)
(2.40–
5.51)
(1.07–
1.98)
Urban
location
1.63
< .001
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(1.30–
2.05)
>3 children in
home
-
-
0.77
.088
-
-
-
-
0.63
0.017
-
-
-
-
-
-
(.57–
1.04)
(0.42–
0.92)
Has birth
certificate
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.49
.025
(0.26–
0.91)
Female
caregiver
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Missing >2
necessities
1.95
< .001
-
-
-
-
-
-
-
-
4.13
< .001
-
-
-
-
(1.52–
2.51)
(1.89–
9.00)
Informal
housing
1.61
< .001
1.45
.018
-
-
1.62
.006
-
-
1.76
.082
-
-
-
-
(1.26–
2.06)
(1.06–
1.97)
(1.15–
2.28)
(0.93–
3.34)
Job in
household
0.83
.010
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(0.72–
0.96)
Constant
0.18
< .001
0.12
< .001
-
-
0.12
< .001
0.09
< .001
0.04
< .001
-
-
0.38
.005
Step 3
SDG 2.1
Hunger
SDG 3.3 AIDS
SDG 3.3 TB
SDG 3.4
Mental Health
SDG 3.5
Substance
Abuse
4.1 School non-
enrolment
5.2 Sexual
violence or
exploitation
16.1 Violence
perpetration
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
AOR
p
(95%
CI)
(95%
CI)
(95%
CI)
(95%
CI)
(95%
CI)
(95% CI)
(95%
CI)
(95%
CI)
Cash
-
-
0.69
.021
-
-
0.67
.027
0.61
.011
0.1
< .001
-
-
0.67
.016
(0.50–
0.95)
(0.47–
0.96)
(0.42–
0.89)
(0.05–
0.19)
(0.48–
0.93)
Care
0.5
< .001
0.56
< .001
-
-
-
-
0.36
< .001
-
-
-
-
0.59
.001
(0.34–
0.73)
(0.41–
0.77)
(0.23–
0.57)
(0.43–
0.81)
Baseline
outcome
1.98
< .001
5.59
< .001
-
-
2.78
< .001
2.22
< .001
56.26
< .001
-
-
1.58
.009
(Continued) PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 10 / 20 Social Protection and the Sustainable Development Goals Table 2. (Continued)
(1.57–
2.51)
(3.93–
7.95)
(1.87–
4.15)
(1.53–
3.22)
(16.12–
196.36)
(1.12–
2.22)
Older than 13
years
-
-
4.2
< .001
-
-
-
-
3.63
< .001
-
-
-
-
1.45
.017
(3.05–
5.80)
(2.40–
5.51)
(1.07–
1.98)
Urban
location
1.59
< .001
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(1.27–
2.01)
>3 children in
home
-
-
-
-
-
-
-
-
0.63
.017
-
-
-
-
-
-
(0.42–
0.92)
Has birth
certificate
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.49
.025
(0.26–
0.91)
Female
caregiver
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Missing >2
necessities
1.53
.008
-
-
-
-
-
-
-
-
5.13
< .001
-
-
-
-
(1.12–
2.09)
(2.40–
10.95)
Informal
housing
1.61
< .001
1.5
.010
-
-
1.62
.006
-
-
-
-
-
-
-
-
(1.26–
2.05)
(1.10–
2.03)
(1.15–
2.28)
Job in
household
0.83
.010
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(0.72–
0.96)
Care*
Missing >2
necessities
1.84
.013
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(1.14–
2.98)
Constant
0.22
< .001
0.07
< .001
-
-
0.12
< .001
0.09
< .001
0.03
< .001
-
-
0.38
.005
Note. For each outcome, interactions between (a) cash and care and (b) each of cash and care and the covariates in the model were tested. None were
statistically significant except for the interaction between care and missing necessities for the outcome hunger, as shown in step 3, and thus only significant
effects are illustrated. 0.63 CI 0.51–0.77); reduced HIV-risk behaviour (OR 0.67 CI 0.49–0.91); reduced substance use
(OR 0.32 CI 0.19–0.53); and reduced sexual exploitation (OR 0.71 CI 0.52–0.98). No interactive effects between cash and care were shown for girls, but additive effects of
cash and care were associated with maximised risk reductions in three SDG indicators (see Fig
1). Among girls, substance use incidence in the past year was 10.5% without cash or care provi-
sion, 5.1% with cash provision, 3.6% with care, and 1.7% with cash plus care. For HIV-risk
behaviour, incidence was 14.5% without cash and care, 10.1% with cash provision, 9.7% with
care, and 6.7% with cash plus care. For sexual exploitation, incidence was 14.3%, without cash
or care, 10.9% with cash provision, 10.3% with care and 7.7% with cash plus care. 11 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Social Protection and the Sustainable Development Goals Fig 1. Predicted percent probabilities of SDG indicators when ‘cash,’ ‘care,’ ‘cash and care,’ or ‘no provision’ (‘none’) are received, holding all
other covariates in the logistic regression model at their average levels (see Tables 2 and 3 for variables included in final predictive model for
each outcome). Error bars indicate 95% confidence intervals. doi:10 1371/journal pone 0164808 g001 Fig 1. Predicted percent probabilities of SDG indicators when ‘cash,’ ‘care,’ ‘cash and care,’ or ‘no provision’ (‘none’) are received, holding all
other covariates in the logistic regression model at their average levels (see Tables 2 and 3 for variables included in final predictive model for
)
f Fig 1. Predicted percent probabilities of SDG indicators when ‘cash,’ ‘care,’ ‘cash and care,’ or ‘no provision’ (‘none’) are received, holding all
other covariates in the logistic regression model at their average levels (see Tables 2 and 3 for variables included in final predictive model for
each outcome). Error bars indicate 95% confidence intervals. doi:10.1371/journal.pone.0164808.g001 doi:10.1371/journal.pone.0164808.g001 There was only one statistically significant interaction of cash or care with a socio-demo-
graphic covariate: poverty x care on the SDG indicator of school dropout. Among girls who
were poorer, care had a greater effect on reducing school dropout (4.4% to 1.4%) than among
girls who were less poor (1.1% to 1.5%) (see Fig 2B). PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Discussion The SDGs represent opportunities for improving the health of our highest-risk populations. But to achieve these goals, interventions are preferable that have effects across multiple out-
comes [17]. This study examines the contribution of social protection provision, a core policy
target of the SDGs, on indicators across five SDG goals, amongst deprived South African
adolescents. Findings show a remarkable range of associations with improved SDG outcomes. In 12 of
17 gender-disaggregated indicators cash and/or care were associated with significant reduc-
tions in adolescent health-related risks. These positive associations spanned all five goals for
which measures were available: hunger (SDG 2), health (SDG 3), education (SDG 4), gender
equality (SDG 5) and peaceful societies (SDG 16). Thus, social protection seems to positively
impact multiple domains of adolescent health and wellbeing. This study also provides evidence on components and combinations of social protection. Cash support had independent risk reduction effects for ten SDG indicators, and care support
had independent risk reduction effects for eight SDG indicators. However, for many SDG 12 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Social Protection and the Sustainable Development Goals Fig 2. Panel A shows the predicted percent probabilities of adolescent hunger among boys for the interaction between missing necessities and receipt
of ‘care’ provisions. Panel B shows the predicted percent probabilities of school non-enrolment among girls for the interaction between missing
necessities and receipt of care provisions. Fig 2. Panel A shows the predicted percent probabilities of adolescent hunger among boys for the interaction between missing necessities and receipt
of ‘care’ provisions. Panel B shows the predicted percent probabilities of school non-enrolment among girls for the interaction between missing
necessities and receipt of care provisions. doi:10.1371/journal.pone.0164808.g002 health-related targets, strong additive effects were shown of combining cash plus care: with
cumulative risk reduction effects shown on six SDG indicators. Overall, combination social
protection may be an effective way to maximise health and wellbeing benefits for high-risk
adolescents. There are a number of potential mechanisms for the impacts of social protection on multi-
ple health-related outcomes. Prior research has shown that cash transfers work in multiple
ways within a household and are primarily used by families for food and education costs [39]. Qualitative and quantitative evidence suggests that they reduce need for adolescents to have
transactional sexual relationships with older partners who provide food and essential financial
support to the household [12]. PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Social Protection and the Sustainable Development Goals Table 3. Logistic regression models showing associations between social protection receipt and SDG indicators among girls (N = 1926). Discussion There is also evidence that improved supervision and monitor-
ing of adolescents is associated with lower exposure to community violence, risk of sexual
exploitation and pregnancy [40]. Finally, evidence from the parenting support literature has
demonstrated that improved caregiver-child relationships can lead to improved mental health
outcomes within the family [41]. Evidence regarding combination ‘cash plus care’ mechanisms
is still emerging, but one study of HIV risk reduction suggests that the two types of support
work in complementary ways and may target different stages in pathways to risk–for example
cash reduces the impact of structural deprivations on family psychososial problems (such as
abuse, child behaviour problems), and care targets those psychosocial problems directly [42]. Elucidating the mechanisms of cash plus care for other outcomes is clearly an area for future
research. In the course of checking for possible interaction effects, it emerged that the effect of care
provision may be modifiedby adolescents’ level of poverty. Amongst boys, care provision was
associated with reduced hunger, but only for those at lower levels of poverty. It may be that
improved parental care resulted in greater food allocation to children in the family, but where
poverty was very severe, even the most caring of parents did not have opportunities to reduce
their adolescents’ hunger levels. Amongst girls, those living in severe poverty were most likely
to drop out of school, but for these girls, care provision had the greatest effect, reducing school PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 13 / 20 Step 1
SDG 2.1
Hunger
SDG 3.3
AIDS
SDG 3.3 TB
SDG 3.4
Mental
Health
SDG 3.5
Substance
Abuse
4.1 School
non-
enrolment
5.2 Sexual
violence or
exploitation
5.6
Pregnancy
16.1 Violence
perpetration
AOR
AOR
AOR
AOR
AOR
AOR
AOR
AOR
AOR
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
Cash
0.98
.887
0.64
.008
1.09
.809
1.05
.775
0.45
< .001
0.13
< .001
0.68
.030
0.49
.011
1.13
.554
(0.77–1.26)
(0.45–0.89)
(0.56–2.10)
(0.76–1.45)
(0.29–0.70)
(0.08–0.23)
(0.47–0.96)
(0.28–0.85)
(.76–
1.68)
Care
0.63
< .001
0.69
.023
1.10
.718
0.94
.664
0.33
< .001
0.52
.017
0.71
.042
0.63
.116
0.92
.922
(0.51–0.76)
(0.50–0.95)
(0.65–1.88)
(0.71–1.24)
(0.20–0.55)
(0.30–0.89)
(0.51–0.99)
(0.36–1.12)
(.67–
1.28)
Baseline
outcome
2.06
< .001
7.23
< .001
1.94
.115
3.31
< .001
2.16
< .001
7.46
< .001
4.07
< .001
15.77
< .001
1.31
.235
(1.69–2.53)
(5.06–10.34)
(0.85–4.43)
(2.49–4.40)
(1.40–3.34)
(3.30–16.88)
(2.74–6.06)
(7.75–32.09)
(.84–
2.06)
Older than 13
years
1.10
.352
4.45
< .001
0.85
.560
1.27
.089
2.33
< .001
2.74
.001
2.49
< .001
11.58
< .001
1.24
.187
(0.90–1.34)
(3.07–6.45)
(0.50–1.45)
(0.97–1.66)
(1.47–3.70)
(1.54–4.86)
(1.76–3.53)
(4.12–32.58
(.91–
1.70)
Urban location
1.38
.002
1.12
.485
0.74
.276
0.82
.154
0.88
.547
1.20
.462
1.14
.434
0.72
.258
1.20
.267
(1.13–1.69)
(0.82–1.52)
(0.43–1.27)
(0.63–1.08)
(0.58–1.34)
(0.73–1.97)
(0.82–1.58)
(0.41–1.27)
(.87–
1.66)
>3 children in
home
1.08
.433
0.77
.092
1.78
.038
0.87
.302
0.66
.056
1.01
.975
0.99
.972
0.72
.239
0.61
.002
(0.89–1.32)
(0.57–1.04)
(1.03–3.05)
(0.67–1.13)
(0.43–1.01)
(0.62–1.65)
(0.73–1.36)
(0.42–1.24)
(.44-
.83)
Has birth
certificate
1.05
.832
1.21
.546
0.69
.489
0.65
.647
1.24
.620
0.39
.008
0.86
.635
0.83
.677
1.03
.935
(0.67–1.64)
(0.65–2.24)
(0.24–2.00)
(0.38–1.09)
(0.53–2.86)
(0.20–0.78)
(0.46–1.60)
(.34–
2.04)
(.50–
2.12)
Female
caregiver
0.92
.617
0.96
.879
1.94
.115
1.24
.378
1.11
.781
0.81
.596
0.73
.216
2.67
.131
1.46
.229
(0.65–1.29)
(0.58–1.59)
(0.85–4.43)
(0.77–2.00)
(0.55–2.23)
(0.38–1.75)
(0.45–1.20)
(.75–
9.52)
(.79–
2.69)
Missing >2
necessities
1.86
< .001
1.29
.133
1.33
.329
1.22
.185
1.25
.342
2.22
.008
1.21
.284
1.22
.511
1.01
.948
(1.50–2.31)
(0.93–1.79)
(0.75–2.34)
(0.91–1.63)
(0.79–1.98)
(1.24–4.00)
(0.86–1.70)
(0.68–2.21)
(.72–
1.42)
Informal
housing
1.77
< .001
0.95
.767
0.46
.028
1.04
.797
1.04
.854
1.23
.437
0.78
.181
0.40
.007
1.01
.621
(1.43–2.19)
(0.68–1.33)
(0.23–0.92)
(0.78–1.39)
(0.67–1.64)
(0.73–2.07)
(0.55–1.12)
(0.20–0.78)
(.77–
1.54)
Job in
household
0.95
.379
0.93
.475
0.98
.899
0.95
.545
1.02
.872
0.67
.672
0.98
.845
0.78
.174
0.98
.827
(0.84–1.07)
(0.77–1.13)
(0.72–1.33)
(0.81–1.12)
(0.79–1.33)
(0.48–0.94)
(0.81–1.19)
(0.55–1.11)
(.81–
1.17)
Constant
0.23
< .001
0.51
< .001
0.08
.004
0.18
< .001
0.06
< .001
0.15
.013
0.10
< .001
0.01
< .001
0.05
< .001
Step 2
SDG 2.1
Hunger
SDG 3.3
AIDS
SDG 3.3 TB
SDG 3.4
Mental
Health
SDG 3.5
Substance
Abuse
4.1 School
non-
enrolment
5.2 Sexual
violence or
exploitation
5.6
Pregnancy
16.1 Violence
perpetration
AOR
AOR
AOR
AOR
AOR
AOR
AOR
AOR
AOR
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
Cash
-
-
0.66
.013
-
-
-
-
0.46
< .001
0.14
< .001
0.67
.018
0.46
.004
-
-
(0.48–0.92)
(0.30–0.70)
(0.09–0.23)
(0.48–0.93)
(0.27–0.78)
Care
0.63
< .001
0.67
.012
-
-
-
-
0.32
< .001
0.50
.011
0.71
.038
-
-
-
-
(Continued) Table 3. Logistic regression models showing associations between social protection receipt and SDG Table 3. Logistic regression models showing associations between social pr PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 14 / 20 Social Protection and the Sustainable Development Goals Table 3. (Continued)
(0.51–0.77)
(0.49–0.92)
(0.19–0.53)
(0.29–0.85)
(0.52–0.98)
Baseline
outcome
2.07
< .001
7.36
< .001
-
-
-
-
2.20
< .001
7.84
< .001
4.29
< .001
15.87
< .001
-
-
(1.70–2.54)
(5.18–10.47)
(1.45–3.32)
(3.53–17.42)
(2.90–6.35)
(8.04–31.30)
Older than 13
years
-
-
4.50
< .001
-
-
-
-
2.32
< .001
2.76
< .001
2.53
< .001
12.13
< .001
-
-
(3.10–6.51)
(1.47–3.68)
(1.56–4.89)
(1.79–3.58)
(4.33–33.96)
Urban location
1.37
.002
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(1.13–1.67)
>3 children in
home
-
-
0.77
.083
-
-
-
-
0.65
.050
-
-
-
-
-
-
-
-
(0.57–1.04)
(0.43–1.00)
Has birth
certificate
-
-
-
-
-
-
-
-
-
-
0.38
.006
-
-
-
-
-
-
(0.20–0.76)
Female
caregiver
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Missing >2
necessities
1.87
< .001
-
-
-
-
-
-
-
-
2.35
.004
-
-
-
-
-
-
(1.51–2.31)
(1.32–4.18)
Informal
housing
1.75
< .001
-
-
-
-
-
-
-
-
-
-
-
-
0.48
.025
-
-
(1.42–2.16)
(0.25–0.91)
Job in
household
-
-
-
-
-
-
-
-
-
-
.68
.020
-
-
-
-
-
-
(0.49–0.94)
Constant
0.23
< .001
0.07
< .001
-
-
-
-
0.08
< .001
.17
.001
0.08
< .001
0.01
< .001
Step 3
SDG 2.1
Hunger
SDG 3.3
AIDS
SDG 3.3 TB
SDG 3.4
Mental
Health
SDG 3.5
Substance
Abuse
4.1 School
non-
enrolment
5.2 Sexual
violence or
exploitation
5.6
Pregnancy
16.1 Violence
perpetration
AOR
AOR
AOR
AOR
AOR
AOR
AOR
AOR
AOR
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
Cash
-
-
0.64
.005
-
-
-
-
0.46
< .001
0.14
< .001
0.67
.018
0.46
.004
-
-
(0.46–0.87)
(0.30–0.70)
(0.09–0.24)
(0.48–0.93)
(0.27–0.78)
Care
0.63
< .001
0.67
.010
-
-
-
-
0.32
< .001
1.49
.439
0.71
.038
-
-
-
-
(0.51–0.77)
(.49-
.91)
(0.19–0.53)
(0.54–4.10)
(0.52–0.98)
Baseline
outcome
2.07
< .001
7.30
< .001
-
-
-
-
2.20
< .001
8.60
< .001
4.29
< .001
15.87
< .001
-
-
(1.70–2.54)
(5.14–10.39)
(1.45–3.32)
(3.80–19.49)
(2.90–6.35)
(8.04–31.30)
Older than 13
years
-
-
4.44
< .001
-
-
-
-
2.32
< .001
2.82
< .001
2.53
< .001
12.13
< .001
-
-
(3.07–6.43)
(1.47–3.68)
(1.59–5.00)
(1.79–3.58)
(4.33–33.96)
Urban location
1.37
.002
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(1.13–1.67)
>3 children in
home
-
-
-
-
-
-
-
-
0.65
.050
-
-
-
-
-
-
-
-
(0.43–1.00)
Has birth
certificate
-
-
-
-
-
-
-
-
-
-
0.35
.003
-
-
-
-
-
-
(0.18–0.70)
Female
caregiver
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(Continued) Table 3. (Continued)
(0.51–0.77)
(0.49–0.92)
(0.19–0.53)
(0.29–0.85)
(0.52–0.98)
Baseline
outcome
2.07
< .001
7.36
< .001
-
-
-
-
2.20
< .001
7.84
< .001
4.29
< .001
15.87
< .001
-
-
(1.70–2.54)
(5.18–10.47)
(1.45–3.32)
(3.53–17.42)
(2.90–6.35)
(8.04–31.30)
Older than 13
years
-
-
4.50
< .001
-
-
-
-
2.32
< .001
2.76
< .001
2.53
< .001
12.13
< .001
-
-
(3.10–6.51)
(1.47–3.68)
(1.56–4.89)
(1.79–3.58)
(4.33–33.96)
Urban location
1.37
.002
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(1.13–1.67)
>3 children in
home
-
-
0.77
.083
-
-
-
-
0.65
.050
-
-
-
-
-
-
-
-
(0.57–1.04)
(0.43–1.00)
Has birth
certificate
-
-
-
-
-
-
-
-
-
-
0.38
.006
-
-
-
-
-
-
(0.20–0.76)
Female
caregiver
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Missing >2
necessities
1.87
< .001
-
-
-
-
-
-
-
-
2.35
.004
-
-
-
-
-
-
(1.51–2.31)
(1.32–4.18)
Informal
housing
1.75
< .001
-
-
-
-
-
-
-
-
-
-
-
-
0.48
.025
-
-
(1.42–2.16)
(0.25–0.91)
Job in
household
-
-
-
-
-
-
-
-
-
-
.68
.020
-
-
-
-
-
-
(0.49–0.94)
Constant
0.23
< .001
0.07
< .001
-
-
-
-
0.08
< .001
.17
.001
0.08
< .001
0.01
< .001
Step 3
SDG 2.1
Hunger
SDG 3.3
AIDS
SDG 3.3 TB
SDG 3.4
Mental
Health
SDG 3.5
Substance
Abuse
4.1 School
non-
enrolment
5.2 Sexual
violence or
exploitation
5.6
Pregnancy
16.1 Violence
perpetration
AOR
AOR
AOR
AOR
AOR
AOR
AOR
AOR
AOR
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
(95%
CI)
p
Cash
-
-
0.64
.005
-
-
-
-
0.46
< .001
0.14
< .001
0.67
.018
0.46
.004
-
-
(0.46–0.87)
(0.30–0.70)
(0.09–0.24)
(0.48–0.93)
(0.27–0.78)
Care
0.63
< .001
0.67
.010
-
-
-
-
0.32
< .001
1.49
.439
0.71
.038
-
-
-
-
(0.51–0.77)
(.49-
.91)
(0.19–0.53)
(0.54–4.10)
(0.52–0.98)
Baseline
outcome
2.07
< .001
7.30
< .001
-
-
-
-
2.20
< .001
8.60
< .001
4.29
< .001
15.87
< .001
-
-
(1.70–2.54)
(5.14–10.39)
(1.45–3.32)
(3.80–19.49)
(2.90–6.35)
(8.04–31.30)
Older than 13
years
-
-
4.44
< .001
-
-
-
-
2.32
< .001
2.82
< .001
2.53
< .001
12.13
< .001
-
-
(3.07–6.43)
(1.47–3.68)
(1.59–5.00)
(1.79–3.58)
(4.33–33.96)
Urban location
1.37
.002
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(1.13–1.67)
>3 children in
home
-
-
-
-
-
-
-
-
0.65
.050
-
-
-
-
-
-
-
-
(0.43–1.00)
Has birth
certificate
-
-
-
-
-
-
-
-
-
-
0.35
.003
-
-
-
-
-
-
(0.18–0.70)
Female
caregiver
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(Continued) Table 3. Social Protection and the Sustainable Development Goals Table 3. (Continued) Table 3. (Continued)
Missing >2
necessities
1.87
< .001
-
-
-
-
-
-
-
-
4.51
.001
-
-
-
-
-
-
(1.51–2.31)
(1.93–10.56)
Informal
housing
1.75
< .001
-
-
-
-
-
-
-
-
-
-
-
-
0.48
.025
-
-
(1.42–2.16)
(0.25–0.91)
Job in
household
-
-
-
-
-
-
-
-
-
-
0.69
.024
-
-
-
-
-
-
(0.49–0.95)
Care*Missing
>2 necessities
-
-
-
-
-
-
-
-
-
-
0.20
.012
-
-
-
-
-
-
(0.06–0.70)
Constant
0.23
< .001
0.07
< .001
-
-
-
-
0.08
< .001
0.10
< .001
0.08
< .001
0.01
< .001
-
-
Note. For each outcome, interactions between (a) cash and care and (b) each of cash and care and the covariates in the model were tested. None were
statistically significant except for the interaction between care and missing necessities for the outcome hunger, as shown in step 3, and thus only significant
effects are illustrated. Note. For each outcome, interactions between (a) cash and care and (b) each of cash and care and the covariates in the model were tested. None were
statistically significant except for the interaction between care and missing necessities for the outcome hunger, as shown in step 3, and thus only significant
effects are illustrated. doi:10.1371/journal.pone.0164808.t003 dropout substantially. It may be that–with largely free schooling in these low-income areas–
girls living in high poverty levels require psychosocial support in order to be able to engage
with education. We note however that two significant interactions in a large set of possible
interactions tested may be due to chance and that future research would be needed to confirm
these findings. Findings also show that social protection is not a panacea. There were no statistically signifi-
cant associations with indicators of tuberculosis, nor with girls’ mental health or violence per-
petration, nor with boys’ victimisation by sexual violence, suggesting that more specified
interventions are required. These may require greater expertise and targeting. As patterns of
effect differ by gender, social protection may also require a gendered understanding to inform
and target combinations of interventions. g
There are a number of important limitations to these findings. (Continued) PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 15 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
October 17, 2016 Social Protection and the Sustainable Development Goals SDG 6 ‘sustainable water and sanitation for all’ is a key health-related factor, but is provided in
South Africa at a large-scale level by government servicesand was therefore likely to be weakly
associated with the household-focusedsocial protection provisions examined in this study. SDGs 1 and 10 ‘end poverty’ and ‘reduce inequality’ are broad and multidimensional concepts
that include the aims of other SDGs such as health, education and food security, but are essen-
tial overarching considerations in health. Finally, in any testing of multiple possible outcomes,
there is a risk that findings are due to chance. To mitigate this, while we have reported findings
of .05<p < .01 in the tables for their substantive interest, we note that more than three-quar-
ters of findings for boys and girls alike are significant at the more stringent p< = .01 level. Simi-
larly, the two significant interactions among the large number tested may be due to chance and
further research would be needed to confirm this possible effect modification. SDG 6 ‘sustainable water and sanitation for all’ is a key health-related factor, but is provided in
South Africa at a large-scale level by government servicesand was therefore likely to be weakly
associated with the household-focusedsocial protection provisions examined in this study. SDGs 1 and 10 ‘end poverty’ and ‘reduce inequality’ are broad and multidimensional concepts
that include the aims of other SDGs such as health, education and food security, but are essen-
tial overarching considerations in health. Finally, in any testing of multiple possible outcomes,
there is a risk that findings are due to chance To mitigate this while we have reported findings Despite these limitations, this study has notable advantages, particularly in external validity. It measured real-world interventions provided by an African government, NGOs, and families. The longitudinal data allowed analyses to examine incident outcomes by controlling for base-
line SDG health risks, thus providing stronger causal assumptions. We know that, unlike care-
fully controlled experimental situations, implementation of any social programmes in Sub-
Saharan Africa is administratively challenging and fraught with logistical problems. But, even
in these conditions, and amongst a population of young people who are hardest-hit by poverty
and inequality, social protection provision was associated with substantial positive impacts on
adolescent health and wellbeing. These findings demonstrate the potential of social protection to contribute to multiple
aspects of the SDG agenda. Acknowledgments The authors wish to thank Alice Redfern and our fieldwork teams in the Western Cape and
Mpumalanga. Thanks to Tom Fenn, Anurita Bains, Doug Webb, Patricia Lim ah Ken and
Charles King for early discussions in Melbourne at IAS 2014. Thanks to the kNOw Violence in
ChildhoodGlobal Learning Initiative for discussion and input. Most importantly, we thank all
the participants and their families. They highlight the value of providing ‘care’ as well as ‘cash’, sug-
gesting the importance of resource allocation to psychosocial care in a time of global cuts and
reliance on NGO and soft providers. It is clear that social protection does not solve every socie-
tal problem and that for key outcomes such as tuberculosis and mental health we will need
additional, targeted investments. But the wide-ranging impacts of social protection also pro-
vide a major opportunity. The last decade of programming for children has seen a growth in
global initiatives that provide a blueprint on how evidence can be corralled, synthesized, and
used as a key driver of policy.[43, 44] There is no question that the Sustainable Development
Goals present a challenging set of aspirations. But they also include the potential to contribute
to the health of our next generations. First, social protection access
was not randomized, and although analyses carefully controlled for a range of potential con-
founders, this observational study does not provide the level of causal reliability of a random-
ized trial. We attempted to mitigate this risk by checking for, interaction effects among social
protections and with significant socio-demographiccovariates. Only two such instances were
found, in which the impact of social protection differed by poverty level. Further research
using fixed effects models may be desirable, to better examine how changes in access to social
protection over time are associated with SDG outcomes. Third, the study only has two annual
time-points, which again limits the capacity for causal certainty. It will be essential to examine
longer-term associations of social protection and SDG outcomes as adolescents progress into
adulthood. Fourth, research could valuably examine the levels and length of social protection
exposure required in order to reduce SDG risks. Fifth, this study only takes place in one coun-
try and initially in two provinces. However, a systematic sampling method was followed in
both urban and rural areas, with rigorous follow-up. Further research across other low and
middle-income countries is required in order to understand whether a South African experi-
ence is indicative of other settings in the region or elsewhere. Sixth, the study only examined
indicators in five of the SDG goals, focusing on those that were most directly relevant to adoles-
cent health. Other SDGs, for example SDG 8 ‘economic growth, full and productive employ-
ment, and decent work for all’ become increasingly important as adolescents become adults. 16 / 20 PLOS ONE | DOI:10.1371/journal.pone.0164808
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Green fiscal reform for a just energy transition in Latin America
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Vol. 13, 2019-17 | March 7, 2019 | http://dx.doi.org/10.5018/economics-ejournal.ja.2019-17 V Vol. 13, 2019-17 | March 7, 2019 | http://dx.doi.org/10.5018/economics-ejournal.ja.2019-17 Green fiscal reform for a just energy transition
in Latin America Michael Jakob, Rafael Soria, Carlos Trinidad, Ottmar Edenhofer, Celine Bak,
Daniel Bouille, Daniel Buira, Hernan Carlino, Veronica Gutman, Christian
Hübner, Brigitte Knopf, André Lucena, Luan Santos, Andrew Scott, Jan
Christoph Steckel, Kanako Tanaka, Adrien Vogt-Schilb, Koichi Yamada JEL
H23, E62, Q54, N16, Q48 JEL
H23, E62, Q54, N16, Q48 Keywords
Green fiscal reform; energy subsidies; Latin America; multi-objective climate policy;
sequencing; distribution Received December 2, 2018 Published as Economics Discussion Paper December 17, 2018
Revised February 26, 2019 Accepted March 5, 2019 Published March 7, 2019 © Author(s) 2019. Licensed under the Creative Commons License - Attribution 4.0 International (CC BY 4.0) (Published as Global Solutions Paper) (Published as Global Solutions Paper) Abstract Green fiscal reforms would contribute to climate change mitigation, increase the economic efficiency
of national tax systems and provide additional public revenues. Some countries in Latin America
have already taken first steps towards green fiscal reforms. This outlook article provides an overview
of the major challenges for the successful implementation of such reforms and discusses how they
could be overcome. 1 We follow the International Energy Agency (IEA) and the Organisation for Economic Co-operation and
Development (OECD) definition of fossil fuel subsidies as government support of the consumption or production of
oil, gas or coal that lowers their prices below market prices. This definition excludes un-priced environmental and
social externalities, such as air pollution and related health effects, which are included in some other estimations, as
for example in Coady et al. (2017). Failing to internalize environmental damages related to fossil fuel use also
constitutes a form of a subsidy. Post-tax subsidies (which include climate and health impacts as well as foregone
government revenue due to tax exemptions) are about ten times larger than the pre-tax subsidies considered by the
price-gap approach. 1
Background 1 Putting a price on greenhouse gas (GHG) emissions can help reduce environmental damage
linked to both local pollution and global warming (Bak et al. 2017). However, most countries in
Latin America either have no such price or negative prices in the form of fossil fuel subsidies1
(World Bank, Ecofys, and Vivid Economics 2017). This actively supports the use of fossil fuels,
particularly for large oil producers (Di Bella et al. 2015). Green fiscal reforms that reduce
subsidies for fossil fuels and introduce positive prices on emissions would not only contribute to
climate change mitigation, but could also increase the economic efficiency of national tax
systems and provide additional public revenues that could be employed to advance human
development (Edenhofer et al. 2015). Energy and climate policy is deeply embedded within a broad range of policy targets. For
instance, many Latin American economies are dependent on extractive industries for exports
and fiscal revenues and display high levels of economic inequality. Climate change mitigation
can only be successful if it is part of a ‘just transition’ that fosters human well-being. For
instance, Franks et al. (2018) emphasize the potential to cover financial needs to achieve the
Sustainable Development Goals (SDG) in different countries if all subsidies on fossil fuels were
redirected accordingly. In some Latin America and Caribbean countries (LAC), a fossil fuel
subsidy reform could cover a large part of the required finance to achieve the SDG goals, for
instance, 60% in Bolivia, and 45% in El Salvador. In 2013, when oil prices were relatively high, energy subsidies (including fuels and
electricity) in LAC amounted to $86 billion USD, which represented about 2% of GDP. In
2015, following the decline in oil prices, subsidies in LAC fell to about $45 billion USD (Jewell
et al. 2018). According to Jewell et al. (2018), under a high oil prices scenario, by 2030 LAC
subsidies would grow to between $70 and $140 billion USD, and to $40 to $100 billion under a
low oil prices scenario. The authors estimate that in LAC subsidy removal would lead to
emission reductions of up to 5% below the base line scenario, comparable to the so-called
‘conditional’ NDCs2 (that is, commitments dependent on international action). Authors M. Jakob, Mercator Research Institute on Global Commons and Climate Change (MCC), Berlin,
Germany; R. Soria, Escuela Politécnica Nacional de Ecuador (EPN), Departamento de Ingeniería
Mecánica, Quito, Ecuador, rafael.soria01@epn.edu.ec; C. Trinidad, Sociedad Peruana de Derecho
Ambiental (SPDA), Lima, Perú; O. Edenhofer, Mercator Research Institute on Global Commons and
Climate Change (MCC), Berlin, Germany; C. Bak, Analytica Advisors, Ottawa, Canada; D. Bouille,
Fundación Bariloche, Buenos Aires, Argentina; D. Buira, Tempus Analítica, Ciudad de México,
México; H. Carlino, Fundación Torcuato Di Tella (FTDT), Buenos Aires, Argentina; V. Gutman,
Fundación Torcuato Di Tella (FTDT), Buenos Aires, Argentina; C. Hübner, Konrad-Adenauer-Stiftung
(KAS), Lima, Peru; B. Knopf, Mercator Research Institute on Global Commons and Climate Change
(MCC); A. Lucena, Energy Planning Program (PPE), Graduate School of Engineering, Universidade
Federal do Rio de Janeiro (COPPE/UFRJ), Rio de Janeiro, Brazil; L. Santos, Energy Planning Program
(PPE), Graduate School of Engineering, Universidade Federal do Rio de Janeiro (COPPE/UFRJ), Rio
de Janeiro, Brazil; A. Scott, Overseas Development Institute (ODI), London, UK; J.C. Steckel, Mercator
Research Institute on Global Commons and Climate Change (MCC); K. Tanaka, Center for Low
Carbon Society Strategy (LCS), Japan Science and Technology Agency (JST), Tokyo, Japan; A. Vogt-
Schilb, Inter-American Development Bank, Washington, USA; K. Yamada, Center for Low Carbon
Society Strategy (LCS), Japan Science and Technology Agency (JST), Tokyo, Japan Citation
M. Jakob, R. Soria, C. Trinidad, O. Edenhofer, C. Bak, D. Bouille, D. Buira, H. Carlino, V. Gutman, C. Hübner, B. Knopf, A. Lucena, L. Santos, A. Scott, J.C. Steckel, K. Tanaka, A. Vogt-Schilb,
K. Yamada. Green fiscal reform for a just energy transition in Latin America. Economics: The Open-
Access, Open- Assessment E-Journal, 13 (2019-17): 1–11. h
//d d i
/10 5018/
i
j
l j 2019 17 Citation
M. Jakob, R. Soria, C. Trinidad, O. Edenhofer, C. Bak, D. Bouille, D. Buira, H. Carlino, V. Gutman, C. Hübner, B. Knopf, A. Lucena, L. Santos, A. Scott, J.C. Steckel, K. Tanaka, A. Vogt-Schilb,
K. Yamada. Green fiscal reform for a just energy transition in Latin America. Economics: The Open-
Access, Open- Assessment E-Journal, 13 (2019-17): 1–11. http://dx.doi.org/10.5018/economics-journal.ja.2019-17 2 National Determined Contributions, submitted by countries to the UNFCCC within the framework of the Paris
Agreement. Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers resource wealth with the public, despite their benefits accruing mostly to richer households. Energy subsidies contributed to fiscal deficits, and their costs were comparable to that of
education and health combined. In these countries, energy subsidies significantly constrained
fiscal space and were in most cases not targeted to the poorest and most vulnerable. Energy
subsidies were at times financed off budget via losses at state-owned enterprises, hampering
transparency, increasing uncertainty, constraining energy sector investment and reducing
economic efficiency. On the other hand, some countries in the region have begun to implement deeper fiscal
reforms to tax CO2 emissions, along with new international trends to put a price on carbon. In
some cases, downstream taxes have been promoted aimed at taxing subjects that cause
emissions with the consumption of fossil fuels (Chile). Other countries have maintained the
structure of upstream taxes or taxes at the producer level (Colombia), but have contemplated
innovative payment options that allow linking the carbon tax with carbon markets and cap and
trade schemes (Mexico). At the same time, efforts have also been made to strengthen the development of a regional
carbon market. In June 2017, member countries of the Pacific Alliance committed to intensify
their efforts in the measurement, reporting and verification of CO2 emissions to identify
possible voluntary market mechanisms among countries in the region.3 Besides, in December
the same year, several countries and subnational governments in the region signed the Paris
Declaration on the Price of Carbon in the Americas, which includes commitments to implement
national carbon pricing policies, including the promotion of a market.4 These reforms and initiatives are recent, so it is not yet possible to have an accurate
diagnosis of their effectiveness, but the projections and preliminary results are encouraging. For
this reason, the next section considers how the key challenges for successful implementation of
green fiscal reforms could be addressed. 1
Background Subsidies are larger in energy rich countries and in those that rank lower on measures of
institutional and policy quality, such as budget transparency, rule of law, competitiveness or the
ease of doing business (Di Bella et al. 2015). Among LAC oil producer countries that ranked
lower in measures of institutional quality are Argentina, Belize, Bolivia, Ecuador, Suriname,
and Venezuela (Di Bella et al. 2015). In these countries subsidies are seen as a way of sharing 2 www.economics-ejournal.org Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers 3 https://alianzapacifico.net/?wpdmdl=9850 4 The countries that signed this declaration were: Chile, Colombia, Mexico, Costa Rica, Canada and the states of
California, Washington, Alberta, British Columbia, Nova Scotia, Ontario and Quebec.:
http://www.ieta.org/resources/News/Press_Releases/2017/Declaration%20on%20Carbon%20Pricing_FINAL.pdf 4 The countries that signed this declaration were: Chile, Colombia, Mexico, Costa Rica, Canada and the states
California, Washington, Alberta, British Columbia, Nova Scotia, Ontario and Quebec.: http://www.ieta.org/resources/News/Press_Releases/2017/Declaration%20on%20Carbon%20Pricing_FINAL.pdf 2.1
Identifying favorable political conditions for green fiscal reforms A first step is to develop an understanding of the required enabling conditions for green fiscal
reform. Variables such as the overall state of the economy, internal political stability, and public
debt can be expected to play important roles (Karapin 2016). Developments on the international
level, such as progress in international climate negotiations or introduction of green policies in
other countries, may boost domestic support for green fiscal reforms. Likewise, newly
appointed heads of state may have the clout necessary to successfully foster such reforms,
especially when there is sufficient backing by the general population. Furthermore, trust in
government effectiveness and the expectation that associated revenues would be used in
beneficial ways have been found to be important factors for the successful introduction of green
fiscal reforms (Drews and van den Bergh 2016; Klenert et al. 2018). In Ecuador, the last decade saw favorable political conditions for fostering a green reform. Former President Rafael Correa was relatively popular, the oil price was very high between
2011 and 2013 (over US$ 95 per barrel) (BCE 2019) and there was a large investment in hydro
power plants (MEER 2017). Nevertheless, green fiscal reform was never introduced as the
authorities were afraid of how citizens, especially poor people, would react. Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers academic literature and compiles expert knowledge on selected countries to shed some light on
possibilities for, and limitations to, green fiscal reform. www.economics-ejournal.org 2
Key challenges for green fiscal reform After the adoption of the Paris Agreement, practically all countries in Latin America submitted
their Nationally Determined Contributions (NDCs) that specified their intended climate targets. Green fiscal reform would be a step to move from ambition to partial implementation of these
targets. Even though there are some general insights applicable to all countries (Rentschler and
Bazilian 2017; Withana 2016), the specific national situation will determine the details of policy
design and implementation. The following sub-sections review the evidence provided by the 3 www.economics-ejournal.org Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers Latin American challenges such as informality, inequality, unemployment, air quality, or lack of
national industries to provide capital inputs for renewable energy projects. For instance, Argentina has recurrently aimed to reduce trade and fiscal imbalances,
unemployment, poverty, and inflation while keeping external debt in check. Despite paucity in
achieving those goals, an integrated green fiscal reform could accelerate investment in
innovative technologies that not only reduce environmental impacts, but may also enhance
competitiveness, job creation, poverty alleviation and reduce economic inequality. In Peru, the main environmental problems are closely linked to social and energy problems,
such as a lack of access to electricity and sanitation in rural areas. By replicating the successful
international experiences, a green tax reform in Peru could finance payment schemes for
environmental services to compensate indigenous communities and guarantee the sustainable
use of forests (Trinidad and Vargas 2017). This requires coordination between various public
sector agencies, such as the Ministries of Economy and Finance, Environment, Energy, Social
Inclusion and Agriculture. 2.2
Developing comprehensive reform plans Energy and climate-related policies do not exclusively affect environmental issues; they also
impact areas such as transport, industry, agriculture, finance, trade and social inclusion (Fuso
Nerini et al. 2018). Policy-makers can build on synergies to ensure a just transition and increase
support for reform (Vogt-Schilb and Hallegatte 2017). Policy areas of potential synergies
include energy security (reduced reliance on fossil fuel imports), local environmental benefits
and the potential to diversify the economy. On the other hand, trade-offs may arise in terms of
economic competitiveness due to higher energy prices and adverse effects of renewable energy
use on land-use, food production, and biodiversity (Tanaka 2011). In addition, green fiscal reforms should consider the characteristics of different sources of
CO2 emissions. For Latin American countries, a high share of emissions stems from land use,
land use change and forestry (LULUCF), namely 42% of total emissions (CEPAL 2017).This
level indicates how green fiscal reforms also require fiscal mechanisms to reduce deforestation,
e.g. by channeling some of the revenues of fossil fuel subsidy reform or carbon pricing to
results-based payments for forest protection. The multi-objective nature of energy and climate policies needs to be reflected in
comprehensive strategies that ensure consistency of climate targets with other policies. Such
strategies will need to include all relevant ministries and encourage coordination between
national and subnational public entities. Green reforms should particularly consider important 4 Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers www.economics-ejournal.org Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers situation is structurally stabilized (Gutman 2018), in particular including an equitable fossil fuel
subsidy reform. situation is structurally stabilized (Gutman 2018), in particular including an equitable fossil fuel
subsidy reform. Peru already applies taxes to gasoline and diesel based on the health impacts of its
particulate matter and nitrous oxide emissions. Adding GHG emissions to this index would be a
straightforward way to align the price of transport fuels with their true social costs (Jakob 2018). Likewise, in cost-benefit analyses of public investment, Peru incorporates an accounting price
of about US$ 7 per tCO2. These accounting costs of carbon could be raised gradually in line
with increasingly ambitious national (as well as international) climate targets (Jakob 2018). 2.3
Sequencing of reforms and gradualism In most situations, fiscal reforms cannot be introduced instantly; they normally require a
preparatory phase that lowers the costs of reform, addresses barriers due to market
imperfections and policy inconsistency, and ensures its legitimacy by reducing social cost of
phasing out subsidies, thus reducing political resistance. For instance, fiscal incentives for
renewable energy sources would create groups that would directly benefit from (and which can
hence be expected to lend political support to) green policies (Meckling et al. 2015). Green
fiscal reforms can also be introduced after building administrative capacity to effectively
enforce the policies (e.g. by monitoring fossil fuel sales and tax payments). In many countries price increases for LPG and diesel are politically contentious as these
fuels are important for low-income households, either directly, in the case of LPG, or indirectly
through public or goods transportation. On the other hand, gasoline prices can be less
contentious in terms of aggravating poverty, on countries where they are predominantly
consumed by richer households and are not used to produce basic goods. Suddenly raising
prices for all fossil energy carriers to their desired level could cause substantial economic
problems as firms and consumers require time to adjust (IMF 2013). In 2017, Argentina introduced a tax (of approximately US$ 10 per ton of CO2) on the
carbon content of gasoline, gas oil, fuel oil, coal and other liquid and solid fossil fuels. The
carbon tax has been designed to have no initial impact on the final prices of fuels as it partially
replaces an existing tax on fuels. The intention has been to start preparing the ground and create
consciousness among producers and consumers. As it is known, due to existing infrastructure
limitations, the lack of technological alternatives at affordable prices, and the contentious tariff
reform in place, the carbon tax will not, by itself, generate enough incentives for
decarbonization in the scale and timeframe required. This shift will only be achieved if
Argentina’s carbon pricing policies are accompanied by adequate energy, infrastructure,
industrial, technological and communications policies and, above all, if the macroeconomic 5 5 www.economics-ejournal.org www.economics-ejournal.org Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers 2.4
Understanding distribution and compensation Social expenditure: In Colombia, the income from the national carbon tax finances activities related to
peace building, sustainable rural development and conservation, and environmental sustainability. Box 1: Overview of compensation mechanisms Box 1: Overview of compensation mechanisms
Increases in energy prices are likely to negatively affect the poorest social groups. Compensatory
mechanisms that have been used in some countries in Latin America include:
Vouchers: When LPG subsidies were removed in 2002, Brazil introduced LPG vouchers for households
that were recipients of the Bolsa Família social assistance program. The government later introduced a
conditional cash transfer program to obviate the need for general LPG subsidies (Kojima 2013; Komives
et al. 2008). Cash transfers: The Vale Gás program in Brazil was established in 2001, and is still in operation. It
assists consumption of gas by poor households by subsidizing bottle purchase and direct payment was
made to registered families. Registration is combined with Bolsa Família. Reliance on general social protection programs: In Mexico, LPG prices have been gradually increased
(Toft et al. 2016). These reforms employed transfer mechanisms within existing social welfare
mechanisms (Oportunidades) to mitigate the effects of higher prices. Social expenditure: In Colombia, the income from the national carbon tax finances activities related to
peace building, sustainable rural development and conservation, and environmental sustainability. Increases in energy prices are likely to negatively affect the poorest social groups. Compensatory
mechanisms that have been used in some countries in Latin America include: Vouchers: When LPG subsidies were removed in 2002, Brazil introduced LPG vouchers for households
that were recipients of the Bolsa Família social assistance program. The government later introduced a
conditional cash transfer program to obviate the need for general LPG subsidies (Kojima 2013; Komives
et al. 2008). Cash transfers: The Vale Gás program in Brazil was established in 2001, and is still in operation. It
assists consumption of gas by poor households by subsidizing bottle purchase and direct payment was
made to registered families. Registration is combined with Bolsa Família. Cash transfers: The Vale Gás program in Brazil was established in 2001, and is still in operation. It
assists consumption of gas by poor households by subsidizing bottle purchase and direct payment was
made to registered families. Registration is combined with Bolsa Família. Reliance on general social protection programs: In Mexico, LPG prices have been gradually increased
(Toft et al. 2016). 2.4
Understanding distribution and compensation Phasing out inefficient fossil fuel subsidies has been a long-standing issue in the G20
negotiations. While the G7 has suggested that all countries should phase out subsidies by 2025,
the G20 has not yet agreed on a date. The resistance partly stems from the fear that poor
households would suffer from the phasing out of subsidies. In Latin America, a recent Inter-
American Development Bank study suggests that, with energy subsidies, it costs governments
US$12 to transfer US$1 of income to households in the poorest quintile. Gasoline and diesel are
the most inefficient subsidies, costing US$14 per dollar benefit. The most pro-poor fuels are gas
or LPG, but they still costing about US$9 on average per dollar distributed to poor households
(Feng et al. 2018). Different schemes can protect low-income households from the impacts of higher energy
prices. These include direct cash transfers, in-kind transfers and the provision of health,
education, social security, or public infrastructure, including public transportation (see Box 1). A further option are targeted tax reductions, such as indirect or regressive taxes and taxes on
wages, but only in countries where the poorest households do indeed pay a substantial amount
of taxes. In addition, numerous countries use block-pricing schemes for electricity, which allow
low-income households to consume a specified amount of electricity at a reduced rate. Box 1: Overview of compensation mechanisms
Increases in energy prices are likely to negatively affect the poorest social groups. Compensatory
mechanisms that have been used in some countries in Latin America include:
Vouchers: When LPG subsidies were removed in 2002, Brazil introduced LPG vouchers for households
that were recipients of the Bolsa Família social assistance program. The government later introduced a
conditional cash transfer program to obviate the need for general LPG subsidies (Kojima 2013; Komives
et al. 2008). Cash transfers: The Vale Gás program in Brazil was established in 2001, and is still in operation. It
assists consumption of gas by poor households by subsidizing bottle purchase and direct payment was
made to registered families. Registration is combined with Bolsa Família. Reliance on general social protection programs: In Mexico, LPG prices have been gradually increased
(Toft et al. 2016). These reforms employed transfer mechanisms within existing social welfare
mechanisms (Oportunidades) to mitigate the effects of higher prices. 2.4
Understanding distribution and compensation These reforms employed transfer mechanisms within existing social welfare
mechanisms (Oportunidades) to mitigate the effects of higher prices. Social expenditure: In Colombia, the income from the national carbon tax finances activities related to
peace building, sustainable rural development and conservation, and environmental sustainability. 6 www.economics-ejournal.org Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers In principle, a small fraction of savings from subsidy removal is sufficient to compensate poor
and vulnerable households, since poorer households benefit less than others from fossil fuel
subsidies. For instance, in Ecuador the poorest 40% of the population only receive 20% of every
dollar spent on subsidies for diesel and gasoline, whereas the other 80% accrue to the richest
60% of the population (Table 1). By contrast, redirecting revenues freed up by subsidy reform
to spending on social security could raise the net income of the poorest 20% of the population
by around 5% (Schaffitzel et al. 2018). Table 1: Fraction of government proceeds from subsidy removal or energy taxation on different types o
fuels needed to compensate poor and vulnerable households in 11 LAC countries
(i.e. households in the bottom 40% of the income distribution)
Diesel and gasoline
Electricity
Natural gas and LPG
Argentina
21.9%
27.4%
27.2%
Bahamas
27%
29.6%
Barbados
14.1%
20.5%
Chile
21.4%
27.3%
27.6%
Costa Rica
15.7%
26.7%
33.9%
Ecuador
20%
24.2%
33.6%
Guatemala
14.7%
18.7%
10.6%
Jamaica
19.1%
21.4%
23.2%
Nicaragua
17.5%
21.4%
20.7%
Paraguay
20.6%
21.4%
25.4%
Uruguay
15.1%
21.2%
28.1%
Source: Feng et al. (2018) Table 1: Fraction of government proceeds from subsidy removal or energy taxation on different types of
fuels needed to compensate poor and vulnerable households in 11 LAC countries
(i.e. households in the bottom 40% of the income distribution) www.economics-ejournal.org 3
Conclusions 3 This short article has highlighted several issues that are crucial for the successful introduction
and implementation of green fiscal reforms in the context of Latin American countries. The
country-specific context needs to be considered to overcome political challenges. For instance,
political conditions favorable for green fiscal reform may vary substantially between countries
with different socio-economic conditions and political cultures. Likewise, the entities required
to develop the comprehensive reform plans will crucially depend on the division of power
between different ministries, and the proper sequencing of reform will be influenced by
previous experience with certain policies and constrained by institutional and administrative
capacities. Finally, the compensation schemes can only be implemented to the extent to which
they are politically and technically feasible, i.e. not restricted by public resistance or difficulties
to target recipients. Stakeholder consultations can be an appropriate means to take into account all possible
intricacies of green fiscal reforms. Such consultations would also ensure that all relevant social
groups are represented in the decision-making process and that appropriate measures are
adopted to alleviate excessive adverse impacts on any single group. In particular, green fiscal 7 7 www.economics-ejournal.org Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers Acknowledgments We thank the Think 20 Argentina 2018 (T20) for their support during initial stages
of this work presented for the G20 Argentina 2018 meeting. We declare that there is no financial interest
in the topic of the paper. Thus, there is no conflict of interest. Economics: The Open-Access, Open-Assessment E-Journal 13 (2019-17)
Global Solutions Papers reforms need to include consultation processes to guarantee that traditional rights of the
indigenous peoples living in most Latin American countries are not violated. Such consultations
should not only occur prior to the introduction of a reform, but its impacts and the position of
key stakeholder to the reform should be continuously monitored. reforms need to include consultation processes to guarantee that traditional rights of the
indigenous peoples living in most Latin American countries are not violated. Such consultations
should not only occur prior to the introduction of a reform, but its impacts and the position of
key stakeholder to the reform should be continuously monitored. In addition, the international community could play a key role in supporting green fiscal
reforms. For instance, the G20 could host processes that allow the exchange of experiences to
better understand the relationship between policies, their effects and their contexts. Regional
development banks can play a very useful role in supporting the access of Latin American
countries to international climate finance. Such international climate finance could not only be
used for project finance, but could also cover the macro-economic costs of green fiscal reforms,
e.g. by means of results-based payments that are tied to the introduction of a price on emissions
(Steckel et al. 2017) or de-risking of clean energy and energy efficiency investments (Steckel
and Jakob 2018). Acknowledgments We thank the Think 20 Argentina 2018 (T20) for their support during initial stages
of this work presented for the G20 Argentina 2018 meeting. We declare that there is no financial interest
in the topic of the paper. Thus, there is no conflict of interest. 8 www.economics-ejournal.org 8 References Bak, C., Amar Bhattacharya, Ottmar Edenhofer, and Brigitte Knopf (2017). Towards a comprehensive
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A Systematic Review of Automatic Health Monitoring in Calves: Glimpsing the Future From Current Practice
|
Frontiers in veterinary science
| 2,021
|
cc-by
| 12,198
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A Systematic Review of Automatic
Health Monitoring in Calves:
Glimpsing the Future From Current
Practice We discuss these findings, look into potentials at stage four,
and touch upon the topic of resilience, whereby health-monitoring system might be used
to detect low resilience (i.e., prone to disease but clinically healthy calves), promoting
further improvements in calf health and welfare. Infectious diseases, particularly bovine respiratory disease (BRD) and neonatal calf
diarrhea (NCD), are prevalent in calves. Efficient health-monitoring tools to identify such
diseases on time are lacking. Common practice (i.e., health checks) often identifies sick
calves at a late stage of disease or not at all. Sensor technology enables the automatic
and continuous monitoring of calf physiology or behavior, potentially offering timely and
precise detection of sick calves. A systematic overview of automated disease detection
in calves is still lacking. The objectives of this literature review were hence: to investigate
previously applied sensor validation methods used in the context of calf health, to identify
sensors used on calves, the parameters these sensors monitor, and the statistical tools
applied to identify diseases, to explore potential research gaps and to point to future
research opportunities. To achieve these objectives, systematic literature searches were
conducted. We defined four stages in the development of health-monitoring systems:
(1) sensor technique, (2) data interpretation, (3) information integration, and (4) decision
support. Fifty-four articles were included (stage one: 26; stage two: 19; stage three:
9; and stage four: 0). Common parameters that assess the performance of these
systems are sensitivity, specificity, accuracy, precision, and negative predictive value. Gold standards that typically assess these parameters include manual measurement
and manual health-assessment protocols. At stage one, automatic feeding stations,
accelerometers, infrared thermography cameras, microphones, and 3-D cameras are
accurate in screening behavior and physiology in calves. At stage two, changes in
feeding behaviors, lying, activity, or body temperature corresponded to changes in health
status, and point to health issues earlier than manual health checks. At stage three,
accelerometers, thermometers, and automatic feeding stations have been integrated
into one system that was shown to be able to successfully detect diseases in calves,
including BRD and NCD. We discuss these findings, look into potentials at stage four,
and touch upon the topic of resilience, whereby health-monitoring system might be used
to detect low resilience (i.e., prone to disease but clinically healthy calves), promoting
further improvements in calf health and welfare. Keywords: calf, early disease detection, precision livestock farming, sensor, health monitoring A Systematic Review of Automatic
Health Monitoring in Calves:
Glimpsing the Future From Current
Practice Dengsheng Sun 1*, Laura Webb 2, P. P. J. van der Tol 1 and Kees van Reenen 2,3
1 Farm Technology Group, Wageningen University and Research, Wageningen, Netherlands, 2 Animal Production Systems
Group, Wageningen University and Research, Wageningen, Netherlands, 3 Livestock Research, Research Centre,
Wageningen University and Research, Wageningen, Netherlands 1 Farm Technology Group, Wageningen University and Research, Wageningen, Netherlands, 2 Animal Production Systems
Group, Wageningen University and Research, Wageningen, Netherlands, 3 Livestock Research, Research Centre,
Wageningen University and Research, Wageningen, Netherlands Infectious diseases, particularly bovine respiratory disease (BRD) and neonatal calf
diarrhea (NCD), are prevalent in calves. Efficient health-monitoring tools to identify such
diseases on time are lacking. Common practice (i.e., health checks) often identifies sick
calves at a late stage of disease or not at all. Sensor technology enables the automatic
and continuous monitoring of calf physiology or behavior, potentially offering timely and
precise detection of sick calves. A systematic overview of automated disease detection
in calves is still lacking. The objectives of this literature review were hence: to investigate
previously applied sensor validation methods used in the context of calf health, to identify
sensors used on calves, the parameters these sensors monitor, and the statistical tools
applied to identify diseases, to explore potential research gaps and to point to future
research opportunities. To achieve these objectives, systematic literature searches were
conducted. We defined four stages in the development of health-monitoring systems:
(1) sensor technique, (2) data interpretation, (3) information integration, and (4) decision
support. Fifty-four articles were included (stage one: 26; stage two: 19; stage three:
9; and stage four: 0). Common parameters that assess the performance of these
systems are sensitivity, specificity, accuracy, precision, and negative predictive value. Gold standards that typically assess these parameters include manual measurement
and manual health-assessment protocols. At stage one, automatic feeding stations,
accelerometers, infrared thermography cameras, microphones, and 3-D cameras are
accurate in screening behavior and physiology in calves. At stage two, changes in
feeding behaviors, lying, activity, or body temperature corresponded to changes in health
status, and point to health issues earlier than manual health checks. At stage three,
accelerometers, thermometers, and automatic feeding stations have been integrated
into one system that was shown to be able to successfully detect diseases in calves,
including BRD and NCD. Edited by:
Marcia Endres,
University of Minnesota Twin Cities,
United States Reviewed by:
Joao H. C. Costa,
University of Kentucky, United States
Gillian Scoley,
Queen’s University Belfast,
United Kingdom
Robert Elliott James,
Virginia Tech, United States *Correspondence:
Dengsheng Sun
dengsheng.sun@wur.nl Specialty section:
This article was submitted to
Animal Behavior and Welfare,
a section of the journal
Frontiers in Veterinary Science Specialty section:
This article was submitted to
Animal Behavior and Welfare,
a section of the journal
Frontiers in Veterinary Science Received: 19 August 2021
Accepted: 04 November 2021
Published: 26 November 2021 SYSTEMATIC REVIEW
published: 26 November 2021
doi: 10.3389/fvets.2021.761468 SYSTEMATIC REVIEW Definitions
l
l d d For example, in a study diagnosing
BRD in beef calves using clinical examination, the estimated
sensitivity and specificity were 61.8 and 62.8%, respectively (26). Many sick calves, hence, go undetected, or require re-treatment
due to delayed intervention and inappropriate antimicrobial
dosage for the first case, which makes it difficult to promptly
treat them, leading to greater chances of spread of disease, poorer
animal welfare, and greater negative impacts on economy and
environment, overall leading to poor sustainability of production
systems involving calves. The common practice for identifying diseases in calves is
based on visual appraisal and clinical examinations performed by
farmers and veterinarians (5). This practice is linked to a number
of disadvantages: (1) Calves identified as sick already show clear
clinical symptoms and may have already been sick for a while. For example, clinical signs of BRD might occur later than onset
of fever (24), or even without the occurrence of fever (5), and
clinical signs of NCD are visible when much of the associated
tissue damage to the intestinal submucosa has already occurred
(25). (2) Visual appraisal and clinical examinations are typically
poor at identifying sick calves. For example, in a study diagnosing
BRD in beef calves using clinical examination, the estimated
sensitivity and specificity were 61.8 and 62.8%, respectively (26). Many sick calves, hence, go undetected, or require re-treatment
due to delayed intervention and inappropriate antimicrobial
dosage for the first case, which makes it difficult to promptly
treat them, leading to greater chances of spread of disease, poorer
animal welfare, and greater negative impacts on economy and
environment, overall leading to poor sustainability of production
systems involving calves. We defined four stages of development of a particular sensor
technique for disease detection based on Rutten et al. (31)
(Figure 1): • Stage
one:
SENSOR
TECHNIQUE-applying
sensor
technology to automatically or manually record behavioral
or
physiological
parameters
in
animals,
visualizing
these parameters. • Stage two: DATA INTERPRETATION-changes in data are
detected and connected to changes in behavior and physiology
with an established link to the animal’s health status; • Stage three: INFORMATION INTEGRATION-multiple data
resources, e.g., treatment records and sensor data, are
integrated to direct the farmer to potential problems that
need attention; • Stage four: DECISION SUPPORT–a sensor system that aids to
make a decision, e.g., whether to treat an animal or not; what
to treat the animal for? Citation: Sun D, Webb L, van der Tol PPJ and
van Reenen K (2021) A Systematic
Review of Automatic Health
Monitoring in Calves: Glimpsing the
Future From Current Practice. Front. Vet. Sci. 8:761468. doi: 10.3389/fvets.2021.761468 November 2021 | Volume 8 | Article 761468 Frontiers in Veterinary Science | www.frontiersin.org 1 Automatic Health Monitoring in Calves Sun et al. Definitions
l
l d d Improved methods to detect health problems accurately
and on a timely basis in individual calves are warranted. The
decreasing cost and increasing implementation of electronic
tools allows for the application of “sensing solutions” to
animal farming. Behavioral and physiological parameters can
nowadays be automatically recorded at individual animal level, INTRODUCTION continuously and over long periods of time (27, 28). During the
past decade, various sensor data models have been proposed for
automatic health-monitoring systems in dairy and veal calves. To
date, however, there has been no systematic review presenting
the associated gaps in research, while literature reviews have
previously been done for pigs (29, 30), dairy cows (31, 32),
dairy sheep (33), and dairy calves (27, 28). The objectives
of this literature review were hence: to investigate previously
applied sensor validation methods and gold standards; to identify
sensors used on calves, the parameters these sensors monitor,
and the statistical tools applied to identify diseases; and to
explore potential research gaps to point to opportunities for
future research. Diseases, in particular bovine respiratory disease (BRD) and
neonatal calf diarrhea (NCD), are the most common causes
of morbidity and mortality in veal calves (1), dairy calves (2),
and beef youngstock (3). Despite slightly different prevalence
rates (4), disease types affecting dairy and veal calves are
similar (5–7). BRD symptoms include hampered respiration,
nasal discharge, and coughing (5). A direct symptom of NCD
is extremely watery feces (8). Potential risk factors for BRD
include: inadequate passive transfer of immunity from colostrum
(2, 9); low body weight at arrival in veal calves (10); poor
indoor housing conditions compared to outdoor housing (10);
and management practices such as weaning, comingling, and
castration (11). Potential risk factors for NCD include: high
exposure to pathogens causing NCD; factors related to host
resistance or susceptibility to disease, e.g., low quality and
quantity of colostrum; and factors about the environment that
favor the host or agent, e.g., high stocking density and too high
or too low ambient temperature and air humidity (12, 13). Frontiers in Veterinary Science | www.frontiersin.org Definitions
l
l d d Animals included in this review were bovine animals aged < 1
year; these include “calf” or “calves” (pre-weaned or weaned),
heifers (weaning to 1 year of age), growing bulls (after arrival
at the fattening farm up to 1 year of age), and beef cattle (early
fattening period until 1 year of age). Precision livestock farming
(PLF) is defined based on Berckmans (34) as “measuring variables
on the animals, modeling these data to select information,
and then using these models in real time for monitoring and
control purposes”. We defined the following terms–SENSOR:
an automatic tool capable of recording activities, behaviors,
physiology, and body size of calves continuously; MODEL:
a mathematical tool that describes the relations between the
sensor output and the actual values of the measured parameters
of the physical environment; VALIDATION: the process of
determining the measurement ability of automatic tools relative
to a gold standard using statistics. DISEASE: sickness status of an
animal occurred naturally or induced by disease challenges. Diseases in calves cause significant economic losses (14, 15),
due to treatment (16), impaired growth and mortality (17). Diseases also impaired calf welfare (18). Moreover, antibiotic
resistance, a major concern in human and veterinary medicine
(19), is a serious problem in the veal (20, 21) and dairy industry
(22). In addition, the overuse of antibiotics might result in the
contamination of surface water near farms due to residues in
the urine and feces of animals (23). Given the all-encompassing
impact of calf health on sustainability aspects, it is essential that
we develop accurate, timely, and practical systems to identify sick
calves, in both the dairy and veal sectors. The common practice for identifying diseases in calves is
based on visual appraisal and clinical examinations performed by
farmers and veterinarians (5). This practice is linked to a number
of disadvantages: (1) Calves identified as sick already show clear
clinical symptoms and may have already been sick for a while. For example, clinical signs of BRD might occur later than onset
of fever (24), or even without the occurrence of fever (5), and
clinical signs of NCD are visible when much of the associated
tissue damage to the intestinal submucosa has already occurred
(25). (2) Visual appraisal and clinical examinations are typically
poor at identifying sick calves. RESULTS AND DISCUSSION Following the article-selection process described above, 54
articles were included in this review (Figure 2). As shown in
Figure 3, 26 articles fell into stage one (sensor technique), 19
articles fell into stage two (data interpretation), and 9 articles fell
into stage three (information integration). We found no articles
at stage four (decision support). Data Management Extracted data were entered into and managed in excel
spreadsheets
(version
2016,
Microsoft
Corp.,
Redmond,
WA, RRID:SCR_016137). Search Strategy Systematic searches were conducted using the Web of Science
Core Collection database because it has high coverage rates of
animal behavior and welfare and bio-system engineering journals
with significant PLF contents. Inclusion and Exclusion Criteria Peer-reviewed scientific articles describing applying sensors to
calves were eligible for inclusion. Only articles based upon
original data were included. Included articles were written in November 2021 | Volume 8 | Article 761468 2 Automatic Health Monitoring in Calves Sun et al. FIGURE 1 | Four-stage development approach. FIGURE 1 | Four-stage development approach. sensor technology, or original articles of automatic health-
monitoring systems in mature cattle or other species. English, with complete, full-text documents available. To provide
up-to-date review, only articles published between 2009 and 2021
were included. Manuscripts published after the completion of
the literature search were not included (i.e., after May 10, 2021). Exclusion and inclusion criteria for the systematic review were
based on an previous work by Beaver et al. (35) and agreed upon
by all co-authors. • Step
two. Evaluating
abstracts–identify
and
remove
irrelevant articles. • Step three. Snowballing–checking and selecting references
within selected articles. • Step four. Eligibility. Full texts of the remaining articles were
read in detail. Original experimental studies were excluded if
not aiming at health monitoring in calves aged up to 1 year
using sensor technology. Data Extraction From each included article, where applicable, we recorded
the
objectives,
animal
category,
parameters
measured,
sample size, gold standards for validation, sensors used,
and measurements used to assess the performance of the
sensors or algorithms. Missing information was noted down as
“not available.” The results were pooled in the form of a table
(Supplementary Material). The reliability for data extraction
was tested by author 1 (DS) on a random subset of 20 articles,
with a result of 100% agreement. The following search terms were applied: (calf OR calves
OR dairy calf OR dairy heifer OR heifer calf OR heifers OR
young cattle) AND (BRD OR bovine respiratory disease OR
calf comfort OR calf health OR diarrhea OR group housing
OR health OR precision livestock OR precision livestock
farming OR proneness to disease OR welfare) AND (automatic
OR automated measurement OR automated measures OR
detection OR diagnosis OR disease monitoring OR evaluation
OR
modeling
OR
non-invasive
detection
OR
prediction
OR validation) AND (accelerometer OR activity sensor OR
artificial intelligence OR automatic milk feeder OR bioacoustics
OR computer vision OR electronic monitoring OR infrared
thermography OR low-cost sensor OR non-invasive technology
OR radio frequency identification OR reticulo-rumen bolus OR
statistical process control OR sound analysis OR 3-D sensor). The selection of these search terms was based on initial screening
of relevant articles to gain general background information and
expert opinion. Validation (a) manually collected parameter(s) as gold standard for their
validation, e.g., video observations of lying bouts or rectal body
temperature measurements using a thermometer. Stage two and
stage three studies aim to identify sick calves as early as possible. Stage two and three studies develop and test algorithms applied to
sensor data to accurately detect sick individuals. Manual health-
assessment protocols are typically used as gold standards to
develop and test these algorithms (Table 1). The validation assessments at different stages of studies
share common principles. Validation assessments are typically
calculated via so-called confusion matrices (Table 2) (36). Confusion matrices reveal relationships between the sensor of
interest, the selected gold standard (see the below formulas
for: sensitivity, specificity, accuracy, precision, and negative
predictive values) and the underlying prevalence of the disease
interest. “Positive and negative” show the sensor (or model)
output (a response of “yes” or “no” to the disease detection),
while “true and false” reflects whether the sensor (or model)
output is in line with the gold standards in a pre-specified time
window (i.e., whether the prediction matches the reality). When
comparing article outcomes, it should be noted that sensitivity
and specificity are affected by characteristics of the sensor, while
accuracy, precision, and negative predictive values are affected
by the prevalence of disease or behavior based on the dataset:
the higher the prevalence, the better the accuracy, precision,
and predictive values for the given dataset. Model developments
are usually aimed to enhance the contrast in a sensor system
output for the purpose of threshold evaluations (e.g., sensitivity,
specificity, or accuracy) over a given range. Common methods
used for model developments are correlation, area under curve
(36), and receiver operating characteristic curves (36). We first define what is meant by “validation” in this
review as well as define the terms used in this context,
i.e., sensitivity, specificity, accuracy, and positive and negative
predictive value. We follow up with a description of the
different gold standards that have been used at the different
stages of investigation. Next, we describe the various sensors
that have been used in calf-health-monitoring research, the
parameters these sensors record, and their accuracies in these
recordings. Selection Process The primary outcomes were selected based upon a four-step
screening and appraisal process (Figure 2): Studies at these different stages use different validation
methods and gold standards. Studies at stage one aim to check
that a given sensor is accurately recording a particular behavioral
or physiological parameter of interest. These studies typically use • Step one. Scanning the titles–filter out irrelevant results
such as review articles in automatic detection, original
articles of health monitoring in calves without applying November 2021 | Volume 8 | Article 761468 Frontiers in Veterinary Science | www.frontiersin.org 3 Sun et al. Automatic Health Monitoring in Calves FIGURE 2 | Article selection process. FIGURE 2 | Article selection process. Validation We end by presenting the current research at
stage two (data interpretation) and stage three (information
integration), revealing important knowledge gaps between
stage three and stage four (decision support), suggesting
the direction for future study that will enable the bridging
of these gaps, hence reaching automated health-related data
interpretation and complete decision-support systems for calf
production systems. November 2021 | Volume 8 | Article 761468 Frontiers in Veterinary Science | www.frontiersin.org 4 Sun et al. Automatic Health Monitoring in Calves FIGURE 3 | Distribution of stages of included articles. *In Studds et al. (56) both diarrhea and navel inflammation were studied. GURE 3 | Distribution of stages of included articles. *In Studds et al. (56) both diarrhea and navel inflammation were studied. Sensitivity =
true positives
(true positives + false negatives)
Specificity =
true negatives
(false positives + true negatives)
Accuracy
=
(true positives + true negatives)
(true positives + true negatives + false positives
+ false negatives)
Precision (positive predictive value)
=
true positives
(true positives + false positives)
Negative predictive value =
true negative
(true negative + false negative) intervals, are a commonly used reference for validation. Continuous sampling of focal animals will provide the most
accurate data for calf behavior, but is a time-consuming exercise. For certain, long-term, so-called “state” behaviors, instantaneous
scan sampling at regular intervals may provide an accurate
enough gold standard and is less time consuming, for example,
meal time and frequency over a 3 day period can be detected
accurately with instantaneous scan sampling at short intervals of
30 s and 1 min (37). However, one main disadvantage of video
observation is that it is labor intensive, and requires training to
achieve appropriate observer reliability; and observer error might
occur (38). For sensors recording physiological parameters, manual
measurements of these physiological parameters are also used
as gold standards. Sensors recording body temperature, e.g.,
body surface temperature (39), eye temperature (40, 41),
and rectal area temperature (40), typically use manually
recorded
rectal
temperature
as
gold
standard. When
validating body dimensions in calves and heifers, manual
measurements of body weight and dimensions are common,
including body weight (42, 43), hip height (42), and wither
height (42). Frontiers in Veterinary Science | www.frontiersin.org Gold Standard To obtain a sound validation of a sensor or PLF system, an
objective “gold standard” is needed. In this regard, studies at
stage one to stage four require different gold standards. At stage
one, gold standard means “variables of interest,” i.e., behavioral
or physiological parameters; at stage two, three, and four, gold
standard usually refers to the identification of disease, typically
via a manual “clinical examination.” With increasing research into validating sensors in terms of
how accurately they record behavior or physiological parameters,
previously validated sensors may be used as automated gold
standard to validate new sensors, which significantly reduces
labor required for these types of stage one studies. The Hobo
Pedant G Data Logger, for example, has been previously used as Stage one studies, where sensors are checked directly for their
ability to record behavioral or physiological parameters, tend
to use manual sampling of these behavioral or physiological
parameters. For
sensors
recording
behavioral
parameters,
behavioral observations of videos, continuous or at regular November 2021 | Volume 8 | Article 761468 Frontiers in Veterinary Science | www.frontiersin.org 5 Automatic Health Monitoring in Calves Sun et al. TABLE 1 | Gold standards of studies at stage two and three. No. Reference
Stage
Gold standard
Clinical examination
Blood analysis
Other
1
Borderas et al. (45)
2
Yes (daily)
2
Timsit et al. (24)
2
Yes (twice daily)
Yes
3
Schaefer et al. (16)
2
Yes (daily)
Yes
4
Moya et al. (58)
2
Yes (frequency information not available)
Carcass information, lung
lesions
5
Wolfger et al. (61)
2
Yes (twice daily)
Yes
6
Jackson et al. (55)
2
Yes (at least twice daily)
BW
7
Johnston et al. (52)
2
Yes (modified Wisconsin calf health scoring chart: twice
weekly in pre-weaning and weaning periods and once
weekly in post-weaning period)
Yes
8
Pillen et al. (81)
2
Yes (daily)
Depression score
9
Vandermeulen et al. (51)
2
Yes (Wisconsin calf clinical respiratory score: at least
twice weekly in pre-weaning period and once weekly in
post-weaning period)
Yes
10
Voss et al. (46)
2
Yes (at least twice daily)
11
Knauer et al. (53)
2
Yes (Wisconsin calf clinical respiratory score: daily)
Yes
Calf enrollment, treatment
record, morbidity and
mortality data
12
Swartz et al. (47)
2
Yes (Wisconsin calf health scoring chart: twice daily)
13
Carpentier et al. (65)
2
No examination
Yes
14
Knauer et al. Gold Standard (54)
2
Yes (Wisconsin calf clinical respiratory score: daily)
Calf enrollment, treatment
record, morbidity and
mortality data
15
Oliveira et al. (62)
2
Yes (daily)
Yes
16
Shane et al. (49)
2
Yes (daily)
17
Studds et al. (56)
2
Yes (twice weekly)
18
Kayser et al. (57)
2
Yes (twice weekly)
BW
19
Swartz et al. (63)
2
Yes (Wisconsin calf health scoring chart: twice weekly)
20
Hanzlicek et al. (60)
3
Yes (three times daily)
Yes
21
Szyszka et al. (66)
3
Rectal temperature (day 0, 13, 15, 17, 20, 27, and 31);
fecal samples (day 0, 13, 15, 17, 20, 27)
Yes
BW
22
Toaff-Rosenstein et al. (59)
3
Yes (daily)
Necropsy
23
Toaff-Rosenstein and Tucker (50)
3
Yes (daily)
24
Hixson et al. (48)
3
Yes (Wisconsin calf health scoring chart: twice daily)
25
Sutherland et al. (4)
3
Yes (daily)
Yes
BW
26
Lowe et al. (7)
3
Yes (daily)
27
Kayser et al. (72)
3
Yes (twice daily)
Yes
28
Duthie et al. (64)
3
Yes (modified Wisconsin calf health scoring chart: daily) TABLE 1 | Gold standards of studies at stage two and three. a gold standard to validate another accelerometer, the AfiTag II,
for lying behavior and step count (44). 62). Of all the clinical examination protocols, the (modified)
Wisconsin calf health score chart was the most commonly used
protocol (47, 48, 52, 63, 64). Gold standards without clinical
examination, e.g., from blood analysis (65), or a combination
of BW, biochemical parameters from blood and fecal samples,
and rectal temperature (66) have also previously been used. Clinical examination can be combined with clinical chemistry,
for example, via blood sampling, to improve the accuracy of
health assessment. Stage two and three studies aim to identify sick calves. Here, a clinical examination is the most commonly used
gold standard for disease diagnosis (Table 1) (45–50). Various
protocols have been used in this type of study, such as the
Wisconsin clinical respiratory score (51) and the Wisconsin
calf health scoring chart (52). Frontiers in Veterinary Science | www.frontiersin.org Gold Standard Further information can be
added to these clinical examinations to complement the gold
standard, including metadata such as management information
(e.g., calf registration or enrollment data), morbidity and
mortality data from the farm (53, 54), BW (55–57), post-mortem
examination (58, 59), or blood parameters (16, 24, 51–53, 60– Visual appraisal of disease, e.g., BRD, relies on the experience
of observers, and may have low specificity and be highly variable
between observers based on their level of experience (67). Thus errors from the clinical examinations may transfer to the Frontiers in Veterinary Science | www.frontiersin.org November 2021 | Volume 8 | Article 761468 6 Automatic Health Monitoring in Calves Sun et al. TABLE 2 | Confusion matrix. actual
Predicted
Positive
Negative
Positive
True positive
False negative
Negative
False positive
True negative can measure a wide range of parameters linked to feeding and
drinking patterns, including daily feed intake (4, 45, 47, 52–55,
57, 72–74), frequency and duration of rewarded and unrewarded
visits (4, 45, 47, 52–55, 57, 58, 61, 62, 72, 74), drinking speed
(milk) (47, 52–54), water-drinking behavior (intake, time, and
frequency) (62, 74), and other feeding behaviors (head-down
duration at the AFS) (55, 72), time-to-bunk: time to approach
feeding stations following feed-truck delivery (55, 72), and
duration of unrewarded visit intervals (55). We did not find
validation studies for common systems such as Förster-Technik
GmbH (n = 5; Engen, Germany) or GrowSafe Systems (n =
3; GrowSafe Systems Ltd., Airdrie, AB, Canada). However, we
found a study validating automatic feed and water bin (Intergado
R⃝Ltd, Contagem, Minas Gerais, Brazil), and showed that this
system seem to be able to measure feeding time, water drinking
time, feed intake per visits, and water intake per visits with high
correlation compared with the gold standards (r2 = 0.917, 0.963,
0.973, and 0.986, respectively) (74). corresponding models (58). Improvements are necessary for the
clinical examinations used as gold standards for the development
of algorithms to detect diseases in calves. Firstly, training in
clinical examination and high inter-observer consistency are
required. Secondly, to better relate clinical examinations to model
outcome, consistent and explicit definitions of diseases across the
literature are needed. When performing time-consuming clinical examinations for
use as a gold standard, the frequency of these examinations
needs to be carefully considered. Temperature Sensors Boluses, IRT cameras, and thermometers are used to measure
body temperature. These temperature sensors have been
developed to record body temperature at different anatomical
areas, enabling the measurement of rectal temperature (50, 59)
or temperature around the rectal area (40), reticulo-rumen
temperature (24, 46), eye temperature (7, 16, 40, 41, 82), cheek
temperature (7, 82), back, shoulder, and side temperature (7),
and temperature at the base of the tail (39). These cameras have
also shown high accuracy in measuring cheek temperatures (82),
but have not been found to be highly accurate in measuring
temperature around the rectal area (40) or core body temperature
(41). In terms of eye temperature, IRT cameras seem to show
varying levels of correlation between eye temperature and
rectal temperature, e.g., high correlation (R2 ≥0.99) (82), low
correlation (R2 ≤0.32) (40). This might be partly due to the use of
an detection algorithm (82). A prototype thermometer provided
by Nogami et al. (39) has been found to measure tail temperature
with high correlation compared with rectal temperature in calves. Stage One: Sensor Technology Used in
Calves Data sources used in calves include automatic feeding stations
(AFS), accelerometers, microphones, infrared thermography
(IRT) cameras, temperature sensors (i.e., boluses, thermometers),
radiofrequency identification (RFID) chips, 3-D cameras, and
2-D cameras. Gold Standard As observed by the current
authors, daily clinical examinations of calves can provide better
timely reference, at the cost of disturbance to the group and
high labor requirements. However, a low frequency of clinical
examinations will result in late detection, making it difficult
to develop an early disease detection algorithm (68). Previous
research applied different frequencies–for clinical examinations–
ranging from daily to weekly (Table 1). Clinical examinations
combining two different frequencies applied at different life
stages were also found, e.g., before (twice a week) and after the
weaning period (once per week) of dairy calves (51, 52). To
the author’s knowledge, no study has yet compared the effect of
different frequencies of clinical examinations on the accuracy of
disease-detection models. Accelerometers
l Accelerometers are attached to the body of the calf, generally
to one of the limbs, neck, or ear (tag). They are typically used
to assess various activity-related behaviors. Accelerometers are
accurate in recording calf behaviors, including lying time (44,
75–78), lying bouts (44, 75, 76, 78), standing time (75, 78),
standing bouts (75), step counts (44, 79), locomotion time (78),
gait scoring (79), feeding time (73, 78), sucking behavior from
dams (38), and licking or sucking at objects, other calves’ bodies,
or own body (78). After more than 10 years of development,
accelerometers are now used to record a broader variety of
behaviors and more detailed behavioral patterns, e.g., recognizing
between galloping, trotting, and walking (79), and recording
behaviors such as eating, water drinking, chewing, positive social
interactions, self-grooming, and inactivity (80). Step counts were
originally measured by pedometers (60). This activity parameter
was later integrated into accelerometers (44, 47, 66, 79, 81). In summary, clinical examination is the most common gold
standard used in the development of algorithms to identify sick
calves. The Wisconsin calf health score chart was identified
as a commonly used protocol for clinical examination in this
context, with a sensitivity of 62.4, and specificity of 74.1% (69). Clinical references with high accuracy, consistent guidelines,
and easy-to-follow protocols are needed for disease detection in
calves. A standardized clinical scoring system will benefit the
validation of the sensors and algorithms, making it easier to
compare the performance of different algorithms. In addition,
ultrasonographic assessment of the thorax could be a useful tool
to assess BRD detection in calves (70, 71). Changes in Other Parameters Coughing which is a typical symptom of BRD can be detected
using microphones (83). Note that as opposed to activity and
feeding behaviors, coughing has so far only been measured at
group level. An increased coughing frequency was found to be
correlated to BRD occurrence in group-housed calves (51, 65). Changes in Feeding Behaviors Changes in Feeding Behaviors
Feeding behaviors and patterns, including intake, frequency,
speed, and duration at various time ranges, are commonly used
parameters for the early detection of disease in calves (91, 92). Note that most studies look at feeding behaviors aggregated at
a daily level. With the application of RFID, individual calves
are identified at AFS, whereby individual feeding behaviors can
be recorded. For example, pre-weaned calves diagnosed with
BRD drank less milk on the day of clinical examination (47)
and on the first day of treatment (53), drank milk slower 4
days prior to the clinical examination (53), and performed fewer
unrewarded visits to the milk dispenser 3 days prior to (52),
and on the first day of treatment (53). Moreover, net daily
energy intake (calculated for each calf by summing daily milk
replacer and concentrate intake values) (52) and DMI (55), were
reduced in BRD-infected calves in the time prior to the clinical
examination, e.g., 3 days in Johnston et al. (52) and 6.8 days in
Jackson et al. (55). In calves diagnosed with NCD, daily milk
intake and time at water trough dropped 4 days prior to clinical
examination (7). With the application of approaches such as computer vision
or machine learning, an even broader range of parameters might
be recorded with the available sensors. For example, Carslake
et al. (89) applied machine-learning approaches to multi-class
behavior identification (including locomotor play, self-grooming,
ruminating, non-nutritive suckling, nutritive suckling, active
lying, and non-active lying) as well as behavior quantification
(i.e., behavior distribution) using a single sensor (comprised of
an accelerometer and gyroscope) in calves. Computer vision
allowed 2-D cameras to identify multiple behaviors, e.g., pen
entering, pen leaving, standing or lying static behavior, turning,
and feeding and drinking behaviors (90). 3-D cameras can
monitor growth and morphology (i.e., BW, body mass, hip
height, and wither height) in young calves and heifers (42,
43). Knowing
which
parameters
that
sensors
(or
sensor
combinations) can accurately measure can contribute to
the development of an efficient sensor system at stage two and
three. For example, accelerometers are not accurate in screening
rumination time in calves (73), but this can instead be achieved
by microphones (84, 85). Both accelerometers and AFS can
record feeding and water-drinking behaviors, but AFS can
record these behaviors directly without having to apply statistical
models and are non-intrusive, i.e., not attached to the animal
(80). Changes in Activity Changes in activity parameters, such as step counts and lying
behaviors, are used to detect sick calves. In calves diagnosed with
BRD, for example, step counts (<6 days), lying bouts (<5 days),
standing time (< 1 day) were reduced (81). In calves diagnosed
with NCD, results are inconclusive regarding activity: lying bouts
were found to both decrease (<7 days) (7) and increase (<7
days to 3 days) (63), and lying durations were found to both
decrease (<6 days to 3 days) (63) and increase (<7 days) (7). Finally, calves with inflamed navels show reduced lying time at
day level after arrival at fattening farms compared with healthy
calves (56). To sum up, available sensors (AFS, accelerometers, IRT
cameras, microphones, and 3-D cameras) are accurate in
measuring different behavioral or physiological parameters in
calves, and approaches such as machine learning and computer
vision broaden the range of behaviors that sensors can record. Future work should further develop behavior classification
and quantification by applying computer-vision and machine-
learning approaches. Automatic Feeding Stations AFS, such as automated milk dispensers for pre-weaned calves
and automatic concentrate bunks for post-weaned calves, and
water bins, have been used in studies aimed at automated health
monitoring in young calves, hence stage two research. These AFS November 2021 | Volume 8 | Article 761468 Frontiers in Veterinary Science | www.frontiersin.org 7 Automatic Health Monitoring in Calves Sun et al. Changes in Feeding Behaviors In addition, no further hardware is needed when extracting
data from AFS compared with accelerometers. Other Sensors and Techniques the accuracy (or performance) of algorithms in detecting sick
calves based on changes in these behavioral and physiological
parameters. In this section, we highlight the algorithms that
can detect diseases prior to clinical confirmation (Table 3), and
summarize changes in behavioral and physiological parameters
in response to disease as well as time course: disease states
in animals typically lead to both behavioral and physiological
changes over time. Microphones, when integrated into sound-acquisition systems,
can detect abnormal cough sounds (51, 65, 83) and rumination
sounds in calves (84–86). The performance of microphones
varied in calves of different ages. Microphones accurately
recorded rumination time in pre-weaned calves (85), but
overestimated rumination time in weaning calves (86). RFID ear
tags can be applied to monitor grooming behavior (measured
via proximity to a brush) in heifers (87). IRT cameras have also
been used to assess respiration rate in calves, at a high level of
accuracy (88). Stage Two: Data Interpretation–Outcomes
of Algorithms In order to develop a sensor-based system that detects sick
calves, i.e., sensor technology combined with algorithms, stage
two studies must follow three steps: (1) identify how behavioral
or physiological parameters change with disease, identified via
a gold standard (this includes the selection of both parameters
of interest and corresponding sensors); (2) investigating how
these behavioral and physiological changes vary at which stage of
disease they can first be detected; and (3) developing and testing Changes in body temperature can be used to detected sick
calves before clinical examination. BRD-diagnosed calves showed
increases in orbital (eye plus 1 centimeter surrounding the eye)
maximum temperature (16) and reticulo-ruminal temperature,
e.g., −136 to −12 h (24) and −3.5 days (46) relative to diagnosis. One important methodological consideration with thermometers
is that recorded temperatures differ based on the body area that
is investigated. For example, skin temperature was consistently November 2021 | Volume 8 | Article 761468 Frontiers in Veterinary Science | www.frontiersin.org 8 Automatic Health Monitoring in Calves Sun et al. TABLE 3 | Performance of algorithms and models. No.References
Features
Performance
Sea (%)
Spb (%)
Accuracy
(%)
PPVc (%)
NPVd (%)
Other
parameters
Days prior
(best)
Days
(lea
1
Jackson et al. (55)
Feeding behavior
−14.2
−1
2
Kayser et al. (57)-univariate factors
Feeding behavior
48.7–80.1
−10.2
−0
3
Wolfger et al. (61)
Feeding behavior
−7
4
Lowe et al. (7)
Feeding behavior, lying behavior,
body temperature
−7
−4
5
Swartz et al. (63)
Activity, lying behavior
−7
−6
6
Jackson et al. (55)
DMI
−6.8
7
24
Reticulo-rumen temperature
0.91 (r), 0.82
(r2)
−5.7
−0
8
Sutherland et al. (4)
Feeding behavior, lying behavior
−5
0
9
Pillen et al. (81)
Activity
−5
−
10 Kayser et al. (72)
Feeding behavior
0.61–0.89
−4.5
11 Kayser et al. (72)
Rumen temperature
0.78
−4.5
12 Sutherland et al. (4)
Feeding behavior
−4
0
13 Knauer et al. (53)
Feeding behavior, activity
−4
7
14 Voss et al. (46)
Reticulo-ruminal temperature
71
98
86
98
0.855 (area
under curve)
−3.5
15 Moya et al. (58)-model 33
Feeding behavior
66.7
58.3
62.5
−3.1
16 Moya et al. (58)-model 66
Feeding behavior
75
50
50
−3.1
17 Knauer et al. (54)
Feeding behavior
56.4
49.5
66.6
49.5
−3.1
18 Knauer et al. (54)
Feeding behavior
70.9
32.9
65.3
38.7
−3.1
19 Knauer et al. Stage Two: Data Interpretation–Outcomes
of Algorithms (54)
Feeding behavior
74.9
27.1
64.6
37.4
−3.1
20 Johnston et al. (52)
Feeding behavior
−3
21 Oliveira et al. (62)
Feeding behavior
−3
4
22 Duthie et al. (64)
Feeding behavior, activity
−3
−
23 Moya et al. (58)-model 14
Feeding behavior
58.3
83.3
70.8
−2.4
24 Kayser et al. (57)-multivariate
factors
Feeding behavior
84
−2.1
−2
25 Shane et al. (49)
Social network patterns
17.9–100
<10
>90
−2
0
26 Swartz et al. (47)
Behavior, activity
−2
27 Sutherland et al. (4)
Feeding behavior
−2
0
28 Sutherland et al. (4)
Lying behavior
−2
0
29 Toaff-Rosenstein and
Tucker (50)
Rectal temperature
−2
30 Moya et al. (58)-model 3
Feeding behavior
50
100
75
−1
31 Oliveira et al. (62)
Feeding behavior
−1
1
32 Toaff-Rosenstein and
Tucker (50)
Feeding behavior
0
arefer to sensitivity. brefer to specificity. crefer to positive predictive value. drefer to negative predictive value. TABLE 3 | Performance of algorithms and models. arefer to sensitivity. brefer to specificity. crefer to positive predictive value. drefer to negative predictive value. arefer to sensitivity. brefer to specificity. crefer to positive predictive value. drefer to negative predictive value. calves. In calves diagnosed with NCD, the temperature of the
side flank and shoulder increased at least seven days prior to
diagnosis (7). 2 to 3◦C lower than the rectal temperature (39), while reticulo-
rumen temperature was consistently 0.57◦C higher than rectal
temperature (24). As long as these differences between recorded
temperature and body temperature are consistent, this should not
affect the detection of temperature increases due to diseases in Changes in social behaviors were also detected in sick calves. In a modeled disease challenge study (calves were infected Frontiers in Veterinary Science | www.frontiersin.org November 2021 | Volume 8 | Article 761468 9 Automatic Health Monitoring in Calves Sun et al. with Mannheimia haemolytica), sick calves were found to show
decreases in daily social grooming time and daily social lying time
(lying within one body length of another calf) (48). with Mannheimia haemolytica), sick calves were found to show
decreases in daily social grooming time and daily social lying time
(lying within one body length of another calf) (48). Given that many sensors and techniques are already
commercially available, it is crucial to choose an appropriate
sensor system when recoding certain parameters. Stage Two: Data Interpretation–Outcomes
of Algorithms Ideally,
models at this stage include a minimum number of sensors per
animal, which is advantageous in terms of costs and maintenance
labor but also in maintaining the integrity and freedom of
movement of calves. Stage Four: Decision Support–Automation
At stage four, decision support means that the integrated system
can identify which disease is occurring based on the developed
model. Farmers can refer to the decision made by the system
as support. To the author’s knowledge, no such systems are
available for early disease detection in calves. An example of a
stage four system in dairy cows is estrous detection and automatic
identification of the best way to inseminate the cow (99). In the
situation of early disease detection in calves, however, so far only
alerts are available. For the future, automation is crucial–a decision-support
system with an easy-to-operate user interface is what farmers
need for an easy identification of sick calves. Current models
may give some form of alert, yet cannot give automatic
decision support. Another important characteristic of such
systems is the possibility for the farmers to enter feedback
(e.g., whether the identified individual was truly ill with the
suspected disease and whether the treatment was efficient)
so that the system can continuously learn and adapt to
the specific farming conditions. Knight (98) suggested a
business model that bridges information integration and decision
support. In the provided business model, farmers are buying
a service from service providers. A service provider purchases
the technologies from different developers, and provides the
service of installation, maintenance, data collection, and data
integration, thereby providing decision support to the farmers. However, at the same time we believe that technology should
not replace but, rather, support management decisions made by
the farmer. Frontiers in Veterinary Science | www.frontiersin.org Stage Three: Information To date, sensor fusion (i.e., two or multiple sensors) was applied
in a number of studies (n = 9, Figure 3), in which data from
accelerometers, thermometers, and AFS are integrated into one
model to identify diseases including BRD (64, 72) and NCD (4, 7). Information integration, however, means more than a multiple-
sensor tool. First, “integration” does not mean accumulating all
the data obtained from different sources. In the design of systems
at stage three, redundancy needs to be reduced for a disease-
detection model. To reach this, data mining (98), which allows
for a more complete understanding of different parameters in
relation to disease occurrence, is a prerequisite. Data mining
allows for the selection of the key parameters, the variation of
which reflects health status with high accuracy. In this way,
by reducing the redundancy, the number of sensors used and
possibly attached to a calf will be reduced. Second, multiple
data sources mean that sensor data are not the only sources of
data. Economic insights, for example, were also suggested to be
considered for the treatment decisions (24, 57). Stage Two: Data Interpretation–Outcomes
of Algorithms For example,
the combination of video cameras and sensors (including
thermometers, accelerometers, or AFS), although popular for
research (n = 5), seems impractical for on-farm settings. This might be due to the number of cameras required and
the time-consuming process of analyzing the video footage. However, a recent study show that artificial intelligence is
able to identify the physiology and behavior of animals
using video footages with high accuracy (90), allowing for
less labor. Some other behavioral parameters can be well recorded by
sensors, but their potential in early disease detection is yet
unknown. These include sucking behavior (38), rumination time
(86), and play behavior (93–95). Further research into the link
between these parameters and disease is warranted. As explained above, behavior and physiology change with
disease, and these changes can be detected using sensors and
algorithms. Theoretically, “behaviors that are less critical for
immediate survival and primarily support long-term fitness are
most affected by disease” (96), such as play and exploratory
behaviors (97). In practice, the type of diseases and the age
of the animals also need to be taken into consideration as
they might influence behavioral deviations. For example, in
parasitized beef steers (aged between 4 and 11 months) and
BRD-infected dairy calves, changes in activity (e.g., lying,
standing, and step counts) enabled a better disease detection
than feeding behaviors such as frequency and duration of
feeding and drinking (66) and feed intake (47). In identifying
NCD-infected calves and BRD-infected steers, however, feeding
behaviors (e.g., the number of unrewarded visits to an
automated milk dispenser, DMI, and bunk visit duration)
permitted a more accurate detection of disease compared with
activities such as lying and standing duration (4, 72). In
addition, certain diseases result in behavioral changes that are
easier to detect at an earlier stage. NCD-diagnosed calves,
for example, displayed earlier and more consistent changes
in feeding behaviors compared with BRD-diagnosed calves
(53). Further research is hence needed into identifying the
best, most sensitive behavioral and physiological parameters
that can identify specific diseases or diseased state on a
generic level. Therefore, “information integration” means selecting as
few meaningful parameters indicative of diseases as possible
when developing models (thereby avoiding redundancy). The
integrated systems will give an alert when the current status of
a calf deviates from its earlier patterns, i.e., being sick. CONCLUSIONS This review summarized the literature on sensor systems
so far studied in the context of health monitoring in
calves between 2009 and 2021, and revealed the current
phase of development by categorizing each study based on
a four-stage system (sensor technology, data interpretation,
information integration, decision support). Our literature search
demonstrated that most studies up to now are at stage one
(sensor technique) or stage two (data interpretation), and a
few studies are at the beginning of stage three (information
integration). Accelerometers, IRT cameras, microphones, and
3-D cameras can be accurate in measuring behavioral and
physiological parameters in calves (at stage one). Deviations in
behaviors (e.g., feeding, lying, and social behaviors), activity,
and body temperature can be detected prior to the clinical
examination (at stage two and three), and are promising for
developing algorithms. To develop a health detection model
with a minimal number of sensors, it is crucial to select
appropriate sensor systems that can record the most relevant
parameters that show clear changes in response to diseases in
calves. Clear gaps in research include stage three (information
integration) and stage four (decision support) systems, as
well as forecasting methods via the identification of low-
resilience animals. Resilience in farm animals has been defined as “the capacity
of the animal to be minimally affected by a disturbance or to
rapidly return to the physiological, behavioral, cognitive, health,
affective and production states that pertained before exposure
to a disturbance” (100). Calves falling sick can be equaled to
a complex system transiting from one stable state (healthy)
to another (unhealthy), with the return to the original state
being more difficult than the simple cancellation of factors that
caused the change in state. Such shifts in complex systems
have been termed “critical transitions” or “tipping points” (101). When such complex systems are close to tipping points, the
recovery rate of that system from small perturbations becomes
very slow, and this is known as “critical slowing down” (CSD)
(102). For example, a cow showing “CSD” before parturition,
in this case by using an accelerometer to assess activity (e.g.,
low average eating time, a disturbed circadian rhythm, and
variance in ear temperature), is likely to develop periparturient
disorders (103). CSD, which can be revealed through dynamic
aspects of sensor data, is here seen as an increase in variance
in the activity data, hence a loss of regularity. CONCLUSIONS CSD, therefore,
reflects a loss of resilience (101, 102). In still clinically healthy
individuals, CSD reflects the animal’s vulnerability to pathogens
prior to the disease, and hence reflects a state of low resilience. Identifying CSD in sensor data patterns of “low-resilient”
individual animals, would enable, for example, timely change
of the environment of this animal in an attempt to increase its
resilience (e.g., by removal of stressors, or the improvement of
nutrition, etc.). FUNDING This
study
was
financially
supported
by
Stichting
Brancheorganisatie Kalversector (SBK), the Dutch Ministry
of Agriculture, Nature and Food Quality. This
study
was
financially
supported
by
Stichting
Brancheorganisatie Kalversector (SBK), the Dutch Ministry
of Agriculture, Nature and Food Quality. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding authors. AUTHOR CONTRIBUTIONS DS wrote the review (main body) and LW, PT and KR
contributed during the discussion and reviewed the preliminary
versions of the manuscript. All authors contributed to the article
and approved the submitted version. Introducing “Resilience” Theory Following the above four-stage approach, the decision-support
system is regarded as a screening tool that aims to detect
diseases at an early stage and provides a short list of “positive November 2021 | Volume 8 | Article 761468 Frontiers in Veterinary Science | www.frontiersin.org 10 Automatic Health Monitoring in Calves Sun et al. cases” of animals that have a sufficiently high chance of being
prone to develop the targeted disease. Hence the farmer and
the care taker is provided a window of opportunity to check
for the clinical status. Additionally some preventive measures
can be taken or a predefined health protocol can be applied. A negative outcome of a decision-support system predicts
the likelihood of a clinically healthy animal of becoming
diseased in the near future, i.e., its predisposition to diseases
then is generally speaking a balance between environmental
infection pressure and its immune system functioning. In a
paper discussing sensor technologies in dairy farming, Knight
argued that “the focus is on improving overall husbandry,
rather than “solving” specific disease problems” (98). The
same focus should apply to the dairy and veal industry
as well. We therefore introduce “resilience” theory, through
which the developed system might be able to quantify the
resilience of individual animals, thereby identifying animals
in a low-resilience state. This potentially allows for early
intervention in the husbandry system, whereby the environment
or management are specified in such a way that low-resilient
individuals and the herd as a whole can maintain relatively
healthy states. eventually also be applicable to advanced and innovative calf
health management systems. Frontiers in Veterinary Science | www.frontiersin.org REFERENCES Marcato F, van den Brand H, Kemp B, van Reenen K. Evaluating potential
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moving beyond classification in precision livestock. Sensors. (2021) 21:88. doi: 10.3390/s21010088 90. Guo Y, He D, Chai L. A machine vision–based method for monitoring. Animals. (2020) 10:190. doi: 10.3390/ani10020190 Copyright © 2021 Sun, Webb, van der Tol and van Reenen. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
terms. 91. Johnson RW. The concept of sickness behavior: a brief chronological account
of four key discoveries. Vet Immunol Immunopathol. (2002) 87:443–50. doi: 10.1016/S0165-2427(02)00069-7 92. Svensson C, Jensen MB. Short communication: identification of diseased
calves by use of data from automatic milk feeders. J Dairy Sci. (2007)
90:994–7. doi: 10.3168/jds.S0022-0302(07)71584-9 92. Svensson C, Jensen MB. Short communication: identification of diseased
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90:994–7. REFERENCES doi: 10.3168/jds.S0022-0302(07)71584-9 November 2021 | Volume 8 | Article 761468 Frontiers in Veterinary Science | www.frontiersin.org 14
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Ampliación de la distribución del Armadillo Cola de Trapo Transandino Cabassous centralis (Cingulata: Chlamyphoridae), para el Alto Magdalena en Colombia
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Mammalogy notes
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NOTAS
Mammalogy Notes ISSN 2382-3704
https://doi.org/10.47603/mano.v7n1.220
Extensiones de distribución
Ampliación de la distribución del Armadillo Cola de
Trapo Transandino Cabassous centralis (Cingulata:
Chlamyphoridae), para el Alto Magdalena en
Colombia
Diego A. Gómez-Sánchez1*
, Sebastián García-Restrepo2
, Carlos Bravo-Vega3
1 Rey Zamuro – Matarredona (RN), San Martín de los Llanos, Dpto. Meta, Colombia.
2 Museo de Historia Natural C.J.Marinkelle, Departamento de Ciencias Biológicas, Universidad de los Andes. Carrera 1 #18A12, M 101, Bogotá D.C., Colombia.
3 Grupo de investigación en Biología Matemática y Computacional (BIOMAC), Departamento de ingeniería biomédica,
Universidad de los Andes, Carrera 1 #18A-12, Bogotá D.C., Colombia.
* Correspondencia: diegophidio@hotmail.com
Resumen
Colombia es uno de los países con mayor riqueza de armadillos al contar con seis especies,
pertenecientes a tres géneros, que abarcan la mayor parte del territorio nacional, siendo Cabassous
centralis la única especie trasandina. En este estudio buscamos aportar al conocimiento de la
distribución brindando nuevos registros para el departamento del Tolima. Durante un muestreo
nocturno de serpientes, en enero de 2019, se observó y fotografió un individuo de Cabassous
centralis en el oriente del departamento, en estribaciones de la Cordillera Oriental. Este registro,
junto con un espécimen preservado en la colección de mamíferos del Instituto de Investigación de
Recursos Biológicos Alexander von Humboldt, confirman la presencia de la especie en el Tolima y
son los registros más al sur en su distribución dentro del Valle del Magdalena.
Palabras clave: coletrapo, nuevo registro, bosque seco tropical, Valle del Magdalena, Tolima
Abstract
Colombia is a country with a great richness of armadillos, with six species belonging to three genera,
covering most of the national territory. One of them, Cabassous centralis, is the only trans-andean
species. An individual of Cabassous centralis was found and photographed during a snakesearching in January of 2019. The casual encounter occurs at the eastern Tolima department, in the
slopes of Eastern Cordillera. This record, together with a specimen preserved in the mammal
collection of the Alexander von Humboldt Biological Resources Research Institute, confirm the
presence of the species in the department and are the southernmost records in its distribution
within the Magdalena Valley.
Key words: northern naked-tailed armadillo, new record, tropical dry forest, Magdalena Valley,
Tolima
Mammalogy Notes 2021, 7(1), 220; https://doi.org/10.47603/mano.v7n1.220
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Cabassous centralis (Miller, 1899) es una de las seis especies de armadillos presentes en
Colombia, con una distribución estrictamente transandina, y cuyo rango global abarca
Centroamérica y parte del norte de Suramérica entre 0 y 3018 m.s.n.m. (Cruz-Rodríguez et
al. 2011; Solari et al. 2013; Tirira et al. 2014; Ramírez-Chaves et al. 2016; Soriero et al. 2018;
Aya-Cuero et al. 2019; Sánchez-Brenes & Monge 2019; Sánchez-Soto et al. 2020). En
Colombia se distribuye en las provincias biogeográficas de: Chocó-Magdalena, Cinturón
Árido Caribeño, Macizo de la Sierra Nevada de Santa Marta y en gran parte de la Provincia
Norandina (Hernández et al. 1992; Alberico et al. 2000; Cruz-Rodríguez et al. 2011; Tirira et
al. 2014; Aya-Cuero et al. 2019; Chacón-Pacheco et al. 2019; Santos et al. 2019; Figura 1). Esta
especie cohabita con Dasypus novemcinctus en la región transandina de Colombia y se
diferencian porque C. centralis es robusto, ligeramente aplanado dorsoventralmente,
posee un hocico corto y robusto, las orejas se encuentran bien distanciadas entre sí por
tres o más placas, posee uñas hipertrofiadas en las manos (ver Figura 2) y una cola
desnuda con pequeñas placas muy espaciadas entre sí. Por su parte, D. novemcinctus tiene
un hocico largo y angosto, orejas muy juntas y cola cubierta por placas dérmicas
(Rodríguez-Mahecha et al. 2006; Gardner 2008; Hayssen et al. 2013).
Existen notables vacíos dentro de la distribución nacional de C. centralis, específicamente
en la cuenca del río Magdalena donde se ha reportado en el Medio y Bajo Magdalena, con
solo un par de registros al extremo norte del Alto Magdalena en el departamento del
Tolima (Aya-Cuero et. al. 2019; Figura 1) pero sin evidencia de su presencia al interior de
este (García-Herrera et al. 2015; Aya-Cuero et al. 2019; García-Herrera et al. 2019). En la
presente contribución se reportan los primeros registros de C. centralis para el interior del
Valle Alto del Magdalena.
El primer registro se realizó por encuentro ocasional en campo y el segundo corresponde
a un espécimen preservado, no publicado previamente, encontrado en la colección de
mamíferos del Instituto de Investigación de Recursos Biológicos Alexander von Humboldt,
Villa de Leyva-Boyacá (IAvH-M-5407). Se descartó el reporte de un espécimen, de la misma
colección (IAvH-M-7056), proveniente de una localidad cisandina en el departamento de
Norte de Santander (que podría corresponder a C. unicinctus) debido a que no se pudo
corroborar su determinación. Finalmente, se elaboró un mapa (Figura 1) con el programa
QGIS (Open Source Geospatial Fundation Project) y se utilizó Google Earth Pro ©2021
Google para medir las distancias geográficas en línea recta aérea (km) entre los nuevos
registros y los conocidos previamente (Aya-Cuero et al. 2019; Santos et al. 2019).
El 30 de enero de 2019, a las 21h15, se observó de forma incidental a un individuo de C.
centralis (Figura 2), el cual se encontraba aparentemente forrajeando entre la hojarasca.
Fue capturado de forma manual por el baquiano (guía local) quien consideraba
consumirlo, sin embargo, se liberó rápidamente al notar que no correspondía a la especie
D. novemcinctus, momento en que los autores realizaron el registro fotográfico. Esta
observación tuvo lugar en el Alto Magdalena, en el Valle Apicalá a una altitud de 427
m.s.n.m. (4.063889, -74.758306 WGS84; Figura 1), específicamente en la quebrada La Caja, en
una finca privada en la localidad conocida como La Cajita, vereda Cuatro Esquinas,
municipio de Carmen de Apicalá, al oriente del departamento del Tolima, Colombia. El área
es un agroecosistema compuesto por praderas para cría de ganado vacuno, con quebradas
de cobertura arbórea y arbustiva. Se encuentra al borde de los remanentes de Bosque
Seco Tropical (BST) que aún persisten, con enclaves semi-húmedos de bosques de ribera,
con alta intervención local de subsistencia.
Mammalogy Notes 2021, 7(1), 220; https://doi.org/10.47603/mano.v7n1.220
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FIGURA 1. Distribución actualizada de Cabassous centralis en Colombia. Círculos: registros
conocidos previamente (Aya-Cuero et al. 2019; Santos et al. 2019). Estrellas: nuevos registros para
el Alto Magdalena (Tolima-CO). Estrella blanca: Coyaima (IAvH-M-5407). Estrella Roja: Carmen de
Apicalá.
El espécimen IAvH-M-5407 (Figura 2), es un caparazón preparado en seco, recolectado en
1986 por Humberto Piñeros, proveniente también del Alto Magdalena a 357 m.s.n.m.
(3.797618, -75.153116 WGS84; Figura 1), y específicamente de la vereda Zaragoza-Tamarindo
situada entre los ríos Saldaña y Magdalena, en el municipio de Coyaima, centro del
Mammalogy Notes 2021, 7(1), 220; https://doi.org/10.47603/mano.v7n1.220
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departamento del Tolima, Colombia. Desconocemos el panorama ambiental al momento
de la recolección, sin embargo, actualmente toda el área está cubierta por
agroecosistemas abiertos principalmente para uso ganadero. La zona que abarca estos
dos registros se caracteriza por poseer una precipitación anual de 1630 mm en un ciclo
bianual con una temperatura media anual de 28 °C (IDEAM 2017).
FIGURA 2. Nuevos registros de Cabassous centralis para el Alto Magdalena en Tolima - Colombia.
a-c encuentro directo en el municipio de Carmen de Apicalá. d-f Espécimen IAvH-M-5407
proveniente del municipio de Coyaima (sentido antero-posterior de izquierda a derecha; escala 10
cm).
Estos registros confirman la presencia de C. centralis en el departamento del Tolima, y
amplían la distribución conocida de la especie dentro del valle seco del Alto Magdalena,
sumando 131.95 km en línea recta aérea hacia el sur y 90 km en línea recta aérea hacia el
oriente desde los registros más cercanos, ubicados en el valle del Magdalena y el flanco
Mammalogy Notes 2021, 7(1), 220; https://doi.org/10.47603/mano.v7n1.220
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oriental de la Cordillera Central, respectivamente (Figura 1). De esta manera corroboran su
presencia, hasta ahora, en gran parte del área total que abarca el BST en el Alto Magdalena,
comparado con menos del 1% estimado previamente (Aya-Cuero et. al. 2019). No obstante,
con relación a la representatividad de esta especie en la Cordillera Oriental, es relevante
destacar el registro del Carmen de Apicalá, el cual expande la distribución de C. centralis
aproximadamente 287 km en línea recta aérea, en sentido suroccidente, desde el registro
más cercano situado sobre el flanco occidental de la Cordillera Oriental (Chucurí Santander). Sin embargo, la Cordillera Oriental aún posee la más baja representatividad
de la distribución conocida para el Coletrapo en los Andes de Colombia (Figura 1).
El registro del Carmen de Apicalá es el primero para el oriente del Tolima, así mismo es el
registro más al sur sobre las estribaciones occidentales de la Cordillera Oriental, al igual,
el espécimen IAvH-M-5407 es el registro más al sur dentro de su distribución general en el
valle seco del Alto Magdalena. Por consiguiente, es probable que su distribución se
extienda por todo el Alto Magdalena, dada la afinidad ecosistémica presente y la aparente
ausencia de barreras geográficas y biológicas/ecológicas (relaciones interespecíficas) que
pudieran limitar su distribución en esta ecorregión. Finalmente, se recomienda realizar
inventarios enfocados en estimar su densidad poblacional y así, a futuro, reevaluar su
estado de amenaza que actualmente se encuentra en Datos Insuficientes (DD) a nivel
global y nacional (Rodríguez-M et al. 2006; Tirira et al. 2014; Sánchez-Soto et al. 2020).
AGRADECIMIENTOS
A Lusmila, Tulio y Nelson por su hospitalidad y colaboración durante el trabajo de campo.
Al Dr. Juan Cordovéz, por su apoyo y por su calidez humana, quien nos introdujo al Valle
de Apicalá. Al Sr. José G. Cordovéz, la Sra. Gabriela Álvarez y su familia, por facilitarnos las
instalaciones necesarias para nuestra estancia durante el trabajo de campo. A Juan
Esteban Villota por compartir bibliografía precisa al caso y María José Andrade Erazo por
realizar comentarios valiosos para mejorar del manuscrito. A Nicolás Reyes Amaya, Daniela
García Cobos y Alejandra Jaramillo Marín por facilitar el acceso a la información y
fotografías del espécimen IAvH-M 5407. Agradecemos, a la convocatoria 727 del 2016 para
estudiantes doctorales de Colciencias por financiar las salidas de campo del proyecto.
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Aya-Cuero C, Trujillo F, Mosquera-Guerra F, Chacón-Pacheco J, Caicedo D, Franco-León N, & Superina
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areas inhabited. Mammal Research. 64: 569-580. https://doi.org/10.1007/s13364-019-004354
Chacón-Pacheco, J, Ballesteros-Correa J, Racero-Casarrubia J. 2019. Primer registro en cámaras
trampas del armadillo coletrapo Cabassous centralis (Cingulata, Chlamyphoridae) en el
departamento de Córdoba, Colombia. Oecología Australis. 23 (1): 150-155
Cruz-Rodríguez C, Larrotta L, González-Maya J, Zárrate-Charry D, Cepeda A, Balaguera-Reina S, AngeJaramillo C, Zamora A, Castaño-Uribe C. 2011. New records for the northern naked-tailed
armadillo Cabassous centralis (Cingulata: Dasypodidae) in tropical dry forest of the
department of Sucre, Colombian Caribbean. Revista Mexicana de Mastozoología. 15: 39-45.
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García-Herrera L, Ramírez-Fráncel L, Reinoso-Flórez G. 2015. Mamíferos en relictos de seco tropical
del Tolima, Colombia. Mastozoología Neotropical. 22 (1): 11-21
García-Herrera L, Ramírez-Fráncel L, Reinoso-Flórez G. 2019. Mamíferos del departamento del
Tolima: distribución y estado de conservación. Revista U.D.C.A Actualidad & Divulgación
Científica. 22. https://doi.org/10.31910/rudca.v22.n2.2019.1100
Gardner AL. (ed). 2008. Mammals of South America. Volumen 1: Marsupials, xenarthrans, shrews and
bats. The University of Chicago Press, 669 pp.
Hayseen V, Ortega J, Morales-Leyva A, Martínez-Mendez N. 2013. Cabassous centralis (Cingulata:
Dasypodidae). Mammalian Species. 45: 12-17. https://doi.org/10.1644/898.1
Hernández-Camacho J, Hurtado AG, Rosario Ortiz Q, Walschburger T. 1992. Unidades biogeográficas
de Colombia. Pp. 105-151. En: Halffter IG, (ed). La Diversidad Biológica de Iberoamérica. Acta
Zoológica Mexicana, Instituto de Ecología, A.C., México, México.
IDEAM. Promedios climatológicos (1971-2000 y 1981-2010). Colombia. 2017.
Disponible en:
http://www.ideam.gov.co/web/tiempo-y-clima/clima Acceso el 18 Marzo 2021.
Ramírez-Chaves HE, Suárez-Castro AF, González-Maya JF. 2016. Cambios recientes a la lista de los
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Rodríguez-Mahecha JV, Alberico M, Trujillo F, Jorgenson J. (eds.). 2006. Libro Rojo de los Mamíferos
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Colombia. 429 pp
Sánchez-Brenes R, Monge J. 2019. El armadillo, Cabassous centralis (Cingulata: Chalyphoridae) en
agroecosistemas con café de Costa Rica. UNED Research Journal, 11 (3), 436-443.
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Sánchez-Soto S, Ramírez-Arenas C, Esteban-Guzmán N, Sánchez-Gómez W.S. 2020. Registro notable
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Northern Naked-tailed Armadillo, Cabassous centralis Miller, 1899 (Mammalia, Cingulata,
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Acceso 15 Abril 2021.
Editor: Diego J. Lizcano
Recibido 2021-03-30
Revisado 2020-04-05
Aceptado 2021-05-08
Publicado 2021-05-09
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Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology
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Strojniški vestnik
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Design of a Self-Folding Composite Variable-Diameter
Wheel Structure based on 4D Printing Technology
Zh
W
G
Z
Li D Wencai Zhang1 – Zhenghao Ge1 – Duanling Li1,2,*
1 Shaanxi University of Science and Technology, College of Mechanical and Electrical Engineering, China
2 Beijing University of Posts and Telecommunications, School of Automation, China The conventional variable-diameter wheel’s complex control system and structure seriously affect its mobility and dependability in unstructured
terrain. Based on 4-dimensional (4D) printing technology, this work proposes a self-folding composite variable-diameter wheel consisting
of a self-folding structure and an outer hub that can self-adjust the wheel diameter under thermal stimulation, avoiding the drawbacks of
conventional structures. The structure integrates the control system and variable-diameter mechanical structure using 4D printing. The design
and construction of the self-folding structures are introduced, and the mathematical model and design parameters for self-folding motion
are obtained using kinematic analysis. Based on the above research and material properties analysis, a programmable morphing structural
design and morphing influence investigation based on manufacturing parameters are carried out for the self-folding rod that controls the
contraction of this structure. The digital model and prototype have been developed to verify the feasibility of the design and the correctness of
the theoretical analysis and to realize the self-adjusting wheel diameter under thermal stimulation. g
Keywords: self-folding, smart materials, 4D printing, variable-diameter wheel g
g
•
A conventional variable-diameter wheel’s complex structure is simplified using 4D printing technology to integrate the control
system and the variable-diameter mechanical structure. •
A conventional variable-diameter wheel’s complex structure is simplified using 4D printing technology to integrate the control
system and the variable-diameter mechanical structure. •
A conventional variable-diameter wheel’s complex control system is simplified using smart materials to cont
changes under external thermal stimulation. •
Conventional mechanical structure design, smart materials, and fabrication are integrated via 4D printing. T
mechanical structure design extends to a programmable morphing structure design. •
Conventional mechanical structure design, smart materials, and fabrication are integrated via 4D printing. T
mechanical structure design extends to a programmable morphing structure design. Received for review: 2022-10-05
Received revised form: 2022-12-05
Accepted for publication: 2023-02-10 Received for review: 2022-10-05
Received revised form: 2022-12-05
Accepted for publication: 2023-02-10 Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195
© 2023 The Authors. CC BY 4.0 Int. Licensee: SV-JME
DOI:10.5545/sv-jme.2022.359 Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195
© 2023 The Authors. CC BY 4.0 Int. Licensee: SV-JME
DOI:10.5545/sv-jme.2022.359 Original Scientific Paper *Corr. Author’s Address: Shaanxi University of Science & Technology, Weiyang District, Xi’an, China, liduanlini@163.com 0 INTRODUCTION Based on mechanical structure design methods
and principles, kinematic analysis introduces
to obtain a mathematical model of self-folding
motion. 3. The structural design based on programmable
morphing and research of morphing influence
based on manufacturing parameters is conducted
for the self-folding rod to control the contraction
of this structure. 4. Constructed simulations and experiments achieve
the drivable self-adjustment of the wheel size
ratio under predetermined thermal stimuli. 0 INTRODUCTION necessitate integrating both the control system and
the variable-diameter mechanical structure to switch
between different environments to improve passing
capability. However, these mechanical structures are
often less reliable and more difficult to control due
to their complex structures and control systems [13]
to [15]. As a result, variable-diameter wheels must
retain their original powerful passing capability
while maintaining a simple structure with low control
complexity and improved reliability. This urgent issue
must be addressed. Robots have been increasingly used in aerospace,
industrial production, geological exploration, and other
fields. When carrying out the design of robots (except
fixed-position robots), the traveling mechanism, as
the crucial system for performing tasks, is mainly
wheeled traveling device, legged traveling device,
crawler traveling device, or composite traveling
device [1] to [3]. Wheeled traveling devices are widely
used because of their adaptability, reliable operation,
and easy control. However, with the expanding scope
of human research, engineering, and habitat, complex
and harsh application scenarios require wheeled
mechanisms with enhanced environmental adaptability
[4]. Therefore, the variable-diameter wheel, which
changes the diameter to cope with different terrain
changes and improves the passing capability, has
been created [5] to [7]. Commonly variable-diameter
wheels can be divided according to their deformation
modes: inflatable and mechanical [8] to [9]. Among
these, the mechanical type gained the attention of
many scholars because of its simple design concept,
high stiffness, and good movement efficiency [10] to
[12]. Mechanical variable-diameter wheels typically The
emergence
of
smart
materials
and
4-dimensional (4D) printing technology provides
a new idea for the design of variable-diameter
wheels. By changing smart materials’ distribution
and geometric parameters, combining 4D printing
technology with conventional mechanical structure
design methods creates a structure with a controlled
self-driven deformation or transformation function
under predetermined structural excitation conditions. The single mechanical structure design extends to a
programmable morphing structure design. Applying
this new idea will effectively circumvent the defects
of the conventional variable-diameter wheel. This
work’s main contributions are listed as follows: 185 Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195 Fig. 1. Schematic; a) self-folding composite variable-diameter
wheel structure, b) outer hub, c) self-folding structure,
and d) structural contraction process 1. Based on 4D printing technology, this work
proposes a novel self-folding composite variable-
diameter wheel structure. The structure comprises
a self-folding structure and an outer hub. 2. 1 DESIGN OF A SELF-FOLDING COMPOSITE
VARIABLE-DIAMETER WHEEL STRUCTURE This section consists of three parts. First, it gives the
general design concept and operation mode of the self-
folding composite variable-diameter wheel structure. Second, it introduces the composition of the core
self-folding structure that attains the wheel diameter
change function. Finally, there are discussions on
the design differences between self-folding and
conventional mechanical structures. Fig. 1. Schematic; a) self-folding composite variable-diameter
wheel structure, b) outer hub, c) self-folding structure,
and d) structural contraction process Fig. 1. Schematic; a) self-folding composite variable-diameter
wheel structure, b) outer hub, c) self-folding structure,
and d) structural contraction process Second, it introduces the composition of the core
self-folding structure that attains the wheel diameter
change function. Finally, there are discussions on
the design differences between self-folding and
conventional mechanical structures. Fig. 2. Schematic of self-folding structure;
a) angulated scissor rod, b) self-folding rod, c) assembly structure,
and d) self-folding structure A self-folding composite variable-diameter wheel
structure with thermally stimulated deformation
response property is designed (see Fig. 1a). The
structure consists of a self-folding structure (see Fig. 1c) and an outer hub (see Fig. 1b), prepared with a
high-precision 3D printer. Usually (T<Tg), the self-folding structure unfolds
and works as a moving wheel. At that moment, the
wheel diameter reaches its maximum value. Applying
an external thermal stimulus (T>Tg), the self-folding
structure contracts along the track and into the outer
hub and uses the outer hub as a moving wheel. At this
moment, the wheel diameter reaches its minimum
value (see Fig. 1d). Fig. 2. Schematic of self-folding structure;
a) angulated scissor rod, b) self-folding rod, c) assembly structure,
and d) self-folding structure The response of the self-folding structure to
external thermal stimulus is crucial to attaining the
change in wheel diameter. For this purpose, the self-
folding structure consisting of a self-folding rod and
an angulated scissor rod is designed (see Fig. 2). When an external thermal stimulus is applied, the self-
folding rod changed from an unfolded state to a folded
state according to the pre-programmed design and
drives the angulated scissor rod to contract the entire
structure during the traveling process (see Fig. 1d). Fig. 2. 2 ANALYSES OF SELF-FOLDING STRUCTURE ∵The lines nh and kh are the perpendicular bisector
of △dhb and △pha
∴△dhn = △nhb = △phk = △kha ∵The lines nh and kh are the perpendicular bisector
of △dhb and △pha
∴△dhn = △nhb = △phk = △kha ∵The lines nh and kh are the perpendicular bisector This section contains two subsections. First, there are
analyses of the self-folding structure’s design methods
and structural principles. Second, kinematic analysis
is carried out to obtain the equation and structural
design parameters for drivable self-folding motion. ∴△dhn = △nhb = △phk = △kha ∵∠dhn = ∠nhb = ∠phk = ∠kha
∴2∠nhb + ∠bhp = 2∠phk + ∠bhp = α
∴∠nhb+∠bhp+∠phk=α
∵∠onh+∠okh+∠nhk+∠nok = 2π
∵∠onh = ∠okh = π/2
∴∠nok = ∠doa = π-α p
p
∵∠onh+∠okh+∠nhk+∠nok = 2π
∵∠onh = ∠okh = π/2
∴∠nok = ∠doa = π-α 1 DESIGN OF A SELF-FOLDING COMPOSITE
VARIABLE-DIAMETER WHEEL STRUCTURE Schematic of self-folding structure;
a) angulated scissor rod, b) self-folding rod, c) assembly structure,
and d) self-folding structure Because of the distinct features of controlled
structural transformation in response to predetermined Because of the distinct features of controlled
structural transformation in response to predetermined 186 Zhang, W. – Ge, Z. – Li, D. Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195 The perpendicular lines are made through point h
to line db and the x-axis, with the intersection points
n and k. The top angle α and the length B of dhp and
bha are manufacturing parameters (see Fig. 2a) whose
values are constants. We can prove the following: thermal stimuli, the critical structural design
parameters and mathematical model required for
deformation control need to be deduced in order
to investigate the deformation control relationship
between the angulated scissor rod and the self-folding
rod. However, the conventional mechanical structure
design method cannot be used as a single basis for
this research, which aims to lay part of the foundation
for the next advancement of conventional structural
design to programmable morphing structural design. ∵∠dhp = ∠bha = α
∵∠bhp is the common angle
∴∠dhb = ∠dhp –∠bhp = ∠bha – ∠bhp = ∠pha
∵dh = bh = ph =ah ∵∠dhp = ∠bha = α ∵∠bhp is the common angle ∵∠bhp is the common angle p
g
∴∠dhb = ∠dhp –∠bhp = ∠bha – ∠bhp = ∠pha
∵dh = bh = ph =ah ∴∠dhb = ∠dhp –∠bhp = ∠bha – ∠bhp = ∠pha
∵dh = bh = ph =ah ∴△dhb and △pha are congruent isosceles triangles ∴△dhb and △pha are congruent isosceles triangles 2.1 Design Method and Structural Principle Two angulated scissor rods (dhp and bha) and two
self-folding rods (tpq and uaj) are extracted from
the self-folding composite variable-diameter wheel
structure to establish a rectangular coordinate system
(see Fig. 3a). Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology Let α be Let Rmin be the minimum RLCM =
1
1
0
0
0
1
1
0
0
0
1
0
0
0
m
,
(2) In the triangle doa, ad
od
oa
od oa
2
2
2
2
cos
.
(4) (4) (2) When the folding angle reaches a minimum value,
the structure is contracted. Let Rmin be the minimum
circumcircle radius. Let βmin be the minimum folding
angle. In the triangle doa, it can be seen that Rmin is
equal to ao and od, bringing Eq. (3) into Eq. (4); we
know that: Let F is the degree of freedom. According to Eq. (2): Let F is the degree of freedom. According to Eq. (2): (2): F
1 1 1 1 1 1 1
1. ad
R
R
R
ad
B
Z
2
2
2
2
2
2
2
min
min
min
min
cos(
),
cos
.
From the above calculations, we conclude that the
self-folding structure has single degrees of freedom. The previous paragraph analyses the self-folding
structure’s design methods and structural principles. On this basis, this structure’s contraction change
process is explained (see Fig. 4). Let Eq. (3) equal Eq. (4), and we can solve the
following: 4
2
2
1
2
2
B
Z
R
cos
(
cos
). min
min
(5) Fig. 4. Contraction process of the self-folding structure (5) According to Eq. (5), we can solve the following: According to Eq. (5), we can solve the following: R
B
Z
min
min
cos
cos
,
2
2
1
(6)
Z
R
B
min
min
arccos
cos
.
2
2
(7) (6) (7) Fig. 4. Contraction process of the self-folding structure Combined with Fig. 4, it can be seen that Rmin
is equal to B. Bringing Eq. (7), we can solve the
following: Fig. 4. Contraction process of the self-folding structure The self-folding rod folded under external
thermal stimulation, and the folding angle change
process is β1→β2→β3. The self-folding rod midpoint
p and a move along the line pa, which drives the
angulated scissor rod between the hinge point h along
the path of the connecting line h1h3 movement. The
diagonal pa changes throughout the process. Let α be One large
and one small circle in different states are formed by
connecting a series of diagonal endpoints h in the
unfolded state to a series of diagonal endpoints a in
the folded state, which obtains structural contraction
changes. Z
m
min
,
2
2
2
(8)
min
min
. 2
2
4
Z
m
(9) (8) (9) When the folding angle reaches a maximum value,
the structure is unfolded. Let Rmax be the maximum
circumcircle radius. Let βmax be the maximum folding
angle. In the triangle oha, it can be seen that Rmax is
equal to oh; we know that: R
B
Z
max
max
sin
.
1
2
(10) (10) Let α be
2
3
m
m
k
k
Z
,
,
(1) (1) where m is the number of rod groups required to
construct the self-folding structure, there are 2m self-
folding rods and 2m angulated scissor rods. Fig. 3. Schematic, a) coordinate systems of the self-folding
structure, b) structured expansion kinetic chain The above proof concludes that the angle in
a circular segment (such as ∠doa) corresponding
to each group of angulated scissor rods is always
constant. Its value is only related to the number of
rod groups m. The value of m also determines the top
angle α of the angulated scissor rods. Thus, using m
rod groups, a ring-shaped self-folding structure can
be constructed. The specific construction method
is as follows (see Figs. 2 and 3): hinges connect the
angulated scissor rods at the top angles h and g. The
self-folding rods are bonded at the limit blocks t, v,
u, and j. By analogy, a self-folding structure can be
constructed consisting of m rod groups. The purpose
of the limit blocks is to prevent uneven force or
collision interference between the self-folding rods
and the angulated scissor rods due to the inaccurate
positioning of the bond. Fig. 3. Schematic, a) coordinate systems of the self-folding
structure, b) structured expansion kinetic chain In this work, the value of m for the designed self-
folding structure is set to 3. The structure is expanded
into a plane kinetic chain along the line connecting
point p and point a (see Fig. 3b). Applying the loop
connectivity matrix (LCM) [16], we know that: Fig. 3. Schematic, a) coordinate systems of the self-folding
structure, b) structured expansion kinetic chain 187 Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195 RLCM =
1
1
0
0
0
1
1
0
0
0
1
0
0
0
m
,
(2)
In the triangle doa,
ad
od
oa
od oa
2
2
2
2
cos
.
(4)
When the folding angle reaches a minimum value,
the structure is contracted. 2.2 Kinematic Analysis Combined with Fig. 4, it can be seen that angle
haf is equal to angle foa, and we can solve the
following: The angles hpg and hag are defined as folding angle
β (see Fig. 4). The angulated scissor rod length is
defined as B (see Fig. 2a). In the triangle dha, let angle hpa is the Z, the
length of the line ad is (see Fig. 3): Zmax
. 2
2
2
(11)
max
max
. 2Z
(12) (11) ad
B
ahd
B
Z
2
2
2
cos
cos
.
(3) (3) (12) 188 Zhang, W. – Ge, Z. – Li, D. Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195 Let χ be the structural contraction ratio, bringing
Eq. (6) and Eq. (10), and we can solve the following: the material’s thermo-mechanical properties and
provide a relevant basis for subsequent research. Four
commercial
elastomer
materials
are
selected, thermoplastic polyurethane (TPU) (Dake,
Shenzhen, China), and one shape memory polymer
material, polylactic acid (PLA) (Raise Premium,
Shanghai, China). The dynamic thermo-mechanical
properties of these five materials are analysed using
a dynamic thermo-mechanical analyser (DMA-Q800,
New Castle, United States), selected tensile mode. The practical test length of the PLA and TPU printed
filaments is 10 mm, and the diameter is 1.75 mm. The
test loading temperature range is 25 °C to 90 °C. The
accuracy of the temperature loading is ±0.2 °C. The
temperature rise rate is controlled by 2 °C/min during
the test. The dynamic axial stretching rate is 1 Hz. The dynamic thermo-mechanical analyser (DMA) test
results (see Fig. 5) included the changes in the storage
modulus G and dissipation factor angle Tan δ with
temperature T. The Ti, Tg, and Th of PLA are 61.96 °C,
68.02 °C, and 73.57 °C, respectively. The G values
for PLA corresponding to the three temperatures are
2458.760 MPa, 1375.287 MPa, and 637.7503 MPa. The subscripts i, g, and h represent the beginning,
transition, and end of PLA’s glass transition phase. Similarly, the DMA test results for TPU show that the
Tg of TPU is below room temperature, and G values
of TPU decrease slowly with increasing temperature.
R
R
m
min
max
sin
. (13) (13) From the above calculations, we conclude that
the m value determines the structure’s construction,
contraction process, and contraction ratio. 2.2 Kinematic Analysis As a result,
the crucial structural design parameter required for
deformation control is m. Taking the structure with an
m value equal to 3 as an example, the contraction ratio
χ of this structure is about 0.866, and its folding angle
β varies from 180° to 120°. 3.2 Structural Design Based on Programmable Morphing The self-folding rod drives the whole structure to
produce contraction by bending. Therefore, this
section discusses how to program the self-folding rod
to produce bending by structural design. 3 PROGRAMMABLE MORPHING RESEARCH
OF SELF-FOLDING RODS In this section, we research the programmable
morphing of the self-folding structure after obtaining
the design method and structural principle. The self-
folding rod is a component with an integrated motion
actuator and driver. It is the core part that controls the
contraction of the self-folding structure. This section contains three subsections. First,
it tests the thermo-mechanical properties of the
materials required to manufacture the self-folding
rod. Second, it researches programmable morphing
based on various materials’ structural design and
thermo-mechanical properties. Finally, it researches
the influence of folding morphing based on the
adjustment of manufacturing parameters. Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology 3.1 Characterization of Material Properties The manufacturing and control of self-folding rods can
utilize the thermo-mechanical properties of different
materials. In addition, heat stimulation is used as a
means of structure activation in this work. Therefore,
material property tests are conducted to characterize A self-folding rod structure based on thermal
stimulus-response is designed. The structure is
manufactured using a fused deposition modelling
(FDM) dual-nozzle printer (Raise E2, Shanghai,
China) utilizing TPU and PLA material (see Fig. 189
Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology
Fig. 5. DMA test results, a) storage modulus of TPU, b) storage modulus and dissipation factor angle of PLA Fig. 5. DMA test results, a) storage modulus of TPU, b) storage modulus and dissipation factor angle of PLA 189 Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195 Fig. 6. Schematic of the manufacturing process of the self-folding rod; a) nozzle A print the first layer,
b) nozzle B prints the second layer, c) nozzle A print the second layer, and d) self-folding rod he self-folding rod; a) nozzle A print the first layer, Fig. 6. Schematic of the manufacturing process of the self-folding rod; a) nozzle A print the first layer,
b) nozzle B prints the second layer, c) nozzle A print the second layer, and d) self-folding rod of TPU is generally lower than room temperature. The
TPU elastic modulus is relatively stable over the Th
temperature range from room temperature to PLA, and
it is assumed that it cannot contract; it can only bend
and slightly elongate. Using these properties, PLA and
TPU are combined in layers to form a combination
structure. TPU converts the unpredictable flex-
torsion of PLA into bending. Although the TPU plays
a restricted role in the structure, its filling patterns
also affect the morphing. The experimental results
reveal that when the filling patterns of the TPU layers
are perpendicular to the PLA layers, and there is no
separation layer, the structure exhibited the best
bending (see Fig. 7). 6). The structure consists of 6 layers, 4 of which are
continuous and 2 of which are split. The separation
layers are designed to control the deformed part’s
width and compensate for the edge bending generated
by the PLA layer. First, we explain the principle of the morphing
produced by the self-folding rod. Parameters After the self-folding rod bends by structural design
to meet the required folding angle β and driving of
the self-folding structure as much as possible, this
section discusses the morphing influence of the self-
folding rod under different manufacturing parameters. The previous discussion shows that releasing stored
prestress in the PLA layer and the TPU layer’s
restriction controls the self-folding rod’s bending. Therefore, if the morphing influence of the self-
folding rod is obtained, it is necessary to research the
effect of the restricted capability of the TPU or the
prestress storage capability of the PLA on the change
of the folding angle β. Table 2. Self-folding rod of sample structure size, printing, and
experimental parameters Table 2. Self-folding rod of sample structure size, printing, and
experimental parameters Structure size
[mm]
H
C
L2
L1
L
1.2
10
10
45
100
Printing
parameters
Layer height [mm]
0.2
Infill amount [%]
100
Extrusion width [mm]
0.4
Nozzle diameter [mm]
0.4
Printing platform temperature [ºC]
30
PLA Printing temperature [ºC]
235
TPU Printing temperature [ºC]
200
Experimental
parameters
Activation medium
Water
Water bath temperature [ºC]
68
Water bath time [s]
≥180 First, the restricted capability of the TPU is
discussed. Four self-folding rods are printed and
experimented with using four TPU materials. Hot
water is chosen as the activation medium for the
experiments to ensure a uniform, accurate and fast
heat application on the samples [21]. The glass
transition temperature Tg of the PLA material selected
from Fig. 5b, and the printing speed are 30 mm/s. The temperature setting of the water bath device
(LICHEN-HH4, Shanghai, China) is kept constant. All samples are kept in water for the experiments, and
heating stops when they no longer exhibit visual signs
of deformation. The printing parameters, sample size,
and experimental parameters are shown in Table 2. Fig. 8. Experiment on the influence of TPU layer restriction
capability on the change of folding angle β Experiments find that TPU materials with higher
storage modulus are more resistant to structural
bending. Therefore, the high storage modulus of the
TPU material causes a weak self-folding rod drive
and a large folding angle β (see Fig. 8). According
to another experimental result in the literature [19]
to [20], the lower the percentage of hard polymer
segments supporting TPU materials, the more difficult Fig. 8. 3.1 Characterization of Material Properties Heating and
squeezing the PLA filament during the printing
process cause the polymer chains to stretch and align
in the direction of that path and subsequently generate
stress. They are stored in the printed material due
to the constraining effect of the printing platform
or previous layer. They are fixed layer by layer as
the printing process cools. When the PLA layers are
removed from the printer and reheated above its glass
transition temperature Tg, the PLA layer shortens
along the printing direction and expands slightly along
the other two directions. Thus, the PLA drives the
structure to produce morphing. Fig. 7. Experiments on the influence of different filling patterns of
TPU layer on bending deformation Second, we explain how the structural design
can program the self-folding rod to produce bending. PLA layers with unidirectional filling patterns exhibit
anisotropic deformation behaviour, resulting in more
significant anisotropic behaviours than PLA layers
with multidirectional filling patterns [17] and [18]. For this reason, all PLA layers in this work are always
printed in the same orientation. However, only single-
layer PLA structures are used, which can produce
unpredictable flexural-torsional deformations. The
DMA test finds that the glass transition temperature Fig. 7. Experiments on the influence of different filling patterns of
TPU layer on bending deformation 190 Zhang, W. – Ge, Z. – Li, D. Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195 191
Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology 3.3 Morphing Influence based on Manufacturing
Parameters it is to print them. After considering the printing
quality and material properties, this work chose a
single TPU-90A material to print the self-folding rod. Parameters Experiment on the influence of TPU layer restriction
capability on the change of folding angle β Second, the prestress storage capability of the
PLA is discussed. The print speed adjustment causes
different stretching of the PLA material during the
extrusion process, resulting in different residual 191
Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology
Fig. 9. 3D optical scanning processes, a) model scanning, b) model extraction, c) model measurement Fig. 9. 3D optical scanning processes, a) model scanning, b) model extraction, c) model measurement 191
Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology 191
Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology 191 Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195 1. Printing PLA materials at faster print speeds
allow for more significant stretching polymer
chain during extrusion. This approach allows
the self-folding rod to maintain higher residual
stresses, resulting in a broader range of folding
angle variations and stronger drive capability. 1. Printing PLA materials at faster print speeds
allow for more significant stretching polymer
chain during extrusion. This approach allows
the self-folding rod to maintain higher residual
stresses, resulting in a broader range of folding
angle variations and stronger drive capability. stresses stored in the material. For each group of
five samples, PLA layers were printed at 150 mm/s
and 30 mm/s, and TPU layers at 30 mm/s. The other
parameters were the same as those shown in Table
2, except for adjusting the PLA printing speed. The
experimental method was the same as described
before. 2. The folding angle of the self-folding rod
gradually decreases as the water bath time
increases. Throughout the process, the bending
is most evident in the first minute. After three
minutes, the self-folding rods no longer produce
significant bending, keeping their folding angle
stable. Therefore, the folding capability of the
self-folding rod continuously decreases with the
increased water bath time. An optical 3D scanner (MetraSCAN 3D, Lévis,
Canada) was used to measure the folding angle β
after deformation for each experiment group, aiming
to assess the experimental results more accurately
and quantitatively. The samples are removed from
the constant temperature water bath, cooled to room
temperature, and placed on a scanning test bench to
capture the surface shape. Parameters The collected data are
combined to create a 3D model for quantitative
deformation assessment (see Fig. 9). 4 MANUFACTURE AND EXPERIMENTS
OF THE SELF-FOLDING COMPOSITE VARIABLE-DIAMETER
WHEEL STRUCTURE Fig. 10. Experiment on the influence of PLA layer prestress
storage capability on the change of folding angle β This section contains two subsections. The self-
folding composite variable-diameter wheel structure
with an m value of 3 is named the M3 structure. First,
the M3 structure’s manufacturing is discussed, and
there are simulations of the design’s viability. Second,
it creates the prototype and verifies the feasibility of
the M3 structural design analysis and self-adjusting
wheel diameter under thermal stimulation through
experiments. Fig. 10. Experiment on the influence of PLA layer prestress
storage capability on the change of folding angle β 4.1 Manufacture of Self-Folding Composite Variable-
diameter Wheel Structure The self-folding composite variable-diameter wheel
structure is rapid-prototyped using a 3D printer. The
M3 structure is constructed with 6 angulated scissor
rods, 6 self-folding rods, and 2 outer hubs. Fig. 10. Experiment on the influence of PLA layer prestress
storage capability on the change of folding angle β The manufacturing process needs to be described
because it contains the drivable self-adjustment of
the self-folding structure. The M3 structure’s folding
angle β variation ranges between 180° and 120°. According to the experimental results provided in the The average measured results of the folding
angle β are shown in Fig. 10. The experimental results
indicate the following: Table 3. Manufacturing parameters of the self-folding rods
Structure
parameters
Height, H [mm]
Width, C [mm]
Separation layer width,
L1 [mm]
Separation layer spacing
distance, L2 [mm]
Length, L [mm]
1.2
10
45
10
100
Printing
parameters
Number of
rod groups,
m
Range of folding angle
variation of 150 mm/s
printing, B [º]
Range of folding
angle variation for
M3 structure, B [º]
PLA layer printing
TPU layer printing
Printing platform
temperature, [ºC]
speed
[mm/s]
temperature
[ºC]
speed
[mm/s]
temperature
[ºC]
3
[180º, 5º]
[180º, 120º]
150
235
30
200
30 Table 3. Manufacturing parameters of the self-folding rods Zhang, W. – Ge, Z. – Li, D. 192 Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195 By the above manufacturing parameters, a digital
M3 structure model is established, and the drivable
self-adjustment of the wheel during travel is verified
using Solidworks Motion. The gravitational load is
9806.65 mm/s², indicated by the green arrow. The
outer hub with a rotational speed is 5 r/min, indicated
by the red arrow. The ground material is chosen to be
the same polycarbonate material as the outer hub, with
a friction coefficient of 0.429 between each other [22]. A simplified constant force replaces the self-folding
rod drive force for ease of calculation with a value
of 0.75 N [23], which loads on the surface where the
angulated scissor rods bond to the self-folding rods,
indicated by the blue arrow. The simulation results
indicate that the structure with the above designs can
achieve self-folding of the wheel during travel (see
Fig. 11). previous section, the self-folding rod at either of the
two printing speeds can meet the design requirements
of the M3 structure for the range of folding angle
variations. 4.1 Manufacture of Self-Folding Composite Variable-
diameter Wheel Structure However, in this work, a printing speed
parameter of 150 mm/s is used to manufacture the
self-folding rod and drive the deformation of the
M3 structure to ensure that the structure obtains a
significant driving force. The specific manufacturing
parameters are shown in Table 3. p
The angulated scissor rod and outer hub are 3D
printed using a common high-temperature resistant
polycarbonate material (Raise Premium, Shanghai,
China). The angulated scissor rod’s length B is first
determined. Eq. (1) is then used to calculate the top
angle α of the angulated scissor rod based on the value
of m. Eq. (6) and Eq. (8) are used to calculate the
radius of the theoretical unfolding and contraction of
the circumcircle based on the above two parameters. Because of the thickness K limitation (see Fig. 2a),
the structure does not reach the theoretical state. As a
result, the radius can be increased appropriately based
on the actual situation to determine the appropriate
circumcircle unfolding and contraction radius. The
actual circumcircle unfolding radius determines the
value of Rmax in the manufacturing of the angulated
scissor rod (see Fig. 2a), and the actual circumcircle
contraction radius determines the value of Rmin in
the manufacturing of the external hub. The specific
manufacturing parameters of the angulated scissor rod
and the outer hub in this work are shown in Table 4. 6 ACKNOWLEDGEMENTS This work is funded by the National Natural Science
Foundation of China (Grant No. 52175019), Beijing
Natural Science Foundations (Grant No.3212009 and
No.L222038), and Beijing Municipal Key Laboratory
of Space-ground Interconnection and Convergence of
China. Fig. 12. Experiments of the self-folding composite variable-
diameter wheel structure Fig. 12. Experiments of the self-folding composite variable-
diameter wheel structure 4.2 Experiments of Self-Folding Composite Variable-
diameter Wheel Structure The M3 structure is manufactured according to the
above parameters. The experimental verification
conditions and parameter settings are consistent with
previous experiments. The experimental contraction
procedure is shown in Fig. 12. Before thermal stimulation, the self-folding rod
is flat, and the folding angle β reaches its maximum
value. The self-folding structure unfolds in the outer
hub, moving under normal wheel diameter conditions. At this moment, the circumcircle diameter of the M3 193
Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology
Table 4. Manufacturing parameters of the angulated scissor rods and outer hub
Structure
parameters
Number of
rod groups
m
Top angle
α [°]
Circumcircle radius
Side length
B [mm]
Thickness
K [mm]
Width
S [mm]
Theoretical value
Actual value
Rmin [mm]
Rmax [mm]
Rmin [mm]
Rmax [mm]
3
60
60
69.282
62
70
60
3
5
Printing
parameters
Printing platform
temperature [ºC]
Printing
speed [mm/s]
Layer height
[mm]
Infill
amount
Extrusion width
[mm]
Printing temperature
[°C]
110
60
0.2
15%
0.4
235
Fig. 11. The self-folding of the structure during the travelling process is obtained via simulation Table 4. Manufacturing parameters of the angulated scissor rods and outer hub Fig. 11. The self-folding of the structure during the travelling process is obtained via simulation Design of a Self-Folding Composite Variable-Diameter Wheel Structure based on 4D Printing Technology 193 Strojniški vestnik - Journal of Mechanical Engineering 69(2023)3-4, 185-195 structure is 140 mm (see Fig. 12a). After thermal
stimulation, the self-folding rod is controllable
bending, and the folding angle β reaches its minimum
value. The self-folding structure moves along the
outer hub track and into it. wheel, the self-folding composite variable-diameter
wheel structure designed using this new idea has less
structural complexity and control difficulty. The limitations of realistic application scenarios
need to be considered. The self-folding rod within
the prototype needs to be redesigned to enable bi-
directional self-folding characteristics. The driving
force of the self-folding rod must be redesigned
to obtain bi-directional self-adjustment of the
wheel diameter. The prototype should be tested in
unstructured terrain and data collected on speed, time,
and centrifugal force to determine parameters such as
driving speed. Fig. 12. Experiments of the self-folding composite variable-
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Diagnostic performance of fractional excretion of urea in the evaluation of critically ill patients with acute kidney injury: a multicenter cohort study
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RESEARCH Open Access Abstract Introduction: Several factors, including diuretic use and sepsis, interfere with the fractional excretion of sodium,
which is used to distinguish transient from persistent acute kidney injury (AKI). These factors do not affect the
fractional excretion of urea (FeUrea). However, there are conflicting data on the diagnostic accuracy of FeUrea. Methods: We conducted an observational, prospective, multicenter study at three ICUs in university hospitals. Unselected patients, except those with obstructive AKI, were admitted to the participating ICUs during a six-month
period. Transient AKI was defined as AKI caused by renal hypoperfusion and reversal within three days. The results
are reported as medians (interquartile ranges). Results: A total of 203 patients were included. According to our definitions, 67 had no AKI, 54 had transient AKI
and 82 had persistent AKI. FeUrea was 39% (28 to 40) in the no-AKI group, 41% (29 to 54) in the transient AKI
group and 32% (22 to 51) in the persistent AKI group (P = 0.12). FeUrea was of little help in distinguishing
transient AKI from persistent AKI, with the area under the receiver operating characteristic curve being 0.59 (95%
confidence interval, 0.49 to 0.70; P = 0.06). Sensitivity was 63% and specificity was 54% with a cutoff of 35%. In the
subgroup of patients receiving diuretics, the results were similar. Conclusions: FeUrea may be of little help in distinguishing transient AKI from persistent AKI in critically ill patients,
including those receiving diuretic therapy. Additional studies are needed to evaluate alternative markers or
strategies to differentiate transient from persistent AKI. Keywords: acute kidney failure, ICU, fractional excretion of sodium, acute tubular necrosis, diuretics, sensitivity and
ifi it Keywords: acute kidney failure, ICU, fractional excretion of sodium, acute tubular necrosis, diuretics, sensitivity and
specificity Diagnostic performance of fractional excretion of
urea in the evaluation of critically ill patients with
acute kidney injury: a multicenter cohort study Michael Darmon1,2,3*, Francois Vincent4, Jean Dellamonica2,5, Frederique Schortgen6, Frederic Gonzalez4,
Vincent Das7, Fabrice Zeni1,3, Laurent Brochard2,8, Gilles Bernardin5, Yves Cohen4,9 and Benoit Schlemmer7 © 2011 Darmon et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. * Correspondence: michael.darmon@chu-st-etienne.fr
1Medical-Surgical Intensive Care Unit, Saint-Etienne University Hospital, and
Jean Monnet University, Avenue Albert Raymond, F-42270 Saint-Etienne,
France
Full list of author information is available at the end of the article Darmon et al. Critical Care 2011, 15:R178
http://ccforum.com/content/15/4/R178 Introduction persistent AKI in critically ill patients is believed to be
acute tubular necrosis (ATN) [4,5]. It is usually assumed
that there is a continuum that leads from prerenal AKI
to ATN [4-6]. Many publications in the fields of internal
medicine, nephrology and critical care still advocate the
use of urinary indices, such as the fractional excretion
of sodium (FeNa), to differentiate transient from persis-
tent AKI [4,5,7-10]. However, diuretic therapy or sepsis
may affect these indices [11-13]. Since urea reabsorption
occurs mainly at the proximal segment of the nephron
and is unaffected by diuretic intake, the fractional excre-
tion of urea (FeUrea) may be more reliable than FeNa
[11,12,14]. Studies evaluating the performance of FeUrea
have produced discordant results [11,12,14]. In addition, Acute kidney injury (AKI) is common and associated
with high mortality in critically ill patients [1-3]. The
causes of AKI other than urinary tract obstruction are
usually divided into two categories: prerenal causes, in
which low renal perfusion leads to promptly reversible
renal dysfunction, and intrinsic causes with renal tissue
damage and persistent renal dysfunction. Although
pathological studies are lacking, the leading cause of Page 2 of 8 Darmon et al. Critical Care 2011, 15:R178
http://ccforum.com/content/15/4/R178 Page 2 of 8 a 50% or greater decrease in serum creatinine [18], and/
or return of serum creatinine to the baseline value
(whether measured or estimated using the MDRD for-
mula [16,17]). Persistent AKI was defined as renal dys-
function without recovery within three days. Oliguria
was defined as urine output < 0.5 mL/kg/hour for six
hours or more. no study specifically designed to evaluate FeUrea in cri-
tically ill patients has been conducted. A recent review
underlined the lack of evidence supporting the use of
usual urinary indices in critically ill patients and in
patients with sepsis [15]. However, distinguishing transi-
ent AKI from persistent AKI can help the clinician to
choose the optimal treatment for critically ill patients. The FeNa percentage was calculated as ([urinary
sodium/serum sodium]/[urinary creatinine/serum creati-
nine]) ×100. The FeUrea percentage was calculated as
([urinary urea/serum urea]/[urinary creatinine/serum
creatinine]) ×100. Our primary objective in this study was to evaluate
the performance of FeUrea as a tool for distinguishing
transient from persistent AKI in a cohort of critically ill
patients. Materials and methods
Patients The study was approved by the institutional review
board of the French Society for Intensive Care Medicine
(SRLF-CE-07-212), which waived the need for signed
informed consent. Patients and their next of kin were
informed, however, and none refused to participate. Three ICUs in university hospitals participated in the
study between April and September 2008. Patients
admitted to the participating ICUs were included, except
those younger than 18 years of age, pregnant women,
patients receiving dialysis for an underlying chronic kid-
ney disease and patients with evidence of obstructive
renal failure. Patients from whom urine could not be
collected during the first six hours were excluded from
this study. Statistical analysis
P Patients remaining in the ICU for < 72 hours were sec-
ondarily excluded from the analysis, since they could
not be classified as having transient or persistent AKI
according to our definition. The results are reported as
medians and interquartile ranges (IQRs), numbers and
percentages or as means ± standard deviations (SD) to
express the percentage changes. Categorical variables
were compared using Fisher’s exact test, and continuous
variables were compared using the nonparametric Wil-
coxon signed-rank test or the Mann-Whitney U test for
pairwise comparisons. The Friedman test was used to
compare continuous variables across the three groups. Protocol Each patient was assessed during the first 12 hours fol-
lowing ICU admission. Plasma sodium, urea and creati-
nine levels were measured at ICU admission, and urine
was collected over the next six hours. To determine how well FeUrea distinguished transient
from persistent AKI (our primary objective), we plotted
the receiver-operating characteristic (ROC) curves of the
proportion of true positives against the proportion of
false positives, depending on the prediction rule used to
classify patients as having persistent AKI. A 2 × 2 table
was established to determine the sensitivity and specifi-
city of FeUrea in diagnosing persistent AKI. Cutoff
values, defined as threshold values that maximized the
sum of sensitivity and specificity, were determined on
the ROC curves. The positive and negative likelihood
(LH) ratios were computed. The same strategy was used
to assess our secondary objectives, namely, the perfor-
mance of the usual urinary indices in these patients and
the performance of the usual urinary indices and of
FeUrea in the subgroup of patients receiving diuretics. Introduction The secondary objectives were to evaluate the
performance of the usual urinary indices in these
patients and to evaluate the performance of the usual
urinary indices and FeUrea in the subgroup of patients
receiving diuretics. The Logistic Organ Dysfunction (LOD) score and the
Simplified Acute Physiology Score version II (SAPS II)
score were calculated at study inclusion [19,20], and the
Knaus scale score was determined to evaluate chronic
health status at ICU admission (A: no limitation of
activity, B: moderate limitation, C: severe limitation, and
D: bedridden or institutionalized) [21]. Sepsis was diag-
nosed using the criteria developed by the American Col-
lege of Chest Physicians/Society of Critical Care
Medicine consensus conference [22]. Individual organ
failure was defined as a LOD score greater than 1 point
for each system except the kidney [19]. Diagnostic performance of urinary indices in patients
undergoing diuretic therapy At ICU admission, the median SAPS II score was 46
(34 to 60) and the median LOD score was 6 (4 to 9). Most patients were admitted for medical conditions
(91.1%). The main risk factors for AKI were sepsis
(67.5%), aminoglycoside therapy (20.7%), chronic heart
failure (19.8%), chronic kidney disease (16.3%) and expo-
sure to iodinated contrast agents (8.9%). Overall, 67 patients (33%) received diuretics before or at
ICU admission. Among them, 17 had no AKI (25.4% of
patients without AKI), 18 had transient AKI (33.3% of
patients with transient AKI) and 32 had persistent AKI
(39% of patients with persistent AKI). The performance
characteristics of urinary indices in patients undergoing
diuretic therapy are reported in Table 2. As with the
overall population, the performance of FeUrea in this
patient subgroup was poor (ROC curve AUC 0.58 (0.41
to 0.75)). The U/P urea ratio performed satisfactorily in
differentiating transient from persistent AKI (ROC curve
AUC 0.82 (0.70 to 0.94)). With a U/P urea ratio cutoff
of 10, sensitivity was 72%, specificity was 69%, positive
LH was 2.32 and negative LH was 0.41. At the time of the study, no patient was being treated
with renal replacement therapy (RRT). Forty-five
patients required RRT during their ICU stay, usually
during the first three days in the ICU (41 of 45
patients). Each of the patients requiring RRT during the
first three days in the ICU had persistent AKI, whereas
the remaining four patients had no AKI at ICU admis-
sion and required RRT later during their ICU stay. Study population During the study period, 203 patients with a median age
of 61 years (46 to 73) were included. Their main charac-
teristics are reported in Table 1. According to our defi-
nitions, 67 patients (33%) had no AKI, 54 patients
(26.6%) had transient AKI and 82 patients (40.4%) had
persistent AKI. Diagnostic performance of other urinary indices g
p
y
The performance characteristics of classical urinary
indices for detecting persistent AKI are reported in
Table 2, with the usual and optimal cutoffs in the study
population. Performance was best for the U/P urea ratio
(ROC curve area under the curve (AUC) 0.71 (0.62 to
0.80)) (Figure 2). A U/P urea ratio < 12 had 66% sensi-
tivity and 66% specificity for persistent AKI (positive
LH, 1.94; negative LH, 0.52). When entered into a
regression logistic model, none of these urinary indices
were independently associated with persistent AKI. Three variables were found to be associated with persis-
tent AKI: chronic kidney disease (OR 11.89, 95% CI 2.52
to 56.24; P = 0.02), need for vasopressors at ICU admis-
sion (OR 2.60, 95% CI 1.15 to 5.91) and oliguria at ICU
admission (OR 2.50, 95% CI 1.11 to 5.63). The model
had good calibration (goodness of fit P = 0.88). FeUrea
was then forced into the final model and was not
selected. All tests were two-sided, and P values < 0.05 were
considered statistically significant. Statistical tests were
performed using the SAS version 6.12 software package
(SAS Institute, Cary, NC, USA). Definitions AKI was defined according to the Acute Kidney Injury
Network classification scheme [16] as a serum creatinine
level increase of 26.4 μmol/L or more, a serum creati-
nine increase ≥150% from baseline or urine output <
0.5 mL/kg/hour for six hours or more. For patients
whose baseline serum creatinine level was unknown,
this variable was estimated using the Modification of
Diet in Renal Disease (MDRD) formula [16,17]. Transient AKI was defined as AKI (of any stage) with
a cause of renal hypoperfusion (that is, shock; dehydra-
tion; a medication interfering with renal perfusion, such
as angiotensine-converting enzyme inhibitor; and so on)
and recovery within three days. Recovery was defined as
reversal of oliguria (in the absence of diuretics), and/or Page 3 of 8 Darmon et al. Critical Care 2011, 15:R178
http://ccforum.com/content/15/4/R178 Darmon et al. Critical Care 2011, 15:R178
http://ccforum.com/content/15/4/R178 Last, to confirm the input of urinary indices to detect
persistent AKI, we performed logistic regression analyses
to identify variables significantly associated with persis-
tent AKI measured by the estimated odds ratio (OR)
with the 95% confidence interval (95% CI). Variables
yielding P values < 0.20 in the bivariate analyses were
entered into a backward stepwise logistic regression
model in which persistent AKI was the variable of inter-
est. The covariates were entered into the model with
critical entry and removal P values of 0.2 and 0.1,
respectively. Last, since the performance of FeUrea was
the primary objective of this study, this variable was
forced into the final model. Colinearity and interactions
were tested. The Hosmer-Lemeshow test was used to
check the goodness of fit of the logistic regression. and 54% specificity (Table 2), yielding a positive LH of
1.37 and a negative LH of 0.68. In the study population,
the optimal cutoff was 37%. However, the performance
of FeUrea at this cutoff was poor (66% sensitivity and
53% specificity) (Table 2). Diagnostic performance of FeUrea Median FeUrea was 37% (26 to 49) overall, 39% (28 to
40) in patients without AKI, 41% (29 to 54) in patients
with transient AKI and 32% (22 to 51) in patients with
persistent AKI (P = 0.12). Figures 1a, b and 1c show the
distributions of FeNa, FeUrea and urine/plasma (U/P)
urea ratios, respectively, in each group. Overall, 137 patients (67%) had sepsis at ICU admission. Among them, 43 had no AKI (64.2% of patients without
AKI), 33 had transient AKI (61.1% of patients with tran-
sient AKI) and 61 had persistent AKI (74.4% of patients
with persistent AKI). The performance characteristics of
urinary indices in patients with sepsis are reported in
Table 2. As with the overall population, the performance
of FeUrea in this patient subgroup was poor (ROC curve
AUC 0.56 (0.43 to 0.68)). The performance of other The area under the ROC curve was 0.59 (95% CI 0.49
to 0.70; P = 0.06) (Figure 2). At the usual cutoff (35%),
FeUrea predicted persistent AKI with 63% sensitivity Darmon et al. Diagnostic performance of FeUrea Critical Care 2011, 15:R178
http://ccforum.com/content/15/4/R178 Page 4 of 8 Page 4 of 8 Table 1 Characteristics of patients without AKI, with transient AKI and with persistent AKIa
Demographics
No AKI (n = 67)
Transient AKI (n = 54)
Persistent AKI (n = 82)
P valueb
Patient characteristics
Male gender
34 (50.7%)
32 (59.3%)
56 (68.3%)
0.15
Age, years
50 (40 to 60)
71 (49 to 76)
66 (56 to 74)
< 0.0001
Weight, kg
68 (57 to 85)
75 (64 to 85)
80 (68 to 89)
0.006
Knaus score C or D [21]
21 (31.3%)
21 (38.9%)
40 (48.8%)
0.09
LOD score at ICU admission [19]
4 (2 to 7)
6 (5 to 9)
8 (5 to 9)
< 0.0001
SAPS II score at ICU admission [20]
35 (27 to 47)
50 (39 to 62)
52 (39 to 62)
< 0.0001
Risk factors for AKI
Chronic heart failure
8 (11.9%)
14 (26.4%)
18 (22.0%)
0.15
Chronic kidney diseasec
1 (1.5%)
3 (5.6%)
23 (28.0%)
< 0.0001
Sepsis
43 (64.2%)
33 (61.1%)
61 (74.4%)
0.12
Aminoglycosides
8 (11.9%)
9 (16.7%)
25 (30.5%)
0.2
Ionidated contrast agents
6 (9.0%)
3 (5.6%)
9 (11.0%)
0.55
Organ failure at ICU admission
Medical condition
62 (92.5%)
51 (94.4%)
72 (87.8%)
0.36
Acute respiratory failure
54 (80.6%)
39 (72.2%)
61 (74.4%)
0.51
Coma
22 (32.8%)
24 (44.4%)
29 (35.4%)
0.34
Shock
22 (32.8%)
28 (51.9%)
43 (52.4%)
0.03
Treatments in the ICU
Need for vasoactive drugs
20 (29.9%)
23 (42.6%)
43 (52.4%)
0.02
Mechanical ventilation
43 (64.2%)
34 (63.0%)
52 (63.4%)
0.99
Noninvasive mechanical ventilation
21 (31.3%)
14 (25.9%)
21 (25.6%)
0.73
Renal replacement therapy
4 (6.0%)
0
41 (50.0%)
< 0.0001
Diuretics (at admission)
17 (25.4%)
18 (33.3%)
32 (39.0%)
0.21
Renal function at admission
Diuresis, mL/kg/hourd
0.69 (0.59 to 0.99)
0.45 (0.32 to 1.11)
0.40 (0.21 to 0.72)
< 0.0001
Plasma urea, mmol/L
5.4 (3.4 to 7.9)
13.1 (6.8 to 17.3)
17.4 (10.9 to 25.1)
< 0.0001
Serum creatinine, μmol/L
68 (59 to 78)
124 (98 to 164)
220 (138 to 360)
< 0.0001
Urinary indices
Urine Na+/urine K+
1.8 (0.35 to 1.75)
1.0 (0.4 to 1.8)
1.3 (0.5 to 2.4)
0.01
FeNa, %
0.5 (0.3 to 1.3)
0.5 (0.2 to 1.3)
0.8 (0.4 to 4.0)
0.004
FeUrea, %
39 (28 to 40)
41 (29 to 54)
32 (22 to 51)
0.12
U/P urea
30 (19 to 39)
16 (9 to 25)
7 (4 to 14)
< 0.0001
U/P creatinine
83 (52 to 127)
47 (25 to 76)
30 (11 to 58)
< 0.0001
Outcomes
ICU mortality
7 (11.7%)
13 (25%)
37 (48.1%)
0.0002
Hospital mortality
14 (20.9%)
15 (27.8%)
42 (51.2%)
0.0003
aAKI: acute kidney injury; LOD: Logistic Organ Dysfunction score, which can range from 0 to 22; SAPS II: Simplified Acute Physiology Score version II; FeNa,
fractional excretion of sodium ([urine sodium/serum sodium]/[urine creatinine/serum creatinine]) ×100; FeUrea, fractional excretion of urea ([urine urea/serum
urea]/[urine creatinine/serum creatinine]) ×100; U/P urea, urine urea/plasma urea; U/P creatinine, urine creatinine/serum creatinine. aAKI: acute kidney injury; LOD: Logistic Organ Dysfunction score, which can range from 0 to 22; SAPS II: Simplified Acute Physiology Score version II; FeNa,
fractional excretion of sodium ([urine sodium/serum sodium]/[urine creatinine/serum creatinine]) ×100; FeUrea, fractional excretion of urea ([urine urea/serum
urea]/[urine creatinine/serum creatinine]) ×100; U/P urea, urine urea/plasma urea; U/P creatinine, urine creatinine/serum creatinine. Data are medians (IQR) or
number of patients (%). bP values represent comparisons across the three patient groups. cChronic renal failure was defined as creatinine clearance < 60 mL/
minute before ICU admission. dDiuresis represents diuresis per kilogram and per hour during the first six hours following inclusion Diagnostic performance of FeUrea Data are medians (IQR) or
number of patients (%). bP values represent comparisons across the three patient groups. cChronic renal failure was defined as creatinine clearance < 60 mL/
minute before ICU admission. dDiuresis represents diuresis per kilogram and per hour during the first six hours following inclusion Table 1 Characteristics of patients without AKI, with transient AKI and with persistent AKIa racteristics of patients without AKI, with transient AKI and with persistent AKIa Persistent AKI (n = 82)
P valueb aAKI: acute kidney injury; LOD: Logistic Organ Dysfunction score, which can range from 0 to 22; SAPS II: Simplified Acute Physiology Score version II; FeNa,
fractional excretion of sodium ([urine sodium/serum sodium]/[urine creatinine/serum creatinine]) ×100; FeUrea, fractional excretion of urea ([urine urea/serum
urea]/[urine creatinine/serum creatinine]) ×100; U/P urea, urine urea/plasma urea; U/P creatinine, urine creatinine/serum creatinine. Data are medians (IQR) or
number of patients (%). bP values represent comparisons across the three patient groups. cChronic renal failure was defined as creatinine clearance < 60 mL/
minute before ICU admission. dDiuresis represents diuresis per kilogram and per hour during the first six hours following inclusion Darmon et al. Critical Care 2011, 15:R178
http://ccforum.com/content/15/4/R178 Page 5 of 8 Page 5 of 8 (a)
(b)
(c)
Figure 1 (a) Boxplot of the fractional excretion of sodium (FeNa) in the overall study population according to renal function. The
dotted line represents FeNa of 1% (P = 0.04). (b) Boxplot of the fractional excretion of urea (FeUrea) in the overall study population according
to renal function. The dotted line represents FeUrea of 35% (P = 0.12). (c) Boxplot of the urine/plasma (U/P) urea ratio in the overall study
population according to renal function. The dotted line represents a U/P urea ratio of 10 (P < 0.0001). (a) (c) (b) Figure 1 (a) Boxplot of the fractional excretion of sodium (FeNa) in the overall study population according to renal function. The
dotted line represents FeNa of 1% (P = 0.04). (b) Boxplot of the fractional excretion of urea (FeUrea) in the overall study population according
to renal function. The dotted line represents FeUrea of 35% (P = 0.12). (c) Boxplot of the urine/plasma (U/P) urea ratio in the overall study
population according to renal function. The dotted line represents a U/P urea ratio of 10 (P < 0.0001). Diagnostic performance of FeUrea 1.0
0.8
FeUrea
U/PUrea
Referenceline
0.6
0.4
Sensitivity
0.2
S
0.0 0.2 0.4 0.6
0.8 1.0
1- Specificity
0.0
Figure 2 Receiver-operating characteristic (ROC) curve depicting the ability of the fractional excretion of urea (FeUrea) and urine/
plasma (U/P) urea ratio to detect persistent AKI in the subgroup of patients with AKI. The ROC curve shows the relationship between the
proportion of true positives (Sensitivity) and the proportion of false positives (1-Specificity) with various FeUrea and U/P urea ratio cutoffs. Diagonal segments are produced by ties. The area under the ROC curve is 0.59 (95% confidence interval, 0.49 to 0.70; P = 0.06) for FeUrea. The
area under the ROC curve is 0.71 (95% confidence interval, 0.62 to 0.80; P = 0.04) for U/P urea ratio. 1.0
0.8
0.6
0.4
Sensitivity
0.2
S
0.0 0.2 0.4 0.6
0.8 1.0
1 Specificity
0.0 FeUrea
U/PUrea
Referenceline Figure 2 Receiver-operating characteristic (ROC) curve depicting the ability of the fractional excretion of urea (FeUrea) and urine/
plasma (U/P) urea ratio to detect persistent AKI in the subgroup of patients with AKI. The ROC curve shows the relationship between the
proportion of true positives (Sensitivity) and the proportion of false positives (1-Specificity) with various FeUrea and U/P urea ratio cutoffs. Diagonal segments are produced by ties. The area under the ROC curve is 0.59 (95% confidence interval, 0.49 to 0.70; P = 0.06) for FeUrea. The
area under the ROC curve is 0.71 (95% confidence interval, 0.62 to 0.80; P = 0.04) for U/P urea ratio. Figure 2 Receiver-operating characteristic (ROC) curve depicting the ability of the fractional excretion of urea (FeUrea) and urine/
plasma (U/P) urea ratio to detect persistent AKI in the subgroup of patients with AKI. The ROC curve shows the relationship between the
proportion of true positives (Sensitivity) and the proportion of false positives (1-Specificity) with various FeUrea and U/P urea ratio cutoffs. Diagonal segments are produced by ties. The area under the ROC curve is 0.59 (95% confidence interval, 0.49 to 0.70; P = 0.06) for FeUrea. The
area under the ROC curve is 0.71 (95% confidence interval, 0.62 to 0.80; P = 0.04) for U/P urea ratio. Darmon et al. Diagnostic performance of FeUrea Table 2 Performance of usual urinary markers for detecting patients with persistent AKI among patients with AKI,
with the usual and optimal (*) cutoff valuesa al urinary markers for detecting patients with persistent AKI among patients with AKI,
*) cutoff valuesa urinary indices was similar to that in the overall patient
population. In addition, the study populations were poorly described
but include both critically ill patients and patients in
wards. Therefore, selection bias and differences between
the institutions and study populations may explain the
discrepancies [11,12]. Furthermore, FeUrea reflects the
ratio of urea clearance over creatinine clearance ratio. Variations in creatinine clearance may therefore modify
FeUrea. In the study that found good performance of
FeUrea [11,12], wide differences in creatinine clearance
can be suspected between patients with transient AKI
and those with persistent AKI: serum creatinine levels
were 140 ± 22 μmol/L and 520 ± 22 μmol/L (means +/-
SD) in these two groups, respectively. Diagnostic performance of FeUrea Critical Care 2011, 15:R178
http://ccforum.com/content/15/4/R178 Page 6 of 8 Page 6 of 8 Table 2 Performance of usual urinary markers for detecting patients with persistent AKI among patients with AKI,
with the usual and optimal (*) cutoff valuesa
Patient groups
FeNa
> 1%
FeNa*
> 0.58%
FeUrea
< 35%
FeUrea*
< 37%
U/P urea
< 10
U/P urea* < 12
U/P creat < 20
U/P creat* < 12
All patients with AKI (n = 136; persistent AKI prevalence = 60.3%)
Sensitivity (%)
0.48
0.63
0.63
0.66
0.74
0.66
0.79
0.59
Specificity (%)
0.7
0.61
0.54
0.53
0.57
0.66
0.39
0.59
Positive predictive value
0.71
0.71
0.67
0.68
0.72
0.74
0.66
0.68
Negative predictive value
0.47
0.47
0.47
0.51
0.59
0.56
0.55
0.48
Positive likelihood ratio
1.6
1.61
1.37
1.4
1.72
1.94
1.3
1.44
Negative likelihood ratio
0.74
0.61
0.68
0.64
0.45
0.52
0.54
0.69
Younden’s index
0.18
0.24
0.17
0.19
0.31
0.32
0.18
0.18
ROC AUC
0.62 (0.52 to 0.72)
0.59 (0.49 to 0.70)
0.71 (0.62 to 0.80)
0.62 (0.53 to 0.72)
Patients taking diuretics (n = 50; persistent AKI prevalence = 64%)
Sensitivity (%)
0.75
0.62
0.61
0.61
0.72
0.61
0.78
0.89
Specificity (%)
0.56
0.56
0.47
0.59
0.69
0.75
0.5
0.38
Positive predictive value
0.71
0.71
0.67
0.68
0.81
0.81
0.73
0.72
Negative predictive value
0.47
0.47
0.49
0.51
0.58
0.52
0.56
0.66
Positive likelihood ratio
1.7
1.41
1.15
1.49
2.32
2.44
1.56
1.44
Negative likelihood ratio
0.47
0.68
0.83
0.66
0.41
0.52
0.44
0.29
Younden’s index
0.31
0.18
0.08
0.2
0.41
0.36
0.28
0.27
ROC AUC
0.69 (0.54 to 0.81)
0.58 (0.41 to 0.75)
0.82 (0.70 to 0.94)
0.71 (0.56 to 0.86)
Patients with sepsis (n = 94; persistent AKI prevalence = 65%)
Sensitivity (%)
0.5
0.65
0.63
0.63
0.8
0.67
0.87
0.93
Specificity (%)
0.86
0.56
0.52
0.57
0.63
0.63
0.42
0.37
Positive predictive value
0.87
0.73
0.71
0.73
0.8
0.63
0.74
0.73
Negative predictive value
0.48
0.46
0.43
0.45
0.63
0.51
0.63
0.74
Positive likelihood ratio
3.57
1.48
1.31
1.47
2.1
1.81
1.5
1.48
Negative likelihood ratio
0.58
0.63
0.71
0.65
0.32
0.52
0.31
0.19
Younden’s index
0.36
0.19
0.15
0.2
0.43
0.3
0.29
0.3
ROC AUC
0.67 (0.56 to 0.79)
0.56 (0.43 to 0.68)
0.71 (0.60 to 0.82)
0.65 (0.53 to 0.77)
aAKI: acute kidney injury; ROC: receiver operating characteristic; AUC: area under the curve. Discussion studies have had several limitations: most of them were
single-center case series or retrospective studies, the
definition of AKI varied across the studies and the defi-
nition of transient AKI also varied, being subjective in
most instances [24-30]. Several of these studies included
patients who did not have critical illnesses [11,12,28]. In
addition, we chose a definition allowing for a distinction
between transient and persistent AKI. Our study was
therefore not designed to evaluate the interest of these
indices in distinguishing prerenal and intrinsic AKI. This point may partly explain the poor performance of
the urinary indices in our study. Last, most of the stu-
died patients had sepsis or shock at ICU admission. This condition is frequently associated with renal hand-
ling of sodium or water independently of an underlying
AKI. This may also explain the poor performance of
these indices in the studied population. Nevertheless,
taking these factors into account, and although the
usual urinary indices were able to differentiate transient
from persistent AKI, the accuracy of the indices was
poor and none of them were independently associated
with the diagnosis of persistent AKI, indicating a need
to identify other biomarkers. other urinary indices. Therefore, these variations need
to be investigated to determine the optimal time for
renal assessment in critically ill patients. Last, few of our patients received diuretics. The poor
performance of urinary indices in this subgroup may
therefore be related to low statistical power. Acknowledgements We thank A Wolfe, MD, for helping with this manuscript. Financial support
consisted of a grant from the Assistance-Publique Hôpitaux de Paris (CRC 07
011), a nonprofit, government-funded organization. Abbreviations
AKI
t
kid AKI: acute kidney injury; AUC: area under the curve; CI: confidence interval;
FeNa: fractional excretion of sodium; FeUrea: fractional excretion of urea; MV:
mechanical ventilation; OR: odds ratio; ROC: receiving operator characteristic;
U/P creat: urine/serum creatinine ratio; U/P urea: urine/plasma urea ratio. Key messages • FeUrea performed poorly in separating transient
from persistent AKI in critically ill patients. • Although the usual urinary indices (FeNa, U/P
urea ratio or U/P creatinine ratio) are able to differ-
entiate transient from persistent AKI, their accuracy
remains poor in this setting. • The high incidence of situations that may induce
renal handling of water or sodium (that is, sepsis or
shock) may explain the poor performance of urinary
indices in this setting. y
Our study has several limitations. First, our definition
of transient AKI was mainly based on renal function
recovery. Indeed, an accurate definition of prerenal AKI
would have required a highly subjective definition based
on clinical histories, physical examinations and physi-
cians’ judgments [11,15]. In addition, AKI is mainly due
to sepsis in critically ill patients, and in this setting there
is frequently a continuum between volume depletion and
persistent kidney injuries rather than two distinct entities,
with the two mechanisms being frequently associated. Therefore, we chose a definition that relies only on an
objective criterion. This point needs to be taken into
account to interpret our findings. In the same way, the
course of kidney function may have been modified by
factors following study inclusion. However, in the ICU
setting, predicting which patients will have persistent
AKI may help to optimize treatment, such as by promptly
restoring renal perfusion, limiting fluids or starting RRT. Our definition was highly sensitive for detecting patients
with transient AKI (none of the patients in this group
required RRT) but lacked specificity, since only 50% of
patients in the persistent AKI group required RRT. Addi-
tional studies may help to determine the definition that
best matches the need for RRT. • Additional studies are needed to evaluate alterna-
tive markers of renal injury or strategies for differen-
tiating transient from persistent AKI. Conclusions In summary, we found that FeUrea and the usual urin-
ary indices performed poorly in separating transient
from persistent AKI in an unselected population of criti-
cally ill patients. Additional studies are needed to evalu-
ate alternative markers of renal injury or strategies for
differentiating transient from persistent AKI. Discussion In critically ill patients, FeUrea was not helpful in differ-
entiating transient AKI from persistent AKI. Both in the
overall population and in the subgroup of patients
receiving diuretics, FeUrea performed less well than
FeNa or the U/P urea ratio. There is little scientific evidence to support the use of
FeUrea. Only three studies have evaluated the accuracy
of FeUrea in distinguishing transient from persistent
AKI [11,12,14]. Their results are conflicting. In one
study, FeUrea was 90% sensitive and 96% specific in dif-
ferentiating transient from persistent AKI when a cutoff
of 35% was used [11]. Conversely, another study found
very poor diagnostic accuracy of FeUrea [12]. Several
factors may explain these discordant results. First, these
studies were single-center cohort studies and included
only patients who were referred to nephrologists [11,12]. Interestingly, the performance of urinary indices in
our study was poor. Several factors may explain this
finding. First, although many publications have advo-
cated the use of urinary biochemistry indices to differ-
entiate transient from persistent AKI, these indices have
not been extensively studied in critically ill patients or
in patients with sepsis [4,5,23]. The few published Darmon et al. Critical Care 2011, 15:R178
http://ccforum.com/content/15/4/R178 Page 7 of 8 studies have had several limitations: most of them were
single-center case series or retrospective studies, the
definition of AKI varied across the studies and the defi-
nition of transient AKI also varied, being subjective in
most instances [24-30]. Several of these studies included
patients who did not have critical illnesses [11,12,28]. In
addition, we chose a definition allowing for a distinction
between transient and persistent AKI. Our study was
therefore not designed to evaluate the interest of these
indices in distinguishing prerenal and intrinsic AKI. This point may partly explain the poor performance of
the urinary indices in our study. Last, most of the stu-
died patients had sepsis or shock at ICU admission. This condition is frequently associated with renal hand-
ling of sodium or water independently of an underlying
AKI. This may also explain the poor performance of
these indices in the studied population. Nevertheless,
taking these factors into account, and although the
usual urinary indices were able to differentiate transient
from persistent AKI, the accuracy of the indices was
poor and none of them were independently associated
with the diagnosis of persistent AKI, indicating a need
to identify other biomarkers. Darmon et al. Critical Care 2011, 15:R178
http://ccforum.com/content/15/4/R178 Darmon et al. Critical Care 2011, 15:R178
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1 1Medical-Surgical Intensive Care Unit, Saint-Etienne University Hospital, and
Jean Monnet University, Avenue Albert Raymond, F-42270 Saint-Etienne,
France. 2INSERM Unit 955, Paris-12 University, 51 Avenue du Marechal De
Lattre de Tassigny, F-94010 Créteil, France. 3Thrombosis Research Group, EA
3065, Saint-Etienne University Hospital, and Saint-Etienne Medical School,
Avenue Albert Raymond, F-42270 Saint-Etienne, France. 4Medical-Surgical
Intensive Care Unit, Avicenne University Hospital, APHP, 125, rue de
Stalingrad, F-93009 Bobigny, France. 5Medical Intensive Care Unit, Archet
University Hospital, Nice, France; and Nice University, UFR de Médecine, 151
Rte Saint Antoine Ginestiere, F-06202 Nice, France. 6Medical Intensive Care
Unit, AP-HP, Albert Chenevier-Henri Mondor University Hospital, and Paris-12
University, 51 Avenue du Marechal De Lattre de Tassigny, F-94010 Créteil,
France. 7Medical ICU, Saint-Louis University Hospital, APHP, Avenue Claude
Vellefaux, F-75010 Paris, France; and UFR de Médecine, Paris-7 Paris-Diderot
University, Avenue Claude Vellefaux, F-75010 Paris, France. 8Medical-Surgical
Intensive Care Unit, Hôpitaux Universitaires de Genève, 24, Micheli-du-Crest,
CH-1211 Genève 14, Suisse. 9Paris-13 University, 125, rue de Stalingrad, F-
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a prospective analysis. J Nephrol 2005, 18:54-60. References Submit your next manuscript to BioMed Central
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Submit your manuscript at
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and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
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• Immediate publication on acceptance
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• Research which is freely available for redistribution
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Mucosal Melanoma of the Head and Neck pT3 TNM Finding v8
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Open Peer Review on Qeios Open Peer Review on Qeios Mucosal Melanoma of the Head and Neck
pT3 TNM Finding v8 National Cancer Institute Source National Cancer Institute. Mucosal Melanoma of the Head and Neck pT3 TNM Finding
v8. NCI Thesaurus. Code C133178. Qeios · Definition, February 2, 2020 Qeios ID: T14LPO · https://doi.org/10.32388/T14LPO National Cancer Institute. Mucosal Melanoma of the Head and Neck pT3 TNM Finding
v8. NCI Thesaurus. Code C133178. Mucosal melanoma of the head and neck with tumor limited to the mucosa and
immediately underlying soft tissue, regardless of thickness or greatest dimension; for
example, polypoid nasal disease, pigmented or nonpigmented lesions of the oral cavity,
pharynx, or larynx. (from AJCC 8th Ed.) Qeios ID: T14LPO · https://doi.org/10.32388/T14LPO 1/1
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https://openalex.org/W4213345992
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https://mrj.ima-press.net/mrj/article/download/1254/1212
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Russian
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Association of rs7574865 G/T polymorphism of STAT4 gene with juvenile onset of systemic lupus erythematosus
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Sovremennaâ revmatologiâ
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Анализ распределения частот аллелей между альтернативными группами пациентов с разными фенотипи-
ческими проявлениями юСКВ (их наличием или отсутствием) показал связь данного полиморфизма с артритом. Ключевые слова: системная красная волчанка с ювенильным началом; ген STAT4; полиморфизм rs7574865; фенотипы системной
красной волчанки; артрит. Контакты: Михаил Юрьевич Крылов; mekry@yandex.ru Для ссылки: Крылов МЮ, Каледа МИ, Гусева ИА и др. Ассоциация полиморфизма rs7574865 G/T гена STAT4 с ювенильным началом
системной красной волчанки. Современная ревматология. 2022;16(1):68–72. DOI: 10.14412/1996-7012-2022-1-68-72 Крылов М.Ю.1, Каледа М.И.1, Гусева И.А.1, Коновалова Н.В.2, Варламов Д.А.2
1ФГБНУ «Научно-исследовательский институт ревматологии им. В.А. Насоновой», Москва;
2ФГБНУ «Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии», Москва
1Россия, 115522, Москва, Каширское шоссе, 34А; 2Россия, 127550, Москва, ул. Тимирязевская 42 Системная красная волчанка (СКВ) с ювенильным началом (юСКВ) – комплексное аутоиммунное заболевание с вовлечением многих
органов и систем. Однонуклеотидный полиморфизм rs7574865 гена STAT4 (сигнального переключателя и активатора транскрипции 4)
связан с риском развития нескольких аутоиммунных заболеваний, включая СКВ у взрослых. Цель исследования – проверка гипотезы о связи полиморфизма rs7574865 гена STAT4 с предрасположенностью к юСКВ. Пациенты и методы. В настоящем исследовании случай-контроль полиморфизм rs7574865 был изучен у 50 детей с юСКВ и 103 здо-
ровых волонтеров контрольной группы с помощью полимеразной цепной реакции в реальном времени. Результаты и обсуждение. Распределение частот генотипов среди пациентов имело статистически значимые различия по сравне-
нию с контролем (р=0,005). Частоты генотипов GG, GT, TT, GT + TT у пациентов c юСКВ и в контрольной группе составили 36,0
и 63,1% (р=0,003); 54,0 и 33,0% (р=0,021); 10,0 и 3,9% (р=0,153); 64,0 и 36,9% соответственно (p=0,003). Частота мутантного
аллеля Т изученного полиморфизма была выше у пациентов с юСКВ по сравнению с контролем (соответственно 37 и 20,4%; р=0,002). Исследована ассоциация аллеля Т с клиническими, лабораторными и иммунологическими фенотипами юСКВ. Заключение. Полученные данные указывают на значение полиморфизма rs7574865 гена STAT4 как важного фактора риска предрас-
положенности к юСКВ. Анализ распределения частот аллелей между альтернативными группами пациентов с разными фенотипи-
ческими проявлениями юСКВ (их наличием или отсутствием) показал связь данного полиморфизма с артритом. Результаты и обсуждение. Распределение частот генотипов среди пациентов имело статистически значимые различия по сравне-
нию с контролем (р=0,005). Частоты генотипов GG, GT, TT, GT + TT у пациентов c юСКВ и в контрольной группе составили 36,0
и 63,1% (р=0,003); 54,0 и 33,0% (р=0,021); 10,0 и 3,9% (р=0,153); 64,0 и 36,9% соответственно (p=0,003). Частота мутантного
аллеля Т изученного полиморфизма была выше у пациентов с юСКВ по сравнению с контролем (соответственно 37 и 20,4%; р=0,002). Исследована ассоциация аллеля Т с клиническими, лабораторными и иммунологическими фенотипами юСКВ. Заключение. Полученные данные указывают на значение полиморфизма rs7574865 гена STAT4 как важного фактора риска предрас-
положенности к юСКВ. Анализ распределения частот аллелей между альтернативными группами пациентов с разными фенотипи-
ческими проявлениями юСКВ (их наличием или отсутствием) показал связь данного полиморфизма с артритом. Заключение. Полученные данные указывают на значение полиморфизма rs7574865 гена STAT4 как важного фактора риска предрас-
положенности к юСКВ. ОРИГ ИНАЛЬНЫ Е ИССЛЕД ОВАНИЯ / ORIGIN AL IN VESTIGATION S Некото-
рые проявления СКВ, такие как антитела к двуспиральной
ДНК (дсДНК), наличие других аутоантител и нарушение
функции почек, были связаны с полиморфизмами гена
STAT4 [19]. Системная красная волчанка (СКВ) – хроническое вос-
палительное и сложное системное аутоиммунное заболева-
ние неизвестной этиологии, которое характеризуется
системным иммуновоспалительным (аутоиммунным) пора-
жением жизненно важных органов и разнообразием клини-
ческих проявлений [1, 2]. Примерно у 10–20% больных СКВ
первые симптомы заболевания развиваются до наступления
совершеннолетия [3]. По данным L.T. Hiraki и соавт. [4], рас-
пространенность СКВ в детском возрасте (СКВ с ювениль-
ным началом, юСКВ) составляет в среднем 9,73 случая на
100 тыс. детского населения. В исследовании N.A. Sinicato и
соавт. [5] этот показатель варьируется от 4 до 250 случаев на
100 тыс.; авторы указывают, что при юСКВ чаще выявляются
семейная агрегация и высокая частота рецидивов. Согласно
современным представлениям, СКВ – полигенная генети-
ческая модель, которая может включать до 100 генов, и каж-
дый ген вносит только умеренный вклад в общую
генетическую составляющую [6]. Ранее проведенные исследования однонуклеотидных
полиморфизмов генов IL6, ILIα, IL1β, IL1R и IL1Ra пока-
зали положительную ассоциацию между промоутерной
последовательностью гена IL1β в положении -511 и геноти-
пом ТТ, который был признан фактором риска для юСКВ. Ни один из других полиморфизмов не был значимо ассоции-
рован с предрасположенностью к юСКВ [7]. В исследовании
A. Rezae и соавт. [8] была изучена связь полиморфизмов двух
противовоспалительных цитокинов: интерлейкина (ИЛ) 10
и трансформирующего фактора роста β (ТФРβ) с юСКВ. Ав-
торами была выявлена значимая ассоциация только с поли-
морфизмом гена IL10. Имеется ограниченное число исследований полимор-
физма rs7574865 гена STAT4 при юСКВ, при этом в России
такие работы не проводились. Цель исследования – изучение ассоциации полимор-
физма rs7574865 гена STAT4 с предрасположенностью к
юСКВ, а также возможной связи этого полиморфизма с кли-
ническими и серологическими проявлениями юСКВ. Пациенты и методы. Группы обследованных. В исследова-
ние включено 50 пациентов с юСКВ и 103 здоровых нерод-
ственных волонтера (студенты колледжа, контрольная группа). В группе пациентов с юСКВ было 38 девочек и 12 мальчиков
в возрасте от 3 до 17 лет (средний возраст – 12,0±3,4 года) со
средней длительностью заболевания 4,1±2,4 года. Все пациенты
проходили лечение в детском отделении ФГБНУ «Научно-ис-
следовательский институт ревматологии им. В.А. Насоновой»
(НИИР им. В.А. Насоновой) в 2017–2020 гг. В качестве конт-
роля для генотипирования полиморфизма STAT4 rs7574865
G/T были исследованы 103 образца ДНК здоровых волонте-
ров, не имеющих аутоиммунных заболеваний. ОРИГ ИНАЛЬНЫ Е ИССЛЕД ОВАНИЯ / ORIGIN AL IN VESTIGATION S Contact: Mikhail Yurievich Krylov; mekry@yandex.ru
For reference: Krylov MYu, Kaleda MI, Guseva IA, et a Contact: Mikhail Yurievich Krylov; mekry@yandex.ru
For reference: Krylov MYu, Kaleda MI, Guseva IA, et al. Association of rs7574865 G/T polymorphism of STAT4 gene with juvenile onset of sys-
temic
lupus
erythematosus. Sovremennaya
Revmatologiya=Modern
Rheumatology
Journal. 2022;16(1):68–72. DOI: 10.14412/1996-7012-2022-1-68-72 Contact: Mikhail Yurievich Krylov; mekry@yandex.ru
For reference: Krylov MYu, Kaleda MI, Guseva IA, et al. Association of rs7574865 G/T polymorphism of STAT4
temic
lupus
erythematosus. Sovremennaya
Revmatologiya=Modern
Rheumatology
Jou
DOI: 10.14412/1996-7012-2022-1-68-72 Contact: Mikhail Yurievich Krylov; mekry@yandex.ru
For reference: Krylov MYu, Kaleda MI, Guseva IA, et al. Association of rs7574865 G/T polymorphism of STAT4 gene with juvenile onset of sys-
temic
lupus
erythematosus. Sovremennaya
Revmatologiya=Modern
Rheumatology
Journal. 2022;16(1):68–72. DOI: 10 14412/1996-7012-2022-1-68-72 типа I [17]. STAT4 был идентифицирован как ген предраспо-
ложенности к СКВ в белой европеоидной и азиатской по-
пуляциях [18, 19]. K. Bolin и соавт. [20] при анализе когорт
люпус-нефрит/контроль выявили значимую ассоциацию
между четырьмя полиморфизмами гена STAT4: rs11889341,
rs7574865, rs7568275 и rs7582694 и развитием люпус-нефрита. Показано, что полиморфизм rs7574865 G/T гена STAT4 был
связан с ЮИА в белой популяции [21]. Изучение другого по-
лиморфизма гена STAT4 – rs7601754 T/C – показало, что ал-
лель
Т
дикого
типа
может
быть
аллелем
риска
предрасположенности к СКВ, а минорный мутантный ал-
лель С является протективным аллелем для СКВ [22]. Иссле-
дование полиморфизмов rs7574865 и rs7601754 гена STAT4 у
иранских пациентов с СКВ установило значимую связь
между болезнью и полиморфизмом rs7574865 [23]. Некото-
рые проявления СКВ, такие как антитела к двуспиральной
ДНК (дсДНК), наличие других аутоантител и нарушение
функции почек, были связаны с полиморфизмами гена
STAT4 [19]. типа I [17]. STAT4 был идентифицирован как ген предраспо-
ложенности к СКВ в белой европеоидной и азиатской по-
пуляциях [18, 19]. K. Bolin и соавт. [20] при анализе когорт
люпус-нефрит/контроль выявили значимую ассоциацию
между четырьмя полиморфизмами гена STAT4: rs11889341,
rs7574865, rs7568275 и rs7582694 и развитием люпус-нефрита. Показано, что полиморфизм rs7574865 G/T гена STAT4 был
связан с ЮИА в белой популяции [21]. Изучение другого по-
лиморфизма гена STAT4 – rs7601754 T/C – показало, что ал-
лель
Т
дикого
типа
может
быть
аллелем
риска
предрасположенности к СКВ, а минорный мутантный ал-
лель С является протективным аллелем для СКВ [22]. Иссле-
дование полиморфизмов rs7574865 и rs7601754 гена STAT4 у
иранских пациентов с СКВ установило значимую связь
между болезнью и полиморфизмом rs7574865 [23]. Современная ревматология. 2022;16(1):68–72 Association of rs7574865 G/T polymorphism of STAT4 gene with juvenile onset
of systemic lupus erythematosus 134A, Kashirskoe Shosse, Moscow 115522, Russia; 242, Timiryazevskaya street, Moscow 1 Systemic lupus erythematosus (SLE) with juvenile onset (jSLE) is a complex autoimmune disease involving many organs and systems. The single
nucleotide polymorphism rs7574865 of the STAT4 (signal switch and transcription activator 4) gene is associated with the risk of developing
several autoimmune diseases, including SLE in adults. g
s of the association of the rs7574865 polymorphism of the STAT4 gene with a predisposition to jSLE. g
Objective: to verify the hypothesis of the association of the rs7574865 polymorphism of the STAT4 gene with a predisposition to jSLE. Patients and methods. In the present case-control study, the rs7574865 polymorphism was studied in 50 children with jSLE and 103 healthy
control volunteers using real-time polymerase chain reaction. j
fy
yp
f
f
p y
p
f
g
p
p
j
Patients and methods. In the present case-control study, the rs7574865 polymorphism was studied in 50 children with jSLE and 103 healthy
control volunteers using real-time polymerase chain reaction. Results and discussion. The distribution of genotype frequencies among patients had statistically significant differences compared to controls
(p=0.005). The frequencies of GG, GT, TT, GT + TT genotypes in patients with jSLE and in the control group were 36.0 and 63.1% (p=0.003);
54.0 and 33.0% (p=0.021); 10.0 and 3.9% (p=0.153); 64.0 and 36.9% respectively (p=0.003). The frequency of the mutant T allele of the
studied polymorphism was higher in patients with jSLE compared with controls (37 and 20.4%, respectively; p=0.002). The association of the T
allele with clinical, laboratory, and immunological phenotypes of jSLE was studied. Conclusion. The obtained data indicate the significance of the rs7574865 polymorphism of the STAT4 gene as an important risk factor for sus-
ceptibility to jSLE. An analysis of the distribution of allele frequencies between alternative groups of patients with different phenotypic manifes-
tations of jSLE (their presence or absence) showed an association of this polymorphism with arthritis. Keywords: systemic lupus erythematosus with juvenile onset; STAT4 gene; polymorphism rs7574865; phenotypes of systemic lupus erythematosus;
arthritis thematosus with juvenile onset; STAT4 gene; polymorphism rs7574865; phenotypes of systemic lupus erythematos Современная ревматология. 2022;16(1):68–72 68 ОРИГ ИНАЛЬНЫ Е ИССЛЕД ОВАНИЯ / ORIGIN AL IN VESTIGATION S Таблица 1. Демографические и клинико-лабораторные характеристики пациентов с юСКВ (n=50)
Table 1. Characteristics of patients with jSLE (n=50)
Показатель Значение
Возраст дебюта, годы, М±σ
12,0±3,4
Продолжительность заболевания на момент обследования, годы, М±σ
4,1±2,4
Женский пол, n (%)
38 (76,0)
Артрит, n (%)
37 (74,0)
Алопеция, n (%)
23 (46,0)
Поражение почек, n (%)
21 (42,0)
Лейкопения, n (%)
33 (66,0)
Гипокомплементемия, n (%)
29 (58,0)
Позитивность по анти-дсДНК, n (%)
42 (84,0) Частота генотипа GG в контроль-
ной группе была принята за референс-
ный показатель. Частота гетерозигот-
ного генотипа GT, объединенных гено-
типов GT + TT и мутантного аллеля T
у больных юСКВ была выше, чем в
контроле: 54,0 и 33,0% (р=0,021); 64,0
и 36,9% (р=0,003); 37 и 20,4% (р=0,002)
соответственно. Частота мутантного
генотипа TT также была выше у паци-
ентов с юСКВ по сравнению с контро-
лем, однако эти различия не достигали
статистической значимости. Носители
мутантного аллеля Т имели почти в
2,3 раза более высокий риск развития
СКВ. При анализе распределения частот
аллелей между группами пациентов с
разными фенотипическими проявле-
ниями юСКВ была выявлена связь изу-
ченного полиморфизма с предрасполо-
женностью к артриту (табл. 3). Таблица 2. Распределение частот аллелей и генотипов полиморфизма rs7574865 гена STAT4
у пациентов с юСКВ и в контрольной группе, n (%)
Table 2. Distribution of allele and genotype frequencies of the rs7574865 polymorphism of the STAT4
gene in patients with jSLE and in the control group, n (%) Таблица 2. Распределение частот аллелей и генотипов полиморфизма rs7574865 гена STAT4
у пациентов с юСКВ и в контрольной группе, n (%) Table 2. Distribution of allele and genotype frequencies of the rs7574865 polymorphism of the STAT4
gene in patients with jSLE and in the control group, n (%)
Аллель/ генотип Больные юСКВ Контрольная OШ (95% ДИ) р
(n=50) группа (n=103)
Примечание. Значимые различия между сравниваемыми показателями выделены жирным
шрифтом. * – референсное значение. STAT4:
G
63 (63,0)
64 (79,6)
1,0*
T
37 (37,0)
42 (20,4)
2,29 (1,31–4,02)
0,002
GG
18 (36,0)
65 (63,1)
1,0*
GT
27 (54,0)
34 (33,0)
2,38 (1,13–5,04)
0,021
TT
5 (10,0)
4 (3,9)
2,75 (0,56–14,44)
0,153
GT + TT
2 (64,0)
38 (36,9)
3,04 (1,43–6,55)
0,003 В группах пациентов с/без арт-
рита частота генотипа GT и TT суще-
ственно не различалась. ОРИГ ИНАЛЬНЫ Е ИССЛЕД ОВАНИЯ / ORIGIN AL IN VESTIGATION S В то же время
у детей с артритом по сравнению с па-
циентами без артрита отмечались
значимое снижение частоты генотипа
GG (соответственно 24,3 и 69,2%;
р=0,006) и накопление носителей ге-
нотипа GT + TT (р=0,006). Сходные
данные были продемонстрированы
при анализе частот аллелей rs7574865
полиморфизма гена STAT4. У пациен-
тов с артритом аллель Т встречался
значимо чаще, чем в альтернативной группе (р=0,015). У
носителей хотя бы одного аллеля Т риск предрасположен-
ности к артриту был в 7 раз выше, чем у носителей гено-
типа GG
rphism of the STAT4
) р
ыделены жирным
2)
0,002
4)
0,021
44)
0,153
5)
0,003 Примечание. Значимые различия между сравниваемыми показателями выделены жирным
шрифтом. * – референсное значение. tistica 6.0 (StatSoft Inc., США). При нормальных распределе-
ниях количественных показателей применяли t-тест Стью-
дента, результаты представлены в виде средних и
стандартных отклонений (М±σ) или медианы и интерквар-
тильного интервала (Ме [25-й; 75-й перцентили]). При
малых значениях вариабельностей использовали двусторон-
ний критерий Фишера. Анализ корреляций между клиниче-
скими характеристиками и полиморфизмом гена STAT4 был
проведен с помощью непараметрического метода Спирмена. Различия в распределении частот генотипов между группой
больных и контрольной группой оценивали с помощью таб-
лицы сопряженности 2×2 с использованием критерия χ2. Для
оценки меры риска развития юСКВ вычисляли отношение
шансов (ОШ) с подсчетом 95% доверительных интервалов
(ДИ). Статистически значимым считали значение p<0,05. Не выявлено связи полиморфизма rs7574865 гена STAT4
с демографическими показателями и другими клиническими
проявлениями юСКВ (возрастом дебюта, гендерной принад-
лежностью, алопецией, почечной патологией, лейкопенией,
гипокомплементемией и наличием антител к дсДНК). Обсуждение. В настоящем пилотном исследовании впер-
вые в российской детской популяции установлена сильная
ассоциация полиморфизма rs7574865 гена STAT4 с предрас-
положенностью к юСКВ и ассоциированному с заболева-
нием артриту. Ген STAT4 кодирует фактор транскрипции,
который способствует дифференцировке хелперных клеток
Тh1 и активности генов в этих клетках, вырабатывающих
специфические цитокины [25]. Функционируя как транс-
крипционный фактор, STAT4 участвует в ответах на ИЛ12 в
лимфоцитах и облегчает мРНК транскрипцию генов IL23,
IFN и IL17 в иммунных клетках в норме и при аномальных
иммунных реакциях [26, 27]. СКВ представляет собой мно-
гофакторное аутоиммунное заболевание со сложной генети-
ческой наследственностью [28]. Результаты. Частоты аллелей и генотипов. В табл. 1 при-
ведены демографические и клинико-лабораторные характе-
ристики пациентов с юСКВ. Соотношение девочек и
мальчиков в группе пациентов составило 3:1; представлены
изменения, встречавшиеся с частотой более 40%. Распределение генотипов rs7574865 в контрольной
группе не имело существенных отклонений от равновесия
закона Харди–Вайнберга. Анализ распределения частот ге-
нотипов и аллелей (табл. ОРИГ ИНАЛЬНЫ Е ИССЛЕД ОВАНИЯ / ORIGIN AL IN VESTIGATION S Диагноз
юСКВ соответствовал критериям SLICC (Systemic Lupus In-
ternational Collaborating Clinics) 2012 г. [24]. Письменное ин-
формированное согласие было получено от всех родителей
пациентов. Полный дефицит С4-компонента комплемента является
сильным генетическим фактором риска развития СКВ [9]. В исследовании K.M. Pereira и соавт. [10] были идентифици-
рованы и изучены два полиморфизма генов C4A и C4B, ко-
дирующих ген С4-компонента комплемента. Авторы
установили, что низкое содержание общего C4-компонента
комплемента и копий продуктов генов C4A и C4B были ассо-
циированы с сильным риском предрасположенности у па-
циентов с юСКВ в отличие от взрослых больных. Полногеномные исследования (GWAS) и метаанализ по-
казали, что мутантный аллель rs7574865T гена STAT4 пред-
располагает
к
восприимчивости
к
ювенильному
идиопатическому артриту (ЮИА), СКВ, ревматоидному арт-
риту (РА), сахарному диабету 1-го типа, системному скле-
розу,
первичному
антифосфолипидному
синдрому,
воспалительным заболеваниям кишечника (язвенному ко-
литу) и первичному синдрому Шёгрена [11–13]. Наши по-
следние
исследования
также
подтвердили
участие
мутантного аллеля rs7574865T гена STAT4 в формировании
предрасположенности к ЮИА, РА и анкилозирующему
спондилиту [14–16]. Генетический анализ в режиме реального времени. У всех
участников были взяты образцы венозной крови. Геномная
ДНК была выделена из свежих или замороженных образцов
крови с помощью набора «Проба-ГС-генетика» («ДНК-Тех-
нология»,
Россия). Генотипирование
полиморфизма
rs7574865G/T гена STAT4 было проведено с использованием
аллель-специфической полимеразной цепной реакции в ре-
альном времени. Меченые праймеры и зонды были разрабо-
таны и синтезированы в компании «Синтол» (Россия). Условия амплификации соответствовали рекомендациям
производителя. Исследование проведено с использованием
амплификатора ДТ-96 («ДНК-Технология», Россия). Ген STAT4 расположен на хромосоме человека 2q32.3 и
включает 24 экзона, охватывающих область в 120 килобаз. Ген кодирует фактор транскрипции, который может быть ак-
тивирован IL12 и IL23 и играет центральную роль в передаче
сигналов посредством рецептора интерферона (ИФН) Статистические методы. Статистическая обработка
данных выполнена с использованием пакета программ Sta- 69 ОРИГ ИНАЛЬНЫ Е ИССЛЕД ОВАНИЯ / ORIGIN AL IN VESTIGATION S Таблица 3. Распределение частот аллеля Т полиморфизма rs7574865 гена STAT4 у больных юСКВ с артритом и бе
Table 3. Frequency distribution of the T allele of the rs7574865 polymorphism of the STAT4 gene in patients with jSLE wi Клинический Генотип Аллель
фенотип GG GT TT GT + TT G T
Примечание. Жирным шрифтом выделены сравниваемые показатели. * – ОШ – 7,00 (95% ДИ 1,45–37,43), р=0,006; ** – ОШ – 4,43 (95%
ДИ 1,30–19,16), p=0,015. Артрит:
наличие (n=37)
9 (24,3)
23 (62,2)
5 (13,5)
28 (75,7)*
41 (55,4)
33 (44,6)**
отсутствие (n=13)
9 (69,2)
4 (30,8)
0 (0,0)
4 (24,3)
22 (74,0)
4 (26,0) ассоциированные с СКВ у европеоидов, также являются
факторами риска у азиатов: гены HLA класса II, STAT4,
BANK1, BLK, IRF5, TNFSF4, ITGAM и т. д. Наши данные от-
личаются от результатов небольшого числа исследований, в
которых изучалась связь полиморфизмов гена STAT4 с пред-
расположенностью к СКВ у детей. Так, в иранской когорте
детей не установлено ассоциации полиморфизма rs7574865
гена STAT4 с риском развития СКВ у подростков и тяжелыми
клиническими и лабораторными проявлениями, несмотря
на связь данного полиморфизма с риском развития СКВ у
взрослых иранцев. Авторы выдвигают гипотезу о различиях
генетического фона при юСКВ и СКВ у взрослых в одной и
той же популяции [29]. При изучении связи другого поли-
морфизма гена STAT4rs –7582694, – наиболее часто иссле-
дуемого при СКВ, египетские ученые нашли ассоциацию
гомозиготных генотипов с тяжестью заболевания у больных
юСКВ [30]. Полиморфизм rs7582694 C/G гена STAT4 был ис-
следован в когорте польских пациентов (n=253) с СКВ. Была
выявлена ассоциация гомозиготного генотипа СС с повыше-
нием риска развития СКВ в 1,58 раза [31]. Генотипы СС и CG
были связаны с почечной патологией (ОШ 2,26), вовлече-
нием центральной нервной системы (ОШ 2,88), наличием
антител к snRNP и Sle70, позитивностью по антинуклеарным
антителам, анти-дсДНК и снижением уровня С3- и С4-ком-
понента комплемента. Для валидации полученных данных необходимы даль-
нейшие исследования взрослых пациентов с ранним нача-
лом СКВ, разной тяжестью заболевания и клиническими
характеристиками, специфичными для СКВ. Заключение. Настоящее пилотное исследование под-
твердило высокий риск предрасположенности к юСКВ, ас-
социированный с носительством аллеля Т rs7574865
полиморфизма гена STAT4 в русской детской популяции. Впервые установлено, что у пациентов с юСКВ полимор-
физм rs7574865 гена STAT4 соотносится также с риском раз-
вития артрита. hematosus. 2016 ACR/ARHP Annual Me-
eting. Abstract Number: 1392. 6. Yuan YJ, Luo XB, Shen N. Current advan-
ces in lupus genetic and genomic studies in
Asia. Lupus. 2010 Oct;19(12):1374-83. doi: 10.1177/0961203310376639. 7. Ziaee V, Tahghighi F, Moradinejad MH,
et al. Interleukin-6, interleukin-1 gene cluster
and interleukin-1 receptor polymorphisms in
Iranian patients with juvenile systemic lupus
erythematosus. Eur Cyrokine Netw 2014 Jun;
25(2):35-40. doi: 10.1684/ecn.2014.0352. 8. Rezae A, Ziaee V, Sharabian FT, et al. Lack
of association between interleukin-10, trans-
forming growth factor-beta gene polymorp-
hisms and juvenile-onset systemic lupus
erythematosus. Clin Rheumatol. 2015 Jun;
34(6):1059-64. doi: 10.1007/s10067-015-
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SLE susceptibility in European Americans. Am J Hum Genet. 2007 Jun; 80(6):1037-54. doi: 10.1086/518257. 10. Pereira KM, Faria AG, Liphaus BL, et al. Low C4, C4A and C4B gene copy numbers
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are stronger risk factors for juvenile-onset than
for adult-onset systemic lupus erythematosus. Rheumatology (Oxford). 2016 May;55(5):
869-73. doi: 10.1093/rheumatology/kev436 . 11. Lee HS, Remmers EF, Le JM, et al. Asso-
ciation of STAT4 with rheumatoid arthritis in
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et al. Interleukin-6, interleukin-1 gene cluster
and interleukin-1 receptor polymorphisms in
Iranian patients with juvenile systemic lupus
erythematosus. Eur Cyrokine Netw 2014 Jun;
25(2):35-40. doi: 10.1684/ecn.2014.0352. 3. ОРИГ ИНАЛЬНЫ Е ИССЛЕД ОВАНИЯ / ORIGIN AL IN VESTIGATION S Настоящая работа имела ограничения, связанные с не-
большим числом пациентов, что снижает мощность статисти-
ческого
анализа,
поэтому
необходимы
дальнейшие
репликационные исследования с большим размером выборок
из других популяций, а также изучение других потенциальных
полиморфизмов, которые будут полезны для интерпретации
результатов исследований и формулирования обоснованного
заключения о роли STAT4 в этиопатогенезе юСКВ. Благодарности Благодарности Выражаем признательность врачам детского отделения
(руководитель – к.м.н. И.П. Никишина) ФГБНУ НИИР
им. В.А. Насоновой за помощь в отборе пациентов для настоя-
щего исследования. ОРИГ ИНАЛЬНЫ Е ИССЛЕД ОВАНИЯ / ORIGIN AL IN VESTIGATION S 2) показал статистически значимые
различия между группами пациентов с юСКВ и контроля
(р=0,005). В нескольких работах выявлено существование генети-
ческой гетерогенности при СКВ в разных этнических груп-
пах
[14–16]. Показано,
что
некоторые
гены, Современная ревматология. 2022;16(1):68–72 70 hematosus. 2016 ACR/ARHP Annual Me-
eting. Abstract Number: 1392.
6. Yuan YJ, Luo XB, Shen N. Current advan-
ces in lupus genetic and genomic studies in
Asia. Lupus. 2010 Oct;19(12):1374-83.
doi: 10.1177/0961203310376639.
7. Ziaee V, Tahghighi F, Moradinejad MH,
et al. Interleukin-6, interleukin-1 gene cluster
and interleukin-1 receptor polymorphisms in
Iranian patients with juvenile systemic lupus
erythematosus. Eur Cyrokine Netw 2014 Jun;
25(2):35-40. doi: 10.1684/ecn.2014.0352.
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forming growth factor-beta gene polymorp-
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erythematosus. Clin Rheumatol. 2015 Jun;
34(6):1059-64. doi: 10.1007/s10067-015-
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и др. Полиморфизм rs7574865 гена STAT4
и риск развития раннего ревматоидного
артрита (исследование РЕМАРКА). Научно-практическая ревматология. 2019;
57(1):62-5. STAT4 region. Arthritis Res Ther. 2008; 10(5):
R113. doi: 10.1186/ar2516 20. Bolin K, Sandling JK, Zickert A, et al. Association of STAT4 polymorphism with se-
vere renal insufficiency in lupus nephritis. PLoS One. 2013 Dec 27; 8(12):e84450. doi: 10.1371/journal.pone.0084450. 21. Matur AN, Chang HC, Zisoulis DG, et al. Stat3 and Stat4 direct development of IL-17-
secreting Th cells. J Immunol. 2007 Apr 15;
178(8):4901-7. doi: 10.4049/jimmunol. 178.8.4901. [Guseva IA, Krylov MYu, Demidova NV,
et al. Rs7574865 polymorphism of STAT4
gene and risk of early rheumatoid arthritis
(REMARKA study). Nauchno-prakticheskaya
revmatologiya. 2019;57(1):62-5. (In Russ.)]. 16. Крылов МЮ, Cтаркова АС, Самар-
кина ЕЮ и др. Ассоциация показателей
активности анкилозирующего спондилита
в русской с rs7574865-полиморфизмом [Guseva IA, Krylov MYu, Demidova NV,
et al. Rs7574865 polymorphism of STAT4
gene and risk of early rheumatoid arthritis (REMARKA study). Nauchno-prakticheskaya
revmatologiya. 2019;57(1):62-5. (In Russ.)]. (REMARKA study). Nauchno-prakticheskaya
revmatologiya. 2019;57(1):62-5. (In Russ.)]. 16. Крылов МЮ, Cтаркова АС, Самар-
кина ЕЮ и др. Ассоциация показателей
активности анкилозирующего спондилита
в русской с rs7574865-полиморфизмом
гена STAT4. Современная ревматология. 2019;13(2):55-60. 30. Nageeb RS, Omran AA, Nageeb GS, et al. STAT4 gene polymorphism in two major
autoimmune diseases (multiple sclerosis and
juvenile onset systemic lupus erythematosus)
and its relation to disease severity. Egypt J
Neurol Psychiatr Neurosurg. 2018;54(1):16. doi: 10.1186/s41983-018-0011-5. Epub 2018 May 25. 31. Piotrowski P, Lianeri M, Wudarski M,
et al. Contribution of STAT4 gene single-nuc-
leotide polymorphism to systemic lupus eryt-
hematosus in the Polish population. Mol Biol
Rep. 2012 Sep;39(9):8861-6. doi: 10.1007/
s11033-012-1752-3. 30. Nageeb RS, Omran AA, Nageeb GS, et al. STAT4 gene polymorphism in two major
autoimmune diseases (multiple sclerosis and
juvenile onset systemic lupus erythematosus)
and its relation to disease severity. Egypt J 30. Nageeb RS, Omran AA, Nageeb GS, et al. STAT4 gene polymorphism in two major
autoimmune diseases (multiple sclerosis and
juvenile onset systemic lupus erythematosus)
and its relation to disease severity. Egypt J
Neurol Psychiatr Neurosurg. 2018;54(1):16. doi: 10.1186/s41983-018-0011-5. 22. Заявление о конфликте интересов/Conflict of Interest Statement Заявление о конфликте интересов/Conflict of Interest Statement Исследование выполнено в рамках фундаментальной научной темы № 1021051302580-4. Исследование выполнено в рамках фундаментальной научной темы № 1021051302580-4. Исследование не имело спонсорской поддержки Конфликт интересов отсутствует Авторы несут полную ответственность д
р
фу д
у
Исследование не имело спонсорской поддержки. Конфликт интересов отсутствует. Авторы несут полную ответственность
за предоставление окончательной версии рукописи в печать. Все авторы принимали участие в разработке концепции статьи и
написании рукописи. Окончательная версия рукописи была одобрена всеми авторами. The investigation has been conducted within scientific topic №1021051302580-4. The investigation has been conducted within scientific topic №1021051302580-4. The investigation has not been sponsored. There are no conflicts of interest. The authors are solely responsible for submitting the final
version of the manuscript for publication. All the authors have participated in developing the concept of the article and in writing the
manuscript. The final version of the manuscript has been approved by all the authors. Крылов М.Ю. https://orcid.org/0000-0002-9922-5124
Каледа М.И. https://orcid.org/0000-0002-0513-6826
Гусева И.А. https://orcid.org/0000-0002-4906-7148
Коновалова Н.В. https://orcid.org/0000-0003-4316-1077
Варламов Д.А. https://orcid.org/0000-0001-7004-981Х Крылов М.Ю. https://orcid.org/0000-0002-9922-5124 Каледа М.И. https://orcid.org/0000-0002-0513-6826 Коновалова Н.В. https://orcid.org/0000-0003-4316-1077 Варламов Д.А. https://orcid.org/0000-0001-7004-981Х Современная ревматология. 2022;16(1):68–72 72
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Crystal structure of diaqua-(acetato-κ<sup>3</sup> <i>O</i>,<i>O′</i>:<i>O′′</i>)-(μ<sub>3</sub>-4,6-di(1<i>H</i>-imidazol-1-yl)isophthalato-κ<sup>4</sup> <i>O</i>:<i>O′</i>:<i>O′′</i>,<i>O′′′</i>)lanthanum(III), C<sub>16</sub>H<sub>15</sub>LaN<sub>4</sub>O<sub>8</sub>
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Zeitschrift für Kristallographie. New crystal structures/Zeitschrift für Kristallographie. New crystal structures
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Xue-Guo Liu*, Wei-Hong Yan, Xiao-Jing Xing and Hui-Tao Fan*
Crystal structure of diaqua-(acetato-κ3O,O′:O′′)-
(µ3-4,6-di(1H-imidazol-1-yl)isophthalato-
κ4O:O′:O′′,O′′′)lanthanum(III), C16H15LaN4O8 ,
g
,
J
g
g
Crystal structure of diaqua-(acetato-κ3O,O′:O′′)-
(µ3-4,6-di(1H-imidazol-1-yl)isophthalato-
κ4O:O′:O′′,O′′′)lanthanum(III), C16H15LaN4O8 https://doi.org/10.1515/ncrs-2017-0393
Received January 5, 2018; accepted April 12, 2018; available Table 1: Data collection and handling. Crystal:
Yellow block
Size:
0.20 × 0.18 × 0.16 mm
Wavelength:
Mo Kα radiation (0.71073 Å)
µ:
2.36 mm−1
Diffractometer, scan mode:
SuperNova, ω-scans
θmax, completeness:
25°, >99%
N(hkl)measured, N(hkl)unique, Rint:
9838, 3247, 0.038
Criterion for Iobs, N(hkl)gt:
Iobs > 2 σ(Iobs), 2896
N(param)refined:
263
Programs:
CrysAlisPRO [1], SHELX [2] Crystal:
Yellow block
Size:
0.20 × 0.18 × 0.16 mm
Wavelength:
Mo Kα radiation (0.71073 Å)
µ:
2.36 mm−1
Diffractometer, scan mode:
SuperNova, ω-scans
θmax, completeness:
25°, >99%
N(hkl)measured, N(hkl)unique, Rint:
9838, 3247, 0.038
Criterion for Iobs, N(hkl)gt:
Iobs > 2 σ(Iobs), 2896
N(param)refined:
263
Programs:
CrysAlisPRO [1], SHELX [2] Table 2: Fractional atomic coordinates and isotropic or equivalent
isotropic displacement parameters (Å2). https://doi.org/10.1515/ncrs-2017-0393
Received January 5, 2018; accepted April 12, 2018; available
online May 3, 2018
Abstract
C16H15LaN4O8, monoclinic, P21/c (no. 14), a = 13.2370(6) Å,
b = 15.4658(5) Å,
c = 9.5723(4) Å,
β = 108.834(4)°,
V = 1854.72(14) Å3, Z = 4, Rgt(F) = 0.0342, wRref(F2) = 0.0814,
T = 293(2) K. CCDC no.: 1814866
The crystal structure is shown in the figure (′ = x,
0.5 −y, 0.5 + y; ′′ = 1 −x, 0.5 + y, 0.5 −z). Tables 1 and 2
contain details on crystal structure and measurement condi-
tions and a list of the atoms including atomic coordinates and
displacement parameters. *Corresponding authors: Xue-Guo Liu, Department of Biology
and Chemical Engineering, Nanyang Institute of Technol-
ogy, Nanyang 473004, China, e-mail: huanliu1987@126.com;
and Hui-Tao Fan, College of Chemistry and Pharmaceutical
Engineering, Nanyang Normal University, Nanyang 473061, China,
e-mail: fanhuitao818@163.com
Wei-Hong Yan: Department of Biology and Chemical Engineering,
Nanyang Institute of Technology, Nanyang 473004, China
Xiao-Jing Xing: College of Chemistry and Pharmaceutical
Engineering, Nanyang Normal University, Nanyang 473061, China
Table 2: Fractional atomic coordinates and isotropic or equivalent
isotropic displacement parameters (Å2). Xue-Guo Liu*, Wei-Hong Yan, Xiao-Jing Xing and Hui-Tao Fan*
Crystal structure of diaqua-(acetato-κ3O,O′:O′′)-
(µ3-4,6-di(1H-imidazol-1-yl)isophthalato-
κ4O:O′:O′′,O′′′)lanthanum(III), C16H15LaN4O8 Atom
x
y
z
Uiso*/Ueq
LA1
0.28957(2)
0.22173(2)
0.32527(2)
0.01701(11)
O1
0.4287(3)
0.16423(18)
0.2311(3)
0.0369(8)
O2
0.4427(3)
0.23978(18)
0.0399(3)
0.0337(8)
O3
0.6283(2)
−0.12579(16)
0.2237(3)
0.0253(7)
O6
0.2109(2)
0.09302(18)
0.1587(3)
0.0329(8)
H6A
0.250984
0.080557
0.107839
0.049*
H6B
0.206854
0.049817
0.211519
0.049*
O4
0.7486(2)
−0.12053(16)
0.1100(3)
0.0262(7)
O5
0.3296(3)
0.08962(17)
0.4794(3)
0.0439(9)
H5B
0.340824
0.103359
0.569172
0.066*
H5A
0.276424
0.055549
0.451221
0.066*
C8
0.6397(4)
0.4188(3)
0.2250(6)
0.0459(14)
H8
0.619812
0.466969
0.268213
0.055*
N2
0.6862(4)
0.4222(2)
0.1142(5)
0.0465(11)
N1
0.6672(3)
0.28645(19)
0.1735(4)
0.0251(9)
N3
0.8780(3)
0.0274(2)
0.2101(4)
0.0272(8)
N4
1.0366(3)
0.0062(3)
0.1856(5)
0.0474(11)
C14
0.4758(3)
0.1862(2)
0.1435(4)
0.0248(10)
C12
0.9326(4)
−0.0280(3)
0.3198(5)
0.0511(14)
H12
0.908015
−0.052265
0.391713
0.061*
C10
0.9457(4)
0.0451(3)
0.1343(5)
0.0413(12)
H10
0.928531
0.081711
0.052911
0.050*
C6
0.6719(4)
0.1943(3)
0.1720(4)
0.0206(9)
C3
0.6873(3)
0.0138(2)
0.1770(4)
0.0193(9)
C2
0.5927(3)
0.0553(2)
0.1702(4)
0.0191(9)
H2
0.534354
0.022006
0.170961
0.023*
C4
0.7757(3)
0.0644(2)
0.1877(4)
0.0223(9)
C1
0.5820(3)
0.1453(2)
0.1623(4)
0.0188(9)
C13
0.6881(3)
−0.0839(2)
0.1711(4)
0.0199(9)
C11
1.0285(4)
−0.0404(4)
0.3035(6)
0.0572(15)
Open Access. © 2018 Xue-Guo Liu et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-
NonCommercial-NoDerivatives 4.0 License. https://doi.org/10.1515/ncrs-2017-0393 p
g
Received January 5, 2018; accepted April 12, 2018; available
online May 3, 2018 Received January 5, 2018; accepted April 12, 2018; available
online May 3, 2018 Z. Kristallogr. NCS 2018; 233(4): 631–632 Xue-Guo Liu*, Wei-Hong Yan, Xiao-Jing Xing and Hui-Tao Fan* Table 2 (Continued)
Atom
x
y
z
Uiso*/Ueq
H11
1.081944
−0.075586
0.363679
0.069*
C5
0.7688(3)
0.1535(2)
0.1863(4)
0.0247(10)
H5
0.829189
0.186634
0.194812
0.030*
C7
0.6275(4)
0.3369(3)
0.2612(5)
0.0366(12)
H7
0.597889
0.318009
0.331764
0.044*
C9
0.7022(4)
0.3412(3)
0.0879(5)
0.0377(12)
H9
0.733768
0.323252
0.018958
0.045*
O7
0.1014(3)
0.21291(19)
0.3288(3)
0.0410(9)
O8
0.1984(3)
0.22760(15)
0.5621(3)
0.0285(7)
C16
0.0051(5)
0.2397(5)
0.4905(6)
0.0724(19)
H16A
0.018922
0.255063
0.592099
0.109*
H16B
−0.036457
0.187632
0.468897
0.109*
H16C
−0.033502
0.285610
0.428419
0.109*
C15
0.1101(4)
0.2253(2)
0.4619(5)
0.0300(11) The asymmetric unit contains one La3+ ion, one fully
protonated dicarboxylate ligand, two coordinated water
molecules and one coordinated acetate anion. Adjacent car-
boxylate ligands generate infinite chain via bridging acetic
group chelating carboxylic group (O1—O2). Such chains
linked through another chelating carboxylic group (O3—O4)
to give rise to layered structure, which further linked through
O—H· · · N hydrogen bonds (O6· · · N4, 2.752(5) Å) to give rise
to three-dimensional network. Acknowledgements: We gratefully acknowledge the finan-
cial support by the Science and Technology Research
Key Project Department of Education in Henan Province
(Nos. 16A150018, 15A150066), the National Natural Science
Foundation of China (No. 21505080), and the Scientific and
Technological Project of Henan Province (Nos. 182102110089,
182102310645). References H2L (0.0090 g, 0.03 mmol), and La(CH3COO)3 (0.0095 g,
0.03 mmol) were dissolved in distilled water (8 mL). The
resultant solution was allowed to evaporate slowly at room
temperature. Yellow block single crystals were obtained. 1. Agilent Technologies: CrysAlisPRO Software System, version
1.171.38.41r, Agilent Technologies UK Ltd., Oxford, UK (2011).i 1. Agilent Technologies: CrysAlisPRO Software System, version
1.171.38.41r, Agilent Technologies UK Ltd., Oxford, UK (2011).i 2. Sheldrick, G. M.: Crystal structure refinement with SHELXL. Acta
Crystallogr. C71 (2015) 3–8. 2. Sheldrick, G. M.: Crystal structure refinement with SHELXL. Acta
Crystallogr. C71 (2015) 3–8. 3. Lindoy, L. F.; Atkinson, I.: Self-Assembly in Supramolecular
Systems, Royal Society of Chemistry, Cambridge, UK (2000). CCDC no.: 1814866 632
|
Liu et al.: C16H15LaN4O8 632 Experimental details 4. Joe Zhoua, H.-C.; Kitagawab, S.: Special issue for metal-organic
frameworks. Chem. Soc. Rev. 43 (2014) 5415–6172. 4. Joe Zhoua, H.-C.; Kitagawab, S.: Special issue for metal-organic
frameworks. Chem. Soc. Rev. 43 (2014) 5415–6172. Absorption corrections were applied by using multi-scan pro-
gram. The structure was refined based on F2 with the SHELXL
[2] software package. Hydrogen atoms attached to C of the
title complex located in difference electron density maps, and
treated as riding atoms. The Uiso values of the hydrogen atoms
were set to 1.2Ueq(C). 5. Li, B.; Zang, S. Q.; Wang, L. Y.; Mak, T. C. W.: Halogen bonding:
a powerful, emerging tool for constructing high-dimensional
metal-containing supramolecular networks. Coord. Chem. Rev. 308 (2016) 1–21. 5. Li, B.; Zang, S. Q.; Wang, L. Y.; Mak, T. C. W.: Halogen bonding:
a powerful, emerging tool for constructing high-dimensional
metal-containing supramolecular networks. Coord. Chem. Rev. 308 (2016) 1–21. 6. Hisaki, I.; Nakagawa, S.; Tohnai, N.; Miyata, M.: A C3-symmetric
macrocycle-based, hydrogen-bonded, multiporous hexagonal
network as a motif of porous molecular crystals. Angew. Chem. Int. Ed. 54 (2015) 3008–3012. 6. Hisaki, I.; Nakagawa, S.; Tohnai, N.; Miyata, M.: A C3-symmetric
macrocycle-based, hydrogen-bonded, multiporous hexagonal
network as a motif of porous molecular crystals. Angew. Chem. Int. Ed. 54 (2015) 3008–3012. Comment 7. James, S. L.: π-π Stacking as a crystal eEngineering tool in ency-
clopedia of supramolecular chemistry (Eds.: J. L. Atwood, J. W. Steed), Marcel Dekker Inc., New York (2004) pp. 1093–1109. The construction of metal-containing supramolecular net-
works has been an active field [3–7]. Coordinate covalent
bonding [4] and noncovalent interactions (hydrogen bond-
ing) [5–7] are two well established essentials crystal engineer-
ing. Recently, several groups have utilized hydrogen bonding
interactions to construct new, porous materials with perma-
nent porosity and highly selective gas separation [8, 9]. We,
aim to construct intriguing architectures through coordina-
tive and hydrogen bonding interactions. Thus, we selected a
carboxylate ligand. 8. Li, P.; He, Y.; Guang, J.; Weng, L.; Zhao, J. C. G.; Xiang, S.;
Chen, B.: A homochiral microporous hydrogen-bonded organic
framework for highly enantioselective separation of secondary
alcohols. J. Am. Chem. Soc. 136 (2014) 547–549. 9. Lü, J.; Perez-Krap, C.; Suyetin, M.; Alsmail, N. H.; Yan, Y.;
Yang, S.; Lewis, W.; Bichoutskaia, E.; Tang, C. C.; Blake, A. J.;
Cao, R.; Schröder, M.: A robust binary supramolecular organic
framework (SOF) with high CO2 adsorption and selectivity. J. Am. Chem. Soc. 136 (2014) 12828–12831.
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Implementation of a Smartphone application in medical education: a randomised trial (iSTART)
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BMC medical education
| 2,017
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cc-by
| 6,408
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Martínez et al. BMC Medical Education (2017) 17:168
DOI 10.1186/s12909-017-1010-4 Martínez et al. BMC Medical Education (2017) 17:168
DOI 10.1186/s12909-017-1010-4 © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: felipe.martinez@uv.cl
1Departamento de Salud Pública, Escuela de Medicina, Universidad de
Valparaíso, Hontaneda, 2664 Valparaíso, Chile
2Área de Investigación y Estudios Clínicos, Clínica Ciudad del Mar, Viña del
Mar, Chile
Full list of author information is available at the end of the article Implementation of a Smartphone
application in medical education: a
randomised trial (iSTART) Felipe Martínez1,2*, Catalina Tobar3 and Carla Taramasco4 Felipe Martínez1,2*, Catalina Tobar3 and Carla Taramasco4 Abstract Background: Smartphones are popular technologies that combine telephone communications and informatics in
portable devices. Limited evidence exists regarding their effectiveness in improving academic performance among
medical students. This study aims to assess whether a smartphone application could improve academic performance
in multiple-choice tests. Methods: A double-masked randomised trial was held among interns at the School of Medicine of the Universidad de
Valparaiso. Participants were randomised to receive an application designed to review key concepts in Internal Medicine
and its subspecialties using clinical vignettes. Contents were selected and provided in a format akin to a mandatory
national examination required for practising medicine in Chile. Analyses were undertaken under the intention to treat
principle and missing data were handled using multiple imputation techniques. Results: Eighty interns volunteered to participate in this trial, most were female (48 students, 60%) and had a mean age
of 25.3 ± 2.2 years. Participants showed significant experience with smartphones, with a median use of 4 years (IQR
3–6 years) and 67 (83.7%) reporting routine use in clinical practice. Intention-to-treat analyses showed significant
improvements in performance amongst students allocated to the smartphone application (mean increase of 14.5 ± 8.9
vs 9.4 ± 11.6points, p = 0.03). A reduction in total time and mean time per question was also found, which was
significant in complete-case analyses (p = 0.04). Discussion: Smartphones were popular among medical trainees. Academic performance was significantly improved
by the use of our application, although the overall effect was smaller than expected from previous trials. This study
provides evidence that smartphone-based interventions can assist in teaching internal medicine. Trial registration: ClinicalTrials NCT02723136. Keywords: Medical education, Internal medicine, Smartphones, Student, medical range from undergraduate education to health resource
management [4, 5]. Several studies have shown that
smartphones are frequently used among physicians,
medical students and interns, with overall use rates
reaching 80%. iOs®-based systems, such as the iPhone®,
seem to be the most commonly used platforms [1, 6–8]. The popularity of smartphones is likely to stem from
their versatility. Current devices have a wide variety of
functions, which can assist in medical decision making,
information
searches
and
educational
applications,
among other uses [8]. Use in clinical practice seems to
be more common among women, people with an Interventions Students allocated to receive the active intervention re-
ceived a downloadable application that was installed in
their smartphones. Those allocated to the control group
did not receive any additional training for EUNACOM. The mobile application was devised by a team of infor-
matic engineers and physicians and made available for
free at the App Store® and PlayStore® for both iOs® and
Android® operating systems. In order to monitor adher-
ence, the application required an active internet connec-
tion for operation. Students also received a brief (5-min)
description on functionality that was also made available
in text form as a part of the software. This study aims to determine whether the implemen-
tation of a smartphone application designed to assist in
delivering key concepts relevant to internal medicine
might improve academic performance in EUNACOM. Contents were primarily directed at the area of In-
ternal Medicine, which is the most important specialty
within EUNACOM. It included a series of questions in
the form of brief clinical vignettes constructed in a
format similar to the one described in EUNACOM’s
website [15]. In short, these vignettes correspond to clin-
ical scenarios against which the student must answer a
key aspect relevant to the diagnosis, management or
monitoring of several diseases. These multiple-choice
questions must be answered from five possible options,
with only one being the correct answer. The depth of
knowledge required to answer was established using the Background Smartphones are recent technologies that combine the
capabilities of telephone communications and informat-
ics in small portable devices that allow communications
and information processing even at the patient’s bedside
[1, 2]. As noted in the general public, these devices have
shown significant growth in the international medical
community [3], niche where they perform functions that * Correspondence: felipe.martinez@uv.cl
1Departamento de Salud Pública, Escuela de Medicina, Universidad de
Valparaíso, Hontaneda, 2664 Valparaíso, Chile
2Área de Investigación y Estudios Clínicos, Clínica Ciudad del Mar, Viña del
Mar, Chile
Full list of author information is available at the end of the article Martínez et al. BMC Medical Education (2017) 17:168 Page 2 of 9 interest in new technologies and those with prior experi-
ences with these platforms [7]. non-pharmacological interventions [13, 14]. The complete
protocol was registered in March 2016 at clinicaltrials.gov
(NCT02723136) and can be reviewed at https://clinical-
trials.gov/ct2/show/NCT02723136?term=NCT02723136&r
ank=1. A flowchart describing participant recruitment and
overall study design is shown in Fig. 1. Despite this popularity, there is limited evidence
regarding the effectiveness of smartphone use in improv-
ing academic performance among medical students [9]. While there is a wide availability of applications and
resources available for these platforms, only a few rando-
mised trials have addressed their effectiveness in improv-
ing academic performance. In 2011, Low and coworkers
published one of these studies using objective clinical
competence scores as a primary endpoint [10]. The lat-
ter trial reported a statistically significant improvement
of roughly 15% in the academic performance of students
allocated to receive the application. Similar findings were
seen in a second, before & after, study that was con-
ducted among Obstetrics & Gynecology residents [11]. Participants Eligible participants were medical interns coursing their
last year of training at the School of Medicine of the
Universidad de Valparaiso, who had a personal Smart-
phone
with
an
iOs®-
or
Android®-based
operating
system. Only those that did not wish to participate were
excluded from this study. Informed consent was ob-
tained from all participants. Every student sat a baseline 90-question test aimed to
resemble EUNACOM (see below) and were randomised
to receive a smartphone-based application training after-
wards. Randomisation was carried out using permuted
blocks by a statistician that was unaware of treatment
allocation. Allocation sequences were concealed from
other researchers participating in this study. All partici-
pants were asked to complete an entry form with basic
demographic data, including age, sex, year of training
and prior experiences with smartphones or similar
platforms (i.e. tablets). Data regarding academic per-
formance was obtained from the University, including
qualifications relevant to the area of Internal Medicine. g
y
gy
Since 2003, a national examination for undergraduate
medical students that have completed their internships
is carried out in Chile. This exam (Examen Unico
Nacional de Conocimientos en Medicina - EUNACOM)
is designed to assess the overall knowledge and practical
skills that any medical student should attain before prac-
tising medicine in the country. Its confection and
administration are regulated by law, and its oversight
has been delegated to the Association of Faculties of
Medicine of Chile (ASOFAMECH). EUNACOM is made
of two sections, theoretical and practical, and is consid-
ered qualifying to practise medicine in Chile. The con-
tents of both sections are of public knowledge and
include 1543 items distributed according to the curricu-
lar time spent training in different areas of medicine,
with special emphasis on internal medicine and its sub-
specialties [12]. The theoretical component is evaluated
using 180 multiple-choice questions delivered in two
90-min
sessions. Additionally,
EUNACOM
provides
professional title validation or equivalencies for foreign
physicians who wish to practice medicine in Chile. Given
the importance of this exam, several medical schools have
implemented preparation courses for their students. How-
ever, the methodologies used in the latter courses are
heterogeneous, and uncertainty exists regarding the best
way in which contents should be delivered. Methods iSTART is a double-masked randomised trial that was
held among medical students at the School of Medicine
of the Universidad de Valparaíso, Chile. The study proto-
col has been drafted in compliance with the Consoli-
dated
Standards
of
Reporting
Trials
(CONSORT)
statement as in its version adapted for trials evaluating Martínez et al. BMC Medical Education (2017) 17:168 Page 3 of 9 Fig. 1 CONSORT Study Flowchart. This figure depicts participant’s flow within the iSTART study ig. 1 CONSORT Study Flowchart. This figure depicts participant’s flow within the iSTART study resemble EUNACOM between groups. The final test did
not repeat any of the questions used within the applica-
tion that was delivered to students and was held 4 weeks
after randomisation. This timeframe was selected in
order to allow students to practise and study internal
medicine with the application given the extent of
contents required by EUNACOM. Simulation tests were
used because of the impossibility to use the actual exam
as part of this study, since it is managed independently
from universities and kept in strict reserve by ASOFA-
MECH. However, previous data has shown that both
simulation exams (baseline and final) have good correl-
ation with overall EUNACOM scores (r > 0.7, p < 0.001),
as well as an excellent diagnostic accuracy for detecting
students at risk of failing the exam(area under the ROC
curve 0.95, 95% CI 0.90 to 0.99) and identifying students
that will obtain high scores in the review (AUC 0.80, 95%
CI 0.71 to 0.88, unpublished data). The correction of both
practice tests was undertaken by reseachers that were kept
unaware of allocation. provisions of the EUNACOM agenda [12]. All contents
of the application were designed by two internists with
5 years experience in developing questions for the exam. Examples
of
these
vignettes
are
provided
in
the
Additional file 1. The application had two modes to provide the afore-
mentioned inquiries. In the first, study mode, students
were not given time constraints to answer the clinical vi-
gnettes. Whenever an answer was provided, instant feed-
back was delivered alongside a brief explanation of the
key concept that was being assessed by the inquiry. In
the second, training mode, participants had a restricted
time window to provide answers. This mode was
designed because of a perceived difficulty amongst in-
terns in managing time in answering questions in previ-
ous simulations of the exam. Methods A default of 60 s was
established, but the application allowed the user to mod-
ify this timeframe to 30 or 90 s. Students had knowledge
regarding their individual performance in both modes,
but no additional feedback in terms of concept review
was provided in training mode. A secondary endpoint was to establish differences in
the average time required to answer clinical vignettes. In
order to allow reliable comparisons to be made, exams
were conducted electronically and under supervision by
the research team, thus allowing an objective assessment Analysis plan Basic descriptive statistics (means, medians, proportions,
interquartile ranges -IQR-, etc) were performed to assess
the characteristics of the study sample. Fisher’s exact test
was used to evaluate univariate association of categorical
variables. Quantitative variables were compared using
Mann-Whitney or Student’s T tests according to data
distribution and variances. Ninety-five percent confi-
dence intervals were constructed whenever appropriate. Missing data relevant to the primary and secondary out-
comes were handled using multiple imputation tech-
niques. In order to reduce sampling variability due to
the imputation process, 20 datasets were generated for
every variable with missing data. Predictor variables
were included in this procedure using linear regression
for data showing normal distributions. Predictive mean
matchings were preferred to impute data for variables
with skewed distributions. All analyses were undertaken
by a statistician who was unaware of participant alloca-
tion using Stata v12.0® (StataCorp LP, 1996–2016) under
the
intention-to-treat
principle,
but
complementary
complete-case
analyses
were
conducted
as
part
of
multiple imputation techniques. Intention to treat analyses using multiple imputation
techniques showed significant differences between study
groups. Missing scores were imputed using results from
the baseline test and allocation as independent variables
in linear regression analyses. On average, interns allocated
to the smartphone application had an increase in scores
that was 5 points (9%) higher than those observed in the
no-intervention group (p = 0.03). Similar trends were seen
when complete-case analyses were undertaken. When
overall scores were analysed, an absolute difference of 3.5
points was observed between groups in favour of those al-
located to the smartphone application, but statistical sig-
nificance was not reached (p = 0.22). Study outcomes are
briefly summarised in Table 2 and Fig. 2. Students
allocated
to
the
smartphone
application
showed reductions in the total time needed to complete
the final examination and the mean time spent per ques-
tion. Intention-to-treat analyses showed a nonsignificant
reduction of 8.5 min for the first outcome and 5.7 s for
the latter (p = 0.08 for both). This estimate was calculated
using predictive mean matching due to the skewed nature
of time data, using allocation and both baseline perform-
ance and time required to complete the first examination
as predictor variables. These differences were more
conservative than the ones observed in complete-case
analyses. Outcomes The primary outcome is the mean change in overall
scores
in
a
90-question
practise
test
designed
to Page 4 of 9 Martínez et al. BMC Medical Education (2017) 17:168 Martínez et al. BMC Medical Education (2017) 17:168 of the total time required to complete the review. Data
regarding adherence was also collected. of the total time required to complete the review. Data
regarding adherence was also collected. The most common operating system was Android® (51
students, 63.8%). No relevant imbalances in study groups
were seen at baseline. A detailed description of these
contrasts and additional information regarding study
participants is provided in Table 1. Analysis plan Among participants who attended the second as-
sessment, a 10-min reduction in overall time and a 6.7 s
reduction in mean time per question were found, and
both reached statistical significance (p = 0.04). Total times
spent by participants answering both baseline and final
questionnaires are shown in Fig. 3. Intervention effects The mean score in the baseline test was of 41.1 ± 11.1
points, and mean total time needed for completion of the
latter review was 65.6 ± 27.0 min. Scores and completion
times were similar between groups at baseline. Sixty-five
interns (81.3%) sat the final test 4 weeks after randomisa-
tion. In both groups, a significant increase in overall
scores was seen, which tended to be greater among interns
allocated to receive the smartphone application. Partici-
pants allocated to no intervention showed an increase of
10.6 ± 11.7 points (p < 0.001) from baseline, while interns
who received the smartphone application improved their
scores by 16.2 ± 8.3 points (p < 0.001). Participant characteristics A total of 80 interns were eligible for this study, and all
volunteered
to
participate. Most
were
female
(48
students, 60%) with a mean age of 25.3 ± 2.2 years and
had spent a median of 6 years in medical school (IQR
6–7 years). Eighteen (22.5%) had repeated at least one
course, and the median number of repetitions was 1
(IQR 1–3). The median time using smartphones was of
4 years (IQR 3–6 years). Most interns reported routine
use of smartphone applications in daily practice (67
students, 83.7%), but only a third of them acknowledged
using them for academic purposes (31 students, 38.8%). Statistical analyses
Sample size Sample size was calculated using data regarding overall
perfomance in prior experiences with practise exams
and estimates from a randomised trial [10]. It was calcu-
lated that a sample size of 64 participants (32 per group)
would be required to obtain 80% power to detect an
absolute difference of 5 points between groups, assum-
ing a standard deviation of 7 points for both groups at
standard significance levels (two-tailed α of 5%). In order
to correct for up to 20% losses of follow-up, it was
sought to randomise 75 participants. All estimates were
calculated using nQuery Advisor® 3.0 for windows. Adherence The median number of tests answered by these the intervention (85%, 95% CI 70.2–94.2%). The median
number of questions answered during the 4-week inter-
vention period was 258 (IQR 66–415), and the median
number of completed questionnaires per participant was
15 (IQR 14–21). Participants used the application’s train-
ing mode less frequently, with only 12 students (30%,
95%CI 16.6–46.5%) registering any activity during this
trial. The median number of tests answered by these Adherence The most popular mode amongst participants was study
mode, which was used by 34 participants allocated to Martínez et al. BMC Medical Education (2017) 17:168 Page 5 of 9 Table 1 Baseline Participant Characteristics
Characteristic
Smartphone Application (n = 40)
No Intervention (n = 40)
Total
P-Value
General and Academic characteristics
Mean Age (years) (SD)
25.6 ± 2.7
24.9 ± 1.5
25.3 ± 2.2
0.181
Female sex (n, %)
27 (67.5%)
21 (52.5%)
48 (60%)
0.252
Median time in medical school (years) (IQR)
6 (6–7)
6 (6–7)
6 (6–7)
0.351
Campus Valparaiso (n, %)
28 (70%)
28 (70%)
56 (70%)
12
Course repetition (n, %)
10 (25%)
8 (20%)
18 (22.5%)
0.791
Median number repetitions (IQR)
1.5 (1–3)
1 (1–3)
1 (1–3)
0.263
Internal Medicine Internship Grade (SD)
6.3 ± 0.4
6.3 ± 0.4
6.3 ± 0.4
0.841
Internal Medicine Undergraduate Examination
Grade (SD)
5.4 ± 0.7
5.4 ± 0.7
5.4 ± 0.8
0.911
Experience with Smartphones
Median time using smartphones (years) (IQR)
4 (3–5)
4 (3–6)
4 (3–6)
0.891
Smartphone use in clinical practice (n, %)
34 (85%)
33 (82.5%)
67 (83.7%)
12
Smartphone use for academic purposes (n, %)
16 (40%)
15 (37.5%)
31 (38.8%)
12
Operating system (n, %)
Android®
25 (62.5%)
26 (65%)
51 (63.8%)
12
iOs®
15 (37.5%)
14 (35%)
29 (36.2%)
Performance in Baseline Test
Mean overall score (SD)
40.3 ± 11.0
41.8 ± 11.2
41.1 ± 11.1
0.531
Mean total time (minutes) (SD)
65.2 ± 26.3
66.0 ± 28.0
65.6 ± 27.0
0.891
Mean time per question (seconds) (SD)
44.3 ± 18.6
45.5 ± 21.7
44.9 ± 20.1
0.801
1Student’s T Test. 2Fisher’s Exact Test3Mann-Whitney U TestSD: Standard Deviation. IQR: Interquartile range Student’s T Test. 2Fisher’s Exact Test3Mann-Whitney U TestSD: Standard Deviation. IQR: Interquartile range dent’s T Test. 2Fisher’s Exact Test3Mann-Whitney U TestSD: Standard students was 2 (IQR 1–4), which translated in 90 (IQR
45–180) time-limited questions (Table 3). students was 2 (IQR 1–4), which translated in 90 (IQR
45–180) time-limited questions (Table 3). the intervention (85%, 95% CI 70.2–94.2%). The median
number of questions answered during the 4-week inter-
vention period was 258 (IQR 66–415), and the median
number of completed questionnaires per participant was
15 (IQR 14–21). Participants used the application’s train-
ing mode less frequently, with only 12 students (30%,
95%CI 16.6–46.5%) registering any activity during this
trial. Discussion Thirty-one physicians who had
already completed an advanced life support course within
the previous 4 years were randomised to receive iResus® as
a cognitive aid or no additional support during a simu-
lated cardiac emergency. Performance was measured
using a validated scoring system. Participants allocated to
iResus® showed median scores that were 12.5 (14%) points
higher than those seem among students without any fur-
ther cognitive aids (p = 0.02). Similar findings were seen
in a larger study by Hand an coworkers [16], in which 111
residents
were
randomised
to
a
smartphone-based
decision support tool aimed at improving adherence to
the American Heart Association Guidelines on Periopera-
tive Cardiac Evaluation. Use of the decision support tool
resulted in a 25% improvement in adherence to guidelines
(p < 0.001), and participants made 77% fewer incorrect re-
sponses in two standardised tests. everyday lives. This popularity makes these platforms
attractive targets to design and develop interventions for
medical training. However, only a handful of randomised
trials that address smartphone applications with educational
purposes are available in the literature. Most of them
have been conducted among postgraduate students
undergoing specialty training, and used smartphones
as cognitive aids concomitant to assessments of very
specific competences [10, 16, 17]. We found that the provision of key concepts in internal
medicine using smartphones was a feasible option that
also translated in significant improvements in academic
performance
among
medical
interns. The
observed
progress was significant even for a relatively brief inter-
vention that was also self-administered by our students,
which adds to the relevance of our findings. Our results
are in concordance with the ones observed in similar Fig. 3 Total time spent per questionnaire. These boxplots show the
changes in time required to complete the simulation tests used within
iSTART between study groups Although our findings are similar to the ones seen in
the aforementioned trials, our estimates are far more
conservative than the ones observed by Low [10] and
Hand [16]. This might be explained by the fact that our
intervention was not devised to be used concomitantly
to assessments as a cognitive aid, but rather as a
complementary resource to facilitate study of internal
medicine as a discipline. Discussion Smartphones are commonly used devices among med-
ical trainees. In this study, every eligible student had at
least one of these gadgets at their disposal, and most
reported considerable experience using them in their Table 2 Study Outcomes
Outcome
Smartphone Application
No Intervention
Mean Difference
P-Value
Intention to Treat Analyses (Multiple Imputation)
Mean overall score (points) (SD)
56.1 ± 14.5
52.2 ± 10.3
3.5
0.221
Absolute change in overall score (points) (SD)
14.5 ± 8.9
9.4 ± 11.6
5.0
0.031
Mean total time (minutes) (SD)
62.2 ± 20.4
70.8 ± 21.3
8.5
0.082
Mean time per question (seconds) (SD)
41.2 ± 14.5
46.9 ± 13.6
5.7
0.082
Complete-Case Analyses
Mean overall score (points) (SD)
56.1 ± 12.9
52.2 ± 9.4
3.9
0.172
Absolute change in overall score (points) (SD)
14.6 ± 7.4
9.6 ± 10.5
5.0
0.032
Mean total time (minutes) (SD)
61.5 ± 19.7
71.5 ± 18.7
10.0
0.042
Mean time per question (seconds) (SD)
41.0 ± 13.2
47.7 ± 12.5
6.7
0.042
SD Standard Deviation
1Estimates obtained by pooling results across 20 multiply imputed data sets
2Student’s T Test Martínez et al. BMC Medical Education (2017) 17:168 Page 6 of 9 Table 3 iSTART adherence rates
Endpoint
Smartphone
Application
95% Confidence
Interval
Study Mode
Proportion of students using
this mode (n, %)
34 (85%)
70.2–94.2%
Median number of questions
answered (IQR)
258 (66–415)
96–376
Median number of questionnaires
completed (IQR)
15 (14–21)
13–22
Training Mode
Proportion of students using
this mode (n, %)
12 (30%)
16.6–46.5%
Median number of questionnaires
answered (IQR)
2 (1–4)
1–5
IQR Interquartile range Table 3 iSTART adherence rates Fig. 2 Absolute change in scores between study groups. These
boxplots compare the observed differences in perfomance in two
simulation tests aimed to resemble EUNACOM Fig. 2 Absolute change in scores between study groups. These
boxplots compare the observed differences in perfomance in two
simulation tests aimed to resemble EUNACOM experiences. In 2011, Low and coworkers [10] published a
randomised trial assessing iResus®, an iOs®-based applica-
tion aimed at improving performance of an advanced life
support provider in an emulated medical emergency. The
application was designed to provide a quick reference to
algorithms and drug dosages to assist in the management
of resuscitation efforts. Strengths and limitations Our study is strengthened by randomisation, which
greatly helps controlling biases due to selection and con-
founding. Contents within the application were designed
by internists with experience in developing questions
that resemble those used in EUNACOM. Previous data
available at our centre had shown good correlations with
overall scores and those specific with internal medicine
within the review, which has translated in excellent diag-
nostic accuracy in detecting students at risk of failing
the examination. We also conducted active monitoring
of the application’s use, which greatly helps understand-
ing our results and represents a key element when
evaluating
interventions
that
are
self-delivered
by
students. These data are likely to be helpful for the de-
sign of future versions or similar applications. g
pp
Several limitations need to be taken into consideration
when analysing our results. The first is that a significant
proportion of students did not attend the final examin-
ation (18.7%), which resulted in the loss of key informa-
tion regarding study outcomes. We chose to mitigate this
event by using multiple imputation techniques, which
have been established as one of the best methods available
to handle missing data in randomised trials [18, 19]. Uncertainty always exists when estimates from multiple
imputation are used to allow the conduction of intention-
to-treat analyses. This stems from the fact that the “miss-
ing completely at random” assumption of missing data is
hard to confirm in practice [18]. We did not find any
contrasts between participants who completed our study
and those who did not, and estimates from complete case
analyses were very similar to the ones obtained from mul-
tiple imputation. Both facts bring reassurance regarding
the reliability of our imputed values. Another limitation
stems from the impossibility to mask participants to the
intervention, which could have resulted in the applica-
tion’s contents being shared across study groups. This
would result in a minimisation of the intervention’s effects
between groups, and thus might explain the smaller-than-
expected difference that was found in this trial. Costs are
always a relevant concern when implementing interven-
tions in medical education. In this case, an investment of
50.000USD was required to develop the application and
its key contents, which was covered entirely by the
research team. Most expenses were incurred in human
resource honoraria. Discussion In addition, it should be
considered that the scope of contents established as
key by the designers of EUNACOM is broader than
the
ones
required
by
guidelines
aimed
at
aiding
clinicians in the management of specific healthcare is-
sues; thus resulting in an apparent reduction of the
intervention’s benefits. Fig. 3 Total time spent per questionnaire. These boxplots show the
changes in time required to complete the simulation tests used within
iSTART between study groups Page 7 of 9 Martínez et al. BMC Medical Education (2017) 17:168 Intention-to-treat analyses also showed a nonsignifi-
cant trend towards a reduction in total test times and
mean time spent per inquiry. A post-hoc power calcula-
tion showed that the estimated power for this contrast
was of only 45%, thus making insufficient power a rea-
sonable possibility to explain this observed lack of statis-
tical significance. Nonetheless, the observed reduction of
8.5 min is relevant for interns planning to undertake
EUNACOM, and is likely to be the result of practice in
answering multiple-choice questions. Clinical vignettes
are constructed using certain features that are typical of
certain conditions, thus leading to patterns that students
exposed to the application might have been able to recog-
nise faster than those allocated to the no-intervention
group. It could also be argued that students allocated to
the intervention also had more experience answering
questions on an electronic platform, thus resulting in
familiarity with the interface that might have explained
these findings. However, this explanation seems rather
unlikely considering the vast experience with smartphone
applications that participants had in this study. their strategies. Exploring motivations to use these types
of applications should be considered in future qualitative
research. Strengths and limitations Although this might be seen as a sig-
nificant barrier to implementation, it should be considered
that after this initial investment, the application was inex-
pensive to maintain, only requiring monthly payments for
a
server
and
a
part-time
engineer
to
oversee
its Given that the intervention was devised to be self-
administered by students, adherence was a key aspect to
assess while conducting our study. Thirty-four out of 40
participants (85%) used the application’s study mode to
review internal medicine in this trial, which was very sat-
isfactory. Furthermore, the median number of questions
and questionnaires completed was more than adequate
considering the relatively brief timeframe in which this
study was conducted. Only a minority of students allo-
cated to the intervention (12 students, 30%) used the ap-
plications’ training mode, the sole feature within the
application in which a time restraint to respond clinical
vignettes was applied. This obvious contrast in use rates
reached statistical significance (p < 0.001), and might be
explained by performance pressure. It is possible that in-
terns
felt
discouraged
to
undertake
activities
that
recorded results in a manner similar than the one used
in the actual EUNACOM. Participants could have asso-
ciated underperforming in these exercises with a poten-
tial for poor results in the exam, thus leading to the
observed use rates. Feedback provided by this mode did
not include a revision of the key concept in internal
medicine that was being assessed, thus possibly making
pressure
for
delivering
high
scores
more
tangible. Furthermore, interns were warned that time-limited ex-
ercises were accessible only once during our trial, which
might have led to lesser use rates in order to save this
component
of
the
application
after
the
reviewable
contents (study mode) were completed. Given these ex-
planations and the fact that EUNACOM applies a time
limit of 60 s per question, future interventions aimed at
improving performance in this and/or similar tests
should not disregard applying time restraints as part of Page 8 of 9 Page 8 of 9 Page 8 of 9 Martínez et al. BMC Medical Education (2017) 17:168 Martínez et al. BMC Medical Education (2017) 17:168 Martínez et al. BMC Medical Education (2017) 17:168 Page 8 of 9 functionality and data collection processes. Strengths and limitations Develop-
ment costs can be mitigated by working with volunteers
or in collaboration with other institutions or academic
departments which might use this application as a plat-
form for additional contents. The modular design of our
application allows unrestricted upload of questionnaires
that are not limited to undergraduate training, thus open-
ing a potential for postgraduate and continuous medical
education. In addition, institutions interested in imple-
menting
these
kind
of
applications
might
consider
allowing access to individuals by paying download/sub-
scription fees in order to ensure sustainability over time. Conclusion In summary, this randomised trial showed that the
provision of a smartphone application designed to emu-
late EUNACOM was successfully implemented amongst
medical interns. Its use was associated with significant
improvements in academic performance, and is likely to
be chiefly explained by the direct provision of concepts
in Internal Medicine in form of clinical vignettes. The
observed high adherence rates amongst interns make
these kinds of interventions a promising field to develop
in the future of medical education. 2. Mosa ASM, Yoo I, Sheets L. A systematic review of healthcare applications
for smartphones. BMC Med Inform Decis Mak. 2012;12(1):67. 3. Baumgart DC. Smartphones in clinical practice, medical education, and
research. Arch Intern Med. 2011;171(14):1294–6. 4. Free C, Phillips G, Watson L, Galli L, Felix L, Edwards P, et al. The
effectiveness of mobile-health technologies to improve health care service
delivery processes: a systematic review and meta-analysis. PLoS Med. 2013;10(1):e1001363. 5. Wheeler S, Tavares C, Jones R. How smartphones are changing the face of
mobile and participatory healthcare: an overview, with example from
eCAALYX. Biomed Eng. 2011;10:24. 1-14 6. Shah J, Haq U, Bashir A, Shah SA. Awareness of academic use of
smartphones and medical apps among medical students in a private
medical college? J Pak Med Assoc. 2016;66(2):184–6. Author details
1 1Departamento de Salud Pública, Escuela de Medicina, Universidad de
Valparaíso, Hontaneda, 2664 Valparaíso, Chile. 2Área de Investigación y
Estudios Clínicos, Clínica Ciudad del Mar, Viña del Mar, Chile. 3Departamento
de Medicina Interna, Hospital Gustavo Fricke, Álvares, 1532 Viña del Mar,
Chile. 4Laboratorio de Información y Tecnología, Escuela de Ingeniería
Informática, Universidad de Valparaíso, General Cruz, 222 Valparaíso, Chile. Received: 23 August 2016 Accepted: 13 September 2017 Received: 23 August 2016 Accepted: 13 September 2017 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. References
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in EUNACOM - ST.] Available at: http://www.eunacom.cl/contenidos/
distribucion.html The datasets used and/or analysed during the current study available from
the corresponding author on reasonable request. 13. Boutron I, Moher D, Altman DG, Schulz KF, Ravaud P, CONSORT Group. Methods and processes of the CONSORT Group: example of an extension
for trials assessing nonpharmacologic treatments. Ann Intern Med. 2008;
148(4):W60-6. Authors’ contributions FM designed the study and application contents, conducted statistical analyses
and drafted the manuscript. CTB designed the application’s contents and reviewed
the final version of the manuscript to be published. CTT devised the smartphone
application, acquired the data and reviewed the manuscript prior to publication. All authors approved the final version of the manuscript to be published. 14. Moher D, Hopewell S, Schulz KF, Montori V, Gøtzsche PC, Devereaux PJ, et
al. CONSORT 2010 explanation and elaboration: updated guidelines for
reporting parallel group randomised trials. BMJ. 2010;340:c869. 15. Asociación de Facultades de Medicina de Chile. [Contents of the Examen
Único Nacional de Conocimientos de Medicina (EUNACOM)] Available At
en: http://www.eunacom.cl/contenidos/contenidos.html Additional file 1: Examples to Clinical Vignettes. (DOCX 112 kb) 8. Boruff JT, Storie D. Mobile devices in medicine: a survey of how medical
students, residents, and faculty use smartphones and other mobile devices
to find information. J Med Libr Assoc. 2014;102(1):22–30. Consent for publication
Not applicable. 17. McEvoy MD, Hand WR, Stiegler MP, DiLorenzo AN, Ehrenfeld JM, Moran KR,
et al. A Smartphone-based decision support tool improves test performance
concerning application of the guidelines for managing regional anesthesia Ethics approval and consent to participate The Review Board of the Universidad de Valparaiso reviewed and approved
the conduction of this study. Its registration number is CEC098–16. Informed
consent was obtained from every included participant. 16. Hand WR, Bridges KH, Stiegler MP, Schell RM, DiLorenzo AN, Ehrenfeld
JM, et al. Effect of a cognitive aid on adherence to perioperative
assessment and management guidelines for the cardiac evaluation of
noncardiac surgical patients. Anesthesiology. 2014;120(6):1339–49. quiz1349–53 Abbreviations
ASOFAMECH A ASOFAMECH: Asociación de Facultades de Medicina de Chile; CI: Confidence
Interval; EUNACOM: Examen Unico Nacional de Conocimientos en Medicina;
IQR: Interquartile Range; iSTART: Implementation of a Smartphone Application
in Medical Education: A Randomised Trial 9. Short SS, Lin AC, Merianos DJ, Burke RV, Upperman JS. Smartphones,
trainees, and mobile education: implications for graduate medical
education. J Grad Med Educ. 2014;6(2):199–202. 9. Short SS, Lin AC, Merianos DJ, Burke RV, Upperman JS. Smartphones,
trainees, and mobile education: implications for graduate medical
education. J Grad Med Educ. 2014;6(2):199–202. 10. Low D, Clark N, Soar J, Padkin A, Stoneham A, Perkins GD, et al. A
randomised control trial to determine if use of the iResus© application
on a smart phone improves the performance of an advanced life
support provider in a simulated medical emergency. Anaesthesia. 2011;66(4):255–62. Additional file 7. Sandholzer M, Deutsch T, Frese T, Winter A. Predictors of students’ self-
reported adoption of a smartphone application for medical education in
general practice. BMC Med Educ. 2015;15(1):91. Additional file 1: Examples to Clinical Vignettes. (DOCX 112 kb) Martínez et al. BMC Medical Education (2017) 17:168 in the patient receiving antithrombotic or thrombolytic therapy.
Anesthesiology. 2016 Jan;124(1):186–98.
18.
Sterne JAC, White IR, Carlin JB, Spratt M, Royston P, Kenward MG, et al.
Multiple imputation for missing data in epidemiological and clinical
research: potential and pitfalls. BMJ. 2009;338:b2393.
19.
Ware JH, Harrington D, Hunter DJ, D'Agostino RB. Missing data. N Engl
J Med. 2012;367:1353–4. Competing interests The authors declare that they have no competing interests. Page 9 of 9 Martínez et al. BMC Medical Education (2017) 17:168 in the patient receiving antithrombotic or thrombolytic therapy. Anesthesiology. 2016 Jan;124(1):186–98. 18. Sterne JAC, White IR, Carlin JB, Spratt M, Royston P, Kenward MG, et al. Multiple imputation for missing data in epidemiological and clinical
research: potential and pitfalls. BMJ. 2009;338:b2393. 19. Ware JH, Harrington D, Hunter DJ, D'Agostino RB. Missing data. N Engl
J Med. 2012;367:1353–4. • We accept pre-submission inquiries
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HTLV-1 p30II: selective repressor of gene expression.
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Retrovirology
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Abstract Human T-lymphotropic virus type-1 (HTLV-1) is a complex retrovirus that causes adult T-cell
leukemia/lymphoma (ATL) and is implicated in a variety of lymphocyte-mediated disorders. HTLV-
1 pX ORF II encodes two proteins, p13II and p30II whose roles are beginning to be defined in the
virus life cycle. Previous studies indicate the importance of these viral proteins in the ability of the
virus to maintain viral loads and persist in an animal model of HTLV-1 infection. Intriguing new
studies indicate that p30II is a multifunctional regulator that differentially modulates CREB and Tax-
responsive element-mediated transcription through its interaction with CREB-binding protein
(CBP)/p300 and specifically binds and represses tax/rex mRNA nuclear export. A new study
characterized the role of p30II in regulation of cellular gene expression using comprehensive human
gene arrays. Interestingly, p30II is an overall repressor of cellular gene expression, while selectively
favoring the expression of regulatory gene pathways important to T lymphocytes. These new
findings suggest that HTLV-1, which is associated with lymphoproliferative diseases, uses p30II to
selectively repress cellular and viral gene expression to favor the survival of cellular targets
ultimately resulting in leukemogenesis. [3,4]. Tax orchestrates multiple interactions with cellular
transcription factors and activates transcription from the
viral promoter and modulates the transcription or activity
of numerous cellular genes involved in cell growth and
differentiation, cell cycle control, and DNA repair [5,6]. Recent studies have indicated novel roles for pX ORF I and
II gene products in the replication of HTLV-1 [7-9]. Although the study of these gene products were largely by-
passed by virologists until the mid 1990's, they intensified
when infectious molecular clones provided the tools to
better understand their role in vivo. Both HTLV-1 pX ORF
I and II mRNAs have been detected in infected cell lines
and blood leukocytes from HTLV-1-infected subjects
including ATL and HAM/TSP patients [10,11]. Also, The complex sequence of events set in motion by human
T-lymphotropic virus type 1 (HTLV-1) to cause prolifera-
tion and ultimately transformation of T lymphocytes is
beginning to be unraveled. Only recently has it become
clear that viral encoded proteins, the so-called "accessory"
gene products of this complex retrovirus, play an integral
role in the pathogenic process. BioMed Central BioMed Central Published: 24 November 2004 This article is available from: http://www.retrovirology.com/content/1/1/40 © 2004 Green; licensee BioMed Central Ltd. © 2004 Green; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Commentary
HTLV-1 p30II: selective repressor of gene expression
Patrick L Green*1,2,3,4 Open Access Page 1 of 3
(page number not for citation purposes) y
HTLV-1 p30II: selective repressor of gene expression
Patrick L Green*1 2 3 4 Address: 1Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA, 2Molecular Virology, Immunology, and
Medical Genetics, The Ohio State University, Columbus, OH 43210, USA, 3Center for Retrovirus Research, The Ohio State University, Columbus,
OH 43210, USA and 4Comprehensive Cancer Center, Arthur G. James Cancer Hospital and Solove Research Institute, The Ohio State University,
Columbus, OH 43210, USA Email: Patrick L Green* - green.466@osu.edu * Corresponding author Received: 09 November 2004
Accepted: 24 November 2004 Received: 09 November 2004
Accepted: 24 November 2004 http://www.retrovirology.com/content/1/1/40 Model for HTLV-1 p30II transcriptional and posttranscrip-
tional gene regulation
Figure 1
Model for HTLV-1 p30II transcriptional and posttranscrip-
tional gene regulation. The cell nucleus surrounded by the
nuclear membrane and key components are shown. p30II can
directly interact with CBP/p300 and modulate transcription
of viral and/or cellular genes. At low concentration p30II may
stabilize the transcription complex and potentiate transcrip-
tion, whereas a high concentration it may compete for lim-
ited amounts of CBP/p300 and repress gene expression. p30II
(as well as the homologous p28II of HTLV-2) specifically binds
tax/rex mRNA and block its export, reducing Tax and Rex
and ultimately repressing viral gene expression. This interac-
tion may be directly linked to splicing factors and splicing
and/or the juxtaposition of specific exon/exon junction
sequences. Thus, p30II is a multifunctional protein with tran-
scriptional and post-transcriptional roles in regulating viral
and/or cellular gene expression. Microarrays are important tools to gain insight into
changes in gene expression profiles of virus-infected cells. This approach has been primarily used to investigate gene
expression in HTLV-1-immortalized/transformed cell
lines or in cells from ATL patients [25-29]. In the report by
Michael et al. [30] the authors used the Affymetrix U133A
human gene chip to test the role of HTLV-1 p30II as a reg-
ulator of gene expression in Jurkat T cells. They identified
alterations in gene expression profiles unique to cell cycle
regulation, apoptosis, and T lymphocyte signaling/activa-
tion. Although p30II expression, as might be expected
from earlier reports, resulted in a general repressive pat-
tern of gene expression, their data indicated that the viral
protein selectively spared or enhanced NFAT, NFκB, and
AP-1 mediated transcription in T cells undergoing co-
stimulation. Signaling pathways primarily affected by
p30II as measured by luciferase reporters included both
NFAT and NFκB, which increased from approximately 3
to 11 fold, depending on co-stimulatory treatment. Over-
all, this study supports earlier reports on the repressive
role of HTLV-1 p30II in gene expression [21-24] and
reveals new potential mechanisms by which p30II may
play a role in HTLV-1 replication (figure 1). The effects of
p30II appear to overlap or counteract the influence of
other HTLV-1 regulatory proteins like Tax or other acces-
sory proteins such as p12I. Further studies to test if these
proteins act coordinately or synergistically will undoubt-
edly shed light on this issue. Abstract In addition to the struc-
tural and enzymatic gene products, HTLV-1 encodes regu-
latory and accessory proteins from four open reading
frames (ORF) in the pX region between env and the 3' long
terminal repeat (LTR) of the provirus [1,2]. The well stud-
ied Rex and Tax positive regulators are encoded in the
ORF III and IV, respectively. Rex plays a critical role in
nuclear export of unspliced or singly spliced viral mRNA Page 1 of 3
(page number not for citation purposes) Page 1 of 3
(page number not for citation purposes) http://www.retrovirology.com/content/1/1/40 Retrovirology 2004, 1:40 Model for
tional gene
Figure 1 This interac-
tion may be directly linked to splicing factors and splicing
and/or the juxtaposition of specific exon/exon junction
sequences. Thus, p30II is a multifunctional protein with tran-
scriptional and post-transcriptional roles in regulating viral
and/or cellular gene expression. immune responses of HTLV-1 infected patients and
asymptomatic carriers indicate that these proteins are
expressed in vivo [12-14]. Abbreviations
HTLV-1, human T cell lymphotropic virus type-1 http://www.retrovirology.com/content/1/1/40 It is possible that HTLV-1
employs selective use of these viral proteins during vari-
ous stages of the infection to promote cell proliferation, a
hallmark of the diseases associated with the deltaretrovi-
rus family. Whatever the outcome of these studies, it is
clear that "accessory" proteins, like p30II, may have
"essential" roles in the life cycle of HTLV-1. Model for
tional gene
Figure 1 Model for
tional gene
Figure 1 Model for HTLV 1 p30 transcriptional and posttranscrip
tional gene regulation
Figure 1
Model for HTLV-1 p30II transcriptional and posttranscrip-
tional gene regulation. The cell nucleus surrounded by the
nuclear membrane and key components are shown. p30II can
directly interact with CBP/p300 and modulate transcription
of viral and/or cellular genes. At low concentration p30II may
stabilize the transcription complex and potentiate transcrip-
tion, whereas a high concentration it may compete for lim-
ited amounts of CBP/p300 and repress gene expression. p30II
(as well as the homologous p28II of HTLV-2) specifically binds
tax/rex mRNA and block its export, reducing Tax and Rex
and ultimately repressing viral gene expression. This interac-
tion may be directly linked to splicing factors and splicing
and/or the juxtaposition of specific exon/exon junction
sequences. Thus, p30II is a multifunctional protein with tran-
scriptional and post-transcriptional roles in regulating viral
and/or cellular gene expression. Model for HTLV 1 p30 transcriptional and posttranscrip
tional gene regulation
Figure 1
Model for HTLV-1 p30II transcriptional and posttranscrip-
tional gene regulation. The cell nucleus surrounded by the
nuclear membrane and key components are shown. p30II can
directly interact with CBP/p300 and modulate transcription
of viral and/or cellular genes. At low concentration p30II may
stabilize the transcription complex and potentiate transcrip-
tion, whereas a high concentration it may compete for lim-
ited amounts of CBP/p300 and repress gene expression. p30II
(as well as the homologous p28II of HTLV-2) specifically binds
tax/rex mRNA and block its export, reducing Tax and Rex
and ultimately repressing viral gene expression. This interac-
tion may be directly linked to splicing factors and splicing
and/or the juxtaposition of specific exon/exon junction
sequences. Thus, p30II is a multifunctional protein with tran-
scriptional and post-transcriptional roles in regulating viral
and/or cellular gene expression. Model for HTLV 1 p30 transcriptional and posttranscrip
tional gene regulation
Figure 1
Model for HTLV-1 p30II transcriptional and posttranscrip-
tional gene regulation. The cell nucleus surrounded by the
nuclear membrane and key components are shown. p30II can
directly interact with CBP/p300 and modulate transcription
of viral and/or cellular genes. At low concentration p30II may
stabilize the transcription complex and potentiate transcrip-
tion, whereas a high concentration it may compete for lim-
ited amounts of CBP/p300 and repress gene expression. p30II
(as well as the homologous p28II of HTLV-2) specifically binds
tax/rex mRNA and block its export, reducing Tax and Rex
and ultimately repressing viral gene expression. References Harhaj EW, Good L, Xiao G, Sun SC: Gene expression profiles in
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interests. 21. 21. Zhang W, Nisbet JW, Bartoe JT, Ding W, Lairmore MD: Human T-
lymphotropic virus type 1 p30II functions as a transcription
factor
and
differentially
modulates
CREB-responsive
promoters. J Virol 2000, 74:11270-11277. http://www.retrovirology.com/content/1/1/40 Retrovirology 2004, 1:40 Abbreviations
HTLV-1, human T cell lymphotropic virus type-1 Molecular clones of HTLV-1 with selective mutations of
ORF I and II have revealed the requirement of p12I and
p13II/p30II in the establishment of infection and mainte-
nance of viral loads in a rabbit model of infection [15-17]. The nuclear and nucleolar localizing p30II has minimal
homology to transcription factors Oct-1 and -2, Pit-1, and
POU-M1 [18-21]. In addition, the protein co-localizes
with p300 in the nucleus and physically interacts with
CREB binding protein (CBP)/p300 and differentially
modulates cAMP responsive element (CRE) and Tax
response element-mediated transcription [21,22]. Intrigu-
ing recent reports also indicate a post-transcriptional role
of HTLV-1 p30II and HTLV-2 p28II(homologous protein
encoded in the HTLV-2 pX ORF II region), in repressing
the export of tax/rex mRNA from the nucleus [23,24]. Thus, it appears that HTLV-1 has yet another multifunc-
tional protein with transcriptional and post-transcrip-
tional roles in regulating viral gene expression. ATL, adult T cell leukemia ATL, adult T cell leukemia HAM/TSP, HTLV associated myelopathy/tropical spastic
paraparesis HAM/TSP, HTLV associated myelopathy/tropical spastic
paraparesis ORF II, open reading frame II
LTR, long terminal repeat
CRE, cAMP responsive element
CREB, cAMP response element binding protein
NFAT, nuclear factor of activated T cells
NFκB, nuclear factor kappa B ORF II, open reading frame II
LTR, long terminal repeat
CRE, cAMP responsive element
CREB, cAMP response element binding protein
NFAT, nuclear factor of activated T cells
NFκB, nuclear factor kappa B ORF II, open reading frame II
LTR, long terminal repeat
CRE, cAMP responsive element
CREB, cAMP response element binding protein
NFAT, nuclear factor of activated T cells
NFκB, nuclear factor kappa B ORF II, open reading frame II Page 2 of 3
(page number not for citation purposes) http://www.retrovirology.com/content/1/1/40 Publish with BioMed Central and every
scientist can read your work free of charge J
16. Bartoe JT, Albrecht B, Collins ND, Robek MD, Ratner L, Green PL,
Lairmore MD: Functional role of pX open reading frame II of
human T-lymphotropic virus type 1 in maintenance of viral
loads in vivo. J Virol 2000, 74:1094-1100. J
17. Collins ND, Newbound GC, Albrecht B, Beard J, Ratner L, Lairmore
MD: Selective ablation of human T-cell lymphotropic virus
type 1 p12I reduces viral infectivity in vivo. Blood 1998,
91:4701-4707. 18. Ciminale V, Pavlakis GN, Derse D, Cunningham CP, Felber BK: Com-
plex splicing in the human T-cell leukemia virus (HTLV) fam-
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HTLV type I. J Virol 1992, 66:1737-1745. yp
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19. Koralnik IJ, Fullen J, Franchini G: The p12I, p13II, and p30II pro-
teins encoded by human T-cell leukemia/lymphotropic virus References Pique C, Ureta-Vidal A, Gessain A, Chancerel B, Gout O, Tamouza R,
Agis F, Dokhélar M-C: Evidence for the Chronic In Vivo Produc-
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15. Silverman LR, Phipps AJ, Montgomery A, Ratner L, Lairmore MD:
Human T-cell lymphotropic virus type 1 open reading frame
II-encoded p30II is required for in vivo replication: evidence
of in vivo reversion. J Virol 2004, 78:3837-3845. Publish with BioMed Central and every
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Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD Berbasis Islami
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validitas serta kemenarikan buku ajar yang dikembangkan. Metode penelitian yang digunakan
adalah penelitian dan pengembangan dengan menggunakan desain Borg & Gall. Hasil
penelitian ini menunjukkan bahwa pengembangan buku ajar bahasa inggris memperoleh
kualifikasi yang baik, dibuktikan dengan hasil validasi oleh ahli materi dengan persentase 80%,
ahli desain dengan persentase 92%, guru kelas dengan persentase 94%. Buku ajar memiliki
tingkat kemenarikan yang tinggi dibuktikan dengan hasil wawancara dan angket kepada peserta
didik dengan persentase 90,04%, Sehingga buku ajar telah memenuhi aspek-aspek dalam
pengembangan & kebutuhan dalam proses pelaksanaan pembelajaran. Kata kunci: Pengembangan Buku Ajar, Bahasa Inggris MI/SD, Islami A. Pendahuluan Bahasa inggris di Sekolah Dasar atau Madrasah Ibtidaiyah merupakan
Bahasa asing dikalangan peserta didik karena Bahasa tersebut tidak digunakan
dalam komunikasi sehari-hari atau pengantar dalam pembelajaran. Penguasaan
Bahasa inggris harus dimulai sejak dini terutama pada jenjang sekolah dasar, Abstract The development of textbooks is one of the main factors in the success of learning, this Islamic-
based English textbook has a characteristic that is contextual and Islamic-based material which
is equipped with 4 aspects of language skills. The objectives of this study were to developing
Islamic-based English textbooks and researching the validity and attractiveness of the developed
textbooks. The research method used is research and development using the Borg & Gall design. The results of this study indicate that the development of English textbooks obtains good
qualifications, as evidenced by the results of validation by material experts with a percentage of
80%, design experts with a percentage of 92%, class teachers with a percentage of 94%. Textbooks
have a high level of interest as evidenced by the results of interviews and questionnaires to students
with a percentage of 90.04%, so that the textbooks have met the aspects of development & needs
in the learning process. Keyword: Textbook development, English for Elementary Students, Islamic e-ISSN: 2775-5789
DOI: 10.35878/guru/v1.i2.300
Vol. 1 No. 2 Agustus 2021 e-ISSN: 2775-5789
DOI: 10.35878/guru/v1.i2.300
Vol. 1 No. 2 Agustus 2021 Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD
Berbasis Islami Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD
Berbasis Islami Fira Nadliratul Afrida
Institut Pesantren Mathali’ul Falah Pati
Email korespondensi: firanadliratulafrida@ipmafa.ac.id https://journal.ipmafa.ac.id/index.php/dawuhguru 93 Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD Berbasis Islami karena Bahasa inggris merupakan Bahasa internasional yang digunakan untuk
komunikasi di seluruh dunia. Beberapa keterampilan berbahasa yang harus
dikuasai dalam pembelajaran di sekolah dasar diantaranya: membaca, menulis,
berbicara dan menyimak. Keterampilan tersebut harus ditunjang dengan minat
peserta didik, buku ajar, dan pembelajaran yang berkualitas. Bahan ajar merupakan segala bahan yang disusun secara sistematis
mencakup kompetensi yang akan dikuasai siswa dan digunakan dalam proses
pembelajaran dengan tujuan perencanaan dan penelaahan implementasi
pembelajaran (Prastowo, 2014). Bahan ajar sebagai salah satu media pembelajaran
mempunyai peranan penting dalam proses pembelajaran yaitu sebagai acuan bagi
siswa dan guru untuk meningkatkan efektifitas pembelajaran. Bagi siswa, bahan
ajar menjadi bahan acuan yang diserap isinya dalam proses belajar sehingga dapat
menjadi pengetahuan. Sedangkan bagi guru, bahan ajar menjadi salah satu
penyampaian informasi kepada peserta didik (Belawati, 2003). Pengembangan bahan ajar dalam hal ini adalah buku ajar merupakan usaha
dalam meningkatkan efektivitas dalam pembelajaran Salah satu upaya untuk
peningkatan kualitas pendidikan melalui bahan ajar diharapkan membawa
dampak positif terhadap pendidikan di Indonesia (Prastowo, 2014). Bahan ajar
yang baik akan dapat meningkatkan motivasi dan hasil belajar peserta didik. Selain itu pengembangan bahan ajar juga harus memperhatikan karakteristik dan
kebutuhan peserta didik karena pada dasarnya peserta didik mempunyai
kreatifitas dan kemampuan yang berbeda. Keberadaan bahan ajar yang tepat juga
penting untuk menunjang pelaksanaan kegiatan belajar mengajar (Susilawati et.al. 2020). Pengembangan buku ajar bahasa inggris yang diintegrasikan dengan materi
keislaman bertujuan untuk membentuk jiwa-jiwa spiritualitas serta menanamkan
kecintaan pada Allah, Al-Qur’an dan Nabi Muhammad sejak dini, karena
dilengkapi dengan kisah nabi muhammad, do’a-doa sehari-hari, hadits nabi,
contoh kegiatan-kegiatan berbasis islami seperti membaca Al-Qur’an, sholat
berjama’ah dan lagu-lagu berbahasa inggris berbasis islami. Selain memahami
materi keislaman peserta didik juga dapat memahami materi bahasa inggris secara
kontekstual serta dilengkapi dengan 4 aspek keterampilan berbahasa. Vol. 1 No. 2 Agustus 2021 94 Fira Nadliratul Afrida Hasil penelitian yang dilakukan oleh (Risnawati 2017) dengan judul
Pengembangan Bahan Ajar Bahasa Inggris Berbasis Lingkungan untuk
meningkatkan kosakata dan hasil belajar peserta didik di Sekolah Dasar
menunjukkan bahwa bahan ajar tersebut mampu meningkatkan pemahaman dan
interaksi peserta didik dengan lingkungan sekitar, dengan demikian penggunaan
kosakata peserta didik dalam bahasa inggris terbantu karena siswa belajar dengan
kondisi nyata pada proses pembelajaran. https://journal.ipmafa.ac.id/index.php/dawuhguru Penelitian selanjutnya oleh (Oktariani &
Juwita R.P, 2019) tentang pengembangan bahan ajar bahasa inggris sekolah dasar
menunjukkan bahwa bahan ajar bahasa inggris berbentuk video animasi
permainan tradisional dapat meningkatkan kosakata dan keterampilan berbicara
peserta didik. Berdasarkan hasil observasi peneliti, buku ajar Bahasa inggris yang
digunakan di madrasah Ibtidaiyah atau sekolah dasar hanya berfokus pada materi
pembelajaran saja, maka perlu untuk diterapkan pembelajaran bahasa inggris
dalam konteks nyata atau secara kontekstual dikaitkan dengan hal-hal yang ada di
sekitar peserta didik sehingga bahasa inggris bukan lagi sekedar bahasa asing yang
diterapkan dalam teori tetapi sedikit demi sedikit dapat menjadi bagian alat
komunikasi sehari-hari (Maduwu B., 2016). Buku ajar yang mempunyai ciri khas
tertentu akan diingat dan memberikan motivasi belajar yang lebih kepada peserta
didik, dalam hal ini peneliti ingin mengembangkan buku ajar bahasa inggris yang
diintegrasikan dengan materi keislaman. Penanaman aspek spiritual sejak dini
merupakan cara untuk melatih dan mengembangkan kepribadian serta karakter
islami pada peserta didik. Materi yang dikembangkan dalam buku ajar ini berfokus pada “Daily
Activities” sehingga cocok sekali dikaitkan dengan kegiatan-kegiatan yang berbasis
keislaman yang biasa dilakukan peserta didik di rumah dan di sekolah sehingga
sesuai dengan pembelajaran berbasis kontekstual. Kegunaan pengembangan buku
ajar bagi peserta didik adalah meningkatkan pemahaman materi bahasa inggris
serta menambah kecintaan terhadap islam melalui pembelajaran yang menarik
dan menyenangkan. Bagi guru buku ajar ini dapat dijadikan buku pedoman
tambahan dan sumber belajar yang variatif untuk dapat menciptakan
pembelajaran yang sesuai dengan abad 21. Dawuh Guru: Jurnal Pendidikan MI/SD Dawuh Guru: Jurnal Pendidikan MI/SD 95 Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD Berbasis Islami B. Metode Penelitian Penelitian ini menggunakan metode penelitian dan pengembangan (Research
& Development) dengan desain Borg & Gall. Desain tersebut (Sugiyono, 2018)
memiliki beberapa tahapan yaitu: 1) Pengumpulan data & Informasi
Pada tahap ini peneliti melakukan wawancara kepada guru bahasa inggris dan
melakukan observasi terhadap pembelajaran bahasa inggris di MI/SD. 2) Perencanaan
Pada tahap ini peneliti melakukan perencanaan terhadap buku ajar yang
dikembangkan yaitu menentukan materi pembelajaran, target, integrasi dengan
keislaman dan evaluasi buku ajar. 3) Pengembangan Produk
Tahap pengembangan buku ajar dimulai dari melakukan penataan isi dan
struktur buku ajar, kemudian penyusunan kegiatan pembelajaran yang menarik
dan sesuai dengan karakteristik peserta didik kelas 3, selanjutnya melakukan
penyusunan buku ajar dengan penambahan materi yang lebih luas seperti
praktik dan integrasi materi keislaman berupa doa-doa, hadits, kisah-kisah
nabi, lagu dan kegiatan-kegiatan yang berbasis islami, terakhir melakukan
penyusunan evaluasi. 4) Validasi produk
Validasi yang digunakan pada penelitian pengembangan ini adalah validasi ahli
mata pelajaran bahasa inggris, ahli desain media pembelajaran, guru bahasa
inggris, dan peserta didik sebagai pengguna produk. Tujuan dari validasi ini
untuk memperoleh data berupa saran, arahan, dan penilaian terhadap produk
yang dikembangkan, selanjutnya peneliti bisa melakukan revisi terhadap
produk yang dikembangkan. Subjek validasi pada penelitian ini adalah dosen
yang memiliki latar belakang pengetahuan pada bidangnya masing-masing
serta guru bahasa inggris yang telah berpengalaman mengajar. 5) Revisi produk
Produk yang telah divalidasi oleh ahli kemudian peneliti melakukan revisi
untuk perbaikan serta saran-saran dari validator digunakan untuk 5) Revisi produk
Produk yang telah divalidasi oleh ahli kemudian peneliti melakukan revisi
untuk perbaikan serta saran-saran dari validator digunakan untuk Vol. 1 No. 2 Agustus 2021 96 Fira Nadliratul Afrida meningkatkan kualitas produk yang dikembangkan yaitu buku ajar bahasa
inggris. 6) Uji coba produk
Uji coba dirancang untuk mengetahui kelayakan produk yang telah
dikembangkan. Tahapan dalam uji coba ini yaitu melakukan uji coba pada
skala kecil setelah itu peneliti melakukan uji coba skala besar pada peserta didik
kelas 3. Teknik analisis data yang digunakan untuk menganalisis data kuantitatif dari
hasil validasi dengan teknik perhitungan nilai rata-rata. Rumus perhitungan nilai
rata-rata sebagai berikut: x 100%
Keterangan:
P
: Kelayakan
∑ x
: Jumlah jawaban penilaian
∑ xi
: Jumlah jawaban tertinggi Tabel 1.1
Kualifikasi Tingkatan Kelayakan berdasarkan Persentase
Persentase (%)
Kriteria kelayakan
Keterangan
80-100
Valid
Tidak revisi
60-79
Cukup valid
Tidak revisi
40-59
Kurang valid
Sebagian revisi
0-39
Tidak valid
Revisi total Tabel 1.1 Dawuh Guru: Jurnal Pendidikan MI/SD C. Hasil dan Pembahasan Hasil pengembangan buku ajar bahasa inggris berbasis islami ini didasarkan
pada kenyataan bahwa belum tersedia buku ajar bahasa inggris yang berbasis islam
untuk madrasah ibtidaiyah dan sekolah dasar. Produk pengembangan buku ajar ini
telah divalidasi oleh ahli materi, desain, dan pembelajaran sehingga bisa
dimanfaatkan dan digunakan secara luas oleh peserta didik. Buku ajar bahasa inggris ini memuat materi-materi keislaman diantaranya
doa-doa, hadits, kisah-kisah nabi, lagu dan kegiatan-kegiatan yang berbasis islami. Tidak hanya itu buku ajar ini dilengkapi dengan gambar dan ilustrasi visual yang
islami sesuai dengan adat istiadat muslim di Indonesia. Harapan dari peneliti
melalui pengembangan buku ajar ini adalah peserta didik dapat menanamkan nilai-
nilai ajaran Islam sejak dini kepada peserta didik dan mampu meneladani dari Dawuh Guru: Jurnal Pendidikan MI/SD Dawuh Guru: Jurnal Pendidikan MI/SD 97 Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD Berbasis Islami kisah nabi yang dipelajari serta dapat menerapkan kegiatan-kegiatan berbasis
spiritual sehari-sehari. Buku ajar ini disesuaikan dengan karakteristik peserta didik MI/SD yaitu
adanya minat kehidupan sehari-hari yang konkrit, hal ini diselaraskan dengan buku
ajar yang kembangkan berupa kegiatan yang biasa dilakukan peserta didik sehari-
hari. Peserta didik usia sekolah dasar/madrasah ibtidaiyah berada pada tahap
operasional konkret, yaitu anak mengembangkan pemikiran logis, terikat pada
fakta-fakta perseptual. Artinya anak mampu berpikir logis tetapi masih terbatas
pada objek-objek konkrit dan mampu melakukan konservasi (Septianti et al., 2020). Faktor penting dalam pembelajaran bahasa inggris di madrasah ibtidaiyah &
sekolah dasar adalah membangun minat dan motivasi belajar karena bahasa inggris
bukan merupakan bahasa ibu yang biasa digunakan untuk komunikasi sehari-hari,
sehingga perlu adanya kegiatan yang sangat menarik dan efektif dalam proses
pembelajaran. Buku ajar yang baik harus mengandung aspek motivation to learn,
yaitu ketika peserta didik membaca sebuah buku ajar, anak akan termotivasi belajar
tanpa harus dipaksakan oleh guru karena buku adalah media belajar (Jannah R.,
2016). Buku ajar yang dikembangkan oleh peneliti berisi aktivitas dan kegiatan yang
membantu penguasaan kosakata (Vocabulary), Berbicara (Speaking), Menulis
(Writing) & Mendengarkan (Listening) sehingga tercipta pembelajaran yang efektif,
efisien, menyenangkan. Tidak hanya itu buku ajar bahasa inggris ini mampu
meningkatkan hasil belajar peserta didik karena dikemas dengan bahasa yang
sederhana sesuai dengan perkembangan bahasa usia SD/MI. Dawuh Guru: Jurnal Pendidikan MI/SD Hasil Validasi Ahli Analisis hasil validasi ahli desain media terhadap buku bahasa inggris berbasis
islami menunjukkan persentase 92% dengan kriteria: 1) kesesuaian gambar, warna tulisan pada buku ajar yang terintegrasi dengan
keislaman; 2) pengemasan desain cover sangat menarik; 2) pengemasan desain cover sangat menarik; 3) layout, huruf, dan tulisan sangat sesuai dengan karakteristik peserta didik
MI/SD. 3) layout, huruf, dan tulisan sangat sesuai dengan karakteristik peserta didik
MI/SD. Vol. 1 No. 2 Agustus 2021 98 Fira Nadliratul Afrida Buku ajar yang telah divalidasi oleh ahli desain berada pada kualifikasi sangat
valid sehingga buku ajar tidak perlu dilakukan revisi. Desain layout harus saling
berkaitan dan memberi tekanan, penekanan bisa berupa pemberian warna yang
berbeda dan ukuran font yang lebih besar (Kurniawan et al., 2019). Layout pada
buku ajar terbukti dapat mempengaruhi motivasi dan pemahaman peserta didik. (Yvonne B. 2016) menjelaskan bahwa buku ajar yang baik didukung oleh desain
layout yang menarik, hal ini dikarenakan keserasian antara desain layout buku
yang diatur dan seberapa jelas elemen visual yang dirancang dapat mempengaruhi
tingkat perhatian partisipan. Validasi ahli materi terhadap buku ajar bahasa inggris berbasis islami
dilakukan dua tahap. Tahap pertama memperoleh saran dari validator yaitu:
penambahan lebih detail dan jelas pada Glosarium yang ada di buku ajar, aspek
Listening dapat digunakan sebagai panduan untuk speaking, sehingga lebih baik
meletakkan aspek listening sebelum speaking. Selanjutnya peneliti melakukan revisi
terhadap buku ajar yang telah dikembangkan sebelumnya kemudian dilakukan
validasi tahap kedua dengan kriteria: 1) Kesesuaian buku ajar bahasa inggris berbasis islami dengan kurikulum 2013; 2) Kesesuaian Bahan ajar bahasa inggris berbasis islami dengan keterampilan
berbahasa; 3) Kesesuaian tujuan pembelajaran Bahan ajar bahasa inggris berbasis islami; 4) Kesesuaian materi yang disajikan dengan silabus Bahasa Inggris; 5) Kesesuaian Bahan ajar bahasa inggris berbasis islami dengan keadaan siswa SD
kelas 3; 6) Kesesuaian bahasa yang dipakai dalam Bahan ajar bahasa inggris berbasis
islami; 7) Kesesuaian jenis-jenis dan bentuk gambar Bahan ajar bahasa inggris berbasis
islami; 8) Penyajian materi dan petunjuk penampilan mudah dipahami bagi siswa; 9) Kesesuaian isi Bahan ajar bahasa inggris berbasis islami dengan materi
pembelajaran; 10) Penyajian materi sesuai dengan karakter mata pelajaran. Dawuh Guru: Jurnal Pendidikan MI/SD 99 Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD Berbasis Islami Buku ajar yang telah divalidasi oleh ahli desain berada pada kualifikasi sangat
valid sehingga buku ajar tidak perlu dilakukan revisi. Hasil Validasi Ahli Validasi ahli pembelajaran
bahasa inggris dilakukan kepada guru bahasa Inggris yang telah berpengalaman
mengajar sehingga mengetahui kebutuhan dan karakteristik siswa kelas 3 MI. Adapun kriteria penilaian pada buku ajar diantaranya: (1) kesesuaian tampilan fisik
bahan ajar bahasa inggris berbasis islami (2) kejelasan tujuan pembelajaran bahan
ajar bahasa inggris berbasis islami (3) kejelasan materi bahan ajar bahasa inggris
berbasis islami (4) kesesuaian antara gambar dan materi dalam bahan ajar bahasa
inggris berbasis islami (5) kejelasan tugas dan latihan dalam bahan ajar bahasa
inggris berbasis islami (6) kemudahan memahami materi dalam bahan ajar bahasa
inggris berbasis islami (7) kemudahan bahasa yang digunakan dalam bahan ajar
bahasa inggris berbasis islami (8) kemenarikan isi dalam bahan ajar bahasa inggris
berbasis islami (9) kesesuaian bahan ajar digunakan dalam pembelajaran (10)
bahan ajar dapat memberikan motivasi belajar bahasa inggris peserta didik. Buku ajar yang telah divalidasi oleh ahli pembelajaran berada pada persentase
94% dengan kualifikasi sangat valid sehingga buku ajar tidak perlu dilakukan revisi. Bahan ajar memiliki pengaruh dan peran sangat tinggi dalam proses pembelajaran,
hal ini disebabkan adanya hubungan guru, sumber belajar dan peserta didik (Walid
et.al. 2017). Kebutuhan buku ajar pada saat ini masih menjadi prioritas utama dalam
melaksanakan kegiatan belajar mengajar. Buku ajar tidak hanya menjadi rujukan
pokok bagi peserta didik dalam belajar, melainkan juga menjadi acuan bagi para
guru dalam proses pembelajaran (Nurdeani, N. 2014). Tujuan pengembangan
buku ajar bahasa inggris berbasis islami selain memberikan penanaman nilai-nilai
spiritual sejak dini yang dikemas dengan kegiatan pembelajaran yang menarik
dengan menggunakan bahasa inggris sebagai pengantarnya juga sebagai acuan
guru dalam peningkatan kualitas pembelajaran. Tingkat kemenarikan Buku Ajar Bahasa Inggris berbasis Islami Hasil validasi buku ajar terhadap pengembangan buku ajar bahasa inggris
berbasis islami dengan subjek kelas 3 MI/SD ini memperoleh persentase 90,04%. Hasil penilaian uji coba lapangan pada setiap komponen sebagaimana dianalisis
secara kuantitatif dapat diinterpretasikan sebagaimana berikut: Vol. 1 No. 2 Agustus 2021 100 Fira Nadliratul Afrida a. Tampilan sampul dalam buku ajar bahasa inggris
berbasis islami memperoleh skor 96% sehingga dapat
disimpulkan bahwa sampul ini sangat menarik
perhatian peserta didik; b. Kemudahan dalam belajar menggunakan buku ajar
bahasa inggris berbasis islami memperoleh persentase
92% sehingga menunjukkan bahwa buku ajar dapat
memudahkan peserta didik dalam belajar; c. Penggunaan
buku
ajar
bahasa
inggris
dapat
memberikan semangat belajar peserta didik memperoleh penilaian dengan
persentase sebesar 94,6%; d. Buku ajar bahasa inggris berbasis islami memudahkan peserta didik memahami
bahan pelajaran memperoleh penilaian dengan persentase sebesar 90,6%
sehingga buku ajar ini dapat digunakan peserta didik dalam memahami materi
bahasa inggris serta dapat diterapkan dalam kehidupan sehari-hari khususnya
pada sisi spiritual; e. Soal-soal yang terdapat pada buku ajar bahasa inggris berbasis islami dapat
mudah dipahami dan dikerjakan oleh peserta didik memperoleh penilaian
dengan persentase sebesar 90,6%; f. Jenis dan ukuran huruf yang terdapat pada buku ajar bahasa inggris berbasis
islami memudahkan peserta didik untuk membaca dan memahami materi. Pada aspek ini mendapat penilaian sebesar 98,6%; g. Kata-kata yang digunakan dalam buku ajar bahasa inggris berbasis islami sesuai
dengan karakteristik peserta didik MI/SD, pada aspek ini peserta didik perlu
untuk memperdalam vocabulary yang ada di buku ajar, hal ini diperoleh
penilaian sebesar 84%; h. Bahasa yang yang digunakan dalam buku ajar bahasa inggris berbasis islami
mudah dipahami memperoleh penilaian dengan persentase sebesar 98,6%; i. Penggunaan buku ajar memerlukan bantuan orang lain seperti teman, guru
atau orangtua memperoleh penilaian dengan persentase 74,6%. Hal ini
dikarenakan bahasa inggris bukan merupakan bahasa yang digunakan oleh Dawuh Guru: Jurnal Pendidikan MI/SD 101 Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD Berbasis Islami peserta didik sehingga perlu adanya arahan & bantuan dari guru atau orangtua
untuk mendampingi proses belajar; j. Buku ajar bahasa inggris berbasis islami sangat menarik untuk dipelajari
memperoleh penilaian dengan persentase 90,4%. j. Buku ajar bahasa inggris berbasis islami sangat menarik untuk dipelajari
memperoleh penilaian dengan persentase 90,4%. Tingkat kemenarikan Buku Ajar Bahasa Inggris berbasis Islami Daya tarik peserta didik terhadap buku ajar bahasa inggris dibuktikan dari
hasil wawancara kepada beberapa peserta didik yang menyatakan bahwa buku ajar
ini berbeda dengan buku ajar yang lain karena memiliki desain yang atraktif,
menarik, dan mampu membangkitkan motivasi peserta didik untuk belajar karena
dilengkapi dengan do’a-doa sehari-sehari, lagu islami, dan kegiatan-kegiatan yang
bersifat kontekstual dekat dengan peserta didik. Daya tarik peserta didik juga terlihat pada saat observasi atau pengamatan
saat pembelajaran sedang berlangsung peserta didik sangat antusias dan tertarik
untuk mempelajari buku bahasa inggris berbasis islami. Mereka sangat semangat
untuk praktik berbicara bahasa inggris di depan kelas sehingga tercipta suasana
pembelajaran yang menyenangkan dan bermakna. Kemenarikan bahan ajar juga
dapat menjadi rangsangan bagi peserta didik untuk membaca dan mempelajari
bahan ajar karena aspek kemenarikan dapat menentukan tersampainya isi dari
buku ajar (Amelia, D.J, 2018). D. Kesimpulan Berdasarkan proses pengembangan dan hasil penilaian terhadap buku ajar
bahasa inggris berbasis islami untuk siswa kelas III MI/SD ini dapat disimpulkan
bahwa buku ajar bahasa inggris berbasis islami untuk siswa kelas III MI/SD
memperoleh kualifikasi yang baik, dibuktikan dengan hasil validasi oleh ahli
materi dengan persentase 80%, ahli desain dengan persentase 92%, guru kelas
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Education. 1(2). http://dx.doi.org/10.32934/jmie.v1i2.45 103 Dawuh Guru: Jurnal Pendidikan MI/SD Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD Berbasis Islami Pengembangan Buku Ajar Bahasa Inggris Kelas 3 MI/SD Berbasis Islami 104 Vol. 1 No. 2 Agustus 2021
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Scoring method of a Situational Judgment Test: influence on internal consistency reliability, adverse impact and correlation with personality?
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Citation for published version (APA):
De Leng, W. E., Stegers-Jager, K. M., Husbands, A., Dowell, J. S., Born, M. P., & Themmen, A. P. N. (2017).
Scoring method of a Situational Judgment Test: influence on internal consistency reliability, adverse impact and
correlation with personality? Advances in Health Sciences Education, 22(2), 243-265.
https://doi.org/10.1007/s10459-016-9720-7 University of Dundee Scoring method of a Situational Judgment Test Link to publication in Discovery Research Portal Citation for published version (APA):
De Leng, W. E., Stegers-Jager, K. M., Husbands, A., Dowell, J. S., Born, M. P., & Themmen, A. P. N. (2017). Scoring method of a Situational Judgment Test: influence on internal consistency reliability, adverse impact and
correlation with personality? Advances in Health Sciences Education, 22(2), 243-265. https://doi.org/10.1007/s10459-016-9720-7 General rights
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DOI 10.1007/s10459-016-9720-7 1
Institute of Medical Education Research Rotterdam (iMERR), Erasmus MC, Room AE-239,
PO Box 2040, 3000 CA Rotterdam, The Netherlands
2
Medical School, University of Buckingham, Buckingham, UK
3
School of Medicine, University of Dundee, Dundee, UK
4
Department of Psychology, Erasmus University Rotterdam, Rotterdam, The Netherlands
5
Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands Background Selection into medical school has been dominated by cognitive-based measures which are
predictive for academic performance, but are less predictive for clinical performance
(Ferguson et al. 2002; Salvatori 2001). Adding non-cognitive-based measures to cognitive-
based measures may improve the predictive quality of a selection procedure (Kulatunga-
Moruzi and Norman 2002; Lucieer et al. 2015; Powis 2015). Non-cognitive-based selec-
tion instruments with good validity and reliability are essential for this purpose, because
selection into medical school is highly competitive, with the number of applicants greatly
exceeding the number of available places. An upcoming non-cognitive-based measure for selection into medical school is the
Situational Judgment Test (SJT). An SJT presents applicants with several situations that
they may encounter during the job (or at medical school), followed by a number of possible
responses to that situation. Respondents are instructed to judge the appropriateness of these
responses by stating what they would or should do in the described situation (Motowidlo
et al. 1990; Weekley and Ployhart 2013). Administering SJTs in work-related selection
procedures has several beneficial characteristics: (1) good predictive validity with regard to
job performance (McDaniel et al. 2001), (2) incremental validity over and above cognitive
ability and personality (Clevenger et al. 2001), (3) less adverse impact than cognitive
measures (McDaniel and Nguyen 2001), (4) higher favorability ratings by candidates than
in cognitive tests (Lievens 2013) and (5) more efficient administration to large groups of
applicants than other non-cognitive-based instruments (e.g., assessment centers) (Mo-
towidlo et al. 1990). Previous studies on the use of SJTs for selection into medical school have shown that
these beneficial characteristics of SJTs also apply in a medical school context (Koczwara
et al. 2012; Lievens 2013; Lievens et al. 2005; Lievens and Sackett 2012; Patterson et al. 2009, 2011, 2015). Despite the good qualities mentioned above, some aspects of SJTs require more
research. One of these aspects is the scoring method (Whetzel and McDaniel 2009). Scoring an SJT is more complicated than scoring a traditional knowledge test because there
are no objectively correct answers, since SJTs consist of dilemmas with no clear-cut
solutions (Bergman et al. 2006). Different researchers and practitioners have used different
methods to convert the judgments on an SJT to a score, which has led to a large variety of
scoring methods. Scoring method of a Situational Judgment Test: influence
on internal consistency reliability, adverse impact
and correlation with personality? W. E. De Leng1 • K. M. Stegers-Jager1 • A. Husbands2 •
J. S. Dowell3 • M. Ph. Born4 • A. P. N. Themmen1,5 Received: 12 February 2016 / Accepted: 6 October 2016
The Author(s) 2016. This article is published with open access at Springerlink.com Received: 12 February 2016 / Accepted: 6 October 2016 Received: 12 February 2016 / Accepted: 6 October 2016
The Author(s) 2016. This article is published with open access at Springerlink.com y
p
The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Situational Judgment Tests (SJTs) are increasingly used for medical school
selection. Scoring an SJT is more complicated than scoring a knowledge test, because there
are no objectively correct answers. The scoring method of an SJT may influence the
construct and concurrent validity and the adverse impact with respect to non-traditional
students. Previous research has compared only a small number of scoring methods and has
not studied the effect of scoring method on internal consistency reliability. This study
compared 28 different scoring methods for a rating SJT on internal consistency reliability,
adverse impact and correlation with personality. The scoring methods varied on four
aspects: the way of controlling for systematic error, and the type of reference group,
distance and central tendency statistic. All scoring methods were applied to a previously
validated integrity-based SJT, administered to 931 medical school applicants. Internal
consistency reliability varied between .33 and .73, which is likely explained by the
dependence of coefficient alpha on the total score variance. All scoring methods led to
significantly higher scores for the ethnic majority than for the non-Western minorities, with
effect sizes ranging from 0.48 to 0.66. Eighteen scoring methods showed a significant
small positive correlation with agreeableness. Four scoring methods showed a significant
small positive correlation with conscientiousness. The way of controlling for systematic
error was the most influential scoring method aspect. These results suggest that the
increased use of SJTs for selection into medical school must be accompanied by a thorough
examination of the scoring method to be used. 123 W. E. De Leng et al. Keywords Situational Judgment Test Scoring method Medical school selection
Internal consistency reliability Adverse impact Integrity Big Five Keywords Situational Judgment Test Scoring method Medical school selection
Internal consistency reliability Adverse impact Integrity Big Five Aspect 1: controlling for systematic error SJTs with a rational scoring key and a Likert scale response format can be scored using
raw, standardized, and dichotomous consensus (McDaniel et al. 2011). Raw consensus
computes the distance between the applicant’s rating and the mean rating of the reference
group using the raw data. Standardized consensus calculates the distance after conducting a
within-person z standardization such that each applicant has a mean of zero and a standard
deviation of one across the SJT items. Dichotomous consensus divides the Likert scale in
the middle. Points are awarded when an applicant’s position on the Likert scale is on the
same side as the reference group. Some dichotomous scoring methods increase the scoring
range by applying a negative correction by subtracting points when applicants are on the
other side of the Likert scale. By standardizing or dichotomizing the data, McDaniel et al. (2011) attempted to control
for systematic error. Systematic error in an SJT score may be caused by response ten-
dencies or coaching in strategies on how to use the Likert scale, for example only opt for
the extremes or only opt for the middle of the scale (McDaniel et al. 2011). Moreover,
response tendencies are influenced by ethnic differences. For example, Black and Hispanic
Americans are more inclined to use the extremes of a Likert scale than White Americans
(Bachman and O’Malley 1984; Hui and Triandis 1989). By standardizing or dichotomizing
the data, these cultural differences in the use of a Likert scale no longer influence the SJT
score. Raw consensus does not control for systematic error. McDaniel et al. (2011) examined the effect of these three scoring methods on the
concurrent validity in two studies, using scores on a biodata scale measuring quitting
tendencies and supervisory ratings of job performance as criterion. Higher concurrent
validity was found for the standardized consensus and dichotomous consensus scales than
for the raw consensus scale, which they explained by the removal of systematic error from
the SJT score. In addition, the standardized and dichotomous consensus scales resulted in
substantially smaller differences between White and Black respondents than the raw
consensus scale, which they attributed to the removal of ethnic differences in the use of a
Likert scale. Similarly, Legree et al. (2010) found a higher concurrent validity for a
standardized scale than a raw scale. Background This study will investigate the effect of these various scoring methods on
three psychometric qualities (i.e., internal consistency reliability, adverse impact and
correlation with personality). For this purpose, we used a previously validated integrity-
based SJT (Husbands et al. 2015) for the selection of medical school applicants at a Dutch
medical school. Choice of scoring method depends on the type of scoring key and response format of an
SJT. This study will focus on scoring methods for SJTs that use a rational scoring key and a
Likert scale response format. A rational scoring key uses the judgments of a reference 123 123 Scoring method of a Situational Judgment Test: influence on… group of Subject Matter Experts (SMEs) to determine the ‘‘correct’’ answer. SMEs are
individuals highly experienced in the relevant domain (Bergman et al. 2006). The Likert
scale response format instructs the respondents to rate the appropriateness of each response
option on a rating scale (Weekley et al. 2013). group of Subject Matter Experts (SMEs) to determine the ‘‘correct’’ answer. SMEs are
individuals highly experienced in the relevant domain (Bergman et al. 2006). The Likert
scale response format instructs the respondents to rate the appropriateness of each response
option on a rating scale (Weekley et al. 2013). Scoring methods The scoring methods in this study differ on four aspects: the way of controlling for
systematic error, the type of reference group, the type of distance and the type of central
tendency statistic. Aspect 2: reference group A second aspect on which scoring methods may differ is the reference group. As stated
above, a rational scoring key uses the judgments of a group of SMEs to determine the
‘‘correct’’ answer on an SJT. Most SJT scoring methods use SMEs because it is expected
that they have knowledge about what behavior is effective and ineffective in their field
(Motowidlo and Beier 2010). However, a number of SJT studies have used the group of
respondents itself as a reference, a procedure called Consensus Based Measurement
(CBM). Legree et al. (2005) argued that this procedure may be more appropriate for
constructs for which no clear SMEs can be identified. A study on an SJT used for the US
Airforce found that the mean ratings of the SMEs strongly correlated with the mean ratings
of the group of respondents (Legree 1995; Legree and Grafton 1995). Similar results were
found for an SJT measuring Tacit Knowledge of Military Leadership comparing lieu-
tenants (i.e., SMEs) with cadets (Hedlund et al. 2003). Comparison of two SJT scoring
keys based on either novices’ or experts’ mean effectiveness ratings found a correlation of
.75 between the two keys (Motowidlo and Beier 2010). In addition, both scoring keys
resulted in scores that had similar criterion-related validity coefficients. These results were
explained by novices’ possession of a different, more general type of knowledge outside
the specific job context. Furthermore, Lineberry et al. (2014) stated that for script con-
cordance tests used for assessing clinical reasoning skills, having experience does not
indicate that someone is an infallible expert and that residents (i.e., novices) can outper-
form most panelists (i.e., SMEs). We are not aware of any previous research on the effect
of using a less experienced reference group in a medical selection context. Aspect 1: controlling for systematic error Next to using raw, standardized and dichotomous consensus, a score on an SJT with a
rational scoring key and Likert scale response format can also be calculated using percent
agreement (Legree et al. 2005). Percent agreement uses the endorsement ratios among the
SMEs to determine the score corresponding to each rating. Percent agreement, like raw
consensus, does not control for systematic error. An example of a scoring method using percent agreement assigns two points to the
Likert scale point endorsed by 50 % or more of the SMEs and one point to the scale point 12 123 W. E. De Leng et al. endorsed by 25–50 % of the SMEs (Chan and Schmitt 1997). Another example assigns a
score to each Likert scale point depending on the proportion of the reference group that
endorsed that rating point (Lievens et al. 2015). endorsed by 25–50 % of the SMEs (Chan and Schmitt 1997). Another example assigns a
score to each Likert scale point depending on the proportion of the reference group that
endorsed that rating point (Lievens et al. 2015). Aspect 3: distance A third aspect on which scoring methods may differ is the type of distance that is cal-
culated between an applicant’s rating and the overall rating of the reference group (SMEs
or respondents). Some SJT studies have used the squared distance (McDaniel et al. 2011),
whereas others have used the absolute distance (Legree 1995). Squaring the distance gives
more weight to ratings that deviate more from the reference group (Legree et al. 2005). Present study The first goal of this study was to investigate the effect of scoring method on the internal
consistency reliability of an SJT score. The appropriateness of internal consistency as a
reliability estimate for SJT scores is often called into question (Catano et al. 2012). Internal
consistency reliability estimates, such as coefficient alpha, are based on the assumption that
all items measure the same latent trait on the same scale, i.e., that the same latent trait
equally contributes to all item scores (Yang and Green 2011). The multidimensional nature
of SJTs violates this strict assumption resulting in an inaccurate estimate of reliability
(Graham 2006). However, the integrity-based SJT used in this study was designed to
measure one dimension, which might lead to a less serious violation of the assumption of
unidimensionality. This is supported by a meta-analysis of Campion et al. (2014) that
reported a mean alpha of .57 across 129 coefficients (range 0–.92). In addition, it was
shown that coefficient alpha was significantly higher for SJTs that had a larger focus on
one dimension. The focus of the current integrity-based SJT on one dimension may support
the use of internal consistency reliability. So, given the anticipated unidimensionality of
the SJT used in this study and because coefficient alpha is still commonly reported in the
SJT literature, we chose it as a measure of comparison between scoring methods. To the
best of our knowledge, this will be the first study to investigate the effect of different
scoring methods on the internal consistency reliability. The second goal of this study was to examine the effect of scoring method on adverse
impact, by analyzing the differences between Dutch and non-Western minority applicants. Adverse impact will be examined because SJTs may play an important role in promoting
fairness in medical school selection, since SJT scores potentially demonstrate lower ethnic
subgroup differences than cognitive ability test scores. On cognitive ability tests, White
test takers have been shown to score approximately one standard deviation higher than
non-White test takers (De Soete et al. 2013). A meta-analysis on ethnic subgroup differ-
ences across 32 SJTs—mainly originating from the US—showed that White test takers
score approximately 0.38 standard deviation higher than Black test takers, 0.24 standard
deviation higher than Hispanic test takers and 0.29 standard deviation higher than Asian
test takers (Whetzel et al. 2008). Aspect 4: central tendency statistic A fourth aspect on which SJT scoring methods may differ is the manner of how the
judgments of the reference group are summarized (i.e., central tendency statistic). Most
SJT scoring methods have used the mean as a central tendency statistic, whereas some
studies have used the mode (De Meijer et al. 2010; Lievens et al. 2015). Scoring methods
using the mode assign points to the Likert scale point that most of the people in the
reference group endorse. Besides the mean and mode, another widely used central ten-
dency statistic is the median, which reflects the number at the central point when the data
are ranked in numerical order (McCluskey and Lalkhen 2007). To our knowledge, the
median has so far never been used for scoring SJTs. For the sake of completeness, this
study will include all three central tendency statistics. 12 Scoring method of a Situational Judgment Test: influence on… Procedure The SJT was administered during the selection procedure for the Erasmus MC Medical
School in 2014 and 2015 (N = 1025). The administration was solely for research purposes
and participation was voluntarily. The Erasmus MC Medical School selects students on
their participation in extracurricular activities, their performance on five cognitive tests
during three on-site testing days (Urlings-Strop et al. 2009) and their pre-university Grade
Point Average (GPA). The administration of the SJT was conducted during the on-site
testing days, using paper-and-pencil. An additional questionnaire was administered
regarding applicants’ demographic characteristics. A personality questionnaire was
administered online when applicants registered for the selection procedure. The applicants
were informed that the SJT and questionnaires were administered solely for research
purposes and that their answers would not influence the outcome of the selection proce-
dure. Participation was voluntarily. Present study 2010; McDaniel et al. 2011). We
are unaware of any previous studies that have investigated the effect of type reference group,
distance and central tendency statistic on the correlation of an SJT score with personality. Measures Integrity-based Situational Judgment Test Present study A Dutch study also found that the ethnic subgroup
difference in an integrity SJT score (d = 0.38) was lower than in a cognitive ability test
score (d = 0.48) (De Meijer et al. 2010). Selection on only cognitive ability test scores
might lead to the rejection of more ethnic minority applicants than ethnic majority
applicants, whereas selection on SJT scores may increase the admission rate among ethnic
minorities, resulting in a more culturally diverse medical student population. To promote
the expected positive influence of an SJT on fairness, it is crucial to investigate the
potential influence of scoring method on adverse impact. In line with the findings of
McDaniel et al. (2011), we expect that scoring methods controlling for systematic error
(i.e., standardized and dichotomous consensus) will lead to smaller ethnic differences than
scoring methods that do not (i.e., raw consensus and percent agreement). The other scoring
method aspects (i.e., type of reference group, distance and central tendency statistic) have
not been studied in combination with adverse impact before. The third goal of this study was to investigate the effect of scoring method on the corre-
lation between the SJT score and three of the Big Five personality traits. The Big Five
describes someone’s personality using five broad dimensions: neuroticism (i.e., emotional
instability), extraversion (i.e., outgoing and energetic), openness to experience (i.e., intel-
lectual curiosity), agreeableness (i.e., altruistic and compassionate) and conscientiousness 123 123 W. E. De Leng et al. (i.e., organized and persistent) (Costa and MacCrae 1992). The correlation with the Big Five
was examined because three of the five dimensions (i.e., conscientiousness, emotional sta-
bility and agreeableness) have been shown to moderately and positively correlate with SJT
scores (McDaniel et al. 2007) and integrity test scores (Marcus et al. 2007). Moreover, the
validity and reliability of thescores on the Big Five measure used inthis study [i.e.,NEO-PI-R
(Costa and MacCrae 1992)] has repeatedly been demonstrated (Costa and McCrae 2008),
including in samples of adolescents (De Fruyt et al. 2000). It is therefore expected that the
integrity-based SJT will becorrelated to these three BigFive dimensionsand that theresulting
correlation coefficients will provide a good measure of comparison between the scoring
methods. We hypothesize that scoring methods that control for systematic error will lead to
higher correlation coefficients, because the influence of response tendencies regarding the use
of Likert scales is removed from the SJT score (Legree et al. 123 Personality questionnaire In 2014, the Dutch version of the NEO-PI-R was administered to assess the applicants’
standing on the Big Five personality traits (Costa and MacCrae 1992; Hoekstra et al. 1996). The questionnaire consisted of 240 statements that applicants had to judge on a five-point
Likert scale (1 Strongly disagree–5 Strongly agree). The five personality subscales
demonstrated good internal consistency reliabilities (coefficient alpha): .92 for neuroti-
cism, .87 for extraversion, .85 for openness, .87 for agreeableness and .88 for conscien-
tiousness. Due to the length of the questionnaire, the NEO-PI-R was not administered in
2015. Demographics An applicant was considered a non-Western minority when one of his/her parents was born
outside Europe or North-America (Statistics Netherlands; www.cbs.nl). An applicant was considered a non-Western minority when one of his/her parents was born
outside Europe or North-America (Statistics Netherlands; www.cbs.nl). The socio-economic status of an applicant was determined by the level of education of
his/her parents. A division was made between first-generation and non-first-generation
university students. First-generation university students were defined as students whose
parents did not attend university (either a research university or a university of applied
science). Integrity-based Situational Judgment Test The integrity-based SJT used in this study was developed in the United Kingdom (UK)
(Husbands et al. 2015). The authors translated this SJT to Dutch. This translation was
validated using the back translation procedure described by Brislin (1970). The back
translation was conducted by an independent commercial translation office. The authors
discussed and made appropriate changes to the translated version. The SJT consisted of ten scenarios describing problematic situations that could occur
during medical school. Each scenario was followed by five response options. The
respondents had to judge the appropriateness of each response option on a four-point Likert
scale (1 Very inappropriate–4 Very appropriate) in terms of what should be done given the
situation [i.e., knowledge-based instructions (Ployhart and Ehrhart 2003)]. An example of
an SJT item is presented in Appendix 1. 123 123 Scoring method of a Situational Judgment Test: influence on… A rational scoring key for this SJT was developed based on the judgments of 16 SMEs
(75 % female). The mean age of this group was 40.8 years (SD = 11.1). The SMEs were
individuals involved in teaching professionalism in the medical curriculum. Two of the
SMEs were medical doctors. The mean number of years of experience with profession-
alism in the medical curriculum of this group was 6.4 (SD = 5.9). All SMEs were native
Dutch. The intraclass correlation coefficient (ICC) among the SMEs was .65, indicating a
moderate agreement (two-way mixed model, absolute agreement). Scoring methods In preparation for this study we combined the four aspects on which scoring methods can
differ; this yielded 28 scoring methods to be tested (Fig. 1). These scoring methods fol-
lowed the categorization into raw, standardized and dichotomous consensus scoring
methods as proposed by McDaniel et al. (2011). Within each of the raw and standardized scoring methods, the distance (absolute or
squared) was calculated between the applicant’s rating and the overall rating of the ref-
erence group on the Likert scale. The reference group was either made up of the 16 SMEs
or of the group of respondents itself. The overall rating of this reference group was
reflected by either the mean, median or mode. In addition to the raw and standardized consensus scoring methods, the dichotomous
consensus scoring method was applied. The reference group consisted of either the SMEs
or the group of respondents itself. Another variation was applied by either assigning zero
points to or subtracting one point from applicants whose rating was located on the opposite
side of the Likert scale than the reference group. The 24 scoring methods based on either raw, standardized or dichotomous consensus
were complemented with four scoring methods based on percent agreement (Legree et al. 2005). These scoring methods used either the 25–50 % endorsement rule used by Chan and 123 123 W. E. De Leng et al. Fig. 1 Schematic representation of the 28 scoring methods. SMEs Subject Matter Experts Fig. 1 Schematic representation of the 28 scoring methods. SMEs Subject Matter Experts Schmitt (1997) or assigned a score to each Likert scale point corresponding to the pro-
portion of subjects in the reference group who endorsed that point (Lievens et al. 2015). The reference group consisted of either the SMEs or the respondents. The correlations between the 28 scoring methods are presented in Appendix 2. Although
some correlation coefficients indicated a large overlap between the scoring methods (i.e.,
within the raw consensus scoring method set), other scoring methods showed less overlap
(i.e., between the raw and dichotomous scoring method sets). To our knowledge, of half of these scoring methods no results have been published in
the context of application to an SJT (i.e., scoring methods using the median, scoring
methods calculating the distance from the group mode, dichotomous scoring methods
using the SMEs, percent agreement scoring methods using the endorsement rate of the
group and the proportions of the SMEs). 123 Participants Nine-hundred thirty-one medical school applicants responded (response rate = 90.8 %). The demographic characteristics of this sample are depicted in Table 1. The two cohorts
(2014 and 2015) were similar with regard to gender, age and ethnicity. Cohort 2015
consisted of significantly more first-generation students than cohort 2014, but the size of
this effect was small [X2(1) = 6.02, p = .014, u = .08]. Personality data were obtained
from 73.3 % of the participants from cohort 2014. SJT scores did not significantly differ
between respondents and non-respondents to the personality questionnaire. Statistical analysis Both SPSS (IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.)
and R (Version 3.1.0) were used to convert the judgments on the SJT to a score, using the
different scoring methods. The raw and standardized consensus scoring methods that used
the group of respondents itself as a reference were conducted using a leave-one-out method
(Hastie et al. 2009). This method removes the applicant whose score needs to be calculated
from the dataset, and calculates the summary statistic across the remaining group members. The distance between the applicant and the remaining group members composes the
applicant’s score. Coefficient alpha was used as an estimate of internal consistency reliability (Cronbach
1951). Independent t-tests were used to examine the 28 different SJT scores on disparities
between first-generation and non-first-generation university applicants and between Dutch
and non-Western minority applicants. The effect sizes of the social and ethnic disparities
were reflected by Cohen’s d (Cohen 1988). A stricter alpha level (a = .001) was used
because of the large number of comparisons. 123 123 Scoring method of a Situational Judgment Test: influence on… For each scoring method, Pearson correlations were used to determine the correlation
between the SJT score and the three Big Five personality traits for which we expected a
correlation. General linear models were used to examine which scoring method aspects significantly
influenced the outcome measures (i.e., coefficient alpha, effect size and correlation coef-
ficient). For each outcome measure, four general linear models were tested, namely one
model for each scoring method aspect. The four aspects were tested in separate models
because the small number of data points (i.e., 28) did not allow entering all four aspects in
one model. The effect sizes were corrected for the reliability of the scoring method by
dividing Cohen’s d by coefficient alpha, since low reliability may obscure subgroup dif-
ferences (Lievens et al. 2008). Internal consistency reliability Coefficient alpha varied from .33 to .73 depending on the scoring method (Table 2). The
lowest coefficient alpha was found for the scoring method that calculated the absolute
distance from the mean of the group of respondents itself using standardized consensus. The highest coefficient alpha was found for the scoring method that calculated the absolute
distance from the mean of the group of respondents itself using raw consensus. For the general linear models with coefficient alpha as dependent variable, the way of
controlling for systematic error was the only significant factor with a very large effect size,
F(3, 24) = 40.05, p \ .001, g2 = .83. Raw consensus led to a significantly higher coef-
ficient alpha than the other three methods of controlling for systematic error. In addition, Table 1 Demographic characteristics of the participants in this study for each cohort
2014 (N = 521)
2015 (N = 410)
Gender (% female)
64.1
62.7
Age [mean (SD)]
19.1 (1.9)
19.2 (1.9)
Ethnicity
% Dutch
58.3
57.2
% non-Western minority
31.3
32.2
% Western minority
10.4
10.6
SES (% first-generation university students)
24.0
31.6
SD standard deviation, SES socio-economic status Table 1 Demographic characteristics of the participants in this study for each cohort W. E. De Leng et al. Table 2 Descriptive statistics and internal consistency reliability (alpha coefficient) for the 28 rate-SJT
scoring methods
Scoring method
M (SD)
Min.–Max. Alpha
Raw consensus
1. Absolute distance—SME mean
34.32 (6.02)
20.01–64.99
.67
2. Absolute distance—SME median
33.11 (6.61)
13.50–66.50
.56
3. Absolute distance—SME mode
32.95 (6.52)
14.50–65.50
.55
4. Squared distance—SME mean
36.25 (12.50)
11.48–107.72
.67
5. Squared distance—SME median
42.44 (13.27)
12.75–122.75
.61
6. Squared distance—SME mode
41.81 (13.18)
13.25–121.25
.60
7. Absolute distance—Group mean
31.26 (6.31)
16.32–63.09
.73
8. Absolute distance—Group median
28.93 (7.00)
11–63
.61
9. Absolute distance—Group mode
29.07 (6.99)
11–63
.59
10. Squared distance—Group mean
30.35 (11.56)
8.47–100.35
.73
11. Squared distance—Group median
35.67 (12.85)
11–113
.65
12. Squared distance—Group mode
36.28 (13.01)
11–115
.63
Standardized consensus
13. Absolute distance—SME mean
32.86 (4.63)
21.24–51.67
.44
14. Absolute distance—SME median
33.52 (4.68)
19.09–51.54
.41
15. Squared distance—SME mean
34.46 (9.57)
14.31–34.46
.49
16. Squared distance—SME median
36.29 (9.61)
13.47–79.99
.45
17. Absolute distance—Group mean
30.42 (3.91)
20.99–50.67
.33
18. Absolute distance—Group median
29.91 (4.57)
18.27–51.00
.43
19. Squared distance—Group mean
29.11 (7.77)
13.58–74.24
.45
20. Squared distance—Group median
30.08 (8.89)
12.63–79.44
.51
Dichotomous consensus
21. Internal consistency reliability SME as reference
34.34 (3.55)
21–44
.34
22. SME as reference—negative correction
18.78 (7.04)
-8–38
.34
23. Group as reference
37.56 (3.59)
22–47
.34
24. Group as reference—negative correction
25.21 (7.11)
-6–44
.34
Percent agreement
25. Endorsement rate—SME
54.23 (7.32)
29–74
.49
26. Endorsement rate—Group
47.53 (5.17)
26–60
.46
27. Proportions—SME
19.39 (2.16)
11.18–25.59
.54
28. Proportions—Group
18.84 (1.55)
11.34–22.63
.58
M mean, SD standard deviation, SME Subject Matter Expert, Min. minimum, Max. maximum standardized consensus and percent agreement yielded a significantly higher coefficient
alpha than dichotomous consensus. standardized consensus and percent agreement yielded a significantly higher coefficient
alpha than dichotomous consensus. Adverse impact All scoring methods led to significantly higher scores for the Dutch majority than for the
non-Western minorities (Table 3). The effect sizes (d) of these differences ranged from
0.48 to 0.66 (medium effect). The largest differences were found for the scoring methods 123 Scoring method of a Situational Judgment Test: influence on… Table 3 Results of the independent t tests for Dutch versus non-Western differences in SJT scores gen-
erated by the 28 different scoring methods
Scoringmethod
Dutch (N = 490)
Non-Western (N = 269)
d
Raw consensus
1. Absolute distance—SME mean
32.88 (5.38)
36.51 (6.50)
0.61
2. Absolute distance—SME median
31.47 (5.92)
35.58 (7.05)
0.63
3. Absolute distance—SME mode
31.34 (5.82)
35.37 (6.95)
0.63
4. Squared distance—SME mean
33.28 (10.86)
40.82 (13.98)
0.60
5. Squared distance—SME median
39.24 (11.50)
47.39 (14.84)
0.61
6. Squared distance—SME mode
38.70 (11.37)
46.67 (14.74)
0.61
7. Absolute distance—Group mean
29.95 (5.61)
33.16 (7.03)
0.50
8. Absolute distance—Group median
27.29 (6.27)
31.31 (7.49)
0.58
9. Absolute distance—Group mode
27.37 (6.21)
31.51 (7.48)
0.60
10. Squared distance—Group mean
27.94 (9.88)
33.96 (13.37)
0.51
11. Squared distance—Group median
32.66 (11.06)
40.02 (14.35)
0.57
12. Squared distance—Group mode
33.13 (11.10)
40.86 (14.59)
0.60
Standardized consensus
13. Absolute distance—SME mean
31.69 (4.23)
34.52 (4.43)
0.65
14. Absolute distance—SME median
32.30 (4.25)
35.22 (4.60)
0.66
15. Squared distance—SME mean
32.07 (8.52)
37.80 (9.36)
0.64
16. Squared distance—SME median
33.88 (8.51)
39.69 (9.56)
0.64
17. Absolute distance—Group mean
29.53 (3.63)
31.55 (3.72)
0.55
18. Absolute distance—Group median
28.83 (4.25)
31.30 (4.34)
0.58
19. Squared distance—Group mean
27.47 (7.14)
31.13 (7.40)
0.50
20. Squared distance—Group median
28.10 (8.11)
32.52 (8.58)
0.53
Dichotomous consensus
21. SME as reference
35.07 (3.32)
33.43 (3.46)
0.48
22. SME as reference—negative correction
20.22 (6.59)
16.98 (6.86)
0.48
23. Group as reference
38.31 (3.37)
36.69 (3.44)
0.48
24. Group as reference—negative correction
26.70 (6.66)
23.49 (6.79)
0.48
Percent agreement
25. Endorsement rate—SME
56.04 (6.76)
51.72 (7.35)
0.61
26. Endorsement rate—Group
48.74 (4.71)
45.78 (5.11)
0.60
27. Proportions—SME
19.93 (1.99)
18.66 (2.16)
0.61
28. Proportions—Group
19.20 (1.37)
18.32 (1.63)
0.58
All differences were significant (p \ .001)
SME Subject Matter Expert, d Cohen’s d (effect size) that calculated the absolute distance from the SME median using standardized consensus. The smallest ethnic difference was observed for all scoring methods that used dichotomous
consensus. that calculated the absolute distance from the SME median using standardized consensus. Table 4 Pearson correlation
coefficients between the SJT
score and the three Big Five
personality dimensions for which
we expect a correlation with the
integrity-based SJT assessed by
the NEO-PI-R in cohort 2014
only (N = 382) Adverse impact The smallest ethnic difference was observed for all scoring methods that used dichotomous
consensus. For the general linear models with the corrected effect size as dependent variable, the
way of controlling for systematic error was again the only significant factor with a very
large effect size, F(3,24) = 15.54, p \ .001, g2 = .66. Raw consensus led to smaller
corrected effect sizes than standardized and dichotomous consensus, but not percent
agreement. 123 W. E. De Leng et al. None of the scoring methods led to significant differences between first-generation
university applicants and non-first-generation university applicants (data available upon
request). Due to the lack of significant differences, no general linear models were tested. Correlation with personality Eighteen scoring methods resulted in an SJT score that had a significant but small positive
correlation with agreeableness (Table 4). The largest correlation coefficients were found
for scoring methods calculating the distance from the SME mean using standardized
consensus. In addition, four scoring methods resulted in an SJT score that had a significant
but small positive correlation with conscientiousness. The largest correlation coefficients Scoringmethod
N
A
C
Raw consensus
1. Absolute distance—SME mean
-.03
-.11
-.04
2. Absolute distance—SME median
.01
-.11
-.07
3. Absolute distance—SME mode
0
-.11
-.06
4. Squared distance—SME mean
-.03
-.12
-.04
5. Squared distance—SME median
-.01
-.12
-.06
6. Squared distance—SME mode
-.01
-.12
-.05
7. Absolute distance—Group mean
-.06
-.07
.02
8. Absolute distance—Group median
-.06
-.08
0
9. Absolute distance—Group mode
-.03
-.08
0
10. Squared distance—Group mean
-.06
-.09
0
11. Squared distance—Group median
-.06
-.11
0
12. Squared distance—Group mode
-.05
-.11
-.01
Standardized consensus
13. Absolute distance—SME mean
0
-.15
-.12
14. Absolute distance—SME median
-.01
-.12
-.12
15. Squared distance—SME mean
0
-.15
-.10
16. Squared distance—SME median
0
-.13
-.11
17. Absolute distance—Group mean
.01
-.10
-.07
18. Absolute distance—Group median
0
-.11
-.06
19. Squared distance—Group mean
.02
-.10
-.06
20. Squared distance—Group median
.01
-.11
-.06
Dichotomous consensus
21. SME as reference
-.07
.07
.10
22. SME as reference—negative correction
-.07
.07
.10
23. Group as reference
.02
.14
.05
24. Group as reference—negative correction
.02
.14
.05
Percent agreement
25. Endorsement rate—SME
0
.10
.05
26. Endorsement rate—Group
.04
.06
.01
27. Proportions—SME
.03
.11
.05
28. Proportions—Group
.04
.08
.01 Bold coefficients reflect a
significant relationship. For the
scoring methods using distance
metrics (number 1 to 20), a
negative correlation coefficient
reflects a positive relationship
and vice versa
N neuroticism, A agreeableness,
C conscientiousness, SME
Subject Matter Expert 12 3 Scoring method of a Situational Judgment Test: influence on… were found for scoring methods calculating the absolute distance from the SME mean and
median both using standardized consensus. Due to the low effect sizes and the small range
of significant correlation coefficients, no general linear models were tested. were found for scoring methods calculating the absolute distance from the SME mean and
median both using standardized consensus. Due to the low effect sizes and the small range
of significant correlation coefficients, no general linear models were tested. Discussion This study shows that the psychometric quality of an SJT greatly depends on the choice of
scoring method, specifically in the way the scoring method controls for systematic error. Firstly, the way of controlling for systematic error strongly affects the internal consistency
reliability of an SJT score, with higher reliability estimates for scoring methods that use
raw consensus. Secondly, the way of controlling for systematic error influences the adverse
impact of the SJT score, with a lower adverse impact for scoring methods that use raw
consensus compared to dichotomous and standardized consensus. Lastly, the different
scoring methods had a minor influence on the correlation with agreeableness and con-
scientiousness, but the practical significance of these correlations was negligible. Internal consistency reliability Our first finding was that the way a scoring method controls for systematic error strongly
influences the internal consistency reliability. This strengthens the concerns about the use
of coefficient alpha as a reliability estimate for an SJT score. Changing only the scoring
method could alter the acceptability of the resulting reliability estimate from poor to
sufficient, even for an SJT that was specifically constructed to measure one dimension. This large variety in internal consistency reliability is likely explained by the dependence
of coefficient alpha on the total score variance (Streiner 2003). Standardized and
dichotomous consensus and percent agreement were associated with a reduction in total
score variance, which is demonstrated by the lower standard deviations in Table 2. This
reduction in total score variance will most likely lead to a lower coefficient alpha. This line of reasoning implies that coefficients alpha reported in previous studies on
SJTs may be strongly influenced by irrelevant aspects, such as the total score variance
generated by the scoring method used. Assuming that most studies on SJTs arbitrarily
choose one scoring method rather than another, choice of scoring method contributes to the
limited usefulness of coefficient alpha as a reliability estimate for SJTs. Future studies
should investigate whether the large variation in coefficient alpha caused by different
scoring methods also occurs in other reliability estimates (e.g., alternate forms reliability)
to find out whether this large variation is an artifact of coefficient alpha only. A more accurate reliability estimate might be obtained by a combination of a more
thoroughly construct-based SJT development (Christian et al. 2010) and a reliability
estimate that takes into account the imposed factor structure of the SJT, for example a
structural equation modeling (SEM) reliability estimate (Yang and Green 2011) or strat-
ified alpha (Catano et al. 2012). Future research is required on the application of construct-
based development methods and alternative internal consistency estimates for SJTs. Adverse impact Although all scoring methods led to significant ethnic differences in SJT score, the way a
scoring method controlled for systematic error influenced the size of these effects. 123 12 W. E. De Leng et al. Specifically, the effect size decreased when using raw consensus instead of standardized or
dichotomous consensus. This result is not in line with the findings of McDaniel et al. (2011) who found lower ethnic subgroup differences for scoring methods that controlled
for systematic error (i.e., standardized and dichotomous consensus), which they explained
by the removal of ethnicity related response tendencies in the use of Likert scales. However, the uncorrected effect sizes do show some support for this line of reasoning with
the lowest effect sizes reported for the scoring methods using dichotomous consensus. The
absence of lower effect sizes for standardized consensus might be caused by the low
number of scale points (i.e., four) on the Likert scale that was used. Narrow Likert scales
may not be as strongly affected by response tendencies as Likert scales with more scale
points (Flaskerud 1988), resulting in no differences when controlling for the response
tendencies. A study on script concordance tests recommended a reduction of the Likert
scale from five to three points in order to decrease the influence of construct-irrelevant
factors such as examinee response styles (Lineberry et al. 2013). Dichotomizing the Likert
scale does seem to have some effect on adverse impact, but at the cost of low internal
consistency reliability, leading to a similar issue as the diversity–validity dilemma (De
Soete et al. 2013). Another noteworthy finding is that adverse impact was similar for both reference groups
(SMEs and respondents). Previous studies which compared different reference groups
found similar validity coefficients for the scores of both groups (Legree et al. 2005;
Motowidlo and Beier 2010), but did not study the effect of the reference group on adverse
impact. Most SJTs use SMEs as a reference group under the assumption that they have
considerable experience in a relevant setting and therefore know what kind of behaviors
are appropriate in the described situations. Our results suggest that the use of a reference
group of inexperienced respondents (i.e., secondary school students) does not affect the
adverse impact of an SJT. A possible explanation for this comparable adverse impact is the better representa-
tiveness of the group of respondents with respect to ethnicity. Adverse impact All our SMEs in this study
were native Dutch, while only 57 % of the applicants were native Dutch. Little is known
about the cultural susceptibility of integrity. However, medical professionalism has been
found to depend on cultural context (Chandratilake et al. 2012; Jha et al. 2015) and since
integrity is an important aspect of medical professionalism, it too might depend on cultural
context (Arnold and Stern 2006). A reference group that is more representative of the
demographic characteristics of the applicant group may lead to a more accurate mea-
surement of the targeted construct and may therefore result in equal or less adverse impact. Future research should investigate the effect of the demographic composition of the ref-
erence group on the psychometric quality of an SJT. Another explanation for the equal adverse impact for both type of reference groups
might be that there were too few SMEs to be able to achieve proper consensus on the
difficult dilemmas described in the scenarios. This was reflected by the non-perfect
agreement in the SMEs’ evaluation of the response options (ICC = .65). A group of 931
individuals might result in more meaningful consensus. This contention is supported by
Legree et al. (2005), who stated that in light of equal validity coefficients, an examinee-
based scoring standard gives more reliable values than an expert-based scoring standard,
due to the larger number of examinees. 123 123 12 Scoring method of a Situational Judgment Test: influence on… Correlation with personality Our last finding was that 18 scoring methods showed a correlation with agreeableness and
four scoring methods showed a correlation with conscientiousness, which was in line with
previous research (Marcus et al. 2007; McDaniel et al. 2007). However, these correlations
must be interpreted with caution, since all correlation coefficients represent small effects
and it is likely that the large sample size has contributed to the statistical significance of
these small effects. The larger number of significant correlations among scoring methods
using standardized consensus is in line with the findings of McDaniel et al. (2011) and
might be explained by the removal of systematic error from the SJT score. However, the
small effect size of these correlations between the integrity-based SJT score and the three
Big Five personality traits precludes any conclusive statements about the effect of scoring
method on the correlation with personality. The small number of significant correlations between the SJT score and the Big Five
personality traits is in consonance with a previously reported non-association between the
Big Five personality traits and the score on a multiple mini interview (MMI), another
widely used selection instrument for medical school (Kulasegaram et al. 2010). This non-
association might be explained by the fact that personality tests assess non-cognitive traits,
whereas MMIs and SJTs assess non-cognitive behaviors. Non-cognitive behaviors are
more dependent on situational factors than personality traits (Eva 2005). This is in line
with a previous study which demonstrated that a contextualized personality measure had
higher criterion validity for academic performance and counterproductive academic
behavior than a generic personality measure (Holtrop et al. 2014). The lack of contextu-
alization of the NEO-PI-R limits the usefulness of personality tests in medical school
selection and may be an explanation for the absence of any meaningful correlations
between the SJT score and personality. Scoring method aspects revisited Four scoring method aspects were examined. Differences in internal consistency reliability
and adverse impact were found for only one aspect: the way of controlling for systematic
error, with raw consensus leading to scores with the highest coefficient alpha and the
smallest ethnic subgroup differences. As mentioned above, these differences might be
explained by the effect of this scoring method aspect on the total score variance and the
negligible effect of response tendencies due to the narrow Likert scale used in this study. No differences were found for the other three aspects (i.e., reference group, distance and
central tendency statistic). As stated before, the absence of differences for reference group might be caused by the
larger size and better representativeness of the group of respondents itself, which might
remove the benefits of using a highly experienced but small group of SMEs. Another
potential reason is that integrity-related issues in the beginning stage of medical school do
not require specific knowledge but more general knowledge which can be possessed by
both reference groups, which is reflected by a correlation of .90 between the group of
SMEs and group of respondents itself in their average rating. The absence of differences for the scoring method aspect of distance (absolute vs. squared) may be explained by the low number of scale points on the Likert scale (i.e.,
four), which means that the maximum distance between an applicant’s rating and the
overall rating can never exceed three. This may not be sufficient to get a significant 3 W. E. De Leng et al. difference in the outcome measure when squaring the distance between both ratings. Future
research should examine the scoring method aspect of distance for SJTs using Likert scales
with more scale points. difference in the outcome measure when squaring the distance between both ratings. Future
research should examine the scoring method aspect of distance for SJTs using Likert scales
with more scale points. Lastly, the similar results for the three different central tendency statistics may be
explained by the distribution of the ratings across the Likert scale. Data with a symmetric
distribution are best summarized using the mean. Since the mean is strongly influenced by
extreme scores (Field 2013), asymmetrically distributed data are better summarized using
the median or mode. Practical implications The most important practical implication of this study is that it creates awareness about
the importance of carefully considering the immense number of possibilities for con-
verting the judgments on an SJT to a score. Instead of arbitrarily choosing one of the
many existing methods, researchers and practitioners should accompany the development
of an SJT with a thorough examination of the scoring method to be used. In addition, this
study demonstrated that the results when using the group of respondents itself are similar
to those obtained when using a group of SMEs as reference. Using the group of
respondents has practical and economic advantages, since the collection of data from
SMEs can be difficult. Unfortunately, this study does not allow any conclusive statements about which scoring
method is best, because the findings are highly dependent on this particular SJT measuring
this particular construct in this particular setting. Firstly, this study was conducted in the
Netherlands, where medical school applicants are relatively young (17–18 years). The use
of more mature applicants may lead to different results for scoring methods that use the
group of respondents itself as a reference. Secondly, the cultural context may influence the
way the reference group judges integrity-related dilemmas (Chandratilake et al. 2012; Jha
et al. 2015). Finally, SJTs measuring other constructs than integrity might be differentially
influenced by changing the scoring method. Future research should replicate this study
with other SJTs measuring different constructs in other settings to investigate the gener-
alizability of these findings and to provide clarity on which scoring method is best for
which situation. Scoring method aspects revisited A four-point Likert scale precludes extreme scores leading to similar
values for the mean, median and mode and likely causes the comparable results for this
scoring method aspect. Conclusion In conclusion, although the SJT scoring method is often chosen arbitrarily, this study
shows that changing the scoring method strongly influences the internal consistency reli-
ability and adverse impact of an SJT score. The most influential characteristic of a scoring
method is the way of controlling for systematic error. Given the increasing use of SJTs for
selection into medical school, it is crucial to thoroughly examine which scoring method is
best to use. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Inter-
national License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,
and reproduction in any medium, provided you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if changes were made. Strengths and limitations To our knowledge, this is the first study to compare such a large number of scoring
methods, varying not only the way of controlling for systematic error and the type of
reference group, but also the type of distance and central tendency statistic. Next to the
large number of scoring methods examined, this study also contributes to previous research
by the examining the effect of scoring method on internal consistency reliability. Embedding the administration of the SJT into the selection procedure led to a very high
response rate, ensuring that our results were not influenced by a volunteer bias. The
credibility of our results is further supported by a relatively small restriction of range. Unlike many other selection procedures, the current selection procedure was not preceded
by a pre-selection on cognitive competencies. 123 123 12 Scoring method of a Situational Judgment Test: influence on… Although this study compared a large number of scoring methods, we do not claim that
this list is exhaustive. Examples of other approaches for scoring SJTs are the squared
Mahalanobis distance (Barbot et al. 2012) and the use of paired comparisons (Gold and
Holodynski 2015). It seems that the possibilities are endless and future studies should
investigate these other scoring methods. For practical reasons, the number of scoring
methods in this study was limited to 28. Appendix 1: example scenario Michael questions Sarah, a fellow medical student about extreme and provocative com-
ments about individuals’ sexual preferences on her Facebook page. Sarah argues she
should be free to express her personal views. She also insists that her personal views have
no bearing on her performance as a medical student or patient care. How appropriate are each of the following responses by Michael in this situation? 1. Advise Sarah to remove all controversial comments from her Facebook page 2. Alert Facebook that Sarah’s page contains potentially inappropriate content as they
could remove it 3. Ask Sarah to ensure her privacy settings are restricted so her page is inaccessible to
patients or the general public 4. Inform a member of staff about Sarah’s Facebook comments 4. Inform a member of staff about Sarah’s Facebook comments 5. Withhold advice to Sarah as her views do not affect patient care or performance as a
medical student Appendix 2 See Table 5. 123 W. E. De Leng et al. .95
.94
.99
.98
.93
.92
.95
.96
.95
.97
.95
.95
.96
.97
.99
.91
.83
.84
.89
.85
.86
.86
.83
.84
.85
.84
.84
.93
.86
.85
.86
.85
.84
.85
.92
.98
.91
.84
.85
.92
.88
.89
.98
.92
.91
.87
.84
.84
.89
.87
.88
.92
.96
.95
.95
.87
.85
.86
.89
.88
.89
.91
.95
.96
.95
.99
.67
.72
.74
.66
.69
.70
.50
.60
.63
.54
.63
.64
.70
.78
.79
.70
.73
.74
.50
.59
.62
.54
.61
.64
.98
.65
.70
.72
.68
.70
.72
.48
.58
.61
.55
.64
.66
.96
.94
.65
.72
.74
.68
.72
.74
.47
.57
.60
.54
.62
.64
.95
.95
.61
.64
.68
.61
.63
.66
.61
.70
.72
.63
.71
.72
.88
.84
.60
.64
.67
.60
.62
.65
.60
.75
.76
.62
.74
.74
.87
.82
.57
.62
.65
.60
.62
.65
.53
.64
.65
.61
.70
.70
.87
.84
.58
.62
.66
.60
.62
.65
.53
.67
.69
.60
.73
.73
.87
.83
-.58
-.62
-.61
-.57
-.58
-.58
-.31
-.37
-.39
-.34
-.39
-.41
-.74
-.82
-.59
-.63
-.61
-.57
-.58
-.58
-.31
-.37
-.39
-.34
-.39
-.41
-.75
-.82
-.51
-.52
-.56
-.51
-.51
-.54
-.49
-.63
-.63
-.49
-.61
-.61
-.75
-.71
-.52
-.53
-.56
-.51
-.51
-.54
-.50
-.63
-.63
-.50
-.61
-.61
-.75
-.71
-.88
-.91
-.93
-.84
-.84
-.85
-.75
-.77
-.80
-.74
-.76
-.77
-.75
-.78
-.73
-.74
-.77
-.72
-.72
-.75
-.73
-.80
-.80
-.75
-.79
-.79
-.75
-.74
-.90
-.91
-.92
-.86
-.86
-.87
-.78
-.80
-.82
-.77
-.79
-.80
-.77
-.80
-.82
-.81
-.83
-.82
-.81
-.83
-.87
-.91
-.91
-.87
-.90
-.89
-.71
-.70 Scoring method of a Situational Judgment Test: influence on… Table 5 continued
Method
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. .99
17. .87
.85
18. .85
.83
.97
19. .91
.89
.96
.93
20. .91
.88
.95
.95
.99
21. -.74
-.77
-.58
-.57
-.60
-.60
22. -.75
-.77
-.58
-.57
-.60
-.60
1
23. -.73
-.70
-.81
-.85
-.76
-.80
.61
.60
24. -.74
-.71
-.81
-.86
-.76
-.80
.60
.60
1
25. Appendix 2 -.71
-.71
-.67
-.67
-.64
-.65
.67
.67
.59
.58 Method
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. .99
17. .87
.85
18. .85
.83
.97
19. .91
.89
.96
.93
20. .91
.88
.95
.95
.99
21. -.74
-.77
-.58
-.57
-.60
-.60
22. -.75
-.77
-.58
-.57
-.60
-.60
1
23. -.73
-.70
-.81
-.85
-.76
-.80
.61
.60
24. -.74
-.71
-.81
-.86
-.76
-.80
.60
.60
1
25. -.71
-.71
-.67
-.67
-.64
-.65
.67
.67
.59
.58 12 W. E. De Leng et al. Table 5 continued
Method
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 26. -.76
-.71
-.85
-.85
-.82
-.81
.56
.55
.69
.68
.77
27. -.74
-.74
-.71
-.71
-.67
-.68
.68
.68
.64
.63
.97
.81
28. -.71
-.70
-.83
-.83
-.78
-.79
.49
.47
.69
.68
.82
.94
.85
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The Role of Deep Learning in Parking Space Identification and燩rediction燬ystems
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The Role of Deep Learning in Parking Space Identification and Prediction
Systems
Rasheed, Faizan; Saleem, Yasir; Yau, Kok Lim Alvin; Chong, Yung Wey; Keoh, Sye Loong The Role of Deep Learning in Parking Space Identification and Prediction
Systems
Rasheed, Faizan; Saleem, Yasir; Yau, Kok Lim Alvin; Chong, Yung Wey; Keoh, Sye Loong Rasheed, Faizan; Saleem, Yasir; Yau, Kok Lim Alvin; Chong, Yung Wey; Keoh, Sye Loong Citation for published version (APA):
Rasheed, F., Saleem, Y., Yau, K. L. A., Chong, Y. W., & Keoh, S. L. (2023). The Role of Deep Learning in
Parking Space Identification and Prediction Systems. Computers, Materials and Continua, 75(1), 761-784. https://doi.org/10.32604/cmc.2023.034988 Citation for published version (APA):
Rasheed, F., Saleem, Y., Yau, K. L. A., Chong, Y. W., & Keoh, S. L. (2023). The Role of Deep Learning in
Parking Space Identification and Prediction Systems. Computers, Materials and Continua, 75(1), 761-784. https://doi.org/10.32604/cmc.2023.034988 Document License
CC BY Citation for published version (APA):
Rasheed, F., Saleem, Y., Yau, K. L. A., Chong, Y. W., & Keoh, S. L. (2023). The Role of Deep Learning in
Parking Space Identification and Prediction Systems. Computers, Materials and Continua, 75(1), 761-784.
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• You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal The Role of Deep Learning in Parking Space Identification
and Prediction Systems sheed1, Yasir Saleem2, Kok-Lim Alvin Yau3,*, Yung-Wey Chong4,* and Sye Loong Keoh 1School of Physics, Engineering and Computer Science, University of Hertfordshire, Hatfield, AL109AB, UK
2Department of Computer Science, Aberystwyth University, Aberystwyth SY23 3FL, UK
3Lee Kong Chian Faculty of Engineering and Science, Universiti Tunku Abdul Rahman,
Kajang, 43200, Selangor, Malaysia
4National Advanced IPv6 Centre (NAv6), Universiti Sains Malaysia, Penang, 11800, Malaysia
5School of Computing Science, University of Glasgow Singapore, 737729, Singapore
*Corresponding Authors: Kok-Lim Alvin Yau. Email: yaukl@utar.edu.my; Yung-Wey Chong. Email: chong@usm.my
Received: 03 August 2022; Accepted: 23 November 2022 1School of Physics, Engineering and Computer Science, University of Hertfordshire, Hatfield, AL109AB, UK
2Department of Computer Science, Aberystwyth University, Aberystwyth SY23 3FL, UK
3Lee Kong Chian Faculty of Engineering and Science, Universiti Tunku Abdul Rahman,
Kajang, 43200, Selangor, Malaysia
4National Advanced IPv6 Centre (NAv6), Universiti Sains Malaysia, Penang, 11800, Malaysia
5School of Computing Science, University of Glasgow Singapore, 737729, Singapore
*Corresponding Authors: Kok-Lim Alvin Yau. Email: yaukl@utar.edu.my; Yung-Wey Chong. Email: chong@usm.my
Received: 03 August 2022; Accepted: 23 November 2022 Abstract: In today’s smart city transportation, traffic congestion is a vexing
issue, and vehicles seeking parking spaces have been identified as one of the
causes leading to approximately 40% of traffic congestion. Identifying parking
spaces alone is insufficient because an identified available parking space may
have been taken by another vehicle when it arrives, resulting in the driver’s
frustration and aggravating traffic jams while searching for another parking
space. This explains the need to predict the availability of parking spaces. Recently, deep learning (DL) has been shown to facilitate drivers to find park-
ing spaces efficiently, leading to a promising performance enhancement in
parking identification and prediction systems. However, no work reviews DL
approaches applied to solve parking identification and prediction problems. Inspired by this gap, the purpose of this work is to investigate, highlight, and
report on recent advances in DL approaches applied to predict and identify the
availability of parking spaces. A taxonomy of DL-based parking identification
and prediction systems is established as a methodology by classifying and
categorizing existing literature, and by doing so, the salient and supportive
features of different DL techniques for providing parking solutions are pre-
sented. Moreover, several open research challenges are outlined. This work
identifies that there are various DL architectures, datasets, and performance
measures used to address parking identification and prediction problems. Take down policy Take down policy
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email: is@aber.ac.uk Download date: 24. Oct. 2024 Computers, Materials & Continua
DOI: 10.32604/cmc.2023.034988
Article Computers, Materials & Continua
DOI: 10.32604/cmc.2023.034988
Article Computers, Materials & Continua
DOI: 10.32604/cmc.2023.034988
Article ech
T
Press
Science This work is licensed under a Creative Commons Attribution 4.0 International License,
which permits unrestricted use, distribution, and reproduction in any medium, provided
the original work is properly cited. Keywords: Convolutional neural network; deep learning; neural networks;
parking identification; parking prediction; smart city Keywords: Convolutional neural network; deep learning; neural networks;
parking identification; parking prediction; smart city 1 Introduction Smart city promises to achieve sustainable development goals, particularly the sustainable cities
and communities goal, which focuses on making cities and human settlements inclusive, safe, resilient,
and sustainable. The evolution of the Internet of Things (IoT) and big data has been playing a vital
role in the feasibility of smart city initiatives [1–3]. Finding an available parking space is one of the
most challenging tasks in today’s smart city transportation system [4]. According to [5], approximately
40% of the traffic congestion in cities is caused by vehicles seeking parking spaces. Consequently,
traffic congestion, pollution emission, and traffic accidents are becoming severe. Identifying parking
spaces using real-time parking occupancy data provided by sensors has been proposed; however,
identification alone is insufficient because an available parking space identified for a vehicle may have
been taken by another vehicle when it arrives, resulting in the driver’s frustration. Searching for another
parking space can aggravate traffic jams. This explains the need to predict the availability of parking
spaces [6,7]. Research has been undertaken to predict and identify the availability of parking spaces within
a short time to facilitate drivers to find parking spaces efficiently. Deep learning (DL) has been
the preferred approach. Traditionally, DL has a deep neural network (DNN), such as multilayer
perceptron (MLP), and it comprises three main types of layers, namely the input, hidden, and output
layers [8,9]. Fig. 1 presents an example of the identification of the availability of parking spaces in
open parking lots using the DL approach. DL, specifically a convolutional neural network (CNN),
identifies whether or not a vehicle is present in each parking space. There are various DL architectures
applied to parking identification and prediction systems (see Section 2.1). Figure 1: An example of the identification of the availability of parking spaces in open parking lo
using DL [10]. The green and red rectangles show the availability and unavailability of parking space Figure 1: An example of the identification of the availability of parking spaces in open parking lots
using DL [10]. The green and red rectangles show the availability and unavailability of parking spaces Figure 1: An example of the identification of the availability of parking spaces in open parking lots
using DL [10]. The green and red rectangles show the availability and unavailability of parking spaces The Role of Deep Learning in Parking Space Identification
and Prediction Systems Moreover, there are some open-source implementations available that can be
used directly either to extend existing works or explore a new domain. This is
the first short survey article that focuses on the use of DL-based techniques
in parking identification and prediction systems for smart cities. This study
concludes that although the deployment of DL in parking identification and
prediction systems provides various benefits, the convergence of these two
types of systems and DL brings about new issues that must be resolved in
the near future. This work is licensed under a Creative Commons Attribution 4.0 International License,
which permits unrestricted use, distribution, and reproduction in any medium, provided
the original work is properly cited. 762 CMC, 2023, vol.75, no.1 1.2 Potential Applications of Parking Identification and Prediction Systems Parking space identification and prediction systems can be applied in a variety of applications. Parking space identification has been applied in: (a) autonomous valet parking systems to find a
parking space without human involvement [19]; (b) traffic light control systems to identify traffic
density on roads [20]; (c) autonomous electric cars to seek parking spaces equipped with the charging
facility [21]; (d) smart cars or robots to seek safer places during a disaster (e.g., flood) [22]; (e)
smart port to seek available places for cargo storage [23]; and (f) industry 4.0 to provide automatic
inventory storage [24,25]. Meanwhile, parking space prediction has been applied in driverless cars to
autonomously predict the availability of parking spaces which is useful in route planning [26,27]. 1.1 Impact of Parking Identification and Prediction Systems on Smart Cities Improving mobility and transportation is one of the major initiatives with significant contribu-
tions to the successful implementation of smart cities. Drivers looking for a parking space may cause
traffic congestion, so parking identification and prediction systems are related to route planning, CMC, 2023, vol.75, no.1 763 traffic management, and parking space management. According to [11], drivers who use information
related to the availability of parking spaces in making decisions are 45% more successful in availing
of parking spaces than those without. Hence, parking identification and prediction systems have
significant impacts on the efficiency of smart cities. DL has been achieving promising results in the
identification and prediction of the availability of parking spaces [12–18], which makes significant
impacts on smart cities. 1.3 Comparison with Existing Surveys 764 CMC, 2023, vol.75, no.1 Finally, in [32], a short survey focusing on enabling technologies to monitor, reserve, and manage
dynamic pricing in smart parking systems is presented. The survey includes categories, functionalities,
and the latest developments of smart parking systems, as well as a comparison of existing solutions
in terms of sensors, deployment, connectivity, services, and processing. None of the surveys focuses
on the DL-based parking identification and prediction system, which is the main focus of this
paper. In general, this paper discusses DL techniques, datasets, performance metrics, open-source
implementations, and open research directions for DL-based parking identification and prediction
systems. Inspired by previous review papers [4,28–31], this paper follows their structures and organizations. In general, the problems (i.e., parking space identification and prediction systems) and general
solutions (i.e., DL) are first introduced, followed by the specific description of proposed schemes in the
literature. Next, open issues and challenges are identified and elaborated. Finally, concluding remarks
are presented. More details about the organization of this paper are presented in Section 1.3. This paper
presents a review of state-of-the-art schemes proposed in research papers mainly published from 2019
to 2021. In short, while general reviews of designing parking management systems using IoT [29,30],
information and communication technology [4], algorithms [28] (e.g., static, dynamic, and real-time
algorithms), and automated valet parking [31], have been presented, this paper complements the
existing works by focusing on the DL approach, particularly on how DL models can be applied to
formulate the parking identification and prediction problems, and how the strengths of various DL
approaches can provide added advantages in addressing the challenges brought about by parking
management systems. Hence, this is the first review paper that provides a systematic and extensive
synthesis, analysis, and summary of DL approaches applied to parking identification and prediction
problems. This helps to identify research gaps in existing works and explore future research directions. 1.3 Comparison with Existing Surveys While there are some surveys in the literature focusing on different aspects of parking systems,
this is the first paper presenting a review of DL-based parking identification and prediction systems. In this section, existing surveys in the domain of parking systems are reviewed. Majority of surveys in the literature focus on smart parking systems rather than DL techniques. For instance, various types of smart parking systems and vehicle detection techniques, which mainly
use sensing outcomes from sensors, are presented in [28]. On the other hand, this paper presents a
survey of the literature based on four main aspects: (a) the types of available smart parking systems
in the literature; (b) the types of vehicle detection techniques used in smart parking systems; (c)
methods and algorithms; and (d) three stages of the implementation of smart parking systems, namely
proposal, simulation, and implementation. Based on the real-world implementation perspective, the
review of prediction models focusing on design factors and enabling technologies (e.g., sensors) has
been presented in [29,30]. In [20], smart parking systems are classified, and various sensors used in
smart parking systems are presented. Security and privacy are considered in interoperability and data
exchange. There are two types of design factors affecting the smart parking system performance: (a)
soft design factors include software, data processing, security, and privacy; and (b) hard design factors
include communication networks, errors, and reliability. In [21], the architecture of smart parking
systems is presented, and their components (e.g., sensors, communication protocols, and software
solutions) are explained and compared (e.g., in terms of usage trends). In [4], a survey of smart parking
systems focusing on three main topics, namely data collection, parking system deployment, and service
dissemination, is presented. Data collection is made through information sensing which depends on
sensor connectivity, parking meters, crowdsourcing, and shared parking. Parking system deployment
includes software systems, large-scale deployment, and the prediction of available parking spaces. Service dissemination disseminates information based on drivers’ behaviors and their competition in
getting a parking space. In [31], a survey of automated valet parking systems focusing on enabling
technologies, including platforms, sensors, localization, maps, and environmental models, is presented. A comparison between the Carnegie Mellon platform and the V-charge test vehicle is made covering
the features of LIDARs, radars, and cameras. The platforms include 3D geometric and semantic maps,
LIDAR- and video-based localizations, and environmental models with static and dynamic obstacles. 1.4 Novelty, Contributions, and Organization of this Paper Based on the literature search, there is a lack of research papers focusing on DL approaches
applied to solving parking identification and prediction problems. This paper is novel in two ways. Firstly, this is the first review paper that extensively covers the application of DL approaches to parking
systems to solve identification and prediction problems by presenting a comprehensive taxonomy. Secondly, this paper identifies various research gaps and challenges that open doors for future research
and advancements in the area of parking identification and prediction systems. Hence, the contributions of this work are as follows: Hence, the contributions of this work are as follows: • The roles played by DL in parking identification and prediction systems are highlighted, and
a review of recent premier advances in DL approaches applied to predict and identify the
availability of parking spaces is presented. • The roles played by DL in parking identification and prediction systems are highlighted, and
a review of recent premier advances in DL approaches applied to predict and identify the
availability of parking spaces is presented. • A taxonomy is devised to categorize and classify proposed schemes in the literature. • Open-source implementations of DL-based approaches for parking identification and predic-
tion systems are presented. • Open-source implementations of DL-based approaches for parking identification and predic-
tion systems are presented. y
p
• Open research challenges are identified and elaborated. This paper is organized as follows. Based on DL-based parking identification and prediction
systems, Sections 2 and 3 present a taxonomy and the role of DL, respectively. Section 4 presents recent
works. Section 5 presents open-source implementations. Section 6 presents open issues and research
challenges. Section 7 concludes this paper. 765 CMC, 2023, vol.75, no.1 2 Taxonomy of DL-Based Parking Identification and Prediction Systems
Fig. 2 presents the taxonomy explained in the rest of this section. Figure 2: Taxonomy of DL-based parking identification and prediction systems 2 Taxonomy of DL-Based Parking Identification and Prediction Systems Figure 2: Taxonomy of DL-based parking identification and prediction systems 2.1 DL Architectures There are seven architectures for DL-based parking identification and prediction systems as
follows: A.1 Multilayer perceptron (MLP) provides a continuous function to process data for performing
pattern classification, recognition, prediction, and approximation tasks [9,18]. MLP has been
adopted in [13] to predict the availability of parking spaces. In [13], the MLP architecture
consists of an input layer with four neurons, three fully-connected (FC) hidden layers with
five neurons each, and an output layer with five neurons as shown in Fig. 3. Data flows from
the input layer to the output layer. p
y
p
y
A.2 Convolutional neural network (CNN) uses convolutional layers to process visual data for
performing image processing, classification, and segmentation tasks [33]. CNN has been
adopted in [12] to identify the availability of parking spaces. CNN has two types of layers,
namely the convolutional layer and the traditional FC layer. In [12], the CNN architecture
consists of an input layer, three convolutional layers, an FC layer, and an output layer as
shown in Fig. 4. Each convolutional layer consists of convolution, pooling, and the activation
function, such as rectified linear unit (ReLU). Data flows from the input layer to the output
layer. 766 CMC, 2023, vol.75, no.1 766 Figure 3: An example of an MLP architecture [13]
Figure 4: An example of a CNN architecture [12] Figure 3: An example of an MLP architecture [13]
Figure 4: An example of a CNN architecture [12] Figure 3: An example of an MLP architecture [13] Figure 4: An example of a CNN architecture [12] Figure 4: An example of a CNN architecture [12] A.3 Region-based convolutional neural network (RCNN), which is based on CNN (A.2), provides
region proposals from images to perform object detection efficiently [34,35]. The region
proposals identify a set of candidate detections present in an image, such as a car and a truck
in an image. The RCNN architecture consists of an input layer, region proposals, a CNN
architecture, the FC layer, which is split into two separate streams, and an output layer for the
two streams as shown in Fig. 5. The input layer represents an image, which is first divided into
approximately 2,000 region proposals, and then CNN is applied to each region respectively. 2.1 DL Architectures Subsequently, bounding boxes, which are rectangles that serve as points of reference to detect
objects, are generated, and objects, such as a car or a truck, are classified in the output layer. RCNN has been adopted in [18] to identify the availability of parking spaces. Data flows from
the input layer to the output layer. Figure 5: Traditional RCNN architecture consists of a CNN architecture (see Fig. 4) [34] Figure 5: Traditional RCNN architecture consists of a CNN architecture (see Fig. 4) [34] re 5: Traditional RCNN architecture consists of a CNN architecture (see Fig. 4) [34] A.4 Graph neural network (GNN) provides graphs to process data and perform node-, edge-, and
graph-level prediction tasks [36]. GNN has been adopted in [15] to identify the availability of
parking spaces. In [15], the GNN architecture consists of an input layer, two hidden layers,
and an output layer, as shown in Fig. 6. There are two parts in each hidden layer, namely
graph and activation function (i.e., ReLU). Each graph is represented by vertices and links, A.4 Graph neural network (GNN) provides graphs to process data and perform node-, edge-, and
graph-level prediction tasks [36]. GNN has been adopted in [15] to identify the availability of
parking spaces. In [15], the GNN architecture consists of an input layer, two hidden layers,
and an output layer, as shown in Fig. 6. There are two parts in each hidden layer, namely
graph and activation function (i.e., ReLU). Each graph is represented by vertices and links, 767 CMC, 2023, vol.75, no.1 whereby each link is either directed or undirected between the vertices. In each graph, each
vertex learns information about the vertices in its neighborhood. Data flows from the input
layer to the output layer. Figure 6: An example of a GNN architecture [15] Figure 6: An example of a GNN architecture [15] A.5 Recurrent neural network (RNN) provides an internal memory to store previous inputs and
perform sequence prediction tasks. However, RNN can process short sequences only. The
traditional RNN architecture consists of an input layer It, hidden layers H, and an output
layer Ot as shown in Fig. 7. In Fig. 7, the input I0 is processed to generate output O0 in the
current time step. Subsequently, the output O0 is combined with the input I1 in the next time
step. 2.1 DL Architectures The output O1 is combined with the input I2 in the subsequent time step and so on. By doing so, the current input and output are obtained based on the previous input during
training. RNN has been adopted in [37–39] to predict the availability of parking spaces. Data
flows from the input layer to the output layer. Figure 7: Traditional RNN architecture [39] Figure 7: Traditional RNN architecture [39] A.6 Long short-term memory (LSTM), which is based on RNN (A.5), also provides memory with
gates to store previous inputs efficiently and perform sequence prediction tasks [40]. LSTM is
suitable for processing long sequences. The LSTM architecture consists of an input layer, an
LSTM layer, and an output layer as shown in Fig. 8. The LSTM layer has a memory cell that
consists of an input gate g, an output gate h, two multiplication nodes X, a summation node
+, and two activation function gates δ. The multiplication and summation nodes perform
multiplication and addition computation tasks, respectively. The activation function gates
transform data into a value within the range of 0 and 1. LSTM has been adopted in [16]
to predict the availability of parking spaces. Data flows from the input layer to the output
layer. 768 768 CMC, 2023, vol.75, no.1 Figure 8: Traditional LSTM architecture [40] Figure 8: Traditional LSTM architecture [40] A.7 Dilated convolutional neural network (DCNN), which is based on CNN (A.2), replaces the
simple convolutional layer with the dilated convolutional layer in its architecture. The dilated
convolutional layer expands the input (or kernel) by inserting holes between its consecutive
elements. Compared to the traditional CNN (A.2) architecture, DCNN has a larger receptive
field covering a large area of input images. The difference between a simple convolutional
layer and a dilated convolutional layer is depicted in Fig. 9. DCNN has been adopted in [41]
to identify the availability of parking spaces. Figure 9: The illustration of the convolutional and dilated convolutional layers [42]. The dilated
convolutional layer has a larger receptive field Figure 9: The illustration of the convolutional and dilated convolutional layers [42]. The dilated
convolutional layer has a larger receptive field 1https://web.inf.ufpr.br/vri/databases/parking-lot-database/
2https://cslinzhang.github.io/deepps/
3http://claudiotest.isti.cnr.it/park-datasets/CNR-EXT/
4https://www.sfmta.com/demand-responsive-parking-pricing
5https://data.smgov.net/ 2.2 Systems There are two systems used to find the availability of parking spaces in the context of DL as
follows: S.1 The identification system identifies and updates the availability of parking spaces in a real-
time manner. For instance, the system identifies an available parking space when a vehicle
arrives at a parking lot [12,15]. p
g
[
,
]
S.2 The prediction system predicts the availability of parking spaces. For instance, the system
predicts the availability of every single parking space in a parking lot [13]. p
g
[
,
]
S.2 The prediction system predicts the availability of parking spaces. For instance, the system
predicts the availability of every single parking space in a parking lot [13]. CMC, 2023, vol.75, no.1 769 2.3 Datasets p
g p
p
y
D.6 CNRPark + EXT 3 [12] is a dataset composed of around 150,000 labeled images of 164 available
and unavailable parking spaces in a parking lot. The images were collected using 9 cameras
under different weather conditions from November 2015 to February 2016. D.6 CNRPark + EXT 3 [12] is a dataset composed of around 150,000 labeled images of 164 available
and unavailable parking spaces in a parking lot. The images were collected using 9 cameras
under different weather conditions from November 2015 to February 2016. D.7 Birmingham [38] is a dataset composed of the occupancy of 29 parking spaces operated by
National Car Parks in Birmingham, UK. Data was updated every 30 min from 8:00 am to
4:30 pm. The dataset is licensed under the Open Government Licenses v3.0. D.8 San Francisco4 is a dataset composed of the sensing outcomes of 8,200 parking sensors
deployed at on-street metered parking lots and gate-controlled off-street locations in San
Francisco. The dataset was collected from 12th August 2013 to 22nd September 2013, and it
was updated every 15 min. There are 3,948 records for each of the 572 street blocks totaling
3,948 × 572 = 2,258,256 records. D.9 Melbourne [44] is a dataset composed of the sensing outcomes of 7,114 parking sensors (i.e.,
in-ground sensors) deployed along 75 streets (or around 270 street blocks) throughout 24 areas
in the Melbourne central business district. The dataset was collected from 1st October 2011 to
30th September 2012 totaling 12,208,178 records of parking events. D.9 Melbourne [44] is a dataset composed of the sensing outcomes of 7,114 parking sensors (i.e.,
in-ground sensors) deployed along 75 streets (or around 270 street blocks) throughout 24 areas
in the Melbourne central business district. The dataset was collected from 1st October 2011 to
30th September 2012 totaling 12,208,178 records of parking events. D.10 Santa Monica5 [45] is a dataset collected from 9 parking lots in Santa Monica, California from
7:00 am on 11th May 2018 to 9:50 pm on 11th June 2018. Data was collected every 5 min. D.10 Santa Monica5 [45] is a dataset collected from 9 parking lots in Santa Monica, California from
7:00 am on 11th May 2018 to 9:50 pm on 11th June 2018. Data was collected every 5 min. 2.3 Datasets A parking lot is a reserved area with parking spaces, which are slots for vehicles to park. There are
eleven main parking lot datasets used in the literature to evaluate and validate the proposed schemes
based on DL, which are as follows: D.1 PKLot1 [10] is a dataset composed of 12,417 images of parking lots and 695,899 images of
parking spaces, which were captured using three different camera views at two parking lots. These parking lots were located at the Pontifical Catholic University of Parana (PUCPR) and
the Federal University of Parana (UFPR) in Curitiba, Brazil. y
(
)
D.2 PS2.02 [43] is a dataset composed of 9,827 training images of parking lots and 9,476 images of
parking spaces. It has 2,338 testing images of parking lots and 2,168 images of parking spaces. The parking lot is located at Tongji University, Shanghai, China. D.2 PS2.02 [43] is a dataset composed of 9,827 training images of parking lots and 9,476 images of
parking spaces. It has 2,338 testing images of parking lots and 2,168 images of parking spaces. The parking lot is located at Tongji University, Shanghai, China. p
g
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y
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D.3 PSV is a dataset composed of 4,200 images of parking spaces. The parking lot was located at
Tongji University in Shanghai, China. D 4 Santander’s dataset is a dataset composed of the measurements collected by almost 400 PSV is a dataset composed of 4,200 images of parking spaces. The parking lot was loca
Tongji University in Shanghai, China. D.4 Santander’s dataset is a dataset composed of the measurements collected by
parking sensors deployed in the main parking lot of Santander in Spain. D.5 Sii-Mobility [37] is a dataset collected from 12 parking spaces located at three different
locations (i.e., downtown, hospitals, and outskirts such as parks and ride systems) within 81
days between 5th January 2017 and 26th March 2017 in Florence, Italy. The status of each
parking space was updated every 15 min. D.5 Sii-Mobility [37] is a dataset collected from 12 parking spaces located at three different
locations (i.e., downtown, hospitals, and outskirts such as parks and ride systems) within 81
days between 5th January 2017 and 26th March 2017 in Florence, Italy. The status of each
parking space was updated every 15 min. 2.3 Datasets D.10 Santa Monica5 [45] is a dataset collected from 9 parking lots in Santa Monica, California from
7:00 am on 11th May 2018 to 9:50 pm on 11th June 2018. Data was collected every 5 min. y
p
y
D.11 Self-created dataset is a dataset composed of a large number of images or measurements of
sensors, which were generated and annotated manually rather than using any publicly available
datasets. y
p
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D.11 Self-created dataset is a dataset composed of a large number of images or measurements of
sensors, which were generated and annotated manually rather than using any publicly available
datasets D.11 Self-created dataset is a dataset composed of a large number of images or measurements of
sensors, which were generated and annotated manually rather than using any publicly available
datasets. 770 CMC, 2023, vol.75, no.1 p
P.2 Precision Precision =
Tp
Tp + Fp Precision =
Tp
Tp + Fp
(2) Precision =
Tp
Tp + Fp (2) where Tp represents the number of true positive values, and Fp represents the number of false
positive values. where Tp represents the number of true positive values, and Fp represents the number of false
positive values. P3 R
ll p
P.3 Recall p
P.3 Recall p
P.3 Recall Recall =
Tp
Tp + Fn
(3) Recall =
Tp
Tp + Fn (3) where Fn represents the number of false negative values. where Fn represents the number of false negative values where Fn represents the number of false negative values. P4 M
b
l t
(MAE) p
P.4 Mean absolute error (MAE) p
P.4 Mean absolute error (MAE) MAE =
|Pv −Av|
ns AE =
|Pv −Av|
n MAE =
|Pv −Av|
ns
(4) |Pv −Av|
ns
(4) (4) where Pv represents the predicted value, and Av represents the actual value. where Pv represents the predicted value, and Av represents p
p
p
P.5 Root mean square error (RMSE) is a quadratic scoring fu P.5 Root mean square error (RMSE) is a quadratic scoring function as follows: 2.4 Performance Measures There are eight performance measures achieved by various DL approaches to predict and identify
the availability of parking spaces. The self-explanatory equations for the performance measures are as
follows: P.1 Accuracy P.1 Accuracy
Accuracy = np
ns
(1) y
uracy = np
ns Accuracy = np
ns (1) where np represents the total number of correct predicted values, and ns represents the total
P.2 Precision where np represents the total number of correct predicted values, and ns represents the total
P.2 Precision p
P.2 Precision MASE = mean
Ov −Pv
1
np−1
i=2
np Ovi −Ovi−1
RMSE =
(Pv −Av)
2
ns
P.6 Mean absolute percentage error (MAPE)
MAPE = 1
ns
|Pv −Av|
Av
P.7 Root relative squared error (RRSE)
RRSE = RMSE
(Pv−v)2
ns
where v represents the last known value.
P.8 Mean absolute scaled error (MASE)
MASE = mean
Ov −Pv
1
np−1
i=2
np Ovi −Ovi−1
where Ov represents the observed value RMSE =
(Pv −Av)
2
ns
(5)
Mean absolute percentage error (MAPE)
MAPE
1 |Pv −Av|
(6) (5)
s
P.6 Mean absolute percentage error (MAPE)
s
P.6 Mean absolute percentage error (MAPE) (6) P.7 Root relative squared error (RRSE) P.7 Root relative squared error (RRSE) (7) where v represents the last known value. where v represents the last known value. p
P.8 Mean absolute scaled error (MASE) p
P.8 Mean absolute scaled error (MASE) (8) where Ov represents the observed value CMC, 2023, vol.75, no.1 771 3 The Roles of DL Architectures in Parking Identification and Prediction Systems 3 The Roles of DL Architectures in Parking Identification and Prediction Systems DL plays an important role in parking identification and prediction systems. There are seven main
DL architectures, namely MLP (A.1), CNN (A.2), RCNN (A.3), GNN (A.4), RNN (A.5), LSTM
(A.6), and DCNN (A.7), applied to identify (S.1) and predict (S.2) the availability of parking spaces in
the literature. The DL architectures are essential to cater to the continuous and high-dimensional data
related to the system. Fig. 10 presents the general framework of DL applied to parking identification
and prediction systems. Table 1 summarizes various enhancements of DL architectures to address the
challenges of parking identification and prediction systems. Figure 10: The general framework of DL applied to parking identification and prediction systems. The input has a data processing module processing dataset and raw data. The input is fed into the DL
architecture for identification and prediction. The output module provides an available parking space. If there is no parking space available, the car may either look at the same or another parking lot Figure 10: The general framework of DL applied to parking identification and prediction systems. The input has a data processing module processing dataset and raw data. The input is fed into the DL
architecture for identification and prediction. The output module provides an available parking space. If there is no parking space available, the car may either look at the same or another parking lot Table 1: Summary of enhancements in DL architectures to address the challenges of parking identifi-
cation and prediction systems
DL architectures
Enhancements
Layers in architecture
Strength
A.1 MLP
Provides a continuous
function to process data and
perform pattern classification,
recognition, prediction, and
approximation tasks. Uses FC layers. Performs prediction
efficiently. A.2 CNN
Provides convolutional layers
to process visual data and
perform image processing,
classification, and
segmentation tasks. Uses convolutional and
FC layers. Performs
classification. A.3 RCNN
Provides region proposals of
images to perform object
detection efficiently. Uses region proposals,
convolutional, and FC
layers. Performs
classification and
detection with higher
accuracy. A.4 GNN
Provides graphs to process
data and perform node-,
edge-, and graph-level
prediction tasks. Uses graph layers. Performs graph-level
prediction efficiently. (Continued) 772 CMC, 2023, vol.75, no.1 Table 1: Continued Table 1: Continued
DL architectures
Enhancements
Layers in architecture
Strength
A.5 RNN
Provides memory to store
previous inputs and perform
sequence prediction tasks. Uses FC layers. Provides memory to
store previous inputs
and perform
prediction. 3 The Roles of DL Architectures in Parking Identification and Prediction Systems A.6 LSTM
Provides memory to store
previous inputs efficiently and
perform sequence prediction
tasks more efficiently. Uses an LSTM layer
that consists of a
memory cell. Provides memory to
store previous inputs
and perform
prediction with
higher accuracy. A.7 DCNN
Provides dilated convolution
to expand inputs and perform
object detection efficiently. Uses dilated
convolutional and FC
layers. Performs
classification and
detection with higher
accuracy. Uses an LSTM layer
that consists of a
memory cell. Uses dilated
convolutional and FC
layers. Uses dilated
convolutional and FC
layers. 3.1 The DL Approach Based on the MLP Architecture The DL approach based on the MLP architecture (A.1) (see Fig. 3) has been applied in [13] to
predict parking space availability (S.2). The MLP architecture consists of: (a) the input layer represents
input data comprised of the measurements collected by sensors; (b) the hidden layer(s) have FC layers
and activation functions (e.g., ReLU); and (c) the output layer predicts the availability of every single
parking space in a parking lot. 3.2 The DL Approach Based on the CNN and RCNN Architectures The DL approach, which is based on the CNN (A.2) (see Fig. 4) and RCNN (A.3) (see Fig. 5)
architectures, has been applied in [12,14,17] to identify parking space availability (S.1). The CNN
architecture consists of: (a) the input layer represents an input image of a parking lot (or parking
spaces); (b) the convolutional layer has the convolution and pooling layers, and activation functions
(e.g., ReLU); (c) the FC layers; and (d) the output layer classifies objects, such as a car and an empty
parking space. 3.4 The DL Approach Based on the RNN Architecture The DL approach based on the RNN architecture (A.5) (see Fig. 7) has been applied in [38] to
predict parking space availability (S.2). The RNN architecture consists of: (a) the input layer represents
input data comprised of the measurements collected by sensors; (b) the hidden layer(s) have FC layers
and activation functions (e.g., ReLU); and (c) the output layer predicts the availability of every single
parking space in a parking lot. 3.2.1 Enhancements There are two main enhancements to the RCNN (A.3) (see Fig. 5) architecture. Firstly, region
proposals are provided to identify a set of candidate detections present in an image. Region proposals
allow the DNN network to identify where to look in an image and detect the exact objects. By using
region proposals, DNN enhances the accuracy of object detection. Secondly, the output layer is split
into two separate streams to generate bounding boxes and classify objects, such as a car and an
empty parking space. This enhancement allows DNN to classify objects accurately and tell DNN
where objects are located in an image by generating bounding boxes around objects. In contrast, the
traditional CNN only predicts the class of an object. This enhancement enables DNN to increase its
accuracy in object classification. While these enhancements increase the accuracy of detection and
classification, particularly when the number of classes (e.g., a car, a truck, an empty parking space,
and so on) is large, they increase computational cost and processing time. This limitation, which 773 CMC, 2023, vol.75, no.1 is associated with the RCNN architecture, has been solved using Fast RCNN although it has not
been investigated in the context of parking identification and prediction systems. Compared with
RCNN, the Fast RCNN architecture has convolutional layers before the region proposals [46]. Fast
RCNN architecture uses convolutional, region proposals, and FC layers. It performs classification and
detection tasks with higher speed and accuracy as compared to RCNN [46]. The main objective of Fast
RCNN is to reduce computational cost and processing time, and hence it can predict the locations of
different objects in an image quickly and accurately. 3.3 The DL Approach Based on the GNN Architecture The DL approach based on the GNN architecture (A.4) (see Fig. 6) has been applied in [15]
to identify parking space availability (S.1). The GNN architecture consists of: (a) the input layer
represents an input image of a parking lot (or parking spaces); (b) the hidden layer(s) have graphs
and activation functions (e.g., ReLU); and (c) the output layer classifies objects, such as a car and an
empty parking space. 3.5 The DL Approach Based on the LSTM Architecture The DL approach based on the LSTM architecture (A.6) (see Fig. 8) has been applied in [16]
to predict parking space availability (S.2). The LSTM architecture consists of: (a) the input layer
represents input data comprised of the measurements collected by sensors; (b) the LSTM layer consists
of a memory cell; and (c) the output layer predicts the availability of every single parking space in a
parking lot. 3.2.2 Applications The enhancements have been applied to RCNN in [18] to identify parking space availability (S.1),
whereby the RCNN architecture consists of five types of layers: (a) the input layer; (b) the region
proposals layer divides an input image into approximately 2,000 region proposals and identifies a set
of candidates detected in an image; (c) the convolutional layers; (d) the FC layers; and (e) the output
layer has two streams to generate the bounding boxes and classify the objects, such as a car and an
empty parking space. 4 Recent Works on the Deployment of DL in Parking Identification and Pre 4 Recent Works on the Deployment of DL in Parking Identification and Prediction Syst This section presents a review of some recent works on DL applied to parking identification and
prediction systems. Table 2 presents a comparison of the attributes of DL-based approaches in parking
identification and prediction systems. Table 3 presents a comparison of key contributions and future
directions of parking identification and prediction system investigations. 4.1 Deployment of the MLP Architecture In [13], a comparative analysis of the MLP approach (A.1) and other machine learning techniques
(i.e., K-nearest neighbors, decision tree, random forest, and voting classifier) is presented for the
prediction (S.2) of parking space availability. Analysis was conducted for a validity period of 10 to
20 min using the Santander’s dataset (D.4), and it has been shown to achieve a high accuracy rate
(P.1), high precision rate (P.2), and high recall rate (P.3) in predicting the availability of parking spaces. In [47], two types of prediction approaches (S.2) are proposed to predict parking space availability. The first approach is based on the probability that an available parking lot continues to be available in
the coming time intervals. The second approach is based on the prediction of the short-term occupancy
of parking lots in specific regions. Using MLP (A.1), predictions for the next 1, 5, 15, and 30 min were
based on the 1-min parking data of Santander’s dataset (D.4). It has been shown to achieve better
MAE (P.4), RMSE (P.5), MAPE (P.6) and RRSE (P.7) in predicting the availability of parking spaces. 3.6 The DL Approach Based on the DCNN Architecture The DL approach based on the DCNN architecture (A.7) (see Fig. 9) has been applied in [41]
to identify parking space availability (S.1). The DCNN architecture consists of: (a) the input layer
represents an input image of a parking lot (or parking spaces); (b) the dilated convolutional layer
consists of the dilated convolution and pooling layers, and activation functions (e.g., ReLU); (c) the
FC layer; and (d) the output layer classifies objects, such as a car and an empty parking space. 774 CMC, 2023, vol.75, no.1 CMC, 2023, vol.75, no.1 4.2 Deployment of the CNN Architecture In [12], a CNN-based approach (A.2) runs on a Raspberry Pi platform equipped with a smart
camera module for the identification (S.1) of parking space availability. The main deployment
challenge is the visual classification problem caused by shadows and obstacles (i.e., trees, lamps, or
people), which occupy parking spaces. The proposed scheme uses the redundancy of overlapping
parking spaces to reduce the effects of obstacles (e.g., trees). Based on the PKLot (D.1) dataset, the
approach has been shown to achieve a high accuracy rate (P.1) in identifying the availability of parking
spaces. The work presented in [17] is an extension of [12]. In [17], a CNN-based approach (A.2) acquires
local and global information simultaneously and integrates them for the identification (S.1) of parking
space availability. The local information includes the orientation and location of junctions in parking
lots, and the global information includes the occupancy (e.g., a car or a truck) and types (e.g., a
parallel or perpendicular parking space). Based on the PS2.0 (D.2) dataset, the approach achieves
a high accuracy rate (P.1), precision rate (P.2), and recall rate (P.3) in identifying the availability of
parking spaces. The work presented in [14] is an extension of [12]. In [14], a CNN-based approach (A.2) converts
the empty parking space identification task into a two-step problem: (a) parking space identification;
and (b) occupancy classification for the identification (S.1) of parking space availability. The approach
also updates the PS2.0 (D.2) and PSV (D.3) datasets by labeling the type of parking space in each image. Based on the datasets, the approach has been shown to achieve a high precision rate (P.2) and recall
rate (P.3) in identifying the availability of parking spaces. CMC, 2023, vol.75, no.1
775
Table 2: Summary of the DL-based approaches attributes in parking identification and prediction systems
DL archi-
tectures
Reference
Sys. Datasets
Performance
S.1 Identification
S.2 Prediction
D.1 PKLot
D.2 PS2.0
D.3 PSV
D.4 Santander
D.5 Sii-Mobility
D.6 CNRPark + EXT
D.7 Birmingham
D.8 San Francisco
D.9 Melbourne
D.10 Santa Monica
D.11 Self-created
P.1 Accuracy
P.2 Precision
P.3 Recall
P.4 MAE
P.5 RMSE
P.6 MAPE
P.7 RRSE
P.8 MASE
A.1 MLP
Amato et al. (2020) [13]
×
×
×
×
×
Vlahogianni
et al. (2016) [47]
×
×
×
×
×
×
A.2 CNN
Amato et al. (2017) [12]
×
×
×
×
Suhr et al. (2021) [17]
×
×
×
×
×
Li et al. 4.2 Deployment of the CNN Architecture (2020) [14]
×
×
×
×
×
Yu et al. (2020) [48]
×
×
×
Zhang et al. (2018) [43]
×
×
×
×
Huang et al. (2019) [49]
×
×
×
×
Zheng et al. (2015) [44]
×
×
×
×
×
A.3 RCNN
Zinelli et al. (2019) [18]
×
×
×
×
A.4 GNN
Min et al. (2021) [15]
×
×
×
×
×
A.5 RNN
Camero et al. (2018) [38]
×
×
×
Badii et al. (2018) [37]
×
×
×
×
A.6 LSTM
Hochreiter et al. (1997) [16]
×
×
×
Feng et al. (2021) [45]
×
×
×
×
×
A.7 DCNN
Nurullayev et al. (2019) [41]
×
×
×
× 775 CMC, 2023, vol.75, no.1 776 CMC, 2023, vol.75, no.1 CMC, 2023, vol.75, no.1 Other CNN-based approaches have been proposed. In [48], CNN (A.2) performs the identification
(S.1) of parking space availability using the PS2.0 dataset (D.2). It detects the corners and line features
of parking lots. It has been shown to achieve a high accuracy rate (P.1) of around 98% in identifying the
availability of parking spaces. In [49], CNN (A.2) performs the identification (S.1) of parking space
availability using directional marking-point regression (DMPR-PS) and the PS2.0 dataset (D.2). It
has been shown to achieve a high precision rate (P.2) and recall rate (P.3) in identifying the availability
of parking spaces. In [43], CNN (A.2) performs the identification (S.1) of parking space availability,
namely DeepPS. The investigation uses a new large-scale labeled dataset, namely PS2.0 (D.2), which
has been released for public use. DeepPS has been shown to achieve a high precision rate (P.2) and
recall rate (P.3) in identifying the availability of parking spaces. In [44], CNN (A.2) is incorporated
with two other machine learning techniques, namely regression tree and support vector regression, for
the prediction (S.2) of parking space availability using the San Francisco (D.8) and Melbourne (D.9)
datasets. It has been shown to achieve better MAE (P.4) and RMSE (P.5) in predicting the availability
of parking spaces. Table 3: Summary of key contributions and future directions of DL-based approaches in parking
identification and prediction systems
DL architectures
Reference
Key contributions
Future directions
A.1 MLP
Amato et al. (2020) [13]
Compares MLP with
other artificial
intelligence (AI)
techniques. Extensions with: (a)
emerging DL approaches;
and (b) recommender
systems. Vlahogianni et al. (2016) [47]
Investigates two types of
prediction systems (S.2). Embeds the proposed
approach into AI chips.
Establishes a more
extensive scale dataset, and
improves the accuracy of
the proposed scheme. 4.3 Deployment of the RCNN Architecture The work presented in [18] is an extension of [17] (see Section 4.2). In [18], the RCNN-based
approach (A.3) is suitable for handling generic quadrilaterals and rectangular bounding boxes [12]
while detecting parking spaces for the identification (S.1) of parking space availability. Specifically, it
can process images of parking spaces, which are in both quadrilateral and rectangular shapes, captured
from different angles. Self-created dataset (D.11), which is composed of around 400 bird’s eye view
(BEV) images, was used and it has been shown to achieve a high accuracy rate (P.1) and precision rate
(P.2) in identifying the availability of parking spaces. 4.2 Deployment of the CNN Architecture Extension with the
detection of complex
parking spaces (e.g.,
oblique and trapezoid). A.5 RNN
Camero et al. (2018) [38]
Investigates RNN. Explores other applications
(e.g., energy consumption
forecast). Badii et al. (2018) [37]
Compares RNN with
support vector
regression. A.6 LSTM
Hochreiter et al. (1997)
[16]
Investigates LSTM. Uses context information
to guide drivers in route
planning and car parking. Feng et al. (2021) [45]
Investigates a hybrid
DL mode based on
CNN (A.2) and LSTM
(A.6). A.7 DCNN
Nurullayev et al. (2019)
[41]
Investigates DCNN. Improves the accuracy of
image classification. Table 3: Continued Table 3: Continued Uses context information
to guide drivers in route
planning and car parking. 4.2 Deployment of the CNN Architecture (a) Uses a dataset
comprised of parking
occupancy and duration;
and (b) uses complex DL
approaches. A.2 CNN
Amato et al. (2017) [12]
Investigates CNN. Suhr et al. (2021) [17]
Integrates local and
global information. Embeds the proposed
approach into AI chips. Li et al. (2020) [14]
Converts the parking
space identification task
into a two-step problem. Establishes a more
extensive scale dataset, and
improves the accuracy of
the proposed scheme. Yu et al. (2020) [48]
Investigates CNN to
provide a faster speed
and a smaller size. Huang et al. (2019) [49]
Investigates the
directional
marking-point
regression (DMPR-PS). Extension with
regression-based parking
space detection
approaches. of key contributions and future directions of DL-based approaches in parking
ediction systems Embeds the proposed
approach into AI chips. Establishes a more
extensive scale dataset, and
improves the accuracy of
the proposed scheme. Extension with
regression-based parking
space detection
approaches. (Continued) 777 CMC, 2023, vol.75, no.1 Table 3: Continued
DL architectures
Reference
Key contributions
Future directions
Zhang et al. (2018) [43]
Investigates CNN and
the PS2.0 (D.2) dataset. Enlarges a dataset to
provide a better
benchmark. Zheng et al. (2015) [44]
Compares CNN with
other AI techniques. Incorporates other factors
(e.g., social events). A.3 RCNN
Zinelli et al. (2019) [18]
Investigates RCNN and
BEV images. Uses original images
instead of BEV images. A.4 GNN
Min et al. (2021) [15]
Investigates GNN. Extension with the
detection of complex
parking spaces (e.g.,
oblique and trapezoid). A.5 RNN
Camero et al. (2018) [38]
Investigates RNN. Explores other applications
(e.g., energy consumption
forecast). Badii et al. (2018) [37]
Compares RNN with
support vector
regression. A.6 LSTM
Hochreiter et al. (1997)
[16]
Investigates LSTM. Uses context information
to guide drivers in route
planning and car parking. Feng et al. (2021) [45]
Investigates a hybrid
DL mode based on
CNN (A.2) and LSTM
(A.6). A.7 DCNN
Nurullayev et al. (2019)
[41]
Investigates DCNN. Improves the accuracy of
image classification. Table 3: Continued
DL architectures
Reference
Key contributions
Future directions
Zhang et al. (2018) [43]
Investigates CNN and
the PS2.0 (D.2) dataset. Enlarges a dataset to
provide a better
benchmark. Zheng et al. (2015) [44]
Compares CNN with
other AI techniques. Incorporates other factors
(e.g., social events). A.3 RCNN
Zinelli et al. (2019) [18]
Investigates RCNN and
BEV images. Uses original images
instead of BEV images. A.4 GNN
Min et al. (2021) [15]
Investigates GNN. 4.6 Deployment of LSTM Architecture In [16], an LSTM-based approach (A.6) uses a mobile application to check the live status
of parking spaces and book parking spaces in advance for the prediction (S.2) of parking space
availability. The approach uses a self-created dataset (D.11), which is comprised of the number of
parking spaces, occupancy status, date, and time, and it has been shown to achieve a high accuracy rate
(P.1) in predicting the availability of parking spaces. In [45], a DL model, which is based on CNN (A.2)
and LSTM (A.6), is proposed to predict (S.2) the availability of parking spaces using the Santa Monica
dataset (D.10). By combining two DL architectures (i.e., CNN (A.2) and LSTM (A.6)), the proposed
scheme can accurately make short-term (i.e., ≤30 min) and long-term (i.e., >30 min) predictions of the
parking spaces. The proposed scheme has been shown to achieve better MAE (P.4), RMSE (P.5), and
MAPE (P.6) in predicting the availability of parking spaces. 4.5 Deployment of the RNN Architecture 4.5 Deployment of the RNN Architecture In [38], an RNN-based approach (A.5) for the prediction (S.2) of parking space availability has
been conducted using the Birmingham dataset (D.7). It has been shown to achieve a better MAE (P.4)
in predicting the availability of parking spaces. In [37], a comparative analysis of RNN (A.5) and a
machine learning technique called support vector regression using the Sii-Mobility dataset (D.5) is
performed for the prediction (S.2) of parking space availability. It has been demonstrated that RNN
outperforms others and achieves better RMSE (P.5) and MASE (P.8) in predicting the availability of
parking spaces. 4.4 Deployment of the GNN Architecture In [15], a GNN-based approach (A.4) uses the link information (e.g., location) of the marking
points in an image to identify image features (e.g., empty parking spaces). The marking points (or
vertices (A.4)) are represented as graph-structured data, which consists of vertices and links processed
using GNN. Based on the PS2.0 (D.2) and PSV (D.3) datasets, the approach has been shown to achieve
high precision (P.2) and recall (P.3) rates in identifying the availability of parking spaces. 778 CMC, 2023, vol.75, no.1 CMC, 2023, vol.75, no.1 CMC, 2023, vol.75, no.1 4.7 Deployment of DCNN Architecture In [41], a DCNN-based approach (A.7) for the identification (S.1) of parking space availability
has been conducted using the PKLoT (D.1) and CNRPark + EXT (D.6) datasets. It has been shown
to achieve a high accuracy rate (P.1) in identifying the availability of parking spaces. 5 Open-Source Implementations of DL-Based Parking Identification and Prediction Systems Open-source implementations are mainly useful for two purposes. Firstly, researchers who plan
to extend existing works can use open-source codes and work on their extensions without the
need of reinventing the wheel. Secondly, researchers who are new to the field can use open-source
codes as baselines for their understanding before starting to implement their own approaches, which
may be based on open-source codes. Due to the importance of open-source implementations, such
implementations related to DL-based parking identification and prediction systems are discussed. For parking space identification (S.1), in [43], DeepPS is proposed, and it has been widely used
in the literature for CNN-based approaches (A.2). An open-source implementation of DeepPS in
MATLAB and PS2.0 (D.2), and a large-scale labeled dataset, are provided in the website6. The work
presented in [49] is an extension of [43]. In [49], using directional marking-point, DMPR-PS, which is
a CNN-based (A.2) multi-attribute regression model, is proposed to identify parking spaces based on
the PS2.0 dataset (D.2). Its source code in Python is available at GitHub7. For parking space prediction (S.2), in [38], an open-source implementation of DLOPT, which is
an RNN-based approach (A.5), is proposed to predict the availability of parking spaces based on the
Birmingham dataset (D.7). Its source code in Python is available at GitHub8. 779 CMC, 2023, vol.75, no.1 6 Looking Forward: Open Research Challenges This paper has presented a review of some recent premier advances in DL approaches applied
to predict and identify the availability of parking spaces. Based on the recent works, open research
challenges in the deployment of DL in parking identification and prediction systems are highlighted
in this section. 6.1 Considering Environmental Conditions Considering real-world environmental conditions while training DL models helps to reflect real-
world scenarios. In [14], the effects of environmental conditions, such as rainfall and shadows, have
been considered. However, other environmental conditions, such as snowfall, darkness, and foggy
conditions, should be considered in the training of DL models for more accurate results. The main
challenge brought about by these environmental conditions is the reduced accuracy of object detection
and classification due to the need to detect and classify a diverse range of objects, including cars, trucks,
and empty spaces, under dark, snowfall, and foggy conditions. In real-world scenarios, there are various types of parking spaces, such as on-street, off-street,
parallel, perpendicular, 30-degree angle, and 45-degree angle parking spaces [50]. In [17], parallel and
perpendicular parking spaces have been considered. However, other types of parking spaces should be
considered to reflect real-world scenarios. The main challenge is that considering a diverse range of
parking spaces takes a longer time in object detection and classification. 6.2 Integration of Future Generation Technologies Future-generation technologies, such as autonomous vehicles equipped with cameras, sensors,
lidars, and radars [51], can be integrated with DL to provide performance enhancement in automation. For instance, in the literature, autonomous vehicles have not been considered while deploying DL
models to identify and predict parking space availability. The use of autonomous vehicles, instead of
vehicles with human drivers, can reduce human errors leading to improvement in overall performance
and safety [52]. Nevertheless, autonomous vehicles may not make optimal decisions in all real-
world environmental conditions, such as darkness, snowfall, and fog [53]. Hence, DL must address
the challenge of object detection and classification, such as a reduced accuracy of detecting and
classifying a diverse range of objects, such as cars, trucks, and empty spaces, under unpredictable real-
world environments. However, training DL models under such circumstances often require a dataset
consisting of a large collection of images covering all kinds of states, which may also increase the
training duration of the DL models. Fast RCNN and Faster RCNN techniques, which are based on
CNN [12], have been introduced to increase the speed and accuracy of DL models. Based on Fast
RCNN, the Faster RCNN technique enhances both RCNN and Fast RCNN, whereby its architecture
uses a separate network to predict the region proposals of images [54]. While these techniques can
be adopted for the deployment of parking identification and prediction systems to achieve higher
learning speed and accuracy of object detection and classification among autonomous vehicles,
further investigation can be pursued to integrate autonomous vehicles with DL models in parking
identification and prediction systems. 6.4 Reducing the Computational Cost of Deployment Higher computational cost is an important issue when DL is deployed in a real-world environ-
ment. For instance, in [14,15,17], the DL models have been deployed in the real-world environment
for the identification (S.1) of parking space availability. The main issue is the high computational
cost. One possible solution is that the DL model can be compressed and embedded in edge artificial
intelligence chips, which have been released recently [57]. By using these chips, the computational cost
can be reduced significantly when deploying DL, which requires a large amount of data for training
and testing purposes. However, the main challenge of using these chips is achieving lower computing
power requirements and energy consumption despite processing a large amount of data. The proposed
solution to address this challenge is to adopt a lightweight DL model, which has recently been shown
to achieve promising results in human activity recognition tasks. In [58], a lightweight DL model is
proposed, and it is a combination of RNN and LSTM with two hidden layers and 30 neurons in each
hidden layer. This model can be adopted to achieve lower computing power requirements and energy
consumption in parking identification and prediction systems. 6.3 Integration of Recommendation Systems and IoT Systems A recommendation system can be integrated with the DL model to improve driver experience. For
instance, in [13,16], the parking prediction system (S.2) has been used. In these works, recommendation
systems are adopted to predict parking spaces to provide performance enhancement, such as finding 780 CMC, 2023, vol.75, no.1 CMC, 2023, vol.75, no.1 CMC, 2023, vol.75, no.1 the nearest parking space to the driver’s destination within the shortest time. Thus, a recommendation
system understands a driver’s needs and provides recommendations accordingly. Nevertheless, the
recommendation system requires a large amount of data to facilitate different kinds of drivers’ needs
and make effective recommendations, so it must address the challenge of an increased computational
cost. The proposed solution in [55] uses binary neural networks to reduce the computational cost, and
it has been shown to achieve promising results in reducing the computational cost of DL. Future
investigations could be pursued to reduce the computational cost of DL while being deployed in
parking identification and prediction systems. Moreover, privacy can be considered. Some existing
works on privacy preservation in parking recommender systems [56] can serve as a guide to improve
privacy in DL-based parking space identification and prediction systems. Additionally, it is also interesting to integrate the DL model with IoT systems (e.g., smart parking)
because DL alone may not be sufficient in providing an accurate prediction of parking spaces. IoT
systems provide additional information to predict parking spaces, such as traffic congestion and air
pollution. 6.5 Detecting Illegal Parking Illegal parking is one of the main causes of congestion. It affects the accuracy of parking identi-
fication and prediction systems causing false positives and false negatives. Therefore, the detection of
illegal parking and its consideration in parking identification and prediction systems is an interesting
research challenge. 6.6 Investigating Deep Learning in Indoor Parking There are two types of parking lots in the real world, namely outdoor and indoor parking lots. In most of the proposed schemes in the literature, such as [12,18], the outdoor parking lot has been
considered. However, indoor parking lots have not been investigated in the context of DL. Future
investigations could be pursued in indoor parking lots in the context of DL to reflect real-world
scenarios. CMC, 2023, vol.75, no.1 781 7 Concluding Remarks In this paper, recent premier advances in deep learning (DL) approaches applied to identify and
predict the availability of parking spaces are analyzed, highlighted, and reported. Then, the literature
is categorized by devising a taxonomy, which is used to uncover the features of DL techniques
beneficial to the parking paradigm. The DL roles in parking identification and prediction systems
are highlighted. Open research challenges critical to the successful deployment of DL in parking
identification and prediction systems are identified and discussed. Although the deployment of DL
in parking identification and prediction systems provides various benefits, the convergence of these
two types of systems and DL brings about new issues that must be resolved in the near future. Funding Statement: This research was supported by Universiti Tunku Abdul Rahman (UTAR), and
the Publication Fund under Research Creativity and Management Office, Universiti Sains Malaysia. Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the
present study. Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the
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networks in the era of the internet of things and industry 4.0,” IEEE Industrial Electronics Magazine, vol. 11, no. 1, pp. 17–27, 2017. [28] O. Diaz, G. Mathias, R. Fabregat and S. Aciar, “Survey of smart parking systems,” Applied Sciences, vol. 10, no. 11, pp. 1–45, 2020. [29] F. Al-Turjman and A. Malekloo, “Smart parking in IoT-enabled cities: A survey,” Sustainable Cities and
Society, vol. 49, pp. 1–20, 2019. [30] J. J. Barriga, J. Sulca, J. L. Leon, A. Ulloa, D. Portero et al., “Smart parking: A literature review from the
technological perspective,” Applied Sciences, vol. 9, no. 21, pp. 1–34, 2019. [31] H. Banzhaf, D. Nienhuser, S. Knoop and J. M. Zollner, “The future of parking: A survey on automated
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https://rapidreviewscovid19.mitpress.mit.edu/pub/inotpnls/download/pdf
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English
| null |
Review 2: "Sensitivity of SARS-CoV-2 antigen-detecting rapid tests for Omicron variant"
| null | 2,022
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cc-by
| 1,028
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Rapid Reviews COVID-19
Review 2: "Sensitivity of
SARS-CoV-2 antigen-
detecting rapid tests for
Omicron variant"
Monika Klimek-Tulwin1
1Medical University of Lublin
Published on: Mar 03, 2022
License: Creative Commons Attribution 4.0 International License (CC-BY 4.0) Rapid Reviews COVID-19 Review 2: "Sensitivity of
SARS-CoV-2 antigen-
detecting rapid tests for
Omicron variant" Review 2: "Sensitivity of SARS-CoV-2 antigen-detecting rapid tests for Omicron variant Review 2: "Sensitivity of SARS-CoV-2 antigen-detecting rapid tests for Omicron variant Rapid Reviews COVID-19 RR:C19 Evidence Scale rating by reviewer: Potentially informative. The main claims made are not strongly justified by the
methods and data, but may yield some insight. The results and conclusions of the
study may resemble those from the hypothetical ideal study, but there is substantial
room for doubt. Decision-makers should consider this evidence only with a thorough
understanding of its weaknesses, alongside other evidence and theory. Decision-
makers should not consider this actionable, unless the weaknesses are clearly
understood and there is other theory and evidence to further support it. *************************************** *************************************** Review: In the introduction, the authors rightly pointed out that SARS-CoV-2 antigen-detecting
rapid diagnostic tests are characterised by lower sensitivity than RT-PCR. rapid diagnostic tests are characterised by lower sensitivity than RT PCR. Nevertheless, due to current public health needs and the need to identify infectious
and non-infectious patients, the use of SARS-CoV-2 antigen-detecting rapid diagnostic
tests is widely applicable in clinical practice. Unfortunately, with the emergence of new
mutations in the genes encoding the virus proteins, the effectiveness of vaccination is
decreasing, and the risk of re-infection is increasing. In addition, there is the question
of the sensitivity of antigen tests validated with earlier variants of the virus. Has their
sensitivity remained constant despite the new coronavirus variants? Researchers are
trying to answer this question. No studies are available to date on the sensitivity of Ag-
RDT against specific virus variants that include the Omicron variant hence why this
topic is important and valuable. It is worth noting that only symptomatic patients were included in the study. Thus, we
are talking here about sensitivity towards symptomatic patients and not in general,
"COVID-19 infected population." Furthermore, the number of samples tested is small,
so the results must be interpreted with caution. The study included as many as eight commercially available Ag-RDT products, which is
impressive. Although some of these results have been published before (data
concerning analytical sensitivity for Alpha, Beta, Gamma, and Delta variants), in the
presented study, these results were presented again to compare them with data 2 Review 2: "Sensitivity of SARS-CoV-2 antigen-detecting rapid tests for Omicron variant Rapid Reviews COVID-19 concerning the Omicron variant. It is worth noting that these earlier papers are the
work of the same authors, which increases the reliability of this comparison. In the results, the authors write, “Two tests showed a slightly higher sensitivity for
Omicron than for Delta (Test V and VII)” [V - Beijng Tigsun Diagnostics Co. Ltd
(Tigsun); VII - ACON biotech (Flowflex)]. However, the chart shows that it is IV [2019-
nCoV Antigen test (Wondfo)] and VII. Was the error in the text or in the graph? An interesting finding is that overall test positivity for Omicron was much lower when
compared to Delta (49.2% vs. 65.5%) (z = -3.65, p<.001). Moreover, when comparing
sensitivity for Delta vs. Review: Omicron for each Ag-RDT, four Ag-RDTs showed significantly
lower sensitivity (p<0.001) [Panbio COVID-19 Ag Rapid test device (Abbott); Standard
Q COVID-19 Ag (SD Biosensor/Roche); Sure Status (Premier Medical Corporation);
Onsite COVID-19 Ag Rapid Test (CTK Biotech)] and three tests showed comparable
performance [2019- nCoV Antigen test (Wondfo); Beijng Tigsun Diagnostics Co. Ltd
(Tigsun); ACON biotech (Flowflex]. Sensitivity ranged between 22.2% and 88.9% for
Omicron and 52.9% to 91.2% for Delta, confirming the high variability of sensitivity
between the different tests. The authors themselves point out the limitations of their work, to which special
attention must be paid when interpreting the results: “The volume of viral transport
medium added to the buffer was lower than what was recommended by some
manufacturers, and for some Ag-RDTs there was no recommendation on the use of
swab samples in VTM. Therefore, viral loads of the original sample and sensitivities
observed in our sample collection cannot be compared to results obtained from clinical
validations performed on fresh samples and our results should be interpreted as a
comparison between Ag-RDTs and not as sensitivity thresholds for absolute viral loads
and/or presence of infectious virus. Rather, we have investigated the lower end of
sensitivity in the Ag-RDTs tested. Therefore, a reduced sensitivity in some tests, but
not complete failure to detect Omicron could be of higher relevance in the beginning
of the infection, when viral loads are still on the rise, and of less relevance once peak
viral loads are reached.” The results of the study still need to be confirmed. Nevertheless, despite the
limitations mentioned above, I consider the work to be highly valuable; the study
contributes important new knowledge to science, and (although with caution) it can be
used in practice. 3 Review 2: "Sensitivity of SARS-CoV-2 antigen-detecting rapid tests for Omicron variant Rapid Reviews COVID-19 Considering possible publication in a peer-reviewed journal, a few points for correction
should be noted: Considering possible publication in a peer-reviewed journal, a few points for correction
should be noted: • The part of the introduction, "In addition, a deletion (Del31-33) is found in the
nucleocapsid of Omicron, as well as another mutation P13L" requires citation of
reference. • The study group was not described. Review: While the lack of a detailed description of the
study group can be explained by the fact that it is not particularly relevant in the
context of these results, it should at least be written out of obligation how many people
were included in the study - this information appears only in the discussion. • There are 27 publications in the bibliography - 8 are websites, 11 are preprints, and
only 8 are publications in peer-reviewed journals. I realise that this topic requires
rapid publication. Perhaps the authors did not find any other available papers in peer-
reviewed journals, but this somewhat reduces the reliability of the content of the
reviewed manuscript.
|
https://openalex.org/W4235543165
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https://www.biodiversitylibrary.org/itempdf/118847
|
English
| null |
The Protozoa,
| null | 1,901
|
public-domain
| 188,076
|
CORNELL UNIVERSITY.
THE
Roswell P. Flower Library
7
THE GIFT OF
ROSWELL P. FLOWER
FOR THE USE OF
* THEN. Y. STATE VETERINARY COLLEGE
1897 2787 ‘ornell Universit:
The Protozoa, ‘ornell Universit:
The Protozoa, Date Due
This boo’,
ce ,
7 he kept = —— Date Due Cornell University
Library The original of this book is in
the Cornell University Library. The original of this book is in
the Cornell University Library. There are no known copyright restrictions in
the United States on the use of the text. THE PROTOZOA Columbia Gniversity Biological Series. EDITED BY
HENRY FAIRFIELD OSBORN
AND
EDMUND
B. WILSON. 1, FROM THE GREEKS TO DARWIN. By Henry Fairfield Osborn, Sc.D. Princeton. 2. AMPHIOXUS AND THE ANCESTRY OF THE VERTEBRATES. By Arthur Willey, B.Sc. Lond. Univ. 3. FISHES, LIVING AND FOSSIL. An Introductory Study. By Bashford Dean Ph.D. Columbia. 4. THE CELL IN DEVELOPMENT AND INHERITANCE. By Edmund B. Wilson, Ph.D. J.H.U. 5. THE FOUNDATIONS OF ZOOLOGY. By William Keith Brooks, Ph.D. Harv., LL.D. Williams. 6. THE PROTOZOA. By Gary N. Calkins, Ph.D. Columbia. COLUMBIA UNIVERSITY BIOLOGICAL SERIES.
VI.
THE PROTOZOA
BY
GARY
N. CALKINS, Pu.D.
INSTRUCTOR
IN ZOOLOGY, COLUMBIA
UNIVERSITY
“Lies dieses Buch, und lern dabey,
Wie gros Gott auch im Kleinem sey.”
D, G. L. Huth: Résel von Rosenhof.
¢ unne
Netw Work
THE MACMILLAN COMPANY
LONDON: MACMILLAN
& CO., LTD. ~
IQgOI COLUMBIA UNIVERSITY BIOLOGICAL SERIES. VI. e
¢ unne~ Nortwoot ¥ress
J. 8. Cushing & Co. — Berwick & Smith
Norwood Mass. U.S.A No. VAI
COPYRIGHT, I90I,
By THE MACMILLAN COMPANY. No. VAI
COPYRIGHT, I90I, Ts
EDMUND
B. WILSON
IN GRATEFUL RECOGNITION OF HIS VALUABLE ADVICE
AND FRIENDLY INTEREST Ts
EDMUND
B. WILSON
IN GRATEFUL RECOGNITION OF HIS VALUABLE ADVICE
AND FRIENDLY INTEREST By THE MACMILLAN COMPANY. Nortwoot ¥ress
J. 8. Cushing & Co. — Berwick & Smith
Norwood Mass. U.S.A PREFACE THE Protozoa not only claim the interest of the professional
naturalist, but also that of a wider circle of nature students who,
with the aid of the microscope, have always found here a fascinating
field for observation and research. In writing the present volume,
embodying a summary of the more recent discoveries concerning
these minute animals, I have aimed to keep in mind the needs of
the latter class of naturalists,
as well as those who search more
deeply in the unicellular organisms for the solution of many morpho-
logical problems which remain unsolved in the higher animals, or for
vital processes which afford a transition from the manifestations of
life in its simplest expression to life as seen in the lower forms
of invertebrates. The subject-matter of the volume is treated from three points The subject-matter of the volume is treated from three points
of view: (1) The historical, to which the first chapter is devoted. (2) The comparative, to which five chapters are given: one to the
group of Protozoa as a whole, the other four to the main classes. (3) The general, to which three chapters are devoted. One of these
is given to the phenomena of old age or senile degeneration in
Protozoa and renewal of youth through the union of two individuals,
and to the bearing of these phenomena upon sexual reproduction in
general. Another is given to the special structures of nuclei and
centrosomes of the Protozoa; this, the most technical chapter in the
book, is introduced because of the growing importance which the
Protozoa have in the problems of cellular biology, especially with
those dealing with the origin of the division-centre and its accom-
panying structures in the cells of the Metazoa. The last chapter
is devoted to a consideration of the physiology of the Protozoa, with
especial reference to the Protozoa as organisms endowed with the
powers of codrdination and of adaptation, which up to the present
time have eluded physical and chemical analysis. Every one who works with the Protozoa is mindful of the debt
we owe to Professor Otto Biitschli, whose indefatigable labors of
vil vil INTRODUCTION AND CHAPTER I FIG. PAGE
1. Types of Protozoa . . . :
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73. Gonium pectorale in division
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CHAPTER V
97. Life history of a gregarine; schematic
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143. xiv
LIST OF FIGURES
FIG,
PAGE
35. Types of marine rhizopod shells. :
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36. Polythalamous shell types schematized
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37. A complex polythalamous shell. :
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40. CHAPTER II Types of spicules in Heliozoa
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41. Skeleton formation
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43. Plagiocarpa procortina
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44. Types of pseudopodia
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45. Camplonema nutans. ‘i
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*
-
. E
:
:
. 81
46. Ciliophrys, pseudopodia and asia aicibs
,
. 5
:
P
:
. a. 82
47. Ameba proteus pseudopodium, diagram
:
‘
:
;
. :
é
. 85
48. Ameba verrucosa, ectoplasm and vacuoles. F
3
Pi
r
é
‘
3
85
49. Contractile vacuole of Ameba proteus
. :
:
‘
F
,
. 89
50. Gromia oviformis . :
:
:
:
2
oF
os
4
s
:
:
-
91
51. AMicrogromia socialis in division
. ‘
‘
‘
F
4
2
z
i
«
OF
52. Paramaba etlhardi
“
‘
‘
‘
‘i
a
‘
3
F
F
. -
94
53- Spore-formation in Heliozoa . . ‘
é
A
5
Fi
‘
5
‘i
-
96
54. Conjugation of Actinophrys sol. :
;
‘
-
‘
:
‘
‘
. 98
55. Dimorpha nutans . :
‘
,
‘3
é
:
. ‘
:
,
2
. 100
56. Nuclearia delicatula
. ;
:
E
4
7
F
‘
a
. 102
57. Protozoa with pseudopodia and flagélts
,
-
‘
7
2
j
:
»
103
58. Membrane in Actinospherium eichhornit
. c
:
°
. . . »
104
CHAPTER IV
59. Proterospongia haeckelt
. és
2
:
-
‘
a
:
-
7
F
»
113. 60. Codonaca costata
. ,
‘
Z
-
é
Z
:
‘
z
:
:
. 11g
61. Dinobryon sertularia
. 5
7
é
é
A
:
. . . . »
IIs
62. Phacotus lenticularis
. :
<
‘
A
F
:
-
a
‘
. 116
63. Distephanus speculum
. . 3
é
‘
:
3
‘
‘
. ~
FD
64. Gymnodinium ovum and Peridinium dusdmmens
‘
c
. e
:
‘
»
118
65. Synura uvella
. :
:
:
:
r
:
5
. CHAPTER II Ciliophrys, pseudopodia and asia aicibs
,
. 5
:
P
:
. a. 82
47. Ameba proteus pseudopodium, diagram
:
‘
:
;
. :
é
. 85
48. Ameba verrucosa, ectoplasm and vacuoles. F
3
Pi
r
é
‘
3
85
49. Contractile vacuole of Ameba proteus
. :
:
‘
F
,
. 89
50. Gromia oviformis . :
:
:
:
2
oF
os
4
s
:
:
-
91
51. AMicrogromia socialis in division
. ‘
‘
‘
F
4
2
z
i
«
OF
52. Paramaba etlhardi
“
‘
‘
‘
‘i
a
‘
3
F
F
. -
94
53- Spore-formation in Heliozoa . . ‘
é
A
5
Fi
‘
5
‘i
-
96
54. Conjugation of Actinophrys sol. :
;
‘
-
‘
:
‘
‘
. 98
55. Dimorpha nutans . :
‘
,
‘3
é
:
. ‘
:
,
2
. 100
56. Nuclearia delicatula
. ;
:
E
4
7
F
‘
a
. 102
57. Protozoa with pseudopodia and flagélts
,
-
‘
7
2
j
:
»
103
58. Membrane in Actinospherium eichhornit
. c
:
°
. . . »
104 CHAPTER IV
59. Proterospongia haeckelt
. és
2
:
-
‘
a
:
-
7
F
»
113. 60. Codonaca costata
. ,
‘
Z
-
é
Z
:
‘
z
:
:
. 11g
61. Dinobryon sertularia
. 5
7
é
é
A
:
. . . . »
IIs
62. Phacotus lenticularis
. :
<
‘
A
F
:
-
a
‘
. 116
63. Distephanus speculum
. . 3
é
‘
:
3
‘
‘
. ~
FD
64. Gymnodinium ovum and Peridinium dusdmmens
‘
c
. e
:
‘
»
118
65. Synura uvella
. :
:
:
:
r
:
5
. F
i
»
11g
66. Primitive forms of Dinoflagellidia :
:
2
:
‘
:
. A
:
-
125
67. Phalansterium digitatum
. :
. . . :
. . :
-
122
68. Types of collars in Chomeilagclias
‘
3
:
. :
. :
‘
-
123
69. A Choanoflagellate type
F
:
‘
:
ss
:
:
:
:
‘
¢
(128
70. Nuclear division in Moctiluca miliaris
. :
:
3
<
as
:
‘
«
T24s
71. Megastoma entericum
. -
:
:
‘
;
é
:
‘
. CHAPTER II 10. Protoplasmic structure in different Protozoa
. é
. ‘
:
-
36
11. Flagellates with stigmata
:
:
A
‘
bo
Z
. . ‘
2
Bz. 12. Ectoplasmic modifications. :
:
;
z
,
. . é
é
-
39
13. Shells andtests
. . :
:
:
:
:
:
. :
:
i
. 4! 14. Types of nuclei. 5
F
:
F
5
7
‘
. 2
eS
»
42
15. Types of pseudopodia
. ‘
‘
:
A
‘
:
3
:
:
:
-
44
16. Cilia and myonemes of Infusoria
. 5
. :
:
5
. ‘
‘
»
45
17. Types of cysts
2
:
:
3
:
. :
Z
. . . :
-
47
18. Food-taking
. g
‘
7
:
4
‘
. F
‘
P
. 49
19. Actinobolus radians
F
. . i
a
:
:
‘
:
‘
-
51
20. Tentacles of Suctoria
. . . 2
:
:
‘
:
;
:
‘
-
52
21. Frontonia leucas
. zg
4
. . 3
:
. -
:
3
3
»
53
22. Division of Euplotes. i
A
:
c
:
é
7
:
‘
:
-
54
23. Budding of Zuglypha alveolata
. ‘
a
4
:
3
é
:
:
»
55
24. Microgromta socialis, a gregaloid colony. :
. . :
. . -
56
25. Uroglena americana
. 5
Fi
‘
is
:
3
F
‘
-
56
26. Codosiga cymosa, an aibeatid ddloay
. :
‘
:
2
‘
:
S
-
57
27. Elermocystis polymorpha, a catenoid colony
. F
:
. . z
z
w
58
28. Exogenous budding in Ephelota biitschliana
. ‘
‘
-
2
‘
,
-
58
29. Degeneration in Onychodromus grandis
‘
-
:
. ,
3
:
-
59
30. Conjugation of Onychodromus grandis
. :
F
:
:
:
. . . 60
31. Internal parasites
. . F
. :
:
:
‘
. ‘
:
:
»
63
CHAPTER III
32. Actinophrys sol. ‘
;
‘
j
3
‘
‘
. . 68
33. The protoplasmic regions of a \eatlioledlan
‘
7
s
3
‘
‘
é
. 69
34. Central capsules of Radiolaria
:
. :
. . . . :
~
go
xiil LIST OF FIGURES xiv
LIST OF FIGURES
FIG,
PAGE
35. CHAPTER II Types of marine rhizopod shells. :
:
F
c
:
‘
r
F
“9
36. Polythalamous shell types schematized
. ,
:
‘
E
‘
7
. :
92
37. A complex polythalamous shell. :
:
*
2
:
. :
. 2
Y3
38. Megalospheric and microspheric shells
. ‘
‘
3
5
:
3
7
-
74
39. Clathrulina elegans
2
2
5
. 4
. . i
:
a
:
«
95
40. Types of spicules in Heliozoa
,
‘
é
‘
‘
<
3
. ‘
. 76
41. Skeleton formation
:
‘
:
p
. . 7
-
:
:
,
2. BF
42. Lichnaspis giltochit
:
5
‘
-
:
e
. . 3
7
:
. 78
43. Plagiocarpa procortina
. 5
. :
Z
z
:
|
F
:
‘
-
79
44. Types of pseudopodia
. 2
is
:
:
a
‘
3
5
:
:
. 8
45. Camplonema nutans. ‘i
:
*
-
. E
:
:
. 81
46. Ciliophrys, pseudopodia and asia aicibs
,
. 5
:
P
:
. a. 82
47. Ameba proteus pseudopodium, diagram
:
‘
:
;
. :
é
. 85
48. Ameba verrucosa, ectoplasm and vacuoles. F
3
Pi
r
é
‘
3
85
49. Contractile vacuole of Ameba proteus
. :
:
‘
F
,
. 89
50. Gromia oviformis . :
:
:
:
2
oF
os
4
s
:
:
-
91
51. AMicrogromia socialis in division
. ‘
‘
‘
F
4
2
z
i
«
OF
52. Paramaba etlhardi
“
‘
‘
‘
‘i
a
‘
3
F
F
. -
94
53- Spore-formation in Heliozoa . . ‘
é
A
5
Fi
‘
5
‘i
-
96
54. Conjugation of Actinophrys sol. :
;
‘
-
‘
:
‘
‘
. 98
55. Dimorpha nutans . :
‘
,
‘3
é
:
. ‘
:
,
2
. 100
56. Nuclearia delicatula
. ;
:
E
4
7
F
‘
a
. 102
57. Protozoa with pseudopodia and flagélts
,
-
‘
7
2
j
:
»
103
58. Membrane in Actinospherium eichhornit
. c
:
°
. . . »
104
CHAPTER IV
59. Proterospongia haeckelt
. és
2
:
-
‘
a
:
-
7
F
»
113. 60. Codonaca costata
. CHAPTER II F
i
»
11g
66. Primitive forms of Dinoflagellidia :
:
2
:
‘
:
. A
:
-
125
67. Phalansterium digitatum
. :
. . . :
. . :
-
122
68. Types of collars in Chomeilagclias
‘
3
:
. :
. :
‘
-
123
69. A Choanoflagellate type
F
:
‘
:
ss
:
:
:
:
‘
¢
(128
70. Nuclear division in Moctiluca miliaris
. :
:
3
<
as
:
‘
«
T24s
71. Megastoma entericum
. -
:
:
‘
;
é
:
‘
. :
2
126:
72. Gonium pectorale
. :
7
:
‘
‘
4
;
‘
:
:
:
. 128
73. Gonium pectorale in division
:
:
5
:
F
:
-
‘
:
»
129
74. Budding in Noctiluca miliaris. is
-
:
:
«
F382,
75. Cercomonas crassicauda, division and spore- femation
:
:
‘i
3
:
-
133. 76. Pandorina morum, conjugation
. :
‘
. . :
. zi
:
«
134
CHAPTER V
97. Life history of a gregarine; schematic
. . ;
;
;
:
:
. -
141
78. Pfeifferia tritonis in Triton cells
. ‘
‘
‘
‘
‘
:
A
‘
. 142
79. Leptotheca agilis,a Myxospore
. ‘
:
,
:
-
. é
:
»
143. xiv FIG,
PAGE
35. Types of marine rhizopod shells. :
:
F
c
:
‘
r
F
“9
36. Polythalamous shell types schematized
. ,
:
‘
E
‘
7
. :
92
37. A complex polythalamous shell. :
:
*
2
:
. :
. 2
Y3
38. Megalospheric and microspheric shells
. ‘
‘
3
5
:
3
7
-
74
39. Clathrulina elegans
2
2
5
. 4
. . i
:
a
:
«
95
40. Types of spicules in Heliozoa
,
‘
é
‘
‘
<
3
. ‘
. 76
41. Skeleton formation
:
‘
:
p
. . 7
-
:
:
,
2. BF
42. Lichnaspis giltochit
:
5
‘
-
:
e
. . 3
7
:
. 78
43. Plagiocarpa procortina
. 5
. :
Z
z
:
|
F
:
‘
-
79
44. Types of pseudopodia
. 2
is
:
:
a
‘
3
5
:
:
. 8
45. Camplonema nutans. ‘i
:
*
-
. E
:
:
. 81
46. CHAPTER II ‘
‘
,
. 198
111. Endogenous budding in Suctoria
é
4
z
3
:
:
. é
. 199
112. Multicilia lacustris. :
‘
. . :
. . 200
113. Illustrating Biitschli’s origin ‘of Hiypoteiohida
‘
‘i
B
,
5
F
. 201
114. Illustrating Biitschli’s origin of the Vorticellide
. *
‘
. ‘
‘
+
202
115. Ciliates with tentacles
. :
. ‘
:
. ‘i
:
. . . »
205
CHAPTER VII
116. Conjugation in Cercomonas . F
:
é
3
-
‘
‘
. ,
»
285 go. Life history of Plasmodium malaria
. ,
‘
‘i
;
F
‘
és
. 163,
CHAPTER VI
gt. Types of Ciliata
. :
:
B
2
F
:
-
. . :
z
=
172:
92. Dileptus anser
. :
z
. eS
ce
fs
:
5
-
:
-
173
93. Coleps hirtus
:
;
:
-
:
2
7
:
:
F
-
é
-
176
94. Lacrymaria coronata
. 3
:
‘
a
‘
‘
‘
i
«
077
95. Supposed shifting of the mouth ae Ciliata
:
-
:
Fi
7
F
j
. 178
96. Zoothamnium arbuscula, myonemes
. 3
‘
5
:
:
F
. -
179
97. Myonemes and cilia. :
‘
7
‘
‘
:
é
‘
a
. . 180
98. Schematic hypotrichous ciliate. :
x
‘
é
F
. :
:
. 182
99. Urocentrum turbo
F
. :
a
é
5
:
:
5
‘
‘
. 183
100,
Actinobolus radians. é
k
‘
%
. é
j
é
‘
. . 184
tor. Buccal apparatus of ciliates
. 4
-
Z
Z
;
F
. :
‘
. 186
102. Anterior end of Ophrydium eichhornti
a
F
a
F
. ;
i
. 187
103. Types of macronuclei
. F
5
F
‘
5
¢
‘
$
3
=
. 189
104. Loxophyllum meleagris, nucleus
. 3
f
. :
-
:
a
é
-
190
105. Spirochona and Parameciunt, mitosis . :
:
:
:
. :
. -
191
106,
. Stentor reseliz, division
. . :
:
:
5
‘
:
. A
-
193
107. L£pzstylis umbellaria, conjugation
c
:
‘
é
:
:
a
:
-
194
108. .Tentacles of Suctoria
. . ‘
2
‘
F
‘
‘
. . ;
. 196
109. CHAPTER II :
2
126:
72. Gonium pectorale
. :
7
:
‘
‘
4
;
‘
:
:
:
. 128
73. Gonium pectorale in division
:
:
5
:
F
:
-
‘
:
»
129
74. Budding in Noctiluca miliaris. is
-
:
:
«
F382,
75. Cercomonas crassicauda, division and spore- femation
:
:
‘i
3
:
-
133. 76. Pandorina morum, conjugation
. :
‘
. . :
. zi
:
«
134
CHAPTER V
97. Life history of a gregarine; schematic
. . ;
;
;
:
:
. -
141
78. Pfeifferia tritonis in Triton cells
. ‘
‘
‘
‘
‘
:
A
‘
. 142
79. Leptotheca agilis,a Myxospore
. ‘
:
,
:
-
. é
:
»
143. LIST OF FIGURES XV FIG. PAGE
80. Clepsidrina munieri, myonemes
. é
,
F
. <
5
‘
. . 145
81. Lymphosporidium Trutte
. -
:
:
-
:
:
. 5
. . 147
82. Movement of a gregarine
. z
:
j
. ‘3
‘
:
7
A
. 148
83. Types of spores
. i
:
‘
. a
Zi
é
F
:
‘
:
. 150
84. Sporulation in a gregarine, schematic
. :
. . -
7
z
2
. 152
85. Gamocystis tenax, sporoducts. 5
x
‘i
‘
:
,
:
-
153
86. ALyxobolus, capsule formation
. :
3
. 3
:
i
. :
-
155
87. Aonocystis ascidie, conjugation
. . 5
-
,
:
A
‘
:
«
IS7
88. Life history of a Coccidium
. é
g
’
‘
é
‘
:
2
:
. 160
89. Klossia helicina, conjugation
‘
,
‘
e
:
:
:
:
:
. I61
go. Life history of Plasmodium malaria
. ,
‘
‘i
;
F
‘
és
. 163, FIG. PAGE
80. Clepsidrina munieri, myonemes
. é
,
F
. <
5
‘
. . 145
81. Lymphosporidium Trutte
. -
:
:
-
:
:
. 5
. . 147
82. Movement of a gregarine
. z
:
j
. ‘3
‘
:
7
A
. 148
83. Types of spores
. i
:
‘
. a
Zi
é
F
:
‘
:
. 150
84. Sporulation in a gregarine, schematic
. :
. . -
7
z
2
. 152
85. Gamocystis tenax, sporoducts. CHAPTER II 5
x
‘i
‘
:
,
:
-
153
86. ALyxobolus, capsule formation
. :
3
. 3
:
i
. :
-
155
87. Aonocystis ascidie, conjugation
. . 5
-
,
:
A
‘
:
«
IS7
88. Life history of a Coccidium
. é
g
’
‘
é
‘
:
2
:
. 160
89. Klossia helicina, conjugation
‘
,
‘
e
:
:
:
:
:
. I61
go. Life history of Plasmodium malaria
. ,
‘
‘i
;
F
‘
és
. 163,
CHAPTER VI
gt. Types of Ciliata
. :
:
B
2
F
:
-
. . :
z
=
172:
92. Dileptus anser
. :
z
. eS
ce
fs
:
5
-
:
-
173
93. Coleps hirtus
:
;
:
-
:
2
7
:
:
F
-
é
-
176
94. Lacrymaria coronata
. 3
:
‘
a
‘
‘
‘
i
«
077
95. Supposed shifting of the mouth ae Ciliata
:
-
:
Fi
7
F
j
. 178
96. Zoothamnium arbuscula, myonemes
. 3
‘
5
:
:
F
. -
179
97. Myonemes and cilia. :
‘
7
‘
‘
:
é
‘
a
. . 180
98. Schematic hypotrichous ciliate. :
x
‘
é
F
. :
:
. 182
99. Urocentrum turbo
F
. :
a
é
5
:
:
5
‘
‘
. 183
100,
Actinobolus radians. é
k
‘
%
. é
j
é
‘
. . 184
tor. Buccal apparatus of ciliates
. 4
-
Z
Z
;
F
. :
‘
. 186
102. Anterior end of Ophrydium eichhornti
a
F
a
F
. ;
i
. 187
103. Types of macronuclei
. F
5
F
‘
5
¢
‘
$
3
=
. 189
104. Loxophyllum meleagris, nucleus
. 3
f
. :
-
:
a
é
-
190
105. Spirochona and Parameciunt, mitosis . :
:
:
:
. :
. -
191
106,
. Stentor reseliz, division
. . :
:
:
5
‘
:
. A
-
193
107. L£pzstylis umbellaria, conjugation
c
:
‘
é
:
:
a
:
-
194
108. .Tentacles of Suctoria
. . ‘
2
‘
F
‘
‘
. . ;
. 196
109. Dendrosoma radians
. . :
3
5
:
:
#
a
-
197
110. Lphelota biitschliana, exogenous padding
:
‘
A
. CHAPTER VI gt. Types of Ciliata
. :
:
B
2
F
:
-
. . :
z
=
172:
92. Dileptus anser
. :
z
. eS
ce
fs
:
5
-
:
-
173
93. Coleps hirtus
:
;
:
-
:
2
7
:
:
F
-
é
-
176
94. Lacrymaria coronata
. 3
:
‘
a
‘
‘
‘
i
«
077
95. Supposed shifting of the mouth ae Ciliata
:
-
:
Fi
7
F
j
. 178
96. Zoothamnium arbuscula, myonemes
. 3
‘
5
:
:
F
. -
179
97. Myonemes and cilia. :
‘
7
‘
‘
:
é
‘
a
. . 180
98. Schematic hypotrichous ciliate. :
x
‘
é
F
. :
:
. 182
99. Urocentrum turbo
F
. :
a
é
5
:
:
5
‘
‘
. 183
100,
Actinobolus radians. é
k
‘
%
. é
j
é
‘
. . 184
tor. Buccal apparatus of ciliates
. 4
-
Z
Z
;
F
. :
‘
. 186
102. Anterior end of Ophrydium eichhornti
a
F
a
F
. ;
i
. 187
103. Types of macronuclei
. F
5
F
‘
5
¢
‘
$
3
=
. 189
104. Loxophyllum meleagris, nucleus
. 3
f
. :
-
:
a
é
-
190
105. Spirochona and Parameciunt, mitosis . :
:
:
:
. :
. -
191
106,
. Stentor reseliz, division
. . :
:
:
5
‘
:
. A
-
193
107. L£pzstylis umbellaria, conjugation
c
:
‘
é
:
:
a
:
-
194
108. .Tentacles of Suctoria
. . ‘
2
‘
F
‘
‘
. . ;
. 196
109. Dendrosoma radians
. . :
3
5
:
:
#
a
-
197
110. Lphelota biitschliana, exogenous padding
:
‘
A
. ‘
‘
,
. 198
111. Endogenous budding in Suctoria
é
4
z
3
:
:
. é
. 199
112. Multicilia lacustris. :
‘
. . :
. . 200
113. Illustrating Biitschli’s origin ‘of Hiypoteiohida
‘
‘i
B
,
5
F
. 201
114. Illustrating Biitschli’s origin of the Vorticellide
. *
‘
. ‘
‘
+
202
115. Ciliates with tentacles
. :
. ‘
:
. ‘i
:
. . . »
205
CHAPTER VII
116. Conjugation in Cercomonas . F
:
é
3
-
‘
‘
. CHAPTER II Dendrosoma radians
. . :
3
5
:
:
#
a
-
197
110. Lphelota biitschliana, exogenous padding
:
‘
A
. ‘
‘
,
. 198
111. Endogenous budding in Suctoria
é
4
z
3
:
:
. é
. 199
112. Multicilia lacustris. :
‘
. . :
. . 200
113. Illustrating Biitschli’s origin ‘of Hiypoteiohida
‘
‘i
B
,
5
F
. 201
114. Illustrating Biitschli’s origin of the Vorticellide
. *
‘
. ‘
‘
+
202
115. Ciliates with tentacles
. :
. ‘
:
. ‘i
:
. . . »
205
CHAPTER VII
116. Conjugation in Cercomonas . F
:
é
3
-
‘
‘
. ,
»
285
117. Conjugation in Zetramitus rostratus
. 3
;
‘
F
A
:
-
216
118. Conjugation in Rhizopoda
. :
:
‘
i
:
. 7
. F
. 217
119. Conjugation in 4rcella vulgaris
. 7
:
‘
. 7
:
z
‘
. 218
120, Conjugation in Polyloma uvella
. :
:
ZA
:
r
:
~
a
2
222
121. Conjugation in Lagenophrys ampulla
. . 7
:
2
7
-
. 35223
122. Conjugation in Afistylis umbellaria
. :
5
‘
3
. i
:
-
224
123. Conjugation in Chlorogonium euchlorum
. :
. :
:
:
. -
225 CHAPTER VI ,
»
285
117. Conjugation in Zetramitus rostratus
. 3
;
‘
F
A
:
-
216
118. Conjugation in Rhizopoda
. :
:
‘
i
:
. 7
. F
. 217
119. Conjugation in 4rcella vulgaris
. 7
:
‘
. 7
:
z
‘
. 218
120, Conjugation in Polyloma uvella
. :
:
ZA
:
r
:
~
a
2
222
121. Conjugation in Lagenophrys ampulla
. . 7
:
2
7
-
. 35223
122. Conjugation in Afistylis umbellaria
. :
5
‘
3
. i
:
-
224
123. Conjugation in Chlorogonium euchlorum
. :
. :
:
:
. -
225 LIST OF FIGURES Xvi FIG,
PAGE
124. Conjugation of Monocystis ascidie
;
ee
th
ee
. »
220
125. Division of Gontum pectorale
‘
c
‘
7
P
‘
‘
9
¥
. 227
126. Conjugation of Pandorina morum
. 7
;
is
‘
3
. 228
127. Formation of microgametes in Alossia
. 7
:
. . z
‘
. 230
128. Life history of a Coccidium
. . :
. -
‘
P
‘
2
-
. 231
129. Conjugation of Zuglypha alveolata
. ®
$
_
‘
:
F
i
. 235
130. Conjugation of Actinophrys sol. ‘
‘
i
;
2
7
5
:
. 236
131. Conjugation of Paramecium caudatum
‘
r
5
‘
‘
. . . 239
132. Onychodromus grandis, degeneration
. s
:
zi
-
é
5
‘
. 241 CHAPTER VIII 133. Diagram of a cell
-
:
:
Z
ee
F
‘5
‘i
2
a
-
247
134. Types of protozoan nuclei
. :
‘. :
:
. . z
:
. . 251
135. Nucleus and karyosome in AVossia_. . i
F
. :
5
2
+
255
136. Mitosis in Euglena. . . 3
. ss
F
F
‘
é
3
-
260
137. Division in Ameba crystalligera
. F
. 3
:
:
7
:
‘
. 261
138. Various nuclei in division
. <
F
$
c
. 7
. :
5
. 262
139. Mitotic division in the Infusoria
. 5
. 5
P
3
. P
3
»
263
140. Nuclear division in Actinospherium
. . é
é
‘
c
a
. 264
141. Mitosis in Moctiluca miliaris
:
. . . E
. . 267
142. Parameba eilhardi, sporulation and division
a
3
3
‘
;
. . 269
143. Mitosis in Ze¢ramitus chilomonas
i
. ni
. . 2
. +
270
144. Nuclear division and spore-formation in Helioti
. . . . . 2°
271
CHAPTER IX
145. Starch grains in Ciliata after partial digestion
. ps
Fs
5
3
-
. 282
146. Digestion in Reticulariida
. ‘
:
é
2
F
5
7
c
é
. 284
147. Digestion in Carchesium
. F
:
. :
. a
. . . . 285
148. Excretory granules in Paramecium
. 2
g
:
. F
5
:
. 287
149. Shell-formation in Gromia fluviatilis
. 2
7
. :
. :
. -
293
150. Phosphorescence in Woctiluca miliaris
. ‘
é
. . s
-
294
151. Motor response in Paramecium
. . . :
:
5
S31
aie
:
+
299
152. Isolated nucleus of 7’%alassicolla nucleata
. . :
. :
‘
:
»
303
153. Reactions of Ameba verrucosa and of fluids
:
:
. :
. 2
+
307 CHAPTER IX 145. Starch grains in Ciliata after partial digestion
. ps
Fs
5
3
-
. 282
146. Digestion in Reticulariida
. ‘
:
é
2
F
5
7
c
é
. 284
147. Digestion in Carchesium
. F
:
. :
. a
. . . . 285
148. Excretory granules in Paramecium
. 2
g
:
. F
5
:
. 287
149. Shell-formation in Gromia fluviatilis
. 2
7
. :
. :
. -
293
150. Phosphorescence in Woctiluca miliaris
. ‘
é
. . s
-
294
151. Motor response in Paramecium
. . . :
:
5
S31
aie
:
+
299
152. Isolated nucleus of 7’%alassicolla nucleata
. . :
. :
‘
:
»
303
153. Reactions of Ameba verrucosa and of fluids
:
:
. :
. 2
+
307 INTRODUCTION INTRODUCTION “In the clearest waters and in muddy pools, in acid as well as alkaline waters, in brooks,
lakes, rivers and seas, often, also, in the interior fluids of living plants and animals, abun-
dantly in living men, and periodically borne on the dusts and vapors of our atmosphere,
there exists a world unknown to the ordinary senses of man, of minute, peculiar forms of
life.”
—C, G. EHRENBERG, 1838. Beyonp the ordinary range of unaided vision there exists a world
of minute animal organisms, technically known as the Protozoa. They abound in the dust of the air, in the sea, in freshet and ditch,
in brackish and potable waters — wherever, in short, there is air and
moisture, while even air is apparently superfluous for the vast majority
of parasitic forms which make their homes in the living bodies of
higher plants and animals. Their beauty, their varied modes of life,
the suddenness of their appearance and disappearance, the simplicity
of their structure, and modes of reproduction, combine to make them,
even to the superficial observer, a fascinating group. Apart from
their superficial attraction, however, the Protozoa have a deeper sig-
nificance to the student of zodlogy. As the name Protozoa indicates,
they are primitive animals, and in the scale of living things they are
not far removed from the colorless bacteria on the one hand, and the
primitive green plants on the other. Their chief significance, how-
ever, and the main feature which distinguishes them from the higher
animals or Afetazoa, centres in the fact that they consist of but a sin-
gle cell within the confines of which are carried on all of the essential
vital functions which characterize the highest many-celled animals. In their main characteristics these cells do not differ from those In their main characteristics these cells do not differ from those
which make up the tissues and the body of higher animals. Like a
tissue-cell the protozoén consists
of protoplasm differentiated into
nucleus and cell-body or cytoplasm, both parts being variously modi-
fied in the several types (Fig. 1). Unlike tissue-cells, however, the
Protozoa are not specialized for the performance of any one function. They invite
attention,
therefore, from both the morphological or
structural and the physiological or functional points of view. 1 The term Protozoa was first used in its modern sense by von Siebold (45).
B
I INTRODUCTION Mor-
phologically they are equivalent to the isolated epithelial, muscle- or
nerve-cell;
physiologically, they are equivalent not merely to the
muscle- or nerve-cell, but to the entire group of cells which collec- B I THE PROTOZOA 2 tively constitute a higher animal. The Protozoa are, in short, com-
plete, but unicellular organisms, and are to be regarded as the most
generalized of single cells. Considered as a complete animal, the protozoon cell at once arouses Considered as a complete animal, the protozoon cell at once arouses
the inquiry as to the nature of the organs by means of which the vital
functions are carried on. Lending themselves readily to the experi-
mental method of investigation, the Protozoa have already contributed
not a little to knowledge of the localization of function in the cell. The importance of the nucleus in the economy of cell-life, which
Barry and Goodsir early pointed out in animal tissues, has been fully
confirmed by the researches of Gruber, Balbiani, Hofer, Verworn, and
others upon the Protozoa. From the structural point of view, the
protozo6n nucleus with its accompanying structures must ultimately
throw considerable light on the vexed questions connected with the
finer structures of metazoan cells. As the sequel will show, consider-
able advance has already been made in this direction through the
efforts of Biitschli, Schaudinn, Balbiani, R. Hertwig, and many others. Here, the generalized structures, especially those elements concerned
in cell-division,
although
difficult
of analysis,
must, when
more
thoroughly studied, aid the interpretation
of the more specialized
structures in Metazoa which are now involved in some of the most
deeply-lying problems of biology. Physiology likewise has been and is still to be greatly enriched Physiology likewise has been and is still to be greatly enriched
by the study of unicellular animals. Bichat’s theory of tissues, pro-
pounded at the very outset of the last century (1801), formed the
basis of Virchow’s development of the cell-theory along physiological
lines (758). It was Virchow who put on a working basis Schwann’s
conception that the vital activities of an animal are the sum of all of
its parts, and that each part, a cell, or, as Briicke suggested, an
“elementary organism,” performs all of the characteristic activities
of life. 1 Lee (’98), p. 50. INTRODUCTION Thus while the older physiologists were satisfied with the
knowledge
that
the
function
of the kidney is to secrete
urine
containing the waste matters of living activity, the modern problems,
as Virchow intimated, centre more especially in the inquiry as to the
activity of the kidney cells as such. Again, the modern physiological
problem of contractility or of nervous action is concerned with the
muscle- and
ganglion-cell,
and
is therefore
a cell-problem. For
investigations upon cellular physiology there are obvious advantages
in studying the unicellular organisms, which, says Verworn, “seem
to have been created by nature for the physiologists, for, besides their
great capacity for resistance, of all living things they have the invalu-
able advantage of standing nearest to the first and the simplest forms
of life.”? Fig. 1.— Types of Protozoa. A. Ameba proteus, a rthizopod. B. Peranema trichophorum, a flagellate. [BUTSCHLI.]
C. Stylonychia mytilus, a ciliate. [BUTSCHLI.]
D. Pyxinia sp. a sporozoén,
[WASIELEWSKY.]
£. Tokophrya quadripartita, a suctorian. [BUTSCHLI.]
c. contractile vacuole; ¢. epithelial host-
cell; 2. nucleus; v. food or gastric vacuole. Fig A. Ameba proteus, a rthizopod. B. Peranema trichophorum, a flagellate. [BUTSCHLI.]
C. Stylonychia mytilus, a ciliate. [BUTSCHLI.]
D. Pyxinia sp. a sporozoén,
[WASIELEWSKY.]
£. Tokophrya quadripartita, a suctorian. [BUTSCHLI.]
c. contractile vacuole; ¢. epithelial host-
cell; 2. nucleus; v. food or gastric vacuole. THE PROTOZOA 4 Although, as Virchow pointed out, each cell of a tissue is a complete
organism performing all of the functions of living matter, some
one of these functions predominates over the others and gives to the
cell and to the tissues of which it forms a part its special character-
istics. In these specialized cells the secondary functions, z.e. those
acting only for the good of the cell itself, fall into the background
and are not readily investigated. In the Protozoa, on the other hand,
no one function predominates, and despite their primitive nature, the
protoplasm of which they are composed appears quite similar to that
of the most highly specialized tissue-cell. In it, however, lies the
secret of digestion and assimilation, of the kidney’s secretion, and of
muscular contraction. The Protozoa invite attention from still another point of view. As The Protozoa invite attention from still another point of view. A. HISTORICAL REVIEW The Dutch microscopist, Anton von Leeuwenhoek (1632-1723),
using crude lenses of his own make, was one of the first to apply the
microscope to scientific investigation. His contributions to micro-
scopic anatomy and to physiology, inaugurating as they did the
invaluable services of the microscope in biological research, marked
an epoch in the history of science. An ardent follower of Harvey,
he was one of the first to offer experimental evidence against the
current belief that many of the lower organisms arise by spontaneous
generation, and on every occasion he sought to establish the truth of
Harvey’s dictum ex ovo omnia. In 1675, while searching for evi-
dence of spontaneous generation, Leeuwenhoek discovered
“ living
creatures in Rain water, which had stood but four days in a new
earthen pot, glased blew within.” “This invited me,” he continues, “to view this water with great attention, espe-
cially those little animals appearing to me ten thousand times less than those repre-
sented by Mons. Swamerdam, and by him called Water-fleas or Water-lice, which
may be perceived in the water with the naked eye. The first sort by me discover’d
in the said water, I. divers times observed to consist of 5, 6, 7, or 8 clear globuls,
without being able to discern any film that held them together, or contained them. When these azzmalcula or living Atoms did move, they put forth two little horns,
continually moving themselves. The place between these two horns was flat, though
the rest of the body was roundish, sharp’ning a little towards the end, where they
had a tayl, near four times the length of the whole body, of the thickness (by my
Microscope) of a Spider’s-web ; at the end of which appear’d a globul, of the bigness
of one of those which made up the body; which tayl I could not perceive, even in
very clear water, to be mov'd by them. INTRODUCTION As
the lowest animals they show the beginnings of sex differentiation, of
maturation, or the changes which the germ-cells undergo before fer-
tilization, and of fertilization, while their union into cell-aggregates
or colonies with incipient division of labor among the constituent cells,
points the way toward the Metazoa, and makes them significant in
the light of evolution. As complete primitive organisms, therefore, the Protozoa are impor- As complete primitive organisms, therefore, the Protozoa are impor-
tant from many points of view: structurally, they contain in simple
form the elements which in higher tissue-cells are moulded into more
complicated organs of the cell; functionally, they epitomize the life
activities of even the highest many-celled animals, but their vital pro-
cesses are more easily observed and correlated ; theoretically, they
occupy a prominent place in questions of phylogeny, of sex, and of
reproduction, and finally, placed as they are at the lowest limit of
animal life, they must ever be closely connected with problems con-
cerning its origin. With this conception of the Protozoa in mind the present volume
has been written. The work makes no pretence of a comprehensive
description of the Protozoa or of any one group, but aims rather to
give an intelligible idea of the main types, to point out the problems
of biology with which the Protozoa are most closely connected, and,
so far as possible in a limited space, to survey the work already
accomplished. In the present introductory chapter there is a brief historical review In the present introductory chapter there is a brief historical review
of the stages by which the Protozoa have come to be regarded as single
cells, and at the same time as complete animal organisms. Here, too,
is a short account of the interesting position which the Protozoa have
held in the time-honored dispute over the limitations of the anima]
and plant kingdoms, and in theories of spontaneous generation. The
second chapter deals with the general structures and functions of the
Protozoa as a group, and introduces the four following chapters, which INTRODUCTION 5 are devoted to the structural and functional adaptations of the organ-
isms in each class. The last three chapters, finally, deal with the rela-
tions of the Protozoa to more general problems. 1 See Phil. Trans., London, Vol. XII., 1677, p. 821. A. HISTORICAL REVIEW These little creatures, if they chanced to
light upon the least filament or string, or other such particle, of which there are many
in water, especially after it hath stood some days, they stook entangled therein,
extending their body in a long round, and striving to dis-intangle their tay]; whereby
it came to pass, that their whole body lept back towards the globul of the tayl, which
then rolled together Serpent-like, and after the manner of Copper or Iron-wire that
having been wound about a stick, and unwound again, retains those windings and
turnings. This motion of extension and contraction continued awhile; and I have
seen several hundreds of these poor little creatures, within the space of a grain of
gross sand, lye cluster’d together in a few filaments.” + This is the first description of a protozoon; and although the descrip-
tion is incomplete, it undoubtedly refers to a species of Vorticclla. Leeuwenhoek observed several other forms at the same time, but
for the most part their identity is uncertain. THE PROTOZOA 6 At this period, although the term ce// had already been used by
Robert Hooke (1665), the idea of simplicity of organization, apart
from minuteness of the organs, was unknown, and until the cell-
theory was established in 1838, the Protozoa were regarded as com-
plex animals having all of the parts and organs, although of micro-
scopic size, found in Metazoa. Leeuwenhoek allowed his imagination
to see what his eyes could not. “ When we see,” said he, ‘“ the sper-
matic animalcula [spermatozoa] moving by vibrations of their tails, we
naturally conclude that these tails are provided with tendons, muscles,
and articulations, no less than the tails of a dormouse or rat, and no
one will doubt that these other animalcula which swim in stagnant
waters [Protozoa], and which are no longer than the tails of the sper-
matic animalcula, are provided with organs similar to those of the
highest animals. How marvellous must be the visceral apparatus
shut up in such animalcula!”?! A. HISTORICAL REVIEW The minute size of the Protozoa made it impossible for the early The minute size of the Protozoa made it impossible for the early
investigators with their crude instruments, to follow out any life-cycle,
and the prodigious numbers and the sudden appearance of certain
forms in stagnating waters led to the belief already current in respect
to other forms, that they arose de novo. Two misconceptions thus
sprang up almost at the beginning of our knowledge of the Protozoa:
one, that Protozoa are provided with organs like higher animals; and,
two, that they arise by spontaneous generation; and one of the main
tasks of research on the Protozoa down to our own times has been
the correction of these early errors. It is not strange that Leeuwenhoek and his immediate followers It is not strange that Leeuwenhoek and his immediate followers
considered Protozoa as complicated organisms. Organisms without
organs were as novel to them as animals without cells would be to
us, and they described only what experience had taught them to
expect. With increasing knowledge of many forms and with con-
stantly improving microscopes, the conception of simplicity of organ-
ization gradually gained ground until Dujardin, about 1840, defined
the Protozoa as simple, slightly differentiated structures composed of
a fundamental living substance to which he gave the name of savcode. Despite the crudity of their instruments, the early microscopists It is not strange that Leeuwenhoek and his immediate followers
considered Protozoa as complicated organisms. Organisms without
organs were as novel to them as animals without cells would be to
us, and they described only what experience had taught them to
expect. With increasing knowledge of many forms and with con-
stantly improving microscopes, the conception of simplicity of organ-
ization gradually gained ground until Dujardin, about 1840, defined
the Protozoa as simple, slightly differentiated structures composed of
a fundamental living substance to which he gave the name of savcode. Despite the crudity of their instruments, the early microscopists
obtained wonderful results. Leeuwenhoek himself, although study-
ing these low forms only incidentally, gave recognizable descriptions
of twenty-eight species, and in addition, noted the rapid increase of
some of the larger forms, saw conjugation or the temporary union of
two individuals, and discovered so-called embryos. The early litera-
ture soon became crowded with notices of new and interesting forms,
found in all sorts of hitherto unthought-of localities. 1 Quoted from Dujardin (’41), pp. 21, 22. A. HISTORICAL REVIEW Forms with Despite the crudity of their instruments, the early microscopists
obtained wonderful results. Leeuwenhoek himself, although study-
ing these low forms only incidentally, gave recognizable descriptions
of twenty-eight species, and in addition, noted the rapid increase of
some of the larger forms, saw conjugation or the temporary union of
two individuals, and discovered so-called embryos. The early litera-
ture soon became crowded with notices of new and interesting forms,
found in all sorts of hitherto unthought-of localities. Forms with INTRODUCTION 7 whip-like appendages, or flagella ; with cz/ia, or motile appendages,
similar in general form to eyelashes; with changeable processes, or
pseudopodia ; or with no motile apparatus whatsoever; forms of the
most diverse size and shape, including many higher Metazoa, such as
worms, rotifers, ctenophores, crinoids, and crustacean larvz, as well
as many plants, were all described as “ animalcula.’’
Descriptions of
internal organs soon began to accompany the descriptions of types. The contractile vacuole, a characteristic pulsating vesicle of the Pro-
tozoa, was discovered by Joblot (1754~'55), who also showed that
cilia
on
Infusoria
have
a
definite
arrangement
in
different
species, and that many forms are provided with cuticular stripings. All
of
these
forms,
sometimes
called
insects
and
sometimes
fish, were still generally supposed to be microscopic reproductions of
higher animals. Dujardin’s criticism of Joblot’s work might well be
applied to that of many others of this early period: “ Several of the
figures which he gives,” says Dujardin, “bear the impression of a
too lively imagination for scientific purposes, and are frequently so
bizarre and fantastic as to discredit the use of the microscope.”! It
is easy to understand this criticism when we think of Joblot’s picture
of the worm Anguzl/ula with a serpent’s head, or the flagellated pro-
tozoon Luglena with a broad mouth, flagellum, and well-developed
mammalian eyes. ‘For his picture of the ciliate Paramecium aure-
fia,’ says Dujardin, ‘he apparently used his own slipper as a model.”
The life history of a protozodn was first made out by Trembley The life history of a protozodn was first made out by Trembley
(1744-47), who saw the microgonidia or young spore-forms of cer-
tain Vorticellidz leave the parent-colony and begin the formation of
new colonies by longitudinal division. 1 Dujardin (’41), p. 7. A. HISTORICAL REVIEW The discoveries made by means of the microscope were regarded The discoveries made by means of the microscope were regarded
with complete scepticism by Linnzeus in his earlier scientific works,
and the very existence of Leeuwenhoek's animalcula was at first
denied by him, but in the later editions of his Systema Nature they
were grudgingly admitted under the significant generic name of
Chaos [Chaos proteus (Ameba), Chaos redivivum, etc.]. The organ-
ized nature of Volvox globator, a form which had been discovered and
fairly well described by Leeuwenhoek, was admitted at this time, and
finally, in the twelfth edition (1767), the animal nature of a Vorticella. Many of the early investigators studied the Animalcula from the
physiological standpoint, and attempted to ascertain the functions of
many of the so-called organs. Theire efforts were often strikingly
successful and have been confirmed by later observations. Corti
(1774), Spallanzani (1776), and Gleichen (1778) are the most familiar
names in this line of research. Corti and Gleichen compared the The discoveries made by means of the microscope were regarded
with complete scepticism by Linnzeus in his earlier scientific works,
and the very existence of Leeuwenhoek's animalcula was at first
denied by him, but in the later editions of his Systema Nature they
were grudgingly admitted under the significant generic name of
Chaos [Chaos proteus (Ameba), Chaos redivivum, etc.]. The organ-
ized nature of Volvox globator, a form which had been discovered and
fairly well described by Leeuwenhoek, was admitted at this time, and
finally, in the twelfth edition (1767), the animal nature of a Vorticella. Many of the early investigators studied the Animalcula from the Many of the early investigators studied the Animalcula from the
physiological standpoint, and attempted to ascertain the functions of
many of the so-called organs. Theire efforts were often strikingly
successful and have been confirmed by later observations. Corti
(1774), Spallanzani (1776), and Gleichen (1778) are the most familiar
names in this line of research. Corti and Gleichen compared the THE PROTOZOA 8 contractile vacuole of Vorticella, with its regular pulsations, to a
beating heart, while Spallanzani, distinguishing the vacuole from its
canals, assigned to it the function of respiration. The mouth was
found in a number of forms, by Gleichen, who first used the now
common experiment of feeding the Protozoa with minute particles
of colored substances, such as carmine, indigo, etc. 1 Cf. Biitschli (’83), p. 1129. A. HISTORICAL REVIEW INTRODUCTION 9 Unlike his predecessors, Miiller did not regard the Protozoa as
complicated animals, but considered them as the simplest of all living
things, composed of a homogeneous gelatinous substance, a view in
which he was followed by a majority of the ‘ Nature-philosophers ”
(Lamarck, Schweigger, Treviranus, Oken), most of whom gave little
or no study to the Protozoa, but, accepting Miiller’s work as final,
based many of their speculations upon it. It is difficult to understand why, after Miiller’s work, the next great It is difficult to understand why, after Miiller’s work, the next great
authority, C. G. Ehrenberg (1795-1876), the renowned Berlin micro-
scopist, using much finer achromatic lenses, should have returned to
the crude view of Leeuwenhoek, assigning to the Protozoa a system of
minute but complete organs. His conclusions on Protozoa were
brought together in one great work, the title of which alone shows his
point of view: “The Infusoria as Complete Organisms” (Die /nfu-
stonsthierchen als vollkommene Organismen). He was primarily a
student of their finer structure, and the details of organization, although
erroneously interpreted, were clearly described. In working out the
internal structures he made use of Gleichen’s experiments in feeding. The animals were seen to ingest the powdered carmine, so that the
boundaries of the internal gastric vacuoles were clearly marked. He
followed these particles as they passed from the mouth into the cesoph-
agus and thence into one of the many digestive or gastric vacuoles
found in the inner plasm of nearly all Protozoa. He saw that the
particles followed clearly defined paths which might be straight or
curvilinear, or otherwise varied in different forms, but which always
ended in a more or less clearly marked anal opening. He saw also that
the parts of the supposed tract nearest the mouth fill first; that they
become globular, and that successive reservoirs become filled, down to
the posterior end of the body. He inferred from this the existence
of a digestive tract, concluding that the parts thus filled were stomachs. As soon as the first was filled, the overflow of food passed on into
another stomach. From the supposed possession of many stomachs
Ehrenberg gave to this group the name Polygastrica or Magenthicre,
making it a sharply defined class in the animal kingdom. A. HISTORICAL REVIEW A considerable
knowledge of reproduction was also obtained. Longitudinal division,
discovered by, Trembley (1744), was confirmed by Spallanzani, who, in
addition, observed transverse division in no less than fourteen species,
while his friend Saussure followed out for the first time the division
of an encysted Col/goda;
an observation confirmed by Corti and
Gleichen as well as by Spallanzani himself, who saw a Colpoda slip
out of its cyst, which he not unnaturally mistook for an egg-case. These early discoveries were, in most cases, so bound up with fan- These early discoveries were, in most cases, so bound up with fan-
tastic speculations that their zodlogical value was greatly impaired. Many of these early inaccuracies were, however, weeded out by
Otto Friedrich Miiller (1786), to whom we are also indebted for the
scientific naming of the Animalcula, which up to his time had been
called by long descriptive names given according to the fancy of each
observer, and often based
on far-fetched
resemblances. Miller,
adopting the Linnzean binomial nomenclature, described and named
some 378 species, of which about 150 are retained to-day as Protozoa. His classification was the first successful attempt to bring order out
of the heterogeneous collection of forms included under the name
Animalcula. He used Ledenmiiller’s (1760-63) term /xzfusorza, for
the name of the entire group, which he placed as a class of the
worms.! While he eliminated the inaccuracies, he confirmed the
substantial observations of the earlier observers, extending many of
them to all groups of the Protozoa. He ascertained the presence of
an anus, showed that many Infusoria are carnivorous, and observed
the process of conjugation, his description of the latter being more
accurate than that of any of his predecessors or followers until the
time of Balbiani in 1858-59. Like his predecessors, Miller included among Like his predecessors, Miller included among the Protozoa many
other organisms; placing here diatoms, nematode worms, Déstomum
larvee, and larval forms of ccelenterates and molluscs, as well as the
rotifers. The majority of these miscellaneous forms were, however,
properly classified before 1840. The larvz of molluscs and ccelente-
rates, and the worms were the first to be removed from the “animalcula,”
while finally spermatozoa (discovered by Ludwig Hamm, who is said
to have been a pupil of Leeuwenhoek), which had been universally
regarded as Protozoa inhabiting the seminal fluid, were withdrawn
during the present century. A. HISTORICAL REVIEW To all
forms in which he could find no stomachs, but in which he supposed
that mouth and anus were the same opening, he gave the name of
Anentcra
(gutless), while to forms with many stomachs he gave the
name Exterodela (gut-bearing). The red pigment spots of many forms were interpreted as true eyes,
but as eyes could not be conceived without an accompanying nervous
system,
he sought for nerve-ganglia
in different organisms, and
supposed he found what he was looking for in a species of Asvasza. He described the eye in this form, as seated upon a “spherical
glandular mass,” which he considered equivalent to the supra-pharyn- THE PROTOZOA 1ce) geal ganglion of the rotifers
(cf. Fig. 11). He discovered the
myonemes or muscular elements in the stalks of Vorticella, in Stentor,
and in certain other Ciliata, and interpreted them as muscles. He
discovered that the flagellum of the flagellates is the motile organ,
but explained its vibrations as due to the action of exquisitely fine
muscle-fibres. Pigment spheres and protoplasmic granules were de-
scribed
as ovaries,
the nucleus
as a testis, while the contractile
vacuole was at first regarded as a respiratory organ. With this latter
conclusion he could not harmonize his subsequent observations, and
finally decided that the vacuoles have the same functions as in the
rotifers. Ehrenberg’s strong position as an investigator of Protozoa is due to
his remarkable powers of observation, especially of the finer structure
of flagellates and ciliates, which in many cases he described and
accurately figured, and these justify the tribute which Bitschli pays
him: — . “The great service which Ehrenberg did in furthering the knowledge of these
forms cannot be clearly enough recognized. After a naturally somewhat difficult
comparison, I find among the species described in 1838 a few more than 100 Infusoria
(in the present sense), of which five are Suctoria. Also his system of classification”
was much more natural than that of any of his forerunners, and formed the basis of
all subsequent efforts. Many of his genera had correct limitations which hold even
to-day, although many indeed cannot be sustained. . . . With astonishing assiduity
he sought to collect, study, and systematically interpret everything that had been
done upon the Infusoria.”
(°83, p. A. HISTORICAL REVIEW 1145.) Ehrenberg’s interpretations, however, were not as successful as his
collection of data, and it is to be regretted that throughout his life he
obstinately clung to his view of the Poluasaiica, even after the period
of the complete establishment of their unicellular nature. It was
surely the irony of fate which led to the publication of his immense
work on the Polygastrica the same year (38) that the Dutch botanist
Schleiden made the greatest advance in the conception of the cell as
the unit of structure. A formidable opponent of Ehrenberg soon appeared in France, — A formidable opponent of Ehrenberg soon appeared in France, —
Felix Dujardin,
— who, influenced by long study of the RAzzopoda,(or
Protozoa with changeable processes), came to the conclusion, in 1835,
that the marine forms (Foraminifera), which, up to that time, had
been classed with cephalopod molluscs, are in reality the simplest of
organisms, composed of a simple, homogeneous substance which he
called sarcode. He showed that the many stomachs, which, according
to Ehrenberg, constituted the digestive tract, were mere vacuoles
without definite walls, which become filled with water taken in from
the outside with the food. He denied Ehrenberg’s assertion that an
anus terminates the digestive tract, although in some cases his gener- INTRODUCTION II alization was made without adequate observation, for at first he
denied the presence of a mouth as well as anus. Dujardin further showed that the motile organs of the Protozoa, alization was made without adequate observation, for at first he
denied the presence of a mouth as well as anus. Dujardin further showed that the motile organs of the Protozoa, Dujardin further showed that the motile organs of the Protozoa,
whether cilia, flagella, or pseudopodia, are mainly the prolongations
of the outer coatings of the organism, and are in no sense similar to
the hairs of higher animals, and he even suggested the transition,
which has since been shown to occur, between the simplest of pseu-
dopodia and the more complex flagella. He contradicted Ehrenberg’s
theory as to the function of the contractile vacuole, reverting to the
interpretation given by Spallanzani. He also denied the complexity
of the reproductive organs, as described by Ehrenberg, but made a
singular mistake in regarding the granules inside of the body as
germs. Many besides Dujardin had begun to criticise Ehrenberg’s theory. Many besides Dujardin had begun to criticise Ehrenberg’s theory. 1Cf. Biitschli (’83), p. 1153. 1L. Agassiz (’57), adopting a point of view which has appeared sporadically since von
Siebold announced that the Protozoa are single-celled animals, advocated the abandonment
of the Protozoa as a group, placing some divisions with the’ lower plants, others with the
larvee of worms, and still others with the Bryozoa.
His most curious error was in placing
Trichodina pediculata, one of the higher forms of Protozoa, as the medusa-generation of the
fresh-water polyp //ydra.
“TI have seen for instance,” says Agassiz, “a Planaria lay eggs,
out of which Paramecium were born, which underwent all of the changes these animals.
are known to undergo up to the time of their contraction into a chrysalis state; while the
Opatina is hatched from Distomum-eggs” (’57, p. 182).
Similar views were held by Alder
(’51), Burnett (754), and even recently by Lameere (’91), and by Villot (’91). A. HISTORICAL REVIEW Carus (’32) insisted, on purely theoretical grounds, that animals must
exist whose structure is as simple as that of an egg, since all animals
begin with the simple egg structure. Two years later, he criticised
Ehrenberg’s theory, on the ground that inner circulation of the plasm
in Paramecium (discovered by Gruithuisen, ’12), which resembles
the circulation in the plant Chara, does not accord with Ehrenberg’s
description
of the digestive apparatus. A similar objection was
raised by Focke (36), based on observations upon the streaming
plasm in Vaginicola and Paramecium. The first suggestion that Protozoa might be single cells was made The first suggestion that Protozoa might be single cells was made
by Meyen (’39), who compared the entire infusorian body to a single
plant-cell. The cell-theory, according to Biitschli, however, was first
applied directly to the Protozoa by Barry (’43), who asserted that
Monas and its allies among the Flagellidia are single cells, and that
the nucleus found within them is the equivalent of the cell-nucleus
of higher animal forms. At the same time Barry expressed the view
that cells increase only by division, and he compared the processes of
multiplication in Volvor and Chlamydomonas with the cleavage of
eggs which he, with Schwann, regarded as single cells. Barry’s view was accepted in part by Owen, who thought, however, Barry’s view was accepted in part by Owen, who thought, however,
that the Infusoria could not be included with the Flagellidia as single
cells, because of their higher differentiation. It was von Siebold
(’48), however, who finally asserted the unicellular nature
of all
Protozoa. Ehrenberg’s theory was not given up without a struggle, and, Ehrenberg’s theory was not given up without a struggle, and,
among others, we find Schmidt (’49) coming to his support with the
fact that the zvzchocysts, or stinging threads of the Infusoria, found by
Ellis (1769), and by Spallanzani (1776), and the contractile vacuoles THE PROTOZOA 12 with their canals, show the strongest similarity to the corresponding
structures in flatworms. The trichocysts were particularly difficult
for the opponents of Ehrenberg to explain. Stein (’56) regarded
them as ‘“‘taste-bodies” (7astkirperchen), and we find even Leydig
(57) regarding them, together with the microsomes in the stalks of
Vorticella, as the nuclei of very minute cells. A. HISTORICAL REVIEW Kolliker ('48, ’49), following von Siebold, but at first almost alone, Kolliker ('48, ’49), following von Siebold, but at first almost alone,
strenuously maintained that all Protozoa are single-celled animals, in
spite of severe criticism, especially by the ardent and brilliant young
naturalists, Claparéde and Lachmann, who, not able to make out the
cellmembranes and nuclei in many cases, placed the Protozoa with
the Hydroida, making them a subdivision of the Ccelenterata. It
should be noted, in justice to Claparéde, that later he admitted his. error. The opponents of Kolliker were, however, gradually convinced. The opponents of Kolliker were, however, gradually convinced. Max Schultze (’63) showed the identity of Dujardin’s sarcode with
protoplasm; Stein (’67) vigorously assailed the objections of Leydig
and Haeckel, while the latter (’73) brought back his Infusoria from
the Articulata, to which he had consigned them in 1866, and became
an ardent advocate of Kolliker’s theory. The next few years saw
the remarkable researches of Biitschli, Engelmann, and Hertwig, upon
the physiology and finer organization of the Protozoa, and their work,
with that of the hosts of others since them, aided by modern tech-
nique, has fully demonstrated the unicellular nature of all Protozoa.? The theory of alternation of generations (the alternation of a sexual The opponents of Kolliker were, however, gradually convinced. Max Schultze (’63) showed the identity of Dujardin’s sarcode with
protoplasm; Stein (’67) vigorously assailed the objections of Leydig
and Haeckel, while the latter (’73) brought back his Infusoria from
the Articulata, to which he had consigned them in 1866, and became
an ardent advocate of Kolliker’s theory. The next few years saw
the remarkable researches of Biitschli, Engelmann, and Hertwig, upon
the physiology and finer organization of the Protozoa, and their work,
with that of the hosts of others since them, aided by modern tech-
nique, has fully demonstrated the unicellular nature of all Protozoa.? The theory of alternation of generations (the alternation of a sexual
with an asexual method of reproduction) also became curiously in-
volved in the foregoing controversy. Steenstrup (’42) applied his
discovery of alternation of generations to the Protozoa, regarding the
parasitic Infusoria which he found in certain molluscs, as the larval
form of the liver-fluke, Dzstomum. The same view was found in
various forms in the works of Claparéde and Lachmann, Perty, and
Kolliker, and finally as the “ Acineta-theory” in the works of Fr. Stein. A. HISTORICAL REVIEW This theory was based upon the supposed metamorphosis of
one form of Protozoa into another. The first suggestion of such a
metamorphosis seems to have been given by Pineau (’45), who ob- The theory of alternation of generations (the alternation of a sexual
with an asexual method of reproduction) also became curiously in-
volved in the foregoing controversy. Steenstrup (’42) applied his
discovery of alternation of generations to the Protozoa, regarding the
parasitic Infusoria which he found in certain molluscs, as the larval
form of the liver-fluke, Dzstomum. The same view was found in
various forms in the works of Claparéde and Lachmann, Perty, and
Kolliker, and finally as the “ Acineta-theory” in the works of Fr. Stein. This theory was based upon the supposed metamorphosis of
one form of Protozoa into another. The first suggestion of such a
metamorphosis seems to have been given by Pineau (’45), who ob- INTRODUCTION 13 served that decaying flesh apparently breaks into minute granules
(bacteria). Influenced no doubt by the teachings of the nature-phi-
losophers, Buffon and Oken, he further thought that he had observed
the formation of larger forms of life from these minute granules, and
among them, some Podophryas, which changed into Vorticellas, and
these again into Oxytrichinas. Stein’s famous Acineta-theory was first brought out in his work of Stein’s famous Acineta-theory was first brought out in his work of
1849, in which he described the many division phases of Vorticella,
and gave a very good account of the process of encystment. The
encysted animal, he thought, breaks down into a great number of
minute particles, having at first the form of certain flagellates, which
develop into young Vorticellas. Later he adopted a second hypothe-
sis, equally untenable, vzz. that Aczueta is derived from the cysts of
Vortecella. This conclusion was based upon the fact that he had seen
the preparatory stages of encystment of the Suctorian Podophrya,
which he thought were transition phases from the encysted condition
to the adult free Podophrya, while the cysts from which he supposed
they had come he thought were formed by Vorteced/as. Generalizing
from this supposed fact, and seeing supposed confirmation in many
different directions, he finally regarded the entire division of the Suc-
toria as merely reproductive phases of the genus
Vordice/la. A. HISTORICAL REVIEW An
apparent support for his theory was found in 1854, when he discov-
ered the ciliated embryos in Suctoria, which closely resemble the
Vorticellidae. Not once did he follow out the development of these
embryos by actual observation; it was purely hypothetical, and the
discovery of Aczzeta embryos, since found to be parasites’ in various
other ciliates, only strengthened him in this point of view. Stein’s theory soon found opponents. Perty (’52) feebly opposed Stein’s theory soon found opponents. Perty (’52) feebly opposed
it, while Johannes Miiller and his pupils, Claparéde and Lachmann,
and Cienkowsky (’55) traced the development of the supposed young
Vorticellas to the adult forms of Suctoria. The theory was finally
completely overturned by Lachmann (’56) and Balbiani (’60), the
former showing by actual observation that neither does Vortdcella
develop into Acznefa, nor do embryos of the latter develop into Vor
ticella, while the latter discovered that the supposed embryos are in
reality parasitic Suctoria, a view in which he was ably supported by
Metchnikoff (54) and Kolliker (’64). Balbiani’s researches in the life history of the Protozoa at first led Balbiani’s researches in the life history of the Protozoa at first led
him into a curious error, a reminiscence apparently of Ehrenberg’s
and the older point of view. O. F. Miiller had observed and cor-
rectly interpreted conjugation in different forms, but his successors
down to Balbiani regarded this interpretation as incorrect, maintain-
ing that he had seen only stages in simple division. Balbiani (’61)
returned to Miiller’s view, and clearly stated that, in addition to THE PROTOZOA 14 simple division, another and a sexual method of reproduction occurs. His interpretation of the sexual organs of the Protozoa was given in
1858, when he maintained that the larger of the two kinds of nuclei
of Infusoria, the macronucleus, is the ovary, and the smaller one, or
micronucleus, the testis. He saw and pictured the striped appear-
ance of the micronucleus prior to division, and interpreted the stripes
as spermatozoa. The eggs were said to be formed in the macronu-
cleus, to be fertilized and then deposited on the outside, where they
develop into new ciliates. Stein at first opposed this assumption, but
in the second volume of his work on the-Infusoria, misled by his
Acineta-theory, he practically adopted it, maintaining, however, that
the embryos develop in the nucleus first, and only later leave the
mother organism. A. HISTORICAL REVIEW Biitschli (73) was apparently the first to point
out Balbiani’s error, and in his epoch-making work of 1876, after
demonstrating the ‘‘striped” appearance of many egg-cells during
division (mztotic figure), he concluded that the stripings which Bal-
biani held to be spermatozoa were no other than this striated con-
dition of the nucleus during division. He held, therefore, that in
addition to the macronucleus there is a second and a smaller nucleus
in Infusoria, and to this he gave the name Wedenkern or micronucleus
('76). Biitschli further showed at the same time that during conjuga-
tion the macronucleus or larger nucleus disintegrates, and that the
parts which Balbiani regarded as eggs are eliminated, to be replaced
by one of the subdivisions of the Mebenkern (micronucleus). His
interpretation of the process was equally happy. After observing
that a continued asexual division of certain forms resulted in decreased
size and a general “ lowering of the life energy,’’ he concluded that
the function of conjugation is to bring about a re/uvenescence.( Ver-
Jjungung) of the participants. He called attention to the similarity
between conjugation and fertilization of the egg in animals and plants,
and, at the same time, made the classic comparison between the body
of the metazoon and the chain of individuals which arise from one
individual protozoén subsequent to conjugation. simple division, another and a sexual method of reproduction occurs. His interpretation of the sexual organs of the Protozoa was given in
1858, when he maintained that the larger of the two kinds of nuclei
of Infusoria, the macronucleus, is the ovary, and the smaller one, or
micronucleus, the testis. He saw and pictured the striped appear-
ance of the micronucleus prior to division, and interpreted the stripes
as spermatozoa. The eggs were said to be formed in the macronu-
cleus, to be fertilized and then deposited on the outside, where they
develop into new ciliates. Stein at first opposed this assumption, but
in the second volume of his work on the-Infusoria, misled by his
Acineta-theory, he practically adopted it, maintaining, however, that
the embryos develop in the nucleus first, and only later leave the
mother organism. 1 Engelmann (’76), p. 628. A. HISTORICAL REVIEW Biitschli (73) was apparently the first to point
out Balbiani’s error, and in his epoch-making work of 1876, after
demonstrating the ‘‘striped” appearance of many egg-cells during
division (mztotic figure), he concluded that the stripings which Bal-
biani held to be spermatozoa were no other than this striated con-
dition of the nucleus during division. He held, therefore, that in
addition to the macronucleus there is a second and a smaller nucleus
in Infusoria, and to this he gave the name Wedenkern or micronucleus
('76). Biitschli further showed at the same time that during conjuga-
tion the macronucleus or larger nucleus disintegrates, and that the
parts which Balbiani regarded as eggs are eliminated, to be replaced
by one of the subdivisions of the Mebenkern (micronucleus). His
interpretation of the process was equally happy. After observing
that a continued asexual division of certain forms resulted in decreased
size and a general “ lowering of the life energy,’’ he concluded that
the function of conjugation is to bring about a re/uvenescence.( Ver-
Jjungung) of the participants. He called attention to the similarity
between conjugation and fertilization of the egg in animals and plants,
and, at the same time, made the classic comparison between the body
of the metazoon and the chain of individuals which arise from one
individual protozoén subsequent to conjugation. In the same year Engelmann (’76) obtained very similar results. In the same year Engelmann (’76) obtained very similar results. Quite independently of Biitschli he also proved the error of Balbiani’s
view, and came _to a conclusion not far different from Biitschli’s. “The conjugation of the Infusoria,” he said, “does not lead to repro-
duction through ‘eggs, ‘embryonic spheres,’ or any other kind of germ,
but to a peculiar developmental process of the conjugating individual,
which may be designated as reorganization.”
(Reorganization.)}
Neither observer noted the conditions which induce conjugation or Neither observer noted the conditions which induce conjugation or
the mutual interchange of parts of the micronuclei, although both,
indeed, suspected that the latter might take place. The actual inter- 1 Engelmann (’76), p. 628. INTRODUCTION 15 change was first made out by Balbiani (’82), by Jickeli (’84), by Gruber
(86, ’871*), and by Hertwig (’89), and in great detail by Maupas
(88, ’89), by whom the conditions leading to conjugation were for
the first time made known. A. HISTORICAL REVIEW change was first made out by Balbiani (’82), by Jickeli (’84), by Gruber
(86, ’871*), and by Hertwig (’89), and in great detail by Maupas
(88, ’89), by whom the conditions leading to conjugation were for
the first time made known. 1 Stein, Organismus, etc., II. (67), p. 22. B. MODERN CLASSIFICATION OF THE PROTOZOA Although the Protozoa are the simplest forms of animal life and the
most generalized of cells, it does not follow that they are simply
organized and devoid of complicated structures. On the contrary, in
many cases they are highly differentiated, and in the Infusoria, the
highest group of the Protozoa, they become so complex, that Stein,
who was never an ardent advocate of the simplicity of Protozoa, re-
marked:
‘“ The adult Infusoria must ever be considered doubtful
single-celled organisms, for they are not simply cells which have
undergone further development, but the original cell-structure has
given place to an essentially different organization entirely foreign to
typical cells.”+
On the other hand, there are simpler forms of
Protozoa in which the undifferentiated protoplasm falls within the
description of sarcode, as given by Dujardin in 1835. Between the
two extremes of structure lie the vast majority of Protozoa, showing
among them all gradations from extremely simple to extremely com-
plex forms. A partial explanation of their frequent complexity of
structure lies in the fact that, unlike tissue-cells, they live free and
usually motile lives, and, like other independent organisms, are subject
to changes of form and to intracellular modifications in response to
their mode of life. Notwithstanding the innumerable forms and the various intracellular Notwithstanding the innumerable forms and the various intracellular
modifications, differentiation, as a rule, has followed comparatively
few general lines (Fig. 1). It thus becomes possible to arrange the
Protozoa in groups
or classes, with numerous
divisions and sub-
divisions. The four classes which are now generally recognized are
the Sarcodina, the Mastigophora, the Sporozoa, and the /nfusoria. The first attempt to classify the Protozoa was made by O. F. Notwithstanding the innumerable forms and the various intracellular
modifications, differentiation, as a rule, has followed comparatively
few general lines (Fig. 1). It thus becomes possible to arrange the
Protozoa in groups
or classes, with numerous
divisions and sub-
divisions. The four classes which are now generally recognized are
the Sarcodina, the Mastigophora, the Sporozoa, and the /nfusoria. The first attempt to classify the Protozoa was made by O. F. Miiller in 1786. Rude and simple, and based upon the presence of
visible motile organs (Bullaria), or upon their absence (Infusoria
s. B. MODERN CLASSIFICATION OF THE PROTOZOA str.), this nevertheless was retained as the chief system of classifica-
tion until the time of Ehrenberg, who made use of it in his own
system, based upon the presence or absence of “stomachs.”
Despite the progress made by Dujardin, his classification, in its The first attempt to classify the Protozoa was made by O. F. Miiller in 1786. Rude and simple, and based upon the presence of
visible motile organs (Bullaria), or upon their absence (Infusoria
s. str.), this nevertheless was retained as the chief system of classifica-
tion until the time of Ehrenberg, who made use of it in his own
system, based upon the presence or absence of “stomachs.”
Despite the progress made by Dujardin, his classification, in its Despite the progress made by Dujardin, his classification, in its
main divisions, based upon unnatural differences of symmetry was
equally imperfect. His two divisions were very unequal, the “asym- 1 Stein, Organismus, etc., II. (67), p. 22. THE PROTOZOA 16 metrical’’ Infusoria including all but one or two known forms. The
subdivisions were, however, remarkably happy, and were based upon
natural lines which have never been displaced. While Ehrenberg’s
subdivisions were based upon gross external characters, such as the
presence of hairs, the position of the mouth, etc., Dujardin’s were
based upon the means of locomotion, and in this early grouping we
see the first use of our modern terms “ rhzzopod,” “ flagellate,” and
“ciliate.”
“Von Siebold (’45) was the first to divide all Protozoa into Fig. 2. — Actinophrys sol Ehrenberg, a heliozoén,
[After GRENACHER from BUTSCHLI.]
An individual with a large gastric vacuole (g), contractile vacuole (c), and axial filaments (2)
in the ray-like pseudopodia. Fig. 2. — Actinophrys sol Ehrenberg, a heliozoén,
[After GRENACHER from BUTSCHLI.]
An individual with a large gastric vacuole (g), contractile vacuole (c), and axial filaments (2)
in the ray-like pseudopodia. two classes, Rhizopoda and Jnfusoria, a system which formed the
basis of our modern classification; the Mastigophora or flagellates,
regarded by von Siebold as plants, found no place in his zoology. Three of the four great classes recognized Three of the four great classes recognized to-day were thus out-
lined by Dujardin in 1841. The modern Rhizopoda (Sarcodina) were
characterized as “animals provided with variable
processes”; the
Mastigophora as “animals provided with one or several flagelliform
filaments, serving as motile organs” ; and the Ciliata as “ciliated
animals” (Fig. 1 The term “ pseudopodia” was given by von Siebold to replace Dujardin’s more descriptive
phrase “ changeable processes” (expansions variables).
c B. MODERN CLASSIFICATION OF THE PROTOZOA Ehrenberg did great service in describing the
skeletons of many Radiolaria, especially of the fossil forms, but he
had no conception of their organization, and placed them with the
Bryozoa, Rotifera and Echinodermata as a special class ( 7dz/ata). Under the name, Actinophryens, Dujardin
grouped the Heliozoa,
together with a modern subdivision of the Infusoria (Swctorza), as
“forms with slowly contractile appendages.”
The structure of the
Radiolaria was first made out by Huxley (’51), who recognized them
as Protozoa, and correctly compared 7halassicolla with the heliozoon
Actinospherium. The pseudopodia, however, were not recognized,
and he was inclined to regard these forms as higher in organization
than a single cell, and placed them between the Protozoa and the
Sponges. Johannes Miiller(’55—’58) first saw the resemblance between
the fine ray-like pseudopodia of the Radiolaria and of the Heliozoa,
and his pupils, Claparéde and Lachmann (’58), discovered the same
granule-streaming in their pseudopodia that Schultze had observed in
some
of the Rhizopoda. With
these
data, Miller included
the
Radiolaria and the Heliozoa in the class Rhizopoda of von Siebold,
under the name Rfzsopoda radiaria, which was modified
into its
modern form, Radiolaria, by another of his pupils, Ernst Haeckel
(62). Four years later Haeckel (’66) separated the Radiolaria from
the similar fresh-water forms, to which he gave the name Heliozoa. The further subdivisions
of the subclass Rhizopoda have been the forms which are now recognized as Heliozoa or Radiolaria, were
variously interpreted. Ehrenberg did great service in describing the
skeletons of many Radiolaria, especially of the fossil forms, but he
had no conception of their organization, and placed them with the
Bryozoa, Rotifera and Echinodermata as a special class ( 7dz/ata). Under the name, Actinophryens, Dujardin
grouped the Heliozoa,
together with a modern subdivision of the Infusoria (Swctorza), as
“forms with slowly contractile appendages.”
The structure of the
Radiolaria was first made out by Huxley (’51), who recognized them
as Protozoa, and correctly compared 7halassicolla with the heliozoon
Actinospherium. The pseudopodia, however, were not recognized,
and he was inclined to regard these forms as higher in organization
than a single cell, and placed them between the Protozoa and the
Sponges. Johannes Miiller(’55—’58) first saw the resemblance between
the fine ray-like pseudopodia of the Radiolaria and of the Heliozoa,
and his pupils, Claparéde and Lachmann (’58), discovered the same
granule-streaming in their pseudopodia that Schultze had observed in
some
of the Rhizopoda. 1 Thalamophora, R. Wertwig (74); Foraminifera, VOrbigny (’26). B. MODERN CLASSIFICATION OF THE PROTOZOA 1). The further subdivisions, which, little by little,
have been developed along the lines laid down by Dujardin, have
brought order out of this heterogeneous group of organisms, which at
the present time includes nearly sixteen hundred genera and many
thousands of species. As the name of a class, the term Rhizopoda, INTRODUCTION 17 as used by von Siebold, may be replaced by the more comprehensive
term Sarcodina, given by Biitschli (’83), while the term Rhizopoda
may be applied to one group (subclass), characterized by an amceboid
or changeable adult condition, and with lobose or reticulate motile
processes or psewdopodia.1
Two other subclasses are now universally
included in the Sarcodina, the He/zocoa (Haeckel), or “sun animal- Ratmemedeagne
5
An
enemy
Pa
NeN
ashi
Fig. 3. —A radiolarian, 4e¢issa princeps Haeck. [HAECKEL.]
The central capsule (c) separates the inner protoplasm (v) containing the nucleus (z) with its
nucleolus (2), from the outer protoplasm which gives rise to the pseudopodia (/). cula”’ (Fig. 2), characterized by fine ray-like pseudopodia, which often Fig. 3. —A radiolarian, 4e¢issa princeps Haeck. [HAECKEL.]
The central capsule (c) separates the inner protoplasm (v) containing the nucleus (z) with its
nucleolus (2), from the outer protoplasm which gives rise to the pseudopodia (/). Fig. 3. —A radiolarian, 4e¢issa princeps Haeck. [HAECKEL.]
The central capsule (c) separates the inner protoplasm (v) containing the nucleus (z) with its
nucleolus (2), from the outer protoplasm which gives rise to the pseudopodia (/). Fig. 3. —A radiolarian, 4e¢issa princeps Haeck. [HAECKEL.]
The central capsule (c) separates the inner protoplasm (v) containing the nucleus (z) with its
nucleolus (2), from the outer protoplasm which gives rise to the pseudopodia (/). cula”’ (Fig. 2), characterized by fine ray-like pseudopodia, which often
contain
a central
axial thread of stiffened protoplasm, and the
Radiolaria
(Haeckel),
characterized,
in addition
to the
ray-like
pseudopodia, by the possession of a central portion of protoplasm,
which is surrounded bya perforated membrane, the “ central capsule”
(Fig. 3). |
|
Before the modern system of classification was established, many of Before the modern system of classification was established, many of 1 The term “ pseudopodia” was given by von Siebold to replace Dujardin’s more descriptive
phrase “ changeable processes” (expansions variables). c THE PROTOZOA 18 the forms which are now recognized as Heliozoa or Radiolaria, were
variously interpreted. B. MODERN CLASSIFICATION OF THE PROTOZOA With
these
data, Miller included
the
Radiolaria and the Heliozoa in the class Rhizopoda of von Siebold,
under the name Rfzsopoda radiaria, which was modified
into its
modern form, Radiolaria, by another of his pupils, Ernst Haeckel
(62). Four years later Haeckel (’66) separated the Radiolaria from
the similar fresh-water forms, to which he gave the name Heliozoa. The further subdivisions
of the subclass Rhizopoda have been The further subdivisions
of the subclass Rhizopoda have been
made upon two bases having almost equal value. In one system
they are divided according to the nature of the pseudopodia into the
orders Lobosa (Amebea of Ehrenberg) and the Reticularitda (Rett-
cularta of Carpenter, 62). In the other they are subdivided accord-
ing to the absence or presence of a shell, into the orders Amwbida
(Ehbg.) and TZestacea (M. Schultze, ’54).1- The former system is
adopted by Delage and Hérouard, by Lankester and the English
zoodlogists generally ; the latter by Biitschli. A third order under the
name Mycetozoida, is usually included with the Amcebida and the
Reticulariida. Although generally recognized in part at least, by
zodlogists as Protozoa, the taxonomic
position of the organisms
included in this order is in dispute. Under the name MJyromycetes
they are included with the fungi by most botanists, while by the zodl-
ogists they are usually placed as a class of the Rhizopoda under the
name MMycetozoa (de Bary, ’59). The relation to the fungi is claimed
on account of their saprophytic mode of life (terrestrial forms), and
their mode of spore-formation in sporangia which are often compli-
cated by the presence
of stalks, columelle, and other plant-like INTRODUCTION i9 structures such as elastic capillitia for dispersion of the spores. The
relation to the Protozoa, on the other hand, is claimed on account of
the unicellular nature, development of the swarm-spores, and occa-
sional holozoic mode of nutrition. The spores leave the sporan-
gium as amoeboid
or flagellated organisms and may increase by
simple division during the swarming stage. If flagellated, the spore
after a time loses the flagellum and becomes amceboid, in which con-
dition division may again occur;
finally numerous amceboid indi- structures such as elastic capillitia for dispersion of the spores. The
relation to the Protozoa, on the other hand, is claimed on account of
the unicellular nature, development of the swarm-spores, and occa-
sional holozoic mode of nutrition. B. MODERN CLASSIFICATION OF THE PROTOZOA The spores leave the sporan-
gium as amoeboid
or flagellated organisms and may increase by
simple division during the swarming stage. If flagellated, the spore
after a time loses the flagellum and becomes amceboid, in which con-
dition division may again occur;
finally numerous amceboid indi- c
Fig. 4.—Flagellidia. [STEIN.]
A. Chrysomonas (Chromulina) flavicans, Ehy. with chromatophores and an engulfed diatom
(2d). &. The same encysted. C. Phacus longicaudus Duj. c
Fig. 4.—Flagellidia. [STEIN.]
A. Chrysomonas (Chromulina) flavicans, Ehy. with chromatophores and an engulfed diatom
(2d). &. The same encysted. C. Phacus longicaudus Duj. viduals group themselves together, forming a colony or plasmodium. In some cases the fusion is complete, in others the outlines of the
individual Amcebe persist. In view of the questionable position which these forms occupy, In view of the questionable position which these forms occupy,
there is some danger of their being neglected altogether, the botan-
ists refusing them because of their animal characteristics, the zodlo-
gists because of their plant-like features. No harm can be done by
including them in both kingdoms, for on purely @ prior? grounds it is
to be expected that some organisms should be on the boundary line
between artificial groups such as the unicellular animals and plants. The present group and the Phytoflagellida among the Mastigophora
appear to occupy such a position, and it is advisable to include them
as provisional groups of the organisms with which they show the
greatest number of common points. With our present knowledge, THE PROTOZOA 20 the majority of Mycetozoa undoubtedly resemble fungi more than they
do Protozoa, and will not be further considered in the present work ;
the Phytoflagellina have, on the other hand, so many obvious connec-
tions with the animal flagellates that they cannot well be omitted. the majority of Mycetozoa undoubtedly resemble fungi more than they
do Protozoa, and will not be further considered in the present work ;
the Phytoflagellina have, on the other hand, so many obvious connec-
tions with the animal flagellates that they cannot well be omitted. Fig. 5.— Dinoflagellidia. [ScHUTT.]
A. Gymnodinium ovum, Schiitt. B. Peridinium divergens Ehy. f, the transverse furrow. The flagellated organisms now included under Diesing’s name,
Mastigophora, fall naturally into three subclasses: (1) the Flage/lidia
(Fig. 4) (flagellates in a strict sense), recognized by Dujardin and 0,
aad,
ONS
Res
uy
os £
a8
Jez! Hes
Fig. B. MODERN CLASSIFICATION OF THE PROTOZOA 6.— Coccidiida in epithelial cells. [LABBE.]
The coccidium, a species of the genus AZyxinia, is supposed to have divided in one case (to
the right). c¢, the sporozoén; , the nucleus of an epithelial cell. Fig. 6.— Coccidiida in epithelial cells. [LABBE.]
species of the genus AZyxinia, is supposed to have divided in one case (to
rozoén; , the nucleus of an epithelial cell. Fig. 6.— Coccidiida in epithelial cells. [LABBE.]
The coccidium, a species of the genus AZyxinia, is supposed to have divided in one case (to
the right). c¢, the sporozoén; , the nucleus of an epithelial cell. named by Cohn (’53); (2) Dinoflagellidia (Biitschli) (Fig. 5), which
were first seen by O. F. Miiller (1773) and later fairly well described named by Cohn (’53); (2) Dinoflagellidia (Biitschli) (Fig. 5), which
were first seen by O. F. Miiller (1773) and later fairly well described INTRODUCTION 21 by Ehrenberg, but curiously misinterpreted as ciliated forms (a mis-
take rectified only during the last twenty years), which led: Clapa-
réde and Lachmann (’58), R. S. Bergh (84), and Saville Kent (’81) to
regard these organisms, under the name
Ci/io-flagellata, as inter-
mediate forms between the Ciliata and the Mastigophora; (3) Cysto-
Jlagellidia (Haeckel), including two genera, Noctiluca and Leptodiscus,
the former observed during the eighteenth century, the latter dis-
covered by R. Hertwig (’77). by Ehrenberg, but curiously misinterpreted as ciliated forms (a mis-
take rectified only during the last twenty years), which led: Clapa-
réde and Lachmann (’58), R. S. Bergh (84), and Saville Kent (’81) to
regard these organisms, under the name
Ci/io-flagellata, as inter-
mediate forms between the Ciliata and the Mastigophora; (3) Cysto-
Jlagellidia (Haeckel), including two genera, Noctiluca and Leptodiscus,
the former observed during the eighteenth century, the latter dis-
covered by R. Hertwig (’77). yaar
o
es
Fe
BiB
oe Eig
TElR
EO
eer
fl
Get
ae
Fig. 7. — Two forms assumed by Leffotheca agilis,
a myxospore. [DOFLEIN.] Fig. 7. — Two forms assumed by Leffotheca agilis,
a myxospore. [DOFLEIN.] The history of the Sporozoa as a class dates from Kolliker’s (’45-
48) and Stein’s (’48) works, although the name Grvegarine now used
as the title of an order (Gregarinida) goes back to Leon Dufour
(728), and the first observation to Rediin the seventeenth century.! 1 Cf. Diesing, p. 183. B. MODERN CLASSIFICATION OF THE PROTOZOA The different kinds of Sporozoa were
first grouped together by
Leuckart (’79) under the present name, and he subdivided the group
into the Gregarinida (Fig. 1, D) and the Cocctdiida (Fig. 6), the
former dwelling in cavities of various invertebrate hosts, the latter
inside epithelial cells in, chiefly, vertebrate hosts. Under the term
psorosperms (Joh. Miiller, ’41),
a number of fish parasites belong-
ing
to
the
Sporozoa
were
known
early in
the
century,
and
these were grouped together by Biitschli under the term J7/yxospo- THE PROTOZOA 22 vidia (Fig. 7), and in the present classification form a fourth order of
the Sporozoa. A third order under the name Hemosporidii
7a (Labbe,
94), includes the sporozoan parasites dwelling in blood-cells and
plasm of different vertebrates (Fig. 8). The Infusoria finally have been variously classified
since von The Infusoria finally have been variously classified
since von
Siebold restricted the term as given by Ledenmiller (1760-63) to
its modern significance. Until their unicellular structure was defi-
nitely established, the various types were placed among the higher
animals, sometimes with the worms, sometimes with the Coelenterata. Even Perty (’52), who was the first to bring the ciliated forms together
under their present name Ciliata, believed them to be combinations of Fig. 8.— A blood parasite or Heemospore, Plasmodium malarig. Amceboid, spore-forming and
sexual phases are shown. [WASIELEWSKY.] .— A blood parasite or Heemospore, Plasmodium malarig. Amceboid, spore-forming and
sexual phases are shown. [WASIELEWSKY.] cells. He included the Suctoria and the Ciliata as subdivisions of
the Infusoria. Stein (’§7), who put the classification of the Ciliata
upon its final modern basis, had previously confused the relations of
Suctoria and Ciliata in his Acineta-theory. Claparéde and Lachmann
(58-61), after showing that Stein’s interpretation was incorrect, raised
those Infusoria which are provided with suctorial tentacles and are
ciliated only during the embryonic phases, to the grade of a separate
subclass to which they gave the modern name Swetoria (Fig. 1,
CG £)A Since Stein’s work there have been but few important changes in
the classification
of the Infusoria. Biitschli (83-88) divided the
Ciliata into two unequal groups distinguished by the nature of the
mouth parts, the Gymnostomata and the Trichostomata. Stein’s sub-
divisions based upon the arrangement of the cilia are more simple,
however, and the advantage of Biitschli’s division is somewhat ques-
tionable. 1Cf. Stein, II (67), p. 142. C. ANIMALS AND PLANTS In determining the boundaries of the subkingdom Protozoa, two
very interesting controversies have arisen, one relating to the boun-
dary between animals and plants, the other to the relations of Protozoa
to Metazoa. The modern attitude toward the first of these problems
is well expressed by Delage (’96), who says: “The question is not so
important as it appears. From one point of view, and on purely INTRODUCTION 23 theoretical grounds, it does not exist, while from another standpoint
itisinsoluble. If one be asked to divide living things into two distinct
groups of which the one contains only animals, the other only plants,
the question is meaningless, for plants and animals
are concepts
which have no objective reality, and in nature there are only indi-
viduals. If, in considering those forms which we regard as true
animals and plants, we look for their phylogenetic history, and decide
to place all of their allies in one or the other group, we are sure to
reach no result; such attempts have always been fruitless.” ? No one at the present time denies the extremely close relation No one at the present time denies the extremely close relation
which Huxley (’76) has so clearly pointed out between the lower
algze and some of the flagellates, and it is the general opinion that no
one feature separates the lowest plants from the lowest animals, and
the difficulty —in many cases the impossibility
— of distinguishing
between them is clearly recognized. Curiously enough, this modern
idea was early expressed by Buffon at the time when Aristotle’s view
of the plant-like nature of some animals (Zodphyta) was still accepted
in regard to the Ccelenterata. Buffon wrote as follows in 1749:
“From this investigation we are led to conclude
that there is no
absolute and essential distinction between the animal and vegetable
kingdoms; but that Nature proceeds from the most perfect to the
most imperfect animal, and from that to the vegetable.”
This state-
ment might have been written in 1899, but Buffon unfortunately goes
onto say: “Hence the fresh-water polypus (/Zydra) may be regarded
as the last of animals and the first of plants.” ? Ehrenberg included a large number of plant forms among his Ehrenberg included a large number of plant forms among his
Infusoria, most of which Dujardin threw out, restricting the group,
practically, to the Protozoa as known to-day. 1(’96), p. 518. 2 Edition 1812, p. 357. 1(’96), p. 518.
2 Edition 1812, p. 357. C. ANIMALS AND PLANTS But the discovery of
flagellated swarm-spores of algee cast doubt on the animal nature of
the organisms which Dujardin had described as flagellates. Von
Siebold (’45) was thus led to retain only the families Astasiidee and
the Peridinidze in his zodlogy, removing the Mastigophora, as a group,
to the botanical side. In this he was followed by Bergmann and
Leuckart (’56), while Cienkowsky (65) placed them as an intermediate
group between animals and plants. Others went to the opposite
extreme and actually excluded the algal swarm-spores from the plants,
on the ground that they were merely flagellated parasites living on the
plant-cells (Diesing, 65). Still others, noting that some of the flagel-
lates are animal and some vegetable in their nature, undertook the
impossible task of finding a single distinguishing character. The
presence of green coloring matter or chlorophy/, upheld by Cohn (’7€)
and others as a characteristic vegetable feature, seemed to be a good 2 Edition 1812, p. 357. 1(’96), p. 518. THE PROTOZOA 24 test; Oersted (’73), however, showed that in the lower plants there
are forms differing only in the presence or absence of chlorophyl. These forms may be arranged in a series as follows :1— With chlorophyl
Without chlorophyl
With chlorophyl
Without chlorophyl
Oscillaria. Beggiatoa. Spirulina. Spirocheta. Leptothrix. Leptomitus. Palmellacee. Chroccoccacez. Chlamydomonas. Chlamydomonas hyalina. Synedra. Synedra putrida. With chlorophyl
Without chlorophyl
Oscillaria. Beggiatoa. Leptothrix. Leptomitus. Chlamydomonas. Chlamydomonas hyalina. A similar series can be arranged among the Protozoa, including
forms which cannot be genetically separated, though some contain
chlorophyl, and some are colorless. In the first of these, nutrition is
holophytic or of the green plant type, in the second saprophytic or of
the fungus type. The chlorophyl differential, if used here, would
separate closely allied and in other respects identical forms, always to
be found among the Mastigophora, and would lead to confusion. Furthermore, the chlorophyl differential would cause confusion in the
classification of the fungi, where colorless representatives of several
families of the Phycomycetes reproduce by colorless swarm-spores. Again, some of the Mastigophora with chlorophyl are not dependent
upon this substance for their nutriment, but may combine t =< plant
type with the animal type of food-getting (e.g. Chromulina and some
Dinoflagellidia, Fig. 4). 1See Entz (’S8). C. ANIMALS AND PLANTS Stein sought a differential in the presence of contractile vacuoles Stein sought a differential in the presence of contractile vacuoles
and of nuclei, which, he maintained, are not found in vegetable swarm-
spores, but are characteristic of all animal cells. This view has not
been supported by later discoveries, for not only have vegetable spores
been found to possess nuclei, but many of them are also provided with
contractile vacuoles. Haeckel bases the classification of animals and plants upon nutri- Haeckel bases the classification of animals and plants upon nutri-
tion, which differs but little from the earlier chlorophyl differential. All forms with the power of absorbing carbon dioxide, water, and
nitrogen compounds, and of combining them into proteids, he calls
plants, those without this power, animals, but he considers that this
division, though logical, is at best only artificial, and gives no clue to the
actual phylogenetic relations of Protozoa and Protophyta. Asa
single
differential, however, the method of nutrition is probably as satisfac-
tory as any, for there are only a few forms which combine the two
modes of food-getting. If rigorously applied, however, it cannot fail
to shock the prejudices of both botanists and zodlogists in claiming for
the animal kingdom forms which have usually been identified with the
vegetable kingdom, and wee versa. Although Haeckel states that the
dividing line is purely arbitrary and does not represent genetic affinity INTRODUCTION 25 in the least, animal forms being derived from plants in a polyphyletic
series, he does not hesitate to rank certain of the fungi, together
with the Sporozoa and bacteria, as animal forms; the majority of
chlorophyl-bearing Protozoa, on the other hand, are placed with the
plants. Another differential, which, perhaps, has been the most widely
accepted, is the power of spontaneous motion. It is supported to-day Fig. 9.— A spheroidal colony, Uroglena americana Calkins, consisting of monads embed-
ded in a gelatinous matrix. Fig. 9.— A spheroidal colony, Uroglena americana Calkins, consisting of monads embed-
ded in a gelatinous matrix. as the most universal of the arbitrary differentials by Biitschli, Bergh,
and Delage. Briefly stated,
all forms, which
are
freely-motile
in their adult life, are animals, while stationary forms are plants. This distinction is applied only to the lower forms, and not to the
higher groups, but even as thus limited, this differential would neces-
sitate some striking changes in existing schemes of classification. C. ANIMALS AND PLANTS The freely-moving diatoms, which, since the time of Nitsch (’38)
have been classed with the unicellular plants, would be included
among the Protozoa, while the majority of Sporozoa, which are almost
devoid of motion, would be excluded. The point of view which demands the strict separation of animals The point of view which demands the strict separation of animals
and plants has, however, little utility save perhaps to determine the
limits of a text-book or monograph. Many observers, recognizing this
truth, have included all forms in which the transition from plants to
animals is shown, in a special group of the Protozoa, and usually with
some heading which gives a clue to their position. This is first seen
in Aristotle’s Zodphyta (Coelenterata); again in a more modern form
in Perty’s Phytomastigoda, and in the Phytoflagellida of Delage. Haeckel (’66) made a group of equivocal forms large enough to include
all of the Protozoa, and, under the name Pvof7sta, vainly attempted
to establish a third kingdom between the animal and the plant. THE PROTOZOA 26 The arbitrary dividing line between the Metazoa and the Protozoa
can be much more sharply drawn than that between animals and
plants. The Protozoa are usually defined as single-celled animals,
the Metazoa as many-celled; but this definition is not strictly accurate,
for many forms of Protozoa live in aggregates, or colonies in which
specialization and division of labor have progressed to a considerable
degree ( Volvox, Uroglena, Magosphera, etc.; Fig. 9). As a rule,
however, colonies do not form a distinct tissue of cells as in the blas-
tula stage of Metazoa, while a still stronger point is that they never
form a diblastic embryo.! 1See Saville-Kent (’81) for the obsolete theory that Sponges are colonial Protozoa. 1 Spallanzani’s work has hardly been sufficiently recognized by later writers.
Never car-
ried away by enthusiasm, but describing only what he saw, he placed himself outside the
current of popular favor by opposing the tempting hypothesis of the nature-philosophers.
He seems to have combined his power of observation with a remarkable breadth of view,
which in some cases gave rise to daring conceptions.
Thus, in 1776, he wrote:
“ Pour des D. GENERATION DE NOVO Leeuwenhoek’s discovery of the Protozoa had a marked effect
upon current thought, some speculative writers seeing in these minute
organisms the hypothetical units of organic structure, which, from the
time of Democritus to that of Descartes, had been a subject of
philosophical discussion. The rapid and incomprehensible increase
of Protozoa in standing water could apparently best be explained by
a theory of spontaneous generation; Leeuwenhoek, nevertheless, was
convinced that their origin would be found in minute eggs or germs
which are carried through the air as dust, or brought from place to
place by birds, etc., thus showing his firm belief in Harvey's axiom,
ex ovo omnia. He was supported in this view by Joblot (1718), whose
experiments led him to the conclusion that the lower stratum of the
air is filled with the germs of various kinds of animalcula, while
Réaumur (1738) asserted that the dust of the air also contains dis-
ease germs, which are the cause of epidemics. These men were,
however, in the minority, and until the last fifty years only an occa-
sional observer opposed the theory of spontaneous generation, as
applied to these minute organisms. Even in Leeuwenhoek’s time it was well known that dead organic Even in Leeuwenhoek’s time it was well known that dead organic
matter of any kind, when left exposed in water, gradually decom-
poses, while the water, at first clear, becomes murky, and minute
organisms of various kinds develop in it. Adopting the view that
higher organisms are composed of organic units, speculative writers
inferred that the small animals discovered by Leeuwenhoek were
the units which had again become freed from the aggregated condi-
tion. This is the key-note of Buffon's (1749) famous theory of gen-
eration, which, in one form or other, persisted well into the 19th
century. Briefly stated, Buffon believed
that all organisms are
composed
of an
infinite number
of organic
particles. The
In- INTRODUCTION 27 fusoria
he believed
to be nothing
but
these
particles become
free. ‘The destruction of organized bodies is only a separation of
the organic particles of which they are composed. These particles
continue separate till they be again united by some active power. When, however, a man’s body has nearly attained its full size, he does
not require the same quantity of organic particles; the surplus is,
therefore, sent from all parts into reservoirs destined for their recep-
tion. D. GENERATION DE NOVO These reservoirs are the testes and seminal reservoirs” (page
397). ‘The different parts of the body are, however, built up of dif-
ferent kinds of organic units, so that upon disintegration there are
different forms of animalcula, which are in no respect different from
the spermatozoa of the same animal. The freed units are therefore
neither animals nor plants, but the formative elements
of both. Arising as the disintegrated parts of dead organisms, or rather as
elements which never die, they are organisms which pass from one
living state into another.”
This view was carried further by Need-
ham (1748), and as the Buffon-Needham hypothesis, was generally
accepted. Thus the early advocates of the theory of spontaneous gen-
eration did not maintain that living things arise from not-living sub-
stances, but that all organisms are derived from parts of those living
before, —a sort of transmigration. Spallanzani, however, to whom so
much credit is due for our early knowledge of the Protozoa, adhered to
the view of Leeuwenhoek that the Infusoria are not the units which
constitute higher organisms, but distinct forms of life which, like other
organisms, are derived from definite germs. Furthermore, he vigor-
ously upheld their animal nature against Buffon and his school,
basing his arguments upon their voluntary movements, changes of
direction when moving, food taking, and upon
their relations
to
moisture and dryness, warmth and cold, to which they reacted like
higher animals. He found that Infusoria do not develop in a vacuum,
and must, therefore, come from germs contained in the air. Seeing
a Colpoda emerge from its cyst, he concluded that the cysts were
eggs, mistaking the cyst-case for the egg-membrane. He separated
the large from the small forms of Infusoria, a separation which was
the first attempt to distinguish the Protozoa from bacteria, and which
was destined to have great effect upon the theory of spontaneous
generation, for it is a significant fact that the forms which have been
supposed
to arise by spontaneous generation have always been
those approaching the limits of vision. THE PROTOZOA 28 The distinction which Spallanzani drew between large and small
forms was also adopted by O. F. Miiller in his classification of the
Protozoa. The latter maintained, however, that the lower of his two
groups (Infusoria) were formed according to Buffon’s view, from the
disintegrated
parts of higher organisms. animaux inférieurs, le changement de demeure, de climat, de nourriture, doit produire pet
a@ peu dans les individus, et ensuite duns Véspece, des modifications tres considérables qui
deguisent a nos yeux les formes primitives” (cited by Dujardin (’41) from Spallanzani).
1The name “ Protozoa,” given by Goldfuss (’20), meant the same as Oken’s “ Urthiere.”
It did not acquire its present significance until 1845, when von Siebold gave it a new meaning. 2The supposed genus Zozedz (“dawn of life’?) was discovered by Logan, of the Geologic
Survey of Canada
in 1865, and the name
was
given by Dawson
in the same year..
There has been a lengthy dispute, however, in regard to this supposed fossil, some asserting
that it is the earliest known foraminiferon, others that it is entirely inorganic.
The former
Opinion was held by Carpenter (’65, 66, etc.) and Dawson (’65, 75, etc.), the latter by the
majority of geologists and petrologists, beginning with King and Rowney (’66) and followed
by Mobius and others.
Biitschli, while admitting that Dawson and Carpenter had a certain
amount of evidence, inclines to the opposite view, while petrologists maintain that the same
structure as that of Eozoén has been frequently observed in minerals forming parts of rocks.
of undoubted igneous origin. D. GENERATION DE NOVO These parts, after the
disintegration, collect, forming a slimy scum on the surface of the in-
fusion (Zodglwa), which formed
a most important adjunct in all
subsequent theories of spontaneous generation. Minute vesicles
arise later from this scum, and these remain as living organisms in
the form of “Infusoria,” which included bacteria, spermatozoa, and
the smallest forms of flagellates. This mode of origin he limited to
those Protozoa which have no visible organs or means of locomotion. The
other
group
(Bullaria),
which
included
the
larger of the
animalcula (worms, ciliated Protozoa, rotifers, etc.), he maintained
were formed, as in the higher animals, fromeggs. Miiller also held
that these units mix with the inorganic particles to form the solid and
fluid portions of the body of higher forms, while alone, and without
contact with foreign matter, they form the nerves and “soul.”
Oken (1805), another advocate of the theory of spontaneous gen- Oken (1805), another advocate of the theory of spontaneous gen-
eration, held that all Protozoa arose in a similar manner. Since all
plants and animals were built up of these Infusoria, he named the
latter
Urthzere, although,
like
Buffon, he
held
that
they were
neither plants nor animals. Infusions demonstrated to him that all
plants and animals could disintegrate into Infusoria. Small Infusoria
at first joined together to form larger ones, and out of their union
arose the polyps and higher forms. While the outline of Oken’s
view would seem to indicate a prophecy of the cell-theory, it is quite
evident from his book on creation that he had little real conception
of what we now regard as the essence of that theory. The majority of contemporary naturalists followed Buffon and The majority of contemporary naturalists followed Buffon and
Oken, either absolutely or with slight modifications. Among these
Treviranus (703), Goldfuss (’20), and Carus (’23) accepted Oken’s
views, while Lamarck (’15), Blainville (’22), and Bory de St. Vin-
cent (’24) followed Miiller in restricting spontaneous generation to
the simpler and smaller forms. Dallinger and Drysdale (’73—75), taking turns over the microscope
by day and night, followed out the life-histories of many of these
simpler forms through the process of division and spore-formation,
thus showing that the monads arise, as do the higher Protozoa, from INTRODUCTION 29 ancestors similar to themselves. 1For a discussion of this topic the reader is referred to the essays of Huxley, Tyndall, and
Haeckel, and to Verworn’s Allgemeine Physiologie, pp. 298-319.
Lee’s translation, pp.
297-319.
2The supposed genus Zozedz (“dawn of life’?) was discovered by Logan, of the Geologic D. GENERATION DE NOVO Haeckel and Nageli argue with Huxley, and the argument is of great THE PROTOZOA 30 weight, that since organized living bodies are composed of the same
materials as unorganized or lifeless bodies, and after death are again
resolved into those same lifeless materials, it may be logically assumed
that in the beginning and under certain conditions, simple materials
were combined into new compounds having the properties which we
know to-day as life. Haeckel at first held that these complex com-
pounds were primitive organisms which he called A/onera or organisms
consisting of homogeneous plasm without differentiations of any kind,
since differentiation follows the localization
of function and can
originate only as a result of living activity. In his later work, how-
ever, Haeckel (’96) follows Nageli in postulating simple structural
units as the primitive forms of life instead of the homogeneous and
formless lumps of proteid. Nageli maintained that two stages must be distinguished between Nageli maintained that two stages must be distinguished between
inorganic matter and the lowest organisms known to us. The first
consisted of the synthesis of the albumin compounds and the organi-
zation
of these into micellaz which
constitute
primordial plasm. The second stage was the transformation of the primordial plasm
into the simplest of living organisms. Haeckel’s hypothetical A/onera,
if they existed, would approach most closely to these primordial
forms
of living matter, being described
as “organisms
without
organs.”
They could be called structureless, however, only from the
anatomical standpoint. Physically, the earliest organisms must have
been already complex, for, chemically considered, an albumin mole-
cule is an extremely complex substance, and every unit of plasm
which Nageli calls a micella must have had, and now has, that same
complex composition. Somewhere in the obscurity of this early period came the change Somewhere in the obscurity of this early period came the change
from the plant to the animal mode of nutrition. The latter must
have begun at an early time, although the possibility of change at
any time from plant to animal nutrition is not excluded, as shown by
the numerous instances among the higher plants of adaptation to a
parasitic or a saprophytic mode of life. D. GENERATION DE NOVO They found that the spores which
burst from the encysted forms were at first far beyond the limits of
vision even with the high powers of the microscope at their command,
but remained together in the form of a “glairy’”’ mass
in which
minute specks soon appeared, and these specks were watched until
they had become full-grown monads similar to the original form. In later years the theory of spontaneous generation has been limited In later years the theory of spontaneous generation has been limited
almost exclusively to the bacteria, but even here it has been energeti-
cally and successfully opposed by Pasteur, Tyndall, Milne-Edwards,
Claude-Bernard, Quatrefages, and others, against a constantly de-
creasing number of advocates. No one is in a position to assert,
however, that it does not take place in some organisms, although such
a view is highly improbable; nor can it be maintained that it never
has taken place in the past. Many theories of “archigony” (Haeckel),
or the first origin of life by spontaneous generation, have been held
by modern naturalists ; but all such theories are of a purely inferential
character and lack substantial foundation. Without attempting to
discuss these! it may be pointed out that the eminent botanist Nageli
has advocated an hypothesis which suggests that of Buffon. Assum-
ing that protoplasm consists of minute structural units or “ micelle,”
he suggests that such micelle were
first formed from not-living
matter and secondarily united
into organisms. Nageli does not
hesitate to say that the evolution of the simplest protozoén from
inorganic compounds involved a far greater step than from the first
organism to man, and in accordance with this idea Haeckel places the
beginning of life in the oldest known geologic age and in the oldest
period of that age, the Laurentian. This, again, is entirely specula-
tive; for if we except the questionable form /ozodx, the rocks of the
Laurentian contain no recognizable records of past life.? The rocks of the period after the Laurentian, however, the Cam- The rocks of the period after the Laurentian, however, the Cam-
brian, possess a great number of well-marked
types, families, and
genera, thus indicating, even at this time, a considerable antiquity. 1See Lankester (’91), Klebs (’92), and én/ra, p. 99. D. GENERATION DE NOVO So too, among the Protozoa,
the acquisition of a cannibalistic mode of life, or, as Haeckel calls it,
metasitism, may have required, and probably did require, a long
period, and there Is little reason to doubt Haeckel’s view that the Pro-
tozoa are polyphyletic in their origin. We possess no positive data
for the conclusion as to which of the Protozoa were the most primi-
tive. In considering this question, it must not be overlooked that,
during the eras
that have passed, the Protozoa may have been
adapted and re-adapted many times over to changing conditions of
environment, and living species have, in all probability, not come
unchanged from that remote past.! 1See Lankester (’91), Klebs (’92), and én/ra, p. 99. INTRODUCTION 31 GENERAL SKETCH It is a widely accepted opinion among men of science that life
originated in the sea, and here to-day are found the great majority of
species of Protozoa. In the littoral regions, particularly in the super-
ficial slime and upon submerged water-plants, are found a profusion of
Rhizopoda. Farther out, Radiolaria and shelled Rhizopoda belong-
ing to the order Reticulariida float upon the surface or at varying
depths below it, while their empty shells, settling slowly to the bottom,
have added little by little to the accumulations of the past, until to-day,. under the names Radiolarian ooze and Globigerina ooze, they form
vast areas, miles in extent, and often attaining a depth of many feet. By the agency of earthquakes or slow upheavals, these beds have
beconie exposed from time to time, and we recognize the Barbadoes as
composed in large part of the skeletons of Radiolaria, or the chalk
cliffs of England as built up of the lime shells of reticulate Rhizopoda. Apart from the Sarcodina, the majority of Protozoa leave no memo- Apart from the Sarcodina, the majority of Protozoa leave no memo-
rial in stone of their past existence. Pelagic forms such as Dinofla-
gellidia and Cystoflagellidia, living near the shores, and often drawn
together into great aggregates by currents, winds, etc., become the
food of whales, fishes, and other marine animals. Many Rhizopoda,
Ciliata, and Suctoria are attached by mineral secretions, or by stalks,
to rocks, submarine plants, etc. Others are parasites upon the out-
sides of fish and other animals, while still others are parasites within. The fresh-water Protozoa, while less rich in species, are much The fresh-water Protozoa, while less rich in species, are much
better known than the marine forms, for their modes of life, habitats,
and life histories
are more easily observed and controlled. Many
kinds of Rhizopoda, Heliozoa, and Ciliata are found both in fresh
water and in salt; and numerous experiments by Verworn, Gruber,
and others have shown that some forms can live either in salt water
or in fresh; the change from one to the other usually results, how-
ever, in modifications of structure. In general, the Protozoa abound
in fresh water which contains enough food material for their growth
and reproduction, but the widespread belief that each drop of drink-
ing water
contains countless myriads
of microscopic forms has
absolutely no foundation. SPECIAL BIBLIOGRAPHY I Biitschli, O0.—Protozoa. In Bronn’s Klassen und Ordnungen
des Thierreichs.:
Leipzig, 1883-1888. Dujardin, F. — Les Infusoires. Parzs, 1841. Dujardin, F. — Les Infusoires. Parzs, 1841. Entz, Geza — Protistenstudien. Ludapesth, 1 Entz, Geza — Protistenstudien. Ludapesth, 1888. Huxley, T. H.—On the Border Territory between Huxley, T. H.—On the Border Territory between the Animal and the Vegetable
Kingdoms. Collected Essays, D. Appleton & Co. Vol. 8, Edition 1896. Kent, W. Saville. — A Manual of the Infusoria. London, 1881. Kent, W. Saville. — A Manual of the Infusoria. London, 1881. Stein, Fr.— Organismus der Infusionsthiere. Lecpzgg. Abth. I Stein, Fr.— Organismus der Infusionsthiere. Lecpzgg. Abth. I., 1861; II., 1867;
Ill., 1878. GENERAL SKETCH Protozoa cannot live in chemically pure
water, and on the other hand, many of them cannot live in foul wateis.. 32 32 GENERAL SKETCH 33 Thus, in sewage, one finds occasional ciliates and flagellates, but no
such numbers as are sometimes found even in good drinking waters,
where, obtaining their food as do the green plants through the agency
of their green or yellow coloring matter (chlorophyl), the Protozoa
sometimes become a source of annoyance. They thrive in standing Thus, in sewage, one finds occasional ciliates and flagellates, but no
such numbers as are sometimes found even in good drinking waters,
where, obtaining their food as do the green plants through the agency
of their green or yellow coloring matter (chlorophyl), the Protozoa
sometimes become a source of annoyance. They thrive in standing
waters where the accumulation of bacteria gives food for numerous
ciliates and rhizopods;
the decomposing organic matter dissolved in
the water is taken in by saprophytic forms
of flagellates, which
multiply to prodigious numbers, and these in turn may form the food-
supply for predaceous Infusoria and Sarcodina. Rotifers, Crustacea,
molluscs, and worms
prey upon
all forms, and when the cycle is
passed, the water becomes cleared of animal life. In nature, the
pools rarely if ever become thus cleared, because new food is con-
stantly brought in from fresh sources, and the cycle becomes continu-
ous. In such places the superficial slime upon the bottom contains
naked and shelled rhizopods, although the latter are more often found
alive upon the leaves and stems of water-plants; here, too, are colo-
nial Infusoria or single forms attached by their stalks. Suspended in
the water are to be found the majority of species of Flagellidia, a few
Dinoflagellidia, the majority of Heliozoa, many predatory ciliates, and
a few rhizopods, especially certain shelled forms which
secrete a
bubble of gas to buoy them up. Many forms of Protozoa are capable of sustaining life either as Many forms of Protozoa are capable of sustaining life either as
terrestrial or as parasitic organisms. The former, allied to the Myceto-
zoa, grow over damp wood, while a number of rhizopods are almost
able to withstand dryness, for as Dujardin, Ehrenberg, Greeff, and
others early pointed out, they live in damp moss and leaves of the
woods. Forms which have become adapted to a parasitic mode of
life may be found in all classes. 1 «Sarcode.
Je propose de nommer ainsi ce que d’autres observateurs ont appelé une
gelce vivante, cette substance glutineuse diaphane; insoluble dans eau, se contractant en
masses globuleuses, s’attachant aux aiguilles de dissection et se laissant étirer comme du
mucus, enfin se trouvant dans tous les animaux inférieurs interposée aux autres élémens de
structure.”
— DUJARDIN, 735, p. 367. A.
GENERAL MORPHOLOGY As might be expected from the wide distribution of the Protozoa
and their varied modes of life, each of the several classes contains
organisms of varying forms and grades of complexity. In fact, no
one form is characteristic of any group, but in all cases where the
body is plastic and subjected to an even pressure the form is spher-
ical (homaxonic),
readily changing, however, into an elongate or
monaxonic condition. In the higher types, especially those which
are inclosed in a firm membrane, the form is usually asymmetrical,
and cannot be interpreted. as the direct result of mechanical condi-
tions. The homaxonic type prevails among Heliozoa, Radiolaria, and
intra-cellular Sporozoa (Coccidiida), and occurs in the simpler types of
all classes. The monaxonic form prevails among the Mastigophora
and the lumen-dwelling Sporozoa (Gregarinida), while asymmetrical
forms are dominant among the Infusoria. In all classes, when for
any reason the surroundings become unsuitable, or at times as a pre-
liminary to some methods of reproduction, the organisms secrete a
thick and resistant
protective coating
or cyst which
is usually
homaxonic. The various adaptations found in the Protozoa are confined almost
entirely to the outer protoplasm or ectof/asm, the inner portion or
endoplasm remaining approximately similar in structure throughout
the group. The ectoplasm, being in direct contact with the sur-
rounding medium, becomes hardened into ectoplasmic coatings of
various kinds, serving as protective coverings for the inner endo-
plasm. It also becomes differentiated into various external organs
of locomotion, of food-getting, of defence and offence, and, in the
higher types, into organs
of sensation. GENERAL SKETCH Among the Rhizopoda, various
species of intestinal A@be may be found in all sorts of vertebrate
and invertebrate hosts; about twenty species of Flagellidia and many
more of Ciliata live as parasites, some in the blood, some in the intes-
tinal fluids, and others in the cavities of various organs in man and
other hosts. These forms, however, are only occasional parasites,
and are more like commensals than parasites, having little signifi-
cance when compared with the Sporozoa, a ‘class of Protozoa which,
without any
exceptions, are parasitic. These
infest
all animal
forms from Protozoa to man:
one group lives in the digestive tract
and the cavities of the body (Gregarinida); another in the cells of the
digestive organs (Coccidiida); another in the muscle-cells and lymph
surrounding them (Myxosporidiida, Sarcosporidiida); and still another
in the blood-corpuscles and in the blood-plasm (Hzemosporidiida). Of
all Protozoa these are the only forms which are known to menace
the life of man. D THE PROTOZOA 34 1Cf. Chap. IX., p. 286. 1.
Lhe Endoplasm. Examined under the low powers of the microscope, the body of a
protozodn appears to be made up of a gelatinous, diaphanous sub-
stance which, under certain conditions, breaks out of the confines of
the cell-membrane, forming irregular globular masses in the water. This phenomenon was early recognized, and under the term “ difflu-
ence’ was regarded by Dujardin as a special property of sarcode.}
Examined under higher powers of the microscope (c.g. with a one- Examined under higher powers of the microscope (c.g. with a one- GENERAL SKETCH 35 twelfth inch objective), the mass of endoplasm is seen to consist of a
more or less definite matrix, and if the cells be properly fixed and
stained, a distinct structure is visible. This appears to be little more
than a definite meshwork, the meshes of which are sometimes minute,
compressed, and narrow, sometimes large and open. The substance
of the mesh proper appears to differ noticeably from that within its
spaces. The latter is fluid-like, and not infrequently contains gran-
ules of larger or smaller size; the former, also a fluid, appears more
dense, and is made up of exceedingly minute granules (microsomes). Differential stains show that the various granules differ not only in
size, but in chemical composition, and it has been determined that
some are food particles in process of assimilation, and that others
are waste matters. This protoplasmic
structure, which
Biitschli
(92) compares with a foam structure (Schaumplasma), is described
by him as consisting of small drops of a liquid a/veolar substance
inclosed within the meshes of a continuous zyter-alveolar substance,
also liquid, but of a different composition. Each alveolus may be
compared to a bubble in a foam structure;
the air of the bubble
corresponding to the alveolar substance,
the walls
to the inter-
alveolar substance. :
While the endoplasm of all Protozoa is alveolar in structure, there twelfth inch objective), the mass of endoplasm is seen to consist of a
more or less definite matrix, and if the cells be properly fixed and
stained, a distinct structure is visible. This appears to be little more
than a definite meshwork, the meshes of which are sometimes minute,
compressed, and narrow, sometimes large and open. The substance
of the mesh proper appears to differ noticeably from that within its
spaces. 1.
Lhe Endoplasm. The latter is fluid-like, and not infrequently contains gran-
ules of larger or smaller size; the former, also a fluid, appears more
dense, and is made up of exceedingly minute granules (microsomes). Differential stains show that the various granules differ not only in
size, but in chemical composition, and it has been determined that
some are food particles in process of assimilation, and that others
are waste matters. This protoplasmic
structure, which
Biitschli
(92) compares with a foam structure (Schaumplasma), is described
by him as consisting of small drops of a liquid a/veolar substance
inclosed within the meshes of a continuous zyter-alveolar substance,
also liquid, but of a different composition. Each alveolus may be
compared to a bubble in a foam structure;
the air of the bubble
corresponding to the alveolar substance,
the walls
to the inter-
alveolar substance. :
While the endoplasm of all Protozoa is alveolar in structure, there While the endoplasm of all Protozoa is alveolar in structure, there
is considerable variation in density due to the relative sizes of the
alveoli and to the nature of the granules contained within them (Fig. 10, A-D). They vary in size from minute vesicles in Sporozoa (C) to
large vacuoles in many Heliozoa, Radiolaria, and Infusoria. In some
cases, ¢.g. in the heliozoon Actinospharium (D), or the cystoflagellate
Noctiluca, the vacuoles are so large that the protoplasmic structure
appears parenchymatous like a plant-cell. The granules in the walls
of the alveoli are equally variable in size. In some cases they are
exceedingly minute, and correspond apparently to the fine elementary
granules which Altmann (’94) regarded as the basis of all protoplasm
(e.g. Ameba, A); in others they are coarse and obviously of different
kinds (Pelomyxa). The various granules within the alveoli are sometimes inert and The various granules within the alveoli are sometimes inert and
functionless and often crystalline in form.! In other cases they may
have some function to play in the economy of the cell. Thus car-
bohydrates in the form of starch, sugar, or cellulose are generally
present and serve as a reserve store of food, or of building material
for the outer covering. Other granules which are invariably present
may be food particles in various stages of digestion, assimilation, and
excretion, or oil particles of various forms and sizes. Fig. 10.
— Protoplasmic structure in different Protozoa.
[From preparations.]
A, Ameba proteus pseudopodium.
The endoplasm has broken through the ectoplasm, and
is now in advance.
2. Chilomonas paramecium Ehv. a flagellate.
C. Lymphosporidium trutte
Calkins, a sporozoén.
D. Actinospherium EHichhornii, endoplasm and one nucleus.
a, alveoli.
Same magnification throughout. 1.
Lhe Endoplasm. With the exception of the Sporozoa, every class of Protozoa includes With the exception of the Sporozoa, every class of Protozoa includes SEARE
a
+S
va
Bc
Fig. 10. — Protoplasmic structure in different Protozoa. [From preparations.]
A, Ameba proteus pseudopodium. The endoplasm has broken through the ectoplasm,
is now in advance. 2. Chilomonas paramecium Ehv. a flagellate. C. Lymphosporidium tru
Calkins, a sporozoén. D. Actinospherium EHichhornii, endoplasm and one nucleus. a, alve SEA
a
+S
va
Bc
Fig. 10. — Protoplasmic structure in different Protozoa. [From preparations.] ent Pr Fig. 10. — Protoplasmic structure in different Protozoa. [From preparations.]
A, Ameba proteus pseudopodium. The endoplasm has broken through the ectoplasm, and
is now in advance. 2. Chilomonas paramecium Ehv. a flagellate. C. Lymphosporidium trutte
Calkins, a sporozoén. D. Actinospherium EHichhornii, endoplasm and one nucleus. a, alveoli. Same magnification throughout. Fig. 10. — Protoplasmic structure in different Protozoa. [From preparations.]
A, Ameba proteus pseudopodium. The endoplasm has broken through the ectoplasm, and
is now in advance. 2. Chilomonas paramecium Ehv. a flagellate. C. Lymphosporidium trutte
Calkins, a sporozoén. D. Actinospherium EHichhornii, endoplasm and one nucleus. a, alveoli. Same magnification throughout. GENERAL SKETCH 37 some species in which colored masses of protoplasm — chromatophores
—are present, either as living parts of the cell (Mastigophora) or as
commensals or symzbzonts, the protozodn and the plant living together
for mutual benefit (Infusoria, Sarcodina). The chromatophores are
colored by different substances, usually green by chlorophyl (Chloro-
monadina, some Infusoria), or brown by diatomin (Chrysomonadidz
and Dinoflagellidia), and have a definite shape and size for each
species. Brilliantly colored patches of pigment, the so-called eye-
spots or stigmata, are frequently seen, chiefly among the Mastigoph- Fig. 11.— Flagellates with stigmata. [FRANCE.]
A, Euglena Ehrenbergii, Klebs. The color-mass (5) contains several concrements (lenses ?). B, Pandorina morum, Ehr. The color-mass (5) is attached to a single spherical lens. [FRANCE.] Fig. 11.— Flagellates with stigmata. [FRANCE.]
A, Euglena Ehrenbergii, Klebs. The color-mass (5) contains several concrements (lenses ?). B, Pandorina morum, Ehr. The color-mass (5) is attached to a single spherical lens. ora, where they are situated near the base of the flagellum. 1Cf. Pouchet, ’86.
2 Engelmann’s (’82) experiments, on the other hand, tend to show that it is not the
colored body, but the colorless protoplasmic mass in front of the stigma which is particularly
sensitive to light. 1Cf. Pouchet, ’86.
gelmann’s (’82 2.
The Ectoplasm.
In many Protozoa In many Protozoa, especially among the Rhizopoda, there may be
no distinction between ectoplasm and endoplasm. These cases, how-
ever, are exceptions, for in the majority of forms a well-marked ecto-
plasm can be distinguished. In many cases the difference appears
to be only in the presence or absence of granules, and their distribu-
tion depends upon the density of the fluid plasm. No great mor-
phological importance can be attached to this regional difference, for
it appears to be only an index of the physical conditions of the
protoplasm. The body of the common rhizopod Amwéa, for example,
consists of a more or less fluid mass in which lie suspended the
various granules, vacuoles, nuclei, crystals, and food particles, and,
as Griiber (’84) pointed out, if the plasm is thin, z.¢. more fluid, the
contents can spread easily through the whole mass, while if the
plasm is dense and viscous, they will be held back by the resistance,
and a relatively broad ectoplasm may result. The more fluid condi-
tion is seen in rhizopods like Protomyxa and Pelomyxa, the denser in
Ameba proteus, and the majority of fresh-water shell-bearing forms. In some of the latter and in a few Infusoria the distribution according
to density is so marked that several regions can be made out. Thus
Pénard (’90) described no less than four zones in the shelled rhizopod,
Luglypha, while in many Infusoria and in some Sporozoa a mem-
brane, ectoplasm, cortical plasm, and endoplasm, differing from one
another in density, can be distinguished. It is an interesting fact
that in the artificial mixtures which Biitschli has so successfully
made to imitate protoplasm, a similar regional differentiation, at least
as far as ectoplasm and endoplasm are concerned, may be seen. It is perhaps to a tendency of protoplasm to stiffen while in con- It is perhaps to a tendency of protoplasm to stiffen while in con-
tact with water that we can turn for an explanation, first pointed out
by Griiber (81), of the outer condensation of protoplasm resulting in
the numerous membranes and tests of the Rhizopoda, and of the
outer coverings of Protozoa in general. The simplest form of mem-
brane is an almost invisible cuticle of extreme delicacy (pe//écula of
R. S. Bergh) as in the rhizopod Ameba proteus (Griiber, 81). In
other forms of the same genus, however, the outer zone becomes
greatly thickened (A. 1.
Lhe Endoplasm. These
spots are supposed to have some special relation to light,’ an unproved,
though probable, view which is based chiefly upon the fact that in
many of them there is a distinct lens-like body, and other structures
which usually accompany eyes of primitive form in other types of
invertebrates? (Fig. 11). Among other inclusions occasionally found within the endoplasm Among other inclusions occasionally found within the endoplasm
are the peculiar trichocysts found in the holotrichous ciliates (Para-
mecium and its allies). These are minute defensive
or possibly
offensive weapons analogous to the stinging threads of the Celen-
terata. Nematocysts containing a spirally wound thread, as in the
Ccelenterata, are also found in two forms, one a dinoflagellate (Poly-
krikos), the other a ciliate (Epistylis wmbellaria),. while analogous
thread-bearing structures are found in the spores of all Myxospo-
ridiida among the Sporozoa (Fig. 12, C, D). THE PROTOZOA 38 2. The Ectoplasm. In many Protozoa 2.
The Ectoplasm.
In many Protozoa ¢entaculata, A. actinophora, Fig. 12, A), and a
more or less lifeless membrane results. In these thick-skinned forms
the membrane is often perforated by the pseudopodia, which form long
finger-like processes, and when retracted leave minute holes in the
membrane. In these cases
there
is usually
a sharp distinction
between the inner plasm and the cortical part, but in many Infusoria
and Sporozoa there is a gradual increase in density from within
outward, and the outside is covered by living membranes which may
become complicated by the addition of muscular fibrils (azyonemes), of GENERAL SKETCH 39 sensory and tactile organs (czrrz), or protective structures like hooks,
spines, and tentacles (Fig. 12, B-G; see also Fig. 16). Like many of the cells which constitute the tissues of highe sensory and tactile organs (czrrz), or protective structures like hooks,
spines, and tentacles (Fig. 12, B-G; see also Fig. 16). Like many of the cells which constitute the tissues of higher ani- Like many of the cells which constitute the tissues of higher ani- Like many of the cells which constitute the tissues of higher ani-
1b
caer
ae
<= ea
-
are
a
4. Fig. 12. — Ectoplasmic modifications. A. Ameba tentaculata,
[GRUBER]
B. Clepsidrina munieri,
[SCHEWIAKOFF.]
C. Tri-
chocysts. [SCHEWIAKOFF.]
2, Nematocysts from the sporozoén A/yxobolus. [BALBIANL.]
£, F, G, attaching hooks and spines from different Gregarinida. [WASIELEWSKY.]
mals, the protozoan body has the power of forming by chemical pro-
cesses over and above those which relate merely to nutrition, various
products which are secreted just within the peripheral plasm, where Fig. 12. — Ectoplasmic modifications. A. Ameba tentaculata,
[GRUBER]
B. Clepsidrina munieri,
[SCHEWIAKOFF.]
C. Tri-
chocysts. [SCHEWIAKOFF.]
2, Nematocysts from the sporozoén A/yxobolus. [BALBIANL.]
£, F, G, attaching hooks and spines from different Gregarinida. 1Cf. Schewiakoff (’88). 2.
The Ectoplasm.
In many Protozoa [WASIELEWSKY.] mals, the protozoan body has the power of forming by chemical pro-
cesses over and above those which relate merely to nutrition, various
products which are secreted just within the peripheral plasm, where
they usually form a protective armor in the shape of shells, tests, or
“houses.”
The materials thus formed within the cell-body may be
chitin (composed of C, H, N, and O, and supposed to be a deriva-
tive from carbohydrates, but the exact formula is in dispute), ced/zlose mals, the protozoan body has the power of forming by chemical pro-
cesses over and above those which relate merely to nutrition, various
products which are secreted just within the peripheral plasm, where
they usually form a protective armor in the shape of shells, tests, or
“houses.”
The materials thus formed within the cell-body may be
chitin (composed of C, H, N, and O, and supposed to be a deriva-
tive from carbohydrates, but the exact formula is in dispute), ced/zlose THE PROTOZOA 40 (CgH,)0;), calcium carbonate (CaCO), and silica. The secretions
may take the form of plates, of continuous deposits, or of regular
skeletons which are often extremely complex (Fig. 13, D). In the
majority of cases, the secretions are made in the ectoplasm, although
in one well-authenticated case at least (Euglypha alveolata, Fig. 13, A),
the plates destined to form the shell are formed in the endoplasm and
in the immediate vicinity of the nucleus! In other shell-bearing
forms of Rhizopoda, there is usually a basis of chitin, upon which
the various shell-substances are deposited, or the shell may consist of
the chitin alone. In some cases it is no more than a cap covering a
small portion of the body, and into which the entire protoplasmic
mass could not possibly be withdrawn (Pseudochlamys, Fig. 13, C). Here the chitin which forms the shell is perfectly smooth; but in
other forms it may be ornamented in various ways by pits or pro-
tuberances. Again, in many fresh-water Rhizopoda the shell-material
is not secreted, but the test is composed of foreign particles, such
as diatom shells, sand crystals, mud, or detritus of any kind, fused
together and to a chitinous substratum by means of mucilaginous
cement secreted by the organism. 3. Nuclet. Haeckel’s 3. Nuclet.
Haeckel’s Haeckel’s claim (’68) that there are organisms without nuclei
(Monera), although it rests upon negative evidence, cannot be rejected
until all of the forms considered have been shown to possess them. On purely @ priort grounds, it is possible to conceive such organisms,
although the numerous experiments which have been performed dur-
ing the last decade upon nucleated and non-nucleated parts of Pro-
tozoa, show, in these cases at least, the absolute necessity of the
nucleus for the life of the organism. These experiments make it
probable that the so-called Monera have in reality some structure or
structures which perform the functions of the nucleus, although a
well-defined nucleus with membrane and other characteristic parts
may be absent. In the majority of Protozoa there is but one nucleus (many Sar- In the majority of Protozoa there is but one nucleus (many Sar-
codina, Mastigophora, Sporozoa), while in some forms two nuclei are
the rule (some Khizopoda). In others, again, there may be a great
number of nuclei, the number varying with the age of the organism
(examples occur in all groups of the Protozoa). In many of the Pro-
tozoa, although not in all, the nucleus is provided with a membrane
and contains two substances; chromatin, staining with certain basic
dyes and consisting largely of nucleinic acid, and achromatin, a sub-
stance which is not stained by the chromatin dyes, in the form of a Fig. 13. — Shells and tests. [4, SCHEWIAKOFF; B, ORIGINAL;
C, BUTSCHLI; D, STEIN.]
A, Euglypha alveolata Duj. The shell consists of oval siliceous plates glued together by a sili-
ceous (?) cement. B. Cochliopodium digitatum, n.sp. The test is membranous and perforated
for pseudopodia. C. Pseudochlamys patella Clp.and Lach,
The test is membranous and shield-
like. D. Ceratium tripos Nitsch. The shell consists of cellulose plates of diverse size and shape. Fig. 13. — Shells and tests. [4, SCHEWIAKOFF; B, ORIGINAL;
C, BUTSCHLI; D, STEIN.]
A, Euglypha alveolata Duj. The shell consists of oval siliceous plates glued together by a sili-
ceous (?) cement. B. Cochliopodium digitatum, n.sp. The test is membranous and perforated
for pseudopodia. C. Pseudochlamys patella Clp.and Lach,
The test is membranous and shield-
like. D. Ceratium tripos Nitsch. The shell consists of cellulose plates of diverse size and shape. THE PROTOZOA 42 network, or of a homogeneous body of considerable size (Karyo-
somes). Fig. 14. — Types of nuclei.
[4. Calcituba polymorpha Roboz, from SCHAUDINN; &. Colpidium
colpoda, from a preparation;
C. Euglena viridis Ehv. from a preparation; D. Tetramitus chilomo-
mas, n.sp.; 4. Noctiluca miliaris Sur., from a preparation.]
A single karyosome (.1) becomes vesicular, and ultimately gives rise to several daughter-karyo-
somes (so-called ‘fragmentation’ Schaudinn).
Several karyosomes in Actiluca (E) hold the
chromatin, the rest of the nucleus is filled with “achromatic” granules.
In Tetramitus chilomonas
(P) the chromatin is scattered throughout the cell; the lighter-colored body in the centre of the
cell is the homologue of the deeply stained central body in Euglena (C). Fig. 14. — Types of nuclei.
[4. Calcituba polymorpha Roboz, from SCHAUDINN; &. Colpidium
colpoda, from a preparation;
C. Euglena viridis Ehv. from a preparation; D. Tetramitus chilomo-
mas, n.sp.; 4. Noctiluca miliaris Sur., from a preparation.]
A single karyosome (.1) becomes vesicular, and ultimately gives rise to several daughter-karyo-
somes (so-called ‘fragmentation’ Schaudinn).
Several karyosomes in Actiluca (E) hold the
chromatin, the rest of the nucleus is filled with “achromatic” granules.
In Tetramitus chilomonas
(P) the chromatin is scattered throughout the cell; the lighter-colored body in the centre of the
cell is the homologue of the deeply stained central body in Euglena (C). 3. Nuclet.
Haeckel’s Several different types of nuclei may be distinguished ;
some of the most
important being: (1) The distributed nucleus, network, or of a homogeneous body of considerable size (Karyo-
somes). Several different types of nuclei may be distinguished ;
some of the most
important being: (1) The distributed nucleus, GENERAL SKETCH 43 consisting of innumerable chromatin granules distributed throughout
the cell (7rachelocerca, Chenia tercs, Holosticha flava, HI. scutellum,
Tetramitus). (2) The homogencous nucleus consisting of a single
mass of chromatin with a homogeneous
structure throughout
all
stages, and with no trace of reticular substance (many Phytoflagel-
lates). (3) Dimorphic nuclei, consisting of a large nucleus called the
macronucleus, and a small one, the micronucleus, in the same individual. The former is generally regarded as functional chiefly in vegetation,
the latter in conjugation. With the exception of Polykrikos among
the Mastigophora, dimorphic nuclei are found only in the Infusoria
(Fig. 14). The typical form of the nucleus is spherical, although it may be The typical form of the nucleus is spherical, although it may be
discoid or ellipsoid, or, in the case of the macronucleus, drawn out
into various fantastic shapes, of which the horseshoe (Vorticellidae),
the beaded (Stentor and Spirostomum, etc.), or branched (Aciveta,
Dendrosoma) are examples} 1Cf. Chapter VII. for further details concerning nuclei. 4. Organs of Locomotion. With very few exceptions, the Protozoa have the power of moving
from place to place. The exceptions are found among the para-
sitic Sporozoa, although even here there is, in some cases, a peculiar
gliding motion. In no adult sporozodn is there a special organ of
locomotion, yet the Gregarinida and Heemosporidiida actually move
from place to place, although very slowly. In some cases,
the
motion
is due
to peculiar
peristaltic waves
of contraction;
in
other cases to the contraction of muscle-like fibrils, the myonemes. An analogous
movement
is also known
in
certain
flagellates
(Euglenide)
and
ciliates
(Heterotrichida). In
the majority
of
Protozoa, however, movement
is accomplished by the activity of
special motor organs, which may be either changeable processes
(pseudopodia) or permanent vibratile appendages (flagella and cilia). The changeable processes or pseudopodia, found chiefly in the Sar-
codina, are sometimes numerous, sometimes few; when few in number
they are usually short, finger-formed, and quick to change in form and
appearance by the flowing protoplasmic substance of which they are
composed (Fig. 1, A, and Fig. 15, 4, 2); when numerous, they are
fine-pointed, and often sticky, so that when two or more come in
contact, they fuse or anastomose (Reticulariida, Fig. 15, ©). Again,
the pseudopodia may be fine and pointed, but rigid in structure and
unchanging in form, a condition brought about by the presence of an
‘axial filament of stiffened protoplasm, which runs down the centre of
each pseudopodium (Heliozoa, Radiolaria, Fig. 15, D). Unlike pseu- THE PROTOZOA 44 dopodia, the protoplasmic filaments, known as flagella and cilia, are
derived solely from the ectoplasm and are constant in their position,
and, save for the occasional absorption within the body, for some rea-
son or other, they are unchangeable. Flagella-motion, characterized
by energetic contractions or undulations, or by rotary motions, differs derived solely from the ectoplasm and are constant in their position,
and, save for the occasional absorption within the body, for some rea-
son or other, they are unchangeable. Flagella-motion, characterized
by energetic contractions or undulations, or by rotary motions, differs
MAS
gant
este
7
nt
CN Rye
eg
Fre
a
RL
IE
Fig. 15.— Types of pseudopodia. A. AmebalimicolaRhmb, [RHUMBLER.]
&. Amoeba dlatte Biitsch, [BUTSCHLI.]
C. Lieber-
Riihnia sp. (VERWORN.]
D. Actinospherium Eich, Ebr, [ORIGINAL.] , the axial filament. 4. Organs of Locomotion. MAS
gant
este
7
nt
CN Rye
eg
Fre MAS
gant
este
7
nt
CN Rye
eg
Fre
a
RL
IE
Fig. 15.— Types of pseudopodia. A. AmebalimicolaRhmb, [RHUMBLER.]
&. Amoeba dlatte Biitsch, [BUTSCHLI.]
C. Lieber-
Riihnia sp. (VERWORN.]
D. Actinospherium Eich, Ebr, [ORIGINAL.] , the axial filament. Fig. 15.— Types of pseudopodia. [RHUMBLER.]
&. Amoeba dlatte Bi Fig. 15.— Types of pseudopodia. A. AmebalimicolaRhmb, [RHUMBLER.]
&. Amoeba dlatte Biitsch, [BUTSCHLI.]
C. Lieber-
Riihnia sp. (VERWORN.]
D. Actinospherium Eich, Ebr, [ORIGINAL.] , the axial filament. Fig. 15.— Types of pseudopodia. A. AmebalimicolaRhmb, [RHUMBLER.]
&. Amoeba dlatte Biitsch, [BUTSCHLI.]
C. Lieber-
Riihnia sp. (VERWORN.]
D. Actinospherium Eich, Ebr, [ORIGINAL.] , the axial filament. A. AmebalimicolaRhmb, [RHUMBLER.]
&. Amoeba dlatte Biitsch, [BUTSCHLI.]
C. Lieber-
Riihnia sp. (VERWORN.]
D. Actinospherium Eich, Ebr, [ORIGINAL.] , the axial filament. entirely from the slow flowing movement of pseudopodia; yet, as
Dujardin
first observed, in some forms pseudopodia change into
flagella, and flagella into pseudopodia. — In structure, flagella are long,
thin, usually pointed threads of protoplasm, which,
as a rule, are
longer than the cell itself; they are typically single, but there may
be two, three, or many. Cilia, on the contrary, are always multiple,
and are never interchangeable with pseudopodia (Fig. 16). Although GENERAL SKETCH 45 characteristic of various epithelial tissues in Metazoa, they are found
only in a single specialized group of the Protozoa, the Infusoria. In
form, they are similar to flagella, but as a rule they are shorter, never
pointed, and more numerous, many of them acting in unison, with
a quick regular motion like a set of oars. In some groups, the cell
is completely clothed with these motile elements (Holotrichida), in
others only a portion is covered either in one or more rings about the
body (Peritrichida), or upon one surface only (Hypotrichida). The
cilia may also become variously modified by fusion with one another, REE
E
oe
; is
Fig. 16.— Cilia and myonemes of Infusoria. [a, 6, and c, JOHNSON;
¢, d, f, g, BUTSCHLI.]
The surface view of Stentor ceruleus (c, e) shows rows of cilia inserted on the borders of canal-
like markings, each of which contains
a myoneme (d). These are more clearly shown in the
optical section (f). In AHolophrya discolor (g) the canals and myonemes are inserted deeper in
the cortical plasm. a@, the membrane of Stentor cwruleus under pressure. 4. Organs of Locomotion. It becomes much more definite in forms provided
with flagella, where, in many cases, a steady progression with the
flagellum in advance is the characteristic motion. In one group of
the Mastigophora, however, the Choanoflagellida, the flagellum, like
the tail of a spermatozoon,
is directed backward during motion. Among the Ciliata, complex movements accompany the high organiza-
tion of the cell, and the change from one form to another, apparently
at the will of the organism, is extremely suggestive of conscious action. Here, in addition to the normal and constant motion of the cilia, are
various
forms of contractile movement varying from the simple
sarcode streaming, which
is characteristic of the Suctoria, to the
definite contraction of distinct muscular elements in the myonemes of
Heterotrichida and Peritrichida. 4. Organs of Locomotion. REE
E
oe
; is Fig. 16.— Cilia and myonemes of Infusoria. [a, 6, and c, JOHNSON;
¢, d, f, g, BUTSCHLI.]
The surface view of Stentor ceruleus (c, e) shows rows of cilia inserted on the borders of canal-
like markings, each of which contains
a myoneme (d). These are more clearly shown in the
optical section (f). In AHolophrya discolor (g) the canals and myonemes are inserted deeper in
the cortical plasm. a@, the membrane of Stentor cwruleus under pressure. giving rise to motile organs of a more complex structure, such as the
membranes, membranelles, and cirri found in different groups of the
ciliated Infusoria. In many Protozoa the adult forms have no distinct motile organs, In many Protozoa the adult forms have no distinct motile organs,
although they may pass through embryonic stages in which such
structures
are present. The Suctoria, for example,
are,
for the
most part, entirely devoid of cilia in the adult stages, although the
embryos possess them. Again, many of the Rhizopoda pass through
flagellated stages before assuming the amceboid condition, and certain
Mastigophora pass through amoeboid swarm-spore
stages. Some
Heliozoa and Radiolaria similarly pass through both flagellated and
amceboid stages before assuming their own adult forms. the part of Protozoa provided with pseudo- Normal movement on the part of Protozoa provided with pseudo- THE PROTOZOA 46 podia consists in the simple protrusion and retraction of the change-
able processes. It becomes much more definite in forms provided
with flagella, where, in many cases, a steady progression with the
flagellum in advance is the characteristic motion. In one group of
the Mastigophora, however, the Choanoflagellida, the flagellum, like
the tail of a spermatozoon,
is directed backward during motion. Among the Ciliata, complex movements accompany the high organiza-
tion of the cell, and the change from one form to another, apparently
at the will of the organism, is extremely suggestive of conscious action. Here, in addition to the normal and constant motion of the cilia, are
various
forms of contractile movement varying from the simple
sarcode streaming, which
is characteristic of the Suctoria, to the
definite contraction of distinct muscular elements in the myonemes of
Heterotrichida and Peritrichida. podia consists in the simple protrusion and retraction of the change-
able processes. B. GENERAL PHYSIOLOGY In all Protozoa, as in higher animals, the functions of nutrition,
respiration, excretion, reproduction, and irritability the analogue of
nerve-response, are indispensable for the life of the organism. When
compared with the vital functions of the higher animals, all of these
processes appear simple; yet the difference is one of degree only, and
among the unicellular animals, as among the multicellular, the func-
tions become more complicated and difficult to analyze as the cell-
structures become more complex. In the simpler forms, the naked
unmodified protoplasm contains the beginnings of the most compli-
cated functions, none of which can be regarded as having a par-
ticular time and place of birth in the series of animal forms; all are
characteristic of the cell, and beyond that, of living protoplasm, of
which they are the distinguishing properties. The most primitive
Protozoa, entirely destitute of organs, feed without mouth or digestive
tract, move without appendages, react to external stimuli, excrete,
and reproduce. In the higher types the cell-organism becomes dif-
ferentiated into special parts for the performance of these various
functions, and the relative position of the organism in the scale of
Protozoa depends upon the degree of this differentiation. Inno class
of animals is the connection between division of physiological labor
and regional
\
differentiation
so clearly marked
as here. This is
especially noteworthy in the outer plasm, which, directly correlated
with the action of the’ environment, has apparently become progres-
sively modified into external coverings, into motile organs, and into
organs of sensation, while the endoplasm retains the same character
throughout the group. One function, that of excystment, is limited almost exclusively to One function, that of excystment, is limited almost exclusively to GENERAL SKETCH 47 the
Protozoa,
although
occasionally
seen in some Metazoa
(e.g. Macrobiotus, or the “water bear,” and some rotifers). This is a
special adaptive process by which
the organisms
are
enabled to
survive when the environment
is unsuitable. If a pool dries up,
becomes too dense, or too foul from putrefaction or other causes, Fig. 17. — Types Fig. 17. — Types of cysts. B. Stylonychia mytilus Ebr the cell draws in its appendages, rounds out into a sphere and secretes
a resisting membrane, within which it can exist for a long period. When first formed, this membrane is a delicate gelatinous substance,
which soon hardens and gradually acquires the peculiar characters of
chitin. Fig. 17.
— Types of cysts.
A, Ameba proteus.
[SCHEEL.]
B. Stylonychia mytilus Ebr.
[BUTSCHLI.]
C. Pleurotricha
grandis St. (BUTSCHLL.]
D. Euglpha alveolata Duj.
[LEIDY.]
£. Actimospherium Lich. Ebr.
(BuTSCHLI.]
/. Colpoda Steinu. [MAUPAS.]
a, gelatinous matrix; 4, outer cyst wall; c, middle
cyst wall; @, inner cyst wall. A, Ameba proteus.
[SCHEEL.]
B. Stylonychia mytilus Ebr.
[BUTSCHLI.]
C. Pleurotricha
grandis St. (BUTSCHLL.]
D. Euglpha alveolata Duj.
[LEIDY.]
£. Actimospherium Lich. Ebr.
(BuTSCHLI.]
/. Colpoda Steinu. [MAUPAS.]
a, gelatinous matrix; 4, outer cyst wall; c, middle
cyst wall; @, inner cyst wall. B. GENERAL PHYSIOLOGY With the exception of the contractile vacuole, which continues
to contract rhythmically for some time, all of the organs of the body
are quiet at this period. The water, expelled by the vacuole, collects
between the cyst and the spherical wall of the animal, the latter 48 THE PROTOZOA becoming smaller and smaller as more and mort water is expelled. The nucleus appears unaltered, except for a very slight reduction in
size (Fig. 17). In this condition the animal can withstand a long
period of desiccation,
or even extreme heat and cold, and, owing to its
minute size, it may be blown hither and thither until it reaches some
favorable spot where it may recommence active life. Water is then
absorbed, the cyst is ruptured, and the former active life begins anew. Encystment may occur in some cases after a particularly heavy meal,
or more frequently, before reproduction by spore-formation or simple
division. 1. Nutrition.
The processe The processes of nutrition, as in the higher animals, may be divided
into three stages:
I, the capture and ingestion of food;
2, the
digestion of the ingested parts; and 3, the ejection or defecation of
the undigested remains. Many of the Protozoa have no special apparatus for seizing and Many of the Protozoa have no special apparatus for seizing and
ingesting food, but absorb it directly from the surrounding medium. Thus many Mastigophora live, like the fungi, by absorption, through
the body walls, of fluids which hold in solution the products of decom-
position of other animal and plant forms. Others, as the Sporozoa
and some Ciliata, live like a tapeworm and other intestinal parasites,
upon the digested foods of the alimentary tract, or in nutritive fluids
in other cavities of different hosts. The Phytoflagellida, also, do not
ingest solid food, but, by the aid of chromatophores, they have the
power of manufacturing their food in the same manner as do the
green plants. The majority of Protozoa, however, take in solid food
through more or less definite regions of the body. In some of the
phytoflagellates there is a distinct mouth-opening, in addition to the
chromatophores, and such forms may combine both animal and plant
modes of nutrition. Food-taking has been carefully examined in connection with the Food-taking has been carefully examined in connection with the
Ciliata, where many species living upon certain specific organisms
apparently select their food. Thus Maupas (’88) distinguishes forms
that are herbivorous, others that are carnivorous, and still others that
are omnivorous. The food that may be thus selected consists of all
sorts of lower plants, such as desmids, diatoms, zodspores, bacteria,
filamentous algze, etc., while among the animals the Mastigophora
and smaller ciliates are the most frequent victims, although rotifers
and small worms are often eagerly seized. Maupas believes that
the cause of these various adaptations in feeding should be sought
in the modifications of the mouth. ‘The mouth is, in short,” he says,
“the dominating organ par crcellence in the morphology and the
physiology of the Ciliata. Nutrition in its manifold phases in these GENERAL SKETCH 49 minute beings absorbs and completes their entire existence. This
function assumes with them an intensity which, I believe, is equalled
nowhere else in the animal kingdom. They are gluttons par excel-
fence, absorbing and digesting night and day without repose. 1 (88), p. 185. 1. Nutrition.
The processe When, however, an Halteria grandinella, with its quick and jerky
movements, approaches the spot, the carnivore is not so peaceful. The trichocysts are discharged with unerring aim, and the Helteria
whirls around in a vigorous, but vain, effort to escape, then becomes
quiet, with
cilia outstretched, perfectly paralyzed. The tentacle,
with its prey fast attached, is then slowly contracted until the victim
is brought to the body, where, by action of the cilia, it is gradually
worked around to the mouth and swallowed with one gulp. Within
the short time of twenty minutes, I have seen an Actinobolus thus
capture and swallow no less than ten Hadterias. find their way by protoplasmic streaming to all parts of the animal. In many Mastigophora and Ciliata, the motile organs create a vor-
tex current in the region of a well-defined mouth, which usually leads
into a distinct pharynx. In some flagellates, the base of the flagel-
lum is an area of soft plasm, through which the food particles can be
readily engulfed as they strike against it, but in others there is a dis-
tinct opening which leads into the endoplasm. In other flagellates
(Noctiluca and the Choanoflagellida), a peculiar protoplasmic funnel-
shaped collar surrounds the region which answers the same purpose. In most of the Ciliata, the buccal region is surrounded by strong
cilia, which are frequently fused to form membranes or membra-
nelles; these send a powerful current of water, containing innutri-
tious as well as nutritious particles, toward the mouth, which receives
all without discrimination. In some cases, as in certain of the holo-
trichous Ciliata, there is a true swallowing or deglutition, by which
solid food is gulped into a capacious pharynx and thence into gastric
vacuoles. Many of the latter forms have offensive trichocysts, resem-
bling the rhabdites of Turbellaria. One of these, upon the approach
of its prey (usually a small ciliate or flagellate), launches its darts,
which penetrate the cuticle and paralyze the prey. The victim is then
swallowed, the mouth of the carnivore enlarging to accommodate it
(Fig. 18, C). This process is strikingly illustrated by the ciliate
Actinobolus radians, which combines the selection of food with the
offensive use of trichocysts. This remarkable organism possesses a
coating of cilia and protractile tentacles, which may be elongated
to a length equal to three times the body-diameter, or withdrawn
completely into the body. 1. Nutrition.
The processe It
results that the apparatus charged with the performance of such
an intense function becomes modified, diversified, and developed to
an astonishing degree, especially striking when it is remembered that
these are unicellular organisms.” +
The capture and ingestion of food, in its simplest form, occurs in minute beings absorbs and completes their entire existence. This
function assumes with them an intensity which, I believe, is equalled
nowhere else in the animal kingdom. They are gluttons par excel-
fence, absorbing and digesting night and day without repose. It
results that the apparatus charged with the performance of such
an intense function becomes modified, diversified, and developed to
an astonishing degree, especially striking when it is remembered that
these are unicellular organisms.” +
The capture and ingestion of food, in its simplest form, occurs in The capture and ingestion of food, in its simplest form, occurs in Fig. 18.—Food-taking. [.4, PENARD; Z and C, BUTSCHLI.]
A. Raphidiophrys elegans Hert. and Lesser. B. Oikomonas termo Ehr. C. Didinum nasutum,
O.F.M. ¥ food particles. teus, any part of the UTSCHLI.] .] Fig. 18.—Food-taking. [.4, PENARD; Z and C, BUTSCHLI.]
A. Raphidiophrys elegans Hert. and Lesser. B. Oikomonas termo Ehr. C. Didinum nasutum,
O.F.M. ¥ food particles. as in Amaba proteus, any part of the Fig. 18.—Food-taking. [.4, PENARD; Z and C, BUTSCHLI.]
A. Raphidiophrys elegans Hert. and Lesser. B. Oikomonas termo Ehr. C. Didinum nasutum,
O.F.M. ¥ food particles. proteus, any part of the the group of Rhizopoda, where, as in Amaba proteus, any part of the
body can act as a mouth. In this form pseudopodia are pushed out
toward the victim (a flagellate, ciliate, minute plant form of any
kind, or even a higher animal, such as a rotifer or worm) and entirely
surround it, together with a certain amount of water, thus forming a
gastric vacuole, or an improvised “stomach.”
When the rhizopod is
provided with a shell, the food-taking area is limited to a mouth-open-
ing in the shell, while in many of the shelled Heliozoa the taking of
food is complicated by the presence of an unbroken coating, and a
special opening must be made for each ingestion (Fig. 18). In the
Reticulariida, the gastric vacuoles are on the outside of the shell, and
are formed in the network produced by the anastomosis of the pseu- 1 (88), p. 185. 1 (88), p. 185. 1. Nutrition.
The processe E E THE PROTOZOA 50 dopodia. Here the prey is digested, and the products of digestion
find their way by protoplasmic streaming to all parts of the animal. In many Mastigophora and Ciliata, the motile organs create a vor- find their way by protoplasmic streaming to all parts of the animal. In many Mastigophora and Ciliata, the motile organs create a vor-
tex current in the region of a well-defined mouth, which usually leads
into a distinct pharynx. In some flagellates, the base of the flagel-
lum is an area of soft plasm, through which the food particles can be
readily engulfed as they strike against it, but in others there is a dis-
tinct opening which leads into the endoplasm. In other flagellates
(Noctiluca and the Choanoflagellida), a peculiar protoplasmic funnel-
shaped collar surrounds the region which answers the same purpose. In most of the Ciliata, the buccal region is surrounded by strong
cilia, which are frequently fused to form membranes or membra-
nelles; these send a powerful current of water, containing innutri-
tious as well as nutritious particles, toward the mouth, which receives
all without discrimination. In some cases, as in certain of the holo-
trichous Ciliata, there is a true swallowing or deglutition, by which
solid food is gulped into a capacious pharynx and thence into gastric
vacuoles. Many of the latter forms have offensive trichocysts, resem-
bling the rhabdites of Turbellaria. One of these, upon the approach
of its prey (usually a small ciliate or flagellate), launches its darts,
which penetrate the cuticle and paralyze the prey. The victim is then
swallowed, the mouth of the carnivore enlarging to accommodate it
(Fig. 18, C). This process is strikingly illustrated by the ciliate
Actinobolus radians, which combines the selection of food with the
offensive use of trichocysts. This remarkable organism possesses a
coating of cilia and protractile tentacles, which may be elongated
to a length equal to three times the body-diameter, or withdrawn
completely into the body. The ends of the tentacles are loaded with
trichocysts (Entz, 83). When at rest (Fig. 19), the mouth is directed
downward,
and the tentacles
are stretched
out in all directions,
forming a minute forest of plasmic processes, amongst which smaller
ciliates, such as Uvocentrum,
Gastrostyla, etc., or flagellates of all
kinds, may become entangled without injury to themselves and
without disturbing the Actzobolus or drawing out the fatal darts. 1. Nutrition.
The processe The ends of the tentacles are loaded with
trichocysts (Entz, 83). When at rest (Fig. 19), the mouth is directed
downward,
and the tentacles
are stretched
out in all directions,
forming a minute forest of plasmic processes, amongst which smaller
ciliates, such as Uvocentrum,
Gastrostyla, etc., or flagellates of all
kinds, may become entangled without injury to themselves and
without disturbing the Actzobolus or drawing out the fatal darts. When, however, an Halteria grandinella, with its quick and jerky
movements, approaches the spot, the carnivore is not so peaceful. The trichocysts are discharged with unerring aim, and the Helteria
whirls around in a vigorous, but vain, effort to escape, then becomes
quiet, with
cilia outstretched, perfectly paralyzed. The tentacle,
with its prey fast attached, is then slowly contracted until the victim
is brought to the body, where, by action of the cilia, it is gradually
worked around to the mouth and swallowed with one gulp. Within
the short time of twenty minutes, I have seen an Actinobolus thus CENERAL SKETCH SI Still another mode of food-taking is found among the Suctoria. Here there is no mouth and no motile organ to create currents, but
the body is provided with distinct tentacle-like processes, through the Z
Fig. 19. — Actinobolus radians St. The organism is represented at rest, with the mouth turned downward, and with the tentacles
widely outstretched. At the base of each tentacle is a brush of 8 to 12 cilia which vibrate like
flagella instead of striking like cilia. Within the body are represented the nucleus, contractile
vacuole, and one Hadlteria. Fig. 19. — Actinobolus radians St. The organism is represented at rest, with the mouth turned downward, and with the tentacles
widely outstretched. At the base of each tentacle is a brush of 8 to 12 cilia which vibrate like
flagella instead of striking like cilia. Within the body are represented the nucleus, contractile
vacuole, and one Hadlteria. ends of which the food substances
are
absorbed
into the body. These tentacles are of various kinds, some sharp-pointed for piercing,
others cup-shaped for attachment by suction, while others are pointed
and spirally wound (Fig. 20). The cuticle of the prey is pierced by the ends of which the food substances
are
absorbed
into the body. 1. Nutrition.
The processe The gastric vacuole, after the contained
food has been digested as far as possible, frequently becomes a
defecatory vacuole, and its contents are expelled to the outside at the
posterior end of the individual. Finally, a distinct and permanent
anal opening is found in the more complex ciliates. 1. Nutrition.
The processe These tentacles are of various kinds, some sharp-pointed for piercing,
others cup-shaped for attachment by suction, while others are pointed
and spirally wound (Fig. 20). The cuticle of the prey is pierced by the THE PROTOZOA 52 sharper tentacles, and its fluid endoplasm passes in a current down
the cavity of the tentacle and into the endoplasm of the suctorian. In other cases, the endoplasm of the tentacle passes into the body of
the prey and there digests the internal substance zw sztu, the digested
parts flowing back into the body of the minute carnivore. A similar
mode of food-taking occurs in some Heliozoa (Vampyrella), where
the parasite penetrates the cells of algae and there digests the proto-
plasm. ithin the endoplasm. In all Protozoa, digestion is accomplished within the endoplasm. The ingested proteid is contained within a gastric vacuole
filled
primarily with water, which is taken in with the food. The water,
however, gradually changes by osmosis with the fluids of the plasm, Fig. 20. — Tentacles of Suctoria. [HERTWIG.]
A. Seizing tentacles of Ephelota. B. Veeding and seizing tentacles of Ephelota. Fig. 20. — Tentacles of Suctoria. [HERTWIG.]
A. Seizing tentacles of Ephelota. B. Veeding and seizing tentacles of Ephelota. among these is a digestive, acid fluid, which reduces the digestible
portions of the food probably to some form of peptone. Then, again
by osmosis, the digested portions are assimilated in all parts of the
endoplasm. The indigestible remains of the food are excreted in
various ways. Sometimes, as in the Rhizopoda, they are voided
from any portion of the body, usually, however, from that part which
at the time is posterior. The gastric vacuole, after the contained
food has been digested as far as possible, frequently becomes a
defecatory vacuole, and its contents are expelled to the outside at the
posterior end of the individual. Finally, a distinct and permanent
anal opening is found in the more complex ciliates. among these is a digestive, acid fluid, which reduces the digestible
portions of the food probably to some form of peptone. Then, again
by osmosis, the digested portions are assimilated in all parts of the
endoplasm. The indigestible remains of the food are excreted in
various ways. Sometimes, as in the Rhizopoda, they are voided
from any portion of the body, usually, however, from that part which
at the time is posterior. 2.
Excretion.
Like all othe however, there are specialized s
outside of the organism a certai
structures
are the contractile
vacuoles, which, with the
ex-
ceptions of the Sporozoa and
the marine forms, are found
in every class of the Protozoa. In the living animal the vacu-
ole is a clear spherical area in
the endoplasm. It is formed
by the slow addition of water
from the endoplasm, and grows
until a maximum size is reached,
when it suddenly disappears,
the
contained
water
being
driven to the outside. Vacu-
oles are frequently variable in
position (Sarcodina), while the
number is, to a certain extent,
dependent upon the condition
of the protoplasm, several ob-
servers having shown that, as
the individuals lose their vital-
ity, the protoplasm
becomes
more and more vacuolated. In
many Cases the vacuole moves
about with
the
endoplasmic
flow until, becoming heavier than
while the rest of the endoplasm
(many Rhizopoda). In this ma
size, is gradually left at the po
where it finally bursts. Again,
the contractile vacuole is a stati
side by a definite pore. Here,
in the form of canals and reserv
the water and waste matters fro of Protozoa there is no known or
removed. In such forms excret
through the walls of the body, i
phytic forms take food. This mu
many marine forms as well as in
no specialized excretory organ. however, there are specialized st
outside of the organism a certain
structures
are the contractile
vacuoles, which, with the
ex-
ceptions of the Sporozoa and
the marine forms, are found
in every class of the Protozoa. In the living animal the vacu-
ole is a clear spherical area in
the endoplasm. It is formed
by the slow addition of water
from the endoplasm, and grows
until a maximum size is reached,
when it suddenly disappears,
the
contained
water
being
driven to the outside. Vacu-
oles are frequently variable in
position (Sarcodina), while the
number is, to a certain extent,
dependent upon the condition
of the protoplasm, several ob-
servers having shown that, as
the individuals lose their vital-
ity, the protoplasm
becomes
more and more vacuolated. In
many Cases the vacuole moves
about with
the
endoplasmic
flow until, becoming heavier than
while the rest of the endoplasm
(many Rhizopoda). In this man
size, is gradually left at the pos
where it finally bursts. Again,
the contractile vacuole is a stati
side by a definite pore. 2.
Excretion.
Like all othe Like all other animals, the protozoén uses a certain amount of pro-
toplasm in the performance of its vital activities. Ina large number GENERAL SKETCH 53 of Protozoa there is no known organ by which the waste products are
removed. In such forms excretion probably takes place by osmosis
through the walls of the body, in the same way possibly that sapro-
phytic forms take food. This must be the case in the Sporozoa and
many marine forms as well as in certain flagellates, in which there is
no specialized excretory organ. In the majority of the Protozoa,
however, there are specialized structures which regularly throw to the
outside of the organism a certain amount of fluid substance. These
structures
are the contractile
vacuoles, which, with the
ex-
ceptions of the Sporozoa and
the marine forms, are found
in every class of the Protozoa. In the living animal the vacu-
ole is a clear spherical area in
the endoplasm. It is formed
by the slow addition of water
from the endoplasm, and grows
until a maximum size is reached,
when it suddenly disappears,
the
contained
water
being
driven to the outside. Vacu-
oles are frequently variable in
position (Sarcodina), while the
number is, to a certain extent,
dependent upon the condition
of the protoplasm, several ob-
servers having shown that, as
the individuals lose their vital-
ity, the protoplasm
becomes
more and more vacuolated. In Fig. 21.—
7) ontonia leucas Ehr. [SCHEWIAKOFF.]
many Cases the vacuole moves
¢, canal; v, vacuole with external pore; A’, macro-
about with
the
endoplasmic nucleus; x, micronucleus. ,
flow until, becoming heavier than the protoplasm, it remains stationary,
while the rest of the endoplasm moves forward with the organism
(many Rhizopoda). In this manner the vacuole, as it attains its full
size, is gradually left at the posterior end of the moving organism,
where it finally bursts. Again, as in some Mastigophora and Ciliata,
the contractile vacuole is a stationary organ connected with the out- of Protozoa there is no known o
removed. In such forms excret
through the walls of the body,
phytic forms take food. This m
many marine forms as well as i
no specialized excretory organ. 2.
Excretion.
Like all othe Here, t
in the form of canals and reservo
the water and waste matters from
them to the contractile vesicle, t
traction of the vacuole, and con Fig. 21.—
7) ontonia leucas Ehr. [SCHEWIAKOFF.]
¢, canal; v, vacuole with external pore; A’, macro-
nucleus; x, micronucleus. , Fig. 21.—
7) ontonia leucas Ehr. [SCHEWIAKOFF.]
¢, canal; v, vacuole with external pore; A’, macro-
nucleus; x, micronucleus. ,
n the protoplasm, it remains stationary, THE PROTOZOA 54 they are in open connection. The canal system, which some observers
(e.g. Fabre Dumergue, ’90) consider widely spread throughout the
Ciliata, is often strikingly developed, as in /vontonza, where there is a
complicated network traversing the entire cell (Fig. 21). While the excretory function of the contractile vacuole is generally While the excretory function of the contractile vacuole is generally
accepted, there have been only a few satisfactory experiments to
demonstrate it, and the possibility of other functions is not excluded. At the present time the balance of evidence is in favor of the view
that the contractile vacuole has both excretory and respiratory func-
tions, inasmuch as it regularly empties a fluid to the outside, which
carries with it the products of destructive metabolism in the form of
wrea, and probably carbon dioxid, although the respiratory function
has never been demonstrated.1 ction of the contractile vacuole, it does
necessary for the life of the organism,
ozoa and the majority of the Sarcodina
). Furthermore, whatever the use of
the vacuole,
it is independent of the
nucleus, non-nucleated fragments form-
ing new vacuoles which pulsate rhyth-
mically for some
time. Hofer (’89)
found that vacuoles in non-nucleated bits
of Ameba proteus would
contract
for
‘fourteen
days. He
also
noted that
whereas the regular period of pulsation
was seven minutes, the periods became
longer and longer, until at the end of the
fourth day there was but one pulsation
every two hours, and even then the con-
tents were not completely expelled, a
reaction which Pénard (’90) formulated
later by the statement that the activity
of the contractile
vacuole
is directly
proportional to that of the entire indi-
vidual. Fig. 22.— Division ot Euplotes. [FROM
A PREPARATION.]
The danghter-cells are almost ready 1Cf. Chapter IX, p. 283. 3. Reproduction.
. Fig. 22.— Division ot Euplotes. [FROM
A PREPARATION.]
The danghter-cells are almost ready Fig. 22.— Division ot Euplotes. [FROM
A PREPARATION.]
The danghter-cells are almost ready ROM
. . ‘
With the exception of the Sporozoa,
eady simple division,
or splitting into two
lei
:
eee
ens
Parts, is the characteristic mode of re-
tric production
in
all Protozoa. In the
eds. Srorezed, aud at times in niost of the
has
nee! ;
other Protozoa, division
is replaced by
1Cf. Chapter IX, p. 283. The danghter-cells are almost ready
to separate;
the daughter-micronuclei
ge cian ees a macronuclens
(m) is not quite divided;
the gastric
vaguoles (7) are equaily aisitibuleds. the two daughter-cells, one of which has
generated the adoral zone (az). The danghter-cells are almost ready
to separate;
the daughter-micronuclei
ge cian ees a macronuclens
(m) is not quite divided;
the gastric
vaguoles (7) are equaily aisitibuleds. the two daughter-cells, one of which has
generated the adoral zone (az). 1Cf. Chapter IX, p. 283. GENERAL SKETCH 55 spore-formation or the breaking up of the body into many small
particles, each the germ of a new organism. While the major-
ity of the Protozoa reproduce asexually in these ways, reproduc-
tion in some is bound up with complete sexual differentiation, and a
series of forms may be selected which indicate the probable develop-
ment of the sexual from the more primitive methods. In numerous
cases the sexual phenomena include many of the preliminary matura-
tion stages shown by the Metazoa, in the formation of polar bodies
and reduction of the quantity of chromatin, etc. Simple division, the most common method of repro and reduction of the quantity of chromatin, etc. Simple division, the most common method of reproduction,
is
usually a separation of the body into two equal parts either longitu- Simple division, the most common method of reproduction,
is
usually a separation of the body into two equal parts either longitu- Fig. 23. — Division (budding) of Euglypha alveolata Duj. [SCHEWIAKOFF.]
The shell-plates which were stored in the endoplasm about the nucleus pass out with the stream-
ing protoplasm (4) to form the shell of the daughter-cell. The nucleus is shown in differem stages
of mitosis. - AKOFF.] dinally (Flagellidia) or transversely (Ciliata). 3. Reproduction.
. It is invariably preceded
by division of the nucleus, and is often accompanied by the equal
division of certain of the internal structures of the cell, such as the
chromatophores, pyrenoids, etc. It may take place either during
active life or under the protection of a cyst. Ciliata in the process of
division may be frequently seen swimming about actively, the con-
necting-strand becoming narrower and narrower, until finally only a
delicate strand of protoplasm separates the daughter-cells, ard this,
after a few energetic contortions, gives way and the young cells are dinally (Flagellidia) or transversely (Ciliata). It is invariably preceded
by division of the nucleus, and is often accompanied by the equal
division of certain of the internal structures of the cell, such as the
chromatophores, pyrenoids, etc. It may take place either during
active life or under the protection of a cyst. Ciliata in the process of
division may be frequently seen swimming about actively, the con-
necting-strand becoming narrower and narrower, until finally only a
delicate strand of protoplasm separates the daughter-cells, ard this,
after a few energetic contortions, gives way and the young cells are THE PROTOZOA 56 free (Fig. 22). The presence of a firm shell or coating complicates
the process, especially if the shell is a secretion (Fig. 23). In many free (Fig. 22). The presence of a firm shell or coating complicates
the process, especially if the shell is a secretion (Fig. 23). In many Fig. 24. — Microgromia socialis Hert., a gregaloid colony. [HERTWIG.] Fig. 24. — Microgromia socialis Hert., a gregaloid colony. [HERTWIG.] cases the new shell-parts are secreted before the act of division be-
gins, and when the protoplasm buds out of the original shell-mouth,
they are carried with it, and the bud is covered with the newly formed Fig. 25.— Uroglena americana Calkins, a spheeroid colony. pieces, which are glued together by means of a chitinous or silicious
secretion. GENERAL SKETCH 57 Simple division frequently leads to colony-formation through incom-
plete separation of the daughter-individuals. Four general types of
these colonies are met with among the Protozoa. Adopting Haeckel’s
terms, they may be designated according to their general structure as
(1) gregaloid, (2) spheroid, (3) arboroid, and (4) catenoid. 3. Reproduction.
. A gregaloid colony is an aggregate A gregaloid colony is an aggregate of Protozoa having a round,
ellipsoidal, or indefinite shape, and usually with a gelatinous basis in
which the single individuals are variously distributed. The colonies
may be formed by incomplete division of the individuals or by partial
union of two or more adults (Fig. 24). A spheroid colony is a globu- Fig. 26. — Codosiga cymosa Sav. K., an arboroid colony of Choanoflagellida. [KENT.] Fig. 26. — Codosiga cymosa Sav. K., an arboroid colony of Choanoflagellida. [KENT.] lar, ellipsoidal, or cylindrical aggregate in which the individual cells
form a superficial layer in a common gelatinous matrix. When these
superficial cells are closely packed together into an almost continuous
layer as in Volvox, Magosphera, or Uroglena, they are extremely sug-
gestive of certain stages in developing Metazoa (Fig. 25). An ardo-
void colony is a tree- or bush-like aggregate arising by the dendritic
or dichotomous branching of a primary stalk or a gelatinous matrix. Such colonies are usually attached by the base to some foreign object
and often resemble hydroids or Bryozoa (Fig. 26). They may, how-
ever, as in Dznobryon, be free-swimming. <A catenoid colony is fili-
form or chain-shaped, arising from the union of cells end to end, or lar, ellipsoidal, or cylindrical aggregate in which the individual cells
form a superficial layer in a common gelatinous matrix. When these
superficial cells are closely packed together into an almost continuous
layer as in Volvox, Magosphera, or Uroglena, they are extremely sug-
gestive of certain stages in developing Metazoa (Fig. 25). An ardo-
void colony is a tree- or bush-like aggregate arising by the dendritic
or dichotomous branching of a primary stalk or a gelatinous matrix. Such colonies are usually attached by the base to some foreign object
and often resemble hydroids or Bryozoa (Fig. 26). They may, how-
ever, as in Dznobryon, be free-swimming. <A catenoid colony is fili-
form or chain-shaped, arising from the union of cells end to end, or THE PROTOZOA 58 side to side, or through the continuous division of the cells in one plane
(Fig. 27). ints
Many intermediate stages between simple binary division and spore- Many intermediate stages between simple binary division and spore-
formation show that the latter method of reproduction was probably
derived from the former. When simple division takes place within Fig. 27.— Eiermooystis
Fig. 3. Reproduction.
. 28.— Exogenous budding in Ephelota Biitschliana Ishik. polymorpha Vég., a cate-
[FROM A PREPARATION, ]
noid colony of Gregarin-
The macronucleus (/V) is continued into the four daughter-
ida. [WASIELEWSKY.]
cells, which appeared first as minute buds. Fig. 27.— Eiermooystis
polymorpha Vég., a cate-
noid colony of Gregarin-
ida. [WASIELEWSKY.] Fig. 28.— Exogenous budding in Ephelota Biitschliana Ishik. [FROM A PREPARATION, ]
The macronucleus (/V) is continued into the four daughter-
cells, which appeared first as minute buds. Fig. 27.— Eiermooystis
polymorpha Vég., a cate-
noid colony of Gregarin-
ida. [WASIELEWSKY.] a cyst, it not unfrequently happens that each of the daughter-cells
divides again, thus forming four daughter-cells within the cyst (some
Mastigophora and Sporozoa). Again, as many as eight or sixteen
may be formed in the same way, and from this it is not a great step:
to the method of reproduction by spore-formation, where
a great
number of young individuals are produced at one time. Another modification of simple division is the process of budding Another modification of simple division is the process of budding GENERAL SKETCH 59 or gemmation which is common among the Suctoria and some of the
Flagellidia, less frequently observed in thg Ciliata, and is possibly
allied to “spontaneous division” among Sporozoa
A piece of the
nucleus of the mother-animal is pinched off and becomes the nucleus
of a much smaller daughter-cell, which usually arises from a certain
definite place on the parent (Fig. 28). When numerous pieces are
thus budded off, and each piece is surrounded by a bit of protoplasm,
the process is again akin to spore-formation (Voctz/uca). Spore-formation may thus be accomplished either by the repeated Spore-formation may thus be accomplished either by the repeated WAN
TAN
1 he
BH
SDS
SEAS
Fig. 29. — Onychodromus grandis Stein. [MAUuPAS.]
A, Normal individual. &. Smaller form without micronuclei; degenerate. C. A still more
reduced and degenerate form. iV, macronucleus; ”, micronucleus. Fig. 29. — Onychodromus grandis Stein. [MAUuPAS.]
A, Normal individual. &. Smaller form without micronuclei; degenerate. C. A still more
reduced and degenerate form. iV, macronucleus; ”, micronucleus. Fig. 29. — Onychodromus grandis Stein. [MAUuPAS.]
A, Normal individual. &. Smaller form without micronuclei; degenerate. C. A still more
reduced and degenerate form. iV, macronucleus; ”, micronucleus. 1 See Chapter IV, p. 159. 3. Reproduction.
. division of the entire animal, repeated division of the nucleus, the
products of which are later surrounded by minute bits of cytoplasm,
or by fragmentation of the nucleus without thé formality of regular
division. In the latter case- the body of the animal breaks up simul-
taneously into many hundreds of small pieces, each surrounding one
of the nuclear fragments and developing later into the parent form
(Parameba). So-called sexual reproduction or some modification of this process So-called sexual reproduction or some modification of this process
is accomplished, either directly or indirectly, by the temporary or
permanent union of two individuals of the same or of dissimilar size. 1 See Chapter IV, p. 159. THE PROTOZOA 60 From an a priori point of view, there is no reason why the reproduction
of Protozoa by simple division should not go on indefinitely. The
mechanism of metabolism, growth, and reproduction is present, and
the cell appears therefore to be sclf-sufficient. Nevertheless, Biitschli,
Maupas,
and many
others
have
shown that, in many cases at least,
these divisions can be maintained
only for a certain period or number
of generations, after which the in-
dividuals become weaker, deformed,
and finally die out, an exhausted
race (Fig. 29). If two individuals
conjugate while in this enfeebled
condition,
the result is a rejuve-
nescence or renewal of youth in
both cases, and each of the conju-
gants enters upon a new cycle of
cell-generations
(Fig. 30). The
union of two cells is accomplished
in various ways. In some cases it
is by the absolute and permanent
fusion of two individuals; in other. ;
_
cases there is a union for a short
coset. 28 petedrames grands Sim time followed by a separation ;
in
division. still other cases the entire cell does
not conjugate, but develops a great
number of swarm-spores, which again may be of equal or unequal
size, and which conjugate usually by total fusion;
finally, sexual
reproduction, almost as highly differentiated as in the Metazoa and
Metaphyta, is found in some Sporozoa and in the complex colonies of
Flagellidia.t
A very interesting controversy has arisen over the question whether
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coset. 28 petedrames grands Sim
division. 1 Cf. Chapter VI, p. 220. 3. Reproduction.
. This sug-
gestion was confirmed by Biitschli (’76) and by Engelmann (’76), who
demonstrated in connection with a number of Ciliata that after a
certain number ot divisions the resulting individuals become reduced
in size and show other evidences of degeneration. Biitschli regarded
this as evidence of old age, and he observed that the normal size and
the general vitality of the reduced organisms are restored by conju-
gation; and he was one of the first to demonstrate that the function
of conjugation is not for purposes of reproduction, but for the renewal
of vitality as expressed
in his term
Verjungung (rejuvenation). Maupas ('88), finally, has confirmed the latter conception of conjuga-
tion, and in a series of brilliantly planned and carefully executed
experiments has shown that Protozoa, contrary to Weismann’s a priori
assumption, may die of old age unless they be reinvigorated by
conjugation. ‘Senescence,’ says Maupas, “appears to be a very
general phenomenon, at least in the animal kingdom. ... It is
inherent in the organism and comes from internal causes which act
independently
of the surrounding conditions... . Its deleterious
action is offset and annulled by sexual rejuvenescence or conjugation.” ! 3. Reproduction.
. WAU
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division. A very interesting controversy has arisen over the question whether
Protozoa ever succumb to old age. Ehrenberg was apparently the
first to suggest that because of their reproduction by division, a process
in which no portion of the original organism is lost, the Protozoa are
potentially immortal. This conception was greatly expanded by
Weismann (84) and formed the basis of a suggestive essay in which
he maintained that the Protozoa are too simply organized to die a
natural death and that death is first observed in multicellular animals
and plants. -Dujardin
(’41), opposed
to so many of Ehrenberg’s
theories, was equally opposed to this, and although he was unable to
disprove the theory, he suggested that simple division cannot continue GENERAL SKETCH 61 indefinitely, but that periods of division recur at intervals. This sug-
gestion was confirmed by Biitschli (’76) and by Engelmann (’76), who
demonstrated in connection with a number of Ciliata that after a
certain number ot divisions the resulting individuals become reduced
in size and show other evidences of degeneration. Biitschli regarded
this as evidence of old age, and he observed that the normal size and
the general vitality of the reduced organisms are restored by conju-
gation; and he was one of the first to demonstrate that the function
of conjugation is not for purposes of reproduction, but for the renewal
of vitality as expressed
in his term
Verjungung (rejuvenation). Maupas ('88), finally, has confirmed the latter conception of conjuga-
tion, and in a series of brilliantly planned and carefully executed
experiments has shown that Protozoa, contrary to Weismann’s a priori
assumption, may die of old age unless they be reinvigorated by
conjugation. ‘Senescence,’ says Maupas, “appears to be a very
general phenomenon, at least in the animal kingdom. ... It is
inherent in the organism and comes from internal causes which act
independently
of the surrounding conditions... . Its deleterious
action is offset and annulled by sexual rejuvenescence or conjugation.” ! indefinitely, but that periods of division recur at intervals. 1°88), p. 272. 4. Lrritability. Unlike the Metazoa, where the phenomena which are characterized
as manifestations of consciousness are expressed through special or-
gans of the nervous system, the Protozoa in the simplest forms have
nothing, so far as we know at present, but the undifferentiated proto-
plasm which at the same time must be the seat of all functions. The sensory phenomena are, however, very little known. All Pro- The sensory phenomena are, however, very little known. All Pro-
tozoa are irritable, reacting in certain definite ways, although in dif-
ferent degrees, toward various external stimuli. All are sensitive to. electrical, mechanical, thermal, and chemical irritations, and many
to light, while few or none are affected by acoustic vibrations. The
reactions to these stimuli are usually expressed in motion of some
sort, which may be either indefinite or definite, —in the latter case,
as a tule, either positive, z.¢. toward the source of irritation, or nega-
tive, ze. away from it. In many Protozoa, particularly in the lower
forms, there seems to be no portion of the cell more sensitive than
others; in the higher forms, however, there is a greater or less degree
of sensory localization. Here, as a rule, the ectoplasm reacts ener-
getically, and, like the cuticle of Metazoa, becomes a general sensory
organ. The appendages frequently serve as special sensory organs. of touch, as in the aboral cirri of the hypotrichous ciliates, while spe-
cial organoids are frequently present in the form of “ eye-spots,”’ etc. 1°88), p. 272. 62 THE PROTOZOA a
C. SOME ECONOMIC ASPECTS OF THE PROTOZOA The Protozoa are frequently objectionable because of the appear-
ances, odors, and tastes which they may impart to water. In the sea
great areas may be colored orange, red, etc., by incalculable numbers of
Noctiluca or Dinoflagellidia (Prorocentrum, Glenodinium), while at
night their presence is indicated by brilliant phosphorescence, the
light being due to the rapid oxidization of a substance created by the
organisms and thrown out by them upon irritation. In Puget Sound
and in Alaska I have seen hundreds of acres of the sea surface
colored orange by Woctiluca miliaris, although the single individuals
are less than one-fiftieth part of an inch in diameter, and Haeckel
(90) graphically compares such masses to “tomato soup”! When
Protozoa occur in great numbers in fresh water, and especially in
drinking water, they may cause considerable annoyance; for by the
color, odor, and taste which they impart they render the water unfit
to drink. The colors are due in the main to the Phytoflagellida, and
only those forms which are capable of making their own food are able
to live in pure drinking waters. The most frequent causes of trouble
in this respect are Uroglena, Peridinium, or its allies, Euglena and
other Euglenoids, and Syzzra, all of which are
flagellates. The
odors and tastes, however, are more offensive than the colors, and as
they are frequently misunderstood and regarded as evidence of pollu-
tion, an explanation may not be out of place. Ehrenberg noticed
that certain flagellates (Chlamydomonas pulvisculus and Chlorogonium)
impart a certain oily odor. Dunal (’38) and Joly (’40) described an
odor like that of violets from the masses of Hematococcus which gave
to a portion of the Mediterranean a distinct red color. The Massa-
chusetts State Board of Health, dealing with this problem of the
drinking waters, have obtained important results in this direction. They have shown that certain of these organisms may have definite
and specific odors which, like the odors of flowers, can be recognized. a
C. SOME ECONOMIC ASPECTS OF THE PROTOZOA An “oily odor” was traced to Syaura and Uroglena, an “Iceland
moss” odor to Perzdinium, a ‘violet odor” to certain Euglenoids,
etc.1
The cause of these odors has been the subject of a number of
investigations, and it has been found that they are “living odors” due
to disintegration of the cells rather than to their decomposition, a view
first advanced, I believe, by Biitschli ('84), who described a highly
characteristic “fishy odor” from Euglena saneuinea, while the cells
were found to be disintegrated, although not decomposed. The
matter was considered more extensively by the writer (92), who found
that in waters infested with colonies of (roglena americana the odor
was not developed until the organisms had passed through the water
1See S. B. IT. (’92). ances, odors, and tastes which they may impart to water. In the sea
great areas may be colored orange, red, etc., by incalculable numbers of
Noctiluca or Dinoflagellidia (Prorocentrum, Glenodinium), while at
night their presence is indicated by brilliant phosphorescence, the
light being due to the rapid oxidization of a substance created by the
organisms and thrown out by them upon irritation. In Puget Sound
and in Alaska I have seen hundreds of acres of the sea surface
colored orange by Woctiluca miliaris, although the single individuals
are less than one-fiftieth part of an inch in diameter, and Haeckel
(90) graphically compares such masses to “tomato soup”! When
Protozoa occur in great numbers in fresh water, and especially in
drinking water, they may cause considerable annoyance; for by the
color, odor, and taste which they impart they render the water unfit
to drink. The colors are due in the main to the Phytoflagellida, and
only those forms which are capable of making their own food are able
to live in pure drinking waters. The most frequent causes of trouble
in this respect are Uroglena, Peridinium, or its allies, Euglena and
other Euglenoids, and Syzzra, all of which are
flagellates. The
odors and tastes, however, are more offensive than the colors, and as
they are frequently misunderstood and regarded as evidence of pollu-
tion, an explanation may not be out of place. Ehrenberg noticed
that certain flagellates (Chlamydomonas pulvisculus and Chlorogonium)
impart a certain oily odor. Dunal (’38) and Joly (’40) described an
odor like that of violets from the masses of Hematococcus which gave
to a portion of the Mediterranean a distinct red color. 1See S. B. IT. (’92). a
C. SOME ECONOMIC ASPECTS OF THE PROTOZOA The Massa-
chusetts State Board of Health, dealing with this problem of the
drinking waters, have obtained important results in this direction. They have shown that certain of these organisms may have definite
and specific odors which, like the odors of flowers, can be recognized. An “oily odor” was traced to Syaura and Uroglena, an “Iceland
moss” odor to Perzdinium, a ‘violet odor” to certain Euglenoids,
etc.1
The cause of these odors has been the subject of a number of
investigations, and it has been found that they are “living odors” due
to disintegration of the cells rather than to their decomposition, a view
first advanced, I believe, by Biitschli ('84), who described a highly
characteristic “fishy odor” from Euglena saneuinea, while the cells
were found to be disintegrated, although not decomposed. The
matter was considered more extensively by the writer (92), who found
that in waters infested with colonies of (roglena americana the odor
was not developed until the organisms had passed through the water 1See S. B. IT. (’92). GENERAL SKETCH 63 pipes, but after such passage the odor was extremely strong and
repulsive, while no colonies could be found. It was suggested at this
time that the odor was one of disintegration, and due to the libera-
tion of minute drops of oil-like substance which become disseminated
through the water, giving it the characteristic Uroglena smell. It was
also suggested that these drops of oil are analogous to the perfume
oils of the fragrant plants, like them having a certain individual odor
often strong enough and characteristic enough to identify the organ-
ism. Similar oil-like inclusions are found in the protoplasm of all
Protozoa, but to be detected through the sense of smell, they must be
present in great numbers. Far more serious noxious effects of the Protozoa are produced Far more serious noxious effects of the Protozoa are produced
through their frequently parasitic mode of life. In all classes there Fig. 31.— Internal parasites. [4,B, LEUCKART;
C, GRASSI; D, BUTSCHLI.]
A. Ameba coli Lésch, a supposed cause of dysentery. B. Monocystis agilis Leuck., a grega-
rine. C, Megastoma entericum Grassi, a flagellate. D. Balantidium entozoin Ehbr., a ciliate. Fig. 31.— Internal parasites. [4,B, LEUCKART;
C, GRASSI; D, BUTSCHLI.]
A. Ameba coli Lésch, a supposed cause of dysentery. B. Monocystis agilis Leuck., a grega-
rine. C, Megastoma entericum Grassi, a flagellate. D. Balantidium entozoin Ehbr., a ciliate. a
C. SOME ECONOMIC ASPECTS OF THE PROTOZOA Among the first to exclude
the Protozoa were Celli and Fiocca (’95), who obtained cases of dysentéry by inject-
ing cats with material from faeces in which the Protozoa had been killed by heat;
the same result was also obtained by injecting material in which both Aveda and
bacteria were absent, the cause evidently being in the poison of the sterilized matter
and not in -d#eba colz. They concluded that the poison is the product of bacteria
(Bacillus coli communis, together with typhus-like bacteria and a streptococcus,
were suggested
as possible causes). This view was supported by a number of
observers, amongst whom may be mentioned Gasser (’95), Cassagrandi and Bar-
bagello (’95), and Petridis (98). The latter especially has shown that dysentery
as observed in Egypt is due to a bacillus and not to Protozoa. He found that S¢ep-
1Cf. Leuckart (79). 2 Page 701. One form, however, Amba coli (Fig. 31, 4), has been long in dispute as the reputed
cause of dysentery. If it is the specific cause of this disease, the animal occupies an
interesting position amongst the intercellular parasites ; for, so far as known, none
other of this kind of protozoan parasites exerts a deleterious effect upon the intestinal
epithelia. Nor is it proven that Awa coli does this in the case of dysentery,
although a belief to that effect is widespread. Briefly reviewing the history of this
belief, it appears that Lambl (’60)1 was the first to observe 47z@ba in the human
intestine, although Lésch (75), who named
it, was the first to consider
it in
connection with dysentery. Many subsequent observers
(Kartulis, Mannaberg,
Cohn, etc.) found Ameba coli in the feces of dysentery patients, Kartulis
(89)
amongst others stating that he found them in no less than five hundred cases. The
belief received a setback, however, by the observations of Cunningham (°81), Grassi
(82), and Calendruccio (’90), who found Asmeba colz in the intestine of sound and
healthy men as well as in dysenteric patients, while still other observers maintained
the entire absence of such an enteric organism. a
C. SOME ECONOMIC ASPECTS OF THE PROTOZOA Councilman (91), in a work which
is certainly as reliable as any that has been undertaken upon this subject, partially
harmonized these views by showing that there are at least three forms of dysentery,
of which one, at least, is characterized by certain definite symptoms and by the
presence of Amba coli, although it was not demonstrated that the rhizopod was the
cause. The entire matter received impartial and critical treatment by Laveran (’93)
in France and by Schuberg (’93) in Germany, and both came to the conclusion that
the cause of dysentery was not yet known, the former basing his opinion largely
upon the absence of Aveda in all but one of ten cases, the latter upon numerous
experiments and observations upon normal and diseased individuals. Schuberg not
only found that Ameéa colz is present in normal men, but also found that there is no
specific difference in the various intestinal Awzewbe which have been described by
various observers as living freely in the intestine in the same way as the commensal
ciliates and flagellates also found there and generally believed to be harmless. He
pertinently says: “If the flagellates are harmless, it is certainly not impossible that
Ameba is also. The increased number of Ameéde in dysenteric patients is not
necessarily evidence that they are the cause of this disease.”2_
Both he and Laveran
expressed the view that all experiments which had been made up to that time had
not excluded the possibility of other causes, ¢.g. bacteria. That dysentery is due to
some specific cause had been early demonstrated by experiment, but in none of these
experiments had it been possible to isolate the Protozoa from bacteria which invyari-
ably accompany them. The reverse experiment is, however, possible, and it is
singular that it has not been made more frequently. Among the first to exclude
the Protozoa were Celli and Fiocca (’95), who obtained cases of dysentéry by inject-
ing cats with material from faeces in which the Protozoa had been killed by heat;
the same result was also obtained by injecting material in which both Aveda and
bacteria were absent, the cause evidently being in the poison of the sterilized matter
and not in -d#eba colz. They concluded that the poison is the product of bacteria
(Bacillus coli communis, together with typhus-like bacteria and a streptococcus,
were suggested
as possible causes). a
C. SOME ECONOMIC ASPECTS OF THE PROTOZOA are certain forms which live as parasites (Fig. 31), and which for
venience may be separated into two groups, the intercellular an
intracellular forms. So far as known these parasites, with few e
tions, do not produce noxious products like bacterial ptomaines are certain forms which live as parasites (Fig. 31), and which for con-
venience may be separated into two groups, the intercellular and the
intracellular forms. So far as known these parasites, with few excep-
tions, do not produce noxious products like bacterial ptomaines, but
whatever damage they may cause is due to the mechanical disturb-
ances set up by their presence. The intercellular parasites infest the
body cavities of various hosts, the cavities of blood-vessels, and ducts
of various glands, or penetrate the spaces between muscle-fibres, while
the intracellular parasites (which belong almost exclusively to the
Sporozoa) bore into cells of epithelia (Gregarines, Coccidia), or the
corpuscles of the blood (Hemosporidiida). From the wide distribu-
tion of the intercellular parasites, it is quite possible that no animal is
entirely free from Protozoa of some kind. Without entering upon a 64 THE PROTOZOA discussion of these forms, it may be stated that Rhizopoda, Flagellidia,
Ciliata, and Sporozoa may be found in the various cavities and canals
in man and the other vertebrates, where they usually give little or no
trouble. One form, however, Amba coli (Fig. 31, 4), has been long in dispute as the reputed
cause of dysentery. If it is the specific cause of this disease, the animal occupies an
interesting position amongst the intercellular parasites ; for, so far as known, none
other of this kind of protozoan parasites exerts a deleterious effect upon the intestinal
epithelia. Nor is it proven that Awa coli does this in the case of dysentery,
although a belief to that effect is widespread. Briefly reviewing the history of this
belief, it appears that Lambl (’60)1 was the first to observe 47z@ba in the human
intestine, although Lésch (75), who named
it, was the first to consider
it in
connection with dysentery. Many subsequent observers
(Kartulis, Mannaberg,
Cohn, etc.) found Ameba coli in the feces of dysentery patients, Kartulis
(89)
amongst others stating that he found them in no less than five hundred cases. a
C. SOME ECONOMIC ASPECTS OF THE PROTOZOA The
belief received a setback, however, by the observations of Cunningham (°81), Grassi
(82), and Calendruccio (’90), who found Asmeba colz in the intestine of sound and
healthy men as well as in dysenteric patients, while still other observers maintained
the entire absence of such an enteric organism. Councilman (91), in a work which
is certainly as reliable as any that has been undertaken upon this subject, partially
harmonized these views by showing that there are at least three forms of dysentery,
of which one, at least, is characterized by certain definite symptoms and by the
presence of Amba coli, although it was not demonstrated that the rhizopod was the
cause. The entire matter received impartial and critical treatment by Laveran (’93)
in France and by Schuberg (’93) in Germany, and both came to the conclusion that
the cause of dysentery was not yet known, the former basing his opinion largely
upon the absence of Aveda in all but one of ten cases, the latter upon numerous
experiments and observations upon normal and diseased individuals. Schuberg not
only found that Ameéa colz is present in normal men, but also found that there is no
specific difference in the various intestinal Awzewbe which have been described by
various observers as living freely in the intestine in the same way as the commensal
ciliates and flagellates also found there and generally believed to be harmless. He
pertinently says: “If the flagellates are harmless, it is certainly not impossible that
Ameba is also. The increased number of Ameéde in dysenteric patients is not
necessarily evidence that they are the cause of this disease.”2_
Both he and Laveran
expressed the view that all experiments which had been made up to that time had
not excluded the possibility of other causes, ¢.g. bacteria. That dysentery is due to
some specific cause had been early demonstrated by experiment, but in none of these
experiments had it been possible to isolate the Protozoa from bacteria which invyari-
ably accompany them. The reverse experiment is, however, possible, and it is
singular that it has not been made more frequently. a
C. SOME ECONOMIC ASPECTS OF THE PROTOZOA This view was supported by a number of
observers, amongst whom may be mentioned Gasser (’95), Cassagrandi and Bar-
bagello (’95), and Petridis (98). The latter especially has shown that dysentery
as observed in Egypt is due to a bacillus and not to Protozoa. He found that S¢ep-
1Cf. Leuckart (79). 2 Page 701. 2 Page 701. 1Cf. Leuckart (79). GENERAL SKETCH 65 tococcus is the most numerous of the micro-organisms and the probable cause of the
disease, for he was able to isolate the bacillus and to produce dysentery in cats by
injecting them with the culture obtained from it. Thus, as the matter stands,
Petridis’s results, the most positive that have yet appeared, together with growing
evidence from the bacteriological side, make it exceedingly probable, although not
definitely established as yet, that bacilli and not Amada coli are the cause of this
disease. While the majority of intercellular parasites are harmless, it is
quite different with the intracellular forms. These, by making their
way into the interior of the cell and growing at the expense of the While the majority of intercellular parasites are harmless, it is
quite different with the intracellular forms. These, by making their
way into the interior of the cell and growing at the expense of the
cell-contents, gradually cause degeneration of the tissues which may
end in death of the host. These parasites belong almost exclusively
to the class Sporozoa of which the Coccidiida and Hemosporidiida are
found in vertebrates, while the Gregarinida are confined to the inverte-
brates, where they are widely distributed. The Coccidiida are found in nearly all of the tissues of the lower The Coccidiida are found in nearly all of the tissues of the lower
vertebrates although rarely in man, unless indeed, as many observ-
ers believe, they are the cause of various tumors and cancers. That
there is some reason for this belief is shown by the fact that in the
lower vertebrates, especially in fishes, the presence of Sporozoa leads
to ulcers and tumors and to the ultimate death of the fish. The sub-
ject, however, as far as man is concerned, is in a very unsatisfactory
state, and opinions differ widely as to the nature of certain elements
found in cancerous growths. By some observers these are regarded
as parasites, by others as disintegrated or pathological cells. 1 Vide infra, pp. 160-165, a
C. SOME ECONOMIC ASPECTS OF THE PROTOZOA Up to
the present time no satisfactory evidence has appeared to prove the
former view, and until such evidence is forthcoming the entire matter
must rest in abeyance. From the pathogenic point of view, the most important protozoén From the pathogenic point of view, the most important protozoén
is the malaria germ (Plasmodium malari@), a form belonging to the
Hemosporidiida. These organisms, in the young stages, move about
by amceboid motion in the blood-vessels of men and birds. They
penetrate the red blood corpuscles, which slowly hypertrophy, until in
one type of the disease, at least, they attain a size three to four times
that of the normal corpuscle, the parasite in the meantime growing at
the expense of the haemoglobin and finally reproducing by spore-
formation. In this form alone there appears to be a poisonous sub-
stance analogous to bacterial ptomaines, which is produced by the
organism and periodically discharged (at spore-formation) into the
blood, thus causing the pyrexial attacks so characteristic of malaria. The recent successful results obtained by Ross, Manson, Koch, Grassi,
and others, in locating the seat of the malaria germ when outside the
human body, leads to the hope that some successful means of guard-
ing against this disease may soon follow.! 1 Vide infra, pp. 160-165, F 66 THE PROTOZOA THE SARCODINA THE term Sarcodina, introduced by Biitschli (’83) as the class
name of the most primitive of the Protozoa, includes all forms which,
like the common fresh-water type Ama@ba proteus, move by the pro-
trusion of protoplasm in the form of broad and finger-like, or sharp
and ray-like, processes called pseudopodia. These forms fall naturally
into three groups readily distinguished by clearly marked differences
in structure,
— the Rhizopoda, Heliozoa, and Radiolaria. Among the Rhizopoda are included forms of Sarcodina with blunt, Among the Rhizopoda are included forms of Sarcodina with blunt,
finger-form or lobose pseudopodia (Ameézda) or with branching and
anastomosing pseudopodia (Reticularizda). They may be naked
(Gymnamebina), or shelled (Thecamebina or Foraminifera). The
pseudopodia may arise from all parts of the body or they may be
limited to special regions; in shelled forms they may pass through
one common opening (Reticulartida imnperforina), or through many
finer openings (Reticularitda perforina). The body form is typically
globular, but may be variable in consequence of amoeboid changes, or
drawn out into a monaxonic form. The material of the shell may be
chitin, silica, foreign particles, or calcium carbonate. The Heliozoa are naked or shelled forms of Sarcodina; they are The Heliozoa are naked or shelled forms of Sarcodina; they are
usually globular with fine ray-like pseudopodia arising from all parts
of the body. The rays are, as a rule, stiffened by an axial filament
formed of modified protoplasm which may be readily dissolved by the
organism. The shells are less compact than those of the Rhizopoda,
and are usually formed of more or less loosely joined silicious spicules. The Radiolaria are similar in form to the Heliozoa. As in the The Radiolaria are similar in form to the Heliozoa. As in the
latter, the pseudopodia arise from all parts of the body and occasion-
ally anastomose. The endoplasm is separated from the outer plasm
by a firm, chitinous, perforated membrane, the central capsule. A
test or skeleton, often of exquisite beauty, is usually present, consist-
ing of isolated spicules of silica, or of a compact skeleton of acanthin
or silica. One or more nuclei are invariably present within the central
capsule. The finer structure of the rhizopod protoplasm has already been The finer structure of the rhizopod protoplasm has already been
mentioned. In many cases, especially in the monothalamous forms,
the plasm is divided into a number of clearly marked zones. SPECIAL BIBLIOGRAPHY
II Biitschli,
0.— Protozoa. In Broun’s Klassen und Ordnungen
des Thierreichs. Leipzig, 1883-1888. Delage et Hérouard. — La cellule et les Infusoires. In Zrasté de Zovlogie concréte. Delage et Hérouard. — La cellule et les Infusoires. In Zrasté de Zovlogie concréte. farts, 1896. Ehrenberg, C. G.— Die Infusionsthierchen als vollkommene Organismen. Le7fzzg, Ehrenberg, C. G.— Die Infusionsthierchen als vollkommene Organismen. Le7fzzg,
1838. Entz, G. — Protistenstudien. Ludapesth, 1888. Lankester, E.R.— Protozoa. In Zodlogical Art Lankester, E.R.— Protozoa. In Zodlogical Articles from the Encyclopedia Britan-
nica. 1891. :
Stein, Fr. — Der Organismus der Infusionsthiere. Lezpzzg, 1861, 1867, and 1878. Stein, Fr. — Der Organismus der Infusionsthiere. Lezpzzg, 1861, 1867, and 1878. THE SARCODINA Griiber found that an Actinophrys when transferred from fresh
into sea water soon loses its vacuoles; and, vice versa, when trans-
ferred back to fresh water, again acquires its vesicular appearance. In general appearance a radiolarian resembles a heliozoon, but there
is a considerable difference in the corresponding regions. A typical
radiolarian can be conceived if we imagine a thick perforated chitinous lives. Griiber found that an Actinophrys when transferred from fresh
into sea water soon loses its vacuoles; and, vice versa, when trans-
ferred back to fresh water, again acquires its vesicular appearance. ferred back to fresh water, again a
In general appearance a radiolaria
is a considerable difference in the c
radiolarian can be conceived if we im
membrane between the ectoplasm
and endoplasm of aheliozoon. The
intra-capsular plasm (Fig. 33, ¢)
contains nuclei, fat particles, and
plastids of one form or another,
and is in communication with the
extracapsular plasm through the
pores in the membrane, although,
as shown by Verworn’s experiments
upon the isolated central capsule,
it can live for a time independently. The outer or extra-capsular plasm
is composed, according to Haeckel,
of four parts. The outermost (¢)
is a zone of pseudopodia ; the latter,
however, originate in the deeper
fourth
zone, forming
a network
through the other extra-capsular
parts. The second zone is of net-
like
(alveolar?)
protoplasm,
the
sarcodictyum. A third zone, the
calymma, is of jelly-like consistency
and forms the bulk of the ecto-
plasm. The fourth and most im-
portant zone, the sarcomatrtx, lies
close against the central capsule,
and is the go-between for the intra-
‘
and extra-capsular portions. The. sarcomatrix
is also the seat of di-
gestion and assimilation,
the food
ij
coming to it through the pseudo-
podia and
the network. As the
. om
means of communication between
the central protoplasm and the sar-
comatrix is of vital importance to the
apertures in the central capsule offer
cation of the Radiolaria. Hertwig(’
divided the group into four legions, In general appearance a radiolaria
is a considerable difference in the c
radiolarian can be conceived if we im
membrane between the ectoplasm
and endoplasm of aheliozoon. The
intra-capsular plasm (Fig. THE SARCODINA Schewi-
67 67 68 THE PROTOZOA akoff (’88) describes three, Pénard (’90) no less than four in Euglypha,
and Rhumbler (’98) the latter number in Cyphoderta. Schewiakoff
(88), apparently on very good grounds, maintained that certain spe-
cific functions characterize each of these zones, indicating, in a general
way, a regional differentiation and division of physiologicallabor. To
the outer zone, which corresponds to the ectoplasm of Ama@ba, he
ascribed a locomotor function, this being the seat of pseudopodia for-
mation ; to the second zone, which contains the nucleus, the function
of assimilation, and to the third zone a reproductive function. Pénard
and Rhumbler separate Schewiakoff’s second zone into two on account
of certain structural differences. According to these observers the Fig. 32. — Actinophrys sol Ehr. [BUTSCHLI after GRENACHER.]
The axial filaments (a) extend through the endoplasm to the membrane of the nucleus; ¢,a
contractile vacuole in the ectoplasm; g, an ingested food particle in a gastric vacuole. Fig. 32. — Actinophrys sol Ehr. [BUTSCHLI after GRENACHER.]
filaments (a) extend through the endoplasm to the membrane of the nucleus; ¢,a
uole in the ectoplasm; g, an ingested food particle in a gastric vacuole. Fig. 32. — Actinophrys sol Ehr. [BUTSCHLI after GRENACHER.] The axial filaments (a) extend through the endoplasm to the membrane of the nucleus; ¢,a
contractile vacuole in the ectoplasm; g, an ingested food particle in a gastric vacuole. outermost zone is distinctly vacuolated, the second contains food-par-
ticles in the process of digestion, the third, granules which represent
waste matter not determined, and the fourth, excretory granules. The appearance of the protoplasm in Heliozoa or Radiolaria is outermost zone is distinctly vacuolated, the second contains food-par-
ticles in the process of digestion, the third, granules which represent
waste matter not determined, and the fourth, excretory granules. The appearance of the protoplasm in Heliozoa or Radiolaria is The appearance of the protoplasm in Heliozoa or Radiolaria is
quite different from that of the Rhizopoda. Ectoplasm and endoplasm
can be distinguished, but unlike the hyaline ectoplasm of Amedéa, the
outer plasm of Heliozoa is made up of vacuoles much larger than
those of the endoplasm, the walls of these vacuoles being distinctly
granular (Fig. 32). The extremely vacuolated appearance, however,
seems to be largely dependent upon the medium in which the animal. THE SARCODINA 69 lives. THE SARCODINA 33, ¢)
contains nuclei, fat particles, and
plastids of one form or another,
and is in communication with the
extracapsular plasm through the
pores in the membrane, although,
as shown by Verworn’s experiments
upon the isolated central capsule,
it can live for a time independently. The outer or extra-capsular plasm
is composed, according to Haeckel,
of four parts. The outermost (¢)
is a zone of pseudopodia ; the latter,
however, originate in the deeper
fourth
zone, forming
a network
through the other extra-capsular
parts. The second zone is of net-
like
(alveolar?)
protoplasm,
the
sarcodictyum. A third zone, the
calymma, is of jelly-like consistency
and forms the bulk of the ecto-
plasm. The fourth and most im-
portant zone, the sarcomatrtx, lies
close against the central capsule,
and is the go-between for the intra-
‘
and extra-capsular portions. The. sarcomatrix
is also the seat of di-
gestion and assimilation,
the food
ij
coming to it through the pseudo-
podia and
the network. As the
. om
means of communication between
the central protoplasm and the sar-
comatrix is of vital importance to the
apertures in the central capsule offer
cation of the Radiolaria. Hertwig(’
divided the group into four legions, Fig. 33.—The protoplasmic regions of a Fig. 33.—The protoplasmic regions of a
‘adiolarian
( Thalassicolla
maculata)
Haeck. [HAECKEL.]
a, large alveoli forming part of the calymma a, large alveoli forming part of the calymma
in which foreign bodies (4) are enclosed, and
whicl is penetrated by meshes constituting the
sacrodictyum; ¢, the central capsule and intra-
capsular plasm; f retracted pseudopodia. The
nucleus () contains a distinct nucleolus (2);
the
sarcomatrix
is
darkened
by pigment
passes (), THE PROTOZOA 7O which the membrane of the central capsule is perforated by pores
arranged regularly about the entire surface (Fig. 34, A); (2) the Acztz-
pylea, in which the pores are arranged in groups over the surface
(B); (3) the M@onopylea, in which
there
is but one
such group
of pores in the membrane. In these forms the perforated disk is con-
nected with the centre of the central capsule by a conical mass of
endoplasm,
the podoconus (DP), rich in food particles
and gran- Fig. 34. — Central capsules of Radiolaria. [HAECKEL.]
A. Thalassolampe maxima Haeck., one of the Peripylea. B. Acanthometron dolichoscion Haeck.,
one of the Actipylea. C. Aulographis candelabrum Haeck., one of the Monopylea,
D. THE SARCODINA Zriptero-
calpis ogneoptera Haeck., one of the Cannopylea. c, central capsules; 7, nuclei. Fig. 34. — Central capsules of Radiolaria. [HAECKEL.] Fig. 34. — Central capsules of Radiolaria. [HAECKEL.]
A. Thalassolampe maxima Haeck., one of the Peripylea. B. Acanthometron dolichoscion Haeck.,
one of the Actipylea. C. Aulographis candelabrum Haeck., one of the Monopylea,
D. Zriptero-
calpis ogneoptera Haeck., one of the Cannopylea. c, central capsules; 7, nuclei. ules ; (4) the Caznopylea, in which the membrane around the pores is
drawn out into funnel-like projections termed astropyles (C). The
central capsule is double in these forms. Haeckel has found that
certain skeletal forms accompany the structure of the membranes, and
he names the above legions respectively as follows: —(1) Spaumel-
laria; (2) Acantharia; (3) Nasselaria, and (4) Pheodaria. In each of the orders of the Sarcodina, and especially in the In each of the orders of the Sarcodina, and especially in the
Radiolaria, there are some forms with symbiotic plant-cells. The THE SARCODINA 71 relationship between the symbionts was worked out by Cienkowsky,
Brandt, Haeckel, and Entz, the latter noting that the plant-cells are
invariably found just outside of the endoplasm, where they do not
“come in contact with endoplasm and its digestive fluids. According
to the more recent observations of Le Dantec (’92), however, the
digestive fluid of these animals is unable to dissolve the cellulose
membranes of the plant-cells, and they remain uninjured in the endo-
plasm, dividing there when the conditions are favorable. relationship between the symbionts was worked out by Cienkowsky,
Brandt, Haeckel, and Entz, the latter noting that the plant-cells are
invariably found just outside of the endoplasm, where they do not
“come in contact with endoplasm and its digestive fluids. According
to the more recent observations of Le Dantec (’92), however, the
digestive fluid of these animals is unable to dissolve the cellulose
membranes of the plant-cells, and they remain uninjured in the endo-
plasm, dividing there when the conditions are favorable. A. SHELLS AND TESTS The ectoplasm of naked protoplasm shows a tendency to
or stiffen when in contact with water, and a cuticle or mem
the result. Amwda proteus, with its differentiation into endop
ectoplasm, shows a primitive stage in the development of su
branes. Here the ectoplasm remains plastic enough to yi
inner pressure of the organ-
ism and to form the first
part of every pseudopodium,
it is rapidly pushed aside,
however, and the endoplasm
becomes the advancing part. In Amba
tentaculata
the
outer layer has become more
firm and the pressure from
within expends itself upon
pseudopodia which are pro-
truded through permanent
holes (Fig. 12, A). The
membrane may become still
Fig. 35.—Types of marine rhizopod shel
riida. [CARPENTER.]
more
firm
through
the
A, Lateral. &. Ventral view of a mo
deposition
of chitin,
until,
shell (Cornuspira foliacea Phillips). C. A
as in the radiolarian central
thalamous shell (Nodosaria hispida D'Orb.)
A
bralina sp., a fossil form,
capsule,
it is an
efficient
means of protection. In addition to the chitin, certain
secrete
a silicious
mucilaginous
material,
which,
like
nous cement, is frequently the means of gluing together
regular plates or disks which the organism also secretes, bu
particles of various kinds. The tests thus made may be e
lime, as in the Reticulariida, or of silica, as in the Radiol
many of the Heliozoa, or of sand crystals, diatom-shells, or d
various kinds. In the lime-shells (Reticulariida or Foraminifera) the se The ectoplasm of naked pr
or stiffen when in contact wit
the result. Amwda proteus, wi
ectoplasm, shows a primitive
branes. Here the ectoplasm
inner pressure of the organ-
ism and to form the first
part of every pseudopodium,
it is rapidly pushed aside,
however, and the endoplasm
becomes the advancing part. In Amba
tentaculata
the
outer layer has become more
firm and the pressure from
within expends itself upon
pseudopodia which are pro-
truded through permanent
holes (Fig. 12, A). The
membrane may become still
more
firm
through
the
deposition
of chitin,
until,
as in the radiolarian central
A
capsule,
it is an
efficient
means of protection. In ad
secrete
a silicious
mucilagi
nous cement, is frequently t
regular plates or disks which
particles of various kinds. lime, as in the Reticulariida,
many of the Heliozoa, or of s
various kinds. In the lime-shells (Reticula R
:
#
Fig. 35.—Types of marine rhizopod shells (Retccudu-
riida. A. SHELLS AND TESTS [CARPENTER.]
A, Lateral. &. Ventral view of a monothalamous
shell (Cornuspira foliacea Phillips). C. A simple poly-
thalamous shell (Nodosaria hispida D'Orb.). D. Verte-
bralina sp., a fossil form,
«
. 7 Fig. 35.—Types of marine rhizopod shells (Retccudu-
riida. [CARPENTER.]
A, Lateral. &. Ventral view of a monothalamous
shell (Cornuspira foliacea Phillips). C. A simple poly-
thalamous shell (Nodosaria hispida D'Orb.). D. Verte-
bralina sp., a fossil form, In the lime-shells (Reticulariida or Foraminifera) the secretion of
calcium carbonate, except for the invariable presence of a mouth- THE PROTOZOA 72 opening, forms an almost complete investment like a cyst. In many
cases this opening is the only means of communication with the sur-
rounding medium (Imperforina), but in other cases the entire shell is
punctured by minute openings through which pseudopodia pass to
the outside (Perforina). These two types of shell are further distin-
guished by their appearance; the Imperforina when seen by reflected
light are opaque and like porcelain, while the shells of the Perforina
are almost transparent (vitreous). Monothalamous
or single-shelled Foraminifera may be either im- Monothalamous
or single-shelled Foraminifera may be either im-
perforate (2g. Sguamulina, Pilulina, or Saccammina) or perforate
(Lagena). In each group a graded series of shells can be arranged,
varying in complexity from the simple monothalamous to the compli- Fig. 36. — Polythalamous shell types schematized. [CARPENTER.]
A. Linear Nodosaria type. B. Frondicularia form of the Nodosaria type. C. Spiral form
of the Modosaria type. ‘ cated polythalamous forms (Polystomella, Calcarina). One of the
simplest of these shells is that of Cornuspira, where the plasm, as it
slowly grows, constantly secretes new shell material and is capable of
unlimited extension (Fig. 35, A). It is never divided by septa into
separate chambers as in the polythalamous shells. A further step,
the simplest of the polythalamous types, is found in shells where the
separate chambers adhere end to end as in Modosaria (C). Here
there may be only a slight septum between adjacent chambers, but
enough to indicate that growth is periodic, and not constant as in
Cornuspira. In these chamber-dwelling animals the plasm, as it grows, extends cated polythalamous forms (Polystomella, Calcarina). One of the
simplest of these shells is that of Cornuspira, where the plasm, as it
slowly grows, constantly secretes new shell material and is capable of
unlimited extension (Fig. 35, A). A. SHELLS AND TESTS [CARPENTER.]
The shell is represented as cut in different planes to show the distribution of the canals
(a’,a"’, a’); c,¢, €, the outer chambers with double walls (d, d,@), one of which is shown in sec-
tion (.g). The chambers communicate by apertures at the inner ends of the septa (e), and by
minute pores (/). The outside (4) of the shell is marked by the radial septa. Fig. 3'7.— A complex polythalamous shell (schematic) of Operculina. [CARPENTER.]
The shell is represented as cut in different planes to show the distribution of the canals
(a’,a"’, a’); c,¢, €, the outer chambers with double walls (d, d,@), one of which is shown in sec-
tion (.g). The chambers communicate by apertures at the inner ends of the septa (e), and by
minute pores (/). The outside (4) of the shell is marked by the radial septa. Fig. 3'7.— A complex polythalamous shell (schematic) of Operculina. [CARPENTER.]
The shell is represented as cut in different planes to show the distribution of the canals
(a’,a"’, a’); c,¢, €, the outer chambers with double walls (d, d,@), one of which is shown in sec-
tion (.g). The chambers communicate by apertures at the inner ends of the septa (e), and by
minute pores (/). The outside (4) of the shell is marked by the radial septa. quently a space filled with a calcareous deposit or what Carpenter
(62) calls the “intermediate skeleton.”
This inter-lamellar deposit is
traversed by a complicated system of canals, and the deposit itself is
frequently carried out into external processes and knobs (Cadécarina). In the annular or discoid types a process of budding takes place
around the entire circumference instead of at a localized area, and
concentric circles of chambers are thus formed (O7ztolites). The character of the mouth-openings between adjacent chambers quently a space filled with a calcareous deposit or what Carpenter
(62) calls the “intermediate skeleton.”
This inter-lamellar deposit is
traversed by a complicated system of canals, and the deposit itself is
frequently carried out into external processes and knobs (Cadécarina). In the annular or discoid types a process of budding takes place
around the entire circumference instead of at a localized area, and
concentric circles of chambers are thus formed (O7ztolites). The character of the mouth-openings between adjacent chambers The character of the mouth-openings between adjacent chambers
depends upon the nature of the outer coating. A. SHELLS AND TESTS It is never divided by septa into
separate chambers as in the polythalamous shells. A further step,
the simplest of the polythalamous types, is found in shells where the
separate chambers adhere end to end as in Modosaria (C). Here
there may be only a slight septum between adjacent chambers, but
enough to indicate that growth is periodic, and not constant as in
Cornuspira. In these chamber-dwelling animals the plasm, as it grows, extends In these chamber-dwelling animals the plasm, as it grows, extends
out of the primary shell-opening and reaches to a certain distance
down the outside; new shell material is then secreted, and the process
is repeated until a chain of chambers is the result (Fig. 36, 4). If THE SARCODINA 73 the plasm extends entirely around the shell, the new chamber almost
incloses the older ones as in Modosarina (B). In other cases the
plasm may extend over one side only of the old shell, and a curvi-
linear axis of growth is the result (Fig. 35, A, B, and 36, C). The
spiral thus formed may be flat or coiled around a longitudinal axis as
in the mollusc Z7ochus, giving an involute shell. This type, the most
highly differentiated of all of the rhizopod shells, exhibits all grades
of complexity (Fig. 37). In the highest forms each new chamber
has a complete wall, so that the septa between the adjacent chambers
consist of two lamella, while between
the lamellae
there
is fre- the plasm extends entirely around the shell, the new chamber almost
incloses the older ones as in Modosarina (B). In other cases the
plasm may extend over one side only of the old shell, and a curvi-
linear axis of growth is the result (Fig. 35, A, B, and 36, C). The
spiral thus formed may be flat or coiled around a longitudinal axis as
in the mollusc Z7ochus, giving an involute shell. This type, the most
highly differentiated of all of the rhizopod shells, exhibits all grades
of complexity (Fig. 37). In the highest forms each new chamber
has a complete wall, so that the septa between the adjacent chambers
consist of two lamella, while between
the lamellae
there
is fre- Fig. 3'7.— A complex polythalamous shell (schematic) of Operculina. A. SHELLS AND TESTS If the lime casing is
perforated by numerous pores through which pseudopodia can be
thrust to collect food, then each chamber is sufficient for itself, and
the so-called mouth-opening is small; but if the perforations are
absent, the mouth-openings are large and allow a free communication 74 THE PROTOZOA‘ between the youngest or external chambers and the oldest or internal. Hence there are morphological and physiological grounds for sepa-
rating the Reticulariida into Perforina and Imperforina. It frequently
happens that the central or original chamber varies in size in the
same species, being large (megalospheric) in some individuals, and
small (szcrospheric) in others (Fig. 38). While the relations of these
two forms have been much discussed, no satisfactory conclusion has
yet been reached. Lister (’95) regards the case as one of alternation
of generations in which spores from individuals A conjugate and form
individuals of the type &, while the latter develops spores which grow
into the form A again. The conjugation of swarmers in these dimor-
phic types is a matter of inference rather than of observation, for the
process has never been seen. between the youngest or external chambers and the oldest or internal. Hence there are morphological and physiological grounds for sepa-
rating the Reticulariida into Perforina and Imperforina. It frequently
happens that the central or original chamber varies in size in the
same species, being large (megalospheric) in some individuals, and
small (szcrospheric) in others (Fig. 38). While the relations of these
two forms have been much discussed, no satisfactory conclusion has
yet been reached. Lister (’95) regards the case as one of alternation
of generations in which spores from individuals A conjugate and form
individuals of the type &, while the latter develops spores which grow
into the form A again. The conjugation of swarmers in these dimor-
phic types is a matter of inference rather than of observation, for the
process has never been seen. [SCHLUMBERGER.]
The dimorphism is shown by the central chamber c. [SCHLUMBERGER.]
The dimorphism is shown by the central chamber c. Among the Heliozoa and Radiolaria,
shell formation
is of
a
somewhat different type, consisting of the deposition of spicules
and rays rather than a continuous layer of material forming a
compact coating. A. SHELLS AND TESTS Even naked forms of Heliozoa, such as Actino-
spherium, secrete these spicules at certain times for the purpose
of encystment, while others have them in greater or less numbers
throughout life. Isolated
spicules are usually retained by a gelat-
inous
mantle, which
covers
the
entire
animal
(Mwclearia,
<Acti-
nolophus, etc.). These spicules are usually curved or straight rods, THE SARCODINA 75 spindles, or blade-shaped plates, and may become firmly attached
to one another, forming latticed skeletons, like those of Radiolaria
(Clathrulina, Fig. 39). Intermediate stages are seen in such forms
as Diplocystis, where the plates are very small and arranged without ystis, where the plates are very small and arranged w
Fig. 39. — Clathrulina elegans Cienk. [GREEFF.] Fig. 39. — Clathrulina elegans Cienk. [GREEFF.] any apparent order
in the gelatinous mantle. In Raphidiophrys
(Fig. 40) the silicious plates are much larger and more regularly
arranged, while in Pizaciophora and Acanthocystis (B, C, D) they be-
come so closely knit that they form an efficient shield. In Acantho-
cystis, each plate is a small rectangular prism, laid tangential to the
surface with sharper spicules arranged at intervals at right angles to any apparent order
in the gelatinous mantle. In Raphidiophrys
(Fig. 40) the silicious plates are much larger and more regularly
arranged, while in Pizaciophora and Acanthocystis (B, C, D) they be-
come so closely knit that they form an efficient shield. In Acantho-
cystis, each plate is a small rectangular prism, laid tangential to the
surface with sharper spicules arranged at intervals at right angles to 76 THE PROTOZOA these, thus forming a bristling coat. Pinactophora is very similar,
but the spicules are not so prismatic. Similarly the Radiolaria may have either simple isolated spicules these, thus forming a bristling coat. Pinactophora is very similar,
but the spicules are not so prismatic. Similarly the Radiolaria may have either simple isolated spicules Similarly the Radiolaria may have either simple isolated spicules
or compact and strong skeletons. In many cases the outer plasm
(calymma) is free from spicules, but in other cases isolated spicules of
sharp and needle-like, or tri- or tetra-radiate form, are present. , The D
Fig. 40. — Types of spicules in Heliozoa. [PENARD.]
A. Raphidiophrys pallida F. E. Sch., with curved silicious rods. B. Pinaciophora rubiconda
Hert. and Less. C. Acanthocystis turfacea Carter. D, Pinaciophora fluviatilis Greeff. D
oa. [PENARD.] Fig. 40. A. SHELLS AND TESTS — Types of spicules in Heliozoa. [PENARD.]
A. Raphidiophrys pallida F. E. Sch., with curved silicious rods. B. Pinaciophora rubiconda
Hert. and Less. C. Acanthocystis turfacea Carter. D, Pinaciophora fluviatilis Greeff. substance of the skeleton of Radiolaria is either silica or acanthin, a
horn-like
modification
of protoplasm. According to Haeckel, the
deposition of silica in many cases occurs only at certain periods, and
an entire skeleton may be laid down at one time (Dictyottc moment,
Haeckel, or Lorication moment, Dreyer). Again, it may be formed
during the entire period of life. The material
of the shell
is
secreted from the sarcodictyum, and as the deposition of the silica THE SARCODINA 77 follows the outlines of the vesicles which form this zone of proto-
plasm, the resulting skeleton forms a reticulum. Growth may take
place more rapidly, however, at certain places, and spines, spicules, or
protuberances of one kind or another are the result. The usual form
of the network upon which the skeleton is deposited is an hexagonal
mesh, but this may become modified in numerous ways, the apertures
becoming either circular, polygonal, or elliptical (Fig. 41). When spines are formed,
a secondary calymma may also be When spines are formed,
a secondary calymma may also be
developed, carrying with it the sarcodictyum, and the latter, in turn,
may give rise to a secondary skeleton outside of the first. This
process may be repeated until there are as many as Six or seven acces-
sory skeletons. : Fig. 41. — Schematic figure illustrating the modifications of skeletons according to mechanical
principles of deposition. [DREYER.]
The secretion is supposed to collect in the interstices between alveoli as at (c), forming simple
spicules, or tri- and tetra-radiate spicules (4). Collecting in the lines of union of six alveoli, the
deposit takes the form of an hexagonal mesh (@), which, by the addition of more material, becomes
changed as at (a), (e), (f), and (g). 41. — Schematic figure illustrating the modifications of skeletons according to mechanical
principles of deposition. [DREYER.] The secretion is supposed to collect in the interstices between alveoli as at (c), forming simple
spicules, or tri- and tetra-radiate spicules (4). Collecting in the lines of union of six alveoli, the
deposit takes the form of an hexagonal mesh (@), which, by the addition of more material, becomes
changed as at (a), (e), (f), and (g). A. SHELLS AND TESTS A very interesting
set of phenomena are connected with the
acanthin
skeletons
where
the spicules
are
not deposited in thé
calymma, but are formed at the centre of the central capsule, growing
out centrifugally into the extra-capsular plasm and resulting in a
skeleton of radiating spines. With a few exceptions these spines are
twenty in number, and are arranged in a certain geometrical order
which has been characterized as the A/#ilerian law. The points of
the spines fall in five circles parallel to the equator, and there are four
spines to each
circle. The spines
are named, according
to this
scheme, polar, tropical, equatorial, sub-tropical, and sub-polar (Fig. 42). The form of the silicious skeleton is quite varied. In its least-dif- A very interesting
set of phenomena are connected with the
acanthin
skeletons
where
the spicules
are
not deposited in thé
calymma, but are formed at the centre of the central capsule, growing
out centrifugally into the extra-capsular plasm and resulting in a
skeleton of radiating spines. With a few exceptions these spines are
twenty in number, and are arranged in a certain geometrical order
which has been characterized as the A/#ilerian law. The points of
the spines fall in five circles parallel to the equator, and there are four
spines to each
circle. The spines
are named, according
to this
scheme, polar, tropical, equatorial, sub-tropical, and sub-polar (Fig. 42). The form of the silicious skeleton is quite varied. In its least-dif-
ferentiated form, as in most Heliozoa, it isa mere collection of loosely
arranged spicules. In other formsa uniform covering of silica covers
the meshes of the sarcodictyum. Such a generalized condition of the
skeleton becomes modified in many ways, the main types being the
“sagittal ring,’ consisting of a simple ring of silica, like a girdle The form of the silicious skeleton is quite varied. In its least-dif-
ferentiated form, as in most Heliozoa, it isa mere collection of loosely
arranged spicules. In other formsa uniform covering of silica covers
the meshes of the sarcodictyum. Such a generalized condition of the
skeleton becomes modified in many ways, the main types being the
“sagittal ring,’ consisting of a simple ring of silica, like a girdle THE PROTOZOA 78 around the body of the organism. A second type consists of a basal
tripod, the arms of which inclose the central capsule (Fig. 43). B. PsEUDOPODIA A pseudopodium is a portion of the body-plasm temporarily pro-
truded. It is most variable in form, and at any moment can be with-
drawn into the body of the animal to be replaced by others. In the
Rhizopoda,
the pseudopodia
are coarse, blunt, and finger-formed
(Ameebida), or fine, and often
forming a network through
anastomosis
(Reticulariida). In the Heliozoa and Radio-
laria they are more rigid and
radiate out from the body in
all directions, forming a pro-
tective coating, and from their
ray-like appearance suggest-
ing the common name “sun-
animalcula.”
There is a difference’ in the A pseudopodium is a portion of
truded. It is most variable in form,
drawn into the body of the animal to
Rhizopoda,
the pseudopodia
are
(Ameebida), or fine, and often
forming a network through
anastomosis
(Reticulariida). In the Heliozoa and Radio-
laria they are more rigid and
radiate out from the body in
all directions, forming a pro-
tective coating, and from their
ray-like appearance suggest-
ing the common name “sun-
animalcula.”
There is a difference’ in the :
7%;
(8.5 Marware preniine Heck, wi There is a difference’ in the
texture as well as in the form
of the lobose and reticulate
pseudopodia of the Rhizop-
oda. Inthe former the hya-
line ectoplasm, which
goes
into the pseudopodia, is ap-
parently
homogeneous
and
:
structureless, although, upon 7%;
(8.5 Marware preniine Heck, wi
critical examination, Biitschli
(92) was able to make out a fibrous structure in some forms, and
in many of them a reticular appearance was obtained upon retrac-
tion. His observations led him to the conclusion that the hyaline
appearance
is due to the close approximation of the walls of the
alveoli, and not to their absence. The outer plasm is certainly more
dense and non-granular than the endoplasm, and protoplasmic stream-
ing is confined to the latter. The outer plasm in the reticulate type,
on the other hand, is granular, while the central portion is denser and
more resisting. Streaming of the granules here takes place in the
ectoplasm, instead of in the endoplasm, and when two or more pseu-
dopodia come in contact, the viscid character of this outer plasm leads
to fusion. The lobose forms, on the other hand, never coalesce. A. SHELLS AND TESTS A
third modification is the simple alteration of the spherical latticed shell around the body of the organism. A second type consists of a basal
tripod, the arms of which inclose the central capsule (Fig. 43). A
third modification is the simple alteration of the spherical latticed shell E
Fig. 42. — Lichnaspis giltochi: Haeck., one af the Acantharia (Actipylea),
[HAECKEL.]
The spines are arranged in accord with the Miillerian Law as follows: a, 2,a, a, northern polar
spines; 4, 6, 4, 6, northern tropical spines; c, ¢, ¢, —, equatorial spines; @, ¢, d, d, southern tropi-
cal spines; and e, ¢, e, —, southern polar spines, E
Fig. 42. — Lichnaspis giltochi: Haeck., one af the Acantharia (Actipylea),
[HAECKEL.]
The spines are arranged in accord with the Miillerian Law as follows: a, 2,a, a, northern polar
spines; 4, 6, 4, 6, northern tropical spines; c, ¢, ¢, —, equatorial spines; @, ¢, d, d, southern tropi-
cal spines; and e, ¢, e, —, southern polar spines, E
Fig. 42. — Lichnaspis giltochi: Haeck., one af the Acantharia (Actipylea),
[HAECKEL.]
The spines are arranged in accord with the Miillerian Law as follows: a, 2,a, a, northern polar
spines; 4, 6, 4, 6, northern tropical spines; c, ¢, ¢, —, equatorial spines; @, ¢, d, d, southern tropi-
cal spines; and e, ¢, e, —, southern polar spines, into elliptical, ovate,
or sub-spherical
forms. Again, the skeleton
may be discoid, or even bivalved, and, in still other types, there may
be a combination of two or more of the above modifications. THE SARCODINA 79 B. PsEUDOPODIA Despite the uncertainty of the
pseudopodia as a basis of classification, their structure among the °
Rhizopoda is frequently so characteristic that the identification of
some species of Ameba is comparatively easy. Thus Ameba proteus
has large and blunt pseudopodia in the adult phase, while the young
form (known
as
A. radiosa)! has
sharper, stiffer, and hyaline
pseudopodia. When
a pseudopodium
of A. proteus
starts from
the periphery,
it continues
as
a stream
until, as a rule, a long,
lobose structure results. When, however, a pseudopodium
of 4. blatte Biitschli or of A. Zmicola Rhumbler starts from the periphery
of the spherical body, it resembles a miniature eruption. A break is character can have but little value. Despite the uncertainty of the
pseudopodia as a basis of classification, their structure among the °
Rhizopoda is frequently so characteristic that the identification of
some species of Ameba is comparatively easy. Thus Ameba proteus
has large and blunt pseudopodia in the adult phase, while the young
form (known
as
A. radiosa)! has
sharper, stiffer, and hyaline
pseudopodia. When
a pseudopodium
of A. proteus
starts from
the periphery,
it continues
as
a stream
until, as a rule, a long,
lobose structure results. When, however, a pseudopodium
of 4. blatte Biitschli or of A. Zmicola Rhumbler starts from the periphery
of the spherical body, it resembles a miniature eruption. A break is Fig. 45.— Camptonema nutans,
[SCHAUDINN.]
The axial filaments extend throughout the endoplasm (4), taking their origin at the nuclear
membrane (8). x, an axial filament highly magnified. Fig. 45.— Camptonema nutans,
[SCHAUDINN.]
The axial filaments extend throughout the endoplasm (4), taking their origin at the nuclear
membrane (8). x, an axial filament highly magnified. Fig. 45.— Camptonema nutans,
[SCHAUDINN.]
The axial filaments extend throughout the endoplasm (4), taking their origin at the nuclea
brane (8). x, an axial filament highly magnified. made on the periphery, and through it the granular endoplasm flows
down the sides of the spherical body instead of outward into elongate
pseudopodia (Fig. 44, 4, B). In such cases the pseudopodia may be
used to identify the organism. The pseudopodia of the Heliozoa and the Radiolaria are far more The pseudopodia of the Heliozoa and the Radiolaria are far more
complicated than those of the Rhizopoda. B. PsEUDOPODIA The resemblance between the central denser strand of protoplasm :
7%;
(8.5 Marware preniine Heck, wi The resemblance between the central denser strand of protoplasm
in the pseudopodia of the reticulate type and the axial filament of the
pseudopodia of Heliozoa and Radiolaria was early recognized by M. Schultze (’63) and critically examined by Biitschli(’92) and Schaudinn
(93), and is now generally recognized. THE PROTOZOA 80 The nature and the number of pseudopodia have frequently been
used as a method of identification of certain species of Rhizopoda. Amba polypodia, A. radiosa, and A. proteus have certain characteris:
tic pseudopodial structures which are seemingly of diagnostic value,
yet A. proteus under the influence of a constant electric current can
be made to assume the forms characteristic of A. polypodia and then ic pseudopodial structures which are seemingly of diagnostic value,
et A. proteus under the influence of a constant electric current can
be made to assume the forms characteristic of A. polypodia and then
0
oper
a
3
a
pT caggc conte
ie oSASS
6k
sa
xa}
Mea, eee
Fig. 44. — Types of pseudopodia. A, Amebalimicola Rhmb. [RHUMBLER.]
B. Ameba dblatte Biitsch. [BUTSCHLI.]
C. Lie-
erkiihnia sp. [VERWORN.]
D. Actinospherium Eich. Ehr,
[ORIGINAL.]
x, axial filament. of A. fémax. Conversely, A. /imax, when placed in an alkaline solu- 0
oper
a
3
a
pT caggc conte
ie oSASS
6k
sa
xa}
Mea, eee 0
oper
a
3
a
pT caggc conte
ie oSASS
6k
sa
xa}
Mea, eee Fig. 44. — Types of pseudopodia. [RHUMBLER.]
B. Ameba dblatte Biitsch. [BUTSCHLI.]
C. Lie- Fig. 44. — Types of pseudopodia. A, Amebalimicola Rhmb. [RHUMBLER.]
B. Ameba dblatte Biitsch. [BUTSCHLI.]
C. Lie-
berkiihnia sp. [VERWORN.]
D. Actinospherium Eich. Ehr,
[ORIGINAL.]
x, axial filament. Fig. 44. — Types of pseudopodia. A, Amebalimicola Rhmb. [RHUMBLER.]
B. Ameba dblatte Biitsch. [BUTSCHLI.]
C. Lie-
berkiihnia sp. [VERWORN.]
D. Actinospherium Eich. Ehr,
[ORIGINAL.]
x, axial filament. of A. fémax. Conversely, A. /imax, when placed in an alkaline solu-
tion of potassium hydrate, becomes transformed into A. frofeus, and
later, into A. radiosa (Verworn, ’94). Whena
change in the surround-
ing medium can so affect the protoplasm that the entire character of
pseudopodia-formation is altered, the specific value of pseudopodia
alone may well be questioned, and species based upon such a variable THE SARCODINA 81 character can have but little value. 1Cf. Scheel (’99). B. PsEUDOPODIA They usually have dis-
tinct axial filaments, consisting of some unknown substance, extending
throughout the entire length, and even into the endoplasm, where
they not infrequently abut against the membrane of the nucleus or
meet at a common centre (Actinophrys, Acanthocystis). The granular
protoplasm which surrounds the axial filament is in constant but slow
streaming motion. The point of interest is the axial filament, which
is not strictly comparable with the skeletal parts, but is probably stif- THE PROTOZOA 82 fened protoplasm similar to the central plasm of the reticulate pseudo-
podia. It is easily softened by the animal, and when the latter is
irritated may be withdrawn into the body. That there is some con-
nection between the axial filament and the nucleus would seem to be
indicated by their invariable propinquity, the nucleus in some cases
being actually surrounded by the substance that forms the filament
and which Schaudinn (’96) thinks is a soft fluid at this time (Campto-
nema nutans, Fig. 45). In other cases the filament appears to end in
a peculiar crescent or spherical capsule which lies within the endo-
plasm (Dimorpha, Fig. 46). In many instances the rays pass com-
pletely across the animal’s body and rest against the nucleus on the
opposite side; in others they are focussed in a central or “ astral” fened protoplasm similar to the central plasm of the reticulate pseudo-
podia. It is easily softened by the animal, and when the latter is
irritated may be withdrawn into the body. That there is some con-
nection between the axial filament and the nucleus would seem to be
indicated by their invariable propinquity, the nucleus in some cases
being actually surrounded by the substance that forms the filament
and which Schaudinn (’96) thinks is a soft fluid at this time (Campto-
nema nutans, Fig. 45). In other cases the filament appears to end in
a peculiar crescent or spherical capsule which lies within the endo-
plasm (Dimorpha, Fig. 46). In many instances the rays pass com-
pletely across the animal’s body and rest against the nucleus on the
opposite side; in others they are focussed in a central or “ astral” Fig. 46.— Flagella (/) and axial filaments of the pseudopodia of Czliophrys (Dimorpha?) Cienk. 1Cf, Chapter VITT. B. PsEUDOPODIA [BLOCHMANN.]
In the Heliozoa stage (4) the ray-like pseudopodia (f) and the flagella (/) are present; in
the flagellate stage (2) the pseudopodia are absent. The axial filaments (.v) and flagella centie
in the excentric nucleus (C). granule (Gymnosphera, Actinophrys, Spherastrum, etc.), which in some
‘cases has been seen to divide like a centrosome and to form an
amphiaster, as in the early stages in cell-division of many cells of the
Metazoa (Acanthocystis, Spherastrum, etc.)
The axial filaments have not been made out in all forms classed granule (Gymnosphera, Actinophrys, Spherastrum, etc.), which in some
‘cases has been seen to divide like a centrosome and to form an
amphiaster, as in the early stages in cell-division of many cells of the
Metazoa (Acanthocystis, Spherastrum, etc.)
The axial filaments have not been made out in all forms classed The axial filaments have not been made out in all forms classed
among Heliozoa, and it is a question whether such forms should be
considered as Heliozoa or as Rhizopoda. Tampyrella and Nuclearia
(Fig. 56), for example, have fine, radiating pseudopodia which change
like those of the Rhizopoda and, as in many Ameebida, are formed
of hyaline ectoplasm. They are placed among the Rhizopoda by
some (Delage) and among Heliozoa by others (Biitschli). The pseud-
opodia occasionally vary in other respects from the sharp radial
forms, as in Actzévolophus, where they end in knobs; or in Camp- 1Cf, Chapter VITT. THE SARCODINA 83 tonema, where there is an elbow or joint which can be bent at right
angles. No entirely satisfactory explanation of pseudopodia formation and gles. No entirely satisfactory explanation of pseudopodia formation and
movement has yet appeared, although the subject has been attacked
on many sides, and by almost all students of the Rhizopoda since the
time of Dujardin. Like the early attempts to explain other phe-
nomena in the Protozoa, the first explanations of pseudopodia-motion
were
based
upon
the
analogy
to
higher
forms. Protoplasmic
contractility, the basis of locomotion
in all higher animals, and
probably
in many Protozoa (Mastigophora
and
Infusoria),
was
early suggested as the cause of the protrusion of pseudopodia. B. PsEUDOPODIA The
majority of casual observers were content with this general explana-
tion; others, more definite, conceived the seat of contraction to be in
the cortical plasm or ectoplasm (Ecker, '49; Dujardin, ’41), which they
compared with the dermal musculature of worms, and which they
supposed forces out the pseudopodia by backward peripheral con-
traction, as water can be forced out of a rubber tube by pressure from
behind. Others, again, imagined that in addition to the contractile
cortex the entire mass of the amceboid body is penetrated by a con-
tractile substance (Cienkowsky, ’63), the sarcous matter of Briicke
(61). Still others conceived a contractile motor apparatus of even
greater complexity. Amongst these, Heitzmann (’73), in working out
his well-known theory of the structure of protoplasm, and adapting
Briicke’s view to his own interpretation, maintained that the body of
Am@ba is composed of contractile fibres and an inter-fibral
‘‘ non-
contractile fluid.”
The protrusion of a pseudopodium, he argued, is
due to the local contraction or stretching of this fibrous framework. Modifications of Heitzmann’s view have frequently appeared in sub-
sequent writings. In connection with the Metazoa it still makes its
appearance in the numerous theories of contractile fibres, especially
in explanation of mitosis (van Beneden, Boveti, Flemming, Reinke,
and many others). In connection with the Rhizopoda, it found its
most ardent advocate in R. Greeff (’91), who described radial, fibrillar,
contractile
structures in the ectoplasm
of many
so-called
Earth
Amebe, and interpreted them as muscle-fibres whose outer ends
are inserted in the ectoplasm with their inner ends attached to the
protoplasmic framework of the endoplasm. Subsequent research has
shown that the supposed muscle-fibres
are bacteria (Bourne, ’91;
Israel, 94; Gould, ’95). Contractility in a somewhat different form was also brought in to
explain pseudopodium formation. In connection with the Protozoa,
the most noteworthy advocate was Engelmann (’79), who conceived
units of contractile substance built up of molecules of protoplasm. To these hypothetical units he gave the name zzotegmata. During THE PROTOZOA 84 rest, Engelmann assumed, each inotogma has an elongated form,
becoming spherical upon contraction. If all contract at the same
time, as upon a sudden shock, the animal assumes a spherical con-
dition; if the inotogmata contract in certain groups, a pseudopodium
is started, although some pseudopodia, notably the fine, thread-like
forms, are due to “relaxation”
of rows
of contracted units. 1 Cf. Biitschli, p. 275. 2 pp. 172-212. 1 Biitschli, Zoc. ci4, English translation, pp. 310-311.
2 Loc. cit, p. 312.
3 Cf. Fig. 10, 4, p. 36. B. PsEUDOPODIA A
considerable uncertainty is attached to Engelmann’s theory, especially
when the attempt is made to explain special cases, and Biitschli (’92)
shows in a very convincing manner that it does not justify the expec-
tations of its originator. }
Wallich ('63) early observed
that the current in a progressive Wallich ('63) early observed
that the current in a progressive
pseudopodium does not begin in the body of the Amada, but at the
periphery, an observation which de Bary (’64) confirmed in Mycetozoa. Biitschli ('73) drew attention to the same fact soon after, and upon
the strength of his observations appeared, even at this early period,
as an opponent of the contractility hypothesis. As stated previously, Biitschli holds that protoplasm is essentially As stated previously, Biitschli holds that protoplasm is essentially
a mixture of liquids consisting of a fluid alveolar substance and an
intra-alveolar fluid of different physical nature. According to this
conception, which is widely accepted, a naked protoplasmic mass such
as Ameba must be subject to the same physical laws as other fluids. The rounding-out of drops of exuded protoplasm was early interpreted
by Hofmeister (67), and by Engelmann (’69) before he adopted the
theory of inotogmata, as the same phenomenon
that causes the
rounding-out of any liquid substance, z.¢. to surface tension. Of late
years, especially since the appearance of Biitschli’s masterly work on
the structure of protoplasm, there has been a general tendency to
abandon the older theory of contractility and to explain the move-
ments of amoeboid bodies through the physical laws of liquids, and in
particular, by the laws of surface tension. Weber (’55) compared the
protoplasmic movements in plant-cells to the streaming, due to surface
tension, in drops of liquid, and subsequently Berthold ('86), Biitschli
(92), Rhumbler (’98), and others, following the same line of investi-
gation, have obtained fruitful results. An excellent account of the
several interpretations along this line of reasoning may be seen in
Biitschli’s Profoplasma,? and it will be sufficient here to give the
most recent explanation
as worked
out by Rhumbler
(’98) upon
the basis of Biitschli’s earlier view. B. PsEUDOPODIA [BUTSCHLI.] very short distance behind the tip, —a circumstance
which in any case is extremely favorable to the rapid outgrowth of
the pseudopodium, in contradistinction to the relations that obtain in
the drops of foam, since the protoplasm that has come to rest is
heaped up and the pseudopodium grows in this way.’’? Rhumbler (’98) attempts to explain the formation of new ectoplasm
and the increase in surface of an advancing pseudopodium through
the hardening effect of water upon protoplasm, a fact which has long
been recognized (Biitschli, Pfeffer). An advancing pseudopodium of
Ameba proteus, if properly fixed and stained, shows an advanced mass
of endoplasm broken through the walls of ectoplasm. ®
The outer ectoplasm has a firm
consistency, and, as Rhumbler dem-
onstrated by treatment with diluted
caustic
potash
(Fig. 48), may
be isolated from the endoplasm. Nevertheless,
it is converted into
streaming endoplasm again. The
Fig. 48.—The ectoplasm
(e) and gastric Conversion of ectoplasm into endo-
racuoes 0) of Aneta
verrucae ate Sa" plasm, which was early noted by
Engelmann (’79) and recently by
Pénard, Pfeffer, Verworn, Biitschli, and others, takes place accord-
ing to Rhumbler
at all times. It is particularly well shown
in
Ameba limicola Rhumbler, or A. dlatte Biitschli, where the eruptive
pseudopodium incloses a definite portion of the old ectoplasm, which
soon disappears and becomes lost in the endoplasm. Both Biitschli Rhumbler (’98) attempts to explain the formation of new ectoplasm
and the increase in surface of an advancing pseudopodium through
the hardening effect of water upon protoplasm, a fact which has long
been recognized (Biitschli, Pfeffer). An advancing pseudopodium of
Ameba proteus, if properly fixed and stained, shows an advanced mass
of endoplasm broken through the walls of ectoplasm. ®
The outer ectoplasm has a firm The outer ectoplasm has a firm
consistency, and, as Rhumbler dem-
onstrated by treatment with diluted
caustic
potash
(Fig. 48), may
be isolated from the endoplasm. Nevertheless,
it is converted into
streaming endoplasm again. The
Fig. 48.—The ectoplasm
(e) and gastric Conversion of ectoplasm into endo-
racuoes 0) of Aneta
verrucae ate Sa" plasm, which was early noted by
Engelmann (’79) and recently by
Pénard, Pfeffer, Verworn, Biitschli, and others, takes place accord-
ing to Rhumbler
at all times. It is particularly well shown
in
Ameba limicola Rhumbler, or A. 2 Loc. cit, p. 312. B. PsEUDOPODIA Biitschli says:
‘The expla-
nation of the processes of movement in Amabe is to be found,
therefore, to my mind, in correspondence with the interpretation of
the phenomena of streaming movements in the drops of foam, in the
fact that, by the bursting of some of the superficial alveoli, enchylema THE SARCODINA 85 is poured out upon the free surface of the protoplasmic body, where
it produces a local diminution of surface tension, and in this way sets
up an extension centre together with forward movement.” ! The origin of a pseudopodium, according to this conception, is in is poured out upon the free surface of the protoplasmic body, where
it produces a local diminution of surface tension, and in this way sets
up an extension centre together with forward movement.” ! The origin of a pseudopodium, according to this conception, is in The origin of a pseudopodium, according to this conception, is in
the ectoplasm, and the rapidity
of a pseudopodium-formation
is
increased by the peculiar “fountain currents”’ char-
acteristic
of most pseudopodia. As observed by
NN
Biitschli,
an advancing
stream
of granules
flows
RY
through the centre or axis of the growing pseudopo-
dium, while near the tip back-running currents like
the falling drops of water in a fountain surround the
central stream (Fig. 47). “In the formation of a
Fig.47.—Diagram
finger-shaped pseudopodium of Amada proteus,” says feo
Biitschli,
‘“‘it can be seen that the current which
ules in an advance
traverses the axis of the pseudopodium and flows ing pseudopodium
:
é
x
of Ameba
proteus. away on all sides from its tip, comes to rest at a [BUTSCHLI.]
very short distance behind the tip, —a circumstance
which in any case is extremely favorable to the rapid outgrowth of
the pseudopodium, in contradistinction to the relations that obtain in
the drops of foam, since the protoplasm that has come to rest is
heaped up and the pseudopodium grows in this way.’’? Rhumbler (’98) attempts to explain the formation of new ectoplasm
‘\ NN
RY
Fig.47.—Diagram
‘\ Fig.47.—Diagram
feo
ules in an advance
ing pseudopodium
of Ameba
proteus. B. PsEUDOPODIA dlatte Biitschli, where the eruptive
pseudopodium incloses a definite portion of the old ectoplasm, which
soon disappears and becomes lost in the endoplasm. Both Biitschli Fig. 48.—The ectoplasm
(e) and gastric
racuoes 0) of Aneta
verrucae ate Sa" Fig. 48.—The ectoplasm
(e) and gastric
racuoes 0) of Aneta
verrucae ate Sa" Fig. 48.—The ectoplasm
(e) and gastric
racuoes 0) of Aneta
verrucae ate Sa" 86 THE PROTOZOA and Rhumbler recognize that the longer the action of water is con-
tinued upon the ectoplasm, the greater the stiffening ;
hence, Rhumbler
argues, the new ectoplasm forming at the edge of the advancing
pseudopodium is less resisting than elsewhere and the forward flow
continues in one direction until the surface tension is equalized. New
material for the advancing pseudopodium
must be supplied from
endoplasm, and
this in turn from the posterior ectoplasm, so the
assumption is made by Rhumbler that there is a continual change
of Amwba’s protoplasm from ectoplasm into endoplasm, and from
endoplasm into ectoplasm. Explanations of this nature, based upon purely physical Jaws of fluid Explanations of this nature, based upon purely physical Jaws of fluid
substances, seem inadequate to explain all types of pseudopodia, the
reticulate and long filamentous forms in particular. Up to the present
time no satisfactory and comprehensive explanation has been made,
and it should be recognized that the theories advanced still remain
only working hypotheses. Hofer (’89) and Verworn
(’91), and
many others have demonstrated that an enucleated amoeboid mass
soon comes to rest and assumes a spherical form. After a few days,
movement recommences, and is interpreted by Hofer as an expression
of the changes in surface tension. Such observations make it prob-
able that the chemical activity, which is constantly operating between
the numerous substances which make up the protoplasm, plays an
important part in pseudopodia-formation, and with our present imper-
fect knowledge of these intra-cellular reactions, it is premature
to
settle upon any one cause, however suggestive and attractive it may
appear, of this widely varied phenomenon. In many cases, especially among the Heliozoa, pseudopodia-motion In many cases, especially among the Heliozoa, pseudopodia-motion
approximates
flagella-motion. In many of the shelled Rhizopoda
(eg. Arcella, or some species of Difiugia), the hyaline pseudopodia
sway backward and forward like thick, slow-moving flagella, while in
some Heliozoa (e.g. Artodiscius) this motion is much more energetic,
causing the organism to dance about likeamonad. B. PsEUDOPODIA The resemblance
is more noteworthy and interesting from a theoretical point of view,
because both the flagellum of Mastigophora and the axial filament of
Heliozoa arise in the same manner in the endoplasm, and both are
apparently connected with a “division
centre,” a central
granule,
which is analogous to the centrosome of metazoan cells. } 1Cf. p. 271. C.
THe Nucieus Nuclei are almost as varied in the different forms of Sarcodina as
are the different types of the animals as a whole. In some cases,
there is no well-defined nucleus, the chromatin being scattered in the
1Cf. p. 271. 1Cf. p. 271. 87 THE SARCODINA form of granules throughout the entire cell, as in some of the Masti-
gophora; again, it is confined to a solid sphere without membrane or
intra-nuclear vacuoles;
or, there may be a membrane and a single
compact mass of chromatin which occupies the centre of the distinct
nucleus, and is separated from the membrane by hyaline matter. In
other
cases, there may be two or more saryosom.s
or chromatin
reservoirs, or there may be a great number ot granules in the nucleus
without the reservoirs (Ameba proteus). In some of the Rhizopoda
(Euglypha) and Heliozoa (Actinophrys
and Actinospherium),
the
nucleus is strikingly similar to that of metazoan cells, consisting of
chromatin in the form of a reticulum and a network of linin (Figs. 14 and 54). The number of nuclei is also quite variable, many forms having The number of nuclei is also quite variable, many forms having
only one (Amba proteus, Actinophrys, etc.); others, two (dmadba b1-
nucleata, Arcella, etc.); while some have many, the giant Ameda,
Pelomyxa, having, according to Bourne (’91), about
ten thousand,
although,
even with this large number, the proportion of nuclear
substance to the total mass of the organism is about the same as in
other cells (Bourne). In almost all of the shelled forms, a multiple
number of nuclei is the rule, but in the many-chambered Reticulari-
ida, every chamber
does
not
possess
a nucleus,
the number of
nuclei being smaller than the number of chambers, thus indicating that
these forms are not colonies, but syzcy¢za, or multinucleate cells. D.
THe CONTRACTILE VACUOLE Contractile vacuoles are almost entirely absent in the marine forms
(Reticulariida, Radiolaria), and in a few of the fresh-water forms
of Sarcodina (Protamwba, Pelomyxa, Myxodictyum, Protogencs, etc.),
but they are generally present in the Amoebida and Heliozoa, some-
times two or three in one organism. The number
of contractile
vacuoles is quite variable. In most of the naked forms there is
but one; this, however, may be of large size, sometimes measuring
one-quarter
of the volume of the organism (Acienophrys, Actino-
spherium). In the shelled forms, on
the other hand, there are
two or more (2-3 in Exg/ypha, 12 or more in Arce//a). The position of the vacuole in the naked forms is also variable, The position of the vacuole in the naked forms is also variable,
but becomes fixed in the shelled forms and in the Heliozoa. In the
shelled forms they sometimes lie in the middle zone about the edge
of the granular region (Evglypha), sometimes around the periphery of
the flattened body, while in other forms they are found now in one
zone, now in another. In all cases, shortly before contraction, they
come to lie close to the outer edge, and in some cases they form
minute wart-like excrescences. THE PROTOZOA 88 Ameba proteus, with its comparatively clear protoplasm and free-
dom from pigments, is one of the most favorable objects for the study
of the contractile vacuole. If a sufficiently high power is used, the
formation and contraction of the vacuole and the expulsion of the con-
tents to the exterior can be followed step by step. At first the vacuole
lies near the nucleus, but as it grows, it becomes separated from the
latter, and at the time of its contraction lies at the end of the body
farthest from the advancing pseudopodia, at what is sometimes called
the posterior end (Fig. 49, /). Its reappearance is always somewhere
near its point of disappearance. While still small it is carried along
by the streaming protoplasm back to a position near the nucleus,
where it completes its development. The increasing weight of the
growing vacuole causes it to lag behind the streaming granules and
nucleus, until at its full growth it is widely separated from the latter
organ. D.
THe CONTRACTILE VACUOLE The vacuole may appear to move in the direction contrary to
that of the protoplasmic streaming, although in reality it is quiescent;
for while it remains in the field of the microscope, the main body of
the animal moves well out of it, until the vacuole is surrounded only
by the posterior end of the animal (G), which is reduced to a thin
layer of granules and a hyaline layer of ectoplasm between the vacu-
ole and the exterior. The granules later move away, passing around
the vacuole,
until
finally there is only a thin
layer of hyaline
plasm between the vesicle and the exterior. Shortly after this the
vacuole bursts and disappears, in most cases a distinct bulge toward
the outside preceding contraction. Contraction always begins on
one side of the vacuole, and is carried across it toward the outer
edge (/7). Stokes (’93) asserts that there is no bursting of the wall, but that Stokes (’93) asserts that there is no bursting of the wall, but that
minute pores are formed through which the contents of the vacuole
are forced to the outside. In some instances the contents of the
vacuole are not completely emptied, but as much as half may be left,
the vacuole then rounding out to be carried back by the streaming
plasm to the nucleus, where it completes its growth. In other cases
the contents of the old vacuole may be entirely discharged, with the
exception of a small quantity of liquid retained in exceedingly small
vesicles, each of which may grow to some extent independently,
although they ultimately fuse to form the new vacuole (/). Thus
the new vacuole does not necessarily re-form at the place of dis-
appearance, but may be derived by the coalescence
of a number
of smaller ones which themselves are the remains of an old one. As many as six may unite in this manner to form the new vacuole
(A, B, C.)
These unite two by two in various parts of the plasm,
and the last two may not fuse until in the neighborhood of the
nucleus. : THE SARCODINA 89 In addition to the contractile vacuoles the Sarcodina occasionally
possess gas vacuoles, which were first made out in Avcedla, but which Fig. 49.— Amebu proteus and the contractile vacuole. A, The vacuole (v) is in the form of minute vesicles in the region of contraction. £. D.
THe CONTRACTILE VACUOLE Three
minutes later. C. ‘Two minutes later still (two vesicles have united). D. Two vesicles previous
to union near the nucleus (). £. The single vacuole becoming separated from the nucleus,
/. Fig. 49.— Amebu proteus and the contractile vacuole. A, The vacuole (v) is in the form of minute vesicles in the region of contraction. £. Three
minutes later. C. ‘Two minutes later still (two vesicles have united). D. Two vesicles previous
to union near the nucleus (). £. The single vacuole becoming separated from the nucleus,
/. The vacuole at the posterior end previous to contraction. G,H,/,and ¥. Four stages in the con-
traction of the vacuole. have since been shown to be quite general in the group (Claparéde
and Lachmann, Engelmann, Biitschli, Entz, Rhumbler). The gas Fig. 49.— Amebu proteus and the contractile vacuole. Fig. 49.— Amebu proteus and the contractile vacuole. A, The vacuole (v) is in the form of minute vesicles in the region of contraction. £. Three
minutes later. C. ‘Two minutes later still (two vesicles have united). D. Two vesicles previous
to union near the nucleus (). £. The single vacuole becoming separated from the nucleus,
/. The vacuole at the posterior end previous to contraction. G,H,/,and ¥. Four stages in the con-
traction of the vacuole. A, The vacuole (v) is in the form of minute vesicles in the region of contraction. £. Three
minutes later. C. ‘Two minutes later still (two vesicles have united). D. Two vesicles previous
to union near the nucleus (). £. The single vacuole becoming separated from the nucleus,
/. The vacuole at the posterior end previous to contraction. G,H,/,and ¥. Four stages in the con-
traction of the vacuole. have since been shown to be quite general in the group (Claparéde
and Lachmann, Engelmann, Biitschli, Entz, Rhumbler). The gas THE PROTOZOA 90 appears to be mainly carbon dioxide, and apparently serves an hydro-
static purpose, allowing the heavy forms like Dzfflugza to raise or
lower themselves in the water. appears to be mainly carbon dioxide, and apparently serves an hydro-
static purpose, allowing the heavy forms like Dzfflugza to raise or
lower themselves in the water. E. EncysTMENT Encystment is undoubtedly a widespread phenomenon among the
Sarcodina, although it is apparently absent altogether in the marine
forms (Radiolaria and Reticulariida). With the exception of some
Heliozoa, it has not been extensively studied, and the few observa-
tions are frequently contradictory. There is a general agreement,
however, that its object is to protect the individual during periods of
drought, cold, or during periods of reproduction. Thus a heliozoon,
when its environment becomes unsuitable, draws in its pseudopodia,
loses its ectoplasmic vacuoles, and secretes a double-layered coating,
the inner layer being gelatinous at first, but later like
a membrane. The outer layer is warty, and composed usually of silicious plates,
cemented together by a silicious jelly. If multinucleate, most of the
nuclei are absorbed, about 5 per cent remaining intact (Hertwig, for
Actinospherium). When conditions
are again suitable, the animal
absorbs water, swells, becomes vacuolated, bursts its membrane and
outer cyst, and as a free-swimming heliozoon develops pseudopodia,
and again leads an active life. Brauer (’94) and Hertwig (’98) have
shown that in Actinospherium encystment is accompanied by numer-
ous phenomena of flastogamy and karyogamy. In some forms of
Heliozoa, as in Vampyrella, the outer cyst is composed of other mate-
rial than silica, usually of cellulose, while in the fresh-water Rhi-
zopoda the cyst coatings are chitinous. Here also the conditions are
often changed by the presence of a shell, the cyst membrane in such
cases covering over only the mouth-opening, although in one form at
least (Euglypha)a second cyst membrane envelops the whole animal
inside of the shell (see Fig. 17, p. 47). LCE. pp. 236 and 237. F. NvuTRITION The food of Sarcodina consists of vegetable substances, of flagellates,
Infusoria, and not infrequently of larger animals, such as rotifers or
small Crustacea. In the simpler forms there is no region set aside for
the ingestion of food particles, but any portion of the body-surface can
function as a mouth. When food particles strike the body, the stimu-
lus causes the protrusion of specialized pseudopodia, which gradually
surround the object. Zacharias ('93), upon rather uncertain
data, THE SARCODINA gI suggested that the pseudopodia
prehensile organs, and are with
ridium pedatum). In some cases,
paralyze the prey, for flagel-
lates
or
ciliates,
coming
in
contact with the sharp pseudo-
podial
tips, are immediately
stunned and lie quiet, while
either
the pseudopodia
lose
their rigidity and bend around
them, or smaller and new pseu-
dopodia are formed from the
body-substance, gradually sur-
round the prey, and draw it
into the body (see Fig. 18, p. 49). Inthe shelled forms the
process of engulfing prey is
less simple, and where there is
a distinct cuticle the ingestion
of the food can take place only
by the softening or disappear-
ance
of
some
part
of
the
membrane. In the shell-bear-
ing Rhizopoda (Thecameebina)
food ingestion is confined to
one part of the animal, the
region about the mouth-open-
ing; while in some Reticulari-
ida
the prey
is not carried
inside of the animal at all, but
seizure, ingestion, and diges-
tion all take place in the net-
work of protoplasm formed by
the anastomosed pseudopodia
(Fig.50). In the shell-bearing
Heliozoa
the
outer
coating
must be ruptured for the en-
trance
of the food
particles
(Pénard),. In all cases the food substa suggested that the pseudopodia in some forms are not motile, but
prehensile organs, and are withdrawn after a full meal (Actinosphe-
ridium pedatum). In some cases, at least, the pseudopodia apparently
paralyze the prey, for flagel-
lates
or
ciliates,
coming
in
contact with the sharp pseudo- suggested that the pseudopodia
prehensile organs, and are with
ridium pedatum). In some cases
paralyze the prey, for flagel-
lates
or
ciliates,
coming
in
contact with the sharp pseudo-
podial
tips, are immediately
stunned and lie quiet, while
either
the pseudopodia
lose
their rigidity and bend around
them, or smaller and new pseu-
dopodia are formed from the
body-substance, gradually sur-
round the prey, and draw it
into the body (see Fig. 18, p. 49). F. NvuTRITION Inthe shelled forms the
process of engulfing prey is
less simple, and where there is
a distinct cuticle the ingestion
of the food can take place only
by the softening or disappear-
ance
of
some
part
of
the
membrane. In the shell-bear-
ing Rhizopoda (Thecameebina)
food ingestion is confined to
one part of the animal, the
region about the mouth-open-
ing; while in some Reticulari-
ida
the prey
is not carried
inside of the animal at all, but
seizure, ingestion, and diges-
tion all take place in the net-
work of protoplasm formed by
the anastomosed pseudopodia
(Fig.50). In the shell-bearing
Heliozoa
the
outer
coating
must be ruptured for the en-
trance
of the food
particles
(Pénard),. Fig. 50. — Gromia stiorai Duj. [M. SCHULTZE.]
— Some of the reticulate pseudopodia have captured
2 diatom. Fig. 50. — Gromia stiorai Duj. [M. SCHULTZE.]
— Some of the reticulate pseudopodia have captured
2 diatom. In all cases the food substance is subsequently inclosed within
a water vacuole, the liquid being taken in with the food (Dujardin,
’41; Le Dantec,’90; Metschnikoff, ’83). The fluid of the vacuole, at
first nothing more than water similar to that in which the animal
lives, gradually becomes acid, and in it the food particles are slowly In all cases the food substance is subsequently inclosed within
a water vacuole, the liquid being taken in with the food (Dujardin,
’41; Le Dantec,’90; Metschnikoff, ’83). The fluid of the vacuole, at
first nothing more than water similar to that in which the animal
lives, gradually becomes acid, and in it the food particles are slowly THE PROTOZOA Q2 disintegrated, the digestible portions being transformed into a sort
of chyle which is distributed throughout the protoplasm. The gastric
vacuole with its undigested residue is gradually left behind like a
loaded contractile vacuole, until finally it is expelled to the outside
(Ameba). The Sarcodina, apparently, digest mainly proteids, some
forms of starch, and fats remaining unchanged (Meissner, 88; Green-
wood,
*80; Stolc, ’00). G. REPRODUCTION The Sarcodina reproduce mainly by simple division or spore-for-
mation, either in the free state while active, or when quiet in the
encysted state. The simplest form, consisting of a mere bipartition
of the protoplasm and of the essential body-contents, occurs when the
body is so large that it becomes unwieldy and it divides from sheer
inertia. A well-known example is that of the division of Ama@ba
polypodia (Dactylosphera, F. E. Schultze). Here, as in all cell-divi-
sions, the nucleus divides first, the body then separating into two parts. Simple division becomes more complicated when the organism is Fig. 51.— Microgromia socialis Hert. [HERTWIG.]
Division takes place within the shell, and one of the daughter-individuals migrates, forming a
new shell. Fig. 51.— Microgromia socialis Hert. [HERTWIG.] Fig. 51.— Microgromia socialis Hert. [HERTWIG.]
Division takes place within the shell, and one of the daughter-individuals migrates, forming a
new shell. provided with an outer coating or test, although in the simplest of
such cases, where the coating is flexible and plastic, as in Vampyrella,
the process involves only the partition of the outer membrane. When
the outer covering becomes hard and firm by impregnation with chi-
tinous, silicious, calcareous, or horny materials, the operation is more
complicated. The organism, while still within the shell, may divide
by longitudinal division, one of the daughter-individuals then migrat-
ing from the parent shell and, after a longer or shorter time, settling
down and secreting a new shell for itself, the other daughter-indi- provided with an outer coating or test, although in the simplest of
such cases, where the coating is flexible and plastic, as in Vampyrella,
the process involves only the partition of the outer membrane. When
the outer covering becomes hard and firm by impregnation with chi-
tinous, silicious, calcareous, or horny materials, the operation is more
complicated. The organism, while still within the shell, may divide
by longitudinal division, one of the daughter-individuals then migrat-
ing from the parent shell and, after a longer or shorter time, settling
down and secreting a new shell for itself, the other daughter-indi- THE SARCODINA 93 vidual remaining in the old quarters (AZicrogromia, Fig. 51). A more
complicated process is found in the majority of fresh-water shelled
Rhizopoda, where division is practically a form of budding, the plasm
growing out of the original shell mouth and forming a small bud on
the outside. G. REPRODUCTION This bud grows until it has reached its definitive size
(usually about that of the original cell), when the shell-coating for
the new individual is deposited. The building material for the shell
of the daughter-individual is formed within the protoplasm of the
maternal cell. If regular plates of silica
or
chitin, these plates
are secreted long before division and stored up in the protoplasm
which surrounds the nucleus (Eug/ypha, Quadrula),
If quartz crys-
tals, or any other foreign bodies, these particles are picked up and
stored in a similar manner, to be used later for the test of the daugh-
ter-cell. When the bud has reached a certain size, the plates or par-
ticles which are to form the shell move out through the mouth-opening
of the parent shell and form around the protoplasm of the bud. In
the meantime the nucleus undergoes division, and, in the case of
Euglypha at least, the daughter-nucleus is the last element to leave
the parent organism (see Fig. 23, p. 55). Heliozoa, when preparing for division, become soft, draw in their vidual remaining in the old quarters (AZicrogromia, Fig. 51). A more
complicated process is found in the majority of fresh-water shelled
Rhizopoda, where division is practically a form of budding, the plasm
growing out of the original shell mouth and forming a small bud on
the outside. This bud grows until it has reached its definitive size
(usually about that of the original cell), when the shell-coating for
the new individual is deposited. The building material for the shell
of the daughter-individual is formed within the protoplasm of the
maternal cell. If regular plates of silica
or
chitin, these plates
are secreted long before division and stored up in the protoplasm
which surrounds the nucleus (Eug/ypha, Quadrula),
If quartz crys-
tals, or any other foreign bodies, these particles are picked up and
stored in a similar manner, to be used later for the test of the daugh-
ter-cell. When the bud has reached a certain size, the plates or par-
ticles which are to form the shell move out through the mouth-opening
of the parent shell and form around the protoplasm of the bud. In
the meantime the nucleus undergoes division, and, in the case of
Euglypha at least, the daughter-nucleus is the last element to leave
the parent organism (see Fig. 23, p. 55). G. REPRODUCTION Heliozoa, when preparing for division, become soft, draw in their Heliozoa, when preparing for division, become soft, draw in their
pseudopodia, and round out into a perfect sphere, after which the
nucleus divides by mitosis (Actznophrys), and the cell slowly separates
into two parts. In Muwuclearta, division
is very rapid, the entire
process taking place within one minute. In many cases, division is
incomplete,
the individuals remaining attached
to form
colonies
(Heterophrys, Spherastrum, Raphidiophrys). Swarm-spore formation is widely distributed among the Sarcodina, Swarm-spore formation is widely distributed among the Sarcodina,
usually taking place under the protection of a cyst. The parent
organism divides into a number of daughter-cells, each containing
a part of the original nucleus, and each provided with pseudopodia or
flagella. A good illustration is seen in Parameba Ettlhardt, one of
the naked
Rhizopoda (Schaudinn,
’96). The animal
is flat and
discoid, with short, lobose, finger-formed pseudopodia, and varies in
size from
10 to 90 pw (Fig. 52). It usually increases by simple
division, but at the end of its vegetative life it encysts, and the plasm
divides into a number of pieces. Fragmentation of the nucleus is
preceded by division of a peculiar cytoplasmic body, which Schaudinn
‘terms the Vebenkorper. The contents of the cyst break into as many
pieces as there are divisions
of the Mebenkorper. Finally, each
fragment of the protoplasm, containing a part of the original nucleus
and of the cytoplasmic Vebenkorper, develops two flagella and breaks
out of the cyst as a swarm-spore (2, C, D). The young organisms
swim about in this condition for some time, and may increase by longi-
tudinal division until, finally, losing their flagella, they develop pseudo- THE PROTOZOA 94 podia and become young amoeboid forms. Other cases of swarm-spore
formation have been frequently recorded, in some cases by so many tion have been frequently recorded, in some cases by so many
Fig. 52. — Parameba eilhardi Schaud. [SCHAUDINN.]
Section. &. Sporulation,
C. The flagellated swarm-spore. D-A. Stages in division of
ellated spore. 4, Nedbenkorper ,; n, the nucleus. ent observers that there can be little doubt of their truth. In Fig. 52. — Parameba eilhardi Schaud. [SCHAUDINN.] Fig. 52. — Parameba eilhardi Schaud. [SCHAUDINN.]
A. Section. &. Sporulation,
C. The flagellated swarm-spore. D-A. Stages in div
the flagellated spore. 4, Nedbenkorper ,; n, the nucleus. different observers that there can be little doubt of their truth. G. REPRODUCTION In
the Reticulariida, swarm-spore formation may be considered the typi-
cal method of increase, and it is connected with the dimorphism THE SARCODINA 95 of the shells which Munier-Chalmas (’83) and Schlumberger (’83)
have shown to be widespread throughout the group. The original
nucleus divides into numerous parts, which are spread throughout all
of the chambers. The protoplasm then segregates about them, and
the original mass of plasm becomes divided into as many parts as there
are nuclei. These leave the parent organism either by rupture of
the shell or through the mouth-opening, and soon form new shells
(megalospheric). After a short time they bud, and calcium car-
bonate is secreted around the bud, thus making a two-chambered cell. This process continues until the organism is full grown. Finally, the
pseudopodia are drawn into the shell, and the protoplasm divides
into numerous small swarm-spores, each with two flagella. These
probably conjugate (Lister, ’95; Schaudinn, ’95), the copula giving
rise to individuals with shells of the microspheric form. A very
similar process occurs in the Radiolaria, where the endoplasm within
the central capsule breaks up into swarm-spores, each with a portion
of the original nucleus and each provided with flagella. These finally
break out of the capsule, and, after a short free-swimming period, they
lose their flagella and gradually assume the typical radiolarian form,
passing through Heliozoa stages. In some cases, dimorphic spores
(anzsospores) are formed, which perhaps conjugate, as assumed by
Brandt (’85) and Haeckel (’88), although the process has never been
seen. Here, too, an alternation of generations is assumed by Haeckel
and Brandt, an asexual or zsospore generation alternating with a sexual
anispore generation. In addition to simple division and swarm-spore formation, some of the shells which Munier-Chalmas (’83) and Schlumberger (’83)
have shown to be widespread throughout the group. The original
nucleus divides into numerous parts, which are spread throughout all
of the chambers. The protoplasm then segregates about them, and
the original mass of plasm becomes divided into as many parts as there
are nuclei. These leave the parent organism either by rupture of
the shell or through the mouth-opening, and soon form new shells
(megalospheric). After a short time they bud, and calcium car-
bonate is secreted around the bud, thus making a two-chambered cell. This process continues until the organism is full grown. G. REPRODUCTION In other cases, flagella are formed, and the buds
move about like swarm-spores, although after a couple of days these,
too, become amoeboid. A few days later silicious spicules appear
in the vicinity of the nucleus, and soon after make their way to the
periphery, where the shell is formed (Fig. 53). Clathrulina also
forms buds in a similar manner, and has been observed by Cienkow-
sky, Greeff, Hertwig, and Lesser; the observations thus are as well formed by the same animal. The history of the buds is different in
different individuals. In the simplest cases, the bud merely drops
off the parent and remains on the bottom
for some days in an
ameceboid state. In other cases, flagella are formed, and the buds
move about like swarm-spores, although after a couple of days these,
too, become amoeboid. A few days later silicious spicules appear
in the vicinity of the nucleus, and soon after make their way to the
periphery, where the shell is formed (Fig. 53). Clathrulina also
forms buds in a similar manner, and has been observed by Cienkow-
sky, Greeff, Hertwig, and Lesser; the observations thus are as well Fig. 53. — Nuclear division and spore-formation in Heliozoa. [SCHAUDINN.]
A. A vegetative cell of Spherastrum, with the axial filaments focussed in a central granule
(division-centre or “ centrosome”). S&-ZD. Division of the nucleusin Acanthocysts. LE. F. Flag-
ellated and amoeboid swarm-spores formed by budding. G. Exit of the central granule from the
nucleus. NN.] NN.] ore-f ore-f [SC Fig. 53. — Nuclear division and spore-formation in Heliozoa. [SCHAUDINN.]
. A vegetative cell of Spherastrum, with the axial filaments focussed in a central granul Fig. 53. — Nuclear division and spore-formation in Heliozoa. [SCHAUDINN.]
A. A vegetative cell of Spherastrum, with the axial filaments focussed in a central gra
(division-centre or “ centrosome”). S&-ZD. Division of the nucleusin Acanthocysts. LE. F. F
ellated and amoeboid swarm-spores formed by budding. G. Exit of the central granule from
nucleus. Fig. 53. — Nuclear division and spore-formation in Heliozoa. [SCHAUDINN.]
A. A vegetative cell of Spherastrum, with the axial filaments focussed in a central granu
(division-centre or “ centrosome”). S&-ZD. Division of the nucleusin Acanthocysts. LE. F. Fl A. A vegetative cell of Spherastrum, with the axial filaments focussed in a central granule
(division-centre or “ centrosome”). S&-ZD. Division of the nucleusin Acanthocysts. LE. F. G. REPRODUCTION Finally, the
pseudopodia are drawn into the shell, and the protoplasm divides
into numerous small swarm-spores, each with two flagella. These
probably conjugate (Lister, ’95; Schaudinn, ’95), the copula giving
rise to individuals with shells of the microspheric form. A very
similar process occurs in the Radiolaria, where the endoplasm within
the central capsule breaks up into swarm-spores, each with a portion
of the original nucleus and each provided with flagella. These finally
break out of the capsule, and, after a short free-swimming period, they
lose their flagella and gradually assume the typical radiolarian form,
passing through Heliozoa stages. In some cases, dimorphic spores
(anzsospores) are formed, which perhaps conjugate, as assumed by
Brandt (’85) and Haeckel (’88), although the process has never been
seen. Here, too, an alternation of generations is assumed by Haeckel
and Brandt, an asexual or zsospore generation alternating with a sexual
anispore generation. In addition to simple division and swarm-spore formation, some
Sarcodina reproduce by bud-formation or gemmation. Buck (’77)
early observed a number of small amoeboid germs in the shell of
Arcella (as many as thirty), an observation since confirmed by Cat-
taneo (’78), Biitschli, and recently by Hertwig (’99). Both Buck and
Cattaneo traced the development of the buds up to the formation
of the characteristic shell, while Hertwig has described the nuclear
divisions leading to bud-formation. Biitschli found that the number
of amoeboid buds does not exceed nine. Le Blanc (’92) describes
similar processes in Dzffugia. Somewhat similar buds were observed
inside Pelomyra palustris by Weldon,! although neither the devel-
opment nor origin was made out. Bud-formation has been repeatedly
seen in the Heliozoa as well as in the Rhizopoda. The genus Alcan-
thocystis in particular has been studied in this connection by Hertwig
(74), Korotneff, and more recently by Schaudinn (’96). According
to the latter, the nucleus divides by amitosis, the daughter-nuclei
moving toward the periphery, where they bud off with a small amount
of cytoplasm; in some cases as many as twenty-four buds may be
1 Cf. Lankester (91). 1 Cf. Lankester (91). THE PROTOZOA 96 formed by the same animal. The history of the buds is different in
different individuals. In the simplest cases, the bud merely drops
off the parent and remains on the bottom
for some days in an
ameceboid state. G. REPRODUCTION After twenty-four hours one shell appeared transparent,
the other dense, while all movements had ceased. When the two shells
separated at the end of forty-eight hours, one was empty, its contents
having fused with those of the other shell. Gruber (’87) also reports a
similar conjugation between two Difflugias. Conjugation has been
frequently described in the Heliozoa also, although it is quite possible
that many cases of so-called conjugation are only instances of plas-
togamy, or fusion of the cell-body, and are not followed by union of
the nuclei (karyogamy), as in fertilization. Numerous observations might be cited which seem, at first sight, to supposed that Avcel/a, which he found in pairs, were conjugating, and
later he held the view that the phenomenon is quite widespread. Holman (’86) observed a large Ama@ba surround a small one, the
two remained together for some
time, and after they separated
swarm-spores were formed in each. She regarded this as a possible
case of conjugation. A somewhat similar process occurs in Amoeba
spatula, although here the smaller individual does not regain its iden-
tity, while the larger one appears as before, except for the presence
of two nuclei(Pénard, ’90). Conjugation is apparently more common
among the shelled forms; Pénard (’90) says that, although he has
met with conjugating animals in almost all of the species studied by
him, he cannot cite a single instance where he has seen two animals,
at first free, approach each other and fuse. Jickeli (84) was more
fortunate, for he saw two individuals of Diffugia globulosa fuse by
their mouth-parts, the union being followed by lively pseudopodial
movements. After twenty-four hours one shell appeared transparent,
the other dense, while all movements had ceased. When the two shells
separated at the end of forty-eight hours, one was empty, its contents
having fused with those of the other shell. Gruber (’87) also reports a
similar conjugation between two Difflugias. Conjugation has been
frequently described in the Heliozoa also, although it is quite possible
that many cases of so-called conjugation are only instances of plas-
togamy, or fusion of the cell-body, and are not followed by union of
the nuclei (karyogamy), as in fertilization. G. REPRODUCTION Flag-
ellated and amoeboid swarm-spores formed by budding. G. Exit of the central granule from the
nucleus. established as in Acanthocystis. The number of buds is not so large
as in the latter form, and the process is not unlike simple division. The body divides into three dissimilar pieces, two smaller and one
larger. The latter remains within
the old shell, but the former
develop flagella and swim about like swarm-spores. In about half
an hour they lose their flagella, settle to the bottom, throw out pseu-
dopodia, and develop a stalk. Conjugation has only rarely been seen among the different kinds of established as in Acanthocystis. The number of buds is not so large
as in the latter form, and the process is not unlike simple division. The body divides into three dissimilar pieces, two smaller and one
larger. The latter remains within
the old shell, but the former
develop flagella and swim about like swarm-spores. In about half
an hour they lose their flagella, settle to the bottom, throw out pseu-
dopodia, and develop a stalk. Conjugation has only rarely been seen among the different kinds of Conjugation has only rarely been seen among the different kinds of
Sarcodina, and further observations must be made before it can be
considered a widespread phenomenon. <A few authentic observa-
tions, however, show that it occurs in some cases. Biitschli (’74) THE SARCODINA 97 supposed that Avcel/a, which he found in pairs, were conjugating, and
later he held the view that the phenomenon is quite widespread. Holman (’86) observed a large Ama@ba surround a small one, the
two remained together for some
time, and after they separated
swarm-spores were formed in each. She regarded this as a possible
case of conjugation. A somewhat similar process occurs in Amoeba
spatula, although here the smaller individual does not regain its iden-
tity, while the larger one appears as before, except for the presence
of two nuclei(Pénard, ’90). Conjugation is apparently more common
among the shelled forms; Pénard (’90) says that, although he has
met with conjugating animals in almost all of the species studied by
him, he cannot cite a single instance where he has seen two animals,
at first free, approach each other and fuse. Jickeli (84) was more
fortunate, for he saw two individuals of Diffugia globulosa fuse by
their mouth-parts, the union being followed by lively pseudopodial
movements. G. REPRODUCTION Numerous observations might be cited which seem, at first sight, to Numerous observations might be cited which seem, at first sight, to
show that copulation and conjugation, in Heliozoa, are preliminary
to reproduction by simple division or by spore-formation, but the evi-
dence in most cases is incomplete, and the connection between con-
jugation and reproduction is still largely inferential. The ease with
which large forms like Actinospherium can be artificially reproduced
by breaking them into pieces (Foulke, ’83) is reason enough to excite
caution as to generalizations on the connection between copulation
and increase. The phenomenon is, nevertheless, clearly established
in at least one form (Actznophrys sol, Schaudinn ’96). In this case
two free-swimming individuals come together and fuse; pseudopodia
are drawn in, and the double cell sinks to the bottom, where it becomes
coated by a cyst of silicious plates. Each of the two as yet ununited
nuclei now prepares for division, passing through typical spireme and
spindle stages as in Euglypha. Two of the four nuclei which are
formed by these divisions round out and become normal nuclei, the
others degenerate and finally disappear without playing any further
réle. The phenomenon recalls in a striking manner the formation
of polar bodies among the Metazoa, and obviously represents some
form of maturation (Fig. 54). The two functional nuclei now fuse, H 98
THE PROTOZOA THE PROTOZOA 98 forming a cleavage nucleus, which divides by mitosis, giving rise to
daughter-cysts. C. For-
([SCHAUDIN
P,
polar
body,
B. The
nuclei
during
the
prophas
of
division
Fig. 54. —
Conjugatio
of
-fcfivophry
sol
Ehr. A. Two
individu
fused;
the
axial
filament
abut
against
the
nuclei. The conjugation of swarm-spores has been seen in a few cases. F. First
divis
J. Fusio
of
the
nue'ci
PD. Recon
of
the
nuclei
matio
of
the
first
polar
spindl forming a cleavage nucleus, which divides by mitosis, giving rise to
daughter-cysts. forming a cleavage nucleus, which divides by mitosis, giving rise to
daughter-cysts. C. For-
P,
pol
bod
F. Firs
divi ([SCHAUDI
B. The
nuclei
during
the
propha
of
divisio
Fig. 54. —
Conjugati
of
-fcfivophr
sol
Ehr. A. Two
individ
fused;
the
axial
filamen
abut
against
the
nuclei. The conjugation of swarm-spores has been seen in a few cases. In Vampyrella variabilis, the animal breaks up into swarm-spores
while encysted (Klein). These make their way out of the cyst to
F. First
divi
J. G. REPRODUCTION Fusi
of
the
nue'c
PD. Reco
of
the
nucle
mati
of
the
first
pola
spin The conjugation of swarm-spores has been seen in a few cases. In Vampyrella variabilis, the animal breaks up into swarm-spores
while encysted (Klein). These make their way out of the cyst to THE SARCODINA 99 conjugate, and later form either double or multiple individuals (plas-
modia). The conjugation of swarm-spores of Reticulariida and Radiolaria conjugate, and later form either double or multiple individuals (plas-
modia). The conjugation of swarm-spores of Reticulariida and Radiolaria The conjugation of swarm-spores of Reticulariida and Radiolaria
has not been observed, although the diverse size of the anisospores
in the latter group favors this view. In the fresh-water form, Aya-
lopus, which differs but slightly from the marine forms in regard
to reproduction, the swarm-spores actually conjugate, although the
development of the copula was not observed (Schaudinn, ’94). 1Biitschli enumerates 98 genera of Flagellidia and 174 genera of Rhizopoda; Delage
and Hérouard 314 genera of Rhizopoda and 153 Flagellidia. INTER-RELATIONSHIPS OF THE SARCODINA In drawing conclusions as to the most primitive group of the Pro-
tozoa, there is need of extreme caution. Biitschli long since showed
that the development of the Protozoa, from spores or germs of any
kind, gives but little indication of their genetic affinities, and that
such affinities must be deduced from the study of the group as a
whole. In questions concerning the most primitive Protozoa, the Infusoria In questions concerning the most primitive Protozoa, the Infusoria
are immediately thrown out, for, of all groups of Protozoa, they are the
most highly specialized, and along lines which have carried them
to the highest point of morphological development of the single cell. The Sporozoa also have become highly specialized through their
parasitic mode of life. Neither of these groups therefore can be
said to have been the most primitive forms of Protozoa. Among the
Sarcodina, the Heliozoa and Radiolaria show abundant evidence of
descent from rhizopod-like ancestors, while a similar relation can be
assumed of the Dinoflagellidia and Cystoflagellidia to the Flagellidia. It remains, therefore,
to ascertain
if possible which
of the two
groups, Flagellidia or Rhizopoda, shows the more primitive charac-
teristics. Students
of the Protozoa have differed widely on this
question. Biitschli avoids the difficulty by the assumption that the
beginnings of both are represented by the forms intermediate between
the two, and sees in the members of the family Rhizomastigidze
(Mast gameba and its allies), the common stem-forms of Flagellidia
and Rhizopoda. On the other hand, the Rhizopoda, with their animal
mode of nutrition, must have had other forms of life as their source
of food, and from which they were possibly derived by the process of
metasitism. On this account, Klebs (’92) makes the Flagellidia the
original group, since here are retained forms which live to-day as the
original forms probably did, with the power to manufacture their own
food (Phytoflagellida). Itis extremely difficult to choose between the
two assumptions, although the balance apparently lies in favor of the
view which Klebs advocates. INTER-RELATIONSHIPS OF THE SARCODINA 55, also Fig 46, p. 82). Here the axial filament is homologous with the flagellum, and there dence to show that pseudopodia and flagella are closely related, and
this evidence is strong enough, I believe, to throw additional light
upon the Flagellidia, regarded as the most primitive forms of Proto-
zoa. It has been shown that the flagellum in most cases arises in the
vicinity of the nucleus. This is also the case with the axial filaments
of the Heliozoa, an extremely interesting example being shown in the
form Dimorpha, as described by Gruber (Fig. 55, also Fig 46, p. 82). Here the axial filament is homologous with the flagellum, and there
is ground for believing that the homology can be carried from
Dimorpha to the true Heliozoa, where all of the appendages are simi-
lar to the axropodia of Dimorpha. Thus, in Acanthocystis and Actino-
phrys, the axial filaments radiate from a common
centre
in the
nucleus (Actenophrys, see Fig. 54 A), or in the cytoplasm (Acantho-
cystis). In Actinospherium and Camptonema, they arise from the
nuclei and do not converge at a common point. In these particular
cases, the pseudopodia have little power of vibratile motion, such as
we might expect if the axial filaments are comparable with flagella. In other cases they do possess this power, however, to a certain
degree, as shown by the rolling motion of Acanthocystis, which is able
to travel a distance equal to twelve times its own diameter in one
minute, or by the quick dancing motion of Artodiscus (Pénard). That this motion is due to the vibrations or elasticity of the axial
filaments I think there can be no doubt, and these structures
are
comparable therefore with the flagella of the Mastigophora. Unlike
flagella, however, they are covered by plastic and streaming proto-
plasm, which gives them their pseudopodial character. In those
forms of Heliozoa which are usually regarded as more primitive,
e.g. Nuclearia and Vampyrella, the axial filaments are not formed,
and it is an important question whether these are primitive forms
representing a condition before differentiation of the axial filaments
in other Heliozoa, or are to be considered as degenerate forms in
which the axial filaments have disappeared (Fig. 56). INTER-RELATIONSHIPS OF THE SARCODINA From the variety of forms they assume,
the Flagellidia appear to have a greater power of adaptation than the THE PROTOZOA 100 Rhizopoda, and to this power of change may be due the fact that
fewer species of Flagellidia than of Rhizopoda are known.1
The
development of a flagellate or of a rhizopod throws little light upon
the question, for both flagellates with amoeboid swarm-spores, and
rhizopods with flagellated swarm-spores, are known. The very close
relation of the two groups is also shown by the fact made out by Fig. 55.— Dimorpha mutans Gruber. [GRUBER]
A, Ameeboid phase. Z. Flagellated phase. Fig. 55.— Dimorpha mutans Gruber. [GRUBER]
A, Ameeboid phase. Z. Flagellated phase. numerous observers, that in the same organism pseudopodia may
change into flagella, and flagella into pseudopodia. The relations of pseudopodia to flagella have not hitherto been numerous observers, that in the same organism pseudopodia may
change into flagella, and flagella into pseudopodia. The relations of pseudopodia to flagella have not hitherto been The relations of pseudopodia to flagella have not hitherto been
sufficiently emphasized. Not only do flagella become pseudopodia,
and pseudopodia flagella, in some forms, but in cases where the
mutual change has never been observed, there is morphological evi- THE SARCODINA IOI dence to show that pseudopodia and flagella are closely related, and
this evidence is strong enough, I believe, to throw additional light
upon the Flagellidia, regarded as the most primitive forms of Proto-
zoa. It has been shown that the flagellum in most cases arises in the
vicinity of the nucleus. This is also the case with the axial filaments
of the Heliozoa, an extremely interesting example being shown in the
form Dimorpha, as described by Gruber (Fig. 55, also Fig 46, p. 82). Here the axial filament is homologous with the flagellum, and there
is ground for believing that the homology can be carried from
Dimorpha to the true Heliozoa, where all of the appendages are simi-
lar to the axropodia of Dimorpha. Thus, in Acanthocystis and Actino-
phrys, the axial filaments radiate from a common
centre
in the
nucleus (Actenophrys, see Fig. 54 A), or in the cytoplasm (Acantho-
cystis). In Actinospherium and Camptonema, they arise from the
nuclei and do not converge at a common point. INTER-RELATIONSHIPS OF THE SARCODINA In these particular
cases, the pseudopodia have little power of vibratile motion, such as
we might expect if the axial filaments are comparable with flagella. In other cases they do possess this power, however, to a certain
degree, as shown by the rolling motion of Acanthocystis, which is able
to travel a distance equal to twelve times its own diameter in one
minute, or by the quick dancing motion of Artodiscus (Pénard). That this motion is due to the vibrations or elasticity of the axial
filaments I think there can be no doubt, and these structures
are
comparable therefore with the flagella of the Mastigophora. Unlike
flagella, however, they are covered by plastic and streaming proto-
plasm, which gives them their pseudopodial character. In those
forms of Heliozoa which are usually regarded as more primitive,
e.g. Nuclearia and Vampyrella, the axial filaments are not formed,
and it is an important question whether these are primitive forms
representing a condition before differentiation of the axial filaments
in other Heliozoa, or are to be considered as degenerate forms in
which the axial filaments have disappeared (Fig. 56). If this question
could be answered, it might afford evidence as to whether the Flagel-
lidia or the Sarcodina are the more primitive forms; for if degener-
ate, they point toward the Heliozoa or Radiolaria as the ancestors of
the reticulate Rhizopoda; but if primitive, they point toward the
Rhizopoda as the ancestors of Heliozoa and Radiolaria, and, through
Dimorpha, of the Flagellidia. Evidences of the axial filaments are
found in other Sarcodina than the Heliozoa and Radiolaria. In the
Reticulariida the central plasm of the pseudopodia is denser and more
resisting than the outer plasm, and M. Schultze, Biitschli, Schaudinn,
Rhumbler, and others, assume that it has a contractile function; mor-
phologically and physiologically, therefore, it appears to be similar to
the axial filaments of Heliozoa, and to the flagella of Mastigophora. dence to show that pseudopodia and flagella are closely related, and
this evidence is strong enough, I believe, to throw additional light
upon the Flagellidia, regarded as the most primitive forms of Proto-
zoa. It has been shown that the flagellum in most cases arises in the
vicinity of the nucleus. This is also the case with the axial filaments
of the Heliozoa, an extremely interesting example being shown in the
form Dimorpha, as described by Gruber (Fig. INTER-RELATIONSHIPS OF THE SARCODINA If this question
could be answered, it might afford evidence as to whether the Flagel-
lidia or the Sarcodina are the more primitive forms; for if degener-
ate, they point toward the Heliozoa or Radiolaria as the ancestors of
the reticulate Rhizopoda; but if primitive, they point toward the
Rhizopoda as the ancestors of Heliozoa and Radiolaria, and, through
Dimorpha, of the Flagellidia. Evidences of the axial filaments are
found in other Sarcodina than the Heliozoa and Radiolaria. In the
Reticulariida the central plasm of the pseudopodia is denser and more
resisting than the outer plasm, and M. Schultze, Biitschli, Schaudinn,
Rhumbler, and others, assume that it has a contractile function; mor-
phologically and physiologically, therefore, it appears to be similar to
the axial filaments of Heliozoa, and to the flagella of Mastigophora. THE PROTOZOA 102 As a matter of fact, the beginnings of the various branches of the
Protozoa rest in complete obscurity, and the relationships of the sub- Fig. 56. — Nuclearia delicatula Cienk. A. Heliozoan phase. B&B. Rhizopod phase. groups are almost equally uncertain. There are a few interesting Fig. 56. — Nuclearia delicatula Cienk. A. Heliozoan phase. B&B. Rhizopod phase. groups are almost equally uncertain. There are a few interesting
forms, however, which are generally given as intermediate stages THE SARCODINA 103 between the classes, and are supposed to show genetic relationships
between the groups which they simulate. The close connection between the M The close connection between the Mastigophora and the Sarcodina
has been recognized since the discovery by F. E. Schultze (’75) and
Biitschli (78) of amoeboid forms with flagella. These are unmistak-
ably animals which take in food at any portion of the body by means
of pseudopodia, and move by means of flagella or pseudopodia. In
some instances they are more like an Ameba (Mastigameba, F. E. Schultze, Fig. 57, A); in others they are more
like a heliozoén
(Crltophrys infusionum of Cienkowsky, or Acténomonas of Kent, Fig. 57, B, Mastigophrys of Frenzel, etc.). Their undetermined position Fig. 517. — Protozoa with both pseudopodia and flagella. A. Mastigameba aspera F. E. Sch. (SCHULTZE.]
B. Actinomonas pusilla S. K. [KENT.]
J, flagellum ; z, nucleus; #, pseudopodia, Fig. 517. — Protozoa with both pseudopodia and flagella. A. Mastigameba aspera F. E. Sch. (SCHULTZE.]
B. Actinomonas pusilla S. K. INTER-RELATIONSHIPS OF THE SARCODINA The
close
connection
of
the
Heliozoa with the Mastigophora is shown in other ways than by
the
transitional
forms
Dimorpha,
Actinomonas,
etc. The
finer
structure of the body of the sun-animalcula, the nucleus and archo-
plasmic substances, show
a degree of differentiation approached
Fig. 58. — Actinospherium Eich. Ehr. Section. ozoa. The forms which Pseudospora represents are placed between
the Rhizomastigide as enumerated above, and the Vampyrellide,
largely on account of their methods of reproduction and life history,
which in the majority of the Rhizomastigide are unknown. Vampy-
rella reproduces while encysted, by dividing into a number of parts,
each of which emerges as a small Vampyrella with pseudopodia like
the parent. Profomouas amyli (Haeckel), Monas amyl (Cienkowsky),
and Pseudospora reproduce in the same way, with the exception that
the
swarm-spores
formed
within
the
cyst
are
not
amoeboid, but are provided
with flagella. The swarmers
soon lose their flagella, how-
ever, becoming amceboid, a
condition in which they fuse
together to form larger or
smaller
plasmodia. This
fusion is characteristic of the
Vampyrellide and of My-
cetozoa,
but
not
of
the
Rhizomastigide,
where
it
has never been observed. There are many features ozoa. The forms which Pseudospora represents are placed between
the Rhizomastigide as enumerated above, and the Vampyrellide,
largely on account of their methods of reproduction and life history,
which in the majority of the Rhizomastigide are unknown. Vampy-
rella reproduces while encysted, by dividing into a number of parts,
each of which emerges as a small Vampyrella with pseudopodia like
the parent. Profomouas amyli (Haeckel), Monas amyl (Cienkowsky),
and Pseudospora reproduce in the same way, with the exception that
the
swarm-spores
formed m, membrane between ectoplasm and endoplasm; 7
nuclei; 2, axial filaments,
’
Fig. 58. — Actinospherium Eich. Ehr. Section. There are many features
in this theory of Klebs to
recommend it. It affords a
logical and satisfactory ex-
planation of the relations of
the Mycetozoa to the Sar-
codina, and from the stand-
point of the botanist points
out the relation of this group
m, membrane between ectoplasm and endoplasm; 7
nuclei; 2, axial filaments,
’
to the colorless plants. The
close
connection
of
the
Heliozoa with the Mastigophora is shown in other ways than by
the
transitional
forms
Dimorpha,
Actinomonas,
etc. INTER-RELATIONSHIPS OF THE SARCODINA [KENT.]
J, flagellum ; z, nucleus; #, pseudopodia, in classification is indicated by the fact that sometimes they are in-
cluded with the Sarcodina, while at other times they are regarded as
flagellates. Klebs believes that the connection between
the two
groups is not quite so apparent as the mere description of these inter-
mediate forms would indicate, and places between
the primitive
animal flagellates, the Vampyrellidz, and the Mycetozoa, an inter-
mediate group, that of the Pseudosporez, with the genera Pseudospora
and Protomonas which Biitschli includes with the Flagellidia; while
the Rhizopoda are derived from the Vampyrellide through the Heli- in classification is indicated by the fact that sometimes they are in-
cluded with the Sarcodina, while at other times they are regarded as
flagellates. Klebs believes that the connection between
the two
groups is not quite so apparent as the mere description of these inter-
mediate forms would indicate, and places between
the primitive
animal flagellates, the Vampyrellidz, and the Mycetozoa, an inter-
mediate group, that of the Pseudosporez, with the genera Pseudospora
and Protomonas which Biitschli includes with the Flagellidia; while
the Rhizopoda are derived from the Vampyrellide through the Heli- THE PROTOZOA 104 ozoa. The forms which Pseudospora represents are placed between
the Rhizomastigide as enumerated above, and the Vampyrellide,
largely on account of their methods of reproduction and life history,
which in the majority of the Rhizomastigide are unknown. Vampy-
rella reproduces while encysted, by dividing into a number of parts,
each of which emerges as a small Vampyrella with pseudopodia like
the parent. Profomouas amyli (Haeckel), Monas amyl (Cienkowsky),
and Pseudospora reproduce in the same way, with the exception that
the
swarm-spores
formed
within
the
cyst
are
not
amoeboid, but are provided
with flagella. The swarmers
soon lose their flagella, how-
ever, becoming amceboid, a
condition in which they fuse
together to form larger or
smaller
plasmodia. This
fusion is characteristic of the
Vampyrellide and of My-
cetozoa,
but
not
of
the
Rhizomastigide,
where
it
has never been observed. There are many features
in this theory of Klebs to
recommend it. It affords a
logical and satisfactory ex-
planation of the relations of
the Mycetozoa to the Sar-
codina, and from the stand-
point of the botanist points
out the relation of this group
m, membrane between ectoplasm and endoplasm; 7
nuclei; 2, axial filaments,
’
to the colorless plants. INTER-RELATIONSHIPS OF THE SARCODINA The
finer
structure of the body of the sun-animalcula, the nucleus and archo-
plasmic substances, show
a degree of differentiation approached
only by the Flagellidia and Metazoa, while the axial filaments are
homologous with flagella. It may be pointed out, however, that
Klebs’ theory leaves unexplained the relatively simple nuclear struc-
tures and nuclear processes of division in the Rhizopoda. This objec-
tion is fatal to the view that the Rhizopoda are derived from the
higher types of Heliozoa, and it must be admitted that they arose
from much less specialized forms, perhaps from the Pseudosporez or
Fig. 58. — Actinospherium Eich. Ehr. Section. m, membrane between ectoplasm and endoplasm; 7
nuclei; 2, axial filaments,
’
Fig. 58. — Actinospherium Eich. Ehr. Section. THE SARCODINA 105 other flagellated forms, perhaps from forms like the Vampyrellide. On the whole, there is no conclusive evidence to support the view that
Rhizopoda are more primitive than Flagellidia, or vice versa. Their
mutual affinities are very close, and together they stand as the most
primitive forms of modern Protozoa. The relations of the Radiolaria to the Heliozoa are extremely close, The relations of the Radiolaria to the Heliozoa are extremely close,
and there is abundant evidence to show that the former were derived
from the latter by the acquisition of a chitinous membrane between
ectoplasm and endoplasm, and the retention of a gelatinous mantle
like that of Sphe@rastrim (Haeckel). As first pointed out by Brandt
(85), the young Radiolaria pass through flagellated and amoeboid
swarm
stages, then through Heliozoa stages, until the
definitive
radiolarian structure is attained. Haeckel described the intermediate
forms which are represented in this growth, as flagellate, amoeboid,
Actinophrys, Spherastrum, and Actissa, the last-being the simplest of
the Radiolaria. Although the external appearance of a radiolarian
is strikingly similar to that of a heliozodn, there is no structure in
Heliozoa to compare with the chitinous central capsule of the Radio-
laria. Greeff(’67, 71) described a membrane-like thickening between
the endoplasm and the ectoplasm of Actinospherium, and regarded it
as homologous with the central capsule. Other observers, ¢.g. F. E. Schultze, Hertwig and Lesser, Biitschli, etc., have not seen it, and
the latter, especially, considers Greeff’s contribution of little value. INTER-RELATIONSHIPS OF THE SARCODINA However incorrect his interpretation may have been that Actznosphe-
rium is a fresh-water radiolarian belonging to the Acantharia, Greeff
was not mistaken in his observation, for an occasional specimen is
found which shows such a membrane (Fig. 58). CLASSIFICATION (°74),
fresh water; Chromatella Frenzel (92), fresh water; Stylama@ba Frenzel (’92),
fresh water; Sa/fonella Frenzel (92), fresh water; Zzkenia Frenzel (92), fresh
water; Pelomyxa Greeff (74), fresh water; Amphizonella Greeff (66), fresh
water; Podostoma Clap. & Lach. (’58), fresh water; Arcaothrix Hallez (°85),
cultures of Jscaris megalocephala. uborder 2. THECAMG@BINA. Ameebida
provided with
a shell, with lobose Suborder 2. THECAMG@BINA. Ameebida
provided with
a shell, with lobose
pseudopodia, which may be sharp pointed and branched, and with one or more
nuclei and contractile vacuoles. Family 2. Arcellida. The shell is more or less membranous. Contractile vacuoles. Family 2. Arcellida. The shell is more or less membranous. Contractile vacuoles. are numerous; the nucleus is single or multiple. Genera: Arcella Ehbg. (°38),
fresh water, common; Cochlopodium Hert. & Less. (74), fresh water, common;
Pyaidicula Ehbg. (°38), fresh water; Pseudochlamys Clap. & Lach. (’58), fresh
water ; Hyalosphenia Stein (’57), fresh water; Quadrula F. E. Schultze (’75),
fresh water;
Dzflugza Leclerc
(1 5), fresh
water, common;
Lecguereusia
Schlumberger, fresh water. Family 3. Euglyphida. The shell is formed of regular plates of chitin, or of silica, Family 3. Euglyphida. The shell is formed of regular plates of chitin, or of silica,
and is often provided with spines. The pseudopodia are sharp pointed and
often branching, but do not anastomose. Genera: Luglypha Dujardin (41),
fresh water; Zrzzenza Duj. (°36), fresh water; Cyphoderza Schlumberger (745),
marine and fresh water;
Campascus Leidy (’77), fresh water;
Madznella
Pénard (99). Order 2. RETICULARIIDA. Rhizopoda with fine branching and anastomosing, or Order 2. RETICULARIIDA. Rhizopoda with fine branching and anastomosing, or
reticulate pseudopodia, forming an irregular network erunnd the body, wien
may or may not have a shell. Shells, when present, are calcareous (rarely
silicious) and provided with many pores (Perforina), or without pores (Imper-
forina), and consisting of one chamber (Monothalamous), or of many chambers
(Polythalamous). Suborder 1. NUDA. Shell absent; the pseudopodia are reticulate, and the cell-
body, in many cases,
is apparently without a nucleus;
marine. Genera:
Gymnophrys
Cienkowski
(°76) ; Protomyxa
Haeckel
(°68);
ALyaxodictyum
Haeckel (68) ; Protogenes Haeckel (°64) ; Pontomyxa Topsent (93). Suborder 2. IMPERFORINA. [With but few modifications, the following classifica-
tion of the Reticulariida is taken from Brady (84) and Lankester (’85), after
Carpenter (69)]. Shell-bearing forms;
the shells are calcareous, solid, and
without minute apertures, or they are made up of foreign particles cemented upon
a chitinous base. CLASSIFICATION Cxiass I. SARCODINA. Naked or shelled Protozoa, characterized by the possession
during adult life of movable or changeable processes of protoplasm, the pseudo-
podia, which may be finger-form, reticulate, or ray-like, and which may or may
not have axial filaments. Reproduction is brought about by simple division and
by spore-formation. Subclass I. RHIZOPODA. Naked or shelled Sarcodina having pseudopodia of the Cxiass I. SARCODINA. Naked or shelled Protozoa, characterized by the possession
during adult life of movable or changeable processes of protoplasm, the pseudo-
podia, which may be finger-form, reticulate, or ray-like, and which may or may
not have axial filaments. Reproduction is brought about by simple division and
by spore-formation. Subclass I. RHIZOPODA. Naked or shelled Sarcodina having pseudopodia of the Subclass I. RHIZOPODA. Naked or shelled Sarcodina having pseudopodia of the
lobose (finger-formed) or reticulate (anastomosing) type. The adult form is
amceboid:
the young forms are amceboid or flagellated, and are produced by
spontaneous division of the cell during active phases or during encystment. The adults in some cases fuse to form plasmodia. Order 1. AMGBIDA. Rhizopoda provided with lobose pseudopodia, with or with- Order 1. AMGBIDA. Rhizopoda provided with lobose pseudopodia, with or with-
out a shell, with one or more nuclei, and usually with a contractile vacuole. Suborder 1. GYMNAMCBINA. Naked forms of Amcebida having lobose pseudo- Order 1. AMGBIDA. Rhizopoda provided with lobose pseudopodia, with or with-
out a shell, with one or more nuclei, and usually with a contractile vacuole. Suborder 1. GYMNAMCBINA. Naked forms of Amcebida having lobose pseudo- Suborder 1. GYMNAMCBINA. Naked forms of Amcebida having lobose pseudo-
podia, and with or without nucleus and contractile vacuoles. Family
1. AM@BIDA. The pseudopodia are lobose, occasionally sharp pointed Family
1. AM@BIDA. The pseudopodia are lobose, occasionally sharp pointed
and branched. Genera: 4A@béba, marine and fresh water; Parameba Schau-
dinn (196); Protam@ba
Haeckel
(66), marine
and
fresh water;
Grénga Family
1. AM@BIDA. The pseudopodia are lobose, occasionally sharp pointed
and branched. Genera: 4A@béba, marine and fresh water; Parameba Schau-
dinn (196); Protam@ba
Haeckel
(66), marine
and
fresh water;
Grénga 106 THE PROTOZOA Frenzel (’92), lagoons; Glo¢dium Sorokin (’78), fresh water;
Cheloproteus
(Dinameba
Leidy) Stein
(’57), fresh
water;
Zréchospherium Schneider ;
Pachymyxa Gruber;
f/fyalodiscus Hertwig and
Lesser
(’74), fresh
water;
Plakopus F. EE. Schultze (75), fresh water; Dactylosphera Hert. & Less. CLASSIFICATION Genera: Aawerina d'Orb. (46); Articulina d'Orb. (46). Subfamily 4. /eneroplidine. The shells are plano-spiral or cyclical, and bilaterally Subfamily 4. /eneroplidine. The shells are plano-spiral or cyclical, and bilaterally
symmetrical. Genera: Peneropl’s Montfort (10); Orbiolites Lamarck (1801) ;
Orbiculina Lamarck (716); Cornuspira M. Schultze (°54). Subfamily 5. Adveolintne@. The shell is spiral and elongated in the axis of the Subfamily 5. Adveolintne@. The shell is spiral and elongated in the axis of the
convolution; the chambers are subdivided into secondary chambers. Genera:
Alveolina VOrb (726). Subfamily 6. Keramospherine. The shell is spherical with the chambers in con-
centric layers. Genera: Keramosphera Brady (’84). Family 3. Astrorhizide. The shell is invariably composite, consisting of foreign Family 3. Astrorhizide. The shell is invariably composite, consisting of foreign
particles, such as diatom-cases, spicules, sand grains, etc. It is usually large and
single chambered, frequently branched or even radiate, with usually a single
pseudopodial aperture at the end of each branch. Subfamily 1. Astrorhizin@. The shells have thick walls, consisting of sand or Subfamily 1. Astrorhizin@. The shells have thick walls, consisting of sand or
mud, lightly cemented together. Genera:
Astrorhiiza Sandahl (°57); Den-
drophrya Wright (61); Syrzngammina Brady (84); Pelosina Brady (’79)-
Subfamily 2. /elulintne. The shell consists of one chamber, the walls being thick Subfamily 2. /elulintne. The shell consists of one chamber, the walls being thick
and composed of felted spicules and fine sand. Genera: /elulina Carpenter
(70) ; Bathysiphon Sars ('71). Subfamily 3. Saccamminine. The chambers are Aen spherical, with thin walls Subfamily 3. Saccamminine. The chambers are Aen spherical, with thin walls
composed of closely cemented sand grains. Genera: Saccammina Sars (’65) ;
Psammosphera Schultze (75); Sorosphera Brady (’79). Subfamily 4. Rhabdamminine. The shell is composed of sand grains, firmly Subfamily 4. Rhabdamminine. The shell is composed of sand grains, firmly
cemented together, and often with sponge spicules intermixed. They are
tubular, straight, radiate, branched or irregular, but rarely segmented. Genera:
JSaculella Brady (79); Botellina Carpenter ('70); Haltphysema Bowerbank (’62);
Marstpella Norman (778) ; Rhabdammina Sars (65) ; Aschemonella Brady (’79) ;
Rhizammina Brady (°79); Sagenella Brady (’79). Family 4. Lituolida. The shell is arenaceous, and the septa which imperfectly Family 4. Lituolida. The shell is arenaceous, and the septa which imperfectly
mark the chambers are often incomplete or absent. Subfamily 1. L¢twoline@. The shell is composed of coarse sand grains, is rough Subfamily 1. L¢twoline@. CLASSIFICATION They may have one, two, or many mouth openings, and are
either monothalamous or polythalamous. Family 1. Gromide. The shell is membranous and in the form of a simple sac, Family 1. Gromide. The shell is membranous and in the form of a simple sac,
with a pseudopodial aperture either at one extremity or at each end. The
pseudopodia are long, branching, and anastomosing;
marine and fresh-water
forms. Subfamily 1. Afonostomine. The shell has but one aperture. Genera: Gromia Subfamily 1. Afonostomine. The shell has but one aperture. Genera: Gromia
Duj. (35); Leeberkithnia Clap. & Lach. (758), both found in fresh water, the
former also marine; A/icrogromia R. Hertwig (74), fresh water; Platoum F.E. Schultze (75); Plectophrys Entz (77); Psendodigiugia Schlumberger (’45). brackish and fresh water. Subfamily 2. Amphistomine. With an aperture at each end of the shell. Genera: Subfamily 2. Amphistomine. With an aperture at each end of the shell. Genera:
Diplophrys Barker; Ditrema Archer (70); «laphitrema Archer (70); Shep-
heardella Siddall (80). THE SARCODINA 107 Family 2. Miliolide. The shell is mono- or polythalamous, usually, calcareous and
porcellanous, but may be covered with sand. The polythalamous forms may be
linear, spiral, or a combination of the two. Subfamily 1. Mubecularing. The shell has an irregular and asymmetrical form, Family 2. Miliolide. The shell is mono- or polythalamous, usually, calcareous and
porcellanous, but may be covered with sand. The polythalamous forms may be
linear, spiral, or a combination of the two. Subfamily 1. Mubecularing. The shell has an irregular and asymmetrical form, Subfamily 1. Mubecularing. The shell has an irregular and asymmetrical form,
with the aperture or apertures variously placed. Genera: Ca/ctuba Roboz;
Sguammulina Schultze (54); Mubecularia Dufrance. Subfamily 2. A¢¢dioling. The shell is coil Subfamily 1. Mubecularing. The shell has an irregular and asymmetrical form,
with the aperture or apertures variously placed. Genera: Ca/ctuba Roboz;
Sguammulina Schultze (54); Mubecularia Dufrance. Subfamily 2. A¢¢dioling. The shell is coiled, ei Subfamily 2. A¢¢dioling. The shell is coiled, either symmetrically or asymmetri-
cally, on an elongated axis, with usually two chambers to each convolution. During growth the shell-mouth is alternately at each end of the shell. Genera:
Spiroloculina @Orb. (26);
Biloculina @Orb. (126);
Fabularia Dufrance;
Miliolina Williamson (58). Subfamily 3. Hauerining. The shells are varied, the chambers being partly milio- Subfamily 3. Hauerining. The shells are varied, the chambers being partly milio-
line in their arrangement, partly spiral or linear. CLASSIFICATION The shell is composed of coarse sand grains, is rough
externally, and often labyrinthic. Genera: Aheophar Montfort ('08); Haplo-
phraginium Reuss (60); Coskinolina Stache ; //2plostiche Reuss (61) 3 Lituola
Lamarck (1801) ; Bdelloidina Carter (°77). Subfamily 2. 7rochamminine. The shell is thin, and consists of a chitinous basis
in which are embedded minute sand grains. The outside of the shell is smooth
and often polished ; the interior is smooth or occasionally reticulate, but never
labyrinthic. Genera:
7hurammina Brady (79); Am#modiscus Reuss;
Tro-
chammina Parker and Jones (*59); Hebbina d'Orb. (39); Carterina Brady
(79); Hippocrepina Parker: Hormosina Brady (79). 108 THE PROTOZOA Subfamily 3. Exdothyrine. The shell is more calcareous and less sandy than in
the other Zztvolide, and the septa between the chambers are distinct. Genera:
Nodosinella Brady ; Endothyra Phillips (46); Polyphragma Reuss; Bradyina
Moll. (78) ; Stacheza Brady (’76). Subfamily 4. Loftusing. The shell is large, lenticular, spherical, or fusiform, and Subfamily 4. Loftusing. The shell is large, lenticular, spherical, or fusiform, and
deposited either in concentric layers or spirally. The chambers are occupied
to a large extent by an excessive enlargement of the arenaceous cancellated wall. Genera: Cyclammina Brady (76); Loftusia Brady ('69); Parkeria Carpen-
ter
(69). ek 3. PERFORINA. The shell wall is perforated by numerous minute open- ek 3. PERFORINA. The shell wall is perforated by numerous minute open-
ings through which the pseudopodia can pass as well as through the main
openings. Family 5. Textularide. The shells of the larger species are arenaceous, either with Family 5. Textularide. The shells of the larger species are arenaceous, either with
or without a calcareous matrix ; the smaller forms are hyaline and conspicuously
perforated. The chambers are arranged in alternating series, spirally or without
apparent order. Subfamily 1. Zextularin@. The shells are typically bi- or tri-serial, and are often Subfamily 1. Zextularin@. The shells are typically bi- or tri-serial, and are often
dimorphous. Genera:
Zextwlaria Dufrance (°28): Bzgenerina d'Orb. ('26);
Verneuilina VOrb. ; Czuneoltna d'Orb. (39) ; Pavonina d'Orb. (26); Valvulina
d'Orb. (°26); Chrysalidina VOrb. (46); Zritaxia Reuss; Clavulina d’Orb. Subfamily 2. Bulrminine. The shells are typically spiral, the weaker forms are Subfamily 2. Bulrminine. The shells are typically spiral, the weaker forms are
more or less bi-serial. The main aperture is not round, but elliptical, comma-
shaped, etc. Genera: Vrrgulina d’Orb. (26) ; Bulimina d’Orb. (26) ; Bolivina
d'Orb.; Bzfarinza Parker and Jones. CLASSIFICATION The shell is calcareous, perforated, free, or adherent; it is
typically spiral in form, but irregular forms may be outspread or flaring, acervu- THE SARCODINA 109 line or irregular. Some of the higher types have double walls, with supple-
mental skeleton and a canal system. Subfamily 1. Spzrd/din@. The shell is a flat spiral, without septa; it may be free line or irregular. Some of the higher types have double walls, with supple-
mental skeleton and a canal system. Subfamily 1. Spzrd/din@. The shell is a flat spiral, without septa; it may be free Subfamily 1. Spzrd/din@. The shell is a flat spiral, without septa; it may be free
or attached. Genera: Sprrdlina Ehbg (°41). ;
Subfamily 2. Rotating. The shell is spiral, rotaliform, and rarely evolute or ir- Subfamily 2. Rotating. The shell is spiral, rotaliform, and rarely evolute or ir-
regular. Genera:
Descorbina
Lamarck
('04);
Planorbulina dOrb. (26) ;
Truncatulina VOrb. ('26); Anomalina d’Orb. (126); Rotalia Lamarck (1801) ;
Catarina VOrb. (26) ; Patellina Williamson (’58); Carpenteria Gray (58) ;
etc. etc. Suborder 4. TINOPORINZE. The shell consists of irregularly heaped chambers,
usually with a more or less spiral primordial portion; a main pseudopodial
aperture is usually absent. Genera:
Zzuoforus Carpenter (’57); Folytrema
Risso (’26); Gypszna Carter; Thalamopora Roemer; Aphrosina Carter. Family 10. Nummulinide. The shell is calcareous and finely tubulated;
it is Family 10. Nummulinide. The shell is calcareous and finely tubulated;
it is
typically polythalamous, free, and symmetrically spiral. The higher forms pos-
sess a supplementary skeleton and a well-developed canal system. Subfamily 1. Fvselenine@. The shell
is bilaterally symmetrical, with chambers Subfamily 1. Fvselenine@. The shell
is bilaterally symmetrical, with chambers
extending from pole to pole, so that each convolution completely incloses the
preceding whorl. The septa between the chambers are single as a rule. Genera:
fusulina Fischer ('29); Schwagerina Moller ('77). Subfamily 2. Polystomelling. The shell is bilaterally symmetrical and nautiloid. Subfamily 2. Polystomelling. The shell is bilaterally symmetrical and nautiloid. The simpler forms are without supplemental skeleton; the more complex forms. have a skeleton, and canals leading to the outside at regular intervals along the
external septal depressions. Genera: Polystomella Lamarck (’22); Montonina
d’Orb. (’26). Subfamily 3. Mammulitine.’ The shell is lens-shaped or flattened. Genera: Subfamily 3. Mammulitine.’ The shell is lens-shaped or flattened. Genera:
Archeodiscus Brady; Amphistegina VOrb. (26); Ofperculina d’Orb. ('26) ;
Nummutites Lamarck (1801); Heterostegina d’Orb. ('26). Subfamily 4. Cycloclypeina. CLASSIFICATION Subfamily 3. Cassédulinee. The shell consists of a series of alternating segments Subfamily 3. Cassédulinee. The shell consists of a series of alternating segments
more or less coiled. Genera: Cassidulina Orb. (26); Ehrenbergina Reuss. Family 6. Chilostomellide. The shell is calcareous, finely perforate, and polythala- Family 6. Chilostomellide. The shell is calcareous, finely perforate, and polythala-
mous. The segments follow each other from the same end of the long axis, or
alternately from the two ends, or in cycles of three, which are more or less em-
bracing. The aperture is a curved slit at the extremity of the final segment. Genera: Lillipsotdina Seguenza; Chilostomella Reuss; Allomorphina Reuss. Family 7. Lagenidea. The shell is calcareous and very finely perforated;
it is Family 7. Lagenidea. The shell is calcareous and very finely perforated;
it is
monothalamous or polythalamous. In the latter the chambers may be joined
together in a straight, curved, spiral, or branching series. The aperture is ter-
minal, and may be simple or radiate. The shell is not complicated by inter-
septal skeletons or by canal systems. Subfamily 1. Lagenzne. Shell monothalamous. Genera: Lagena Walker and- Subfamily 1. Lagenzne. Shell monothalamous. Genera: Lagena Walker and-
Boys (1784) ; odosaria Lamarck (16); Lingulina VOrb. (26); Vaginulina
dOrb. (26); Remuhkna VOrb. (26); Frondicularia Defrance; Alarginulina
Orb. (26), etc. Subfamily 2. Polymorphining. The segments composing the shell are arranged Subfamily 2. Polymorphining. The segments composing the shell are arranged
spirally or irregularly around the long axis; they are rarely biserial and alter-
nate. Genera: Polymorphina d’Orb. (126); Uvigerina VOrb. (26); Sagrina
Parker and Jones. Subfamily 3. Ramulinine. The branching shell is composed of long tubulariform Subfamily 3. Ramulinine. The branching shell is composed of long tubulariform
tubes. Genera: Ramulina Rupert Jones. Family 8. Globigerinide. The shell is free, calcareous, and perforated. The con- Family 8. Globigerinide. The shell is free, calcareous, and perforated. The con-
spicuous shell-aperture may be single or multiple. There is no supplementary
skeleton or canal system. The animals are normally pelagic in habit. Genera:
Globigerina VOrb. (26) ; Orbiculina Lam.; Hastigerina Thompson (*76) ; Caz-
deina d’Orb. (°26); Pullenia Park. & Jones (62); Spheroidina VOrb. (°26). Family 9. Rotalide. The shell is calcareous, perforated, free, or adherent; it is Family 9. Rotalide. CLASSIFICATION The shell is flat, with a thickened centre, or lens-. Subfamily 4. Cycloclypeina. The shell is flat, with a thickened centre, or lens-. shaped, and consists of a disc of chambers arranged in concentric annuli with
peripheral thickenings. The septa are double, and furnished with a system of
interseptal canals. Genera: Cycloclypeus Carpenter (756) ; Ordztoides d’Orb. Subclass 1]. HELIOZOA. These are naked or shelled forms of Sarcodina of typi- Subclass 1]. HELIOZOA. These are naked or shelled forms of Sarcodina of typi-
cally spherical form, with but little tendency to change form by amceboid mo-
tion. The pseudopodia, radiating from all parts of the body, are
fine and
ray-like, rarely changeable, and usually provided with an axial filament. Order 1. APHROTHORACIDA. Heliozoa, without a skeleton, but provided with a Order 1. APHROTHORACIDA. Heliozoa, without a skeleton, but provided with a
more or less developed power of amceboid motion, and with plastic (myxopo-
dia) or stiff (axopodia) pseudopodia, the latter possessing axial filaments. Genera:
lampyrella
Cienk. ('65);
Muclearia Cienk. ('65);
Monobia A. Schneider (78) ; AZvxastrum Haeck. (°70) ; Actinophrys Ehr. (730); Actino-
spherium Stein (°57) 3 Actinolophus F. E. Schultze (74). Order 2. CHLAMYDOPHORIDA. Heliozoa, with a soft gelatinous or felted fibrous Order 2. CHLAMYDOPHORIDA. Heliozoa, with a soft gelatinous or felted fibrous
covering. Genera:
Heterophrys Archer (69); Spherastruim Greeff ('73) ;
Astrodisculus Greeff (’69). Order 3. CHALARATHORACIDA. Heliozoa, with a silicious coating composed of Order 3. CHALARATHORACIDA. Heliozoa, with a silicious coating composed of
separate
and
loosely-jointed
spicules. Genera:
Pompholyxophrys
Archer
(69); Raphidiophrys Archer ('70); Pinacocystis Hert. & Less. (74); Pi-
naciophora Greeff ('73) ; Acanthocystis Carter ('63) ;
Dzplocystis Pénard (’90) ;
Cienkowskya Schaudinn; Wagnerella Mereschkowsky (’81). Order 4. DESMOTHORACIDA. Heliozoa, with a shell of one piece perforated by
numerous openings. Stalked or unstalked forms. Genera:
Ordulinella Entz
(77); Clathrulina Cienk. ('67). THE PROTOZOA IIo Subclass II]. RADIOLARIA. Marine forms of Sarcodina, similar to Heliozoa in
having ray-like pseudopodia (axopodia and myxopodia), but provided with a
chitinous capsule which incloses the nuclei. They may or may not have a
skeleton; when present the skeleton is formed of acanthin or of silica. The
group is subdivided into 4 legions, 20 orders, 85 families, 739 genera, and 4318
species. (Haeckel, 1885.)
Legion 1. SPUMELLAPIA (or PERIPYLEA). The central capsule is perforated Legion 1. SPUMELLAPIA (or PERIPYLEA). The central capsule is perforated
by numerous fine pores. CLASSIFICATION A skeleton may or may not be present. Order 1. COLLOIDIDA. Without skeleton. Families:
Thalassicollide (solitary Order 1. COLLOIDIDA. Without skeleton. Families:
Thalassicollide (solitary
forms) ; Collozoide (colonial). Order 2. BELOIDIDA. The skeleton consists of loose silicious needles. Families: Order 2. BELOIDIDA. The skeleton consists of loose silicious needles. Families:
Thalassospharide (single); and Spherozoide (colonial). Order 3. SPHAROIDIDA. The skeleton consists of from one to many concentric Order 3. SPHAROIDIDA. The skeleton consists of from one to many concentric
globular shells. Families: Liospharide (single); Collospheride (colonial) ;
Stylospheride (single); Staurospheride (single); Cubospheride (single) ;
Asirospheride (single). Order 4. PRUNOIDIDA. With ellipsoidal to cylindrical latticed shells and similar
central capsule. Families:
Ellipside ; Druppulide; Sponguride ; Artiscide ;
Cyphinide ; Panartide; Zygartide. Order 5. DISCOIDIDA. Shell and central
capsule are
discoidal
or lenticular. Families:
Cenodiscidea ; Phacodiscide ; Coccodiscide; Porodiscide;
Pylodis-
_
cide; Spongodiscide. Order 6. LARCOIDIDA. The skeleton is irregularly lenticular or discoid. Families: Order 6. LARCOIDIDA. The skeleton is irregularly lenticular or discoid. Families:
Larcaride ; Larnacide ; Pylonide; Tholonide; Zonaride; Lithelide; Streb-
lonide ; Phorticide ; Soreumide. Legion 2. ACANTHARIA (or ACTIPYLEA). The skeleton is formed of acanthin Legion 2. ACANTHARIA (or ACTIPYLEA). The skeleton is formed of acanthin
arranged in radiating spines, usually twenty in number. Order 7. ACTINELIDA. The spines are more than twenty in number. Families: Order 7. ACTINELIDA. The spines are more than twenty in number. Families:
Astrolophide ; Litholophid@ ; Chiastolide. Order 8. ACANTHONIDA. With twenty spines arranged according to Miiller’s Order 8. ACANTHONIDA. With twenty spines arranged according to Miiller’s
law (four equatorial, eight tropical, and eight polar). Families: Astrolonchide ;
Quadrilonchide ; Amphilonchide. Order 9. SPHAHROPHRACTIDA. With twenty equal quadrangular spines and a Order 9. SPHAHROPHRACTIDA. With twenty equal quadrangular spines and a
complete, fenestrated shell. Families: Spharocapside ; Dorataspide ; Phrac-
topeltide. Order
10. PRUNOPHRACTIDA. With ellipsoidal,
flat, or double-coned
shell,
through which twenty spines radiate according to Miiller’s law. Families:
Belonaspide ; Hexalaspide ; Diploconide. Legion 3. NASSELLARIA
(or MONOPYLEA). The skeleton
is silicious and Legion 3. NASSELLARIA
(or MONOPYLEA). The skeleton
is silicious and
rarely absent. The central capsule has a single, limited, perforated area at one
pole; the extracapsular plasm has no pigment. Order 11. NASSOIDIDA. Monopylaria without
a
skeleton. Families: Nasselide. Order 12. PLECTOIDIDA. A complete latticed shell is never formed, but the skele- Order 11. NASSOIDIDA. Monopylaria without
a
skeleton. Families: Nasselide. Order 12. PLECTOIDIDA. CLASSIFICATION A complete latticed shell is never formed, but the skele-
ton consists of three or more spines radiating from one point below the central Order 12. PLECTOIDIDA. A complete latticed shell is never formed, but the skele-
ton consists of three or more spines radiating from one point below the central
capsule, or from a central rod. Families: Plagonide; Plectanide. Order 135
STEPHOIDIDA. The skeleton consists of one or two fused rings which Order 135
STEPHOIDIDA. The skeleton consists of one or two fused rings which
may be connected by a loose network. Families: Stephanide ; Semantide ;
Coronide ; Tympanide. Order 14. SPYROIDIDA. The skeleton consists of a single sagittal ring and a Order 14. SPYROIDIDA. The skeleton consists of a single sagittal ring and a
latticed shell which is furrowed in the sagittal plane. Families: Zygospyride ;
Tholospyride ; Phormospyrid@ ; Androspyride. Order 15. BOTRYOIDIDA. The skeletons are similar to the preceding, but orna- Order 15. BOTRYOIDIDA. The skeletons are similar to the preceding, but orna-
mented by one or more wing-like processes. Families: Cannobotryide ; Litho-
botryidz ; Pylobotryide. THE SARCODINA Ill Order 16. CYRTOIDIDA. Similar to the preceding, but without the sagittal fur-
row. The skeleton is helmet-shaped. Families: Tripocalpide ; Phenocalpide ;
Cyrtocalpide;
Tripocyrtide;
Anthocyrtide;
Sethocyrtide;
Podocyrtide;
Phormocyrtide ; Theocyrtide ; Podocampide ; Phormocampide ; Lithocampide. Legion 4. PHHODARIA (or CANNOPYLEA). The central capsule has a double Legion 4. PHHODARIA (or CANNOPYLEA). The central capsule has a double
membrane, with a spout-like main opening at one pole, and frequently with
accessory openings on each side of the main axis at the opposite pole. The
central capsule may be multiple in number. There is always a pigmented mass
on the outside of the central capsule (the Pr@odium) and covering the main
opening. The skeleton, which is rarely absent, is silicious and always outside
of the central capsule. Order 17. PH OCYSTINIDA. Skeletal structures may or may not be present; Order 17. PH OCYSTINIDA. Skeletal structures may or may not be present;
the central capsule is the centre of the spherical body. Families: Pheodinide ;
Cannoraphide ; Aulacanthide. Order 18. PHAOSPHERIDA. The skeleton is a simple or a double latticed cover- Order 18. PHAOSPHERIDA. The skeleton is a simple or a double latticed cover-
ing; the central capsule is in the centre of the shell. Families: Orospheride ;
Sazospheride ; Aulospheride ; Cannospharide. Order 19. PHAHOGROMIDA. CLASSIFICATION Radiolaria provided with a simple latticed
shell, Order 19. PHAHOGROMIDA. Radiolaria provided with a simple latticed
shell,
having a mouth opening at one (the main) pole. The central capsule is in the
aboral half of the shell. Families:
Challengeridz; Medusettide ; Castanel-
lida ; Cercoporide ; Tuscaroride. Order 20. PHAOCONCHIDA. The shell consists of two latticed valves, one dorsal, Order 20. PHAOCONCHIDA. The shell consists of two latticed valves, one dorsal,
the other ventral (right and
left according to Biitschli). Families:
Con-
charide ; Calodendride ; Celographide. SPECIAL BIBLIOGRAPHY
III Brady, H. B. — Report on the Foraminifera dredged by H.M.S. Challenger: Chad-
lenger Reports, Zool., 1X., 1882-4. Brandt. K. — Koloniebildenden Radiolarien (Sphaerozceén) des Golfes von Neapel: Brandt. K. — Koloniebildenden Radiolarien (Sphaerozceén) des Golfes von Neapel:
fauna and tlora, 1885. Biitschli, 0.— Protozoa;
Sarcodina. In Bronn’s Klassen und Ordnungen
des Biitschli, 0.— Protozoa;
Sarcodina. In Bronn’s Klassen und Ordnungen
des
Thierreichs. Lezpzzg. 1883. Carpenter, W. B. — Introduction
to the Study of the Foraminifera: Ray Soczety, Carpenter, W. B. — Introduction
to the Study of the Foraminifera: Ray Soczety,
London, 1862. Haeckel, E.— The Radiolaria: Challenger Reports. Zool, XVII. and XVIII., 1888. Haeckel, E.— The Radiolaria: Challenger Reports. Zool, XVII. and XVIII., 188
Hertwig, R.— Der Organismus der Radiolarien. Denkschrift. Jenarsch. Akad., Hertwig, R.— Der Organismus der Radiolarien. Denkschrift. Jenarsch. Akad., I1.,
1879. Hertwig und Lesser. — Ueber Rhizopoden und denselben nahe stehende Organismen: Hertwig und Lesser. — Ueber Rhizopoden und denselben nahe stehende Organismen:
Arch. f. mik. Anat. Bd. X., Suppl., 1874. Leidy, Jos. — Fresh-water Rhizopods of North America. Washington, 1879. s of North America. Washington, 1879. podes d’eau douce: AZém. phys. hist. nat. Leidy, Jos. — Fresh-water Rhizopods of North America. Washington, 1879
Pénard, Eug. — Etudes sur les rhizopodes d’eau douce: AZém. phys. hist. nat. Pénard, Eug. — Etudes sur les rhizopodes d’eau douce: AZém. phys. hist. nat. Geneve,
XXXI., 1890. THE MASTIGOPHORA The Cystoflagellidia, a
group consisting of only two genera, Vocteluca and Leptodiscus, are l12 THE MASTIGOPHORA 113 distinguished by the peculiar parenchymatous structure and by the
presence of a tentacle and a collar. distinguished by the peculiar parenchymatous structure and by the
presence of a tentacle and a collar. THE MASTIGOPHORA THE Mastigophora are provided with a motile apparatus in the
form of flagella, which may vary in number from one to many. In the majority of cases, the body is of well-defined and constant
form, and covered with a cuticle, membrane, or shell. They abound
in infusions, in stagnant pools, in clear water, and in the sea, while
many of them are found as parasites in higher animals, where they
live in the cavities and cells of the body. In this class are found many diverse types of unicellular organisms, In this class are found many diverse types of unicellular organisms,
including, at one extreme, primitive forms whose allies are undoubt-
edly among the bacteria and the lowest plants (monads), at the other
extreme, colonial forms, which in the complexity of their structure and
functions are little lower than some of the Metazoa and Metaphyta. It includes forms whose bodies are naked; others that are clothed
with complex membranes, or incased in chitinous, silicious, or cellu-
lose shells. It includes organisms with very different methods of
food-taking : in some forms the food, like that of the green plants,
consists of products made from simple compounds by the organism
itself ; in others, the food, like that of the fungi, consists of dissolved
organic matters; and in still others, the food, as in the higher animals,
consists of solid particles of proteid and other matters. Notwithstanding these many structural and functional differences, Notwithstanding these many structural and functional differences,
there are some
well-defined structural characteristics according to
which the Mastigophora may be subdivided into a number of more
or less homogeneous groups. These groups are the Flagellidia,
Dinoflagellidia, and Cystoflagellidia. The first comprises the least
homogeneous forms; they consist usually of minute cells with a
simple naked body, which may become more or less amceboid, and
with one, two, or several flagella. In some cases, there is a compli-
cated cell-membrane, in others a shell, while colony-formation is fre-
quently seen. The Dinoflagellidia are distinguished by the presence
of one or two furrows, in whieh the flagella find their origin, one to
pass around the organism transversely, the other to vibrate freely in
the surrounding water. The majority are covered by a cellulose
shell, consisting frequently of several plates. A. PROTOPLASMIC
STRUCTURE The relations of the firm case to the gelatinous mantle are shown in
forms like Codonaca, where the chitin-like urn-shaped cup may become
gelatinous (Fig. 60). The organisms are attached to the bottoms of
such goblet-shaped cups by a protoplasmic process, and in no case
does the cup fit the organism as tightly as
a membrane. Colony-
forms also are frequent in these types, arising, in
the simplest cases, by a young individual attaching
itself to the edge of the parent test and there secret-
ing its own covering (Dzzobryon, Fig. 61). The
majority of these colonies are attached, but Dzvod-
ryon is a free-swimming form, usually found in the
clearest waters. Shells are distinguished from tests or houses by
the fact that they completely inclose the animal, the
so-called mouth-opening where the flagellum is in-
=
serted being the only aperture. Both tests and part of the organism, and even diverse modifications of protoplasm,
such as the fine peripheral layer of alveoli (Ped/zcula of Biitschli), and
the complex membranes of Auglena and Astasza (cf. Fig. 10, B). The outer coatings as in all Protozoa, serving probably for the pur-
pose of protection, include houses and tests of all kinds which are
not a living part of the animal. In many cases they are simply jelly-
like coverings, which in many colony-forms also serve to keep the
individuals together (Uvoglena, many Choanoflagellida, Fig. 59; see
also Fig. 25, p. 56). In other cases, the gelatinous mantle becomes
a tube, into which the organism can completely withdraw (some
Choanoflagellida). In
still other
cases, the jelly is apparently
hardened into a well-defined goblet or beaker-shaped cup with the
consistency of chitin (Codoneca, Epipyxis, Dinobryon, Salpingeca, etc.). The relations of the firm case to the gelatinous mantle are shown in
forms like Codonaca, where the chitin-like urn-shaped cup may become
gelatinous (Fig. 60). The organisms are attached to the bottoms of
such goblet-shaped cups by a protoplasmic process, and in no case
does the cup fit the organism as tightly as
a membrane. Colony-
forms also are frequent in these types, arising, in
the simplest cases, by a young individual attaching
itself to the edge of the parent test and there secret-
ing its own covering (Dzzobryon, Fig. 61). The
majority of these colonies are attached, but Dzvod-
ryon is a free-swimming form, usually found in the
clearest waters. A. PROTOPLASMIC
STRUCTURE The alveoli, forming the structural basis of the protoplasm, vary in
size from minute and scarcely visible spaces to large vacuoles. In
the majority of forms, they are arranged in a typical outer layer (Rzn-
denschicht) of small-sized alveoli, surrounding an inner mass of larger
ones (¢.g. Chilomonas). The protoplasm is not equally dense in all Fig. 59. — Proterospongia HeckeliS. K. [S. KENT.] Fig. 59. — Proterospongia HeckeliS. K. [S. KENT.] cases, but, as in the Rhizopoda, may be of variable consistency. It
may be so soft and flexible that, as in Amada, the periphery will give
way, and pseudopodia may be formed at any point in response to
local changes in the surface tension (Euglenoids and forms of Asta-
sta). There is but little tendency to the differentiation into zones,
so frequently seen in Rhizopoda, and only rarely is there a differen-
tiation into ectoplasm and endoplasm (Mastigamwba). Klebs (’92) distinguishes two types of peripheral structures, the Klebs (’92) distinguishes two types of peripheral structures, the
periplasts and outer coats, stalks being included with the latter. The periplasts include all cuticular differentiations which are a living THE PROTOZOA 114 part of the organism, and even diverse modifications of protoplasm,
such as the fine peripheral layer of alveoli (Ped/zcula of Biitschli), and
the complex membranes of Auglena and Astasza (cf. Fig. 10, B). The outer coatings as in all Protozoa, serving probably for the pur-
pose of protection, include houses and tests of all kinds which are
not a living part of the animal. In many cases they are simply jelly-
like coverings, which in many colony-forms also serve to keep the
individuals together (Uvoglena, many Choanoflagellida, Fig. 59; see
also Fig. 25, p. 56). In other cases, the gelatinous mantle becomes
a tube, into which the organism can completely withdraw (some
Choanoflagellida). In
still other
cases, the jelly is apparently
hardened into a well-defined goblet or beaker-shaped cup with the
consistency of chitin (Codoneca, Epipyxis, Dinobryon, Salpingeca, etc.). The relations of the firm case to the gelatinous mantle are shown in
forms like Codonaca, where the chitin-like urn-shaped cup may become
gelatinous (Fig. 60). The organisms are attached to the bottoms of
such goblet-shaped cups by a protoplasmic process, and in no case
does the cup fit the organism as tightly as
a membrane. A. PROTOPLASMIC
STRUCTURE Colony-
forms also are frequent in these types, arising, in
the simplest cases, by a young individual attaching
itself to the edge of the parent test and there secret-
ing its own covering (Dzzobryon, Fig. 61). The
majority of these colonies are attached, but Dzvod-
ryon is a free-swimming form, usually found in the
clearest waters. Shells are distinguished from tests or houses by
the fact that they completely inclose the animal, the
so-called mouth-opening where the flagellum is in-
=
serted being the only aperture. Both tests and
sao”
shells are usually transparent and colorless, although
Fig. 60.— Codoneca
;
“sunita §, Clavk, they may be colored by the presence
of iron, as
[JAMES CLaRK.]
in Trachelomonas, Rhipidodendron, etc., where the
shells, when present in any quantity, give a distinctly
red color to the water. The simplest shells are the cellulose cover-
ings of many Phytoflagellida, which, although lifeless, have the same
general appearance as membranes. The shell, which is frequently
protected by sharp spines (7vachclomonas), may be separated from
the plasm by a considerable space. It is bivalved in Phacotus, the
two parts being easily separated (Fig. 62). In one form only, Jzs-
tcphanus speculum Stohr, there is a silicious skeleton which recalls the
latticed skeletons of Radiolaria (Fig. 63). The most highly differentiated of these outer coatings are found
in the Dinoflagellidia, where the cellulose shells are often composed
of separate plates fitted together with the greatest nicety and often part of the organism, and even diverse modifications of protoplasm,
such as the fine peripheral layer of alveoli (Ped/zcula of Biitschli), and
the complex membranes of Auglena and Astasza (cf. Fig. 10, B). The outer coatings as in all Protozoa, serving probably for the pur-
pose of protection, include houses and tests of all kinds which are
not a living part of the animal. In many cases they are simply jelly-
like coverings, which in many colony-forms also serve to keep the
individuals together (Uvoglena, many Choanoflagellida, Fig. 59; see
also Fig. 25, p. 56). In other cases, the gelatinous mantle becomes
a tube, into which the organism can completely withdraw (some
Choanoflagellida). In
still other
cases, the jelly is apparently
hardened into a well-defined goblet or beaker-shaped cup with the
consistency of chitin (Codoneca, Epipyxis, Dinobryon, Salpingeca, etc.). A. PROTOPLASMIC
STRUCTURE Schiitt (’90), however,
who has made the most complete study of the coloring matter in these The protoplasm of the Mastigophora usually contains chromato-
phores in which one or more deeply staining bodies —the pyrenoids
—may be found, and these
are frequently covered by a
shell of amylum or starch. Paramylum, a food product
allied to starch, and various
particles of oil-like substance
are widely distributed. The
latter are frequently so nu-
merous that the cell is fairly
filled with them. Upon dif-
fluence, these oil-like bodies
run
together, forming glob-
ules of large size;
or they
become finely divided, giving
to the surrounding liquid the
appearance of an emulsion. Not infrequently the oils have
|
a characteristic odor and taste,
A
B
comparable to the scent of oils Fig. 63.—Distephanus speculum Stohr. [BORGERT.]
of
plants
(Uroglena
amert-
A. Lateral view of skeleton,
&. Surface view. cana, Synura uvella, Fig. 65). Chromatophores are widely distributed among the various Flagel-
lidia. They consist of clearly defined, thickened bodies, usually of The protoplasm of the Mas
phores in which one or more
—may be found, and these
are frequently covered by a
shell of amylum or starch. Paramylum, a food product
allied to starch, and various
particles of oil-like substance
are widely distributed. The
latter are frequently so nu-
merous that the cell is fairly
filled with them. Upon dif-
fluence, these oil-like bodies
run
together, forming glob-
ules of large size;
or they
become finely divided, giving
to the surrounding liquid the
appearance of an emulsion. Not infrequently the oils have
|
a characteristic odor and taste,
comparable to the scent of oils
of
plants
(Uroglena
amert-
cana, Synura uvella, Fig. 65). Chromatophores are widely |
A
B
Fig. 63.—Distephanus speculum Stohr. [BORGERT.]
A. Lateral view of skeleton,
&. Surface view. Fig. 63.—Distephanus speculum Stohr. [BORGERT.]
A. Lateral view of skeleton,
&. Surface view. cana, Synura uvella, Fig. 65). Chromatophores are widely distributed among the various Flagel-
lidia. They consist of clearly defined, thickened bodies, usually of
definite size and shape and of different shades of green, yellow, and
brown. Clear green chromatophores, colored by chlorophy]l as in the
plants,
occur
in
Euglenide,
Peranemide,
Chlamydomonadide,
and Volvocina. A. PROTOPLASMIC
STRUCTURE Shells are distinguished from tests or houses by sao”
Fig. 60.— Codoneca
“sunita §, Clavk,
[JAMES CLaRK.] The most highly differentiated of these outer coatings are found
in the Dinoflagellidia, where the cellulose shells are often composed
of separate plates fitted together with the greatest nicety and often Fig. 61.— Dinobryon sertularia Ebr. [STEIN.]
c, chromatophore; ¢, eye-spot or stigma. Fig. 61.— Dinobryon sertularia Ebr. [STEIN.]
c, chromatophore; ¢, eye-spot or stigma. Fig. 61.— Dinobryon sertularia Ebr. [STEIN.]
c, chromatophore; ¢, eye-spot or stigma. THE PROTOZOA 116 complicated by the presence of spines, wing-like processes, and other
appendages, or they may be pitted by minute depressions or pores. After the death of the animal the plates can, as a rule, be separated
by gentle pressure. The substance of the shell is not true plant cel-
lulose, but a modification, the exact nature of which has not been
definitely determined. The furrows in the shells of the Dinoflagellidia, in which the two The furrows in the shells of the Dinoflagellidia, in which the two
flagella lie, are perhaps the most characteristic feature of these forms. Fig. 62. — Phacotus lenticularis Ehr. [BUTSCHLI.]
A, Individual within its bivalved shell. 4. Spore-forming individual. Fig. 62. — Phacotus lenticularis Ehr. [BUTSCHLI.]
A, Individual within its bivalved shell. 4. Spore-forming individual. One runs across the organism, while the other, which may often, how-
ever, be obliterated, is at right angles to this, usually in the direction
of the longitudinal axis. There may be one, two, or many transverse
furrows, the number determining the family to which the organism
belongs. In the genus Hemidinium, the single transverse furrow
begins on the ventral side and runs as far as the middle of the dorsal
side, where it disappears. In the genera Gymnodinium, Glenodinium,
and Peridintum, it runs completely around the organism; while in
Ceratium it may be broken in its course (Fig. 64). The longitudinal
furrow, on the other hand, is invariably confined to the ventral side,
usually to the lower half, but in some cases, (Glenodinium, Peridinium)
it traverses the cross furrow and stretches some distance along the THE MASTIGOPHORA 117 upper half of the shell. A. PROTOPLASMIC
STRUCTURE The two flagella which lie in these grooves
pass from the body-plasm to the outside through a distinct aperture
in the shell, which Stein (’78) called the “ mouth-opening”’; but as
it serves no purpose in food-taking, Biitschli has substituted the better
term of flagellum fissure. The protoplasm of the Mastigophora usually contains chromato- upper half of the shell. The two flagella which lie in these grooves
pass from the body-plasm to the outside through a distinct aperture
in the shell, which Stein (’78) called the “ mouth-opening”’; but as
it serves no purpose in food-taking, Biitschli has substituted the better
term of flagellum fissure. The protoplasm of the Mastigophora usually contains chromato- term of flagellum fissure. The protoplasm of the Mastigophora usually contains chromato-
phores in which one or more deeply staining bodies —the pyrenoids
—may be found, and these
are frequently covered by a
shell of amylum or starch. Paramylum, a food product
allied to starch, and various
particles of oil-like substance
are widely distributed. The
latter are frequently so nu-
merous that the cell is fairly
filled with them. Upon dif-
fluence, these oil-like bodies
run
together, forming glob-
ules of large size;
or they
become finely divided, giving
to the surrounding liquid the
appearance of an emulsion. Not infrequently the oils have
|
a characteristic odor and taste,
A
B
comparable to the scent of oils Fig. 63.—Distephanus speculum Stohr. [BORGERT.]
of
plants
(Uroglena
amert-
A. Lateral view of skeleton,
&. Surface view. cana, Synura uvella, Fig. 65). Chromatophores are widely distributed among the various Flagel-
lidia. They consist of clearly defined, thickened bodies, usually of
definite size and shape and of different shades of green, yellow, and
brown. Clear green chromatophores, colored by chlorophy]l as in the
plants,
occur
in
Euglenide,
Peranemide,
Chlamydomonadide,
and Volvocina. Yellow
chromatophores
(colored
by datomin,
as
in Diatomacez) occur
in Chrysomonadide, Cryptomonadida,
among the Flagellidia, and possibly in some Dinoflagellidia;
but
the yellow color, when
present
in the latter group, frequently
shades off into brown. Bergh (’81), Klebs (’84), and others regarded
the coloring matter of the Dinoflagellidia as pure or slightly mixed
diatomin, which supported the popular view that the Diatomaceze
and the Dinoflagellidia are closely related. 1Cf. p. 62. A. PROTOPLASMIC
STRUCTURE Yellow
chromatophores
(colored
by datomin,
as
in Diatomacez) occur
in Chrysomonadide, Cryptomonadida,
among the Flagellidia, and possibly in some Dinoflagellidia;
but
the yellow color, when
present
in the latter group, frequently
shades off into brown. Bergh (’81), Klebs (’84), and others regarded
the coloring matter of the Dinoflagellidia as pure or slightly mixed
diatomin, which supported the popular view that the Diatomaceze
and the Dinoflagellidia are closely related. Schiitt (’90), however,
who has made the most complete study of the coloring matter in these 1Cf. p. 62. THE PROTOZOA 118 forms, disproved this view by showing that the coloring matter is quite
distinct from diatomin and is peculiar to the Dinoflagellidia. He suc-
ceeded in extracting three substances: (1) piycopyrrin, similar to the
brownish red coloring matter of the Floridez and Phzophycacez
among the plants, and like this, soluble in clear water; (2) peridinin,
like chlorophyl soluble in alcohol, but of quite different spectrum ;
and (3) chlorophyllin, a substance more like chlorophyl, but difficult
to isolate. The shape and size of the chromatophores vary considerably in The shape and size of the chromatophores vary considerably in
different species, but are fairly constant for the same species. They
increase by simple division. The pyrenoids, which seem to be the
centre of starch formation, are sometimes
quite naked (Lug/ena), A
B
Fig. 64.—A. Gymnodinium ovum Schiitt. B. Peridinium divergens Eur,
f, transverse furrow
with (4) flagellum. (SCHUTT.] 4.—A. Gymnodinium ovum Schiitt. B. Peridinium divergens Eur,
f, transverse furrow
with (4) flagellum. (SCHUTT.] sometimes covered by a shell of paramylum, which apparently differs
from starch only in its reaction to iodine. The paramylum granules
are round, rod-like, or ring-form bodies. Pure starch is also recorded
as a product of non-colored, saprophytic forms (Chz/omonas, Poly-
toma, etc.). In many of the Mastigophora, especially in those holding
chromatophores, there may be an intense red coloring matter, in the
form of fine drops, scattered throughout the protoplasm. These consist
of oil particles impregnated with a deep red pigment,
— hematochrome,
—and the same substance is found in the so-called “ eye-spots,” or
stigmata, which are supposed to be more sensitive to light than other
parts of the protoplasm, although Engelmann’s (’82) results show that,
in Exglena at least, the clear plasm just in front of the stigma is more
definitely involved. A. PROTOPLASMIC
STRUCTURE Thelenses
develop by the union
of from six to eight
refringent
corpus-
cles, while the organ-
ism is still encysted
or while undergoing
fission. The choroid
likewise results from
the union of several
of the pigment gran-
ules. © Considerable
doubt has, however,
been
thrown
upon
these
observations
by subsequent
ob-
servers. Other inclusionsof Fig. 65. — Syzura uvella Ehr. Each individual of the colony is surrounded by a gelatinous
membrane, and possessés two chromatophores (¢c) and a nucleus
“: Fig. 65. — Syzura uvella Ehr. al of the colony is surrounded Each individual of the colony is surrounded by a gelatinous
membrane, and possessés two chromatophores (¢c) and a nucleus
“: Other inclusionsof “:
interest
are
the thread-like
structures which are common among
holotrichous ciliates, and which occur sporadically in other Protozoa. Among the Mastigophora they are found in only two cases (Gony-
ostomum Blochmann and Polykrikos Biitschli). In the former they
are trichocysts similar to those of the ciliate Pavamacium and allied
forms, but in the latter they are true nematocysts, comparable to those
of the Ccelenterata. 1Cf, Pénard (’88). A. PROTOPLASMIC
STRUCTURE In many cases the structures accompanying the
stigmata are so strikingly analogous to the visual organs of higher sometimes covered by a shell of paramylum, which apparently differs
from starch only in its reaction to iodine. The paramylum granules
are round, rod-like, or ring-form bodies. Pure starch is also recorded
as a product of non-colored, saprophytic forms (Chz/omonas, Poly-
toma, etc.). In many of the Mastigophora, especially in those holding
chromatophores, there may be an intense red coloring matter, in the
form of fine drops, scattered throughout the protoplasm. These consist
of oil particles impregnated with a deep red pigment,
— hematochrome,
—and the same substance is found in the so-called “ eye-spots,” or
stigmata, which are supposed to be more sensitive to light than other
parts of the protoplasm, although Engelmann’s (’82) results show that,
in Exglena at least, the clear plasm just in front of the stigma is more
definitely involved. In many cases the structures accompanying the
stigmata are so strikingly analogous to the visual organs of higher THE MASTIGOPHORA 119 forms that there is apparently good reason for supposing them to
play a similar physiological réle. In the green flagellates there are
often distinct concretions, regarded by some observers as lenses; and
if Pouchet (’86) is correct, a still more striking differentiation is found
among the Dinoflagellidia. The so-called eye of Gymnodinium con-
sists of a transparent, highly refracting
lens, rounded at its free
extremity, and always directed forward (Pouchet). The inner sur-
face is embedded in
a hemispherical, cap-
like mass of red or
black pigment, which
Pouchet
considered
achoroid. Thelenses
develop by the union
of from six to eight
refringent
corpus-
cles, while the organ-
ism is still encysted
or while undergoing
fission. The choroid
likewise results from
the union of several
of the pigment gran-
ules. © Considerable
doubt has, however,
been
thrown
upon
these
observations
by subsequent
ob-
Fig. 65. — Syzura uvella Ehr. Each individual of the colony is surrounded by a gelatinous
servers. membrane, and possessés two chromatophores (¢c) and a nucleus
Other inclusionsof “: forms that there is ap
play a similar physiolog
often distinct concretion
if Pouchet (’86) is corre
among the Dinoflagellid
sists of a transparent,
extremity, and always
face is embedded in
a hemispherical, cap-
like mass of red or
black pigment, which
Pouchet
considered
achoroid. B.
THE FLAGELLA The most characteristic part of a flagellate is its motile organ, the
flagellum. This consists of a vibratile filament usually tapering to a
fine point, although in some cases (e.g. in all Choanoflagellida) it is THE PROTOZOA 120 of one thickness throughout. In either case the base is inserted in
the vicinity of a pharyngeal depression and usually at the end of the
body. There is good reason to believe with Klebs, Frenzel, Bloch-
mann, and others, that like the axial filaments of the pseudopodia in
Heliozoa and Radiolaria, the flagellum originates at or near the
nuclear membrane, and does not consist exclusively of the outer or
peripheral plasm. Dallinger (’78) asserts
that the newly forming
flagella are smooth and uniform, arising in or near the nucleus. Fischer (’94), however, claims that many of them are provided with
branches like the cilia of a typhoid germ. He finds others which are
not vibratile throughout their entire length, but are rigid and uniform
for a certain distance and then taper to the extremity. Such flagella
resemble a whip stock and its lash, the relative proportion of stock
and lash varying in different flagella, the stock sometimes running
nearly to the end, and again only a short distance from the body. Other forms, especially in the Dinoflagellidia, have spirally rolled
flagella of various kinds, while some have flattened or band forms
(Peridinium tabulatum and P. divergens). A difference of opinion exists as to the ability of the organism to A difference of opinion exists as to the ability of the organism to
absorb
or retract
its flagellum into the body-protoplasm. Most
observers agree with the early observation of Dujardin that there isa
close relation between pseudopodia and flagella, numerous observa-
tions having been recorded of cases where, under certain conditions,
the pseudopodia change into swinging flagella, and flagella into pseu-
dopodia. There is no doubt that flagella can be absorbed after
changing to pseudopodia. Whether the fully formed flagella can be
changed over into plastic material and then withdrawn, is still a sub-
ject of dispute; Fischer (’94) holds that they are invariably discarded
upon irritation, and Schiitt (95) shows that the longitudinal flagellum
in the Dinoflagellidia is thrown off upon irritation, while the horizontal
flagellum is flattened into a band form. A general rule, therefore,
cannot be formulated in regard to the disposal of flagella. B.
THE FLAGELLA In some
cases they are absorbed; in others, thrown off. The action of the flagella varies with the type of structure. In the The action of the flagella varies with the type of structure. In the
simple, straight, or tapering forms the tip moves in a circle while
waves pass from the base to the extremity. In the whip-like flagella
the basal portion moves back and forth or in a circle, while the distal:
region vibrates or. undulates like the snapper of a whip. The band-
formed flagella move by simple undulations. The position of the flagella is extremely variable. When therfe is The position of the flagella is extremely variable. When therfe is
but one, it is found at the anterior end of the cell, —that is, the end
which is directed forward when in motion. When there are two fla-
gella, they may both be directed forward (Chzlomonas, Cryptomonas,
etc.), and may be of equal (Cryptomonas, etc.) or unequal length THE MASTIGOPRORA 121 (Uroglena, etc.). Again, they may be of equal length but turned in
opposite directions (Lodo, etc.). When there are numerous flagella,
they may be distributed about the body, regularly or irregularly or
aggregated at certain points (M/u/ticilia, Tetramitus, etc.). In the Dinoflagellidia, the longitudinal flagellum, a long, fine thread, In the Dinoflagellidia, the longitudinal flagellum, a long, fine thread,
is invariably directed backward or forward, while the other, the trans- °
verse, lies around the body in an equatorial groove. This flagellum
has a simple undulating motion resembling a row of moving cilia, for
which it was at first mistaken.1_
So strictly does the transverse flagel-
lum adhere to the usual direction of motion, that even when the
groove is absent, as in Prorocentrum, where the flagellum no longer
surrounds the body, the motion is retained,
the flagellum being directed outward from
the end of the body for a short distance
and then turned at right angles to forma
circle, with
the customary
undulatory
motion, as though still encircling the body
(Fig. 66). With the exception of the Choano- @
b
Fig. 66.— Primitive forms of Dino-
Re
senate ean
5
Zxuvielia lima Ehr. — With the exception of the Choano-
flagellida, which swim like a spermatozoon
with the flagellum behind (James-Clark,
66), the Mastigophora swim with the
flagellum in advance. 1 Hence the name of the group, — Ci/doflagellata,
— which was in use until a compara-
tively recent date. 1Cf. Delage (’96), p. 305, for a very full discussion. B.
THE FLAGELLA Again, it may be fully or eve
twice as long as the body. In shape it is either like a bowl or el
like a truncated cone (Fig. 68, 4, C). There
may be two of the collars, one within the other
(Liplosiga
Frenzel, &). Biitschli described
a vacuole which appeared to him to move
rapidly around the base of the collar and to
disappear for a short time when a food particle n into the protoplasm of the cell. It
onsiderable that, as in Phalaustertum,
67). Again, it may be fully or even
shape it is either like a bowl or else
4, C). There
thin the other
chli described
him to move
collar and to
a food particle either wholly or partly withdrawn into the protoplasm of the cell. It
is occasionally so small and inconsiderable that, as in Phalaustertum,
it can have little or no use (Fig. 67). Again, it may be fully or even
twice as long as the body. In shape it is either like a bowl or else
like a truncated cone (Fig. 68, 4, C). There
may be two of the collars, one within the other
(Liplosiga
Frenzel, &). Biitschli described
a vacuole which appeared to him to move
rapidly around the base of the collar and to
disappear for a short time when a food particle ape it is either like a bowl or else
C). There
n the other
described
m to move
llar and to
ood particle
Fig. 69.—A
Choanoflagellate
)
B. Dip-
type. [FRANCE.]
eca marinus
¢, collar;
mz, swinging
mem-
brane. Fig. 68. — Types of collars. Fig. 69.—A
Choanoflagellate
A. Codosiga pulcherrimus Jas.Cl. [J CLARK.)
B. Dip-
type. [FRANCE.]
Zosiga socialis Frenz,
[FRENZEL.]
C. Salpingeca marinus
¢, collar;
mz, swinging
mem-
Jas. Cl. [J. CLARK]
brane. is taken in. Entz (’83) and Francé (’94) claim, however, that this
“mouth-opening”’ is not a vacuole at all, but the edge of a swinging
membrane. According to their view the collar is not a continuous
structure with an unbroken wall, but is like a conical roll of paper
with a free edge capable of motion (Fig. 69). Fig. 69.—A
Choanoflagellate
type. [FRANCE.]
¢, collar;
mz, swinging
mem-
brane. Fig. 69.—A
Choanoflagellate
type. [FRANCE.]
¢, collar;
mz, swinging
mem-
brane. Fig. 68. — Types of collars. A. Codosiga pulcherrimus Jas.Cl. [J CLARK.)
B. Dip-
Zosiga socialis Frenz,
[FRENZEL.]
C. B.
THE FLAGELLA The forward move-
ment of most flagellated organisms
is,
therefore, exceedingly difficult to inter-
pret. It is very curious to see the com-
@
b
paratively large body of Peranema, for Fig. 66.— Primitive forms of Dino-
example, drawn steadily forward by the
Re
senate ean
5
minute tip of its rather long flagellum. Zxuvielia lima Ehr. —
No
satisfactory
mathematical
demon-
stration of the application of the force necessary to produce this
motion has been given. Lankester (’91) compared it to the force
produced by a man’s arm and hand when swimming upon
his
side;
Biitschli ('83) offered a simple and apparently reasonable
explanation, showing that the resistance, which is directed at right
angles to the advancing undulation, can be reduced, through the
parallelogram of forces, to a force of rotation and one of translation,
but Delage (’96) holds that while this explanation is perfectly con-
sistent with the mechanism of certain mechanical contrivances, it is
incompatible with the structure of the flagellate body, and that the
explanation
is much more
complicated. Delage’s interpretation
involves the principles of conic sections, the resisting force being Fig. 66.— Primitive forms of Dino-
Re
senate ean
5
Zxuvielia lima Ehr. — Fig. 66.— Primitive forms of Dino-
Re
senate ean
5
Zxuvielia lima Ehr. — 1 Hence the name of the group, — Ci/doflagellata,
— which was in use until a compara-
tively recent date. THE PROTOZOA 122 applied in a very indirect manner, and he calls the resulting move-
ment “conical translation.” ! applied in a very indirect manner, and he calls the resulting move-
ment “conical translation.” ! ment “conical translation.” ! Fig. 67. — Phalansterium digitatum St. [S. KENT.]
J, Collared cells. A peculiar pseudopodial process, the co//ar, is found in one order Fig. 67. — Phalansterium digitatum St. [S. KENT.]
J, Collared cells. A peculiar pseudopodial process, the co//ar, is found in one order
of the Flagellidia, the Choanoflagellida. This collar, which forms a
cup around the base of the flagellum, is extremely thin, delicate, and
transparent, and like a pseudopodium can be altered in shape and 1Cf. Delage (’96), p. 305, for a very full discussion. TIGOPHORA
123 IGOPHORA
12 THE MASTIGOPHORA 123 either wholly or partly withdrawn into the protoplasm of the cell. is occasionally so small and inconsiderable that, as in Phalaustertum
it can have little or no use (Fig. 67). THE PROTOZOA 124 C. Tue Nucieus B.
THE FLAGELLA Salpingeca marinus
Jas. Cl. [J. CLARK] Fig. 68. — Types of collars. iga pulcherrimus Jas.Cl. [J C is taken in. Entz (’83) and Francé (’94) claim, however, that this
“mouth-opening”’ is not a vacuole at all, but the edge of a swinging
membrane. According to their view the collar is not a continuous
structure with an unbroken wall, but is like a conical roll of paper
with a free edge capable of motion (Fig. 69). is taken in. Entz (’83) and Francé (’94) claim, however, that this
“mouth-opening”’ is not a vacuole at all, but the edge of a swinging
membrane. According to their view the collar is not a continuous
structure with an unbroken wall, but is like a conical roll of paper
with a free edge capable of motion (Fig. 69). THE PROTOZOA C.
Tue Nucieus Inclosed in the protoplasm in all Mastigophora is a more or less
clearly defined nucleus. It is variable in position, and although a
multiple number may occur, is generally single. The structure also Fig. '70. — Nuclear division in Noctiluca miliaris,
A. The first changes of the chromatin from the large karyosome condition; concentration of the substance of the d
sion centre (s). &. Further disintegration of the chromatin and arrangement of granules to form the chromosomes (
c. Amphiaster and completed chromosomes. JD. ‘The central spindle in the hollow of the nucleus; the nuclear plate
chromosomes is thus wrapped around the division-centre. . A section through the centre of the long axis of the divisi
centre before division of the chromosomes. /. A section through the dividing chromosomes,
c, the centrosome w
radiating mantle-fibres, Fig. '70. — Nuclear division in Noctiluca miliaris,
omatin from the large karyosome condition; concentr Fig. '70. — Nuclear division in Noctiluca miliaris,
A. The first changes of the chromatin from the large karyosome condition; concentration of the substance of the divi-
sion centre (s). &. Further disintegration of the chromatin and arrangement of granules to form the chromosomes (cA). c. Amphiaster and completed chromosomes. JD. ‘The central spindle in the hollow of the nucleus; the nuclear plate of
chromosomes is thus wrapped around the division-centre. . A section through the centre of the long axis of the division-
centre before division of the chromosomes. /. A section through the dividing chromosomes,
c, the centrosome with
radiating mantle-fibres, is extremely variable. The chromatin may be either in the form of
granules distributed throughout the cell and not confined by a distinct
nuclear membrane ( Zetramitus), or the granules may be aggregated
without a membrane (CAz/omonas), or again, the chromatin may be THE MASTIGOPHORA 125 inclosed by a definite membrane (euglenoids and the majority of
Mastigophora; cf. Figs. 14, C, D, £,and 10, 8). Again, it may be in
the form of a homogeneous mass in which no granular structure can
be seen (many Phytoflagellida), or, as in many Rhizopoda, it may be
massed in several such aggregates (WVoctzluca). Still another arrange-
ment is seen in the Dinoflagellidia, where the chromatin is arranged
in the form of a twisted thread or threads. 1See zzfra, p. 258. C.
Tue Nucieus Finally, in some forms
the resting nucleus closely resembles that of the Metazoa in having a
linin network in which the chromatin granules are suspended. An integral part of the nucleus is the so-called “ nucleolus,” which, An integral part of the nucleus is the so-called “ nucleolus,” which,
however, is not analogous to the nucleolus
of the Metazoa, but
functions as a sphere or the division centre during mitosis.1 Nuclear division in all forms of Mastigophora may be regarded as
more or less simplified mitosis, or indirect division. In the simplest
types the chromatin masses merely draw out and divide into equal
parts, but in the more complicated types, the process closely resembles
that in the Metazoa, the complete mitotic figure consisting, as in the
higher forms, of chromosomes, mantle-fibres, centrosomes, and spheres
(Fig. 70). D.
Foop-TAKING Closely dependent upon the mode of living is the manner of taking
food. Some forms, which live in foul water, are saprophytic like the
colorless plants, and absorb, through the body walls, the substances
which are dissolved out of decaying vegetable matter. Those which
live in pure and clear water generally have chromatophores, colored
by chlorophyl, diatomin, or some allied substances, and have the
power of manufacturing their food from carbon dioxide, water, and
salts;
like the green plants, their nutrition is holophytic. Parasitic
forms
live upon the juices of other living organisms, which
are
absorbed through any part of the body wall (Fig. 71). Finally, some
take in solid food, which is acted upon and digested by the fluids
of their inner plasm, the indigestible portions being excreted as in
the higher animals. In the holophytic forms there is frequently an unbroken shell about In the holophytic forms there is frequently an unbroken shell about
the animal which makes it impossible for solid food to enter (He@mato-
coccus; many Dinoflagellidia). Many of the holozoic forms have a
distinct mouth and cesophagus. In its simplest form the mouth is
merely a softened area about the base of the flagellum, against which
the solid food particles strike (Ozkomonas, see Fig. 18, 6). Others
have a distinct mouth-opening leading into a gullet, which in turn opens
into the fluid endoplasm (Peranema; Petalomonas, see Fig. 1, 6). 126 THE PROTOZOA Where the flagella are in separate groups, there is a mouth at the
base of each group. The collar of the Choanoflagellida is especially
adapted for the collection and direction of the food particles into the
interior. Perty ('52), Kent (’81), Stein (67), Entz (’88), and others have ein (67), Entz (’88), and others have
which they claim have both kinds of
between the holozoic and holophytic
h he admits that food-taking may be
in one form at least (Chromulina flavt-
ell when one or the other mode of
nutrition is prevented,
is inclined to
doubt
the wide distribution
of this
double function. Meyer (’97) main-
tains that in one form (Ochromonas
granulosa) the organism may be either
holozoic, saprophytic, or holophytic in
nutrition. In many of the holophytic
forms, there is a distinct gullet, which
Perty and Kent regarded as a food-
taking organ and, therefore, evidence
of holozoic nutrition. E. VacuoLes Some of the vacuoles which make up the protoplasm of the Masti-
gophora are gastric, while others are contractile. The former are
formed about the food particles, which are probably digested in the
same way as in the Sarcodina, although in this group no experiments
have been made to test the digestive fluids. The contractile vacuoles, acting possibly as respiratory and excre- The contractile vacuoles, acting possibly as respiratory and excre-
tory organs, pulsate rhythmically and at definite rates, varying from
one or two pulsations per hour to five or six a minute, according
to the temperature and nature of the surrounding medium. They are
typically small, single or double in number (multiple in Chloregonium),
and are situated at either end of the body, or near the centre, while in
some cases they move with the granules in cyclosis. In some of the
more complicated types of Euglenide, the vacuole is connected with
the so-called gullet by a minute canal. This canal in some cases
receives its supply of waste matter from a reservoir, which is the
receptacle for the contents of numerous small vacuoles surrounding
it, and which pulsate at regular intervals. D.
Foop-TAKING In Noctiluca, the flagellum brings a current of food toward the collar,
while the tentacle, which constantly heats down into the bottom of
the collar area, drives it into the mouth situated at the bottom of the
pharyngeal groove. The particles are then received into a gastric
vacuole, which, in the vicinity of the relatively large nucleus, per-
forms its function of digestion. D.
Foop-TAKING Biitschli, how-
ever, maintained that it is a part of the
excretory system and connected with
the contractile vacuole (Luglena, Cryp-
tomonas, etc.). A close connection between holozoic Fig. 71. Megastoma entericum
Grassi. Ventral and side views. [GRAssI.] A close connection between holozoic
and holophytic forms is found, not only
in Flagellidia, but in Dinoflagellidia as
well. Possessing chromatophores and a coating of modified cellulose,
these organisms were for a long time regarded as plants, but some
forms among them are known to move about like animals and to ingulf
solid food. Such forms may be either naked, as in Gymnodintui ( Fig. 64, A) and Polykrikos, where food-taking has been actually seen by
a number of observers (Schmarda, Stein, Bergh, Schilling, Dangeard),
or shell-bearing, as in Glenodinium edax (Schilling). It is probable
that they are much more closely related to the animal Flagellidia than
to the Diatomaceze (as Warming maintains), or other plants, although
no hard and fast line can be drawn about any of these groups. The food of the holozoic flagellates consists of bacteria and minute
Fig. 71. Megastoma entericum
Grassi. Ventral and side views. [GRAssI.] Fig. 71. Megastoma entericum
Grassi. Ventral and side views. [GRAssI.] The food of the holozoic flagellates consists of bacteria and minute
bits of disintegrated proteid matter. These in the Rhizomastigide,
as in the Rhizopoda, are surrounded by pseudopodia, and are subse-
quently drawn into the body. In other Mastigophora, the flagellum
is the chief factor in alimentation, and by its vibrations a current is
created
toward
the base, where the mouth
or
its equivalent
is THE MASTIGOPHORA 127 situated. The particles of food brought with the current find their
way into the body-plasm, where an indefinite cyclosis carries them
hither and thither until the digestible portions are separated from
the indigestible, and the latter are finally thrown out. James-Clark
(66), Kent (81), and most observers have maintained that, in the
Choanoflagellida, the food particles strike against the collar, subse-
quently working down on the inside to the mouth, tut Entz (83) and
Francé ('93, 97) claim that the mouth is not within the collar, but
that the so-called vacuole described by Bitschli (84) is a soft ingest-
ing area at the base of the overlapping edge of the collar (see Fig. 69). F. REPRODUCTION Binary fission,
the typical method of reproduction among the
Mastigophora, and the simplest of all modes of increase, is invariably
preceded by division of the nucleus. When chromatophores, eye-
spots, and pyrenoids are present, they also may be halved and THE PROTOZOA 128 equally represented in the daughter-cells, or they may remain whole,
going to that daughter-cell to which they are nearest, the other cell
forming a new set. The flagellum, in some cases, is also divided
throughout the entire length, although in other cases it is thrown off
before division takes place, new ones being formed by the daughter-
cells. In many cases new flagella, as well as all of the important
structures, are pre-formed before division. Such divisions may take
place while the organisms are moving freely about in the water, or Fig. 72.— Gonium pectoraleO. F. M. [STEIN.] Fig. 72.— Gonium pectoraleO. F. M. [STEIN.] while they are quiescent and inclosed in a firm cyst. As a rule,
division is longitudinal, but cases are well known where it is trans-
verse (in most of the Dinoflagellidia, in Epzpyrzs, Stylochrysalts,
Oxyrrihits, etc.). Some forms (e.g. Zrachelomonas) reproduce by simple division Some forms (e.g. Zrachelomonas) reproduce by simple division
while still within the shell, one half making its way out through
the neck or flagella-opening, leaving the other in possession of the
original home. Colony-formation is closely connected with the process of simple Colony-formation is closely connected with the process of simple
division, and nowhere among the Protozoa does it reach such high
grades of differentiation as in this class. The colonies of this group THE MASTIGOPHORA 129 are composed, for the most part, of descendants of one ancestor and
are formed by incomplete division or by subsequent attachment. In
these colonies, which are wonderfully varied, the individual monads
in some cases are embedded in a transparent cellulose jelly secreted
by the cells, and in which they lie freely, or attached to one another Fig. 73.— Division of Gonium pectorale O. F. M. a, 6, and e, undivided cells; c,d, 7, 2, and 4, 4-celled stages; h, z, 7, , 0, 8-celled stages; y,
Sy
m, and p, 12-16-celled stages, Fig. 73.— Division of Gonium pectorale O. F. M. F. REPRODUCTION a, 6, and e, undivided cells; c,d, 7, 2, and 4, 4-celled stages; h, z, 7, , 0, 8-celled stages; y,
Sy
m, and p, 12-16-celled stages, by stalks, while in other cases there is no surrounding matrix, the
individuals remaining connected through incomplete division or by
attachment subsequent to division. Gonzum (Fig. 72), Pandorina,
Uroglena, Proterospongia, Volvox, etc., have the cellulose jelly, while
Dinobryon, Anthophysa, etc., are formed by attachment subsequent to
division. In some of the more complicated colony-forms, especially
in the Phytoflagellida, the adult condition is attained through cleavage
stages as regular as in any metazoan egg. The formation of such a K THE PROTOZOA 130 colony never varies, and the number of individuals
is constant. (In Genium sociale there are 4 individuals; in G. pectorale 16, while in
Eudortna there are from 16 to 32, and in Pandorina 32.)
A Gonium
colony lies in one plane, but this arrangement is brought about by a
secondary shifting of the cells (Fig. 73), while a Eudora colony
retains the spherical form. Pandorina, a similar compact and defi-
nite colony, is derived as in Eudorina, by the regular cleavage of a
single cell. In some colonies the individuals are connected in the centre by
protoplasmic strands, as in Syzura, while in one genus (Uvoglena)
connecting strands may or may not be present. Ehrenberg (’38) de-
scribed U. volvox as a colony form whose peripheral individuals are
connected in the centre by tail-like processes which, except for a much
greater length, are similar to those of Syzura. He was confirmed in
this by Zacharias (’95) and Kent (’81). Biitschli, however, regarded
this central attachment as extremely doubtful. In one form, U. vol-
vox, this connection does actually exist, but in another, UV. americana,
the posterior ends of the cells are rounded and have no trace of a
central filament. The genus Uvog/ena ‘may afford, therefore, a clue
to the phylogenetic relations of the relatively huge gelatinous colonies
which, save for the surrounding matrix, have no means of connection.! Proterospongia, in its general form and structure, agrees with U. americana. In both cases, as far as known, there is an indefinite
number of individuals and no typical method of increase, as in Pan-
dorina, Eudorina, and Gontum. 1Cf. Calkins (gt). F. REPRODUCTION In Dénodryon, a free-
swimming colony of variable size, each monad occupies a small cup
of cellulose (see Fig. 61). They increase by simple longitudinal
division, one daughter-cell remaining in the original house, while the
other moves out to the edge of the parent cup, where it attaches itself
by the posterior end. A cellulose cup is then secreted about the
daughter-cell, remaining firmly attached, however, to the parent cup. The mother-cell may divide again and again, the daughter-cells at-
taching themselves to the edge of the cup already formed until there
are three or more individuals around the edge of the original one. At the same time the daughter-cells may be dividing in a similar
manner, and a much-branched bush-like colony is the result. Other
forms have stalks which in some cases are much longer than the in-
dividuals themselves (Codonocladium, Dendromonas, Codonosiga, etc.). Still another type of colony-formation is found among the Dino- Still another type of colony-formation is found among the Dino-
flagellidia, where from two to eight individuals are connected end to
end by their shell processes (Ceratium). The significance of this
chain-formation (catenation) is not clearly established, many regard-
ing it as the result of incomplete division (Pouchet, ’85), others as
preparatory to conjugation (Biitschli, ’83). The colonies as well as the individuals may increase by division, The colonies as well as the individuals may increase by division,
a purely mechanical process, however, and probably due to the un-
wieldy size of the overgrown aggregate. Zacharias (’95) and others
have seen large colonies of Urog/ena break into two portions through
the asynchronous
action
of flagella in different regions. If the
flagella of one half of the colony vibrate in one direction while those
of the other half vibrate in an opposite direction, the result is a twist-
ing of the entire mass which must ultimately give way. Such division
cannot be regarded as reproduction in a strict sense. Closely allied to simple division is the formation of swarm-spores or Closely allied to simple division is the formation of swarm-spores or
microgonidia. This may occur either in the free motile condition as
in Polytoma or Chlorogonium, or in the encysted and protected state,
as in many Monadida. F. REPRODUCTION The most highly differentiated colonial forms are the genera Volvor The most highly differentiated colonial forms are the genera Volvor
and Afagosphera, which should perhaps be considered simple multi-
cellular forms rather than Protozoa. In Volvoxr the monads (often as many as 12,000 in a single colony) In Volvoxr the monads (often as many as 12,000 in a single colony)
are arranged as in Uvoglena, around the periphery of a gelatinous
mass, and no organized connections with the centre of the cell can be
traced, although they are connected with one another by definite
protoplasmic strands. In Afagosphera the individuals are connected not only by the jelly In Afagosphera the individuals are connected not only by the jelly
matrix, but also, as in Syzura, by protoplasmic stalks, and they are
in close contact at the periphery. In both A/agosphera and I vlvox
the appearance of the peripheral cells is strikingly similar to a pave-
ment epithelium, and the comparison which is so often made between
such colonies and the blastula stage in the development of Metazoa
is certainly justifiable. Stalked colonies have an entirely different mode of origin, being Stalked colonies have an entirely different mode of origin, being
formed by repeated longitudinal division, the daughter-cells remain- THE MASTIGOPHORA 131 ing attached to the stalk by their basal ends. In Dénodryon, a free-
swimming colony of variable size, each monad occupies a small cup
of cellulose (see Fig. 61). They increase by simple longitudinal
division, one daughter-cell remaining in the original house, while the
other moves out to the edge of the parent cup, where it attaches itself
by the posterior end. A cellulose cup is then secreted about the
daughter-cell, remaining firmly attached, however, to the parent cup. The mother-cell may divide again and again, the daughter-cells at-
taching themselves to the edge of the cup already formed until there
are three or more individuals around the edge of the original one. At the same time the daughter-cells may be dividing in a similar
manner, and a much-branched bush-like colony is the result. Other
forms have stalks which in some cases are much longer than the in-
dividuals themselves (Codonocladium, Dendromonas, Codonosiga, etc.). Still another type of colony-formation is found among the Dino- ing attached to the stalk by their basal ends. F. REPRODUCTION The simplest form is seen in such cases as
Polytom7, where, instead of dividing into two portions, the organism
divides into four, eight, or, according to Dallinger and Drysdale, into
sixteen smaller forms. These develop new flagella, make their way
through the parent membrane, and grow to full size. The formation
of similar gametes has been observed in most of the Mastigophora,
either in their resting or in their encysted stages. The flagella are
drawn in, a mantle or cyst is secreted, within which the protoplasm
divides into a number of spores. In some cases swarm-spores, like
those of the Radiolaria, are of different sizes (macro- and micro-
gametes), and these may conjugate. So far as known, the formation of gametes is not accompanied by So far as known, the formation of gametes is not accompanied by THE PROTOZOA 132 complex nuclear changes. As in the Reticulariida and some Spore -
zoa, the chromatin is reduced to minute granules which are spread
throughout the cell, but in the Flagellidia they are so small that their
further history is not known. Not all forms, however, are of this
primitive type; some, as for example Voctzluca miliaris, and some of
the Dinoflagellidia, undergo a complicated mitotic process which in
Noctiluca is repeated until five or six hundred spores are formed
(Fig. 74). The formation of spores or gametes may or may not be preceded (Fig. 74). The formation of spores or gametes may or may not be preceded
by the conjugation of individuals. In those species of Mastigophora
in which spore-formation is preceded by conjugation, a very interest-
ing series of forms may be
selected, showing the grad-
ual development of sex from
types in which there
is a
union of individuals of sim-
ilar form, size, and, appar-
ently, of condition, to the
union of specially developed
male and female reproduc-
tive elements. Cienkowsky
(’56) was the first to observe
the fusion of similar monads,
but the most complete obser-
vations are those of Dallin-
ger and Drysdale (’73), who
watched the fusion of several
Fig. 74. — Noctiluca miliaris Sur. Spore-formation. individuals of Bodo (Cerco-
[ROBIN.]
monas) crassicauda, the en-
cystment of the fused mass,
and the subsequent divisions of the plasm up to the formation of
an immense number of minute spores (Fig. 75). F. REPRODUCTION In another form
(Otkomonas Dallingert) similar spores are formed, but without the
preliminary fusion of two or more small individuals. The gametes
move about until they come in contact with the adult individuals with
which they fuse. The fused mass then encysts and finally breaks up
into minute spores. An advance toward sexual differentiation
is seen in Pandorina The formation of spores or gametes may or may not be preceded
by the conjugation of individuals. In those species of Mastigophora
in which spore-formation is preceded by conjugation, a very interest-
ing series of forms may be
selected, showing the grad-
ual development of sex from
types in which there
is a
union of individuals of sim-
ilar form, size, and, appar-
ently, of condition, to the by the conjugation of individuals. In those species of Mastigophora
in which spore-formation is preceded by conjugation, a very interest-
ing series of forms may be
selected, showing the grad-
ual development of sex from
types in which there
is a
union of individuals of sim-
ilar form, size, and, appar-
ently, of condition, to the
union of specially developed
male and female reproduc-
tive elements. Cienkowsky
(’56) was the first to observe
the fusion of similar monads,
but the most complete obser-
vations are those of Dallin-
ger and Drysdale (’73), who
watched the fusion of several
Fig. 74. — Noctiluca miliaris Sur. Spore-formation. individuals of Bodo (Cerco-
[ROBIN.]
monas) crassicauda, the en-
cystment of the fused mass,
and the subsequent divisions of the plasm up to the formation of
an immense number of minute spores (Fig. 75). In another form
(Otkomonas Dallingert) similar spores are formed, but without the
preliminary fusion of two or more small individuals. The gametes
move about until they come in contact with the adult individuals with
which they fuse. The fused mass then encysts and finally breaks up
into minute spores. An advance toward sexual differentiation
is seen in Pandorina Fig. 74. — Noctiluca miliaris Sur. Spore-formation. [ROBIN.] Fig. 74. — Noctiluca miliaris Sur. Spore-formation. [ROBIN.] cystment of the fused mass,
and the subsequent divisions of the plasm up to the formation of
an immense number of minute spores (Fig. 75). In another form
(Otkomonas Dallingert) similar spores are formed, but without the
preliminary fusion of two or more small individuals. F. REPRODUCTION Four of the
thirty-two cells situated
at the end of the colony
form gametes by re-
peated divisions in one
plane, while the other
twenty-eight
cells
merely develop more
amylum granules and
Fig. 76.— Pandorina morum Ehr. Conjugation. turn darker. The ga- two or more may be formed. These gametes soon divide and form
the typical sixteen-cell Pandorina colony. Thus in Pandor:na, each
of the cells forms both sexual elements, but an advance in differentia-
tion is seen in Evdoriua clegans, where, according to the rather incom-
plete observations of Carter (’58), the thirty-two cells forming the
colony have a different
fate when the conju-
gation
period
comes
around. Four of the
thirty-two cells situated
at the end of the colony
form gametes by re-
peated divisions in one
plane, while the other
twenty-eight
cells
merely develop more
amylum granules and
Fig. 76.— Pandorina morum Ehr. Conjugation. turn darker. The ga-
A. 16-celled colony, ra een Cand £. Fusion
metes which are formed
of macrogamete with microgamete. D and /. Fusion of micro- from
the upper
four
gametes. G. Copula. cells are elongate and
spindle-shaped, with
two flagella, a red eye-spot, and a long tail. The fate of the different
cells was not made out, but there seems reason to believe that if the
observations were correct, the gametes represent male elements, the
other cells female. A still more decided advance is shown by the colonies of lo/vox. Fig. 76.— Pandorina morum Ehr. Conjugation. A. 16-celled colony, ra een Cand £. Fusion
of macrogamete with microgamete. D and /. Fusion of micro-
gametes. G. Copula. Fig. 76.— Pandorina morum Ehr. Conjugation. two flagella, a red eye-spot, and a long tail. The fate of the different
cells was not made out, but there seems reason to believe that if the
observations were correct, the gametes represent male elements, the
other cells female. A still more decided advance is shown by the colonies of lo/vox. A still more decided advance is shown by the colonies of lo/vox. Volvox can scarcely be regarded as a unicellular organism, for differ-
entiation has gone so far that the cells if separated, with the excep-
tion of the reproductive elements, cannot live. The individuals form-
ing the peripheral layer (in Volvox globator about 12,000, Cohn, ’75)
form the sterile vegetative or somatic cells of the aggregate. F. REPRODUCTION Four of the
thirty-two cells situated
at the end of the colony
form gametes by re-
peated divisions in one
plane, while the other
twenty-eight
cells
merely develop more
amylum granules and
Pandorina morum Ehr. Conjugation. turn darker. The ga- THE PROTOZOA 134 two or more may be formed. These gametes soon divide and form
the typical sixteen-cell Pandorina colony. Thus in Pandor:na, each
of the cells forms both sexual elements, but an advance in differentia-
tion is seen in Evdoriua clegans, where, according to the rather incom-
plete observations of Carter (’58), the thirty-two cells forming the
colony have a different
fate when the conju-
gation
period
comes
around. Four of the
thirty-two cells situated
at the end of the colony
form gametes by re-
peated divisions in one two or more may be formed. These gametes soon divide and form
the typical sixteen-cell Pandorina colony. Thus in Pandor:na, each
of the cells forms both sexual elements, but an advance in differentia-
tion is seen in Evdoriua clegans, where, according to the rather incom-
plete observations of Carter (’58), the thirty-two cells forming the
colony have a different
fate when the conju-
gation
period
comes
around. Four of the
thirty-two cells situated
at the end of the colony
form gametes by re-
peated divisions in one
plane, while the other
twenty-eight
cells
merely develop more
amylum granules and
Fig. 76.— Pandorina morum Ehr. Conjugation. turn darker. The ga-
A. 16-celled colony, ra een Cand £. Fusion
metes which are formed
of macrogamete with microgamete. D and /. Fusion of micro- from
the upper
four
gametes. G. Copula. cells are elongate and
spindle-shaped, with
two flagella, a red eye-spot, and a long tail. The fate of the different
cells was not made out, but there seems reason to believe that if the
observations were correct, the gametes represent male elements, the
other cells female. A still more decided advance is shown by the colonies of lo/vox. two or more may be formed. These gametes soon divide and form
the typical sixteen-cell Pandorina colony. Thus in Pandor:na, each
of the cells forms both sexual elements, but an advance in differentia-
tion is seen in Evdoriua clegans, where, according to the rather incom-
plete observations of Carter (’58), the thirty-two cells forming the
colony have a different
fate when the conju-
gation
period
comes
around. F. REPRODUCTION The gametes
move about until they come in contact with the adult individuals with
which they fuse. The fused mass then encysts and finally breaks up
into minute spores. An advance toward sexual differentiation
is seen in Pandorina and the subsequent divisions of the plasm up to the formation of
an immense number of minute spores (Fig. 75). In another form
(Otkomonas Dallingert) similar spores are formed, but without the
preliminary fusion of two or more small individuals. The gametes
move about until they come in contact with the adult individuals with
which they fuse. The fused mass then encysts and finally breaks up
into minute spores. An advance toward sexual differentiation
is seen in Pandorina An advance toward sexual differentiation
is seen in Pandorina
(Pringsheim, ’69), where, after a long period of asexual reproduction
resulting in numerous colonies, the cells separate and begin to form
swarm-spores which may be of the same or of different size. These
spores then swim about until two of them meet and fuse by the color-
less ends into a common body (Fig. 76). Fusion may take place
between two small gametes, or between a large and a small one. THE MASTIGOPHORA 133 Pringsheim regards the larger ones as females, while the smaller ones
may be either male or female. Pringsheim regards the larger ones as females, while the smaller ones
may be either male or female. E
F
Fig. '75.— Cercomenas crassicauda Duj. [DALLINGER and DRYSDALE.]
A, Ordinary forms. &. Division stage. C. Conjugation of two individuals in amceboid con-
dition. D-&, The copula,
. Sporulation. The fused mass (zygospore) encysts and dries, the color changing F F F Fig. '75.— Cercomenas crassicauda Duj. [DALLINGER and DRYSDALE.]
A, Ordinary forms. &. Division stage. C. Conjugation of two individuals in amceb
dition. D-&, The copula,
. Sporulation. The fused mass (zygospore) encysts and dries, the color changing
from green to red. When remoistened, the contents again turn green
and break open the cyst, usually as a single swarm-spore, although THE PROTOZOA
may be formed. These gametes soon divide and form
xteen-cell Pandorina colony. Thus in Pandor:na, each
orms both sexual elements, but an advance in differentia-
Evdoriua clegans, where, according to the rather incom-
tions of Carter (’58), the thirty-two cells forming the
colony have a different
fate when the conju-
gation
period
comes
around. F. REPRODUCTION ‘Fertilization takes place while the egg is
still in the parent colony; the copula forms two membranes about
itself, while the color changes from green to orange. After a consid-
erable resting period, the egg undergoes regular cleavage, forming the
adult colony, in which, even before the embryo leaves the egg, the
cells are differentiated into somatic cells and parthenogonidia. After
these cleavage stages the outer cyst wall is ruptured and the young
colony swims out. F. REPRODUCTION A few
of these cells, which reproduce asexually, are found upon the inside
of the peripheral layer, which protects them like a mantle. Stein
finds eight of these asexual cells or parthenogonidia in V. globator,
and Cohn, one to nine in / mznor. The-parthenogonidia, by repeated division, form daughter-colonies The-parthenogonidia, by repeated division, form daughter-colonies
from one-quarter to two-fifths the size of the parent colony, which
finally make their escape from the latter by rupture of its walls. After a considerable
period of such asexual reproduction, sexual
elements are formed. These are at first similar to the parthenogo-
nidial cells, but are more numerous. Later they can be distinguished
as male (avdrogonidia, Cohn) and female (gynogonidia, Cohn). In
Volvox globator there may be from twenty to forty gynogontdia and THE MASTIGOPHORA 135 from two to five androgonidia, but there may be one hundred andro-
gonidia in 7 mznvr while there are only eight gyxogonidia. Each
female cell becomes at first flask-shaped, then withdraws inside of the
colony and becomes a mature egg. The male cell, at the beginning
about three times the size of the sterile cells, soon begins to divide in
one plane until a bundle of from sixty-four to one hundred and twenty-
eight flagellated, spindle-formed elements results. These, the sperma-
tozoids, gather around the egg-cells, which are fertilized exactly as in
higher animals or plants. ‘Fertilization takes place while the egg is
still in the parent colony; the copula forms two membranes about
itself, while the color changes from green to orange. After a consid-
erable resting period, the egg undergoes regular cleavage, forming the
adult colony, in which, even before the embryo leaves the egg, the
cells are differentiated into somatic cells and parthenogonidia. After
these cleavage stages the outer cyst wall is ruptured and the young
colony swims out. from two to five androgonidia, but there may be one hundred andro-
gonidia in 7 mznvr while there are only eight gyxogonidia. Each
female cell becomes at first flask-shaped, then withdraws inside of the
colony and becomes a mature egg. The male cell, at the beginning
about three times the size of the sterile cells, soon begins to divide in
one plane until a bundle of from sixty-four to one hundred and twenty-
eight flagellated, spindle-formed elements results. These, the sperma-
tozoids, gather around the egg-cells, which are fertilized exactly as in
higher animals or plants. G. INTER-RELATIONS OF THE MASTIGOPHORA The transition from these primitive forms of Dinoflagellidia to the
more complex types with a shell composed of nicely articulated plates,
is much more difficult than the connection between the main groups. Stein maintained that the simplest form is the unshelled Gymno-
dinium, but Biitschli showed that this view is not in harmony with the
other forms of Dinoflagellidia, it being much more obvious to con-
sider the shell of the Peridinide, for example, as arising by the split-
ting up of the bivalve shell of the primitive type, than by the loss of
this shell and the subsequent formation of the articulated forms. Evidence in Biitschli’s favor is seen in the forms where there are but
few plates (e.g. Ceratocorys), although we are inclined to agree with
Klebs that a polyphyletic origin of the group is possible and that
Gymnodinium might have been derived from the Rhizomastigide. The origin of the Cystoflagellidia, composed of Joctiluca and The origin of the Cystoflagellidia, composed of Joctiluca and
Leptodiscus, is to-day generally conceded
to be from
the Dino-
flagellidia, and is supported by direct evidence in the development
of Noctiluca, where the swarm-spores are strikingly similar to Per-
dintum. ‘he relationship to the Dinoflagellidia, as first pointed out by
Allman, was based upon superficial resemblances only, and the first
conclusive observations must be credited to Pouchet (’83) and to Stein
(83), while Biitschli (’85) first applied the theory on .the basis of the
swarm-spore as described by Cienkowsky (’73) and Robin (’78). The
interesting form which Pouchet later described ('92) as Peridinium
pseudonoctiluca is now considered a young stage of Moctzluca. G. INTER-RELATIONS OF THE MASTIGOPHORA Transitional forms between the Mastigophora and the Sarcodina
show how closely the two classes are related. The development,
both in Sarcodina and in Mastigophora, throws little or no light upon
the question as to the more
primitive nature of one or the other. While many Sarcodina have flagellated swarm-spores, many Flagel-
lidia have amceboid spores, and even in the same species both amce-
boid and flagellated swarm-spores
are formed
at the same time
(Acanthocyst?s Schaudinn, see Fig. 53, /). The most primitive forms of Flagellidia suggest the long-disputed The most primitive forms of Flagellidia suggest the long-disputed
question over the boundary-line between animals and plants.!|
Un-
questionably, the most primitive flagellates are those forms which,
while actively motile, possess chromatophores and chlorophyl, and
are able to make their own food, or which, like the bacteria, can sup-
port themselves upon simpler substances than proteid. The living
flagellates which come closest to these primitive types are the monads,
while the Choanoflagellida are probably not far removed. Klebs (’93)
takes the ground that the collar-like process of the Choanoflagellida
is not a sufficient taxonomic differential save for ordinal distinctions,
and recent zodlogists are inclined to accept this view. Stein, Biitschli, and Bergh have been the most active in formulat- Stein, Biitschli, and Bergh have been the most active in formulat-
ing views as to the origin of the Dinoflagellidia, although none of
these is wholly satisfactory. Some authors derive them from the
Phytoflagellida directly (Haeckel), others place them as a group of the 1Cf. p. 22. 136 THE PROTOZOA Diatomaceze (Warming). The majority of observers are agreed, how-
ever, that a connecting link with the Flagellidia is seen in certain
species of the family Prorocentridze and represented among living
forms by Lxuviella and Prorocentrum (Fig. 66). Biitschli (83) and
Klebs (’93) agree that these might be called true Flagellidia; for, as
Delage happily expresses it, they are little more than a chromomonad
in the shell of Pacotus. The shell is bivalve, and perforated by
minute apertures characteristic of the Dinoflagellidia, and there is an
entire absence of longitudinal and transverse furrows, while the flagella
are directed outward from the anterior end. Biutschli, Bergh, and
Klebs derive them from forms like C7yptomonas, where the two flagella
are pointed in the same direction and the chromatophores are yellow. CLASSIFICATION Cuiass I]. MASTIGOPHORA. Protozoa of definite or indefinite form; naked, or
provided with a well-defined membrane. The nutrition is holozoic, parasitic,
holophytic, or saprophytic. The motile organs are flagella, which may vary in
number from one to many. Mouth, contractile vacuole, and nucleus are usually
present. They are usually small forms with a widespread tendency to colony-
formation. THE MASTIGOPHORA 137 Subclass
1. FLAGELLIDIA. These are small organisms possessing usually a
sharply defined, mononucleate body with a definite anterior end in which are
inserted one or more flagella. They are actively motile during the greater
period of life, but all have the power of encystment. Reproduction occurs by
longitudinal division, usually during the flagellated stage, although it may take
place during resting phases. Nutrition
is holophytic, holozoic, parasitic, or
saprophytic. Order 1. MONADIDA. Small forms of Flagellidia having a simple structure. The
body is frequently amoeboid, with one or two flagella at the anterior end. There
is no distinct mouth-opening, but a localized area about the base of the flagella
serves for the ingestion of food particles. Family 1. Rhizomastigidez. Simple, mouthless forms with one or two flagella and
an ameeboid body capable of putting out lobose pseudopodia like a rhizopod, or
stiff radial pseudopodia like a heliozodn. The contractile vacuole is frequently
at the posterior end. Food particles may be ingested at any part of the body
by the aid of the pseudopodia. Genera: Mastigameba F. E. Schultze (75) ;
Ciliophrys Cienk. (76); Dzémnorpha Gruber (°81); Actinomonas Kent (80) ;
Trypanosoma Gruby ('43) 3; Alast7gophrys Frenzel (91). Family 2. Cercomonadide. Oval or elongated forms which are frequently amoeboid Family 2. Cercomonadide. Oval or elongated forms which are frequently amoeboid
or changeable, but unable to form pseudopodia. There is one large flagellum
with a mouth area at its base. The family includes small forms, saprophytic, or
holozoic, or sometimes parasitic in nutrition. Genera:
Cercomonas Dujardin
(41); Herpetomonas Kent (80), parasitic. Ozkomonas Kent (’80) ; «lzcyro-
monas Kent (80); Phyllomonas Klebs (’93)-
Family 3. Codonecide. Small colorless monads which secrete and remain in a Family 3. Codonecide. Small colorless monads which secrete and remain in a
gelatinous or membranous cup. Genera: Codoneca James-Clark (°66) ; Platy-
theca Stein (°78). Family 4. Bikecide. Small monads of peculiar form. They are provided with a Family 4. Bikecide. Small monads of peculiar form. CLASSIFICATION Genera: Bod Stein (78); Phylomilus Stein
(78); Colponema Stein (°78) ; Oxyrrhis Dujardin (*41). Family
2. Trimastigide. With two accessory
flagella. Genera:
Dallingeria Family
2. Trimastigide. With two accessory
flagella. Genera:
Dalli
Kent (81); Zrzmast2r Kent (81). Order4
POLYMASTIGIDA. The body is invariably without a shell, and is pro Order4
POLYMASTIGIDA. The body is invariably without a shell, and is provided
with a delicate membrane, which allows more or less amoeboid movement. The
number of flagella varies from three to many, and the number of mouth open-
ings, or food-taking areas, likewise varies. Nutrition is holozoic. They increase
by longitudinal division. Tribe t. Astomea. Polymastigida with many flagella and without a mouth opening. Tribe t. Astomea. Polymastigida with many flagella and without a mouth opening. Genera: Afulticilia Cienk. (81); Grassia Fisch (85). Tribe 2. Monostomea. The anterior part is provided with a large mouth opening at Tribe 2. Monostomea. The anterior part is provided with a large mouth opening at
the base of the four or six flagella. Genera: Collodictyon Carter (65); Zetra-
mitus Perty (52); Alonocercomonas Grassi (82); Zrechomonas Donné (737);
Megastoma Grassi (’81). Tribe 3. Distomea. The flagella are separated into two symmetrical groups, with a Tribe 3. Distomea. The flagella are separated into two symmetrical groups, with a
mouth area at the base of each group. Genera: Zrzgonomonas Klebs (°93);
Hexamitus Dujardin (°38) 5 Zrepomonas Dujardin (°39); Spzronema Klebs (°93) ;
Vrophagus Klebs (°93). Tribe 4. Trichonymphinea. Polymastigida, of unknown affinities, provided with Tribe 4. Trichonymphinea. Polymastigida, of unknown affinities, provided with
numerous flagella. They are parasites in the rectum of various hosts ( Termites). Genera: Lophomonas Stein ('78); Leedyonella Frenzel (‘91); Zrichonympha
Leidy (77); /e@nza Grassi (85); Pyrsonympha Leidy (77). Order 5. EUGLENIDA. Large forms having one or two flagella, a contractile or firm Order 5. EUGLENIDA. Large forms having one or two flagella, a contractile or firm
body-wall,
a mouth and pharynx at the base of the flagellum, and with a con-
tractile vacuole opening into the pharynx. They frequently form colonies and
are usually provided with chromatophores. Nutrition is holozoic, holophytic, or
saprophytic. Family 1. Euglenide. Elongate forms, with a more or less pointed end and usually Family 1. Euglenide. Elongate forms, with a more or less pointed end and usually
with one flagellum. CLASSIFICATION They are provided with a
cup, to which they are attached by a slender thread. The basal portion is
. broader than the upper part, which bears a curious tentacle-like process. Nutri-
tion is holozoic; the individuals are single or colony-forming. Genera: Brcoseca
James-Clark ('67) ; Potertodendron Stein (°78). Family 5. Heteromonadide. Small colorless monads which have, in addition
to Family 5. Heteromonadide. Small colorless monads which have, in addition
to
the chief flagellum, one or two accessory flagella. They frequently form colonies
upon a common stalk. Increase of the individuals is by longitudinal division. Genera:
Monas Stein (°78); Lendromonas
Stein (78);
Cephalothamnium
Stein ('78); Anthophysa Bory d. St. Vincent (24); pzpyazs Ehr. (738);
Amphimonas Kent (81); Spongomonas Stein ('78); Cladomonas Stein (°78) ;
Rhipidodendron Stein (78); Diplomita Kent (80). Order 2. CHOANOFLAGELLIDA. Flagellidia with one or more collar-like pro- Order 2. CHOANOFLAGELLIDA. Flagellidia with one or more collar-like pro-
cesses about the base of the single flagellum. Family
1. Phalansteride. Colony-forming Choanoflagellida. Each individual is Family
1. Phalansteride. Colony-forming Choanoflagellida. Each individual is
situated in a granular gelatinous tube. The gelatinous tubes form either a dis-
coid colony in which the single tubes are arranged radially, or a dichotomously
branched aggregate. Genera: Phalanstertum Cienk. (’70). Family 2. Craspedomonadide. Solitary or colonial forms. The individuals are Family 2. Craspedomonadide. Solitary or colonial forms. The individuals are
naked, or lie in an incomplete cup, or in a gelatinous mass. Genera: A/ono-
stga Kent (80);
Codosiga, James-Clark (67);
Codonocladium Stein (78) ;
Hirmidiun Perty (52); Proterospongia Kent (81); Spheraca Lauterb. (99) ;
Salpingeca James-Clark (67) ; Polyeca Kent (81); Diploszga Frenzel (’91). Order 3. HETEROMASTIGIDA. A small group with various kinds of flagellated Order 3. HETEROMASTIGIDA. A small group with various kinds of flagellated
organisms, which are sometimes naked and amceboid, sometimes provided with
a complex membrane. The essential character is the possession of two or more 138 THE PROTOZOA flagella, one being directed forward and used in locomotion, the others directed
backward and trailed after the organism. Nutrition is holozvic, and all of the
forms included are colorless. flagella, one being directed forward and used in locomotion, the others directed
backward and trailed after the organism. Nutrition is holozvic, and all of the
forms included are colorless. Family 1. Boconiig. Small naked forms in which there is only a slight difference,
if any, between the flagella. CLASSIFICATION The cuticle is marked with spiral stripings: the contractile
vacuole, or vacuoles, open into a reservoir, which in turn opens into the pharynx. A red eye-spot, or stigma, and green chromatophores, are usually present. Within the body there are discoid, or, occasionally, band-formed chromatophores. Paramylum granules are always present. Genera: Auglena Ehr. (130); Colactumu
Ehr. (°33); Zutreptia Perty (*52); Ascoglena Stein (78); Trachelwmonas Ehv. (33); Lepocinclis Perty (49); Phacus Nitsch (116); Cryptoglena Ehr. (21). Family 2. Astasiide. The body is elongate and usually has a striped membrane. Family 2. Astasiide. The body is elongate and usually has a striped membrane. The anterior end is similar to that of Zzg/ena, but there is no eye-spot. The
body is invariably colorless. Nutrition is saprophytic. Genera: -ls¢aséa Ehr. (38) ; Déstzgma Ehr. (31); Rhabdomonas Fres. (°58) ; Alenotdium Perty (52) ;
Atractonema St. (78): Sphenomonas Stein (78). Familv 3. Peranemide. The body is either stiff or plastic. and usually symmetrical. Familv 3. Peranemide. The body is either stiff or plastic. and usually symmetrical. The anterior end bears either one or two dissimilar flagella, which are more or
less deeply sunk in the body. A distinct mouth is found at the base of the
flagella. Nutrition is holozoic. Genera: A. With plastic body and one flagel-
lum:
Evelenopsis Klebs (93); Peranema Dujardin (41); Urceolus Meresch-
kowsky (77). B. With
a plastic body and
two
flagella:
Aeteronema
Dujardin
(*41); Dena
Perty (76); Zvegoselnis
Duj. (41). C. With a
constant body form and one flagellum: Scy¢omonas Stein (78) ; Petalomonas THE MASTIGOPHORA 139 Stein (’59). D. With a constant body form and two dissimilar flagella: 77o-
pidoscyphus
Stein (78);
Axncsonema
Duj. (41); Lxtostphon
Stein
('78);
Thaumatomastix Lauterb. (’99). Order 6. PHYTOFLAGELLIDA. Flagellated unicellular organisms with chlorophyl Stein (’59). D. With a constant body form and two dissimilar flagella: 77o-
pidoscyphus
Stein (78);
Axncsonema
Duj. (41); Lxtostphon
Stein
('78);
Thaumatomastix Lauterb. (’99). Order 6. PHYTOFLAGELLIDA. Flagellated unicellular organisms with chlorophyl Order 6. PHYTOFLAGELLIDA. Flagellated unicellular organisms with chlorophyl
and holophytic nutrition, or without chlorophyl, and saprophytic in nutrition. They are sometimes classified as plants, sometimes as animals. Suborder 1. CHLOROMONADINA. The body is somewhat plastic and without a dis-
tinct membrane;
with numerous discoid chromatophores but without stigmata. Genera:
Vacuolaria Cienk. (70); C@lomonas Stein (78). Suborder 2. CHROMOMONADINA. Small forms with strong tendency to colony- Suborder 2. CHROMOMONADINA. Small forms with strong tendency to colony-
formation. CLASSIFICATION They are often inclosed in a gelatinous mass, or occupy cups. They
may or may not have chromatophores, which, if present, are yellow or yellowish
brown in color. Nutrition is usually holophytic, but holozoic and saprophytic
forms are occasionally present. There may be one or two flagella, which are
invariably directed forward. Family 1. Chrysomonadidea. The body is rarely naked, but usually covered bya Family 1. Chrysomonadidea. The body is rarely naked, but usually covered bya
gelatinous mass or by a hyaline cup. With one or two flagella at the anterior
end and with or without stigmata. One or two yellowish chromatophores are
invariably present. Nutrition
is holophytic
or
holozoic, sometimes both. Genera:
A. With naked body which may be inclosed during resting stages in
a gelatinous mass. Nutrition either holozoic
or holophytic. C#rysam@ba
Klebs (’90); Chromulina Cienk. (°70); Ochromonas Wysotzki (°87); Stylo-
chrysalis Stein (°78). B. With a shell or lorica in which the individuals
are attached. Nutrition is holophytic. Chrysococcus Klebs (’92); Linobryon
Ehr. (738);
Chrysopyxis
Stein (°78); Mephroseliis St. (78); Hyalobryon
Lauterb. (’99). CC. Individuals protected by a close-fitting membrane. //y-
menomonas Stein (78); Mcraglena
Ehr. (°31); Mallomonas Perty ('76) ;
Synura Ehr. (33); Syuerypta Ehr. (33);
Uroglena Ehr. (33);
Chryso-
spherella Lauterb. (’99)-
Family 2. Cryptomonadide. The body has a firm cuticle and is never amceboid. Family 2. Cryptomonadide. The body has a firm cuticle and is never amceboid. There are two similar flagella, a peculiar cesophagus-like canal, and a contractile
vacuole in the anterior end. Two chromatophores of variable color may or may
not be present. Nutrition is holophytic or saprophytic. Genera: Cryptomonas
Ehr. (31); Chzlomonas Ebr. (31) 3 Cyathontonas Fromentel (’74). Suborder 3. CHLAMYDOMONADINA. Body-form more or less changeable. Color Suborder 3. CHLAMYDOMONADINA. Body-form more or less changeable. Color
usually green, and due to the presence of a large, single chromatophore contain-
ing chlorophyl. A firm shell is usually present. The body has two or four
flagella, one or two contractile vacuoles, and a stigma at the anterior end. Re-
production takes place by continued division within the shell either during active
or resting phases. Macro- and micro-gametes may be formed. Family t. Chlamydomonadida. With a stiff coating perforated only by minute Family t. Chlamydomonadida. With a stiff coating perforated only by minute
apertures for the flagella. Genera: Chlamydomonas Ehr. (°33); Chlorogoniuim
Ehr. (35); Polytoma Ehr. CLASSIFICATION Order 2. DINIFERIDA. Dinoflagellidia with two transverse furrows. Family 1. Peridinidea. The cross furrow is near the middle of the Family 1. Peridinidea. The cross furrow is near the middle of the body, which
may be with or without a shell. The form is extremely variable. Genera:
Podolampas Stein (83); Blepharocysta Ehr. (73); Diplopsalis Bergh (‘82);
Peridinium Ebr. (32);
Gontodoma Stein (83); Gonyaulax Diesing (66);
Ceratium Schrank (1793); Amphidoma Stein (83); Oxytoxum Stein (’83);
Pyrophacus Stein (°83) ; Ptychodiscus Stein (°83) ; Protoceratium Bergh (°82) ;
Glenodinium Ebr. (35); Gymnodinium Stein (78); Hemidinium Stein (°78) ;
Steintella Schiitt (95); Monaster Schiitt (95); Amphitholus Schiitt (95). Family 2. Dinophysida. The cross furrow is above the middle of the body, and yrophacus Stein (°83) ; Ptychodiscus Stein (°83) ; Protoceratium Bergh (°82) ;
lenodinium Ebr. (35); Gymnodinium Stein (78); Hemidinium Stein (°78) ; lenodinium Ebr. (35); Gymnodinium Stein (78); Hemidinium Stein (°78) ;
eintella Schiitt (95); Monaster Schiitt (95); Amphitholus Schiitt (95). Steintella Schiitt (95); Monaster Schiitt (95); Amphitholus Schiitt (95). ily 2. Dinophysida. The cross furrow is above the middle of the body, an Family 2. Dinophysida. The cross furrow is above the middle of the body, and
its edges are raised into characteristic ledges. Marine. Genera: Phalacroma
Stein (83); Lnophysis Ehr. (39); Amphisolenia Stein (’83); Cztharistes
Stein (°83) ; A/stéonets Stein (83); Ornithocercus Stein (83); Amphidinium
Clap. and Lach. (’59); Ceratocorys Stein (’83). Order 3. POLYDINIDA. The order consists of the single genus Polykrikos Biit- Order 3. POLYDINIDA. The order consists of the single genus Polykrikos Biit-
schli (73), which is characterized by a naked body, by several transverse furrows
and flagella, by macro- and micro-nuclei, and by nematocysts. Nutrition is
holozoic. Subclass
III]. CYSTOFLAGELLIDIA. Mastigophora of considerable size, with a Subclass
III]. CYSTOFLAGELLIDIA. Mastigophora of considerable size, with a
single nucleus, parenchymatous protoplasm, and a firm membrane. Nutrition is
holozoic. Marine. Genera: Moctiluca Suriray (36); Leptodiscus R. Hertwig
(77). CLASSIFICATION ('38) ; Hematococcus Agardh (°28) ; Carterza Diesing
(66) 3 Spoxdylomorum Ehr. (48); Chhrangium Stein (78). Family 2. Phacotide. The body of the flagellate corresponds to that of Hemato- Family 2. Phacotide. The body of the flagellate corresponds to that of Hemato-
cocus, and is surrounded by a thick shell membrane which the body does not
fill. The shell is frequently bivalved. Genera: Coccomonas Stein (°78) ; AZeso-
stigma Lauterb
('94); Phacotis Perty (52); Zetratoma Bitschl (85); Pyra-
mimonas Schmarda (’50): Chloraster Ehr. ('48). Suborder 4. VOLVOCINA. Colony forms. The individuals possess two flagella and Suborder 4. VOLVOCINA. Colony forms. The individuals possess two flagella and
chlorophyl-bearing chromatophores. The number of individuals composing the
colony may be constant or variable; when constant, the colony is formed by
regular cleavage, as in the eggs of Metazoa. Reproduction asexual by division 140 THE PROTOZOA or sexual. Genera:
Gonium O. F. Miiller (1773); Stephanosphera Cobn
(53); Pandorina Bory de St. Vincent ('24); Zudorina Ehr. (31);
Volvox
Leeuw. Ehr. (38); #/@odorina Shaw (94); Platydorina. Kofoid ('99). Order7. SILICOFLAGELLIDA. A single genus, Déstephanus Stohr (’81), character- or sexual. Genera:
Gonium O. F. Miiller (1773); Stephanosphera Cobn
(53); Pandorina Bory de St. Vincent ('24); Zudorina Ehr. (31);
Volvox
Leeuw. Ehr. (38); #/@odorina Shaw (94); Platydorina. Kofoid ('99). Order7. SILICOFLAGELLIDA. A single genus, Déstephanus Stohr (’81), character- Order7. SILICOFLAGELLIDA. A single genus, Déstephanus Stohr (’81), character-
ized by the presence of a silicious latticed skeleton like that of the Radiolaria. There is no mouth nor modifications of the plasm whatsoever, but the animal is
colored yellow by (probably) diatomin. Parasitic on Radiolaria. Subclass
I]. DINOFLAGELLIDIA. Naked or shelled Mastigophora. There are Subclass
I]. DINOFLAGELLIDIA. Naked or shelled Mastigophora. There are
usually two flagella, of which one is directed away from the body, the other
around the body; the shell usually has two furrows, one running transversely
around the body, the other vertically. Marine and fresh-water forms. The
nutrition is holophytic or holozoic. Order 1. ADINIDA. The transverse furrow is absent, and the two flagella arise from Order 1. ADINIDA. The transverse furrow is absent, and the two flagella arise from
the anterior end of the body. The shell may be bivalved. Family 1. Prorocentrida. With the characters of the order. Genera: Eauviella Family 1. Prorocentrida. With the characters of the order. Genera: Eauviella
Cienk (82); Prorocentrum Ehr. (733). Order 2. DINIFERIDA. Dinoflagellidia with two transverse furrows. THE SPOROZOA THE Sporozoa are unicellular animal parasites living in the cells,
tissues, and cavities of various hosts and, as the name indicates, char-
acterized by reproduction through spore-formation. If we except
the bacteria, they are the most widely distributed of all parasites, and
are found in every class of animals, frequently in Vermes, Arthrop-
oda, Mollusca, and Vertebrata, rarely in Protozoa, Coelenterata, and
Echinodermata. They may infest the alimentary tract, and
all Fig. 77. — The vegetative phase in the life-history of a
|
gregarine (schematic). [WASIELEWSKY.]
The young sporozoite (A), liberated in the intestine,
enters an epithelial cell (2), where as an intra-cellular
parasite (f) it grows at the expense of the cell-contents,
often forcing the nucleus (z) to a corner of the cell. It
finally grows through the cell-wall (C) and ultimately
drops into the lumen of the organ as a sporont (J). the life-history of a
|
SIELEWSKY.]
ated in the intestine,
as an intra-cellular
of the cell-contents,
rner of the cell. It
(C) and ultimately
a sporont (J). of the connecting organs and ducts; the kidneys and their ducts;
the blood-vessels and the blood; the muscles and connective tissues ;
while even the skin is not exempted. In most instances they are harm-
less, but they may produce morbid and even fatal results, either in-
directly, by increasing to such numbers that the lymph-spaces and
cavities are filled with them, thus preventing nutrition of the cells and
tissues, or directly, by causing atrophy and death of the cells in which
they live. They are usually taken into the system in the spore-stage
with the food of their host, although infection may take place through
the gills or lungs, or even by inoculation from insects. The spore-
membranes are soon dissolved by the fluids of the host, and one or
more germs are thus liberated. These germs, the sporozoites, then
141 of the connecting organs and ducts; the kidneys and their ducts;
the blood-vessels and the blood; the muscles and connective tissues ;
while even the skin is not exempted. In most instances they are harm-
less, but they may produce morbid and even fatal results, either in-
directly, by increasing to such numbers that the lymph-spaces and
cavities are filled with them, thus preventing nutrition of the cells and
tissues, or directly, by causing atrophy and death of the cells in which
they live. SPECIAL BIBLIOGRAPHY IV Biitschli, 0.— Protozoa, Mastigophora. In Bronn’s
Klassen und Ordnungen des
Thierreichs. Lepzzg, 1883-1887. Francé, R. H.— er Organismus der Craspedomonaden. Sudapesth, 1897. Francé, R. H.— er Organismus der Craspedomonaden. Sudapesth, 1897. Kent, W. Saville. — A Manual of the Infusoria. London, 1881. Manual of the Infusoria. London, 1881
nstudien 1. Zest. f. wiss. Zool. Bd. LV Kent, W. Saville. — A Manual of the Infusoria. London, 1881. Klebs, G.— Flagellatenstudien 1. Zest. f. wiss. Zool. Bd. LV., Klebs, G.— Flagellatenstudien 1. Zest. f. wiss. Zool. Bd. LV., pp. 265-445. Schiitt, F.— Die Peridineen der Plankton-Expedition. Th. I. vel and L Schiitt, F.— Die Peridineen der Plankton-Expedition. Th. I. vel and Letpsrg,
1895. Senn, a Flagellata. In A. Engler’s Die naturlichen Pflanzenfamilien. 202 and 203 Senn, a Flagellata. In A. Engler’s Die naturlichen Pflanzenfamilien. 202 and 203
Lieferung. Lezpz7g, 1900. Stein, Fr.— Der Organismus der Infusionsthiere. Le7pzzg, 1878. Senn, a Flagellata. In A. Engler’s Die naturlichen Pflanzenfamilien. 202 and 203
Lieferung. Lezpz7g, 1900. Stein, Fr.— Der Organismus der Infusionsthiere. Le7pzzg, 1878. Lieferung. Lezpz7g, 1900. Stein, Fr.— Der Organismus der Infusionsthiere. Le7pzzg, 1878. Stein, Fr.— Der Organismus der Infusionsthiere. Le7pzzg, 1878. THE SPOROZOA The mature parasites finally may
leave the cell-host and sporulate
in the digestive cavity ne ccelom,
and the spores are then carried
to the outside with the fzeces or
other excreta. <
- p
Fig. 78.—Coccidia in the epitvelial cells of
Triton cristatus. n, nuclei of the tissue cells;
%, the intra-cellular parasite Pfeiferca tritonis. [LABBE.]
aN
fendi Fig. 78.—Coccidia in the epitvelial cells of
Triton cristatus. n, nuclei of the tissue cells;
%, the intra-cellular parasite Pfeiferca tritonis. [LABBE.] THE SPOROZOA They are usually taken into the system in the spore-stage
with the food of their host, although infection may take place through
the gills or lungs, or even by inoculation from insects. The spore-
membranes are soon dissolved by the fluids of the host, and one or
more germs are thus liberated. These germs, the sporozoites, then
141 141 THE PROTOZOA 142 bore into the epithelial cells, where they grow (Fig. 77). All forms,
apparently, begin life as intra-cellular parasites, where, at first, they do
little harm, but as they grow by the absorption of fluids contained
within their cell-hosts, the latter
are improperly
nourished
and,
unless the parasites leave them,
they degenerate and die (Fig. 78). The duration of intra-cellular life
<
varies in different kinds of Spo-
- p roz0a:
some
are permanently
intra-cellular (#onophagous forms,
so-called Cytosporid.a, ctc.); others bore into the epithelial cells, where they grow (Fig. 77). All forms,
apparently, begin life as intra-cellular parasites, where, at first, they do
little harm, but as they grow by the absorption of fluids contained
within their cell-hosts, the latter
are improperly
nourished
and,
unless the parasites leave them,
they degenerate and die (Fig. 78). The duration of intra-cellular life
<
varies in different kinds of Spo-
- p roz0a:
some
are permanently
intra-cellular (#onophagous forms,
so-called Cytosporid.a, ctc.); others
are intra-cellular only in the young
or immature phases(Gregarinida);
while still others pass different
phases of their life-history in dif-
ferent cells (folyshagous forms). The mature parasites finally may
leave the cell-host and sporulate
Fig. 78.—Coccidia in the epitvelial cells of in the digestive cavity ne ccelom,
Triton cristatus. n, nuclei of the tissue cells; and the spores are then carried
%, the intra-cellular parasite Pfeiferca tritonis. to the outside with the fzeces or
[LABBE.]
other excreta. aN
fendi they grow (Fig. 77). All forms,
r parasites, where, at first, they do
he absorption of fluids contained
within their cell-hosts, the latter
are improperly
nourished
and,
unless the parasites leave them,
they degenerate and die (Fig. 78). The duration of intra-cellular life
varies in different kinds of Spo-
roz0a:
some
are permanently
intra-cellular (#onophagous forms,
so-called Cytosporid.a, ctc.); others
are intra-cellular only in the young
or immature phases(Gregarinida);
while still others pass different
phases of their life-history in dif-
ferent cells (folyshagous forms). AA. PROTOPLASMIC STRUCTURE A typical sporozoodn consists of protoplasm and one nucleus. It
has no mouth, anus,
excretory pore,
or other openings. It has
neither gastric nor contractile vacuoles, and has at most a sluggish
movement in the adult stage, although the young forms may be
amoeboid or flagellate. Owing to the number of cytoplasmic granules
which make up the bulk of the animal, the protoplasmic structure of
adult forms can be made out only with the greatest difficulty. Apart
from these granules, however, which are regarded as reserve nutri-
ment, it is probable that, asin all Protozoa, the protoplasm is alveolar. This is certainly the case in Coccidiida (intra-cellular Sporozoa), espe-
cially in the young forms, where Labbé (’96) describes the cytoplasm
as alveolar;
in some forms of Gregarinida (Fig. 87), and in Myxo-
sporidiida as described by Thélohan (’95) and Doflein (98) (Fig. 79). The granules, which are so characteristic of the group, completely
fill the alveolar network, and give to the protoplasm its peculiarly
dense appearance. They differ somewhat in size and shape, and
apparently in chemical composition, and are generally regarded as
food substances reserved for use during the spore-preducing period. THE SPOROZOA 143 Wasielewsky (’96) enumerates the following kinds: (1) Paraglyco-
gen. These form the bulk of the granules in the Gregarinida; they
are distinct refringent granules of variable size and are usually oval or
spherical in form, consisting of a peculiar amyloid substance which
Biitschli (84) regarded as similar to amidon or glycogen. They give
characteristic reactions, staining brown to violet with dilute sulphuric
acid, and dissolving in potassium carbonate and strong mineral acids. (2) Carminophilous granules. These granules, which were first made
out by Schneider (’75), are less numerous than the paraglycogen
granules, but like them variable in size and strongly refractive. eo MG
oe £6
QB
e! oer
fete
eee
SPS
eS
AOTC
642686;
Fig. '79. = Leptotheca agilis Dof., one of the Myxosporidiida. [DOFLEIN.] Fig. '79. = Leptotheca agilis Dof., one of the Myxosporidiida. [DOFLEIN.] They are easily soluble in ammonia, but are not destroyed by alcohol,
embedding in paraffine, etc., and are easily stained by carmine anu
many aniline colors, but not at all by haematoxylin. They consist,
apparently, of albumen. (3) fat. These granules are widely dis-
tributed throughout the entire group, and have about the same
appearance in all types, although they are colored differently in
different species. AA. PROTOPLASMIC STRUCTURE They are soluble in alcohol, ether, and chloroform,
and are stained black by osmic acid. In addition to the above gran-
ules, which are found in most Sporozoa, there are others which have
been found hitherto only in certain subdivisions. Jn the Gregarinida,
pyxinine granules and protein crystals have been observed in certain
species, the former by Frenzel (’85) in Pyxiuta, where they appar- THE PROTOZOA 144 ently take the place of the paraglycogen granules, although of similar
chemical nature, but slightly different in their reactions. The latter
are also rather questionable inclusions in certain Dzdymophyes. In
the Coccidiida all adult forms are characterized by the presence of
so-called plastic granules. These are globular, strongly refractive
granules of slighily variable size which react differently from the
glycogen granules, remaining unchanged in sulphuric acid and stain-
ing yellow with iodine. Here also are found the so-called chromatord
granules, which are distinguished by their affinity for haematoxylin, and
are probably albuminoid in nature. In the Hamosporidiida or blood-
infesting Sporozoa, the effect of the intra-corpuscular life is shown
by the presence of pigmented granules (we/aniz) of black, yellow
ochre, or red color resulting from the disintegration of haemoglobin. In the majority of the Sporozoa the cell-body consists of a more or In the majority of the Sporozoa the cell-body consists of a more or
less sharply differentiated ectoplasm and endoplasm, while even a
third layer, wesoplasm, is said to have been observed in some forms
(Cohn, '96). It is possible that these different zones are function-
ally specialized, a supposition first made by Labbé (’96) in connection
with the Coccidiida, and repeated by Doflein (’98) in connection with
the Myxosporidiida. As in the Sarcodina and Mastigophora, the
ectoplasm may be plastic, yielding to the pressure from within and
thus giving rise to pseudopodia (Monocystis ascidi@, Siedlecki, ’99,
Myxosporidiida), or it may be modified into a hard and tough cuticle,
which offers a good protection for the cell-body within. Again, it
may be modified into a complex membrane, plastic and capable of
various kinds of motion and similar to that of the higher types of
Flagellidia. The most highly differentiated ectoplasm is found in the
Gregarinida, where it forms a dense cortical layer about the body,
while its outermost part is transformed into a complex membrane. AA. PROTOPLASMIC STRUCTURE In some cases the inner cortical layer of the ectoplasm is carried
across the cell, forming a partition dividing the organism into two:
portions which are known as the fprotomerite and the deutomerite,
the nucleus being in the latter. The non-nucleated portion is often
further differentiated into an apparatus called the epzmcrite, which usu-
ally develops hooks or anchors used for attaching the animal to its
cell-host. The organism thus appears to be multi-chambered, and the
presence or absence of such chambers was formerly regarded as a
good basis for classification; but it has been shown, especially by
Léger (’92), that the partitions vary considerably in the same species,
and even in the same individual, at different times, and in the recent
systems of classification this feature has been discarded in determir ing
the limits of the larger divisions. The epimerite, which is so important
in holding the lumen-dwelling parasites in place, may be simple or
branched; plain, like a knob or a rod; branched with filiform, or flat THE SPOROZOA 145 with digitiform, appendages. It may be closely attached to the pro-
tomerite, or carried on a long neck, while variations in all types are
numerous (Fig. 12, £, /, G, p. 39). Under certain conditions, prior
to reproduction, the animal throws off the epimerite which may be left
in the cell-host, and drops into the lumen of the organ in which it
lives. Here it encysts, the protomerite and deutomerite forming one
spore-producing individual. As the attached and the detached stages
in the life-history of the Gregarine are each important, they have
received
special names,
the. former
being known as a cephalont, the latter
——
as a sporont. : with digitiform, appendages. It may be closely attached to the pro-
tomerite, or carried on a long neck, while variations in all types are
numerous (Fig. 12, £, /, G, p. 39). Under certain conditions, prior
to reproduction, the animal throws off the epimerite which may be left
in the cell-host, and drops into the lumen of the organ in which it
lives. Here it encysts, the protomerite and deutomerite forming one
spore-producing individual. As the attached and the detached stages
in the life-history of the Gregarine are each important, they have
received
special names,
the. former
being known as a cephalont, the latter
——
as a sporont. B.
THe Nwcieus B. THe Nwcieus With the exception of the multinucleate Myxosporidiida, the Sporo-
zoa are mononucleate. Schneider (’81) abandoned the attempt to
compare the nuclei in Sporozoa with those of ordinary animal and
plant cells, because of their peculiar structure. In most cases they
consist of a firm and resisting membrane containing a single large
chromatin reservoir or faryosome, and are apparently without a linin
reticulum, such as is found in the nuclei of Metazoa. In some forms
the nucleus is similar to that of the Sarcodina and Mastigophora,
consisting of membrane, reticulum, and one
or more
chromatin
reservoirs. Recent observers have found that the different appear-
ances of the nucleus are characteristic of different stages of nuclear
activity, and that the reticulum, and even the nuclear membrane, are
derived from the karyosome, which in the sporozoite appears as a solid
homogeneous sphere of chromatin.’
In the active phases the nuclei
of the Sporozoa differ widely from those in other Protozoa, the most
striking point of difference being the disappearance of the nuclear
membrane during division. The chromatin reservoirs may divide
directly, thus simulating the entire nucleus, or they may break down
into small chromatin granules, resembling the first stages of chromo-
some-formation in the flagellate Voctz/uca. In the former there is no
distinct spindle, in the latter the completed spindle-figure has two sets
of fibres, although, according to Wolters’s (’91) description, the fibres
seem to have a different function from those in the mitotic figures of
the Metazoa, since there is no connection between them and the
chromatin.” 2 See zufra, p. 259. LCL, infra, p. 253. AA. PROTOPLASMIC STRUCTURE :
Between the cortical ectoplasm and ——
:
r
————
ee
Figs Soh suleinais Beuve ot tie
mvonemes of Clepsedrina munieri; m
the myonemes. [SCHNEIDER,]
a
2
A
SS
= 3 | as a sporont. :
Between the cortical ectoplasm and
the inner endoplasm there is a layer of
r
myonemes, or muscular fibrils similar
————
ee
in all respects to those of the Ciliata
(Fig. 80). These
are
occasionally
found in the Hzemosporidiida, but are
much more characteristic of the Gre-
garinida, where, except in the epimerite,
they form a network about the entire
animal. On the outside of this net-
work, according to Schewiakoff (’94),
there is, at times, a layer of gelatinous
matter apparently secreted by the ecto-
plasm, and this, in turn, is covered by
the
membrane
proper. The mem-
brane is longitudinally striated by rib-
like projections, while the canals or
furrows between them are filled with
Figs Soh suleinais Beuve ot tie
is
a
mvonemes of Clepsedrina munieri; m
jelly from the gelatinous layer below
the myonemes. [SCHNEIDER,]
(see Fig. 82, B). Schewiakoff believes
that the active secretion of jelly in these furrows accounts for the
peculiar gliding motion of certain kinds of Gregarinida. In the region
of the epimerite, the membrane is plain, the ribs and furrows stopping
with the protomerite. The hooks or spines are formed from the
cortical plasm. In one group of Sporozoa, the Sarcosporidiida, the protoplasmic
a
2
A
SS
= 3 | Figs Soh suleinais Beuve ot tie
mvonemes of Clepsedrina munieri; m
the myonemes. [SCHNEIDER,] In one group of Sporozoa, the Sarcosporidiida, the protoplasmic
body is inclosed in a peculiar pouch which appears to be a secretion
from the protoplasm rather than a true cellular membrane. The
mass slowly enlarges by regular growth until it reaches a considerable
length, in some cases several millimetres. It then undergoes spore-
formation. These organisms, known as Razucy's Tubes, are parasites
of sheep, swine, deer, horses, rats, etc., where they infest the muscle-
tissues, causing morbid symptoms, similar to those in trichinosis. L 146 THE PROTOZOA B. THe Nwcieus C.
Foop-TAKING Like all endoparasites, the Sporozoa absorb fluid food through the
body-wall, even when, as in the Myxosporidiida, pseudopodia are
present. There
is probably no specialized area devoted to food-
taking, but all parts are equally receptive. It is believed that in
some cases, notably in the Gregarinida and Myxosporidiida, minute
pores perforate the membrane between the outer markings. Although
the taking of food has never been observed, the indirect effects are
seen in the rapid growth of the parasite when in a suitable medium. Thus a young gregarine, when it penetrates an epithelial cell (Fig. 77, A), is a minute ball of protoplasm; but it rapidly grows until it
occupies the greater part of its host, often forcing the nucleus to one
side. As it continues to grow, the front wall of the cell is pushed
outward until it finally breaks, and the lower portion of the parasite THE SPOROZOA 147 is left exposed in the lumen of the digestive organ (Fig. 77, C). If it
is a polycystic or multi-chambered form, the exposed portion becomes
differentiated
into protomerite
and deutomerite,
while
the intra-
cellular portion remains as the epimerite. After growth, the surplus
food is stored in the endoplasm in the form of granules as described is left exposed in the lumen of the digestive organ (Fig. 77, C). If it
is a polycystic or multi-chambered form, the exposed portion becomes
differentiated
into protomerite
and deutomerite,
while
the intra-
cellular portion remains as the epimerite. After growth, the surplus
food is stored in the endoplasm in the form of granules as described food is stored in the endoplasm in the form of granules as described
Fig. 81. — Lymphosporidium trutte Calkins. A. The young sporozoite and its development. 4. Older forms in the muscle-bundles surround-
ing the intestine. C. Still older amoeboid form prior to, and during, spore-formation. Fig. 81. — Lymphosporidium trutte Calkins. A. The young sporozoite and its development. 4. Older forms in the muscle-bundles surround-
ing the intestine. C. Still older amoeboid form prior to, and during, spore-formation. Fig. 81. — Lymphosporidium trutte Calkins. Fig. 81. — Lymphosporidium trutte Calkins. Fig. 81. — Lymphosporidium trutte Calkins. rozoite and its development. 4. Older forms in the muscle-bundles surround-
Still older amoeboid form prior to, and during, spore-formation. The young sporozoite and its development. 4. Older forms in the muscle-bundles surround-
intestine. C. C.
Foop-TAKING Still older amoeboid form prior to, and during, spore-formation. A. The young sporozoite and its development. 4. Older forms in the muscle-bundles surround-
ing the intestine. C. Still older amoeboid form prior to, and during, spore-formation. above, to be used during the process of spore-formation and encyst-
ment. In Sarcosporidiida and other muscle-infesting Sporozoa, growth above, to be used during the process of spore-formation and encyst-
ment. In Sarcosporidiida and other muscle-infesting Sporozoa, growth In Sarcosporidiida and other muscle-infesting Sporozoa, growth
takes place at the expense of the muscle-cells, although the organisms
are not intra-cellular parasites. Thus, Lymphosporidiuim trutte begins
to grow in the lymph surrounding
the intestine. The sporozoite
develops into a small amceboid form which penetrates the muscle- THE PROTOZOA 148 bundles, and there grows to adult size by absorbing food destined for
the muscles. When mature, it leaves the muscle-bundles and returns
to the lymph-spaces, where it sporulates (Fig. 81). D. Morion Schewiakoff ('94) offered
an
explanation,
based upon
actual observation
and experiment,
and
although very improbable at first sight, it is the only one thus far that
fits the case (Fig. 82). These observations have been confirmed re-
cently by Siedlecki (00), who accepts Schewiakoff's interpretation,
while Lauterborn also gives a similar interpretation of the movement
in diatoms. According to Schewiakoff, the forward, gliding motion
is the result of the active secretion of the gelatinous substance from
the ectoplasm, which accumulates below the membrane to form a
gelatinous layer. The membrane of the cell, as described above, is
marked externally by clear longitudinal grooves, and the gelatinous
substance after filling these grooves, instead of spreading over the
surface of the membrane, flows down and backward in the grooves
to the posterior end of the body, where the secretion from different
furrows unites to form larger currents, and these, in turn, form still
larger streams, which, like a spider’s web, solidify upon leaving the
body(D). Thus, a solid cylinder is formed behind the animal, the pos-
terior end of which fits into the basin-like depression like a cast in its
mould. Theaddition of new jelly by active secretion in the ectoplasm,
and the resistance of the solidified portion, causes a forward move-
ment of the animal. The movement, Schewiakoff further observes,
is only periodic, for the flowing of the jelly is more rapid than the
secretion — a fact which explains the occasional absence of the external
gelatinous layer. D. Morion In addition to the amceboid motion which has already been men-
tioned, there are various movements, due to the contraction of the
myonemes or of the entire ectoplasm. Among these may be men- er
ees
OPER
RS
how
+
a as
4
ee
Fig. 82. — Cortical modifications and movement of a gregarine. [SCHEWIAKOFF.]
A. Moving gregarine with paths of excreted granules. 8 and D, The same, more highly mag-
nified. Cand Z. Details of structure. c, cortical plasm;
7, jelly-layer; ™, myoneme; 5, secretion. er
ees
OPER
RS
how er
ees
OPER
RS
how +
a as
4
ee
Fig. 82. — Cortical modificati +
a as
4
ee
Fig. 82. — Cortical modification Fig. 82. — Cortical modifications and movement of a gregarine. [SCHEWIAKOFF.] Fig. 82. — Cortical modifications and movement of a gregarine. [SCHEWIAKOFF.]
A. Moving gregarine with paths of excreted granules. 8 and D, The same, more highly mag-
nified. Cand Z. Details of structure. c, cortical plasm;
7, jelly-layer; ™, myoneme; 5, secretion. Fig. 82. — Cortical modifications and movement of a gregarine. [SCHEWIAKOFF.]
A. Moving gregarine with paths of excreted granules. 8 and D, The same, more highly mag-
nified. Cand Z. Details of structure. c, cortical plasm;
7, jelly-layer; ™, myoneme; 5, secretion. A. Moving gregarine with paths of excreted granules. 8 and D, The same, more highly mag-
nified. Cand Z. Details of structure. c, cortical plasm;
7, jelly-layer; ™, myoneme; 5, secretion. THE SPOROZOA 149 tioned the peristaltic contraction of certain Gregarinida, or the apeti
getic jerking motion sometimes observed in the same forms. None
of these movements, however, brings about a regular translation from
place to place, and the Sporozoa are regarded as the most sluggish
of the Protozoa. T*ood-seeking, which in free-living animals is the
main occasion for locomotion, is here unnecessary;
for the adult
animals, placed in the chyle of the host, or in the spaces between
cells and tissues, or in the cells themselves, have little occasion for
movement, save that which, in young forms, is necessary to reach the
host, to maintain their positions, and to prevent displacement. There
is, however, in certain forms of Gregarinida, a peculiar gliding motion
on the part of the adult organism. This is accomplished without
apparent exertion of any kind by the animal, and for a long time was
a puzzle to students of the group. D. Morion Schewiakoff ('94) offered
an
explanation,
based upon
actual observation
and experiment,
and
although very improbable at first sight, it is the only one thus far that
fits the case (Fig. 82). These observations have been confirmed re-
cently by Siedlecki (00), who accepts Schewiakoff's interpretation,
while Lauterborn also gives a similar interpretation of the movement
in diatoms. According to Schewiakoff, the forward, gliding motion
is the result of the active secretion of the gelatinous substance from
the ectoplasm, which accumulates below the membrane to form a
gelatinous layer. The membrane of the cell, as described above, is
marked externally by clear longitudinal grooves, and the gelatinous
substance after filling these grooves, instead of spreading over the
surface of the membrane, flows down and backward in the grooves
to the posterior end of the body, where the secretion from different
furrows unites to form larger currents, and these, in turn, form still
larger streams, which, like a spider’s web, solidify upon leaving the
body(D). Thus, a solid cylinder is formed behind the animal, the pos-
terior end of which fits into the basin-like depression like a cast in its
mould. Theaddition of new jelly by active secretion in the ectoplasm,
and the resistance of the solidified portion, causes a forward move-
ment of the animal. The movement, Schewiakoff further observes,
is only periodic, for the flowing of the jelly is more rapid than the
secretion — a fact which explains the occasional absence of the external
gelatinous layer. tioned the peristaltic contraction of certain Gregarinida, or the apeti
getic jerking motion sometimes observed in the same forms. None
of these movements, however, brings about a regular translation from
place to place, and the Sporozoa are regarded as the most sluggish
of the Protozoa. T*ood-seeking, which in free-living animals is the
main occasion for locomotion, is here unnecessary;
for the adult
animals, placed in the chyle of the host, or in the spaces between
cells and tissues, or in the cells themselves, have little occasion for
movement, save that which, in young forms, is necessary to reach the
host, to maintain their positions, and to prevent displacement. There
is, however, in certain forms of Gregarinida, a peculiar gliding motion
on the part of the adult organism. This is accomplished without
apparent exertion of any kind by the animal, and for a long time was
a puzzle to students of the group. E. REPRODUCTION The most characteristic phenomena connected with the Sporozoa
are those
of reproduction and development. The many methods
occurring in the other forms of Protozoa are here limited to spore-
formation, although Labbé describes rather questionable simple divi- Fig. 83. — Types of spores. [WASIFLEWSKY; A. SCHNEIDER; THELOHAN, etc.]
A, Fimerianepe. B,C. Lar oussia orvata,
2, Tailed-spore of Gregarine. £2, F. Ophryocystis
Biitschlii, with multiple epispores. G. Ceratomyxa spherulosa,
HH, Klossia helices. 1. Crysfallo-
spora thélohan,
F. Leptotheca agilis. K. Alvxobolus ellipoides,
L. Crystallospora crystalloides. M. Goussia clupearum,
N, Adelea ovata,
Coiled threads are shown in Gand ¥, the extruded
thread in A’ Fig. 83. — Types of spores. [WASIFLEWSKY; A. SCHNEIDER; THELOHAN, etc.]
Fimerianepe. B,C. Lar oussia orvata,
2, Tailed-spore of Gregarine. £2, F. Ophryocy Fig. 83. — Types of spores. [WASIFLEWSKY; A. SCHNEIDER; THELOHAN, etc.]
A, Fimerianepe. B,C. Lar oussia orvata,
2, Tailed-spore of Gregarine. £2, F. Ophryocystis
Biitschlii, with multiple epispores. G. Ceratomyxa spherulosa,
HH, Klossia helices. 1. Crysfallo-
spora thélohan,
F. Leptotheca agilis. K. Alvxobolus ellipoides,
L. Crystallospora crystalloides. M. Goussia clupearum,
N, Adelea ovata,
Coiled threads are shown in Gand ¥, the extruded
thread in A’ THE SPOROZOA
- 151 sion in Coccidiida (Fig. 6, p. 20). Spore-formation is almost invariably
preceded by encystment, an exception being found in the Gymno-
sporea and Myxosporidiida,
In general, it may be stated that the entire organism takes part in sion in Coccidiida (Fig. 6, p. 20). Spore-formation is almost invariably
preceded by encystment, an exception being found in the Gymno-
sporea and Myxosporidiida,
In general, it may be stated that the entire organism takes part in In general, it may be stated that the entire organism takes part in
the formation of archispores (or sforoblasts), each archispore gives
rise to spores, and each spore to sporozoites, either directly or indi-
rectly. Each spore, containing from one to many sporozoites, is coated
by either a single or a double membrane. When double, the inner
membrane is called the exdospore, and the outer the efzspore (Fig. 83,
f). The spores may be of similar or dissimilar size (#acrospores and
mucrospor:s), they may be ovoid,
spherical,
biconvex,
cylindrical,
crystalline, discoid, etc., in form, and may be provided with diverse
kinds of appendages, ridges, spines, etc., or with polar capsules con-
taining protrusible filaments (Myxosporidiida). In some cases there
is a special apparatus for the dissemination of the spores (sporoducts,
Fig. 1 See, however, Thélohan, ’95. E. REPRODUCTION 85); in other cases, the spores are liberated by the simple
bursting of the outer envelope, or by the rupture of the walls through
swelling of a residual protoplasmic mass termed a pseudocyst. The process of spore-formation in the gregarine of an ascidian The process of spore-formation in the gregarine of an ascidian
— Monocystis ascidieé
— may be given as an example of a type common
to all Sporozoa, although in the several orders the details are vari-
ously modified. Two animals come together and form a common
cyst (Fig. 84, 4). The nucleus of each divides by repeated mitoses
into a great number of daughter-nuclei, which soon arrange themselves
about the periphery (2, C) like the nuclei of a centrolecithal egg of
some Metazoa. A portion of the endoplasm is then budded off about
each of the daughter-nuclei, the buds thus formed becoming conju-
gating gametes. The bulk of the original cells is not used in this pro-
cess, a considerable portion which Labbé (’96) regards as a reserve
store of nutriment! remaining unused (7hetlungskorper, Cystenrest,
Réliquat de ségmentation). During this process the ectoplasm and
the membrane in each cell disappear, leaving the gametes and the cen-
tral residual masses within the cyst(D). The gametes now conjugate
two by two (£) to form the spores (sporocysts). Each of the spores,
which from their peculiar shape are known as pseudonavicclle, now in
its turn secretes two distinct membranes (epispore and endospore), and
within these the nucleus, with its surrounding plasm, divides into eight
parts which are disposed quite regularly in the spore (/). As in the
formation of the archispores, a portion of the plasm is usually left
unused (Sporenrest, Restkorperchen, Réliquat de différentiation). Teach
of these parts is a sporozoite, which, after a developmental period,
reproduces an adult gregarine. When mature, the spores or pseudo-
navicellz are liberated by the bursting of the outer cyst-walls, brought
1 See, however, Thélohan, ’95. - THE PROTOZOA 152 about either by the simple rupture of the wall or by the swelling of
the central mass of useless material. E. REPRODUCTION Sporulation of the Coc- about.a central residual mass, lie in the centre, the unused portion
of the original protoplasm forming a thick layer about them. At
the same time, a third and very delicate membrane, probably com-
posed of the residual peripheral mass, is formed inside of the cyst
and against the second or inner coating. Six to eight radial thicken-
ings can be seen later in this residual portion, and each of these
develops
a distinct lumen, thus becoming tubular and extending
through the residual mass of protoplasm to the new internal mem-
brane. Each tube expands at the extremity into a disc-like cup, while
the inner part of the tube is lost in the central mass of spores. In
some unexplained way the walls of the primary cyst open, leaving the
protoplasm and the spores
inclosed only by the third
membrane. The
tubes
already formed then evag-
inate, and the cylindrical
portion of the
tube
is
thrown
to
the
outside. The tubes act as spore-
ducts for the inner archi-
spores,
each
of
which
contains the definite num-
ber of sporozoites (Fig. 85). Sporulation of the Coc-
cidiida is strikingly similar
to that of the Gregarinida. Here, as a rule, only one
membrane
(capsule)
is
Fig. pope of Gamocystis tenax,
[A. A
CHNEIDER.]
formed around the spheri-
d, spore ducts;
5s, spores
in an external
gelatinous
cal animal; and the nu-_
mantle. cleus,
in
addition
to
division through mitosis, frequently fragments into as many pieces as
there are to be archispores (fragmentation). Before the nucleus divides,
a certain amount of the chromatin is given off, as in the Gregarinida,
to form what Labbé calls the equivalent of the ‘polar body” of
the Metazoa. Again, as in the Gregarinida, the archispores or sporo- about.a central residual mass, lie in the centre, the unused portion
of the original protoplasm forming a thick layer about them. At
the same time, a third and very delicate membrane, probably com-
posed of the residual peripheral mass, is formed inside of the cyst
and against the second or inner coating. Six to eight radial thicken-
ings can be seen later in this residual portion, and each of these
develops
a distinct lumen, thus becoming tubular and extending
through the residual mass of protoplasm to the new internal mem- Fig. pope of Gamocystis tenax,
[A. E. REPRODUCTION The spores are thus freed, but
not the sporozoites; the latter are still confined within their double
walls, and cannot be liberated until they are swallowed by some host,
where, in the digestive tract, the two coatings are dissolved off by the
digestive fluids, and the sporozoites emerge in the form of minute
elliptical bits of protoplasm, each containing a nucleus. rinida. Fig. 84. Scheme of sporulation in gregarinida. A. Union of two individuals in a common cyst. Band C. The formation of gametes of similar
size. DD. Union of the amceboid gametes. # and F. Formation of sporozoites in the fused
gametes. The process of spore-formation in the many-chambered Gregarinida
is more complicated. Thus in C/lepsidrina, a frequent parasite of
insects, the organism when mature throws off the epimerite by which
it is attached to an epithelial cell of its own host and, as a sporont,
secretes its cysts and undergoes nuclear division as in AZonocysits. The encysted animal, however, is carried to the exterior with the
feeces of the host, and sporulation is outside of the host or exogenous,
as opposed to the endogenous sporulation of Monocystis. In these
excreted cysts, according to Schneider (’75) and Biitschli (’84), the
archispores, instead of, as in A/onxocystzs, forming a peripheral layer THE SPOROZOA 193 about.a central residual mass, lie in the centre, the unused portion
of the original protoplasm forming a thick layer about them. At
the same time, a third and very delicate membrane, probably com-
posed of the residual peripheral mass, is formed inside of the cyst
and against the second or inner coating. Six to eight radial thicken-
ings can be seen later in this residual portion, and each of these
develops
a distinct lumen, thus becoming tubular and extending
through the residual mass of protoplasm to the new internal mem-
brane. Each tube expands at the extremity into a disc-like cup, while
the inner part of the tube is lost in the central mass of spores. In
some unexplained way the walls of the primary cyst open, leaving the
protoplasm and the spores
inclosed only by the third
membrane. The
tubes
already formed then evag-
inate, and the cylindrical
portion of the
tube
is
thrown
to
the
outside. The tubes act as spore-
ducts for the inner archi-
spores,
each
of
which
contains the definite num-
ber of sporozoites (Fig. 85). E. REPRODUCTION CHNEIDER.]
d, spore ducts;
5s, spores
in an external
gelatinous
mantle. Sporulation of the Coc-
cidiida is strikingly similar
to that of the Gregarinida. Here, as a rule, only one
membrane
(capsule)
is
Fig. pope of Gamocystis tenax,
[A. A
CHNEIDER.]
formed around the spheri-
d, spore ducts;
5s, spores
in an external
gelatinous
cal animal; and the nu-_
mantle. cleus,
in
addition
to
division through mitosis, frequently fragments into as many pieces as
there are to be archispores (fragmentation). Before the nucleus divides,
a certain amount of the chromatin is given off, as in the Gregarinida,
to form what Labbé calls the equivalent of the ‘polar body” of
the Metazoa. Again, as in the Gregarinida, the archispores or sporo-
cysts are arranged around the periphery, and a residual mass occu-
pies the centre. The archispores which are liberated by simple rupture
of the walls of the cyst, form a definite number of sporozoites, varying
from one (onvzotc) or two (dizorc) to many ( folyzoic). In some forms
of Coccidiida either sporozoites or archispores may be formed directly. The number of spores formed is usually small, as in Cocc¢dium, where
the nucleus divides only twice, producing only four archispores, each Fig. pope of Gamocystis tenax,
[A. CHNEIDER.]
d, spore ducts;
5s, spores
in an external
gelatinous
mantle. THE PROTOZOA 154 of which gives rise to two sporozoites. 1n other cases (viz. Pfeifferta
Labbé), a great number of nuclear divisions may take place, and the
final daughter-nuclei with their surrounding protoplasm form sporozo-
ites directly and without an intervening archispore stage. A similar
direct sporozoite-formation takes place among the Haemosporidiida,
the sporozoites being frequently of two kinds, macrosporozoites ard
microsporozoites. While not established, it is probable that in all
forms this dimorphism in the spores has a sexual significance, the same
individual giving rise to only one form. One peculiarity of these
sporozoites is that the nucleus is apparently never provided with a
nuclear membrane, the chromatin, as in some flagellates, lying freely
in the plasm. Sporulation in the tribe Gymnosporea takes place without the pro-
tection of a cyst. The parasite rounds out, but does not secrete a
membrane. The nucleus divides into a great number of parts, which
migrate
to the periphery as in other forms, and there
divide. Sporozoites are formed directly without preliminary spore-stages. E. REPRODUCTION In most
cases there is no means of exit for the spores from the body of the
host until the latter dies. The archispores thus accumulate until
great cysts, sometimes as large as 30 mm. in diameter (Zschokke, ’98, wound filament is the result. The nuclei of the two polar capsules
soon degenerate and disappear, leaving only the capsules with their
threads, which show a striking similarity to those of the nematocysts
of the Coelenterata. The archispore in many
cases develops a
bivalve shell, and in this condition can remain for some time within
the original spore-forming body (fazn-sporob/ast);
or the original
membrane may be thrown
off, leaving the encapsuled spores sus-
pended freely in the endoplasm of the parent organism. In most
cases there is no means of exit for the spores from the body of the
host until the latter dies. The archispores thus accumulate until
great cysts, sometimes as large as 30 mm. in diameter (Zschokke, ’98, Fig. 86.— Myxodolus ; capsule-formation,
[THELOHAN.]
A-D. Division of the sporoblast nucleus. . The sporoblast is divided into two “ sporogenous
masses” each containing three nuclei. G. Sporogenous mass with protoplasm of the spore and two
masses which are destined to develop into capsules and filaments. A. The threads are first seen
as buds in the vacuole. Fig. 86.— Myxodolus ; capsule-formation,
[THELOHAN.] A-D. Division of the sporoblast nucleus. . The sporoblast is divided into two “ sporogenous
masses” each containing three nuclei. G. Sporogenous mass with protoplasm of the spore and two
masses which are destined to develop into capsules and filaments. A. The threads are first seen
as buds in the vacuole. A-D. Division of the sporoblast nucleus. . The sporoblast is divided into two “ sporogenous
masses” each containing three nuclei. G. Sporogenous mass with protoplasm of the spore and two
masses which are destined to develop into capsules and filaments. A. The threads are first seen
as buds in the vacuole. A-D. Division of the sporoblast nucleus. . The sporoblast is divided into two “ sporogenous
masses” each containing three nuclei. G. Sporogenous mass with protoplasm of the spore and two
masses which are destined to develop into capsules and filaments. A. The threads are first seen
as buds in the vacuole. Myxobolus bicaudatus), are formed within the tissues of their host. 1For discussion of various views see Gurley (’93). E. REPRODUCTION An entirely different mode of sporulation occurs in the Myxospori- An entirely different mode of sporulation occurs in the Myxospori-
diida, where the process is somewhat similar to the internal budding
of some of the Ciliata. In the genus A/yrobolus, for example, one of
the numerous nuclei of the amoeboid form is surrounded by a thick-
ened mass of protoplasm, so that it can be distinguished from the
remainder of the animal. The thickened plasm soon forms a mantle
about the nucleus, which then divides by mitosis until there are ten or
a dozen daughter-nuclei within the specialized protoplasmic region
(Thélohan, ’95; Gurley, ’93). This mass, the sporoblast, which, how-
ever, does not quite correspond to the archispores of preceding types,
now divides into two equal parts, both of which remain inside of the
original protoplasmic mass. Each is an archispore, and each con-
tains three of the ten nuclei. The other four nuclei are left in the
free plasm within the membrane and soon degenerate and disap-
pear, corresponding, apparently, to the residual mass of chromatin
(polar body) of other forms. Each archispore next divides into three
cells (Biitschli, Balbiani for J7yxrobolus), two of which are destined to
form peculiar thread-bearing capsules known as the polar capsules. The other is much larger and represents the definitive spore. Each
sporoblast thus contains one spore, whose nucleus soon divides to
form the two nuclei which characterize the young myxospore. The
formation of the thread in the polar capsules according to Thélohan
((95) seems to be the same in all species; a vacuole appears in each
of the smaller cells of the sporoblast (Fig. 86), then a small knob-like
projection grows up from one side of the vacuole, whose outer walls
harden until a distinct capsule is formed. The bud of protoplasm
within the vacuole now elongates and winds around until a spirally THE SPORKOZOA 155 wound filament is the result. The nuclei of the two polar capsules
soon degenerate and disappear, leaving only the capsules with their
threads, which show a striking similarity to those of the nematocysts
of the Coelenterata. The archispore in many
cases develops a
bivalve shell, and in this condition can remain for some time within
the original spore-forming body (fazn-sporob/ast);
or the original
membrane may be thrown
off, leaving the encapsuled spores sus-
pended freely in the endoplasm of the parent organism. E. REPRODUCTION When taken into a new host, the shell of the archispore under suitable
excitant, either chemical or physical, soon opens, and the filaments
contained within the capsules are thrown out, according to Balbiani,
.through special apertures, and by the pressure of the capsular walls
(Biitschli). These filaments, several times the length of the spore,
remain attached, their free ends being swayed about by the currents
until they come in contact with and penetrate some cell of the mucous
membrane of a new host. The parasite thus anchored retains its
position in the lumen until its bivalve shell is thrown off and it can
move for itself (Leuckart, Biitschli, Gurley ’). In the Myxosporidiida, therefore, sporulation is not the final act of In the Myxosporidiida, therefore, sporulation is not the final act of
a cell-parasite, but takes place while the animal is performing other THE PROTOZOA 156 normal vegetative functions. It is a case of cellular division of labor
in which possibly some of the multiple nuclei are specially differ-
entiated for reproduction. The number of archispores formed in the
pan-sporoblasts varies in the different species and genera. In some
cases
there is but one (Chloromyxum), in others a large number
(Glugea). The polar capsules, which are particularly characteristic of
this type of Sporozoa, also vary in number and in position. They
may both be at one end of the spore (anterior), as in A/yrobolus (Fig. 83, K), at the two ends, as in AZyxzdium, or in the centre, as in
Ceratomyxa (Fig. 83, G). The Sarcosporidiida resemble the Myxosporidiida in forming spores The Sarcosporidiida resemble the Myxosporidiida in forming spores
throughout life. The peculiar pouch, which corresponds to the ame-
boid body of the Myxosporidiida and which may grow to a consider-
able length (up to 16 mm. in sheep), is filled with masses of nucleated. protoplasm which may be called pan-sporoblasts. Those in the
centre of the pouch become coated by a membrane and divide into a
number of germs or sporozoites known as Raiuey'’s Corpuscles, which
in some cases appear to have polar thread-bearing capsules similar to
those of the Myxosporidiida. The life-history and mode of infection
of new hosts is unknown. Conjugation is a well-authenticated phenomenon in at least three Conjugation is a well-authenticated phenomenon in at least three
orders: Hzmosporidiida, Gregarinida, and Coccidiida, although the
observations have not been numerous
enough
to warrant further
generalizations. E. REPRODUCTION Among the Hzmosporidiida, where the intra-cellular
parasites frequently leave their cell-hosts, there is a shorter or longer
period of free life. During this period two individuals, upon meeting,
fuse together, forming one individual (Labbé). The nuclei also
fuse, forming a single nucleus. It is an instance of total conjugation,
similar to the total fusion in some Monadida, but, unfortunately, the
significance of the process and the bearing upon the life-history of
the individuals are entirely unknown. The union of two individuals within a common cyst is not infre- The union of two individuals within a common cyst is not infre-
quently observed among the Gregarinida, and has been a long-known
phenomenon. Two or more individuals may join end to end, pro-
tomerite to deutomerite, or side to side, and so form aggregates (Fig. 27, p. §8). If the individuals thus associated happen to be mature
at the same time, they may develop a common cyst and so give the
appearance of conjugation. Such pseudoconjugation frequently leads
to the formation of catenoid colonies, where the protomerite of one
(satellite) becomes attached to the deutomerite of another ( frimz7te). We are indebted to Wolters (’91), Siedlecki (’96, ’98, ’99), and We are indebted to Wolters (’91), Siedlecki (’96, ’98, ’99), and
Schaudinn
(‘96, ’99) for more complete accounts
of conjugation
among the Gregarinida and Coccidiida. According to the former,
two gregarines (A/onocystis agtlis) place themselves end to end, but THE SPOROZOA 157 without fusing. The nuclei of the two cells then divide by mitosis,
and in each case one of the daughter-nuclei is thrown off as a useless
moiety in the same way asa
polar globule. The other two daughter-
nuclei move toward the partition wall which separates the two
individuals, and meet each other in an opening of this wall. They
fuse, and this fused mass divides by mitosis, one of the daughter-
halves going to each of the conjugants. The nuclei then divide
repeatedly, and spores
are formed in the usual
manner. This
method if correctly observed, in contradistinction to pseudoconjugation
among the Gregarinida and Hzemosporidiida, is nuclear conjugation
as seen in its highest development among the Infusoria; but, unfortu-
nately, there are no observations similar to those of Biitschli, Engel- Fig. 87.— Afonocystis ascidie Lankest. [SIEDLECKI.]
A, Fusion of two individuals. &. Formation of gametes (cy. Fig. 84). C. E. REPRODUCTION Nuclear division after
the fusion of gametes, and sporozoite formation, Fig. 8 Fig. 87.— Afonocystis ascidie Lankest. [SIEDLECKI.]
A, Fusion of two individuals. &. Formation of gametes (cy. Fig. 84). C. Nuclear division after
the fusion of gametes, and sporozoite formation, mann, Maupas, and others on Infusoria to indicate the significance,
and the facts themselves rest upon the observation of a single observer
(Wolters). In a closely related form (Monocysits ascrdie), Siedlecki
(99) describes an entirely different process. Here two individuals
come together in a single cyst, within which each forms a number of
merozoites or gametes. The gametes fuse together, and thus affect
the conjugation of the two original indivicuals (Fig. 87). The greatest advances in our knowledge of the reproduction in The greatest advances in our knowledge of the reproduction in
Sporozoa, during the last ten years, have been in connection with the
Coccidiida, and modern research has shown that the life-history of these
forms is bound up with a complicated alternation of generations, the
product of the union of sex-cells being the permanent spores by
which the infection is carried from one organism to another, while
the products of asexual increase lead to auto-infection within the
same host. Up to the last five years the usual description of the life-history of Up to the last five years the usual description of the life-history of 158 THE PROTOZOA Coccidiida followed that of Leuckart (’79) in the case of Cocctdium
oviformis, a parasite of the rabbit. According to this view, the
adult Cocctdiuim, which consists of a globular or oval mononucleate
parasite, living in the epithelial cells of the digestive tract and the
related organs, encysts and falls into the lumen of the digestive
tract, from which it is defecated with the faeces. Inside of the cyst
the plasm divides into several parts (in Cocczd¢’um four), and these
parts, after the formation of a firm, resisting membrane, form the
permanent spores. Each spore divides into two parts in Coccrdium,
and these two
parts constitute the end product of reproduction,
according to the older view. Each of the parts forms a germ or
sporozoite, which penetrates a new cell-host and develops again to
the adult organism. This cycle, while perfectly logical, left unexplained the immense This cycle, while perfectly logical, left unexplained the immense
multitudes
of parasites
found
in the epithelial
cells
of every
Coccidium-infected
rabbit. E. REPRODUCTION Since then, the fact of fertilization, with the resulting formation of
sporozoites through spores, has been safely established for a number
of species by several different observers, the details alone differing
in the several cases. The microsporozoites thus are not true sporo-
zoites, but gametes having a sexual function. According to these various observations the life-history of the Coc- According to these various observations the life-history of the Coc-
cidiida may now be described as follows: the permanent cysts contain
spores, each of which contains sporozoites which are taken into the
digestive tract with the food. Here the cyst membrane bursts or is
dissolved, and the sporozoites
are liberated. They penetrate the
epithelial cells and grow to the normal size of the adult. They then
undergo repeated nuclear division by a process which resembles frag-
mentation rather than mitosis (Schaudinn), or (possibly) in some cases
by binary division also (Labbé), and the nuclear parts wander out to
the periphery, where small portions of the cytoplasm form around
them and they are pinched off as minute germs which Simond called
merozoites. These differ in several important respects from the
sporozoites, but like them are capable of developing directly into new
adult parasites. This process, which Schaudinn
calls schzzogony,
leads to the increase of parasites within the host (Fig. 88, a-c). Dur-
ing development, some of these merozoites store up reserve nutriment
and form large ovoid cells, while others form the mother-cells of the
microsporozoites or spermatozoids, without storing up a reserved food
supply. The small forms, in some cases, are provided with flagella,
which were first made out by Léger and by Wasielewski. _ Fertiliza-
tion takes place in a manner almost identical with that of the Metazoa
(d
7). In some cases a micropyle is formed in the egg through
which a spermatozo6n can enter, and in all cases after one has
entered, a hard membrane corresponding to the vitelline membrane is
at once formed (£). Complete fusion takes place between the nuclei,
and the cleavage nucleus divides by repeated mitosis to form spores. It is quite probable that the other cases of dimorphism, which have
been recorded from time to time, are instances of similar sex-differen-
tiation. The motile forms especially, which numerous
observers
have recorded, will probably be found
to be similar in function. E. REPRODUCTION The
first attempt
to explain
its wide
distribution
was made by R. Pfeiffer (’92), who insisted that, in
addition to this exogenous spore-formation, there exists an internal
reproduction as well, which leads to further infection in the same
host, or, as he called it, to auto-infection. In contrast with the first
method, this was called the czdogenous sporosoite-formation. This
view was based upon the discovery by Pfeiffer of spore-forming cells
in the tissues of the host, in addition to those in the lumen of the
digestive tract. The majority of investigators along this line have
accepted the latter view. There are two notable exceptions, how-
ever, one of whom is Labbé, who holds that these smaller forms are
only poorly fed individuals and not sporozoites, and explains the
undeniable auto-infection through simple division of the parasites. A large number of papers soon followed, some on Coccidium, others A large number of papers soon followed, some on Coccidium, others
on related forms. Mingazzini (’92) followed out the multiple division
of the nucleus
in the formation
of the endogenous
sporozoites. Podwyssozki (94) made the discovery that there are two kinds of
these endogenous forms, which were accordingly named mcrosporo-
zoites and macrosforozoites. It was Schuberg (95), however, who. first suggested, although he did not confirm the suggestion, that
these two forms of sporozoites conjugate and thus lead to sexual
reproduction. Labbé strongly opposed the latter view, and held that
the larger types of supposed dimorphic spores belong to some un-
known species of Coccidiida, and that the smaller forms are degen-
eration types. The way was thus prepared for the discovery of conjugation among The way was thus prepared for the discovery of conjugation among
the cells of the Coccidiida, a discovery made first by Schaudinn and
Siedlecki (’97). In two different species, Cocczdiuim Schneider and
Adelea ovata, it was found that a large cell, an egg, is fertilized by a THE SPOROZOA 159 small one, which has all of the characteristics of a spermatozoén. In
the same year Simond worked out again the life-history of the Coc-
cidium of the rabbit and described a true copulation between the
microsporozoites. Schaudinn regards this, however, as an error,
holding that copulation takes place between one of the smaller forms
and an enlarged ordinary individual. E. REPRODUCTION Lavéran (98), Bosc (’98), Sjébring (’97), Wasielewsky (’98), Sied-
lecki (’98), and others have recently described them in different
Coccidiida (Fig. 89). 160
_
DHE PR 160
_
DHE PR DHE PROTOZOA 160 Conjugation in the malaria-causing organism (Plasmodium malaria)
is bound up with a change of hosts, thus giving a complicated life-
history, which may, and probably does, occur in other kinds of
Sporozoa as well, although the phenomenon has been only recently
made known. Fig. 88. — Life-history of a Coccidium,
[SCHAUDINN.]
a, 6, c, schizonts and asexual reproduction (schizogony). The merozoites at ¢ repeat the cycle
or pass on to the following stages. d,e, f development of the female or macrogamete. 4, 2, /,
development of the male flagellated gametes; g, copulation of the male and female gametes; &
and J, stages in the formation of the four spores and sporozoites. The sporozodn which is now positively known to be the cause of
“malarial disease,” lives in the human blood under various forms, Fig. 88. — Life-history of a Coccidium,
[SCHAUDINN.]
and asexual reproduction (schizogony). The merozoites Fig. 88. — Life-history of a Coccidium,
[SCHAUDINN.]
a, 6, c, schizonts and asexual reproduction (schizogony). The merozoites at ¢ repeat the cycle
or pass on to the following stages. d,e, f development of the female or macrogamete. 4, 2, /,
development of the male flagellated gametes; g, copulation of the male and female gametes; &
and J, stages in the formation of the four spores and sporozoites. The sporozodn which is now positively known to be the cause of
“malarial disease,” lives in the human blood under various forms,
which may possibly be distinct species differing from one another in
the number of spores produced and in the pathogenic effects. | Sev-
eral varieties at least have been described and specially named on
account of minute differences, but it is probable that these can be THE SPOROZOA 161 reduced to three principal types: Plasmodium malaria, P. vivax, and
Laverania malarie. These all agree in having an intra-corpuscular, reduced to three principal types: Plasmodium malaria, P. vivax, and
Laverania malarie. These all agree in having an intra-corpuscular, reduced to three principal types: Plasmodium malaria, P. vivax, and
Laverania malarie. These all agree in having an intra-corpuscular, ed to three principal types: Plasmodium malaria, P. vivax, and
ania malarie. These all agree in having an intra-corpuscular, e,
Fig. 89. E. REPRODUCTION — Conjugation and sporulation in Avossea helicona, Labbé,
[SIEDLECKI.]
A-E, Formation of microgametes. /. Conjugation. G. Union of the male and female nuclei. #H. Formation of spores. e, Fig. 89. — Conjugation and sporulation in Avossea helicona, Labbé,
[SIEDLECKI.]
A-E, Formation of microgametes. /. Conjugation. G. Union of the male and female nuclei. #H. Formation of spores. amoeboid stage during which the body-plasm becomes stored with
melanin granules or metamorphosed hemoglobin. They agree also
in having an intra-corpuscular spore-forming stage (schzzogony, Fig. 90, F, G, L, M), the spores bringing about auto-infection by pene-
M M 162 THE PROTOZOA trating new blood-corpuscles and there repeating the cycle;. and
under certain conditions they all produce flagellated bodies or spores
(Polymitus form, R). They differ in the number of spores that are
formed, although in the same variety the number appears to be incon-
stant, so that mere difference in number cannot be considered a good
specific character. A much more satisfactory means of distinguishing
them lies in the regular periodicity of spore-formation, which accom-
panies well-marked
morbid symptoms in the patient. Thus, in
Plasmodium vivax, spore-formation occurs every forty-eight hours (ap-
proximately); in P. malari@, every seventy-two hours. The pyrexial
attacks in all cases consist of similar symptoms, a stage of fever
following one of chill and followed by a stage of perspiring. Spore-
formation is the signal for a chill in the patient. The pigment
granules (melanin) become aggregated in the centre of the cell,
while
the protoplasm breaks up into spores about them. The
blood-corpuscle which contains the parasite then disintegrates, and
the spores and melanin are liberated. It is supposed that this dis-
ruption of the corpuscle, by liberating a toxin (melanin) created and
stored up by the parasite, is the direct cause of the attack. If this
hypothesis, which is certainly based upon considerable evidence,
should be verified by future investigation, the malaria-organism will
be the only protozoén known to produce poisonous growth-products. Quinine in the system is apparently fatal to the parasite by preventing
its growth and sporulation. The spores of the malaria organism are not covered by a protective The spores of the malaria organism are not covered by a protective
coating as in the majority of Telosporidia, and are, therefore, unsuited
for an exposed life outside of their host. 1JIn Italy it is estimated that 2,000,000 people are ill every year with malaria (Santori,
1900). Fig. 90. — Life-history of Malaria, causing Sporozoa.
[Ross and FIELDING-OULD.]
A-F, Stages in the development of merozoites.
.4-/. The sporozoite.
4, C, D, and ¥, KL.
The growing sporozoite in blood-corpuscles.
£. Asexual reproduction (schizogony).
F, G, and
MM, Liberation of merozoites and melanin granules.
O-I1’, Stages in the development of sexual
individuals.
FR. Polymitus form.
S, Fully developed microgametes.
7-W. Development of the
female individual (macrogamete).
-\. Fertilization of a macrogamete bya microgamete.
Y. The
fertilized cell copuZa, with its vitelline membrane.
a-e. ‘he copula in the stomach of the mosquito
(Anopheles sp.).
6. The copula penetrating the epithelium which lines the stomach of Anopheles.
6, ¢, d, e. Growth of the copula in the body-cavity of zopheles.
The small sphzerules at /, UW’, and
.V are supposed to be analogous to polar bodies of metazoan eggs. f Sporulation in the body-
cavity of Anopheles.
g. Liberation of the sporozoites.
4. Salivary gland (in section), with sporo-
zoites in the lumen, in the cells, and penetrating the membrane. Fig. 90. — Life-history of Malaria, causing Sporozoa.
[Ross and FIELDING-OULD.]
A-F, Stages in the development of merozoites.
.4-/. The sporozoite.
4, C, D, and ¥, KL.
The growing sporozoite in blood-corpuscles.
£. Asexual reproduction (schizogony).
F, G, and
MM, Liberation of merozoites and melanin granules.
O-I1’, Stages in the development of sexual
individuals.
FR. Polymitus form.
S, Fully developed microgametes.
7-W. Development of the
female individual (macrogamete).
-\. Fertilization of a macrogamete bya microgamete.
Y. The
fertilized cell copuZa, with its vitelline membrane.
a-e. ‘he copula in the stomach of the mosquito
(Anopheles sp.).
6. The copula penetrating the epithelium which lines the stomach of Anopheles.
6, ¢, d, e. Growth of the copula in the body-cavity of zopheles.
The small sphzerules at /, UW’, and
.V are supposed to be analogous to polar bodies of metazoan eggs. f Sporulation in the body-
cavity of Anopheles.
g. Liberation of the sporozoites.
4. Salivary gland (in section), with sporo-
zoites in the lumen, in the cells, and penetrating the membrane. E. REPRODUCTION It was early recognized,
however, that there must be an extra-corporeal period in the life-
history of the parasite in order that the species should be perpetuated. That there actually is such a period is shown by the spread of the
disease throughout a community.! Nevertheless, the whereabouts
of the organism and its form during the extra-corporeal existence
have remained a mystery until within the last few years. The key
to the puzzle has been given by the flagellated body or Polymitus
form. The blood when examined fresh from a malaria patient
shows the ordinary form of the parasite, but after a short period
of exposure
to the
air (10 to
30 minutes,
Manson,
’98), the
parasite develops long flagelliform
processes, which
vibrate with
great vigor and not infrequently break away from the body of
the cell to swim about like Spzri//a in the plasm. Danilewsky
(91) believed this stage to be an independent flagellated parasite
of a special nature, and he named it Polymtus. Lavéran, Metsch- 1JIn Italy it is estimated that 2,000,000 people are ill every year with malaria (Santori,
1900). THE SPOROZOA 163 nikoff, Manson, and others regarded it as an essential develop-
mental stage of the malaria organism, and believed that the free- mental stage of the malaria organism, and believed that the free-
Sry
‘
S
a)
S
#
B
c
D
z
F
G
H
Z-:
Ys
[ace
SS ‘Hage I
ee
ys
Fig. 90. — Life-history of Malaria, causing Sporozoa. [Ross and FIELDING-OULD.]
A-F, Stages in the development of merozoites. .4-/. The sporozoite. 4, C, D, and ¥, KL. The growing sporozoite in blood-corpuscles. £. Asexual reproduction (schizogony). F, G, and
MM, Liberation of merozoites and melanin granules. O-I1’, Stages in the development of sexual
individuals. FR. Polymitus form. S, Fully developed microgametes. 7-W. Development of the
female individual (macrogamete). -\. Fertilization of a macrogamete bya microgamete. Y. The
fertilized cell copuZa, with its vitelline membrane. a-e. ‘he copula in the stomach of the mosquito
(Anopheles sp.). 6. The copula penetrating the epithelium which lines the stomach of Anopheles. 6, ¢, d, e. Growth of the copula in the body-cavity of zopheles. The small sphzerules at /, UW’, and
.V are supposed to be analogous to polar bodies of metazoan eggs. f Sporulation in the body-
cavity of Anopheles. g. Liberation of the sporozoites. 4. E. REPRODUCTION He suggested a
theory that the Polymztus forms are flagellated spores, the extra-
corporeal homologue
of the intra-corporeal
spores. He further
suggested that, since the parasite is normally incased within a blood-
corpuscle, it is unable to leave the host by its own efforts and must
be removed by some blood-eating animal, probably a suctorial insect,
such as a mosquito, common in swampy, malarial regions. At the
same time (’96) Lavéran, in France, proposed an identical hypothesis. Subsequent investigation has given the complete confirmation of this
hypothesis. The work of Major Ross, in India, of Koch, Grassi, and
others, elsewhere, has established beyond a doubt, that extra-corporeal
life of the parasite is spent in the mosquito, and that the disease is
spread by these insects through inoculation. About eight or ten days
after drawing blood from a malaria patient, the insects are able to
transmit the germs to new hosts by inoculation through the proboscis. After a number of experiments, Ross and Grassi found that certain
genera of mosquitoes, e.g. Culex sp., are incapable of fostering the
human parasite, while all species of the genus Axopheles are particu-
larly susceptible. The history of the parasite in the mosquito has been variously in-
terpreted. Manson ('96, ’98), on a@ priori grounds, suggested that the
Polymitus form is developed after the blood is taken from the host
into the colder digestive tract of the insect, the change of medium
acting upon the parasite in the same way that the air does. Here, he
argued, it penetrates an epithelial cell and repeats the life-history
of
an ordinary form, sporulating and increasing by auto-infection. Mac-
Callum (’97), however, described a true conjugation between a Poly. mitus form and a free pigmented parasite in a very similar organism
(Haltertdiuim Labbé), which is parasitic in the blood of the Ameri-
can crow. In this case the impregnated Ha/teridiuim slowly changes
form, becoming elongated and more or less worm-like, and moves
about in the blood-plasm. Thus in this case the Polymdtus form
corresponds to a spermatozoon, and the ordinary individual, as
in
Coccidium, toanegg. Ross and Grassi, finally, have demonstrated the
same relation in Plasmodium malarieé ; the Polymitus form fuses with
an ordinary individual in the intestine of the mosquito, and‘ as in
FHlalteridium,
the copula becomes a motile individual which, after a
short period, penetrates the epithelial cells lining the digestive tract
(Fig. 90, a-e). E. REPRODUCTION Salivary gland (in section), with sporo-
zoites in the lumen, in the cells, and penetrating the membrane. swimming detached processes are germs which reproduce the adult. Sry
‘
S
a)
S
#
B
c
D
z
F
G
H [Ross and FIELDING-OULD.] swimming detached processes are germs which reproduce the adult. Many others (Grassi, Felletti, Celli, Sanfelice, Sacharoff, Labbé, etc.),
however, regarded these forms as degenerating parasites induced
by the abnormal conditions of exposure and not present, normally, in swimming detached processes are germs which reproduce the adult. Many others (Grassi, Felletti, Celli, Sanfelice, Sacharoff, Labbé, etc.),
however, regarded these forms as degenerating parasites induced
by the abnormal conditions of exposure and not present, normally, in THE PROTOZOA 164 the blood. Manson (’96) was one of the first to call attention to the
fact that the formation of these so-called degeneration forms, which
occurs only at the time when the blood is exposed to the air, is
evidence of the beginning of extra-corporeal life. He suggested a
theory that the Polymztus forms are flagellated spores, the extra-
corporeal homologue
of the intra-corporeal
spores. He further
suggested that, since the parasite is normally incased within a blood-
corpuscle, it is unable to leave the host by its own efforts and must
be removed by some blood-eating animal, probably a suctorial insect,
such as a mosquito, common in swampy, malarial regions. At the
same time (’96) Lavéran, in France, proposed an identical hypothesis. Subsequent investigation has given the complete confirmation of this
hypothesis. The work of Major Ross, in India, of Koch, Grassi, and
others, elsewhere, has established beyond a doubt, that extra-corporeal
life of the parasite is spent in the mosquito, and that the disease is
spread by these insects through inoculation. About eight or ten days
after drawing blood from a malaria patient, the insects are able to
transmit the germs to new hosts by inoculation through the proboscis. After a number of experiments, Ross and Grassi found that certain
genera of mosquitoes, e.g. Culex sp., are incapable of fostering the
human parasite, while all species of the genus Axopheles are particu-
larly susceptible. the blood. Manson (’96) was one of the first to call attention to the
fact that the formation of these so-called degeneration forms, which
occurs only at the time when the blood is exposed to the air, is
evidence of the beginning of extra-corporeal life. E. REPRODUCTION Here it resembles one of the Coccidiida, growing at the
expense of the cell-host and finally sporulating. The spores do not
form protective coatings, but divide at once into sporozoites (/, g) THE SPOROZOA 165 These make their way into the body cavity, or lymph spaces, of the
mosquito, and ultimately find their way to the salivary glands, from
which they may be deposited, together with the salivary fluid, in the
blood of man (%). The life-history of a malaria organism thus involves
a complete change of hosts, one phase being in the warm blood of
man, the other a Coccidia-like stage in the Insecta, a group which
above all others is noted for the frequency and number of sporozoan
parasites. It is not improbable that a similar change of hosts occurs
in the parasites belonging to this same group of Hemosporidiida,
which have been observed in birds (Lavéran, Labbé, ’94); in reptiles
(Labbé, ’94; Langmann,
’99); and amphibia (Labbé,
’94; Lang-
mann, ’99); also it is possible that many of the uncertain forms, such
as Serumsporidium of the Crustacea (Pfeiffer, ’95) or Lymmphospo-
vidium of the trout (Calkins, ’99), have a similar complicated life-
history. ‘ Apart from their pathogenic effects
in man, the Sporozoa are
frequently a pest in the lower animals. The Sarcosporidiida have
already been mentioned as producing morbid symptoms resembling
Trichinosis, in the domestic animals often leading to death. The
Myxosporidiida occasion great loss to fish culturists by causing ulcers
which ultimately result in the death of the fish, and to silkworm cul-
turists on account of costly and extensive epidemics produced by
them among the silkworms. These organisms (Glugea bombycis)
were so disastrous to the silk industry during the years 1854-1867,
that a loss was estimated
of at least 1,000,000,000 francs (about
$190,000,000). In regard to this epidemic Huxley ('70) writes: “In
the years following 1853 this malady broke out with such extreme
violence that, in 1858, the silk crop was reduced to a third of the
amount which it had reached in 1853; and, up till within the last year
or two, it has never attained half the yield of 1853. E. REPRODUCTION This means not
only that the great number of people engaged in silk growing are
some 30 millions sterling poorer than they might have been; it means
not only that high prices have had to be paid for imported silkworm
eggs, and that, after investing his money in them, in paying for mul-
berry leaves and for attendance, the cultivator has constantly seen
his silkworms perish and himself plunged in ruin; but it means that
the looms of Lyons have lacked employment, and that, for years,
enforced idleness and misery have been the portion of a vast popula-
tion which, in former days, was industrious and well to do.”
The caterpillars, although infested by the parasites which were The caterpillars, although infested by the parasites which were
frequently so numerous that all of the organs of the body swarmed
with
them, were
nevertheless
able
to produce
the
moth. The
latter, though stunted and undeveloped, could lay eggs which them-
selves contained spores of the organism, and these spread the disease. 166 THE PROTOZOA The disease was checked only by careful examination of the food of
the caterpillar, and by microscopic examination of all eggs and rejec-
tion of the infected ones. No remedy is known for the many other diseases due to Sporozoa, No remedy is known for the many other diseases due to Sporozoa,
especially among domestic animals, or fresh-water fish, and careful
prophylactic measures analogous to those employed in stopping the
silkworm epidemic may be the only means of checking them. Such
measures have already been successfully applied to prevent the spread
of malaria, and the experiments which are now going on in all parts
of the world justify the hope that this disease will be ultimately
stamped out. 1 (83), Pp. 479. 2 (84), p. 807. F. INTER-RELATIONSHIPS OF THE SPOROZOA The Sporozoa, modified beyond doubt by adaptation to a parasitic
mode of life, have ever been a puzzle to systematists. Kolliker (’48)
early suggested that they are single cells, and included them in his
Protozoa. Stein (’48) agreed with him as to their primitive structure,
but was loath to regard them as single animal cells, and compromised
by calling them Symphyta, a group of the Protozoa. Another view,
developed by Henle (’45) and Bruch (’50) and taken up by Leydig
(51) and Leuckart (52), was based upon the superficial resemblance
of the Gregarinida to Nematode worms. It found little support, how-
ever, against Kolliker’s view. Still another theory of the origin of
the Sporozoa has been held by those who, following Gabriel (’75,
’80), regard these forms as plants, placing them with the Mycetozoa,
among the Fungi. Biitschli
at first! favored the view that the
Sporozoa are derived from the Rhizopoda, basing his belief upon the
method of reproduction, general morphology, and physiology. Later,
however,” he considered their relationship to the Flagellidia as much
more close, not to the simplest forms, but to the higher types with a
well-differentiated cuticle. The flagellum and mouth parts, he as-
sumed, became lost with gradual adaptation
to the intra-cellular
mode of life, while the methods of reproduction became specialized in
response to the requirements of a new environment. This view is
strengthened by the close agreement in finer structures of the Grega-
rinida and the Flagellidia, especially as regards the differentiations
of the cuticle and the presence of muscular elements. Their move-
ments, too, recall those of the Flagellidia, especially certain species
of As/asta, where, in the non-flagellated condition, the plasm moves
forward by a peculiar peristalsis, while the secretion of a jelly from
the sub-cuticular or cortical plasm is identical in the two groups. The
nuclei show perhaps a closer resemblance to those of the Rhizopoda 2 (84), p. 807. THE SPOROZOA 167 than to the Flagellidia, but the conjugation processes are much more
like those of the Flageliidia. Haeckel follows Biitschli in regarding
the Sporozoa in this light, and derives them from the Phytoflagellida
through adaptation, first to asaprophytic and then to a parasitic mode
of life. Wasielewsky also favors the flagellate origin, basing his opin-
ion, however, upon the uncertain ground of flagellated swarm-stages
of certain Sporozoa as well as upon the general resemblance to the
Astasiide. CLASSIFICATION Crass III. SPOROZOA. The Sporozoa are Protozoa which are never provided
with flagella or cilia in the adult state. They are always endoparasites in cells,
tissues, or cavities of other animals, and food is taken in by osmosis. Repro-
duction is always by spore-formation, and germs (sforozoites) are produced
either directly from the parent, or indirectly through spores. Subclass I. TELOSPORIDIA. Sporozoa in which spore-formation ends the indi- Crass III. SPOROZOA. The Sporozoa are Protozoa which are never provided
with flagella or cilia in the adult state. They are always endoparasites in cells,
tissues, or cavities of other animals, and food is taken in by osmosis. Repro-
duction is always by spore-formation, and germs (sforozoites) are produced
either directly from the parent, or indirectly through spores. Subclass I. TELOSPORIDIA. Sporozoa in which spore-formation ends the indi- Subclass I. TELOSPORIDIA. Sporozoa in which spore-formation ends the indi-
vidual life, the entire cell then forming spores. Order 1. GREGARINIDA. Telosporidia
possessing a distinct membrane, with Subclass I. TELOSPORIDIA. Sporozoa in which spore-formation ends the indi-
vidual life, the entire cell then forming spores. Order 1. GREGARINIDA. Telosporidia
possessing a distinct membrane, with Order 1. GREGARINIDA. Telosporidia
possessing a distinct membrane, with
myonemes during adult life, locomotion being accomplished mainly by their
contraction. The young stages alone (cepfhalonts) are intra-cellular parasites,
the adults (sforonts) being found in the digestive tract or the body cavities. Sporulation takes place after or without conjugation, but within a cyst which is
never formed while the parasite is intra-cellular. Suborder 1. CEPHALINA. Gregarinida
possessing
an
organ
for attachment Suborder 1. CEPHALINA. Gregarinida
possessing
an
organ
for attachment
(epimerite), and with or without septa dividing the cell into chambers. Tribe 1. Gymnosporea. The adults are solitary or associated; the sporozoites are Tribe 1. Gymnosporea. The adults are solitary or associated; the sporozoites are
formed directly from the adult without encystment. Family 1. Aggregatide. Colonies consisting of two or more individuals. Several Family 1. Aggregatide. Colonies consisting of two or more individuals. Several
residual
protoplasmic
masses
are
found during sporulation in each cyst. Genera: Aggregata Frenzel (’85). Family 2. Porosporide. The individuals
are usually solitary. The sporozoites Family 2. Porosporide. The individuals
are usually solitary. The sporozoites
are arranged in groups around a central residual mass. Genera: Porospora
A. Schn. (75). Tribe 2. Angiosporea. Cephalina with well-developed spores, which are provided Tribe 2. Angiosporea. F. INTER-RELATIONSHIPS OF THE SPOROZOA In general, however, it must be admitted that there is
very little support for any one of these theories, and all attempts to
trace the origin of the Sporozoa upon the mere basis of their present
degenerate condition are highly speculative. than to the Flagellidia, but the conjugation processes are much more
like those of the Flageliidia. Haeckel follows Biitschli in regarding
the Sporozoa in this light, and derives them from the Phytoflagellida
through adaptation, first to asaprophytic and then to a parasitic mode
of life. Wasielewsky also favors the flagellate origin, basing his opin-
ion, however, upon the uncertain ground of flagellated swarm-stages
of certain Sporozoa as well as upon the general resemblance to the
Astasiide. In general, however, it must be admitted that there is
very little support for any one of these theories, and all attempts to
trace the origin of the Sporozoa upon the mere basis of their present
degenerate condition are highly speculative. CLASSIFICATION Cephalina with well-developed spores, which are provided
-with spore-membranes (efispores and exdospores). Family 1. Didymophyide. Chain-forming aggregates, two individuals being so Family 1. Didymophyide. Chain-forming aggregates, two individuals being so
closely joined as to appear like one with three chambers. Genera: Didymophyes
Stein ("48). Family 2. Gregarinide. The individuals are solitary or associated. The epimerite Family 2. Gregarinide. The individuals are solitary or associated. The epimerite
is simple and regular. The cysts may or may not have spore-ducts. Genera:
Gregarina
Dufour
(’28);
Gamocystis
A. Schn. (°75);
A@rmocystis Léger
(92); Myalospora A. Schn. (75); Euspora A. Schn. (75); Sph@rocystis
Léger (’92) ; Cnuemidiophora A. Schn. (82) ; Stenophora Labbé (99). Family 3. Dactylophoride. The epimerite is asymmetrical and irregular. Genera: Family 3. Dactylophoride. The epimerite is asymmetrical and irregular. Genera:
Rhopalonia Léger (93); Echinomera Labbé (‘99) ; Trichorhynchus A. Schn. (82) ; Pterocephalus A. Schn. (87); Dactylophorus Balbiani (°89). Family 4. Actinocephalide. The individuals are always single. The epimerite is Family 4. Actinocephalide. The individuals are always single. The epimerite is 168 THE PROTOZOA simple or lobed and symmetrical. The cysts open by simple dehiscence. The
spores are boat-shaped, bi-conical, or cylindro-conical. Genera: Sciadiophora
Labbé
('99); Anthorhynchus Labbé ('99);
Pileocephalus
A. Schn. (75);
Amphoroides .Labbé ('99);
Discorhynchus Labbé (99);
Stectospora Léger
(93);
Schneiderta
Léger (92);
Asterophora Léger (’92); Stephanophora
Léger (92); Bothriopsis A. Schn. (75); Coleorhynchus Labbé (’99); Actzno-
cephalus Stein (48); Pyacnza Hammerschmidt (38); Légerza Labbé (99) 3
Phialotdes Labbé (’99) ; Belozdes Labbé (’99). F amily 5. Acanthosporidez. Solitary. The spores are provided with equatorial EF amily 5. Acanthosporidez. Solitary. The spores are provided with equatorial
or polar spines. Genera: Corycella Léger ('92); Acanthospora Léger ('92);
Ancyrophora Léger ('92); Cometotdes Labbé (’99). Family 6. Menosporide. Solitary. The epimerite is on a long neck. The spores Family 6. Menosporide. Solitary. The epimerite is on a long neck. The spores
are crescent-shaped. Genera: Menosfora Léger (92); Hoplorhynchus Carus
(°63). Family 7. Stylorhynchide. The spores are formed in chains, and the cysts have a Family 7. Stylorhynchide. The spores are formed in chains, and the cysts have a
double envelope. Genera: Lophocephalus Labbé (’99); Cystocephalus A. Schn. (86); Oocephalus A. Schn. (86); Spherorhynchus Labbé (99); Stylorhyn-
chus Stein (48). Family 8. Doliocystida. Cephalina without septa dividing the cell into protomerite Family 8. Doliocystida. Cephalina without septa dividing the cell into protomerite
and deutomerite, but consisting of a single chamber with epimerite. CLASSIFICATION Genera:
Doliocystis Léger (’93). Suborder 2. ACEPHALINA. Gregarinida consisting of a single chamber, and with- Suborder 2. ACEPHALINA. Gregarinida consisting of a single chamber, and with-
out epimerite. They are parasites in the body cavity or cavities of the various
organs of different
animals. Genera:
Monocystzs
Stein
(’48);
Zygocystis
Stein (48); Zygosoma Labbé ("99); Prerospora Racovitza and Labbé ('96);
Cystobia Mingazzini (91); Léthocystés Giard (76); Ceratospora Léger (’92) ;
Urospora A. “Schn. (75);
Gonospora A. Schn. (°75);
Syucystes A Schn. (86). Order 2. COCCIDIIDA. Telosporidia having a spherical or oval form, without a free Order 2. COCCIDIIDA. Telosporidia having a spherical or oval form, without a free
and motile adult stage, and never amceboid. Sporulation takes place within
cysts formed while the organism is an intra-cellular parasite. Family 1. Disporocystide. The cell forms two sporocysts, each sporocyst forming Family 1. Disporocystide. The cell forms two sporocysts, each sporocyst forming
two or four sporozoites. Genera: Cyclospora A. Schn. (°81), with two sporo-
zoites ; /sospora A. Schn. (’81), and D7plospora Labbé (’93), with four or more
sporozoites. Family 2. Tetrasporocystide. Each organism forms four sporocysts, each of which Family 2. Tetrasporocystide. Each organism forms four sporocysts, each of which
produces two sporozoites. Genera: Coccédium Leuckart (°79) (including Gous-
sza Labbé) ; Crystallospora Labbé (‘96). Family 3. Polysporocystide. Each organism produces an indefinite number of
sporocysts, each of which produces one sporozoite, — [Barrouxia A. Schn. (85),
Diaspora Léger (99)], two sporozoites, — [4delea A. Schn. (°75), and some
species of Hyaloklossia Labbé (’96)], three sporozoites, — [Benedenia A. Schn. (75), or four, Genus A7ossia A. Schn. ((75)]. Order 3. HEMOSPORIDIIDA. Sporozoa of small size living in the blood-corpuscles
or plasm of vertebrates. The adult form is mobile, and in some cases is pro-
vided with myonemes. They reproduce by endogenous or asexual
spore-
formation while in the host, and by exogenous spore-formation after conjugation. Genera: Lankesterella Labbé (99); Caryolysus Labbé ( 94); Hemogregarina
Danilewsky (85); Caryophagus Steinhaus (89); Haltertdium Labbé (94);
Hemoproteus Kruse (90); Plasmodium Marchiafava & Celli (°85); Laverania
Grassi & Feletti (92); Cytamaba Labbé (794). Subclass II. NEOSPORIDIA. Sporozoa which form sporocysts throughout life; the Subclass II. NEOSPORIDIA. Sporozoa which form sporocysts throughout life; the
entire cell is not used in the formation of spores. 169 THE SPOROZOA Order 1. MYXOSPORIDIIDA. Neosporidia of amceboid or spherical shape; multi-
nuclear. CLASSIFICATION The initial free stage is passed in the cavities of the organs, or in the
tissues of the host. In sporulation a definite or an indefinite number of sporo-
blasts is formed, each of which gives rise to one or several spores; the latter are
provided with one or several polar capsules, which contain coiled threads like a
nematocyst. Each spore gives rise to one amoeboid sporozoite. Suborder 1. PHAXNOCYSTINA. Spores with polar capsules distinctly visible when Suborder 1. PHAXNOCYSTINA. Spores with polar capsules distinctly visible when
fresh. Family 1. Myxidiidea. Myxosporidiida forming two or more spores at the same Family 1. Myxidiidea. Myxosporidiida forming two or more spores at the same
time. Spores
variable
in form
inclosing
two
polar capsules. Genera:
Spherospora Thélohan (92); Leptotheca Thél. ('95); CeratomyxaThél. (92) ;
MUyxidium Biitschli (82); Spheromyxa Thél. (92); Cystodiscus Lutz (89):
Myxosoma Thél. (’92). Family 2. Chloromyxide. The spore has four polar capsules. Genera: Chloro- Family 2. Chloromyxide. The spore has four polar capsules. Genera: Chloro-
myxum Mingazzini (90). Family 3. Myxobolide. Adult stages very rare, ordinarily found encysted in the Family 3. Myxobolide. Adult stages very rare, ordinarily found encysted in the
tissues; usually polysporous. The spores have one
or two polar capsules. Genera: Myxobolus Biitschli (82); Henneguya Thél. (’92). Suborder 2. MICROSPORIDIINA. Myxosporidiida in which the spores have but one Suborder 2. MICROSPORIDIINA. Myxosporidiida in which the spores have but one
polar capsule, which is invisible in the fresh state without the use of reagents. Family 1. Mosematéde. With a bivalve spore. Genera: Mosema Nageli (’57) Family 1. Mosematéde. With a bivalve spore. Genera: Mosema Nageli (’57)
(Glugea Thélohan, ’92); Péstophora Gurley (93); Thélohania Henneguy (’92)-
Order 2. SARCOSPORIDIIDA. Sporozoa in which the initial stage is passed in Order 2. SARCOSPORIDIIDA. Sporozoa in which the initial stage is passed in
muscle-cells of vertebrates. The form is usually elongate, tubular or oval, or
sometimes spherical. It forms cysts with a double membrane, in which are
formed kidney-shaped or falciform sporozoites, or else spores (?), provided with
a polar capsule and projectile thread. Genera: Sarcocystzs Lankester (’82). SPOROZOA INCERTA! SEDIS Amebosporidia. Sporozoa possessing an amceboid body, and reproducing either by
division or by spore-formation after conjugation. Genera: Ophryocystis A. Schn. (784). Serumsporidia. Sporozoa which reproduce by division (?) or by spore-formation, Amebosporidia. Sporozoa possessing an amceboid body, and reproducing either by
division or by spore-formation after conjugation. Genera: Ophryocystis A. Schn. (784). Serumsporidia. Sporozoa which reproduce by division (?) or by spore-formation, Serumsporidia. Sporozoa which reproduce by division (?) or by spore-formation,
the sporozoites being minute oval or spherical bodies
They are found in the
cavities or coelomic fluids of Invertebrates and Vertebrates. Genera: Serum-
sporidium L. Pfeiffer (95); Blanchardina Labbé saan Lymphosporidium
Calkins (1900). SPECIAL BIBLIOGRAPHY V Gurley, R. R. — The Myxosporidia, or Psorosperms of Fishes, and the Epidemics pro-
duced by them. Bull. U. S. Fish Comm. X1., 1893. Labbé, A.— Recherches zoologiques, cytologiques et biologiques sur les Coccidies. Gurley, R. R. — The Myxosporidia, or Psorosperms of Fishes, an
duced by them. Bull. U. S. Fish Comm. X1., 1893. Labbé, A.— Recherches zoologiques, cytologiques et biologiqu Labbé, A.— Recherches zoologiques, cytologiques et biologiques sur les Coccidies. Arch. da. zool. expér. et gén. (3) IV., pp. 517-654, 1896. Labbé, A.— Sporozoa. In Das Tierreich, Bertin, 1899. Labbé, A.— Sporozoa. In Das Tierreich, Bertin, 1899. Légér, L.— Recherches sur les Gregarines. Zad/ettes zoolo Légér, L.— Recherches sur les Gregarines. Zad/ettes zoologiques, III., pp. 1-182, 1892. Leuckart R.— Die Parasiten des Menschen. Lezfzzg, 1879. Légér, L.— Recherches sur les Gregarines. Zad/ettes zool
Leuckart R.— Die Parasiten des Menschen. Lezfzzg, 1 Légér, L.— Recherches sur les Gregarines. Zad/ettes zoologiques, III., pp. 1-18
Leuckart R.— Die Parasiten des Menschen. Lezfzzg, 1879. Leuckart R.— Die Parasiten des Menschen. Lezfzzg, 1879. Ross, D. — On Some Peculiar Pigmented Cells found in Two M Ross, D. — On Some Peculiar Pigmented Cells found in Two Mosquitoes fed on Mala-
rial Blood. Brit. Med. and Surg. Jour., 1897. pp. 1786-1788. Schneider, A. — Sur les eae oviformes ou Coccidées. espéces nouvelles ou Schneider, A. — Sur les eae oviformes ou Coccidées. espéces nouvelles ou
peu connues. Arch. d. sool. expér. et gen. (1) 1X., pp. 387-404, 1881. Siedlecki, M. — Etude cytologique et cycle évolutif de la coccidie de laseiche. Anz. Siedlecki, M. — Etude cytologique et cycle évolutif de la coccidie de laseiche. Anz. d. U Inst. Pasteur, X11., 1898. THE INFUSORIA “Die Infusionsthiere gehdren in den Kreis der Protozoen. Innerhalb desselben bilden
sic eine eigene und zwar die am héchsten stehende Klasse.”
— STEIN.1 As was long since clearly recognized by Stein, the Infusoria are
the most highly differentiated of all Protozoa and often attain a
degree of complexity which
is perhaps greater than in any other
cells. Their form varies considerably in the several divisions, but all
are characterized by certain structural features by which they can be
distinguished at a glance. All are provided with cilia which may
be retained throughout
life (Cz/ata), or may be replaced in the
adult phases by suctorial tentacles, cilia being present only during
the embryonic phases (Swctorta); they possess mouth parts which
are adapted for swallowing,
for simple ingestion, for sucking, or
which may be entirely degenerate through parasitism; and they are
provided with two kinds of nuclei, known as macronuclei and mitcro-
nuclet. They reproduce by simple division and by budding, or rarely
by spore-formation. THE PROTOZOA THE PROTOZOA 170 Siedlecki, M.-- Ueber die geschlechliche Vermehrung der Monocystis ascidie R. Lank. Bull. d. ?Acad. d. Sct. a. Cracovie, 1899. Thélohan, P. — Recherches sur les Myxosporidies. Bzd/. Sct. de la France et dela Thélohan, P. — Recherches sur les Myxosporidies. Bzd/. Sct. de la France et dela
Belgique, XXVI, pp. 101-394, 1895. Wasielewsky,
Von. — Sporozoenkunde, ein Leitfaden
fiir Aerzte
Tierarzte
und Wasielewsky,
Von. — Sporozoenkunde, ein Leitfaden
fiir Aerzte
Tierarzte
und
Zoologen. ena, 1896. I. THE CILIATA Among the Ciliata the arrangement of the cilia upon the body
affords a character which was first used by Stein (’59), and is still
retained as a means of distinguishing the subdivisions of this group. In the first and probably the most primitive type, Ho/otrichida, the
cilia are arranged uniformly over the entire body of the animal and
show no regional differentiations (Fig. 91, A). In the second type,
Hieterotrichida, the cilia are uniform over the main portion of the
body, while a specialized set fused into a curved series of firm vibra-
tory plates, or membranelles, are found in an adoral zone about the
mouth (4). In the third type, Hypotrichida,
the body is flattened
dorso-ventrally and the dorsal side is entirely free from cilia, while on
the ventral side the cilia are frequently fused together into stiff seta-
like organs, the czvrz, and as in the Heterotrichida, they may form
a curved
line of membranelles around the mouth
(C)._
Finally,
in the Perttrichida, the highest type of this class, the cilia are reduced
to one or two bands or girdles in addition to the adoral zone (J). Although, with the exception of the motile organs, no single item Although, with the exception of the motile organs, no single item
of structure is found here which is not occasionally met with in other 1 (59)s P» 54. 171 171 THE PROTOZOA 172 classes of Protozoa, yet in no other class are they all present in a
single cell. Each of the different elements thus brought together has
a definite function to play in the life of the organism, and intra-cellular
division of labor is developed to a high degree. Leading an active life and forced to seek food in all sorts of places, Leading an active life and forced to seek food in all sorts of places,
from the clearest waters to the internal fluids of various hosts, the
Ciliata have acquired a very great diversity of form. The simplest
and probably the most primitive forms are monaxonic, the mouth
being anterior and the anus posterior (Fig. 91, 4). Symmetry, how-
ever, is the exception and asymmetry the rule, the latter condition
arising by the gradual shifting of the mouth to'a more or less well- Fig. gt. — Types of Ciliata. [BUTSCHL1.]
A. Prorodon teres Ehr.; an holotrichous form. 8. Climacostomum virens Ehr.;
an hetero-
trichous form. C. I. THE CILIATA Pleurotricha grandis St.,; an hypotrichous form. LD, Vortscedla umbellaria; a
peritrichous form,
2, adoral zone. Fig. gt. — Types of Ciliata. [BUTSCHL1.]
A. Prorodon teres Ehr.; an holotrichous form. 8. Climacostomum virens Ehr.;
an hetero-
trichous form. C. Pleurotricha grandis St.,; an hypotrichous form. LD, Vortscedla umbellaria; a
peritrichous form,
2, adoral zone. defined ventral side, while the anus becomes more or less dorsal. The functional anterior end may thus be either ventral, or superior to
the mouth, when the latter
becomes
sub-terminal. The simple
monaxonic ground-type is subject to other minor variations among
the Holotrichida, which point the way toward the more
striking
deviations among the Heterotrichida and the Hypotrichida. A fre-
quent modification is the anterior prolongation of what might be con-
sidered the upper lip, as in Dilepius or Livnotus (Fig. 92), where
bilaterality and asymmetry are well established. The mouth becomes
more and more ventral in the family Trachelinidz (Holotrichida),
while in the order Hypotrichida it is always ventral and the original
monaxonic structure is replaced by dorso-ventral differentiation and
complete asymmetry. defined ventral side, while the anus becomes more or less dorsal. The functional anterior end may thus be either ventral, or superior to
the mouth, when the latter
becomes
sub-terminal. The simple
monaxonic ground-type is subject to other minor variations among
the Holotrichida, which point the way toward the more
striking
deviations among the Heterotrichida and the Hypotrichida. A fre-
quent modification is the anterior prolongation of what might be con-
sidered the upper lip, as in Dilepius or Livnotus (Fig. 92), where
bilaterality and asymmetry are well established. The mouth becomes
more and more ventral in the family Trachelinidz (Holotrichida),
while in the order Hypotrichida it is always ventral and the original
monaxonic structure is replaced by dorso-ventral differentiation and
complete asymmetry. THE INFUSORIA 173 A. PROTOPLASMIC STRUCTURE There
are
also various fats and
excretory products
either crystalline or granular in form. The
crystals, according to Schewiakoff ('93), are granules of calcium
phosphate."
Among the pigmented inclusions of the cell must be
noted the so-called “ eye-spots”’ or stigmata, which, in some instances,
are accompanied by lenticular differentiations of various kinds. These
pigmented spots are, as a rule, mere heaps of granules colored either
red, brown, black, or orange, and are probably the same in function
as the similar products in Mastigophora. The protoplasm becomes more dense toward the periphery, and, carried food-products in various stages of digestion and assimilation,
as well as excretory products in the form of granules similar in all
respects to those found in other Protozoa. Engelmann (’83) found
that in one of the Vorticellidz (Vorticella campanula) the animal is
colored by diffuse green pigment, which he took to be chlorophyl,
and he further showed that oxygen is generated and that the animal
can assimilate like a plant, but that it is not limited to this kind of
nutrition, since it also takes in solid food through the mouth. Other
colored particles which are found in various kinds of Ciliata, espe-
cially in those forms which subsist upon plant food, are presumably
due to the coloring matter contained in the food. Schewiakoff (’89) has
shown that the colored balls which appear in some cases are merely
fluid drops colored with the pigment contained in Oscz//aria and other
vegetable cells. In many cases, also, green algal cells live as sym-
bionts within the endoplasm. Le Dantec (’92) found that these cells
(Zoochlorella) are apparently taken in as food and become inclosed
within gastric vacuoles, the fluids of which have no effect upon them. Soon the vacuoles disappear and the alge are left free in the plasm,
where they live and multiply. There
are
also various fats and
excretory products
either crystalline or granular in form. The
crystals, according to Schewiakoff ('93), are granules of calcium
phosphate."
Among the pigmented inclusions of the cell must be
noted the so-called “ eye-spots”’ or stigmata, which, in some instances,
are accompanied by lenticular differentiations of various kinds. These
pigmented spots are, as a rule, mere heaps of granules colored either
red, brown, black, or orange, and are probably the same in function
as the similar products in Mastigophora. 1Cf. p. 286, znfra. A. PROTOPLASMIC STRUCTURE As in all other Protozoa, the endoplasm consists of alvecli of vary-
ing size and arrangement, the network being built up of plasm of
greater or less density. Within the endoplasm there is a constant
streaming of the granules, which varies greatly in the different species
and even in the same individual under different conditions. In some AN
LAL
Ltt)
EeCCe
COCO
eC
Orne
Loni
COPPA
OO
N HN
BN
18h
AS:
SEAN
yiN\ _-m
WOON
LH4
Ts
Aa
Ve SSSsEak
Fig. 92.— Dileptus anser O. F. M. [BUTSCHLL]
m, mouth; AV, macronucleus; 4, trichocysts on the tentacle-like end. Fig. 92.— Dileptus anser O. F. M. [BUTSCHLL]
m, mouth; AV, macronucleus; 4, trichocysts on the tentacle-like end. cases the course of the streaming recalls cyclosis in some plant-cells,
and frequently follows a well-defined and unvarying route (Co/poda
cucullus, or Paramecium bursaria). In all cases the current seems
to start from the mouth, passing backward around the cell either to
the right or left, then forward and back to the mouth (Bitschli). In this stream, circulating through the body of the animal,
are THE PROTOZOA 174 carried food-products in various stages of digestion and assimilation,
as well as excretory products in the form of granules similar in all
respects to those found in other Protozoa. Engelmann (’83) found
that in one of the Vorticellidz (Vorticella campanula) the animal is
colored by diffuse green pigment, which he took to be chlorophyl,
and he further showed that oxygen is generated and that the animal
can assimilate like a plant, but that it is not limited to this kind of
nutrition, since it also takes in solid food through the mouth. Other
colored particles which are found in various kinds of Ciliata, espe-
cially in those forms which subsist upon plant food, are presumably
due to the coloring matter contained in the food. Schewiakoff (’89) has
shown that the colored balls which appear in some cases are merely
fluid drops colored with the pigment contained in Oscz//aria and other
vegetable cells. In many cases, also, green algal cells live as sym-
bionts within the endoplasm. Le Dantec (’92) found that these cells
(Zoochlorella) are apparently taken in as food and become inclosed
within gastric vacuoles, the fluids of which have no effect upon them. Soon the vacuoles disappear and the alge are left free in the plasm,
where they live and multiply. A. PROTOPLASMIC STRUCTURE The protoplasm becomes more dense toward the periphery, and, The protoplasm becomes more dense toward the periphery, and,
as in the Sarcodina, it finally becomes too compact for the granules
to penetrate. This outer portion is, therefore, comparable to the
ectoplasm of the less differentiated Protozoa. The importance of
this layer is seen in the fact that nearly all of the organs which
characterize the Ciliata, including the myonemes, cilia, membranes
and membranelles, the trichocysts, nematocysts, and the complex
membranes and tests, are modifications of, or are produced by, the
ectoplasm. In some forms the thickened plasm immediately adjoin-
ing the endoplasm is distinctly marked off from the more external
portions, forming a continuous layer around the entire body. This
layer, which is in reality neither ectoplasm nor endoplasm, but inter-
mediate between the two, is called the cortical plasm (Rindenparen-
chyma, Stein), and is characterized by the reduced size and number of
its vacuoles, by the absence of granules and streaming motion, and THE INFUSORIA 175 by its fixity in the cell. It is occasionally thickened to form the
denser ends of the body, as in the tail of Sveztor. In some cases,
also, processes from the cortical plasm invade the endoplasm to
surround the nucleus and hold it in a fixed position in the body
(Dasytricha). The cortical plasm, furthermore, is the seat of the
peculiar and characteristic offensive and defensive trichocysts. In
some forms, probably representing the primitive condition, these
are distributed about the body (Paramecium), but, even more than
the cilia, they have been subject to reduction in most parts of the
body until, in the majority of forms, they are restricted to a limited
area, while in the Hypotrichida and Peritrichida they occur only
sporadically. In Prorodon (Holotrichida), the trichocysts are found
in the anterior end only, and in the family Zvacheltnide they are
found only on the ventral side, while in Zrachelzus, Dileptus, etc., they
are on the ventral side of the anterior process (Fig. 92). In Lzonotus,
they are reduced to a single line along the ventral side of the anterior
process. The trichocysts are so minute that their finer structure has not The trichocysts are so minute that their finer structure has not
been definitely made out, although a few different types have been
studied (Fig. 12, C, p. 39). 1See ante, p. 50. 1 Cf. Stein (’59), p. 56. A. PROTOPLASMIC STRUCTURE Rod-like forms have been seen in Loxophyl-
lum, Lionotus, and Strombidium, and spindle forms in Paramecium,
Frontonta and Nassula. When protruded from the body they are,
for the most part, apparently of the same size and shape as when
within the ectoplasm. Occasionally when protruded, however, they
have small hooks or swellings on the end (Maupas). They vary in
size from three to twelve microns when within the body, but when
protruded they measure from thirty to sixty microns. The cause of
the protrusion is unknown;
certain reagents
act as irritants and
cause them to explode and throw out the long threads. Their func-
tion, too, is purely conjectural, although it is generally supposed that
they serve as defensive weapons. In some cases they appear to serve
as weapons of offence as well, especially in those ciliates where they
are limited in number to a comparatively few large ones. According
to Maupas (’83), these Infusoria chase their prey and launch their
trichocyst darts, which penetrate the outer coating of the victim and
paralyze
it, possibly through the action
of some
noxious
fluid. Forms much larger than the hunters are frequently brought down in
this way, to be swallowed either whole or piecemeal. The attack is
not necessarily fatal, for the larger forms frequently revive.? The position of the trichocysts is primarily in the cortical plasm, The position of the trichocysts is primarily in the cortical plasm,
but they are rarely entirely immersed, being much more frequently
suspended in the streaming endoplasm. They are occasionally drawn 176, THE PROTOZOA out from the cortical plasm and are then carried about in the
stream. In addition to the trichocysts, some of the Ciliata carry still more In addition to the trichocysts, some of the Ciliata carry still more
effective weapons in the form of nematocysts. In Vorticella umbel-
laria (Clap. & Lach.), Engelmann described from twelve to twenty
pairs of capsules, each of which contained a coiled thread which, as
in the Ccelenterata, could be thrown out upon irritation. The cortical plasm may be considered the inner portion of the in the Ccelenterata, could be thrown out upon irritation. The cortical plasm may be considered the inner portion of the
ectoplasm, the outer portion of which forms the covering of the
animal, the membrane, cuticle, or pellicle. The latter is extremely
variable in thickness and in complexity. A. PROTOPLASMIC STRUCTURE It is apparently homolo-
gous with the ex-
ternal
membrane
of ordinary animal
cells, and, accord-
ing to Biitschli, is
formed by conden-
sation of the pro-
toplasmic
ground
substance, and, as
Stein
(59)
first
maintained,
is in
Ns
no sense a secre-
“® ww
tion.)
Bitschli
:
/ \
(88),
Schuberg
(87),
and
many
C
others regarded it
;
as
the agglutina-
Fig. 93.— Coleps hirtus Ehr. [MAUuPAS.]
tion
uter
A. Side. B&. One of the 15 rows of plates composing the test. . of the
oute
C. Division-phase. thickened lamelle
of
the
external
alveoli into a continuous membrane. In forms where the cortical
plasm is absent, as in Hypotrichida, the cuticle,
or, as
Biitschli
prefers to call it, the fed/cle, forms
a thin coating to the cell
and lies directly upon the endoplasm. In many cases the outer
protoplasm either becomes changed into, or else secretes, an external
casing or house, which may be either loose or tight-fitting. This
covering may be of jelly (¢.g. Ophrydium), or of chitin (e.g. Folli-
culina), or of a horny product without any mineral elements (e.g. Coleps). In Coleps hirtus (Ehr.) the horn-like covering is tight-fitting,
and composed of separate pieces, which form four girdles about the
body (Fig. 93). Each girdle is composed of separate pieces, each of
which is straight on one edge and serrated upon the other in such a
1 Cf. Stein (’59), p. 56. the inner portion of the
forms the covering of the
. The latter is extremely
It is apparently homolo-
gous with the ex-
ternal
membrane
of ordinary animal
cells, and, accord-
ing to Biitschli, is
formed by conden-
sation of the pro-
toplasmic
ground
substance, and, as
Stein
(59)
first
maintained,
is in
s
no sense a secre-
ww
tion.)
Bitschli
/ \
(88),
Schuberg
(87),
and
many
C
others regarded it
as
the agglutina-
tion
uter
the test. . of the
oute
thickened lamelle
of
the
external
forms where the cortical
e cuticle,
or, as
Biitschli
thin coating to the cell
In many cases the outer
or else secretes, an external
oose or tight-fitting. This
), or of chitin (e.g. Folli-
any mineral elements (e.g. ike covering is tight-fitting,
orm four girdles about the
of separate pieces, each of
upon the other in such a
. A. PROTOPLASMIC STRUCTURE THE INFUSORIA 177 manner that, when they are put together, the serrations slightly over-
lap the straight edge of the next adjacent piece, thus leaving open-
ings for the protrusion of the cilia. The lower end is covered by
separate pieces, which open centrally for excretion. On the anterior
end, the mouth, crowned by a ring of large cilia, is protected by a
set of plates with teeth-like projections, which act somewhat like the
similarly arranged teeth of a sea-urchin. In a great many cases the outer body wall is marked by striations In a great many cases the outer body wall is marked by striations
of various kinds showing the lines of insertion of the cilia. A
typical example is shown in the membrane of
Holophrya, one of the Holotrichida (Fig. 91, A,
and 97, g)
Here the cilia are inserted
in
regular lines which run from the anterior to
the posterior end. In other cases, as in Lem-
badion (Holotrichida), the cilia are inserted on
minute papille, which
lie in rows upon the
cuticle with more or less distinct furrows be-
tween them, thus forming secondary, but very
distinct, markings in addition to the primary
lines formed by the insertion points of the cilia. That the striation is due to the ciliation can
be easily seen in cases where the cilia are
absent from one portion of the body and pres-
YS
ent in others, as in many of the Holotrichida. Fig. 94.—Lacrymaria coronata
The rows are not always straight, as in Holo- Se Pusletia et
phrya, but are variously changed through the
alteration of the axial relations. The most
frequent variation from the primitive condition is the spiral arrange-
ment (e.g. Lacrymaria coronata, Fig. 94), where the course of the
cilia has become changed by the alteration in the position of the
mouth. A very curious type of striation
is seen
in Dasytricha
ruminantum (Schuberg, ’88) (Holotrichida), where the striations do
not converge at the mouth as they do in the majority of forms, but
in a line above it. The exception is significant, however, as showing
the line of the shifting of the mouth, the path being marked by the
meeting points of the converging striz (Fig. 95). The external markings were early recognized by Ehrenberg, who A YS
Fig. 94.—Lacrymaria coronata
Se Pusletia et Fig. A. PROTOPLASMIC STRUCTURE The cortical plasm may be considered the inner portion of the
ectoplasm, the outer portion of which forms the covering of the
animal, the membrane, cuticle, or pellicle. The latter is extremely
variable in thickness and in complexity. It is apparently homolo-
gous with the ex-
ternal
membrane
of ordinary animal
cells, and, accord-
ing to Biitschli, is
formed by conden-
sation of the pro- Ns
“® ww
:
/ \
C
;
Fig. 93.— Coleps hirtus Ehr. [MAUuPAS.]
A. Side. B&. One of the 15 rows of plates composing the test. C. Division-phase. C ;
Fig. 93.— Coleps hirtus Ehr. [MAUuPAS.]
A. Side. B&. One of the 15 rows of plates composing the test. C. Division-phase. alveoli into a continuous membrane. In forms where the cortical
plasm is absent, as in Hypotrichida, the cuticle,
or, as
Biitschli
prefers to call it, the fed/cle, forms
a thin coating to the cell
and lies directly upon the endoplasm. In many cases the outer
protoplasm either becomes changed into, or else secretes, an external
casing or house, which may be either loose or tight-fitting. This
covering may be of jelly (¢.g. Ophrydium), or of chitin (e.g. Folli-
culina), or of a horny product without any mineral elements (e.g. Coleps). In Coleps hirtus (Ehr.) the horn-like covering is tight-fitting,
and composed of separate pieces, which form four girdles about the
body (Fig. 93). Each girdle is composed of separate pieces, each of
which is straight on one edge and serrated upon the other in such a alveoli into a continuous membrane. In forms where the cortical
plasm is absent, as in Hypotrichida, the cuticle,
or, as
Biitschli
prefers to call it, the fed/cle, forms
a thin coating to the cell
and lies directly upon the endoplasm. In many cases the outer
protoplasm either becomes changed into, or else secretes, an external
casing or house, which may be either loose or tight-fitting. This
covering may be of jelly (¢.g. Ophrydium), or of chitin (e.g. Folli-
culina), or of a horny product without any mineral elements (e.g. Coleps). In Coleps hirtus (Ehr.) the horn-like covering is tight-fitting,
and composed of separate pieces, which form four girdles about the
body (Fig. 93). Each girdle is composed of separate pieces, each of
which is straight on one edge and serrated upon the other in such a 1 Cf. Stein (’59), p. 56. A. PROTOPLASMIC STRUCTURE 94.—Lacrymaria coronata
Se Pusletia et The external markings were early recognized by Ehrenberg, who
interpreted them as the insertion points of the cilia as described above. Stein, however, held that the markings are invariably due to the pres-
ence of myonemes which form the insertion base of the cilia. Both
observers were right in part, for striations in some of the Heterotri-
chida and Peritrichida are due to the presence of myonemes, but in the
Holotrichida, where, with one or two exceptions (Ho/ophrya, Prorodon),
myonemes do not occur, the markings are unmistakably due to the cilia. N 178 THE PROTOZOA The myonemes are ectoplasmic differentiations which are contrac-
tile in nature and are formed, according to Butschli and Schewiakoff,
from the walls of the alveoli which make up the sub-cuticular layer of
the membrane. Although probably arising in the peripheral alveolar
region, these threads occasionally become separated from this position
and are then found in the cortical plasm or even in the endoplasm. Myonemes are most highly differentiated and are best known in the
Vorticellidz, where the sudden contraction of the bell, or the instan-
taneous rolling-up of the stalk, are due to their action. While the
most conspicuous myonemes in lortecc//a run from the centre of the
disk to the very base of the stalk, Entz (’91) has described additional
fibrils which have a similar but less important function. According The myonemes are ectoplasmic differentiations which are contrac-
tile in nature and are formed, according to Butschli and Schewiakoff,
from the walls of the alveoli which make up the sub-cuticular layer of
the membrane. Although probably arising in the peripheral alveolar
region, these threads occasionally become separated from this position
and are then found in the cortical plasm or even in the endoplasm. Myonemes are most highly differentiated and are best known in the (OG
A
B
G
D
Fig. 95. — Supposed change of position of the mouth in Ciliata. [BUTSCHLI.]
A. Original position (as in Holophrya),
8B. The mouth has become elongated (as in Exchelys
or Spathidium),
C, Similar stage from the ventral side. J, The mouth has become closed be-
hind, leaving the opening away from the body extremity,
The markings on the membrane now
meet in the line represented by the origina] mouth-slit (e.g. in Glaucoma). A. PROTOPLASMIC STRUCTURE The largest and most important of
all of the myonemes are those forming the fourth set. These are
longitudinal muscle-fibres of considerable thickness running from the
centre of the disk radially toward the periphery, then continuing
down the sides of the bell as far as the ciliary girdle ( Wainperring),
where they leave the wall of the body and come together to form the
thick muscle-strand of the stalk. The latter highly contractile organ
consists of a wall and of the central, contractile strands which are
bathed with a fluid contained within the walls of the stalk. The wall
itself, according to Entz, but contrary to Biitschli, is a continuation of
the living wall of the bell, in which membrane and underlying mus- Fig. 96. — Zoothamnium arbuscula Ebr. [ENTZ.]
A. Lower portion of main trunk. 4. One of the branches of the main trunk. a, axoneme;
p, Spasmoneme;
5s, spironeme, Fig. 96. — Zoothamnium arbuscula Ebr. [ENTZ.]
A. Lower portion of main trunk. 4. One of the branches of the main trunk. a, axoneme;
p, Spasmoneme;
5s, spironeme, Fig. 96. — Zoothamnium arbuscula Ebr. [ENTZ.]
A. Lower portion of main trunk. 4. One of the branches of the main trunk. a, axoneme;
p, Spasmoneme;
5s, spironeme, cular structures can be distinguished, as in the main portion of the
body. Biitschli, on a less substantial basis, described the stalk as a
secretion similar to the stalks of the Mastigophora and Sarcodina, and
chitinous in composition. The main strand within the stalk is formed
by the collection of the strands of the inner longitudinal myonemes,
and is covered by a delicate sheath which separates it from the fluid
or gelatinous matter surrounding it. Biitschli regards this sheath as
a continuous coat from the alveolar layer of the bell. The strand has
three threads which Entz calls spasmoneme, spironeme, and axoneme
(Fig. 96). The fibres of the first run to the base of the stalk. The
other two are closely connected, and both are made up of microsomes,
which Entz described as nucleus-like granules (a7yophans )
surrounded
by an ovoid matrix (cyéophan). These granules, so conspicuous in the
stalks of
Vorticella, evidently correspond to the Elementar-Granula
(Greeff, ’71) or cyto-microsomes. Entz figured them as arranged in cular structures can be distinguished, as in the main portion of the
body. A. PROTOPLASMIC STRUCTURE G OG
B OG
B (O
A B A [BUTSCHLI.] to this observer there are two sets of myonemes, one internal, the
other external. Each set includes two groups of myonemes, one cir-
cular in its course,
the other longitudinal. The external
layer,
observed by Lachmann (’56) and Stein (’59, ’67) but denied by many,
is formed of a large, single fibre composed of fibrillae, which winds
spirally about the bell from the junction of the peduncle to the centre
of the disk. It is this myoneme, Entz maintains, which gives the
annulate appearance to the bell, and, like a muscle-fibre, it is charac-
terized by fine transverse striations. A second circular set is formed
by another single fibre, which, however, is confined to the peristome disk,
and is located deep in the ectoplasm (Fig. 91, D),
This fibre takes
only a few spiral turns at the base of the elevated disk and around the
edge of the collar, and functions as a sphincter-muscle to close over
the disk. Two sets of longitudinal myonemes complete the muscular
system. Of these, the external set, lying between the two circular
myonemes, consists of fine fibres running from the peduncle to the to this observer there are two sets of myonemes, one internal, the
other external. Each set includes two groups of myonemes, one cir-
cular in its course,
the other longitudinal. The external
layer,
observed by Lachmann (’56) and Stein (’59, ’67) but denied by many,
is formed of a large, single fibre composed of fibrillae, which winds
spirally about the bell from the junction of the peduncle to the centre
of the disk. It is this myoneme, Entz maintains, which gives the
annulate appearance to the bell, and, like a muscle-fibre, it is charac-
terized by fine transverse striations. A second circular set is formed
by another single fibre, which, however, is confined to the peristome disk,
and is located deep in the ectoplasm (Fig. 91, D),
This fibre takes
only a few spiral turns at the base of the elevated disk and around the
edge of the collar, and functions as a sphincter-muscle to close over
the disk. Two sets of longitudinal myonemes complete the muscular
system. Of these, the external set, lying between the two circular
myonemes, consists of fine fibres running from the peduncle to the THE INFUSORIA 179 disk where their course is radial. A. PROTOPLASMIC STRUCTURE Biitschli, on a less substantial basis, described the stalk as a
secretion similar to the stalks of the Mastigophora and Sarcodina, and
chitinous in composition. The main strand within the stalk is formed
by the collection of the strands of the inner longitudinal myonemes,
and is covered by a delicate sheath which separates it from the fluid
or gelatinous matter surrounding it. Biitschli regards this sheath as
a continuous coat from the alveolar layer of the bell. The strand has
three threads which Entz calls spasmoneme, spironeme, and axoneme
(Fig. 96). The fibres of the first run to the base of the stalk. The
other two are closely connected, and both are made up of microsomes,
which Entz described as nucleus-like granules (a7yophans )
surrounded
by an ovoid matrix (cyéophan). These granules, so conspicuous in the
stalks of
Vorticella, evidently correspond to the Elementar-Granula
(Greeff, ’71) or cyto-microsomes. Entz figured them as arranged in 180 THE PROTOZOA rows like a string of beads. Without going deeply into the subject,
which is far from settled, it will suffice here to state that two views
are now heldas to the seat of contractility in the stalks of Vortzcella. One set of observers hold that the outer membrane of the stalk is the
contractile portion, and that the contained thread merely counteracts
the force of the membrane, which tends to contract and roll up the
stalk. In other words, the myonemes of the spasmoneme are regarded
as elastic and not as contractile fibrils, at rest when the stalk is coiled,
active when the bell is extended (Cohn, 62; Metschnikoff, 63; Rouget, MO
ee MO
ee
Fig. 97.— Myonemes and cilia. [METSCHNIKOFF, BUTSCHLI, and JOHNSON.)
a, 6, d, e. Cuticle and myonemes of Stentor ceruleus. d. More highly magnified piece of
myoneme. g. Optical section through the body wall of Holophrya discolor. MO
ee Fig. 97.— Myonemes and cilia. [METSCHNIKOFF, BUTSCHLI, and JOHNSON.)
a, 6, d, e. Cuticle and myonemes of Stentor ceruleus. d. More highly magnified piece of
myoneme. g. Optical section through the body wall of Holophrya discolor. 61; Schaaffhausen, ’68; Entz,’91). Entz described the membrane of
the stalk and the spasmoneme as antagonistic elements. The former,
which stretches out while at rest and contracts when irritated, opposes
the latter, which acts in the reversed manner. The axoneme he re-
garded as a sort of nerve-centre. A. PROTOPLASMIC STRUCTURE The opponents of this view hold
that the rolling of the stalk is accomplished by the contraction of
the muscle-like spasmoneme (Stein ’67, Clap. & Lachmann
’58,
Engelmann ’76, Biitschli ’88, etc.). The myonemes lie in minute canals according to Biitschli (’88) and The myonemes lie in minute canals according to Biitschli (’88) and
Schewiakoff
(’89) ;
in direct contact with the plasm according to John-
son (’93) and Entz (’91), and they probably vary in position in differ-
entforms. The structure and position of a myoneme in the ectoplasm
can be more easily seen from the accompanying figure than from a
description (Fig. 97). The myonemes lie in minute canals according to Biitschli (’88) and
Schewiakoff
(’89) ;
in direct contact with the plasm according to John-
son (’93) and Entz (’91), and they probably vary in position in differ-
entforms. The structure and position of a myoneme in the ectoplasm
can be more easily seen from the accompanying figure than from a
description (Fig. 97). THE INFUSORIA 181 Other
contractile
elements
are
occasionally found:
the
most
remarkable, perhaps, is the peculiar muscular band which surrounds
the peristome of Bursaria truncatella. This highly differentiated
muscular organ, which functions as a sphincter, is, like the myonemes,
derived from the alveolar layer immediately below the pellicle. The ectoplasm appears throughout to be the seat of motion. Not
only are the contractile myonemes differentiations of this important
layer, but the cilia and all of their modifications are likewise derived
from it. The cilia themselves appear to be mere prolongations of
the alveolar layer. They are minute, probably of similar diameter
throughout, and except for regional differentiation in the vicinity of
the mouth, are of uniform length. Asa
rule, they are inserted upon
minute elevations or papilla on the cuticle and appear to be connected
by minute fibrils with the myonemes. The finer structure of the cilia
has not been satisfactorily made out, but the present results tend to
the view that they are simple, firm threads without differentiations. Unlike flagella, they act in unison, and their motion is that of a paddle
rather than a lash, as in flagella. Jensen (’93) has figured the absolute
lifting power of the ciliary apparatus of Paramecium at 0.00158 milli-
grammes, or nine times the weight of the animal. The cilia are grouped
together in various ways, forming more or less complex motile organs. 1Johnson (’93) alone regards the membranelles in Sven¢or as endoplasmic in origin. A. PROTOPLASMIC STRUCTURE On the con-
—eo
trary, they arise from the ecto-
_p
._plasm as cirri and not as cilia. ;
aoe
c& We
S& Wht
KI
Wh
i 1
a
S N
This objection, however, seems. a
: Sea ae
hardly sufficient
to counter-
28
e-<.S.S}-poc balance the evidence in favor
a
NING
of the concrescence
theory,
4 EN } ts = >
evidence which is strengthened
Be
A = =
by the position
of the cirri
: aN nY
along the lines of the cilia-
6
Y
aN Ss
markings. “Crp,
The membranelles
are
flat
plates of striated appearance
usually
in the form
of
tri-
a
Sy
Aipy
GZ
~
= cok
IH
angles, squares,
or parallelo-
grams. Each membranelle
is. Fig. 98.— Schematic hypotrichous ciliate. inserted
in
a
furrow
below
az, adoral zone; ¢, ventral cirri;
e, endoral mem- which
is
a_
basal
stripe
of
brane; eo, endoral cilia; pv, praeoral membrane; Zo, thickened protoplasm continu-
paroral cilia; foc, preoral cilia,
ous with the longitudinal
cil-
iary markings (Heterotrichida). Like the cirri, they can be readily reduced
to component
fila-
ments resembling
cilia, and there
is, therefore,
every reason
to
suppose that the membranelles which form such a characteristic
differential for all orders save the Holotrichida, are merely the differ- Cirri, membrdnelles, and membranes are each striated, and when
treated with certain reagents (¢.g. gold chloride, Maupas), can be
reduced to separate fibres which are similar to cilia. The simplest of
these aggregations are the cirri. These bundles of threads
are
usually pointed, and either curved or straight, forming the Griffelx
und Haken of Ehrenberg. A simple condition is seen in the tail-like
process of Uvocentrum, which has a distinctly fibrillar structure and
can
be readily reduced
to a brush of very fine hairs (Fig. 99). -
NL
-\--€72
—eo
_p
;
aoe
c& We
S& Wht
KI
Wh
i 1
a
S N
a
: Sea ae
28
e-<.S.S}-poc
a
NING
4 EN } ts = >
Be
A = =
: aN nY
6
Y
aN Ss
“Crp,
a
Sy
Aipy
GZ
~
= cok
IH
Fig. 98.— Schematic hypotrichous ciliate. A. PROTOPLASMIC STRUCTURE These are rarely seen in Holotrichida, but in the other orders they
may be pointed aggregates (cirri), plate-like vibratile organs (mem-
branelles), or broad, undulating membranes. All of these modifica-
tions are found in the Hypotrichida, where the motile apparatus is
especially characteristic. The arched dorsal surface is without cilia,
but occasionally holds a varying number of bristles which have,
possibly, a sensory function. On the flat, ventral side of the most
primitive forms of this order, the cilia are very generally distributed
(Peritromus, Fig. 113, £), but in the more differentiated forms they
are reduced in number, and modified into cirri, membranes, and mem-
branelles. In many forms they may be entirely absent, the only motile
apparatus being the membranelles on the ventral side about the mouth. These form the adoral zone, which stretches from the mouth forward
on the left side of the peristome, and as far as the dorsal anterior region. In some cases a row of cilia stretches along the floor of the peristome
parallel with the membranelles, a single cilium opposite each mem-
branelle. The right border of the peristome (Fig. 98) is continued
into a vibratile membrane, and close to the left of this and running
parallel with it is another row of cilia (preoral cilia foc). In the
centre of the peristome is a second undulating membrane, the endoral
membrane (em), which passes downward and into the pharynx, and
this, also, is sometimes accompanied by a row of cilia even into the
pharynx (endoral cilia ¢o). 182 THE PROTOZOA Cirri, membrdnelles, and membranes are each striated, and when
treated with certain reagents (¢.g. gold chloride, Maupas), can be
reduced to separate fibres which are similar to cilia. The simplest of
these aggregations are the cirri. These bundles of threads
are
usually pointed, and either curved or straight, forming the Griffelx
und Haken of Ehrenberg. A simple condition is seen in the tail-like
process of Uvocentrum, which has a distinctly fibrillar structure and
can
be readily reduced
to a brush of very fine hairs (Fig. 99). Although the striated appear-
ance and the reduction to the
component fibrils make it prob-
able that
cirri arise by the
fusion of cilia, an objection is
-
met in the fact that their de-
NL
velopment, after division, shows. -\--€72 no such origin. A. PROTOPLASMIC STRUCTURE az, adoral zone; ¢, ventral cirri;
e, endoral mem-
brane; eo, endoral cilia; pv, praeoral membrane; Zo,
paroral cilia; foc, preoral cilia, “Crp,
The membranelles
are
flat
plates of striated appearance
usually
in the form
of
tri-
a
Sy
Aipy
GZ
~
= cok
IH
angles, squares,
or parallelo-
grams. Each membranelle
is. Fig. 98.— Schematic hypotrichous ciliate. inserted
in
a
furrow
below
az, adoral zone; ¢, ventral cirri;
e, endoral mem- which
is
a_
basal
stripe
of
brane; eo, endoral cilia; pv, praeoral membrane; Zo, thickened protoplasm continu-
paroral cilia; foc, preoral cilia,
ous with the longitudinal
cil-
iary markings (Heterotrichida). Like the cirri, they can be readily reduced
to component
fila-
ments resembling
cilia, and there
is, therefore,
every reason
to
suppose that the membranelles which form such a characteristic
differential for all orders save the Holotrichida, are merely the differ-
entiated portions of the ciliary rows! The basal stripes of the
membranelles, which are spirally arranged upon the peristome, are in
turn inserted, in some cases at least, in a thick fibrous strand which Fig. 98.— Schematic hypotrichous ciliate. az, adoral zone; ¢, ventral cirri;
e, endoral mem-
brane; eo, endoral cilia; pv, praeoral membrane; Zo,
paroral cilia; foc, preoral cilia, Fig. 98.— Schematic hypotrichous ciliate. az, adoral zone; ¢, ventral cirri;
e, endoral mem-
brane; eo, endoral cilia; pv, praeoral membrane; Zo,
paroral cilia; foc, preoral cilia, THE INFUSORIA 183. runs around the peristome connecting the series, and possibly form-
ing a nervous organ (Delage, ’96; Moore, ’93). The undulating membranes, finally, which are almost always con- The undulating membranes, finally, which are almost always con-
fined to the oral region, and like the membranelles chiefly concerned
with food-taking, have probably a similar origin, although the con-
nection with the cilia is less apparent. They are frequently, as in
the Hypotrichida, placed deep in the vestibule, but in many forms
they are confined to the pharynx itself, as in many of the Holo-
trichida. In addition to cilia, membranelles, and membranes, the ectoplasm. has other modifications, such as pseudopodia (¢.g. Stentor) and tenta-
cles. The pseudopodia are used for anchoring the animal, and are
produced at the posterior end by the so-called foot-disk (Johnson, ’93). The cortical plasm gives rise to these processes, and also to the
peculiar tentacle-like appendages found in some forms. In Ac#tinobo-
lus (Fig. A. PROTOPLASMIC STRUCTURE 100) these pseudopodial tentacles are
particularly well known through the complete
study made by Entz (’82). Here the threads
pass out between the cilia and not infrequently
reach a length of twice or even three times the
body diameter. The threads are of nearly uni-
form thickness, with blunt or slightly knobbed
ends (Entz). These tentacles, while occasion. . ally stiff and unyielding, can be shortened
or lengthened,
or drawn
into the body in
a manner surprisingly suggestive of pseudo- See yee
podia, while the protective and offensive func-
O. F.M,
[BUTSCHLL]
tion is shown by the presence of trichocysts at
their extremities. Similar tentacles are found in Mesodznitum and
Sleonema (see Fig. 115). While the ectoplasm is devoted to the functions of motion and irri- . See yee
O. F.M,
[BUTSCHLL] While the ectoplasm is devoted to the functions of motion and irri-
tability, the endoplasm is charged with digestion and reproduction. Thus the membranelles and membranes are important in creating the
current which brings the food particles; the trichocysts are occasion-
ally developed as food-killing organs, and these, with
the mouth,
vestibule, and pharynx, are ectoplasmic in origin. While all these special modifications are developed for the pur- While all these special modifications are developed for the pur-
pose of food-getting, the endoplasm, with its digestive processes,
shows but little advance, so far as can be made out, over the already
complicated endoplasm in the less highly organized forms. Simi-
lar food substances are treated in similar gastric vacuoles, and the
products of assimilation are carried about in the plasm by similar
cyclosis, while indigestible remains are excreted in the same way. The Protozoa thus offer in the most striking manner an example of THE PROTOZOA 184 how species may have originated through structural adaptations of
the parts (ectoplasm) that are in direct contact with the environment. The mouth parts, which are functionally the beginnings of the diges-
tive system, are formed by the invagination of a limited portion of how species may have originated through structural adaptations of
the parts (ectoplasm) that are in direct contact with the environment. The mouth parts, which are functionally the beginnings of the diges-
tive system, are formed by the invagination of a limited portion of ‘
\
|
\ "
% \
|
XQ
. \
he
|
\
. A. PROTOPLASMIC STRUCTURE As previously indicated, the original course of
these lines is from the anterior to the posterior end, but in numerous
transitional forms in which the mouth has a more or less ventral
position, the markings become curved to agree with the changed
position, while the course which the mouth is supposed to have taken
is shown by the converging lines (Fig. 95). In almost all cases the mouth is not in direct communication with In almost all cases the mouth is not in direct communication with
the endoplasm, but is separated from the latter by a longer or shorter
pharynx, cesophagus, or gullet, which frequently bears cilia, mem-
branes, or membranelles. The cesophagus is likewise an ectoplasmic
invagination, as is also a second cesophageal apparatus, found in
some forms (Vorticellidz), where the mouth leads into a comparatively
large ciliated or membraned space, known as the vestibule
( Fig. 101, C,
PD), and this leads into the cesophagus or gullet proper, which, in turn,
communicates with the endoplasm. This space begins as a wide
tube and gradually narrows down to a more or less narrow aperture
or constriction at the cesophagus. The anus
and the contractile
vacuole, in some forms, open to the exterior through the vestibule. In some
of the
Holotrichida,
the region about the pharynx is
strengthened by accessory apparatus developed in the cortical layer,
which in this region is greatly thickened and which in some cases
contains secretions in the form of bars arranged in a peculiar basket
structure (4, B). The membranelles which surround the mouth are usually in motion, The membranelles which surround the mouth are usually in motion,
as are the membranes and cilia which extend into the vestibule
or cesophagus. Even while the animal is lying quiet, the membra-
nelles continue their active vibrations, keeping a constant current of
water toward the mouth. This current brings a supply of bacteria,
diatoms, algz of various kinds, rotifers of small size, or parts of
animals undergoing
disintegration,
flagellates,
etc. A distinction
can be made
here
between
herbivorous
and carnivorous
forms,
although the differences can hardly extend to structural adaptations,
unless
it be, perhaps, in some
carnivorous
forms, where
special
weapons of offence (the trichocysts) are found. Probably all forms
are more or less omnivorous and make little or no selection of food. A. PROTOPLASMIC STRUCTURE \ ws)
REF j
Vy)
Ly
f
3
~
(\Qw NI "
a
NS
AA
\ “
. \\
=
Ww
ess a
SN
Fig. 100. —-fc/inobolus radians St. Fig. 100. —-fc/inobolus radians St. the ectoplasm in a manner analogous to the formation of the stomo-
deum of Metazoa. The peristome is the beginning of the mouth
depression, becoming more and more deeply depressed as the mouth
region is reached. It is not present in all forms, the mouth in its
original position probably being anterior and terminal in the monaxonic the ectoplasm in a manner analogous to the formation of the stomo-
deum of Metazoa. The peristome is the beginning of the mouth
depression, becoming more and more deeply depressed as the mouth
region is reached. It is not present in all forms, the mouth in its
original position probably being anterior and terminal in the monaxonic THE INFUSORIA 185 body, and leading by a mere passage into the endoplasm below. Mouthless forms are known, but these have degenerated through
parasitism and are not primitive (Opalinide). In the majority of
forms the mouth is displaced from the original terminal position and
has become ventral and central. Biitschli maintains that this change
in the position of the mouth is brought about by the gradual shifting
from the anterior end, as shown by the meeting point of the lines
of ciliary markings. As previously indicated, the original course of
these lines is from the anterior to the posterior end, but in numerous
transitional forms in which the mouth has a more or less ventral
position, the markings become curved to agree with the changed
position, while the course which the mouth is supposed to have taken
is shown by the converging lines (Fig. 95). In almost all cases the mouth is not in direct communication with body, and leading by a mere passage into the endoplasm below. Mouthless forms are known, but these have degenerated through
parasitism and are not primitive (Opalinide). In the majority of
forms the mouth is displaced from the original terminal position and
has become ventral and central. Biitschli maintains that this change
in the position of the mouth is brought about by the gradual shifting
from the anterior end, as shown by the meeting point of the lines
of ciliary markings. A. PROTOPLASMIC STRUCTURE The food particles are thrown by the current of the membranelles
into the peristomial depression and thence
into the vestibule or 186 THE PROTOZOA cesophagus, until they come in contact with the endoplasm at the
base of the latter. Here they are readily absorbed by the endoplasm,
in which, together with a small amount of water, they are confined
in a small gastric vacuole. The vacuole enlarges by the constant
addition of new material, until it is caught up in the current of the
endoplasm and dragged away. In this improvised ‘‘ stomach”
it is
slowly digested, a new drop being formed in the meantime at the
mouth opening. If food is abundant, the animal may become filled
with these gastric vacuoles. The liquid of the vacuole is, at first,
simply water, like the surrounding medium, but gradually becomes
acid through osmosis in the plasm, and the digestible substances are Fig. 101.— Buccal apparatus. [BUTSCHLI.]
A,B. Nassula aurea Ehr. A, From the ventral side. &. The buccal apparatus strongly
magnified. C. Urostyla grandis Ehr. D. Vorticella nebulifera O. F. M. Fig. 101.— Buccal apparatus. [BUTSCHLI.] Fig. 101.— Buccal apparatus. [BUTSCHLI.]
Ehr. A, From the ventral side. &. The buc A,B. Nassula aurea Ehr. A, From the ventral side. &. The buccal apparatus stron
agnified. C. Urostyla grandis Ehr. D. Vorticella nebulifera O. F. M. slowly dissolved, the residue being cast to the exterior through the
anus. slowly dissolved, the residue being cast to the exterior through the
anus. Unlike the mouth, the anus, as a rule, is a simple opening in the
outer wall (Maupas, ’83; Biitschli, ’88), although Stein (67) described
an anal tube in certain forms (Myctotherus). In the Heterotrichida
it is sub-terminal in position. In the Hypotrichida it is never ter-
minal, but usually dorsal, and toward the left edge. In the Peritri-
chida, especially in the Vorticellidz, the anus opens into the vestibule. B.
CONTRACTILE VACUOLES This
is certainly true in /7ox¢onta, where there are
one side and an immense number of feeding
mose and branch to form a complicated network absent) by absorption through the body wall, an
the contractile vacuole to get rid of the surp
variously complicated by the development of a m
series of canals, which
empty in a common
Always situated in the cortical plasm, the con-
tractile vacuoles are fixed in position and com-
municate with the exterior at systole by a
permanent aperture, which, however, becomes
covered internally during filling or diastole. They vary in number from one to a hundred,
or even more, and are absent, apparently, in
only one form (Opaéina), although Vejdovsky
(92) describes contractile vacuoles in a closely
allied form, Monodontophrya longissima, while
even in Ofalina the reminiscence of the vacu-
ole is seen in the remnants of the feeding
canals (Delage, 96). In its simplest form the
vacuole
is single and terminal, a condition
which may be found in each of the four orders. When there
are
more
than one, they are
grouped around the original vacuole in a ter-
minal position, or arranged along one or more
lines upon the dorsal side. In Descophrya and
Hoplitophrya (Holotrichida) there is no regular
vesicle, but a long contracting canal which
runs the length of the body. Spzrostomum
(Heterotrichida) has a terminal vesicle, with
one
long feeding canal, and from
this the
canal system is developed in a variety of ways. Thus there is a vesicle with two feeding canals
in Climacostomum (Fig. 91, 8), one terminal
vesicle and four feeding canals in Urocentrum. In Stentor there is a single vesicle near the
peristome, with two feeding canals, one
of
which runs to the end of the body, while the
<
other runs around the peristome edge. Fabre-
Dumergue (’89) holds that canals, for the most
part invisible, are present in all ciliates. This
is certainly true in /7ox¢onta, where there are o
one side and an immense number of feeding
mose and branch to form a complicated network
body. In some forms the vesicle communicat
directly, but it may be complicated by the f
reservoirs. In the holotrichous form, Lembad aa
we
(vy \
WA
B|
5
4
C---->
Fig. 102. B.
CONTRACTILE VACUOLES A certain amount of water is taken in with the food through the
mouth, and at the same time (as in those cases where the mouth is THE INFUSORIA 187 absent) by absorption through the body wall, an
the contractile vacuole to get rid of the surp
variously complicated by the development of a
series of canals, which
empty in a common
Always situated in the cortical plasm, the con-
tractile vacuoles are fixed in position and com-
municate with the exterior at systole by a
permanent aperture, which, however, becomes
covered internally during filling or diastole. They vary in number from one to a hundred,
or even more, and are absent, apparently, in
only one form (Opaéina), although Vejdovsky
(92) describes contractile vacuoles in a closely
allied form, Monodontophrya longissima, while
even in Ofalina the reminiscence of the vacu-
ole is seen in the remnants of the feeding
canals (Delage, 96). In its simplest form the
vacuole
is single and terminal, a condition
which may be found in each of the four orders. When there
are
more
than one, they are
grouped around the original vacuole in a ter-
minal position, or arranged along one or more
lines upon the dorsal side. In Descophrya and
Hoplitophrya (Holotrichida) there is no regular
vesicle, but a long contracting canal which
runs the length of the body. Spzrostomum
(Heterotrichida) has a terminal vesicle, with
one
long feeding canal, and from
this the
canal system is developed in a variety of ways. Thus there is a vesicle with two feeding canals
in Climacostomum (Fig. 91, 8), one terminal
vesicle and four feeding canals in Urocentrum. In Stentor there is a single vesicle near the
peristome, with two feeding canals, one
of
which runs to the end of the body, while the
<
other runs around the peristome edge. Fabre-
Dumergue (’89) holds that canals, for the most
part invisible, are present in all ciliates. B.
CONTRACTILE VACUOLES — Anterior end of
career erent tg a
© ¢, the reservoir of the vacu-
ole (¥) emptymg through a
long canal into the vestibule; Fig. 102. — Anterior end of
career erent tg a
© ¢, the reservoir of the vacu- © ¢, the reservoir of the vacu-
ole (¥) emptymg through a
long canal into the vestibule;
4 the cesophagus; 2, the nu-
cleus. 188 THE PROTOZOA dorsally in the middle of the body, but is connected with the terminal
aperture by a long canal. In the Peritrichida there are one or two
vesicles, which empty by contraction into special reservoirs, and these,
in turn, empty into the cesophagus (Fig. 102). According to Delage (’96) the contraction of the vesicle is brought According to Delage (’96) the contraction of the vesicle is brought
about by the contractility of the surrounding cortical plasm. Rhum-
bler (’98) has shown, however, that the contained water may so affect
the plasm that it becomes differentiated like ectoplasm, and this gives
some substantial basis for the view that a special membrane surrounds
the vacuole. There is no evidence, however, to show that this modi-
fied protoplasm acts like a sphincter. Biitschli holds that the vesicle
contracts through a mechanical force exerted upon the thin plasmic
layer between the vesicle and the opening of the excretory pore by
the pressure of the filling vacuole and the turgor of the cell. At the
completion of the diastole the pressure becomes too great for the
lamella, and the latter is ruptured, allowing the contained fluid to pass
to the exterior. , C. THe Nuc.Leus The nuclei of the Infusoria show some
of the most striking
structural characteristics connected with the Protozoa. Here there
is a differentiation of the nuclear material into two forms, a larger
macronucleus, and a very much smaller szzcronucleus. With the single
exception of Polykrtkos (Dinoflagellidia), this differentiation of the
nuclei is found nowhere outside of the present group. The functions
of the two kinds of nuclei are supposed to be respectively vegetative
and reproductive
(Biitschli), but this distinction
is, perhaps, too
sweeping. Julin (’93) held that the macronucleus stands not only
for nutrition, movement, sensation, and regeneration, but for asexual
division as well, in fact is a “‘ somatic nucleus,”’ while the micronucleus
functions only as a sexual nucleus. There may be one or many of
each kind in each
cell. The macronucleus, which
is invariably
present, recalls the nucleus of tissue cells. It is usually single, and,
lying in the endoplasm, it may be carried about with the flowing
granules, or maintained in a permanent position in the cortical plasm,
or by processes from this plasm (e.g. /sotricha). Its form is quite
variable and has little significance for systematic work, for in the same
species under certain conditions it may even become amceboid (Loeb &
Hardesty, 95). The usual form is spherical, but it may be elongated
into an oval, or into a flattened rod which may be curved or straight,
or it may be divided into small pieces resembling a string of beads,
connected by a membrane (Fig. 103). It is always provided with a
membrane (Maupas), but the chromatin contained within it is vari- THE INFUSORIA 189 ously distributed. The vesicular structure, in which the nuclear
substance is so distributed as to leave more or less space filled with
“nuclear sap,” is almost never
seen, the macronucleus appearing
solid and completely filled with chromatin. Biitschli described the
finer structure as almost invariably alveolar, the meshes correspond-
ing to those of the surrounding plasm. The entire network stains
deeply with
the nuclear dyes, but
at certain
stages, especially
during division, distinct fine lines can be made out connecting the ously distributed. The vesicular structure, in which the nuclear
substance is so distributed as to leave more or less space filled with
“nuclear sap,” is almost never
seen, the macronucleus appearing
solid and completely filled with chromatin. C. THe Nuc.Leus Biitschli described the
finer structure as almost invariably alveolar, the meshes correspond-
ing to those of the surrounding plasm. The entire network stains
deeply with
the nuclear dyes, but
at certain
stages, especially
during division, distinct fine lines can be made out connecting the Fig. 103.— Types of macronuclei. [SAVILLE KENT.]
A. Macro- and micronucleus of Loxodes rostrum O. F. M. 8. Of Nyctotherus cordiformis
Leidy. C. Macronucleus of Plagiotoma lumbrici Duj. D,. Dendrosoma radians S. K., a young
nucleus,
£. Dendrosoma radians 8. K. F. Stentor polymorphus Ehr. G. Stylonychia mytilus
O. F.M. A. The same in division. J, the macronucleus; x, the micronucleus. [SAVILLE KENT.]
F. M. 8. Of Nyct Fig. 103.— Types of macronuclei. [SAVILLE KENT.] A. Macro- and micronucleus of Loxodes rostrum O. F. M. 8. Of Nyctotherus cordiformis
Leidy. C. Macronucleus of Plagiotoma lumbrici Duj. D,. Dendrosoma radians S. K., a young
nucleus,
£. Dendrosoma radians 8. K. F. Stentor polymorphus Ehr. G. Stylonychia mytilus
O. F.M. A. The same in division. J, the macronucleus; x, the micronucleus. chromatin granules, and corresponding
to the linin reticulum of
most
nucleii
In some
cases
(e.g. Loxrophyllum)
a permanent
spireme is present, as in the nucleus of cells from the salivary gland
of Chironomus larvee, and is transversely striated, indicating disks
which Balbiani (90) thought are alternately chromatin and linin
(Fig. 104). In many cases there are internal modifications of the
nuclear material forming so-called “nucleoli,” although there is a
possibility that these structures are similar to the intranuclear bodies
found in Mastigophora. In many macronuclei a peculiar division of the organ is made by a In many macronuclei a peculiar division of the organ is made by a Igo THE PROTOZOA split, which the Germans call a Kernspalt and the French a fete. While this peculiar feature of the nucleus has not been explained,
what may be an important light has been thrown upon it by Bal-
biani ('95), who described the appearance as due to the presence of
two materials within the nuclei, one
of which
is chromatin, the other,
achromatic material or archoplasm. This interpretation, however, cannot
be accepted as final. While the macronuclei are, as a While the macronuclei are, as a
rule, single in number, the micro-
nuclei are often multiple. C. THe Nuc.Leus Probably
all Ciliata have at least one micro-
nucleus, although the small size and
the extreme
difficulty
in staining
sometimes render it hard to find. In one case at least (Opalina rana-
vum) there is only one kind of nu-
cleus. The number of micronuclei
is usually greater where the macro-
nucleus is elongate, and especially
where it is beaded (Stentor, Spiro-
stomum, etc.). Asarule the micro-
nuclei are closely attached to the
membrane
of
the
macronucleus,
occupying a minute indentation in
the latter, but in some cases they
are well separated. In form they
are round,
ellipsoidal,
or
spindle-
shaped, but the form varies with the
nuclear activity, and does not mean
much in itself. Their longest axis
measures from I » to 10 pw, and like
the macronuclei, they are covered
with
a
distinct
membrane,
while
the chromatin is usually massed at
some part of the nucleus. In cer-
tain cases
the appearance
is like
that of the macronucleus with the
chromatin in the form of a densely
packed reticulum, giving to it a mas-
istinct
portions, the chromatin and
(Fig. 105). ace by mitosis in the micronuclei, and, Fig. 104. — Loxophyllum meleagris O. F. M. [BALBIANI.]
A, Vegetative nuclei with chromatin in the
form of a permanent spireme. 2, CD. The
same in division. nucleus, although the small size and
the extreme
difficulty
in staining
sometimes render it hard to find. In one case at least (Opalina rana-
vum) there is only one kind of nu-
cleus. The number of micronuclei
is usually greater where the macro-
nucleus is elongate, and especially
where it is beaded (Stentor, Spiro-
stomum, etc.). Asarule the micro-
nuclei are closely attached to the
membrane
of
the
macronucleus,
occupying a minute indentation in
the latter, but in some cases they
are well separated. In form they
are round,
ellipsoidal,
or
spindle-
shaped, but the form varies with the
nuclear activity, and does not mean
much in itself. Their longest axis
measures from I » to 10 pw, and like
the macronuclei, they are covered
with
a
distinct
membrane,
while
the chromatin is usually massed at
some part of the nucleus. In cer-
tain cases
the appearance
is like
that of the macronucleus with the
chromatin in the form of a densely
packed reticulum, giving to it a mas-
sive appearance. C. THe Nuc.Leus Here two distinct
portions, the chromatin and
achromatin, can be made out (Fig. 105). Division of the nuclei takes place by mitosis in the micronuclei, and, Fig. 104. — Loxophyllum meleagris O. F. M. [BALBIANI.]
A, Vegetative nuclei with chromatin in the
form of a permanent spireme. 2, CD. The
same in division. Fig. 104. — Loxophyllum meleagris O. F. M. [BALBIANI.]
A, Vegetative nuclei with chromatin in the
form of a permanent spireme. 2, CD. The
same in division. Fig. 104. — Loxophyllum meleagris O. F. M. [BALBIANI.]
A, Vegetative nuclei with chromatin in the
form of a permanent spireme. 2, CD. The
same in division. A, Vegetative nuclei with chromatin in the
form of a permanent spireme. 2, CD. The
same in division. THE INFUSORIA IQI as a rule, by amitosis in the macronuclei. The latter is the simpler;
in many spherical or elliptical nuclei the structure merely draws out
and segments into two equal parts. It is more or less complicated,
however, in different macronuclei, until well-developed mitosis replaces
simple division (¢.g. Spzrochona). There is reason to regard the sim-
ple division of the larger nuclei as the mere degeneration of mitosis,
by a process in which the various stages have gradually disappeared
until only the preliminary stages of such division are to be found. These preliminary stages are seen in the transformation of the reticu-
lum of chromatin into thread-like masses, which recall the spireme of Fig. 105. — Mitotic division of the nuclei of Spirochona and Paramecium. [R. HERTWIG.]
A-C. Macronucleus of Sf:rochona with well-developed pole plates. D-H. Different stages in
division of the micronucleus of Paramecium. ’ Fig. 105. — Mitotic division of the nuclei of Spirochona and Paramecium. [R. HERTWIG.]
A-C. Macronucleus of Sf:rochona with well-developed pole plates. D-H. Different stages in
division of the micronucleus of Paramecium. ’ higher cells; the threads are then divided across into equal parts. In
Spirochona, however, the process of division is strikingly similar to
mitosis in Metazoa (Fig. 105, B, C). Division of the micronuclei is accompanied by the formation of Division of the micronuclei is accompanied by the formation of
polar attraction-spheres, and by the rearrangement of chromatin into
chromosomes. D. ENcySTMENT The phenomenon of encystment may be seen in the Ciliata as in all
other groups of the Protozoa. It occurs when the animal is in danger
of drying, in some cases before division, in others, for the purpose
of digesting a full meal. The cilia are drawn in, the mouth and peri-
stome disappear, the contained body-granules are voided, and a gelati-
nous secretion is poured out from the ectoplasm. The secretion
soon hardens, becoming chitinous. The vacuole continues to pulsate
for some time, and the secretion forms a liquid layer about the animal
under the cyst. The cysts are variously diversified with spines and
processes of different kinds, and are occasionally multiple, the spaces
between the cysts being filled with water (Fig. 17, B, C, F, p. 47). C. THe Nuc.Leus Before
division, the micronucleus swells to nearly
twice its size during resting stages (Hertwig, 77), while the granu-
lar chromatin begins to collect in lines—at first equally thick, but
later concentrated in the equatorial region (Fig. 105, D-H). Divi-
sion then takes place through the centre. THE PROTOZOA 192 E. REPRODUCTION Reproduction among the Ciliata takes place almost exclusively by
simple division or fission. It is practically the same for all forms, the
variations being of minor importance. The nuclei first divide, new
mouth parts are developed in the posterior half, and then the cell
divides. The first indication of division in Stentor, for example, is a
rift in one side of the animal below the adoral zone. This rift rapidly
develops motile organs (membranelles), and acquires the full length
of the lower daughter-individual. The nuclei in the meantime divide,
and the original animal draws out, leaving a slender foot for the upper
or anterior cell,and a swollen portion for the pharyngeal region of the
second individual. The new adoral zone is completely formed before
actual division; the steps in the process are shown in the accompany-
ing diagram from Johnson (’93) (Fig. 106). The new contractile vacu-
ole, according to him, arises de novo in S. ceruleus, and by a dilatation
of the longitudinal canal in S. rese/zz. The new vacuole thus formed
remains in connéction with the longitudinal canal, the upper part of
which becomes drawn out with the torsion of the adoral zone to form
the much-discussed ring-canal discovered by Lachmann. In some forms, as in Sfzrochona, division simulates budding, un- In some forms, as in Sfzrochona, division simulates budding, un-
equal division giving rise to mother- and daughter-cells. When division takes place within the cyst, the various mouth parts When division takes place within the cyst, the various mouth parts
may or may not first be absorbed, but in all cases the vacuole still
continues to pulsate. Here, as a rule, division is double, resulting in
broods of four which escape as embryos, and gradually grow into the
parent form. This condition closely simulates spore-formation, which
results when, as in Ofadina, the number of divisions within the cyst
reaches three, four, five, or six. Nowhere among the Protozoa has the process of conjugation been
so thoroughly studied in connection with the life-history of the organ- THE INFUSORIA 193 ism as in the Ciliata. Worked out first by Biitschli and Engelmann
in 1876, it has since been carefully studied by numerous observers,
and the conditions preliminary to conjugation, during, and subsequent
to it have been made known in a great variety of forms representing
all divisions of the class. E. REPRODUCTION ‘If
the fusion is temporary, as in the majority of forms, the two ecto-
plasms fuse at or near the mouth parts, and a protoplasmic bridge is
formed between the two organisms. The two organisms then become
sluggish, and rest for a considerable time upon the bottom without
movement of any kind. They ultimately separate and begin to
divide. The micronuclei
play the most important part in conjugation. Each divides two or more times to form four or more daughter-nuclei,
some of which de-
generate, while one
divides
again,
one
half to fuse with a
similarly derived nu-
cleus
of the other
organism, while the
other half remains
as the receptive nu-
cleus, or the female
pronucleus. The
two conjugating nu-
clei cannot be distin-
guished from those
which
degenerate,
but are apparently
only those which lie
nearest
the bridge -
joining
the
two
organisms. Mean-
Fig. 107. — Conjugation in Zpistlis umbellaria
while
the
macro-
Greeff. (GREEFF.]
M, macrogamete
; my, microgametes,
nucleus
undergoes
complete degenera-
tion,
breaking
up
into a number of pieces, which are gradually absorbed by the proto-
plasm. The new macronucleus is formed by the enlargement of a
daughter-micronucleus derived from the fusion nucleus. Hoyer (99),
however, asserts that in Co/pzdium colpoda it forms by the union of
two daughter-nuclei. When there are two or more micronuclei in each conjugant, the The micronuclei
play the most important part in conjugation. Each divides two or more times to form four or more daughter-nuclei,
some of which de-
generate, while one
divides
again,
one
half to fuse with a
similarly derived nu-
cleus
of the other
organism, while the
other half remains
as the receptive nu- Fig. 107. — Conjugation in Zpistlis umbellaria
Greeff. (GREEFF.]
M, macrogamete
; my, microgametes, Fig. 107. — Conjugation in Zpistlis umbellaria
Greeff. (GREEFF.]
M, macrogamete
; my, microgametes, into a number of pieces, which are gradually absorbed by the proto-
plasm. The new macronucleus is formed by the enlargement of a
daughter-micronucleus derived from the fusion nucleus. Hoyer (99),
however, asserts that in Co/pzdium colpoda it forms by the union of
two daughter-nuclei. When there are two or more micronuclei in each conjugant, the When there are two or more micronuclei in each conjugant, the
process is repeated for each of them, although
it is not known
whether this holds when, as in Stentor, the number reaches sixty or
seventy. E. REPRODUCTION Biitschli and Engelmann early recognized
that conjugation is necessary for continued life activity of the organ-
ism, and came to the conclusion, which has been fully confirmed by
subsequent observers, that conjugation is a process of rejuvenation or
a renewal of vitality, the need of which is shown by the reduced size Fig. 106. — Diagrams to illustrate the division of Stentor raselit,
[JOHNSON.]
V, the vacuole; A, the ring canal. Fig. 106. — Diagrams to illustrate the division of Stentor raselit,
[JOHNSON.]
V, the vacuole; A, the ring canal. and general degenerate condition of the organism prior to conjuga-
tion. Maupas (’89), in his classical work on conjugation among Infu-
_soria, found that the number of generations which may be formed from
one conjugating period to the next varies with the species, but is
usually between three hundred and four hundred and fifty. He found,
furthermore, that certain conditions are necessary for conjugation. These conditions are: (1) maturity of the organisms, z.e. forms which
have just conjugated will not again conjugate until after a certain
number of generations; (2) partial lack of food, 2.2. if plenty of food is
present, conjugation will not take place even though the individuals
are well along in degeneration ; (3) diverse ancestry, z.e. the conju-
gants must come from different ancestral conjugants. and general degenerate condition of the organism prior to conjuga-
tion. Maupas (’89), in his classical work on conjugation among Infu-
_soria, found that the number of generations which may be formed from
one conjugating period to the next varies with the species, but is
usually between three hundred and four hundred and fifty. He found,
furthermore, that certain conditions are necessary for conjugation. These conditions are: (1) maturity of the organisms, z.e. forms which
have just conjugated will not again conjugate until after a certain
number of generations; (2) partial lack of food, 2.2. if plenty of food is
present, conjugation will not take place even though the individuals
are well along in degeneration ; (3) diverse ancestry, z.e. the conju-
gants must come from different ancestral conjugants. oO oO THE PROTOZOA 194 The two conjugants fuse either temporarily or permanently, and
the external structures, such as the membranelles, are absorbed. E. REPRODUCTION In a few cases (Vorticellidze) the conjugants are of diverse size. In a few cases (Vorticellidze) the conjugants are of diverse size. THE INFUSORIA 195 The larger form or macrogamete is usually a normal-sized individual,
although in some cases it is somewhat larger than the ordinary cells
(Zoothamnium). The microgametes,
on the other hand, are
con-
siderably smaller, and from four to eight are formed by each cell. These never develop a stalk, but leave the parent colony, and swim
about by means of the ring of cilia around the lower pole. They
finally come in contact with the macrogamete and fuse with it, the
union taking place at the lower end of the attached organism, and
near the insertion point of the stalk (Fig. 107). Il. THE SUCTORIA The Suctoria differ decidedly from the Ciliata, from which they
have undoubtedly sprung. With the exception of Hypfocoma (Fig. 115, C ), which remains ciliated throughout life, the Suctoria possess
cilia only during the embryonic stages. They are, for the most part,
sedentary forms, and grow upon a chitinous peduncle, which is at-
tached at the lower end to some foreign object. The upper end
of the peduncle is hollowed out into a bowl, within which the animal
lies. Owing to its attached mode of life, and to the equal pressure
on all sides, the general form of the animal is spherical or radially
symmetrical. In some cases there is a well-defined membrane, but
the various students of the group are not agreed as to its structure. It is never striated, as in the Ciliata, and there is no cortical plasm. The endoplasm shows no differentiations other than the usual food
granules or assimilation products common to all Protozoa. An essential point of difference from the ciliate structure is the An essential point of difference from the ciliate structure is the
presence of ¢ezzacles, which, in the majority of Suctoria, are the only
motile appendages of the adult. In many respects they are similar
to the tentacles of Actznobolus, [leonema, and Mesodinium (Fig. 115),
but differ in the very important fact that they are hollow, while the
extremities bear the mouth openings. There are two general types of tentacles: one, according to Biit- There are two general types of tentacles: one, according to Biit-
schli, captures prey, while the other devours it. Of the latter forms,
there are also two types. One is long and broad, and, like a thorn,
pointed at the extremity; the other is nearly uniform in diameter and
flattened at the top, or hollowed out into a cup-like sucking organ
(Fig. 108). These are distinguished as the sty/zform and capitate
tentacles (Delage). Both sets of tentacles are hollow, and their
lumena open at the ends. There is a difference of opinion, however,
in regard to their inner structure and function. Biitschli holds that
some of them are solid, and others hollow. He maintains that, in
the solid forms, the internal portion is formed of endoplasm, which is
continuous with the inner plasm of the cell. Delage claims that they 196 THE PROTOZOA are allhollow. Il. THE SUCTORIA The function of the endoplasm, according to Biitschli,
differs in the styliform and the capitate tentacles. In the latter, the
prey is retained by the sucking disk at the extremity while the endo-
plasm within the tube meantime works up and down like a pump-
piston, and a vacuum being thus formed, the cuticle of the prey
is burst, and the fluid endoplasm flows down the tentacle canal to
the endoplasm of the captor, where it is digested. Such an explanation of the action of these tentacles is regarded
by most observers as extremely doubtful. Delage finds no motion in
the endoplasm during feeding, save in the rhythmic pulsations of the
contractile vacuole, an organoid which Eismond (‘90) believed is the
cause of the suction. The excretion of water from the vacuole, he
argued, creates a semi-vacuum in the protoplasm, and the pressure Fig. 108. — Tentacles of Suctoria. [R. HERTWIG.]
A. Different types of styliform or piercing tentacles. JS. Capitate and piercing tentacles. Fig. 108. — Tentacles of Suctoria. [R. HERTWIG.]
A. Different types of styliform or piercing tentacles. JS. Capitate and piercing tentacles. Fig. 108. — Tentacles of Suctoria. [R. HERTWIG.] Fig. 108. — Tentacles of Suctoria. [R. HERTWIG.]
A. Different types of styliform or piercing tentacles. JS. Capitate and piercing tentacles. from without forces food or loose particles, etc., through the tentacle
openings to the endoplasm. This explanation, although somewhat
fanciful, is certainly as plausible as the principle of the pump, but
the matter must remain for the present as one of the many unsolved
problems connected with this group. In some cases (7richophrya
angulata) the tentacles are apparently unnecessary for food-taking,
as Dangeard (’90) found that particles are occasionally engulfed, as
in the Rhizopoda, at any point
of the naked body. In the styliform tentacles, on the other hand, the sharp points In the styliform tentacles, on the other hand, the sharp points
pierce the membrane of the prey, while the endoplasm contained
within the tentacle possibly flows into the prey, whose endoplasm is
digested 7 sztz. In some cases they appear to have a paralyzing
effect upon other forms, and ciliates coming in contact with them
have been seen to stop their movements as though stunned. Il. THE SUCTORIA THE INFUSORIA 197 In all cases the tentacles are remarkably like pseudopodia, and may
change their form and their position, and may even be entirely with-
drawn into the body, to reappear, possibly, at some other place. Some forms have the power
of withdrawing their tentacles and
developing cilia, which may be retained
for a longer or shorter ,
x
a
ay /a
Fig. 109. — Dendrosoma radians Ehr. [SAVILLE KENT.]}
z, nucleus. Fig. 109. — Dendrosoma radians Ehr. [SAVILLE KENT.]}
z, nucleus. period. In some cases the tentacles distinctly originate in the endo-
plasm, and penetrate the membrane (Hertwig, ’76; Ishikawa, ’96). The nuclei, like those of the Ciliata, are of two kinds, macro- and The nuclei, like those of the Ciliata, are of two kinds, macro- and
micronuclei. The former are little different from the macronuclei
of the more generalized Ciliata, while very little is known about the
latter. In the colony-form Dendrosoma, where the many branches
suggest a hydroid colony, the macronucleus extends through all the 98
“THE PR 198 “THE PROTOZOA The contractile vacuole never becomes so complicated as in some
Ciliata, but consists, usually,
of a single vesicle, which may b
surrounded by a circle of small vacuoles emptying into it. In some
cases there is a short excretory duct leading from the vesicle to th
excretory pore in th
membrane,
which,
a
‘in the Ciliata, is a per
manent opening. Fig. 110. — Exogenous budding in Ephelota Butschliana Ishi,
XN, nucleus. Fig. 110. — Exogenous budding in Ephelota Butschliana Ishi,
XN, nucleus. swimming period, settles down, loses its cilia, and secretes a stalk. Partial division, or budding, may be either exdogenous or exogenous. The latter is the simpler; an individual prepares as for division, but
instead of dividing into two equal portions, a number of papillae
appear at the outer surface, each becomes a bud, receiving a portion
of the nucleus (Fig. 110). Endogenous division arises by the invagi-
nation of such a budding area, while the walls surrounding it grow THE INFUSORIA 199 together above the developing buds, which, when ripe, break through
the birth-opening left in the covering membrane (Fig. 111). In some
cases the buds are multiple, again single, and a number may develop
at the same time within the brood-sac (Aczneta, Ophryodendron). Il. THE SUCTORIA The embryos thus formed are variously ciliated in the different The embryos thus formed are variously ciliated in the different
genera. In some they are holotrichous, in others hypotrichous, and
in others peritrichous (c, @). Fig. 111. — Endogenous budding in Suctoria. [BUTSCHLI.]
A-B, Two stages in the formation of the bud in Tokophrya guadripartita Cl. and Lach. c. The swarm-spore liberated. C. Buds in Acineta tuberosa Ehr. ad. A swarm-spore liberated. Fig. 111. — Endogenous budding in Suctoria. [BUTSCHLI.] Fig. 111. — Endogenous budding in Suctoria. [BUTSCHLI.] A-B, Two stages in the formation of the bud in Tokophrya guadripartita Cl. and Lach. c. The swarm-spore liberated. C. Buds in Acineta tuberosa Ehr. ad. A swarm-spore liberated. A-B, Two stages in the formation of the bud in Tokophrya guadripartita Cl. and Lach. c. The swarm-spore liberated. C. Buds in Acineta tuberosa Ehr. ad. A swarm-spore liberated. Conjugation occurs here as in the Ciliata, but the process rests
upon the single observations of Maupas, who shows, however, that it
differs in no essential features from that already described. III.
INTER-RELATIONS OF THE INFUSORIA In searching for the origin of the Ciliata, the naturalists
of
thirty years ago had an apparent advantage, in that the supposed
ciliated girdle of the Dinoflagellidia offered a direct transition to the
peritrichous Ciliata, which, accordingly, were regarded as coming
from the flagellate stem at a comparatively late date. Unfortunately
for the theory, however, it was ascertained by Biitschli (85) and
others that the girdle of cilia is only a vibrating flagellum in the
transverse groove. In other directions
the search for the origin
of these forms has been almost equally vain. The singularly con-
servative structure which the ciliate body presents leaves but little
clue to their ancestry. The universal presence of macro- and micro-
nuclei
is paralleled by only one other known
case,
the almost
universal reproduction by transverse division is met with elsewhere
but rarely. The sole possibility which presents itself is that the THE PROTOZOA 200 infusorian stem was derived from the flagellate at a very early period,
and that the side branch became progressively differentiated until the
well-marked characteristics of to-day distinguish the Infusoria as an
entirely independent group. The first forms to diverge from the
flagellate stem may have been like the type described by Cienkowsky,
under the generic name of Mudtictlia (Fig. 112, A), a form with a
number of long flagella. It is thought by Biitschli that the Ciliata
might have been derived from such generalized forms by progressive
increase, with shortening of the motile elements, until cilia were the
\ \
A Pa
Fig. 112. — 4. Multicilia lacustris Lauterb,
[LAUTERBORN.]
B. Fyrsonympha vertens Leidy. [PORTER]
x, the vibrating band in the inner plasm. A Fig. 112. — 4. Multicilia lacustris Lauterb,
[LAUTERBORN.]
B. Fyrsonympha vertens Leidy. [PORTER]
x, the vibrating band in the inner plasm. outcome. There is no close connection, however, between cilia and
flagella, such as exists between the flagella and the pseudopodia. Other forms, more or less similar to Multicilia, have been described
by various observers,
so that the hypothesis of Biitschli
is not
without warrant. Among these forms are Grassia, Trichonympha,
Leidyonella, Lophomonas, Pyrsonympha,
etc., which are placed by
some among the Flagellidia (Delage), by others among the Ciliata
(Biitschli). Another point of view has been based upon the relations
of the Ciliata to the Suctoria, and through them to the Sarcodina outcome. III.
INTER-RELATIONS OF THE INFUSORIA There is no close connection, however, between cilia and
flagella, such as exists between the flagella and the pseudopodia. Other forms, more or less similar to Multicilia, have been described
by various observers,
so that the hypothesis of Biitschli
is not
without warrant. Among these forms are Grassia, Trichonympha,
Leidyonella, Lophomonas, Pyrsonympha,
etc., which are placed by
some among the Flagellidia (Delage), by others among the Ciliata
(Biitschli). Another point of view has been based upon the relations
of the Ciliata to the Suctoria, and through them to the Sarcodina THE INFUSORIA 201 (Entz, Maupas). This view will be more appropriately examined in
connection with the Suctoria. The Holotrichida appear to be the most generalized of the entire
group of Infusoria, but a few forms among them have a slight regional
differentiation of cilia suggesting the characteristics of the Heterotri-
chida(Lembus, Pleuronema, Ophryoglena, etc.). In fact, there appears
to be no sharp line between the two divisions, although the presence
of an adoral band of cilia in the Heterotrichida is a sufficient differen- ww. <<:
ine
fo . a:
’ Ss
ie
. ss
\a
Fame
a
=
Z
z
Py
aS
Nel
SEZZZO
ae
oe 8
=
os
“Tag
tr N)
i
a: »
ww. <<:
ine
fo . a:
’ Ss
ie
. ss
\a
Fame
a
=
Z
z
Py
aS
Nel
SEZZZO
ae
oe 8
=
os
“Tag
tr
Fig. 113. — Illustrating Biitschli’s hypothesis of the origin of the Hypotrichida. [BUTSCHLI.]
A, Stephanopoyon colpoda Entz. B. Peritromus emm@ St. C. Onychodromus grandis St. rope [B Fig. 113. — Illustrating Biitschli’s hypothesis of the origin of the Hypotrichida. [BUTSCHLI.]
A, Stephanopoyon colpoda Entz. B. Peritromus emm@ St. C. Onychodromus grandis St. rope tial. In some forms the uniform coating of cilia is broken in certain
regions, giving characteristic girdled forms, which are included as a
separate order apart from the Holotrichida by some writers (Haeckel). In the Holotrichida, also, there are a few forms which show a distinct
tendency toward bilateral symmetry, due primarily to a bending of
the body, and followed by a reduction of the cilia upon the arched
side (Stephanopogon, Fig. 113, A). III.
INTER-RELATIONS OF THE INFUSORIA form, becoming attached at the posterior half of the ventral surface, THE INFUSORIA 203 with loss of the posterior girdle of cilia, and elevation of the anterior
region bearing the adoral zone, which, as in the other groups of Ciliata,
is turned toward the left. The key to the other group of Peritrichida
is seen, Biitschli maintains, in the family Lichnophoride, where the
individuals closely resemble hypotrichous forms (Fig. 114), being
oval, flattened ventrally, and arched dorsally. The cilia, as in the
Hypotrichida, are limited to the ventral surface, and an adoral zone
is present, which runs from the mouth near the middle of the left
body edge, entirely around the anterior region of the body, to form
an incomplete arc which terminates in the line of the mouth, but on
the right side. Another closed ring of cilia is present in the pos-
terior half of the ventral surface. The anterior and posterior rings
of cilia are separated in Lichnophora by a stretch of plasm in such a
way as quite to divide that surface into a posterior and an anterior
division (£). The posterior part becomes modified
to form an
attaching organ upon which the animal creeps about upon its host;
the anterior region at the same time is elevated, and held in a posi-
tion at right angles to the plane of attachment, the apparent stalk
which supports it being in reality the intermediate plasm between
the posterior and the anterior regions of the ventral surface (J). Thus the curious anomaly arises of an animal whose anterior and
posterior ends represent parts of the same ventral surface. Biitschli derives the entire family of the Vorticellidze from this Biitschli derives the entire family of the Vorticellidze from this
primitive type, through
forms
like
the
Urceolarine,
where
the
attaching disk, primarily, is not so far removed from the peristome,
nor so stalk-like, as it is in the present-day Lichnophoride (DL, £). He argues that the Vortice//a-type is derived from the Urceolaria-
type by the attaching
part of the ventral surface,
7.e. the pos-
terior part being carried outward from the remainder of the ventral
surface, and thus borne upon a platform so that the two portions of
the same surface are no longer in the same plane (4). III.
INTER-RELATIONS OF THE INFUSORIA Biitschli derives the Hypotri-
chida from the Heterotrichida by the supposition of the loss of cilia
upon the arched dorsal side and incomplete closure of the adoral ring THE PROTOZOA 202 are well distributed over the ventral surface, bu
D
E
Fig. 114. — eee ne Biitschli's view of the origin of the Vor-
ticellidze,
[BUYSCHLI.]
The Trichodina form C is supposed to have arisen from the
Lichnophora-like form A by the outgrowth of the lower ciliated
area, first forming an intermediate stage B. This ring of cilia
becomes lost in the Vorticellidae, appearing only when the indi-
viduals are free-swimming. D, £. Side and front of Lichnophora
eee Slay
to the
SS SNANN
EA
s SS SNANN
EA
s SS SNANN
EA
s The
Peritrichida,
finally, show the most
far-reaching _—_ devia-
tions from the holo-
trichous
type, from
which they are prob-
ably derived through
the
Heterotrichida
and the Hypotrichida. In
all members
of
this group the adoral
zone is continued into
a spiral, which may
have as many as five
complete turns (Cam-
panclla). The
chief
interest
concerning
:
this
adoral
zone
is
that
in some forms
the spiral is turned
er groups
of Ciliata,
family of the Vorticel-
sinistral type of the
from an hypotrichous. of the ventral surface, E D
E
Fig. 114. — eee ne Biitschli's view of the origin of the Vor-
ticellidze,
[BUYSCHLI.]
The Trichodina form C is supposed to have arisen from the D
E
Fig. 114. — eee ne Biitschli's view of the origin of the Vor-
ticellidze,
[BUYSCHLI.] D The Trichodina form C is supposed to have arisen from the
Lichnophora-like form A by the outgrowth of the lower ciliated
area, first forming an intermediate stage B. This ring of cilia
becomes lost in the Vorticellidae, appearing only when the indi-
viduals are free-swimming. D, £. Side and front of Lichnophora
eee Slay to the left, similar to that of all of the other groups
of Ciliata,
while in other forms, belonging to the great family of the Vorticel-
lide, the spiral is turned to the right. The sinistral type of the
Peritrichida originates, according to Biitschli, from an hypotrichous. III.
INTER-RELATIONS OF THE INFUSORIA The anterior
ring of cilia is then supposed to have grown around the base of the
elevated portion until the original adoral zone of cilia now forms a ring
about the entire ventral surface. The new arm of this line of cilia
grows on past the mouth-opening and forms a spiral, which, looked
at from the ventral side, turns to the left, as in all other Ciliata seen
from the same surface. Looked at from the other side, however, z.¢. dorsally, the spiral turns to the right (C). This condition is practi-
cally represented by the genus 77ichodina, which moves about on
the skin of various Invertebrata by means of the ciliated or attach-
ment disk, in reality the posterior part of the original primitive
ventral surface; while the other portion is now carried dorsally and
parallel to the attachment disk, the mouth being on the left side of
this anterior part. In the Vorticellidee this posterior or attaching THE PROTOZOA 204 part becomes drawn out into the long contractile
stalk, while the
ciliated condition, as represented by 7richodina, is again brought
about in Vorticella, when the latter breaks away from its stalk,
develops a ciliated band in the posterior region, and swims freely
about. The ciliated band is homologous with the posterior ring of
Lichnophora and the attaching disk of Y7ichodina, while the adoral
zone of cilia conforms to the typical left-handed spiral of the remain-
ing ciliates, when looked at from the same morphological point of
view.,
In Gerda, the peristomial region has degenerated, while the
ciliated disk remains as the organ of locomotion. Returning now to the origin of the Ciliata as a group, quite another
view has been maintained by a number of observers, the essential
point of which is that the Ciliata are connected with the Sarcodina
through the Suctoria, the tentacles in the latter being regarded as
modified pseudopodia. This view was apparently first suggested by
Stein (’54) when he included the Heliozoa and the simpler forms of
Suctoria in the genus Actinophrys. The assumption was taken up
seriously by Maupas (’81), who held that through the Suctoria, the
Ciliata were derived from the Sarcodina, and Pénard ('90) accepted
the same view in regarding Actinolophus capitatus as a connecting
link between the two groups. III.
INTER-RELATIONS OF THE INFUSORIA In Mesodinium, there are only
four of these tentacles, which are arranged about the mouth (Fig. 115,
B). Leonema has only one (A). These processes were considered so
important from the phylogenetic standpoint that Mereschowsky (’82)
formed a special group, the Szctociliata, for their reception. Neither
Entz, nor Stein, nor Mereschowsky, however, regarded the tentacles as
food-taking organs like the tentacles of the Suctoria; the former, at
best, could assign to them no other function than that of assisting in
the capture of aliments. Maupas regarded them simply as pseudo-
podia, and upon them as a basis formulated his view connecting the
Ciliata with the Sarcodina. Biitschli strongly opposed Entz’s view
as to the origin of Suctoria and Ciliata, and believed that there is no or entirely drawn in by the animal. In Mesodinium, there are only
four of these tentacles, which are arranged about the mouth (Fig. 115,
B). Leonema has only one (A). These processes were considered so
important from the phylogenetic standpoint that Mereschowsky (’82)
formed a special group, the Szctociliata, for their reception. Neither
Entz, nor Stein, nor Mereschowsky, however, regarded the tentacles as A
B
Cc
Fig. 115. — Ciliata with tentacles. ©
A. Leonema dispar Stokes. [STOKES]
2B. AMesodinium pulex Clap. and Lach. [ENTz.]
C. Hypocoma parasitica Grub. [ENGELMANN.]
4, tentacles. Cc Fig. 115. — Ciliata with tentacles. ©
A. Leonema dispar Stokes. [STOKES]
2B. AMesodinium pulex Clap. and Lach. [ENTz.]
C. Hypocoma parasitica Grub. [ENGELMANN.]
4, tentacles. direct connection between the tentacles
of the two groups,
but
regarded them as independent adaptations. The hypothesis advanced
by Biitschli is that primitive forms of Suctoria (such as Hypocoma
(C), which has but one suctorial tentacle, and which retains its cilia
throughout life, the cilia being upon the ventral side only, as in
hypotrichous forms of Ciliata)
were
derived
from hypotrichous
ciliates by the mouth portion becoming progressively drawn out into
a tentacle. Haeckel (’96), adopting Biitschli’s view, compared the
simple, single, and terminal mouth-tube of a primitive suctorian with
the long, proboscis-like oral region of certain holotrichous ciliates,
such as Lacrymarta olar or L. phenicopterus. In the closely allied
forms, Didinium and Mesodinium, the oral tube is not ciliated and is
contractile, so that when food is taken in, the tube widens into more
or less of a disk similar to many suctorian tentacles. III.
INTER-RELATIONS OF THE INFUSORIA Claparéde and Lachmann were the
first to deny the connection of Ciliata and Rhizopoda, but made the
even more improbable assertion that the Suctoria are derived from
the Flagellidia through forms like Syucrypta volvox. The close rela-
tion of the Suctoria and the Ciliata was brought into prominence
through Stein’s famous, though erroneous, Acineta-theory, in which
the Suctoria were supposed to be young forms of Ciliata. The con-
nection between the two was, however, first put on a substantial basis
by the discovery of the ciliated embryos of the Suctoria, a connection
which was early accepted by students of the Protozoa, and which was
greatly emphasized by the discovery that, like the Ciliata, the Suctoria
have macro- and micronuclei. At the present time it is almost universally held that the Suctoria At the present time it is almost universally held that the Suctoria
are offshoots of the Ciliata, although the opposite view is maintained
by some observers, who, with Entz (’79, ’82), consider the Ciliata as
permanent forms of the ciliated embryos of Suctoria. Entz himself
regards the matter as insoluble, and believes that the evidence is
about equally balanced. A number of cases certainly gives strength
to Entz’s position, for many of the Enchelinide, in addition to their
cilia, have
distinct
tentacular
processes
(//conema,
Mcsodintuim,
Actinobolus, etc., Fig. 115). Actinobolus, discovered by Stein, and more recently examined by Actinobolus, discovered by Stein, and more recently examined by
Entz, has long tentacle-like threads evenly distributed about and
between the cilia (Fig. 100). They can be lengthened or shortened THE INFUSORKIA 205 or entirely drawn in by the animal. In Mesodinium, there are only
four of these tentacles, which are arranged about the mouth (Fig. 115,
B). Leonema has only one (A). These processes were considered so
important from the phylogenetic standpoint that Mereschowsky (’82)
formed a special group, the Szctociliata, for their reception. Neither
Entz, nor Stein, nor Mereschowsky, however, regarded the tentacles as
food-taking organs like the tentacles of the Suctoria; the former, at
best, could assign to them no other function than that of assisting in
the capture of aliments. Maupas regarded them simply as pseudo-
podia, and upon them as a basis formulated his view connecting the
Ciliata with the Sarcodina. Biitschli strongly opposed Entz’s view
as to the origin of Suctoria and Ciliata, and believed that there is no or entirely drawn in by the animal. CLASSIFICATION Cuass V. INFUSORIA. Protozoa in which the motor apparatus is in the form of
cilia, either simple or united into membranes, membranelles, or cirri. The cilia
may be permanent or limited to the embryonic stages. With two kinds of
nuclei, macronucleus and micronucleus. Reproduction is effected by simple
transverse division or by budding. Nutrition 1s holozoic or parasitic. Subclass I. CILIATA. Infusoria
provided
with
cilia during adult
as well as Cuass V. INFUSORIA. Protozoa in which the motor apparatus is in the form of
cilia, either simple or united into membranes, membranelles, or cirri. The cilia
may be permanent or limited to the embryonic stages. With two kinds of
nuclei, macronucleus and micronucleus. Reproduction is effected by simple
transverse division or by budding. Nutrition 1s holozoic or parasitic. Subclass I. CILIATA. Infusoria
provided
with
cilia during adult
as well as Subclass I. CILIATA. Infusoria
provided
with
cilia during adult
as well as
embryonic life. Reproduction is brought about typically by simple transverse
division. Mouth and anus are usually present. The contractile vacuole is often
connected with a complicated canal system. Order 1. HOLOTRICHIDA. Ciliata in which the cilia are similar and distributed all Subclass I. CILIATA. Infusoria
provided
with
cilia during adult
as well as
embryonic life. Reproduction is brought about typically by simple transverse
division. Mouth and anus are usually present. The contractile vacuole is often
connected with a complicated canal system. Order 1. HOLOTRICHIDA. Ciliata in which the cilia are similar and distributed all Order 1. HOLOTRICHIDA. Ciliata in which the cilia are similar and distributed all
over the body, with, however, a tendency to lengthen in the vicinity of the
mouth. Trichocysts are always present, either distributed about the body or
limited to a special region. Suborder 1. GYMNOSTOMINA. Holotrichida without an undulating membrane Suborder 1. GYMNOSTOMINA. Holotrichida without an undulating membrane
about the mouth, which remains closed except during food-taking intervals. Family 1. Enchelinida. The mouth is always terminal
or sub-terminal, and is Family 1. Enchelinida. The mouth is always terminal
or sub-terminal, and is
usually round or oval in outline. Food-taking is usually a process of swal-
lowing. Genera:
Holophrya Ehr. (31);
Urotricha
Clap. & Lach. (°58) ;
Enchelys Hill (1752), Ehr. (38) ; Spathidium Duj. (41) 3 Chenza Quennerstadt
(68) ; Prorodon Ehr. (°33); Dinophrya Biitschli (88); Lacrymaria Ehr. (’30);
Trachelocerca Ebr. III.
INTER-RELATIONS OF THE INFUSORIA In Alesodindum, direct connection between the tentacles
of the two groups,
but
regarded them as independent adaptations. The hypothesis advanced
by Biitschli is that primitive forms of Suctoria (such as Hypocoma
(C), which has but one suctorial tentacle, and which retains its cilia
throughout life, the cilia being upon the ventral side only, as in
hypotrichous forms of Ciliata)
were
derived
from hypotrichous
ciliates by the mouth portion becoming progressively drawn out into
a tentacle. Haeckel (’96), adopting Biitschli’s view, compared the
simple, single, and terminal mouth-tube of a primitive suctorian with
the long, proboscis-like oral region of certain holotrichous ciliates,
such as Lacrymarta olar or L. phenicopterus. In the closely allied
forms, Didinium and Mesodinium, the oral tube is not ciliated and is
contractile, so that when food is taken in, the tube widens into more
or less of a disk similar to many suctorian tentacles. In Alesodindum, THE PROTOZOA 206 this tube is not only retractile, but is also surrounded by four tentacle-
like processes which simulate some kinds of tentacles in the Suctoria. As the majority of the larvae of the Suctoria are ciliated in girdles,
Haeckel holds that this division of the Holotrichida represents the
nearest allies of the Suctoria, and that the loss of cilia in the adult is
already foreshadowed by the regional loss of cilia in these girdled
forms. The entire matter, finally, of the origin of Infusoria from more The entire matter, finally, of the origin of Infusoria from more
generalized
forms
of Protozoa
remains
unsolved;
the
various
hypotheses are interesting possibilities, but no more can be said for
them. This problem, like that of the origin of the Protozoa, may
never come nearer settlement ; for, without the assistance of palzonto-
logical and embryological evidence, which in other great groups of
the animal kingdom are of inestimable value in tracing ancestors, the
possibility of tracing their origin is reduced to a minimum. CLASSIFICATION (33); Actinobolus
Stein ('67);
Heonema
Stokes (84);
Plagiopogon Stein (59); Coleps Nitsch ('27); Zvarina Bergh ('79); Stepha-
nopogon Entz (84); Dzdinzum Stein (59); Afesodinium Stein (62); Biitschlia
Schuberg (°86). Family 2. Trachelinidea. The body is distinctly bilateral or asymmetrical, with Family 2. Trachelinidea. The body is distinctly bilateral or asymmetrical, with
one side, the dorsal, slightly arched. The mouth may be terminal-or sub-
terminal, or the entire mouth-region may be drawn out into a long proboscis. An cesophagus or gullet may or may not be present; when present, it is usually
supported by a specialized framework. Genera:
<lwphileptus Ehr. (730);
Lionotus Wrzesniowski (’70);
Loxophyllum Duj. (41); Trachelius Schrank
(03) 3 Deleptus Duj. (41); Loxodes Ehr. (730). THE INFUSORIA 207 Family 3. Chlamydodontida. The general form is oval or kidney-shaped. The
mouth 1s almost always in the posterior region. The pharynx is supported by a
rod-apparatus or a smooth, firin tube. Family 3. Chlamydodontida. The general form is oval or kidney-shaped. The
mouth 1s almost always in the posterior region. The pharynx is supported by a
rod-apparatus or a smooth, firin tube. Subfamily 1. Massudine. Ciliation is complete. Genera: Massela Ehr. (’33). Subfamily 1. Massudine. Ciliation is complete. Genera: Massela Eh
Subfamily 2. Chzlodonting. The body is generally flattened, and the Subfamily 1. Massudine. Ciliation is complete. Genera: Massela Ehr. (’33). Subfamily 2. Chzlodonting. The body is generally flattened, and the cilia are
stronger on the dorsal side, or are confined to that region. Genera: Orthodon
Gruber (784);
Chelodan Ehr. (°33);
Chlamydodon Ebr. (35); Opisthodon
Stein (59) ; Phascolodon Stein (’57); Scaphidiodon Stein (°57). Subfamily 3. Zrvilizue@. The cilia are confined to the ventral surface or to a por- Subfamily 3. Zrvilizue@. The cilia are confined to the ventral surface or to a por-
tion of it. The posterior end invariably possesses a movable style arising from
the
posterior
ventral
surface. Genera:
gyria Clap. & Lach. (°58);
Onychodactylus Entz. (84) ; Trochilia Duj. (41); Dysterza Huxley (’57). ;
Suborder 2. TRICHOSTOMINA. In addition to the general coating of cilia there is Suborder 2. TRICHOSTOMINA. In addition to the general coating of cilia there is
an undulating membrane or membranes at the edge of the mouth or in the
pharynx. The mouth is always open. Family 1. Chiliferide. The mouth is in the anterior half of the body or close to Family 1. Chiliferide. CLASSIFICATION The mouth is in the anterior half of the body or close to
the middle
The pharynx when present is short. The so-called “peristome
area” leading to the mouth is absent or only slightly developed. Genera:
Leucophrys Ehr. (30); Glaucoma Ehr. (30); Dallasta Stokes (186); Fron-
tonia \hr. (738); Ophryoglena Ehr. (°31); Colpidium Stein (60); Chasmato-
stoma Engelmann (62); Uronema Duj. (41); Urozona Schewiakoff (Biitschli)
(38); Loxocephalus Kent (°81); Colpoda Miiller (1773). Family 2. Ucccen‘tridea. The mouth, with a long, tubular pharynx, is in the centre Family 2. Ucccen‘tridea. The mouth, with a long, tubular pharynx, is in the centre
of the ventral side. The cilia are confined to two broad zones around the body
at each end. Genera: Urocentrum Nitsch ('27). Family 3. Microthoracida. Small asymmetrical forms, with the mouth invariably Family 3. Microthoracida. Small asymmetrical forms, with the mouth invariably
in the hinder portion. The cilia are always more or less dispersed, sometimes
limited to the oral region. There may be one or two undulating membranes. Genera:
Cinetochilum Perty ('49); A@icrothorax Engelmann (°62); Péychosto-
muim Stein ('60) ; Ancistrum Maupas (°83); Drepanomonas Fresenius ('58). Family 4. Paramecide. The mouth is sometimes in the anterior, sometimes in Family 4. Paramecide. The mouth is sometimes in the anterior, sometimes in
the posterior, half of the body, and is accompanied by a large, triangular “ peri-
stome area” running from the left anterior edge of the body to the mouth. Genera: Paramecium Stein (60). Family 5. Pleuronemide. The mouth is at the end of a long peristome which Family 5. Pleuronemide. The mouth is at the end of a long peristome which
runs along the ventral side; the body is dorso-ventrally or laterally compressed. The entire left edge of the peristome is provided with an undulating membrane
which occasionally runs around the posterior end of the peristome to form a
pocket leading to the mouth. The right edge of the peristome
is pro-
vided with a less developed membrane. There may or may not be a well-
developed pharynx. Genera: Lewbadion Perty ('49); Pleuronema Duj. (41);
Cychidium Ebr. (738), a sub-genus of the preceding;
Calyftotricha Phillips
(82); Lembus Cohn ('65). Family 6. Isotrichida. The body is more or less plastic, but not contractile. The Family 6. Isotrichida. The body is more or less plastic, but not contractile. The
cuticle
is thick and provided with evenly distributed
cilia. Family 7.
Opalinidea.
The form is oval. and the body may be short or drawn out
to resemble a worm.
They are characterized mainly by the absence
of mouth
and pharynx.
Genera: Anoplophrya Stein (60); Hoptitophrya Stein (*60);
Discophrya Stein (60); Opalinopsis Foettinger (81); Ofalina Purkinje and
Valentin (35) ; Alonodontophrya Vejdowsky (*92)-. CLASSIFICATION The mouth is
posterior and accompanied bya
distinct pharynx. They are parasites in the
digestive tract of ruminants. Genera: /sofricha Stein (’59) ; Dasytricha Schu-
berg (°88). Family 7. Opalinidea. The form is oval. and the body may be short or drawn out Family 7. Opalinidea. The form is oval. and the body may be short or drawn out
to resemble a worm. They are characterized mainly by the absence
of mouth
and pharynx. Genera: Anoplophrya Stein (60); Hoptitophrya Stein (*60);
Discophrya Stein (60); Opalinopsis Foettinger (81); Ofalina Purkinje and
Valentin (35) ; Alonodontophrya Vejdowsky (*92)-. THE PROTOZOA 208 Order 2. HETEROTRICHIDA. Ciliata characterized by the possession of a uniform
covering of cilia and an adoral zone, consisting of short cilia fused together into
membranelles. : Suborder 1. POLYTRICHINA. Heterotrichous
ciliates provided with a uniform
coating of cilia. Suborder 1. POLYTRICHINA. Heterotrichous
ciliates provided with a uniform
coating of cilia. Family 1. Plagiotomidea. The peristome is a narrow furrow, which begins, as a Family 1. Plagiotomidea. The peristome is a narrow furrow, which begins, as a
rule, close to the anterior end, and runs backward along the ventral side to the
mouth, which is usually placed between the middle of the body and the posterior
end. A well-developed adoral zone stretches along the left side of the peristome,
and it is usually straight. Genera: Couchophthirus Stein (61); Plagiotoma
Duj. (41); Myctotherus Leidy (49), a sub-genus; Blepharisma Perty (49);
Metopus Clap. & Lach. (58); Spzrostomum Ebr. (’35). Family 2. Bursarida. The body is usually short and pocket-like, but may be Family 2. Bursarida. The body is usually short and pocket-like, but may be
elongate. The chief characteristic is the peristome, which is not a furrow, but a
broad triangular area, deeply insunk, and ending in a point at the mouth. The
adoral zone is usually confined to the left peristome edge, or it may cross over to
the right anterior edge. Genera: Balantidiuim Stein (67); Balantidiopsis
Biitschli (88); Condylostoma Duj. (41); Bursaria O. F. Miiller (1773);
Thylakidium Schewiakoff (’92). Family 3. Stentorida. The peristome is relatively short and limited to the front
end of the animal, so that its plane is nearly at right angles to that of the longi-
tudinal axis of the body. The adoral zone of cilia either passes entirely around
the peristome edge, or ends at the right-hand edge. CLASSIFICATION Euplotide. Hypotrichous ciliates, which are characterized mainly by the Family 3. Euplotide. Hypotrichous ciliates, which are characterized mainly by the
considerable reduction of the cilia, as well as the frontal, marginal, and ventral
cirri; the anal cirri, on the other hand, are always present. The macronucleus
is band-formed. Genera:
Euplotes Stein ('59);
Certesta Fabre-Dumergue
(85); Déophrys Duj. C41) ; Uronychia Stein (57); Aspidisca Ehr. (°30). Order 4. PERITRICHIDA. Ciliata usually of cylindrical or cup-like form, in which Order 4. PERITRICHIDA. Ciliata usually of cylindrical or cup-like form, in which
the cilia are reduced, as a rule, to those which form the adoral zone, but sec-
ondary rings of cilia may be present. Family 1. Spirochonida. Peritrichous ciliates in which the peristome is drawn out Family 1. Spirochonida. Peritrichous ciliates in which the peristome is drawn out
into a curious funnel-like process, either simple or rolled. They are parasitic
forms in which reproduction by budding is characteristic. Genera: Spirochona
Stein (751) ; Kentrochona Rompel (’94); Kentrochonopsis Doflein (’97). Family 2. Lichnophorida. In addition to the adoral zone, there is a secondary Family 2. Lichnophorida. In addition to the adoral zone, there is a secondary
circlet of cilia around the opposite end. The adoral zone is a left-wound spiral. A single genus, Lzchnophora, Claparéde ('67), which is parasitic on various
marine arthropods. ‘
Family 3. Vorticellida. Attached or unattached forms of peritrichous ciliates, in Family 3. Vorticellida. Attached or unattached forms of peritrichous ciliates, in
which the adoral zone, seen from above, forms a right-wound spiral (dexiotropic). A secondary circlet of cilia around the under end may be present either perma-
nently or periodically. Subfamily 1. Urceolaring. Vorticellide having a permanent secondary circlet of cilia Subfamily 1. Urceolaring. Vorticellide having a permanent secondary circlet of cilia
which incloses an adhesive disk, and without a peristome fold. Genera: 7rechodina
Stein (°54); Cyclocheta Jackson ('75); Trechodinopszs Clap. & Lach. ('58). Subfamily 2. Vorticellidine. Peritrichous forms without a permanent secondary Subfamily 2. Vorticellidine. Peritrichous forms without a permanent secondary
circlet of cilia, and provided with a peristome fold which can be contracted
sphincter-like to inclose the peristome. Genera: Scyphidia Lachmann (’56) ;
Gerda Clap. & Lach. (’58);
<stylozodn Engelmann (°62);
Vorticella Ehr. (38) 3 Carchesium Ebr. (30); Zoothamnium Stein (54) ; Glossatella Biitschli
(88); Fpistylés Ehr. (°30); Rhabdostyla Kent ('82); Opercularza Stein (754) ;
Ophrydium Ebr. (38);
Cothurnia Clap. & Lach. (°58); Vaginicola Clap. CLASSIFICATION Almost invariably in these primitive
forms some of the anterior and some of the posterior cilia are fused into large
and more powerful appendages, the cirri, which are distinguished as the frontal
and aval cirri, respectively. In the majority of forms all of the cilia are thus dif-
ferentiated ; strong marginal cirri are formed in perfect rows, and ventral cirri in
imperfect rows. In addition to the adoral zone of membranelles, there is an
undulating membrane on the right side of the peristome, and, in some cases, a
row of cilia between the membrane and the adoral zone. These are the par-
oral cilia, and they form the par-oral zone. Genera:
7rdéchogaster Sterki (°78) ;
Urostyla Ehr. ('30); Kerona Ehr. ('38); Epzclintes Stein (62); Stichotricha
Perty (49); Strongylidium Sterki (78); Amphesia Sterki ('78);
Uroleptus
Stein (59) ; Sparotricha Entz (79); Onychodromus Stein (59); Pleurotricha
Stein (’59); Gastrosivla Engelmann (°62) ; Gonostomum Sterki (78); Urosoma
Kowalewsky (82) ; Oxytricha Ehr. (30); Stylonychia Stein (59) ; Actinotricha
'
Cohn (66); Balladina Kowalewsky (82); Pszlotricha Stein (59); Tetrastyla
Schewiakoff (’92) ; Afolosticha Wrzesniowski (’77). Family 2. Oxytrichidea. The peristome is not always distinctly marked off from
the frontal area. In the most primitive forms the ciliation on the ventral surface
is similar to that of the preceding family. Almost invariably in these primitive
forms some of the anterior and some of the posterior cilia are fused into large
and more powerful appendages, the cirri, which are distinguished as the frontal
and aval cirri, respectively. In the majority of forms all of the cilia are thus dif-
ferentiated ; strong marginal cirri are formed in perfect rows, and ventral cirri in
imperfect rows. In addition to the adoral zone of membranelles, there is an
undulating membrane on the right side of the peristome, and, in some cases, a
row of cilia between the membrane and the adoral zone. These are the par-
oral cilia, and they form the par-oral zone. Genera:
7rdéchogaster Sterki (°78) ;
Urostyla Ehr. ('30); Kerona Ehr. ('38); Epzclintes Stein (62); Stichotricha
Perty (49); Strongylidium Sterki (78); Amphesia Sterki ('78);
Uroleptus
Stein (59) ; Sparotricha Entz (79); Onychodromus Stein (59); Pleurotricha
Stein (’59); Gastrosivla Engelmann (°62) ; Gonostomum Sterki (78); Urosoma
Kowalewsky (82) ; Oxytricha Ehr. (30); Stylonychia Stein (59) ; Actinotricha
'
Cohn (66); Balladina Kowalewsky (82); Pszlotricha Stein (59); Tetrastyla
Schewiakoff (’92) ; Afolosticha Wrzesniowski (’77). Family 3. CLASSIFICATION The surface of the peri-
stome is spirally striated and provided with cilia. Undulating membranes are
absent. Genera: Clzacostomum Stein (759); Stentor Oken (115); Folliculina
Lamarck (16). Genera izcerte sedis: Cenomorpha (Gyrocorys Stein) Perty
(52); Maryna Gruber (’79). Suborder 2. QLIGOTRICHINA. Heterotrichous
ciliates characterized by the re-
duced cilia, which are limited to certain localized areas. Family 1. Lieberkuhnide. This name was given by Biitschli for certain little-known Family 1. Lieberkuhnide. This name was given by Biitschli for certain little-
forms, which were at first considered young Stentors. Family 2. Halteriidea. The peristome has no
cilia, and only a few sca Family 2. Halteriidea. The peristome has no
cilia, and only a few scattered
ones can be found on the ventral and dorsal surfaces. Genera: Strombidium
Clap. & Lach. (58); Halterza Duj. (41). Family 3. Tintinnide. The body is attached bya
stalk toatheca. Inside of the
adoral zone of membranelles is a ring of cilia (par-oral cilia). Genera: Ziutin-
nus Fol. (89); Tintinnidium Kent (81); Zintinnopsis Stein (°67) ; Codonella
Haeckel ('73) ; Dzctyocysta Ehr. (54). Family 4. Ophryoscolecida. Heterotrichous ciliates characterized by a thick cuticle Family 4. Ophryoscolecida. Heterotrichous ciliates characterized by a thick cuticle
and deep funnel-like peristome. The posterior end is provided with distinct
spine-like processes, while the terminal anus is provided with a well-defined anal
tube. Genera: Ophryocolex Stein (59); Antodinium Stein (59); Diplodinium
Schuberg (88). Order 3. HYPOTRICHIDA. Ciliata in which the cilia are limited to the ventral surface Order 3. HYPOTRICHIDA. Ciliata in which the cilia are limited to the ventral surface
of a dorso-ventrally flattened body; they are frequently fused to form larger
appendages, the cirri, and an adoral zone of membranelles. The dorsal surface
is frequently provided with bristles. A pharynx may be absent or but slightly
developed. Family 1. Peritromide. The peristome is but slightly marked off from the remain- Family 1. Peritromide. The peristome is but slightly marked off from the remain-
ing frontal area. The cilia on the ventral surface are uniform in size and
arrangement,
and
are
not
differentiated
into
cirri. Genera:
Perdtromus
Stein (’62). THE INFUSORLA 209 Family 2. Oxytrichidea. The peristome is not always distinctly marked off from
the frontal area. In the most primitive forms the ciliation on the ventral surface
is similar to that of the preceding family. CLASSIFICATION &
Lach. (58); Lagenophrys Stein (’51). Subclass II. SUCTORIA. Infusoria having no cilia during the adult stages, but Subclass II. SUCTORIA. Infusoria having no cilia during the adult stages, but
provided with them during the embryonic period. In a few cases the cilia are
retained. They have tentacles of various kinds, some adapted for sucking, some
for piercing. P THE PROTOZOA 210 Family 1. Hypocomidz. These are unattached forms of Suctoria with a perma-
nently ciliated ventral surface, and with one suctorial tentacle. Reproduction is
effected by cross-division. A single genus, Hyfocoma Gruber (784). Family 2. Urnulide. A family of small attached forms, with or without a cup or Family 2. Urnulide. A family of small attached forms, with or without a cup or
theca; with one or two, rarely more, simple tentacles. Swarm-spores holo-
trichous. Genera: Rhyncheta Zenker (66); Crile Clap. & Lach. (758). Family 3. Metacinetidea. Thecate forms; the base of the cup is drawn out intoa Family 3. Metacinetidea. Thecate forms; the base of the cup is drawn out intoa
long stalk, and the walls are perforated for the exit of the tentacles. A single
genus, A/elacineta Bitschli (’88). Family 4. Podophryida. Stalked or unstalked forms of more or less globular Family 4. Podophryida. Stalked or unstalked forms of more or less globular
shape. The tentacles are numerous and distributed about the entire surface or
limited to the apical region; some of them are knobbed, others pointed and
have a prehensile
function. Genera:
Spherophrya Clap & Lach. (’58);
fndosphera Engelmann (’76); Podophrya Ehr. (38); Aphelota Str. Wright
(58); Podocyathus Kent (’81). Family 5. Acinetida. The individuals
are naked and stalked, or thecate and
stalked or unstalked. The tentacles are numerous, usually knobbed and all
alike. Reproduction is effected by inner or exdogenous budding, which may be
simple or multiple. The swarm-spores are usually peritrichous, but may be
holotrichous or hypotrichous. Genera:
Zokophrya Biitschli (88); Aczneta
Ebr. (733) ; Solenophrya Clap. & Lach. (°58) ; Suctorella Frenzel (’91). Family 6. Dendrosomid#. Suctoria without stalks or theca. The tentacles are Family 6. Dendrosomid#. Suctoria without stalks or theca. The tentacles are
numerous, all alike, and knobbed and grouped in distinct tufts; they may be
simple or branched. Reproduction by endogenous division ; the swarm-spores are
peritrichous. Genera:
Zrichophrya Clap. & Lach. (°58); Dendrosoma Ehr. (38) 3 Staurophrya Zacharias (’93)-
Family 7. Dendrocometida. CLASSIFICATION Sessile Suctoria resting upon the entire basal surface Family 7. Dendrocometida. Sessile Suctoria resting upon the entire basal surface
or upon a portion of it raised asa stalk. The numerous tentacles are short and
knobbed, and distributed over the entire apical surface
or localized upon
branched arms. Spore-formation is endogenous; the swarm-spores peritrichous. Genera: Dendrocometes Stein (67); Stylocometes Stein ('67). Family 8. Ophryodendrida. Stalked or sessile forms possessing numerous long, Family 8. Ophryodendrida. Stalked or sessile forms possessing numerous long,
rarely knobbed tentacles, which are supported upon proboscis-like processes of
the apical side. Reproduction is brought about by endogenous budding. The
swarm-spores are peritrichous. Genera: Opkryodendron Clap. & Lach. (’58). SEXUAL PHENOMENA IN THE PROTOZOA “Die Bedeutung des Konjugationsaktes ist ein Verjiingung der ihn begehenden Tiere. Durch diese Verjiingung erscheinen uns die aus der Konjugation hervorgehenden Individuen
sehr geeignet, zu den Stammvatern einer Reihe durch Theilung sich furtpflanzender Gene-
rationen zu werden,
im Laufe welcher allmahlich ein Sinken der Lebensenergie sich einstellt. Letzter Umstand findet seinen Ausdruck darin, das die Grdsse der Individuen mehr und
mehr sinkt sodass schliesslich eine minimalgrésse erreicht wird, worauf eine neue Kunjuga-
tionsepoche eiptritt.” — BUTSCHLI. THE power of the animal or plant to reproduce its kind froma
por-
tion of its own body is bound up, in the higher forms of life, with sexual
processes and, in its more familiar forms, accordingly, is characterized
as sexual reproduction. It involves the union of two cells having quite
different characteristics ;
the spermatozoén or male cell, being minute
and active, the ovum or female cell, larger and quiescent. Reproduc-
tion of the individual without sexual processes is, however, possible,
even in the higher organism, as we daily witness in plant “cuttings,”
and as is almost equally well known in the higher forms of inverte-
brates
such as insects, where, without fertilization, an ovum may
develop into an adult form. In the lower forms of Invertebrata, such
as the worms and the Ccelenterata, so-called “asexual reproduction ”’
by division or by budding is widespread, and in the Protozoa this
method of reproduction is the usual form. The phenomena of parthenogenesis, or development from the egg The phenomena of parthenogenesis, or development from the egg
without fertilization, and reproduction by simple division or by bud-
ding as seen in the Protozoa and the lower Metazoa, have recently
led to the pertinent query: With what right do we distinguish the phe-
nomena of reproduction as “sexual” and “asexual” ? (Hertwig, ’9Q). In two recent publications R. Hertwig (’98, ’99) has discussed this
question in a very interesting and convincing manner, and he main-
tains that, in order to speak of “sexual” reproduction, it must be
shown that in the Protozoa, for example, fertilization has some direct
effect upon division or that a certain specific form of division results
from fertilization, neither of which, he says, is true in the majority of
cases. SPECIAL BIBLIOGRAPHY VI Biitschli, 0.— Protozoa, Infusoria. In Bronn’s Klassen und Ordnungen des Thier-
reichs. Lezpz7g, 1886-88. Entz, G.— Protistenstudien. Budapesth, 1888. Entz, G.— Protistenstudien. Budapesth, 1888. Kent, W. Saville. — A Manual of the Infusoria. Kent, W. Saville. — A Manual of the Infusoria. Zondon, 1881. Schewiakoff, W.—Beitrage
zur Kenntniss
der Holotrichen Kent, W. Saville. — A Manual of the Infusoria. Zondon, 1881. Schewiakoff, W.—Beitrage
zur Kenntniss
der Holotrichen
Ciliaten. Bzdliotheca
Zoologica, Heft 5, 1889. Schewiakoff, W.—Beitrage
zur Kenntniss
der Holotrichen
Ciliaten. Bzdliotheca
Zoologica, Heft 5, 1889. Stein, Fr.— Der Organismus der Infusionsthiere. Zezpszg, 1863 and 1867. Zoologica, Heft 5, 1889. Stein, Fr.— Der Organismus Stein, Fr.— Der Organismus der Infusionsthiere. Zezpszg, 1863 and 1867. SEXUAL PHENOMENA IN THE PROTOZOA All observers are apparently agreed that asexual reproduction as All observers are apparently agreed that asexual reproduction as
seen in the processes of binary fission and spore-formation
is a
result of growth, usually expressed by the statement that increase 1(’76), p. 421. 211 1(’76), p. 421. 211 211 THE PROTOZOA 212 in volume continually tends to outrun that of surface, for the former
increases as the cube of the diameter, the latter only as the square. The mass of living protoplasm must, therefore, increase more rapidly
than the surface which serves to keep it alive, and the isolated cell
tends to come first to a physiological standstill, and second to a period
of decline, since the surface of nutrition, respiration, and excretion is
incommensurate with the bulk of protoplasm. Cell-division, there-
fore, which Spencer characterized as an indication of the limit of
growth, becomes an apparent necessity. Growth, or the preponderance of constructive processes, leads thus
indirectly to increase of surface by division of the cell, but in conju-
gation or fertilization the very opposite phenomenon occurs, wzs. the
increase in bulk of the cell with a consequent relative decrease in sur-
face because of the union of two cells. Geddes and Thompson (’90),
like Spencer, emphasize the connection between division and the
advent of preponderating katabolism within the cells, and in their
interesting work on the Evolution of Sex, interpret male and female
organisms in terms of relative metabolism, the male being regarded as
relatively katabolic, the female as anabolic. These authors interpret
fertilization as a “ katabolic stimulus to an anabolic cell, and on the
other side, of course, as an anabolic renewal to a katabolic cell, as well
as the union of opposed hereditary characteristics.” 1
If, as Minot (’79) suggested, every newly formed organism be re- If, as Minot (’79) suggested, every newly formed organism be re-
garded as having a certain initial potential energy which is gradually
used up in its life-activities to be restored by conjugation, then the
union of two cells may be interpreted as a renewal of vigor or a “re-
juvenescence” (Maupas), a view of fertilization
first expressed by
Biitschli (76), Engelmann (’76), and Minot (’77, ’79), and apparently
confirmed later by Maupas (’88, ’89) through his admirable observa-
tions on the life-cycle of Infusoria. O. 1 Z00. City. Po 232. SEXUAL PHENOMENA IN THE PROTOZOA If then we define sex as the condi-
tion by which single-celled or many-celled organisms are differentiated
znto male and female, we must admit that the origin of sex is only a
part of the problem, for fertilization occurs between individuals in
which there is no apparent sex-difference, as well as between those
possessing it. a renewed vitality, or in Bitschli’s words (’76) a renewal of youth
( Versiingung), expressed by increased activity in movements
and
reproduction. Conjugation thus, as R. Hertwig insists, is not the
beginning of a series of reproductive acts, but occurs at or near the
end of such a series. Maupas’s results seem to offer conclusive evi-
dence that the absence of conjugation involves a cumulative degener-
ative process which ultimately ends in death. The phenomena of
so-called sexual reproduction and of sex-differentiation have, in all
probability, grown out of this apparently fundamental requirement of
living protoplasm, namely, the periodic union of two cells; and I
believe with
Biitschli,
Engelmann,
Maupas,
Hertwig, and many
others, that it cannot in itself be regarded as primarily a repro-
ductive act. None of the facts that have been determined show that
the morphological distinction of the sexes is a primary attribute or
property of living organisms, nor do any of the dynamic views of
fertilization afford an explanation of sex-differences any more than
does the statement of Geddes and Thompson cited above. Even
though the views of these authors be accepted, we should still have
to admit that no explanation of fertilization can be wholly satisfactory
unless it is equally applicable to forms which cannot be distinguished
as more anabolic or katabolic, z.c. to the conjugation of equal-sized
Infusoria, Mastigophora, or Sporozoa, as well as the union of differ-
entiated male and female cells. If then we define sex as the condi-
tion by which single-celled or many-celled organisms are differentiated
znto male and female, we must admit that the origin of sex is only a
part of the problem, for fertilization occurs between individuals in
which there is no apparent sex-difference, as well as between those
possessing it. In the present chapter I have brought together some of the evi-
dence which bears upon these several points. A number of phe-
nomena which accompany reproduction in the higher animals, and
which have attracted so much attention among biologists of all times,
are seen in simpler forms in Protozoa. SEXUAL PHENOMENA IN THE PROTOZOA Hertwig (’76) also, in con-
nection with fertilization of the metazoan egg, arrived at a similar
dynamic view of fertilization, maintaining that protoplasm gradually
tends toward a state of stable equilibrium expressed by decreased
activity, etc., and that it is restored to a more unstable or more labile
condition by conjugation or fertilization. The force of these views
as to the need of conjugation for different species of Infusoria, at
least, can hardly be questioned ; for, as repeatedly stated in the pre-
ceding chapters, reproduction by simple division may go on for a
certain number of generations, but cannot continue indefinitely, unless
at certain intervals, which Maupas has shown to be more or less
definite, two individuals unite in conjugation. This union, in some
wholly
unexplained
way,
imparts
to
each
of
the
conjugants 1 Z00. City. Po 232. SEXUAL PHENOMENA IN THE PROTOZOA 213 a renewed vitality, or in Bitschli’s words (’76) a renewal of youth
( Versiingung), expressed by increased activity in movements
and
reproduction. Conjugation thus, as R. Hertwig insists, is not the
beginning of a series of reproductive acts, but occurs at or near the
end of such a series. Maupas’s results seem to offer conclusive evi-
dence that the absence of conjugation involves a cumulative degener-
ative process which ultimately ends in death. The phenomena of
so-called sexual reproduction and of sex-differentiation have, in all
probability, grown out of this apparently fundamental requirement of
living protoplasm, namely, the periodic union of two cells; and I
believe with
Biitschli,
Engelmann,
Maupas,
Hertwig, and many
others, that it cannot in itself be regarded as primarily a repro-
ductive act. None of the facts that have been determined show that
the morphological distinction of the sexes is a primary attribute or
property of living organisms, nor do any of the dynamic views of
fertilization afford an explanation of sex-differences any more than
does the statement of Geddes and Thompson cited above. Even
though the views of these authors be accepted, we should still have
to admit that no explanation of fertilization can be wholly satisfactory
unless it is equally applicable to forms which cannot be distinguished
as more anabolic or katabolic, z.c. to the conjugation of equal-sized
Infusoria, Mastigophora, or Sporozoa, as well as the union of differ-
entiated male and female cells. A. PHENOMENA OF CONJUGATION With our present knowledge it is impossible to say that conjuga-
tion is absent in any group of Protozoa, and until the life-cycle of
every genus is fully known, the conservative but logical view which
Biitschli expressed in regard to flagellates, is the most acceptable. He
says: “I, personally, am inclined to the view that the significance of
this process in the life of these organisms is so general and deep-
reaching that the failure to observe it in certain groups up to the
present time is no reason for considering it absent.”2
Nevertheless,
the observations which have been recorded show the greatest diversity
in the process, the variations passing from extremely simple fusion,
which only by stretching the meaning of the term can be called
sexual, to the highly differentiated male and female organisms where,
as in the Metazoa, fertilization is followed by cleavage. Stages in the development of so-called sexual reproduction may be Stages in the development of so-called sexual reproduction may be
considered as follows :—
1. The permanent
or temporary
union
of similar
adult cells 1. The permanent
or temporary
union
of similar
adult cells
(sogamy) (Sarcodina, Sporozoa, Mastigophora, Ciliata). 2. The union of individuals apparently similar in all respects save 1. The permanent
or temporary
union
of similar
adult cells
(sogamy) (Sarcodina, Sporozoa, Mastigophora, Ciliata). 2. The union of individuals apparently similar in all respects save 2. The union of individuals apparently similar in all respects save
size (Auzsogamy) (Sarcodina, Mastigophora, Ciliata). 3. The union of reduced individuals. Swarm-spores (/sogamy or 3. The union of reduced individuals. Swarm-spores (/sogamy or
Anisog my) (Sarcodina, Mastigophora). 4. The union of specialized individuals—male and female cells 3. The union of reduced individuals. Swarm-spores (/sogamy or
Anisog my) (Sarcodina, Mastigophora). 4. The union of specialized individuals—male and female cells 4. The union of specialized individuals—male and female cells
(Spermatozoa and eggs) (Sporozoa, Flagellidia). 4. The union of specialized individuals—male and female cells
(Spermatozoa and eggs) (Sporozoa, Flagellidia). 1. Lhe permanent or temporary union of similar adult individuals
(lsogamy). Thanks to the unbroken observations of Dallinger and Drysdale, the 1. Lhe permanent or temporary union of similar adult individuals
(lsogamy). Thanks to the unbroken observations of Dallinger and Drysdale, the
conjugation and full life-history of some of the lowest forms of Pro-
tozoa (monads) have been made out. SEXUAL PHENOMENA IN THE PROTOZOA Among these the phenomena
of sex-differentiation, of maturation or preparation for fertilization,
and fertilization itself are of paramount interest. The explanation of sex-differentiation is not as yet made more The explanation of sex-differentiation is not as yet made more
easy by the study of Protozoa, and here as in higher animals it must
remain entirely hypothetical until future research throws more light
upon the problem. The conditions accompanying conjugation, how-
ever, have been carefully studied and analyzed in relation to other
vital functions of the cell, and the evidence thus acquired gives a clue,
I believe, to an ultimate explanation of fertilization. An important
underlying principle was first made out by Biitschli (76) and Engel-
mann (’76) upon degenerating Infusoria, and was expressed by Minot THE PROTOZOA 214 three years later in the sentence, “the exhaustion of the rejuvenating
power (z.e. the exhaustion of the initial potential of vitality) becomes
the stimulus for the formation of the sexual products.” } 1(’79), p- 199. A. PHENOMENA OF CONJUGATION All of the forms examined
reproduce by simple division for a few days, and then conjugate. In
Cercommas
longicauda Duj. (typica Kent), one of the Monadida,
reproduction by ordinary fission continues for two to four days, when
the offspring, without losing their flagella, become amceboid and con-
jugate two by two (Fig. 116). The union begins with the fusion of
the pseudopodial processes,
and, as it progresses,
the flagella are
withdrawn. The nuclei finally unite (D), and the product of the
union, or the zygo¢e, forms a thin-skinned globular cyst (£). After a 1(’79), p- 199. 1(’79), p- 199. 2 (83), p. 778. 1(’79), p- 199. 1(’79), p- 199. 2 (83), p. 778. 2 (83), p. 778. SEXUAL PHENOMENA IN THE PROTOZOA 215 short resting period the cyst breaks and an innumerable quantity of
fine spores pour out (/). short resting period the cyst breaks and an innumerable quantity of
fine spores pour out (/). E
F
Fig. 116.— Conjugation of Cercomonas. [DALLINGER and DRYSDALE.]
The vegetative cells increase by transverse division (4. 2). When sexually mature they become
ameebcid, and then fuse (C, D). The result is a zygote (Z), which ultimately bursts and liberates
masses of spores (/*). In Zetramttus, one of the Polymastigida, a similar period of repro-
duction by longitudinal division
finally results in amceboid forms
which conjugate, forming cysts and spores (Fig. 117). The process F F In Zetramttus, one of the Polymastigida, a similar period of repro-
duction by longitudinal division
finally results in amceboid forms
which conjugate, forming cysts and spores (Fig. 117). The process THE PROTOZOA 216 of conjugation is somewhat modified in certain Bodos of the group
Heteromastigida, where, after a period of binary fission, as in Cerco-
monas, the individuals become amoeboid, two or three fusing while in EE
Fig. 117.— Conjugation in Tetramitus rostratus Perty. [STEIN.]
A, B. Individuals from the front and side. C. Amceboidtorm. D. Conjugation of amoeboid
forms. £. Cyst. #, G, H. Development of the spores. EE Fig. 117.— Conjugation in Tetramitus rostratus Perty. [STEIN.]
A, B. Individuals from the front and side. C. Amceboidtorm. D. Conjugation of amoeboid
forms. £. Cyst. #, G, H. Development of the spores. Fig. 117.— Conjugation in Tetramitus rostratus Perty. [STEIN.]
ividuals from the front and side. C. Amceboidtorm. D. Conjugation of amoeboid
yst. #, G, H. Development of the spores. this condition to form a common mass. A. PHENOMENA OF CONJUGATION After a resting period, the
encysted mass breaks up into an immense number of small individuals,
or spores, differing from those in Cercomonas, in that each one is
similar to the parent organism. This union, however, appears to be
purely facultative, for the same process of encystment and spore- this condition to form a common mass. After a resting period, the
encysted mass breaks up into an immense number of small individuals,
or spores, differing from those in Cercomonas, in that each one is
similar to the parent organism. This union, however, appears to be
purely facultative, for the same process of encystment and spore- SEXUAL PHENOMENA IN THE PROTOZOA 217 formation may take place in an isolated individual, and in this case,
at least, reproduction cannot be dependent upon sexual union or
conjugation, although it does not signify that conjugation is not nec-
essary for the continued power of reproducing. Kent (’81), who has
confirmed Cienkowsky’s (’65) observations upon conjugation of Bodo
augustatus, states that a difference exists in the number of spores that
are formed in the fusion and in the solitary cysts, only four spores
arising from the latter. A significant feature in the conjugation of these forms is that the A significant feature in the conjugation of these forms is that the
individuals lose their customary outline and become amoeboid prior to Fig. 118.— Conjugation in Rhizopoda. [RHUMBLER.]
A, B,C. Diffugia lobostoma Duj. D. Aggregated condition of Amweéa verrucosa Ehr. Fig. 118.— Conjugation in Rhizopoda. [RHUMBLER.]
A, B,C. Diffugia lobostoma Duj. D. Aggregated condition of Amweéa verrucosa Ehr. fusion, thus showing that some change has taken place in the con-
sistency of the plasm. A great number of observations have been made among the Rhi- A great number of observations have been made among the Rhi-
zopoda upon so-called conjugation phenomena between similar indi-
viduals, the process varying in complexity from simple contiguity to
the more complicated fusion of cell-bodies. While many of the
earlier observations probably dealt with division phases rather than
with conjugating individuals, a possibility of error first pointed out by
Claparéde and Lachmann (’56), conjugation of shelled forms has been
safely established through the observations of Biitschli (74), Jickeli
(84), Blockmann (’88), Pénard (90), Rhumbler (’98), and
others,
and of unshelled forms by Schultze (Gromza, ’75), Holman (’86), and
Kiihne (64). 2 Cf. union during the ameebvid stage of Cercomonas and other flagellates. 1Cf. Cuénot (’97); Rhumbler (’98).
2 Cf. union during the ameebvid stage 1Cf. Cuénot (’97); Rhumbler (’98).
2 Cf. union during the ameebvid stage of Cercomonas and other flagellates. A. PHENOMENA OF CONJUGATION The phenomena of cyfofropy, or the mutual attraction of two or The phenomena of cyfofropy, or the mutual attraction of two or
more cells, among the Sarcodina at least, if not in all forms, are
probably closely connected with conjugation and may possibly be the 218 THE PROTOZOA first stages in the development of sexual reproduction.! It is certainly
reasonable to argue that the mutual attraction of two previously
separated blastomeres of the frog’s egg (Roux, ’94), or the reunion
of an amputated pseudopodium with the main body of Difiugia
(Verworn, ’88; Rhumbler, ’98), the union of numerous naked Ameba
verrucosa into a common aggregate (Rhumbler, ’98), and the union
of two conjugating individuals, are all phenomena of the same order. The phenomenon in Amwba appears to have no bearing upon the
function of reproduction, for, according to Holman’s and Kiihne’s
accounts, conjugation is not followed by reproduction, while true
conjugation phases may yet be found in other stages of the life-
history of Ame@ba (Fig. 118, D). The discovery by Schaudinn of
swarm-spores
in the allied form Parameba, and of the union of
swarm-spores in the more or less closely allied rhizopod Hyalopus,
makes it not at all improbable that the same
thing may occur in Am@ba verrucosa. Thus cytotropy, leading first to contiguity, Fig. 119. — Conjugation in
Arcella vulgaris Khe. ?. Perinuclear plasm. %
Nuclei. Fig. 119. — Conjugation in
Arcella vulgaris Khe. ?. Perinuclear plasm. %
Nuclei. Fig. 119. — Conjugation in
Arcella vulgaris Khe. ?. Perinuclear plasm. %
Nuclei. Such plastogamic union in the cases cited has apparently no effect
upon the united organisms; both Johnson and Schaudinn found no
changes in the nuclei
in Actrnophrys
and Actinospherium, and SEXUAL PHENOMENA
[IN THE PROTOZOA 219 Rhumbler reached similar results
in the fused Difflugias. The
latter, however, calls attention to the fact that two organisms thus
united are subject to the interchange of substances through osmosis,
and he maintains that such an interchange must take place between
them. This interchange may even extend to the substances of the
nuclei, which are constantly renewed
from, and given off to, the
cytoplasm. Although fusion of the nuclei of the forms just mentioned does not Although fusion of the nuclei of the forms just mentioned does not
take place, the stimulus of the cytoplasmic interchange in some simi-
lar cases is apparently sufficient to bring about reproduction. A. PHENOMENA OF CONJUGATION Thus,
in certain Reticulariida (Patellina corrugata and Discorbina globularis,
Schaudinn, ’95), two, three, four, or even five individuals may fuse
and form embryos without a previous nuclear union. In these cases
plastogamy alone is apparently a sufficient stimulus for reproduction. It is by no means a fanciful assumption to postulate the union of It is by no means a fanciful assumption to postulate the union of
the two nuclei, or karyogamy, through conditions similar to those
which lead to the union of two organisms through cytotropy, 2.e. mutual attraction and consequent fusion when the nuclear plasm is
in the right condition, a condition defined by Dangeard (’99) as
“sexual hunger.”
Almost all cases of karyogamy are complicated by nuclear pro- Almost all cases of karyogamy are complicated by nuclear pro-
cesses analogous to maturation
in Metazoa, a few doubtful cases
among the Mastigophora and Rhizopoda alone indicating that fusion
may take place without a preliminary loss of a portion of the nucleus. Thus Jickeli (84) and Rhumbler (’98) observed two individuals of
Difiugia globulosa with but a single nucleus in conjugation, and
similar observations by Pénard (’90) upon a number of different
species indicate that the phenomenon is widespread (Fig. 118, A). In D. /obostoma, Rhumbler occasionally found two mouth openings
in one shell, and interpreted it as a case of fusion of shells as well
as of protoplasm (8, C). Blochmann (’88) observed the fusion of
two Euglyphas and the formation of a large double shell. In both
cases there was but one functional nucleus observed, although in the
latter form the peculiar behavior of the nucleus in one animal was
very suggestive of maturation (77de zfra). The union of nuclei in temporary conjugants among the Flagel- The union of nuclei in temporary conjugants among the Flagel-
lidia has been observed
in at least one
case,
ANocti/uca
miliaris
(Cienkowsky, 73, and Ishikawa, ’91). Two individuals fuse, their
nuclei come together, but do not fuse, and then separately undergo
mitosis, which results in four daughter-nuclei. These separate and
then fuse in the daughter-cells, two by two; thus nuclear fusion
takes place some time after conjugation. In the simpler flagellates
the nuclei fuse before spore-formation (Dallinger and Drysdale,
"93. THE PROTOZOA 220 Karyogamy
is widely spread throughout the Infusoria, where
conjugation in the different species is characterized by very similar
features. A. PHENOMENA OF CONJUGATION Two individuals unite, usually by the anterior ends, with
the mouth openings apposed. A protoplasmic bridge is formed
between the two, through which there is an interchange of micro-
nuclei. This interchange
is followed by final separation of the
conjugants, each of which regenerates the parts lost during the
period of conjugation (oral cilia, macronucleus, etc.). After careful
observations upon many different species of Infusoria, Maupas (’88,
89) found that certain conditions are apparently necessary in order
that conjugation between two individuals can take place and lead to
fertile results. These conditions are: (1) Sexual maturity, that is,
the individuals must be removed by some generations from the last
conjugating pair. Maupas established the fact that, in Leucophrys
patula, only individuals of the three hundredth to the four hun-
dred and fiftieth generation could be reinvigorated by conjugation;
in Onychodromus -only individuals between
the one hundred and
fortieth and the two hundred and thirtieth generation; and in S¢y/o-
nychia pustilata only individuals between the one
hundred
and
thirtieth and the one hundred and eightieth generations. If these
individuals, when thus mature, are restrained from pairing, they
become over-mature, after which, if they conjugate, the union is
without result, and the individuals finally succumb to what Maupas
calls “senile degeneration.” (2) A second condition is scarcity of food. Maupas also shows
that an over-abundance of food causes the individuals to die from
senile degeneration without developing the “sexual hunger.”
(3) A third condition is diverse ancestry. Maupas arrived at the (2) A second condition is scarcity of food. Maupas also shows
that an over-abundance of food causes the individuals to die from
senile degeneration without developing the “sexual hunger.”
(3) A third condition is diverse ancestry. Maupas arrived at the (3) A third condition is diverse ancestry. Maupas arrived at the
conclusion that two individuals from the same ancestor would not
conjugate. ‘In many pure cultures of nearly related individuals,”
he says, ‘the fast to which I subjected them resulted either in their
becoming encysted, or in their dying of hunger.”
“It was not until
after senile degeneration had already begun to make inroads in the
culture that I noticed that the conjugation of nearly related indi-
viduals occurred in the experimental cultivations. 1 Loc. city p. 41l. 2 (788), p. 1638, 1 Loc. city p. 41l.
2 (788), p. 1638, A. PHENOMENA OF CONJUGATION However, all such
conjugations ended with the death of the Infusoria which had paired,
but which were unable to develop further, or to reorganize themselves
after they had fused. Such pairings are, therefore, pathological
phenomena due to senile degeneration.” 1 (3) A third condition is diverse ancestry. Maupas arrived at the
conclusion that two individuals from the same ancestor would not
conjugate. ‘In many pure cultures of nearly related individuals,”
he says, ‘the fast to which I subjected them resulted either in their
becoming encysted, or in their dying of hunger.”
“It was not until
after senile degeneration had already begun to make inroads in the
culture that I noticed that the conjugation of nearly related indi-
viduals occurred in the experimental cultivations. However, all such
conjugations ended with the death of the Infusoria which had paired,
but which were unable to develop further, or to reorganize themselves
after they had fused. Such pairings are, therefore, pathological
phenomena due to senile degeneration.” 1
The latter conclusion, drawn by Maupas, has not been entirely The latter conclusion, drawn by Maupas, has not been entirely
sustained. Biitschli was one of the first to question it,? and recently
Joukowsky (’98) observed fertile conjugations among descendants of 2 (788), p. 1638, SEXUAL PHENOMENA
OF THE PROTOZOA 221 the same individual. Maupas’s
conclusion, therefore,
that cross-
fertilization is necessary for Infusoria, as for Metazoa, appears to
have been somewhat premature, although in view of the extreme
care
with which
his
observations
and experiments were made,
the objections which have been brought against it are not entirely
conclusive. It is evident from the foregoing review that, with the exception of It is evident from the foregoing review that, with the exception of
sex-differentiation,
all of the essential features which characterize
fertilization are present in those forms of Protozoa where conjugation
takes place between similar adult individuals. Here, also, a hint as
to the significance of fertilization is seen in the fact that the form of
the conjugating individuals is altered, thus indicating some change in
the density of the protoplasm. Thus some Mastigophora and Sarco-
dina become viscous, and some Infusoria show unmistakable signs
of exhaustion. Under these changed conditions the fusion of the
cell-body is possible (plastogamy). This fusion may be partial
(Cystoflagellidia, Gregarinida, Infusoria), or total (Monadida, Heliozoa,
Rhizopoda), and it may or may not be accompanied by nuclear fusion
(karyogamy). A. PHENOMENA OF CONJUGATION The same stages may ‘be conceived for the union of
the nuclei as for the union of the cell-bodies, the evidence appearing
to show that as plastogamy is the outcome of cytotropy, or positive
chemotaxis, so karyogamy is the outcome of karyotropy or nuclear
attraction, and is made possible by plastogamy. 2. The unton of similar but different-sized individuals. 2.
The unton of similar but different-sized individuals. Sexual differentiation is established when the conjugating organ-
isms are of different size. No sharp line, however, can be drawn
between conjugation in isogamous and anisogamous forms, but a
number of instances might be cited in which the union of different-
sized individuals is purely facultative, and the same result is accom-
plished either by the union of similar or of dissimilar forms. Thus,
of two conjugating Bodos (Heteromita), one, which is formed by
transverse division, is motile and becomes attached to a stationary
form resulting from a longitudinal division, and anchored by one of
its flagella. With the exception of these differences, which certainly
indicate some internal difference in the gametes, the conjugants are
identical. Here, for the first time, a distinction can be made between
the more quiescent and the more motile conjugant, although it is not
marked by difference in size. The latter condition, however, exists
in Polytoma, where, according to Krassilstschik (’82) and Dallinger
and Drysdale, normal free-swimming forms unite with smaller ones. Here, however, the process
is purely facultative, for conjugation
between similar-sized individuals also takes place. Biitschli does not THE PROTOZOA 222 consider this an indication of sex-difference, but merely the chance
fusion of two Polytomas of different age. Nevertheless, the phenom-
enon is significant, and indicates that the smaller
or less-grown
individual has the capacity, whatever that may be, of conjugation,
and may be regarded as an intermediate stage, at least, in the devel-
opment of sexually differentiated forms (Fig. 120). A somewhat
analogous process takes place in Codoszga botrytis, one of the Choano-
flagellida, where, as first observed by Stein, an attached form con-
jugates with a free-swimming and somewhat smaller animal. A similar but more definite sex-difference is seen in the peritri- A similar but more definite sex-difference is seen in the peritri-
chous Ciliata, where the individuals are of dissimilar size. In all of
these, with the exception of the genus Zvothamnium, a normal-sized
individual fuses with a smaller one. Engelmann (’76) made the A
B
Cc
D
£E
Fig. 120. — Conjugation of Polytoma uvella Ehr. [DALLINGER and DRYSDALE.] B Cc £E £E B D D B Cc A Fig. 120. — Conjugation of Polytoma uvella Ehr. 2.
The unton of similar but different-sized individuals. [DALLINGER and DRYSDALE.] interesting discovery that the larger form, or macrogamete, is always
one whose sister-buds have given rise by division to smaller forms, or
microgametes, which would certainly suggest that a particular condi-
tion of the plasm accompanies conjugation. In the genus Zvotham-
nium, alone, the macrogametes
are considerably larger than the
normal individuals (Trembley 1747, Ehrenberg, Greeff, Engelmann,
and others). The microgametes, which may arise by budding, as in Lagenophrys The microgametes, which may arise by budding, as in Lagenophrys
ampulla (Fig. 121), or by repeated divisions, as in Epzstylzs (Fig. 122),
swim about freely until they come in contact with macrogametes,
to which
they finally adhere. Upon
fusing, the microgametes
gradually lose their definite structure, until finally they are absorbed. Engelmann (’76), watching the process of sexual union in the ciliate,
Vorticella, records the following interesting observations:
‘‘ The buds,
at the beginning, swarmed about with constant and considerable
rapidity, rotating the while on their axes, but moving more or less in
a straight line through the drop. This lasted from five to ten minutes SEXUAL PHENOMENA IN THE PROTOZOA 223 _or even longer without any special occurrence. Then the scene sud-
denly changed. Happening all at once in the vicinity of an attached
Vorticella, a bud quickly changed its direction with a jerk, and
approached the larger form, fluttering about it like a butterfly over a
flower, and gliding over its surface here and there as though tasting. After this play, repeated upon several individuals, had gone on for
some minutes, the bud finally became firmly attached.”
Again: “I
observed another performance still more remarkable from its physio-
logical and particularly from its psycho-physiological significance. A free-swimming bud crossed the path of a large Vortice/la which
had become free from its stalk in the usual manner and which was
roaming about with great activity. At the instant of the meeting
—there was no trace of a pause—the bud suddenly changed its
direction and followed the Vortccel/a with great rapidity. It developed
into a regular chase which lasted about five seconds, during which time Fig. 121.— Conjugation in Lagenophrys ampulla St. [BUTSCHLI.]
s. Microgamete attached to normal cell (4). &. Fusion of macro- and microgametes. Fig. 121.— Conjugation in Lagenophrys ampulla St. [BUTSCHLI.]
s. Microgamete attached to normal cell (4). &. Fusion of macro- and microgametes. 1 Loc. cit, p. 583. 2.
The unton of similar but different-sized individuals. the bud remained about one-fifteenth of a millimeter behind the Vor-
ticella, although it did not become attached, for it was lost by a sudden
side movement of the larger form. The bud then continued its way
as before. These processes are remarkable, since they demonstrate a
fine and rapid perception, a rapid and safe will determination, and
finely divided motor innervation. They show to what astonishing
height and multiplicity physiological differentiation in animals can go,
even within a single cell.” ! The phenomena which Engelmann observed and regarded
as The phenomena which Engelmann observed and regarded
as
evidence of psychic activity, have been shown to owe their origin for
the most part to chemical and physical stimuli. But the sex-differ-
entiation indicated by the diversity in size and activity of the gametes,
and the fusion of the two cells which he described, are typical of fer- 1 Loc. cit, p. 583. THE PROTOZOA 224 tilization, or of so-called sexual reproduction, throughout both animal
and vegetable kingdoms. 3. The union of swarm-spores (Isogamy and Anisogamy). 3. The union of swarm-spores (Isogamy and Anisogamy). tion described above,
er one, to the condi-
both conditions may
nus Polyfoma, as de-
en two normal-sized
l and a microgamete,
ers of the group to
which Polytoma be-
longs—the Chlamy-
domonadina— show
similar
_indefinite-
ness, and in no case
can it be positively
stated that the union
between
a_
larger
(ovoid) and asmaller
(spermatoid) micro-
gamete
is
obliga-
tory. Goroschankin
(75) maintained that
in Chlorogonium pul-
visculus the sperma-
toid
microgametes
arise by an
eight
division
and
con-
jugate
with
the
ovoid macrogametes
which arise by a two
<#s
“
or four division (Fig. .123), but Reinhardt
(76), on the other
s of the smaller size,
In Po/ytoma, while
duals of diverse size,
obligatory union of
further in Phacotus
ovoid cell arising by
with a minute form
is, however, safely It is but a short step from the primitive condition described above,
where an ordinary individual unites with a smaller one, to the condi-
tion where both conjugants are reduced
— indeed, both conditions may
be present in the same organism. Thus the genus Polyfoma, as de-
scribed above, shows a facultative union between two normal-sized
individuals, or between one normal-sized individual and a microgamete,
or between similar microgametes. Other members of the group to
which Polytoma be-
longs—the Chlamy-
domonadina— show
similar
_indefinite-
ness, and in no case
can it be positively
stated that the union
between
a_
larger
(ovoid) and asmaller
(spermatoid) micro-
gamete
is
obliga-
tory. Goroschankin
(75) maintained that
in Chlorogonium pul-
visculus the sperma-
toid
microgametes
arise by an
eight
division
and
con-
jugate
with
the
ovoid macrogametes
Fig. 122. —
pistylis umbellaria Leeuw. which arise by a two
[GREEFF.]
<#s
“
Macrogametes (7) and microgametes (7). or four division (Fig. .123), but Reinhardt
(76), on the other
hand, observed conjugation between microgametes of the smaller size,
although he noted a frequent difference in size. In Po/ytoma, while
there is a facultative conjugation between individuals of diverse size,
there is also, apparently, a tendency toward an obligatory union of
microgametes. The differentiation goes a step further in Phacotus
lenticularis, where, according to Carter (58), an ovoid cell arising by
two or four division in the normal manner, unites with a minute form
which is the product of a sixty-four division. 3. The union of swarm-spores (Isogamy and Anisogamy). It is but a short step from the primitive condition described above,
where an ordinary individual unites with a smaller one, to the condi-
tion where both conjugants are reduced
— indeed, both conditions may
be present in the same organism. Thus the genus Polyfoma, as de-
scribed above, shows a facultative union between two normal-sized
individuals, or between one normal-sized individual and a microgamete,
or between similar microgametes. Other members of the group to
which Polytoma be-
longs—the Chlamy-
domonadina— show
similar
_indefinite-
ness, and in no case
can it be positively
stated that the union
between
a_
larger
(ovoid) and asmaller Fig. 122. —
pistylis umbellaria Leeuw. [GREEFF.]
Macrogametes (7) and microgametes (7). Fig. 122. —
pistylis umbellaria Leeuw. [GREEFF.]
Macrogametes (7) and microgametes (7). hand, observed conjugation between microgametes of the smaller size,
although he noted a frequent difference in size. In Po/ytoma, while
there is a facultative conjugation between individuals of diverse size,
there is also, apparently, a tendency toward an obligatory union of
microgametes. The differentiation goes a step further in Phacotus
lenticularis, where, according to Carter (58), an ovoid cell arising by
two or four division in the normal manner, unites with a minute form
which is the product of a sixty-four division. The obligatory conjugation
of microgametes
is, however, safely The obligatory conjugation
of microgametes
is, however, safely SEXUAL PHENOMENA IN THE PROTOZOA 225 established in a great many Protozoa, especially among the colonial
forms of Mastigophora, and, to a less extent, in Sarcodina and in
some Sporozoa. In the Sarcodina, macro- and microgametes are
formed by many marine types, including Reticulariida and Radiolaria,
and Brady, Brandt, and Haeckel do not hesitate to say that sexual
reproduction is brought about by their union. In only one case, how-
ever (//yalopus), has conjugation been actually observed. Here the
cell-body
spontaneously
fragments
into isogamous
microgametes
which swim away from the shell and conjugate (Schaudinn). In the
Sporozoa, also, the recent results obtained by Siedlecki (99) show an Cc
Fig. 123. — Conjugation in Chlorogonium euchlorum Ehr. [STEIN.]
A. Adult individual. &. Macro- and C. microgamete formation. D. Conjugation. Fig. 123. — Conjugation in Chlorogonium euchlorum Ehr. [STEIN.]
A. Adult individual. &. Macro- and C. microgamete formation. D. Conjugation. analogous phenomenon, and at the same time they throw considerable
doubt upon Wolters’s (91) conclusions that the nuclei of two conju-
gating Gregarines unite! 1The camera drawing from a permanent preparation shown in Fig, 125 throws consider-
able doubt upon this interpretation of the first three division planes as described by Stein.
According to this one preparation the third division is horizontal, giving four cells above and
four below.
The plate form is assumed in the early sixteen-cell stage. 3. The union of swarm-spores (Isogamy and Anisogamy). According to Siedlecki, two similar in-
dividuals of Monocystis ascidie come together and secrete a common
cyst within which they sporulate, each individual by itself. There
is no union of nuclei as in Actinophrys, nor interchange of parts
of nuclei as in the. Infusoria, but the nuclei rapidly divide, and
the subdivisions ultimately become the nuclei of minute cells resem-
bling spores. These have been repeatedly observed in Gregarinida, 1Cf. p. 157. Q 226 THE PROTOZOA and by most authors are called sporob/asts. The so-called sporoblasts
become motile and move about with considerable freedom, a hitherto
unrecognized phenomenon. But more remarkable still, the sporo-
blasts finally unite two by two, and after complete fusion of nuclei
and cell-plasm, each double cell or copula divides into eight parts,
the sporozoites (Fig. 124). These observations, which are the most
conclusive that have yet appeared, place the Gregarinida in line with
the Reticulariida and Radiolaria, and Siedlecki with Mesnil (’00) sees
in this isogamous union a feature which distinguishes the Gregarinida
from the Coccidiida. The obligatory fusion of microgonidia is widely distributed in the The obligatory fusion of microgonidia is widely distributed in the
colonial forms of Mastigophora, especially in the Phytoflagellida. It
is to be regretted that we do not know the full life-history of the Fig. 124.— Conjugation of Monocystis acidie Lank,
[SIEDLECKI.]
The
two gregarines
unite
(4). The
nuclei
divide
repeatedly, and many gametes are
formed (8). ‘These unite two by two, forming spores. Each spore divides to form eight sporo-
zoites (c). Fig. 124.— Conjugation of Monocystis acidie Lank,
[SIEDLECKI.]
The
two gregarines
unite
(4). The
nuclei
divide
repeatedly, and many gametes are
formed (8). ‘These unite two by two, forming spores. Each spore divides to form eight sporo-
zoites (c). simpler and more indefinite colonies such as Dinxobryon, Anthophysa,
or Synura, where the aggregate arises through continued binary
division, for in the higher types, the fertilized egg, as in Metazoa,
passes by a regular cleavage into the adult form, the cells becoming
separated only in the later stages. Nevertheless, in these more
differentiated types some stages are unquestionably more primitive
than others. In Gonzum pectorale, a colony consisting of sixteen in-
dividuals, the colonies reproduce asexually by simultaneous division
of all of the cells, four successive longitudinal divisions in each cell
resulting in sixteen groups of sixteen cells each, and these groups
form independent colonies (O. F. 3. The union of swarm-spores (Isogamy and Anisogamy). Miller, Cohn, Stein
— Fig. 125). simpler and more indefinite colonies such as Dinxobryon, Anthophysa,
or Synura, where the aggregate arises through continued binary
division, for in the higher types, the fertilized egg, as in Metazoa,
passes by a regular cleavage into the adult form, the cells becoming
separated only in the later stages. Nevertheless, in these more
differentiated types some stages are unquestionably more primitive
than others. In Gonzum pectorale, a colony consisting of sixteen in-
dividuals, the colonies reproduce asexually by simultaneous division
of all of the cells, four successive longitudinal divisions in each cell
resulting in sixteen groups of sixteen cells each, and these groups
form independent colonies (O. F. Miller, Cohn, Stein
— Fig. 125). SEXUAL PHENOMENA IN THE PROTOZOA 227 Under certain conditions some of the adult cells become separated
from the colony and pass into a resting state, during which they divide
into eight biflagellated microgametes, and these, as soon as liberated,
conjugate in pairs (Hieronymous, Rostafinski, 75). There is no size
differentiation between the conjugating microgametes. Nor is there Fig. 125.— Gonium pectorale O. F. M., in division. The third cleavage results in two layers of four cells (¢, 2,0). The flattening occurs in the
I2-16-cell stage (¢, 1, p). Fig. 125.— Gonium pectorale O. F. M., in division. The third cleavage results in two layers of four cells (¢, 2,0). The flattening occurs in the
I2-16-cell stage (¢, 1, p). in the somewhat better-known form, Pandorina morum. In the latter,
which is also a sixteen-cell colony, after a certain number of asexual
generations, a generation appears in which each of the sixteen cells
divides, not into sixteen parts for a new colony, but as in Gozzus into
eight gametes, which ultimately become free, conjugate, and pass into
a resting zygote condition. After a longer or shorter period, either
the cyst bursts, and a naked individual emerges, which by division in the somewhat better-known form, Pandorina morum. In the latter,
which is also a sixteen-cell colony, after a certain number of asexual
generations, a generation appears in which each of the sixteen cells
divides, not into sixteen parts for a new colony, but as in Gozzus into
eight gametes, which ultimately become free, conjugate, and pass into
a resting zygote condition. 3. The union of swarm-spores (Isogamy and Anisogamy). The
sexual
difference
is somewhat
better marked in the genus Euwdorina, although the most reliable
authorities differ as to the details. According to Goroschankin (’76)
and Dangeard (89), the sexually mature colonies are easily distin-
guished as male and female, the latter resembling the ordinary colo-
nies save for a slightly An incipient sexual difference in the colony is thus indicated,
although,
as in chlamydomonads, the size difference
in gametes
is apparently
facultative. The
sexual
difference
is somewhat
better marked in the genus Euwdorina, although the most reliable
authorities differ as to the details. According to Goroschankin (’76)
and Dangeard (89), the sexually mature colonies are easily distin-
guished as male and female, the latter resembling the ordinary colo-
nies save for a slightly
larger size. The male
colonies
are
at
first
quite
similar
to the
ordinary
colony,
but
each of the sixteen cells
divides to form a six-
teenor thirty-two celled
plate, and
each
of
these
cells
gradually
becomes
long
and Fig. 126.— Conjugation of Pandorina morum Ehr. [PRINGSHEIM.]
Large gametes conjugate with small ones (/?, C), or small
ones conjugate with each other (D, £,/). The result is always
azyecte (6), Fig. 126.— Conjugation of Pandorina morum Ehr. [PRINGSHEIM.]
arge gametes conjugate with small ones (/?, C), or Large gametes conjugate with small ones (/?, C), or small
ones conjugate with each other (D, £,/). The result is always
azyecte (6), ovoid
cells. Carter’s (58) description differs so much from this
that Biitschli doubts if he had the same species. He found that
the colonies are hermaphrodite and divided into male and female por-
tions. Four cells at one pole of the oval colony develop into sperma-
tozoids, while the remaining twenty-eight cells become enlarged, and
as ovoid cells, are probably fertilized by the spermatozoids. These
observations, although conflicting, at least show the increasing com-
plexity of the colony, and a differentiation, according to Carter, of
male and female cells in the same multicellular individual, or accord-
ing to Goroschankin, the colonies of Ewdorina, become multicellular
individuals in which the sex is definitely established. In these cases
there is no distinction between germ and somatic
cells, all being
capable of assuming the plasmatic condition necessary for conju-
gation. ovoid
cells. Carter’s (58) description differs so much from this
that Biitschli doubts if he had the same species. 3. The union of swarm-spores (Isogamy and Anisogamy). After a longer or shorter period, either
the cyst bursts, and a naked individual emerges, which by division 228 THE PROTOZOA forms the complete colony;
or the zygote may first divide into
two or three individuals, each of which forms a sixteen-cell colony. Pringsheim (69) stated that a dimorphism exists in the gametes
formed by large colonies and by small ones, and maintained that
the larger gametes never conjugate amongst themselves, while the
smaller ones
can unite with each other or with the larger ones
(Fig. 126). An incipient sexual difference in the colony is thus indicated, incipient sexual difference in the colony is thus indicated,
gh,
as in chlamydomonads, the size difference
in gametes
parently
facultative. The
sexual
difference
is somewhat
marked in the genus Euwdorina, although the most reliable
ties differ as to the details. According to Goroschankin (’76)
angeard (89), the sexually mature colonies are easily distin-
d as male and female, the latter resembling the ordinary colo-
nies save for a slightly
larger size. The male
colonies
are
at
first
quite
similar
to the
ordinary
colony,
but
each of the sixteen cells
divides to form a six-
teenor thirty-two celled
plate, and
each
of
these
cells
gradually
becomes
long
and
spindle-formed and de-
g. 126.— Conjugation of Pandorina morum Ehr. [PRINGSHEIM.]
velops flagella at the
gametes conjugate with small ones (/?, C), or small pointed
end. They
jugate with each other (D, £,/). The result is always
(6),
ultimately become free
and unite with larger
cells. Carter’s (58) description differs so much from this
iitschli doubts if he had the same species. He found that
lonies are hermaphrodite and divided into male and female por-
Four cells at one pole of the oval colony develop into sperma-
, while the remaining twenty-eight cells become enlarged, and
id cells, are probably fertilized by the spermatozoids. These
ations, although conflicting, at least show the increasing com-
of the colony, and a differentiation, according to Carter, of
and female cells in the same multicellular individual, or accord-
Goroschankin, the colonies of Ewdorina, become multicellular
uals in which the sex is definitely established. In these cases
is no distinction between germ and somatic
cells, all being
e of assuming the plasmatic condition necessary for conju- An incipient sexual difference in the colony is thus indicated,
although,
as in chlamydomonads, the size difference
in gametes
is apparently
facultative. 4. The union of eggs and spermatozoa. The union of large with small cells, which is only facultative in the
majority of single Mastigophora, becomes obligatory in the Coccidiida. As in the colonial forms just described, Cocezdiuim (schizont) increases
usually by asexual reproduction (schizogony). A large number of sero-
zottes are produced, each of which may repeat the cycle (Schaudinn). The
asexual
increase
continues
for
five days (Schaudinn,
’oo),
after which the merozoites give rise to sexually differentiated indi-
viduals, some of which are large (macrogametes), others small like
spermatozoa (microgametes). The phenomena of fertilization have
been carefully and independently worked out by Schaudinn (’96) and
Siedlecki (’97) in the forms Ade/ea ovata and Eimeria Schneideri, and
by Siedlecki (98) in K/ossza octopiana (Eberthi) (Fig. 127), and Schau-
dinn (’00) in Cocczdium. The formation of the spermatozoids has
already been described ;1 attention may be called, however, to the pecul-
iar central, residual mass which remains after the microgametes are
formed, suggesting the residual blastophore in the spermatogenesis
of annelids. The microgametes or spermatozoids, when mature, are
mere filaments of chromatin with a minimum of cytoplasm; they are
pointed at each end and may or may not have flagella. They move
very rapidly by serpentine undulations or by means of their flagella,
until they come in contact with a female cell which is fertilized in the
same manner as in the Metazoa. The development of the egg is
similar to that of the microgamete, so far as the nucleus is concerned,
but up to the present no maturation process has been recorded. Sev-
eral microgametes cluster around the macrogamete, as spermatozoa
group themselves around the egg (Fig. 128). The nucleus of the
female cell moves toward the periphery, and one of the microgametes
penetrates the nuclear vesicle, and its chromatin fuses completely
with that of the female pronucleus, while the entire nuclear mass
now moves back toward the centre of the cell. At the same time a
peripheral portion of the cytoplasm becomes more dense, refractile,
and finally thick and resisting, to form the membrane of the fertilized
macrogamete, thus corresponding exactly with the vitelline mem-
brane of a fertilized egg. After a thorough mixture of the chromatin
in the cleavage nucleus, the latter divides by a peculiar method of
mitosis, and a great number of spore-nuclei are formed. SEXUAL PHENOMENA IN THE PROTOZOA 229 4. The union of eggs and spermatozoa. 3. The union of swarm-spores (Isogamy and Anisogamy). He found that
the colonies are hermaphrodite and divided into male and female por-
tions. Four cells at one pole of the oval colony develop into sperma-
tozoids, while the remaining twenty-eight cells become enlarged, and
as ovoid cells, are probably fertilized by the spermatozoids. These
observations, although conflicting, at least show the increasing com-
plexity of the colony, and a differentiation, according to Carter, of
male and female cells in the same multicellular individual, or accord-
ing to Goroschankin, the colonies of Ewdorina, become multicellular
individuals in which the sex is definitely established. In these cases
there is no distinction between germ and somatic
cells, all being
capable of assuming the plasmatic condition necessary for conju-
gation. SEXUAL PHENOMENA IN THE PROTOZOA 1See p. 159. 1Cf. yolk-formation in eggs of Metazoa; see Wilson, Zhe Cell, p. 155. 4. The union of eggs and spermatozoa. In Adelca ovata fertilization is effected by the entrance of a male
cell through a special opening, the mcropyle, while the process is In Adelca ovata fertilization is effected by the entrance of a male
cell through a special opening, the mcropyle, while the process is
further complicated by the preparatory divisions which the nucleus of
the microgamete undergoes before entrance. According to Schaudinn
(’97) it divides twice while in contact with the macrogamete mem- 1See p. 159. THE PROTOZOA 230 brane, and three of the resulting nuclei are eliminated, while one fuses
with the female pronucleus, thus giving a striking analogue of the brane, and three of the resulting nuclei are eliminated, while one fuses
with the female pronucleus, thus giving a striking analogue of the with the female pronucleus, thus giving a striking analogue of the
oe
ae
ee
oe ASE
ye or
Poa)
Cee
Fig. 127.— Formation of microgametes and fertilization of AVossva octopiana Schneid. [SIEDLECKI.]
The chromatin of the nucleus is distributed throughout the cell (.4, 2), finally forming nuclei
of the future gimetes (C, D, £). The mature microgametes (s) swim about, and join a macro-
gamete (/). The nuclei mix (G), and then the cleavage nucleus divides repeatedly by mitosis to
form the spores (//). polar-body formation in Metazoa, or of the degenerate final divisions of
pro-conjugants in the other Protozoa. The formation of both macro- oe
ae
ee
oe ASE
ye or
Poa) Cee
Fig. 127.— Formation of microgametes and fertilization of AVossva octopiana Schneid. [SIEDLECKI.] Cee Fig. 127.— Formation of microgametes and fertilization of AVossva octopiana Schneid. [SIEDLECKI.]
he chromatin of the nucleus is distributed throughout the cell (.4, 2), finally forming nucl The chromatin of the nucleus is distributed throughout the cell (.4, 2), finally forming nuclei
of the future gimetes (C, D, £). The mature microgametes (s) swim about, and join a macro-
gamete (/). The nuclei mix (G), and then the cleavage nucleus divides repeatedly by mitosis to
form the spores (//). polar-body formation in Metazoa, or of the degenerate final divisions of
pro-conjugants in the other Protozoa. The formation of both macro- SEXUAL PHENOMENA IN THE PROTOZOA 231 gametes and microgametes occurs during the same day, and after a
similar period of asexual increase, showing that the same ultimate
causes probably operate in each. 4. The union of eggs and spermatozoa. The macrogamete is distinguished
from the schizonts by the possession of a reserve store of nutriment, Fig. 128. — Life-history of a Coccidium,
[SCHAUDINN.]
a, 6, c, schizonts and asexual reproduction (schizogony). The merozoites at ¢ repeat the cycle
or pass on to the following stages: d, e, 4,
development of the female or macrogamrte; 4, 4,7,
development of the male gametes; ., copulation; 4 and JZ, stages in the formation of the spores
and sporozoites. ‘in the form of granules, which are used in the later development. Fig. 128. — Life-history of a Coccidium,
[SCHAUDINN.]
a, 6, c, schizonts and asexual reproduction (schizogony). The merozoites at ¢ repeat the cycle
or pass on to the following stages: d, e, 4,
development of the female or macrogamrte; 4, 4,7,
development of the male gametes; ., copulation; 4 and JZ, stages in the formation of the spores
and sporozoites. Fig. 128. — Life-history of a Coccidium,
[SCHAUDINN.]
a, 6, c, schizonts and asexual reproduction (schizogony). The merozoites at ¢ repeat the cycle
or pass on to the following stages: d, e, 4,
development of the female or macrogamrte; 4, 4,7,
development of the male gametes; ., copulation; 4 and JZ, stages in the formation of the spores
and sporozoites. ‘in the form of granules, which are used in the later development. The storage of these granules begins in the young merozoite (Fig. 128, e-g) and in the close vicinity of the nucleus! The nucleus is
not essentially different from that of the schizont, but at one stage,
according to Schaudinn, a large amount of chromatin is thrown out of THE PROTOZOA 232 it, a process regarded by him as a kind of reduction and found in
some form
or
other in other Coccidiida (Adelea, Seidlecki, ’99;
Coccidium proprium, ’98, C. lacazetz, Schaudinn,
etc.). The macro-
gamete is thus essentially a normal individual, in which a reserve
store of food is deposited, and in which the quantity of chromatin is
reduced. The microgamete, on the other hand, differs widely from
the macrogamete, the schizont, and the merozoite. The life-history of Cocczdinm is thus similar to that of Gonzum or The life-history of Cocczdinm is thus similar to that of Gonzum or
Panudorina and consists of periods of asexual reproduction, which
alternate with periods of so-called sexual reproduction (Fig. 128). 4. The union of eggs and spermatozoa. This conception of alternation of gencrations may be extended to all
Protozoa, but in only one case ( Vo/vex) is there a differentiation into
germ and somatic cells in,the same individual. Vo/vox affords an
interesting intermediate stage between the generalized Protozoa and
the specialized forms among the Metazoa and Metaphyta. Here the
aggregate is differentiated into somatic and germ cells, although in
the early stages there is no difference between them. Some of the
latter (parthenogonidia)
are sufficiently generalized
to form new
colonies by asexual reproduction, while others, apparently like the
somatic cells, form the sexual reproductive elements; the remaining
peripheral cells are specialized for feeding and motion. It has been determined by Kirschner (’79), Carter (’58), and Stein It has been determined by Kirschner (’79), Carter (’58), and Stein
(’78) that some colonies always produce male and others female. In
the formation of male elements one of the ordinary cells of | o/vox
minor divides into 16 spermatozoids (Kirschner), or from 32 to 128
or more (Goroschankin, ’75; Cohn, ’75; and Stein, 68). The egg-
cell, too, appears at first to differ but slightly from an ordinary cell,
but it rapidly grows in size and becomes entirely different. Fertili-
zation
has been observed
in both Volvox minor and V. globator,
taking place in the inner spaces of the colonies, where the spermato-
zoa work their way to meet the eggs. In no case, however, have the
inner processes of fertilization been observed. Thus, in all Protozoa, with a single exception, apparently, of Volvox, Thus, in all Protozoa, with a single exception, apparently, of Volvox,
where the beginnings of specialization are seen, all of the cells of a
cycle, or of a colony, are equally capable of conjugation and of
rejuvenescence, and the possibility of indefinitely continued life is
open to them all. This power, as Weismann first pointed out, dis-
tinguishes the Protozoa from all higher animals and plants, where
division of labor has resulted in specialization. The majority of the
vegetative or somatic cells which form the organs and tissues of
higher animals and plants, have, with their specialization, lost the
power of rejuvenescence, and when the potential of vitality with
which they start is exhausted they become degenerate through old
age, and finally die. 4. The union of eggs and spermatozoa. The germ cells, on the other hand, like the SEXUAL PHENOMENA IN THE PROTOZOA 233 Protozoa, retain the power to renew their vital activities, and with it
the possibility of continued existence. Thus, the penalty of speciali-
zation appears to be death. 1¥For a discussion of the problems of reduction, see Wilson, Zhe Ced/, 2d ed., 1900, p. 233. B. THE SO-CALLED MATURATION-PHENOMENA IN PROTOZOA It is now a well-established fact that in the higher plants and
animals the nuclei of the germ-cells, when ready for fertilization,
contain only one-half as many chromosomes as the nuclei of the body-
cells. This discovery, made by Van Beneden (’83), has been so
widely extended in the animal and vegetable kingdoms that it is now
generally regarded as a necessary condition of the union of sex-cells;
for by this union, it is argued, the number of chromosomes would be
doubled were it not for the preliminary reduction to one-half. The
phenomena leading to reduction of the chromosomes to one-half the
number characteristic of the species are known as the maturation or
reducing phenomena. In the egg the nucleus divides twice, while
near the periphery of the cell, and two minute cells, abortive eggs or
polar bodies, are formed, which degenerate and disappear without
further action. In the spermatozoon all of the four cells, which are
formed by a similar double division, are functional. In both egg and
spermatozoon
one division of the nucleus, at
least, results
as in
ordinary mitosis, in the equal partition of each chromosome. The
chromosomes
at this period frequently differ in appearance from
the ordinary chromosomes, and from their peculiar shape are known
as vetrads (Vierergruppen). At this point, however, the various
observations differ and an animated controversy, which began with
Weismann, is still vigorously maintained in regard to the exact way
in which these maturation-chromosomes
are formed and divided. The controversy, which in large part involves Weismann’s specula-
tions upon heredity, need not detain us here, however, for with the
Protozoa, only the general aspects of reduction are in question.1
In the Protozoa, with the somewhat doubtful exception of Para- In the Protozoa, with the somewhat doubtful exception of Para-
mactum caudatum (Hertwig, 89), there
is no
case
on record of
reduction in the number of chromosomes, and even in the few cases
where actual chromosomes
occur,
their number
is so great that
counting is impossible. There are, nevertheless, certain phenomena
antecedent to conjugation in Protozoa which warrant the belief that
processes take place, even in these primitive animals, which
are
analogous to the formation of polar bodies in the Metazoa. In
general, these processes consist in the elimination of one or more:
daughter-nuclei prior to conjugation, and are usually the same for
both conjugants. B. THE SO-CALLED MATURATION-PHENOMENA IN PROTOZOA THE PROTOZOA 234 It is a significant fact, first pointed out by Maupas (’89), that
micronuclei in Infusoria which have about exhausted their potential
of vitality, disappear by atrophy and absorption in the cytoplasm,
while the latter continues to live and even to divide for a limited
time (e.g. Onychodromus). Thus, the loss of vitality operates upon
the nucleus as upon the remaining protoplasm, and the final divi-
sions of the nucleus,
like the final divisions
of the cells, result
in daughter-nuclei, which have not the power to live, and which
disappear in the cytoplasm. Furthermore, if two individuals can
fuse only when the body protoplasm is in the proper condition (Dif-
fiugia, Amoeba, etc.), the same may be true of nuclei. It is certainly
true that nuclei of the plasmodia of Mycetozoa, or of Actimophrys in
plastogamy, or of adult multinuclear Actznospheria, do not fuse. But when two Dufflugias are in the proper condition for conjugation,
the nuclei do fuse. When, however, two individuals
are in the
proper condition, so far as the cytoplasm is concerned, it does not
follow that the nuclei are ready, and each nucleus may undergo
division prior to fusion, one of the daughter-nuclei disintegrating
and disappearing
(M/onocystis,
Actinophiys),
or
several
of them
disappearing (Infusoria, Acténospherium). In Cocctdiida a certain
amount of the nuclear material is budded off and disintegrates while
in the plasm, and the conjugating nuclei, without membranes, fuse
to form the cleavage nucleus. Such cases may be interpreted as
evidence of loss of vitality to such an extent that the nucleus has the
power to divide, but not to stimulate division of the cell-body. The loss of vitality is, I believe, the principle which lies at the The loss of vitality is, I believe, the principle which lies at the
bottom of the so-called maturation-phenomena among the Protozoa. Briefly reviewing some of these processes in Protozoa, it will be seen
that in some cases, as in senescent Infusoria, the nucleus atrophies,
while in other cases
the nucleus
divides once
or twice previous
to fusion, thus simulating the maturation of Metazoa. The former
has been observed in Euglypha and Actinospherium, the latter in
Actinophrys, Actinospherium, some Coccidiida, and in Infusoria. In £uglypha, Blochmann (’88) observed the conjugation of two In £uglypha, Blochmann (’88) observed the conjugation of two
individuals, which had become united by their mouth parts. B. THE SO-CALLED MATURATION-PHENOMENA IN PROTOZOA The
cytoplasm of one conjugant passed into the shell of the other and
fused with the cytoplasm there, but the nucleus was left behind. A
pseudopodial process from the fused cytoplasm finally picked up the
rejected nucleus, and its position in the cytoplasm was restored. It
did not live, however, to fuse with the other nucleus, but disintegrated
in the plasm (Fig. 129). These observations have been recently
confirmed
by Prowazek
(’00) and are regarded by Biitschli and
Blochmann as an indication of reduction previous to conjugation; but
it may be interpreted as an instance of degeneration of the nucleus, SEXUAL PHENOMENA
IN THE PROTOZOA 235 while the failure to unite with the other nucleus leads to disintegra-
tion, and may be regarded as facultative reduction. The loss of nuclei through disintegration and absorption is better The loss of nuclei through disintegration and absorption is better
established in the case of Actinospherium. Gruber (’83) and Brauer
(94) maintained that the number of nuclei in old Actnospheria is
reduced by fusion until only a few remain, but Hertwig (’98) shows
that with the exception of about 5 per cent, all of the nuclei atrophy
and are absorbed in the cytoplasm. The remaining nuclei, which he
calls the “sexual nuclei,” then undergo so-called maturation divisions,
and fuse to form the cleavage nuclei of new cycles. Fig. 129. — Conjugation of Zuglypha alveoluta Duj. [BLOCHMANN.]
n, functional nucleus. 2’, degenerating nucleus. alveoluta Duj. [BLOCHMANN.] Fig. 129. — Conjugation of Zuglypha alveoluta Duj. [BLOCHMANN.]
n, functional nucleus. 2’, degenerating nucleus. Fig. 129. — Conjugation of Zuglypha alveoluta Duj. [BLOCHMANN.]
n, functional nucleus. 2’, degenerating nucleus. The simplest case of maturation comparable to that in Metazoa is
shown in two rather widely separated forms, Actenophrys, a heliozoén,
and Monocystis,
a gregarine. In the former, Schaudinn (96) observed
that, although the cytoplasms of two individuals fuse, the nuclei
remain apart. They finally divide by mitosis, and two of the daughter-
nuclei fuse, while the other two (polar bodies) disintegrate and are
absorbed in the cytoplasm (Fig. 130). Similarly in A/oxocystis, Wolters
(91) has shown that two similar individuals join in a common cyst
and that the nuclei divide, one of the daughter-nuclei in each indi-
vidual disappearing in the plasm. In both of these cases, only one daughter-nucleus is eliminated in In both of these cases, only one daughter-nucleus is eliminated in
each conjugant. B. THE SO-CALLED MATURATION-PHENOMENA IN PROTOZOA In the other forms mentioned, there are at least two,
and the process approaches still more closely to maturation in Metazoa. 236
THE PROT THE PROTOZOA 236 In Actinospherium, Hertwig describes a process which may be summa-
rized as follows: after most of the nuclei are absorbed in the cyto- In Actinospherium, Hertwig describes a process which may be summa-
rized as follows: after most of the nuclei are absorbed in the cyto-
Cc. For-
P,
polar
body. [SCHAUDINN
8. The
nuclei
during
the
prophase
of
division. Fusion
of
the
nuclei. Fig. 130. —
Conjugation
of
Actinophrys
sol
Ehr. A. Two
individua
fused;
the
axial
filaments
abut
against
the
nuclei. plasm, the “ mother-cyst” divides into as many parts (primary cysts) as
there are nuclei remaining. In small animals the entire mass may form
¥,
First
divisi
£. YD. Recons
of
the
nuclei. mation
of
the
first
polar
spindle Cc. For-
P,
pol
bod
¥,
Firs
divi plasm, the “ mother-cyst” divides into as many parts (primary cysts) as
there are nuclei remaining. In small animals the entire mass may form
one primary cyst with one nucleus, but large specimens may have plasm, the “ mother-cyst” divides into as many parts (primary cysts) as
there are nuclei remaining. In small animals the entire mass may form
one primary cyst with one nucleus, but large specimens may have plasm, the “ mother-cyst” divides into as many parts (primary cysts) as
there are nuclei remaining. In small animals the entire mass may form
one primary cyst with one nucleus, but large specimens may have SEXUAL PHENOMENA
IN THE PROTOZOA 237 twenty or more primary cysts. Each primary cyst is surrounded bya
special gelatinous mantle; the nucleus of each divides once (primary
mitosis) into two nuclei characterized by radiations at one pole, where
a centrosome is developed from nuclear threads which grow out into
the surrounding cytoplasm. During the formation of the centrosome,
the body of the primary cyst divides, giving rise to the secondary
and mononucleate cysts. Each secondary cyst forms two “polar bodies” in the following Each secondary cyst forms two “polar bodies” in the following
manner. In each the nucleus divides by mitosis into two nuclei, one
of which degenerates while the other prepares for a second division by
mitosis. B. THE SO-CALLED MATURATION-PHENOMENA IN PROTOZOA The active daughter-nucleus
divides again and one of the resultant nuclei migrates to the other
organism, while the other resultant nucleus remains quiescent. The
migrating nucleus in each conjugant unites with the quiescent nucleus
of the other individual, and thus effects fertilization, after which the
organisms separate (Fig. 131). The three daughter-nuclei (corpuscules de rébut) which are elimi- form four daughter-nuclei, of which one only remains active, while the
other three degenerate and disappear. The active daughter-nucleus
divides again and one of the resultant nuclei migrates to the other
organism, while the other resultant nucleus remains quiescent. The
migrating nucleus in each conjugant unites with the quiescent nucleus
of the other individual, and thus effects fertilization, after which the
organisms separate (Fig. 131). The three daughter-nuclei (corpuscules de rébut) which are elimi- The three daughter-nuclei (corpuscules de rébut) which are elimi-
nated in each individual before the union of the “ germ nuclei,” are
undoubtedly analogous to the polar bodies of the Metazoa. The
agreement in number with the polar bodies of the Metazoa is inter-
esting, but it may be doubted whether this agreement has any real
significance, for the number of nuclei eliminated in other genera of
Ciliata varies considerably, e.g. Vorticella
Mark ('82), followed by Biitschli (85), explained the formation of Mark ('82), followed by Biitschli (85), explained the formation of
polar bodies
in the Metazoa as aborted eggs resulting from the
attempt to form many individuals
as the early sperm cells do, and
the latter regarded it asa reminiscence of the colony-formation of the
Protozoa, basing his view upon the sexual relations of Volvox and Pan-
dorina (see above), while Hartog (’91) expressed a somewhat similar
view in the statement “the page of morphological history, revealing
that the oogamete was primitively one of a brood of at least four,
has
not been
obliterated
from
the ontogenetic
records
of the
Metazoa.”’? Richard Hertwig(’98), after summing up the evidence in Protozoa Richard Hertwig(’98), after summing up the evidence in Protozoa
of the homologue of polar bodies, came to the conclusion that in
all cases there is probably a double division before, after, or during
fertilization, which represents a physiological condition of the cell
and without phylogenetic significance. 1 Cf. Maupas. B. THE SO-CALLED MATURATION-PHENOMENA IN PROTOZOA After this division one of the resulting nuclei also degener-
ates ( “2d polar body ” ), leaving one ripe nucleus in each secondary
cyst. Both polar bodies consist exclusively of nuclear matter, and they
rapidly degenerate. Both polar or maturation mitoses are character-
ized by the presence of centrosomes derived from the original centre. After the two secondary cysts formed from the two primary cysts are
ripe, they fuse together, protoplasm with protoplasm, and nucleus with
nucleus, thus re-forming the primary cyst. The fusion product (germ-
sphere) is distinguished from the older primary cyst, not only in the
changes described in ripening and fertilization, which, after the forma-
tion of the polar bodies, would-hardly be noticed, but in the following
additional points: (1) The body concentrates into a denser structure
and smaller circumference. (2) The last remnants of vacuoles disap-
pear. (3) The silicious particles at first diffuse, collect at the periphery
and finally form a compact outer coating. (4) A protective mem-
brane (yolk membrane), extremely impervious to killing agents, is
secreted inside of the protective coating. After a week’s rest the
germ spheres creep out, each animal holding six or eight nuclei,
formed by mitosis. Before beginning to grow, the spheres appar-
ently divide into mononucleate forms. Again in Adelea ovata, one of the Coccidiida,
a somewhat similar Again in Adelea ovata, one of the Coccidiida,
a somewhat similar
elimination takes place, but in only one of the conjugants. Prior to
conjugation, the nucleus of the male cell divides twice, and only one of
the resulting nuclei penetrates and fertilizes the egg, while the other
three disintegrate and disappear (Schaudinn and Siedlecki, 97). This
case is very similar to the well-known maturation-divisions among the
Infusoria, although in the latter both conjugants undergo the same
processes. In the different species of Infusoria the maturation-phe-
nomena, while differing as to details, agree in their essential features,
and for purposes
of illustration Paramecium caudatum may be
selected, for with its single macronucleus and single micronucleus it
is simpler than the majority of other forms. The details of the pro-
cess which have been made out by Maupas (’88, ’89) and Hertwig (’89)
are as follows. The micronucleus of each conjugant divides twice to 238 THE PROTOZOA form four daughter-nuclei, of which one only remains active, while the
other three degenerate and disappear. 22, page 66, B. THE SO-CALLED MATURATION-PHENOMENA IN PROTOZOA THE PROTOZOA 240 number of chromosomes is not reduced in the maturation-divisions
although the amount of chromatin is reduced by half. It must be pointed out also that in many cases
of conjugation number of chromosomes is not reduced in the maturation-divisions
although the amount of chromatin is reduced by half. It must be pointed out also that in many cases
of conjugation It must be pointed out also that in many cases
of conjugation
among Protozoa no maturation processes are known. Thus all of the
Flagellidia, with their complicated division of labor and high grade of
sex-differentiation, offer not a single instance of nuclear reduction,
and the conclusion is suggested that the maturation of forms in other
divisions of the Protozoa show no genetic relation to analogous pro-
cesses in Metazoa, but are independent expressions of the same un-
known vital forces which cause the’ formation of polar bodies, or the
double division of tetrads. B. THE SO-CALLED MATURATION-PHENOMENA IN PROTOZOA Excepting for the Infusoria
the double division is, however, established in only one form (Acfzno-
spherium),
and
it is rather difficult
to believe that observations
have been incomplete in all other reported cases where only one
such division is found. Hertwig’s view, however, that the preliminary
division of the nuclei prior to fertilization represents a physiological
condition of the protoplasm, is in no way opposed to the facts in
Protozoa. Up to the present time no such structures as tetrads have been found Up to the present time no such structures as tetrads have been found
in any Protozoa; indeed, in only a few cases have chromosomes been
observed, and then they are so numerous as to render counting im-
possible. Hertwig (89) somewhat doubtfully asserted that the num-
ber of chromosomes in Paramecium caudatum is reduced from 8 or 9,
to 4 or 6, and, again (’98), he maintained that in Acténospherinum the ff
v4
Fig. 131.— Conjugation
of Paramecium
caudatum,
[A-C, after R. HERTWIG;
D-A,
after
Maupas.]
(The macronuclei dotted in all the figures.)
A, Micronuclei preparing for their first division. 2. Second division. C. Third division;
three polar bodies or “ corpuscules de rébut,” and one dividing germ-nucleus in each animal. D. Interchanve of the germ-nuclei. &, Thesame, enlarged. /. Fusion of the germ-nuclei. G. The
same enlarged. HH. Cleavage-nucleus (c) preparing for the first division. /, ‘The cleavage-nucleus
has divided twice. ¥ After three divisions of the cleavage-nucleus; the macronucleus is breaking
up. A. Four of the nuclei enlarging to form the new macronuclei. ff ff
v4 v4 ff 31.— Conjugation
of Paramecium
caudatum,
[A-C, after R. HERTWIG;
D-A,
after
Maupas.]
(The macronuclei dotted in all the figures.) Fig. 131.— Conjugation
of Paramecium
caudatum,
[A-C, after R. HERTWIG;
D-A,
after
Maupas.]
(The macronuclei dotted in all the figures.)
A, Micronuclei preparing for their first division. 2. Second division. C. Third division; A, Micronuclei preparing for their first division. 2. Second division. C. Third division;
three polar bodies or “ corpuscules de rébut,” and one dividing germ-nucleus in each animal. D. Interchanve of the germ-nuclei. &, Thesame, enlarged. /. Fusion of the germ-nuclei. G. The
same enlarged. HH. Cleavage-nucleus (c) preparing for the first division. /, ‘The cleavage-nucleus
has divided twice. ¥ After three divisions of the cleavage-nucleus; the macronucleus is breaking
up. A. Four of the nuclei enlarging to form the new macronuclei. C. GENERAL CONSIDERATIONS In the foregoing review of the phenomena of conjugation and
maturation, it is only too apparent that many gaps in the series, and
the incompleteness of the observations in various instances
cited,
prevent any far-reaching generalizations. It may be pointed out,
however, that more or less similar conditions characterize all of the
diverse phenomena, and afford a basis for future explanations. The various conjugation-phenomena seen in the Protozoa seem to The various conjugation-phenomena seen in the Protozoa seem to
show that each cycle starts with a certain potential of vitality which
is gradually exhausted in the vegetative activities of the long line of
individuals formed by simple division, or by spore-formation. As
Biitschli long since suggested, such a cycle can be compared to the
ontogeny of a metazoon where the somatic cells, starting with an
initial vitality, finally die from senile degeneration. An important
difference, however,
lies in the fact that each protozoén has the
inherent power of conjugation, and when senile degeneration
has
progressed to a certain extent, the vitality can be restored by this
process. The most careful observations on the Protozoa, as upon all
other animals and plants, have failed to demonstrate the nature of this
renewal of vitality, or the reason why the temporary or permanent
union of two exhausted cells should result in one or two rejuvenated
ones. An explanation of the many diverse modifications in the form of the An explanation of the many diverse modifications in the form of the
conjugants, and of the various maturation-phenomena, does not, how-
ever, appear at the present time quite so far out of reach, for these
phenomena can be observed, and the similar characteristics point
toward a common interpretation. The evidence in Protozoa, while
not conclusive,
certainly
points,
I believe,
as Minot long since
‘suggested, toward the phenomenon
of decreasing
vitality as the
underlying condition which indirectly brings about conjugation, sex-
differentiation, and maturation. SEXUAL PHENOMENA IN THE PROTOZOA 241 Attention may again be called to the fact, repeatedly observed by
Biitschli, Engelmann, Gruber, Maupas, and others, upon the Infuso-
ria, that if conjugation is prevented, continued cell-division leads to
degeneration, and that this process is cumulative, resulting eventually
in the atrophy of external and internal organs (including vibratile
membranes, cilia, micronuclei) and, finally, in death. 1 (88), p. 1638.
2 Cf. Maupas (88), Biitschli (’88).
R 1 (88), p. 1638. C. GENERAL CONSIDERATIONS “There is no
question,” says Biitschli, “that conjugation is a process which, if pre-
vented, leads to death of the ciliates, just as a race of Metazoa dies
out if restrained from sexual reproduction!” The cell-body becomes Sy
St
be SAAD
SAY
Fig. 132.— Onychodromus grandis Stein. [MAUPAS.]
,
A, Normal individual. 2. Smaller form without micronuclei; degenerate. C. A still more
reduced and degenerate form. JV, macronucleus; 7, micronucleus, A, Normal individual. 2. Smaller form without micronuclei; degenerate. C. A still more
reduced and degenerate form. JV, macronucleus; 7, micronucleus, smaller, more plastic, and less resistant, and assumes various irregular
shapes (Fig. 132). If degeneration has not gone too far, the normal size
of the individual, the organs, and the general vitality are restored by
conjugation, which is effected through the union of the soft mouth
parts, while the internal phenomena consist of the interchange of
micronuclei and possibly of some cytoplasm. The phenomena of
degeneration may therefore be considered in two categories: (1) the
effect upon the plasm and the form of the cell-body ; (2) the effect
upon the nuclei. The former, I believe, leads to the reduced size of
the conjugants, the latter to the so-called maturation in Protozoa. smaller, more plastic, and less resistant, and assumes various irregular
shapes (Fig. 132). If degeneration has not gone too far, the normal size
of the individual, the organs, and the general vitality are restored by
conjugation, which is effected through the union of the soft mouth
parts, while the internal phenomena consist of the interchange of
micronuclei and possibly of some cytoplasm. The phenomena of
degeneration may therefore be considered in two categories: (1) the
effect upon the plasm and the form of the cell-body ; (2) the effect
upon the nuclei. The former, I believe, leads to the reduced size of
the conjugants, the latter to the so-called maturation in Protozoa. 2 Cf. Maupas (88), Biitschli (’88). R 42
THE PRO THE PROTOZOA 242 In forms of Protozoa other than the Infusoria, there have been no
careful observations and experiments to determine the limits of the
potential of vitality. Nevertheless, certain facts have been recorded
which appear to be of the same order of phenomena as those seen in
the Infusoria. C. GENERAL CONSIDERATIONS Thus, in some Monadida (Cercomonas,
Tetramitus,
etc.) the loss of the customary body-form and the assumption of a
plastic condition prior to conjugation may be interpreted as an indi-
cation of degeneration, and comparable to the loss of membranelles,
cilia, etc., in the Infusoria. A similar interpretation may account for
the fusion of two cells of Difflugia lobostoma (Fig. 118), which under
normal conditions will not unite, but which fuse readily under “ certain
conditions of the plasm” (Rhumbler). Also in Pol/ytoma and Chlorogo-
nium, where facultative conjugation occurs between two full-sized cells,
between a full-sized and a smaller cell, or between two smaller cells,
the difference in size may be interpreted as an expression of degener-
ation, comparable to that which is indicated by the senescent, small-
sized individuals of Onychodromus or Paramecium, where fusion may
also occur between two small conjugants or between a normal-sized
cell and a small one (Figs. 120 and 132). In all cases the protoplasm
has reached that state which, for the want of a better term, may be
called mature, when conjugation
is possible. The sex-difference,
which is facultative in Polytoma and Chlorogonium, becomes obliga-
tory in the allied form, Phacotus, a condition which may be compared
with that of the peritrichous Ciliata, where, in the Vorticellida, the
free-swimming microgametes are dwarfed forms of the normal indi-
viduals with which they fuse (Fig. 122). In the more complex colonies of Flagellidia, and in the Coccidiida,
the microgametes, although they arise by spore-formation, are to be
compared with spermatozoa of Metazoa, and the macrogametes are
equivalent to eggs. The obligatory sex-difference is here indicated
by the storage of a reserve food supply with concomitant quiescence
in the larger forms (females), and by loss of cytoplasm and concomi-
tant increase of motion in the smaller ones (males). The researches
upon Coccidiida have established the fact that, as in Infusoria, there
is a period of conjugation which alternates with a period of spore-
formation, and the changed form of the conjugants into spermatozoa
and egys may be taken as evidence of exhaustion as
in the less
specialized cases among the Protozoa; although with our present
knowledge an interpretation of these conjugants as degenerate cells
can only be based upon analogy with other forms, where a similar
cycle brings about more or less similar conjugating individuals. 1 Watase’s (’92) interesting suggestion as to sex-differentiation is significant in the light
of the foregoing facts.
Regarding sex-differentiation as a manifestation of irritability (2¢.,
p- 485), he says: “The organism is either a male or a female, not by the difference of * primary
sexual characters’ alone, but by the difference which saturates the whole of its entire structure.
Such a difference is, however, neither absolute nor permanent.
It is a temporary differenti-
ation of protoplasm into one of two different directions, and sooner or Jater comes back to
the original neutral or non-sexual state from which it started, thus manifesting the phenomenon
characteristic of all protoplasmic irritability ” (p. 493). C. GENERAL CONSIDERATIONS It is apparent from the facts cited that the study of Protozoa can It is apparent from the facts cited that the study of Protozoa can
throw but little Jight upon the question of sex-difference. It may be
due either to differences in nutriment or to some more deeply lying SEXUAL PHENOMENA
IN THE PROTOZOA 243 cause in the protoplasm, but in any case, it cannot be of primary
importance, for the one essential requisite demanded by the lowest
flagellate and the highest animal or plant is fusion with another cell,
and by such fusion the restoration of an exhausted vitality.! We must finally, with Hertwig, distinguish clearly between fertili- We must finally, with Hertwig, distinguish clearly between fertili-
zation and reproduction. Fertilization of the ovum in Metazoa is so
closely followed by development of the embryo, that it has grown to
be considered a direct act of reproduction as well as its cause, and the
same view would be equally applicable to the Coccidiida or Infusoria. But, when applied to conjugation in other forms of Protozoa, z.e. to
fertilization in its more general aspects, it is evident that the phe-
nomenon has some deeper significance. Biitschli ('76) long since
pointed out that, during the period of conjugation, the two individuals
of conjugating Paramecia might have formed many others by simple
division, and he, with Engelmann, showed that the phenomenon, in all
probability, could not be a reproductive act. So, too, Ouychodromus
might divide thirteen times during the period of conjugation (Maupas). The facts of facultative conjugation, eg. in Polytoma or in the Rhi-
zopoda, are arguments in the same line, for, in these cases, an individ-
ual may or may not fuse before forming spores. Maupas (89) main-
tained that division is the only method of reproduction, and pointed
out that division is more rapid before conjugation than after, and
Hertwig (’98) showed that, in Actinospherium, reproduction actually
precedes fertilization, and he says:
“A reproductive process
is
bound up with the encystment of Actenospherium, whereby a mother-
cyst gives rise to several primary cysts, each primary cyst to a
germ-sphere, each germ-sphere to new
individuals. C. GENERAL CONSIDERATIONS Reproduction
here precedes fertilization, and the latter has no effect upon the former.”
He further states that fertilization, which has no direct connection
with reproduction, occurs in both animals and plants, and he adds:
“Since we know reproduction without fertilization and fertilization
without reproduction, there must be processes combined in ‘sexual
reproduction’ which in their essence do not belong together. To
distinguish methods of increase as sexual and asexual, awakens false
impressions. There is, in fact, only one kind of reproduction, ze. division in the widest sense of the word. What is known as sexual
reproduction among Metazoa, is division which is combined with 244 THE PROTOZOA sexual activity. The egg is a potential individual as shown by the
facts of parthenogenesis, where, without fertilization, the egg develops
to form a new individual. The first appearance of this individual
goes back to the germ-glands, where it results from the division of an
odgonium. Here is the reproductive act proper ; fertilization is only
the liberation of an inhibited development through which the poten-
tial individual becomes actual. It leads to no increase, but rather to
decrease of individuals, since two potential individuals
— egg-cell and
sperm-cell
— join to form one body.” ? The phrase “liberation of an inhibited development,” if applied to
the effect of conjugation among the Protozoa, might be translated into
Biitschli’s
“ Verjiingung” (’76), or Engelmann’s
“ Reorganization ”
(76), or Maupas’s “ Réjeunissement.”
In no case does word or
phrase explain the phenomenon. From all the facts known at the
present time, the only conclusion that can be drawn is that conjuga-
tion, apparently, is not the cause of reproduction, but as Biitschli,
Engelmann, and Minot long since pointed out, in some unknown
way provides the energy for continuing the functions of the indi-
vidual, including the power of reproducing. SPECIAL BIBLIOGRAPHY VII Biitschli, 0. — Studien iiber die ersten Entwicklungsvorgange der Eizelle, der Zell-
theilung und die Conjugation der Infusorien. Adz. d. Senckenb. naturf. Gesell. Frankfurt a. Main, X., pp. 213-452, 1876. Engelmann,
Th. W.— Ueber Entwickelung und Fortpflanzung von Infusorien. Engelmann,
Th. W.— Ueber Entwickelung und Fortpflanzung von Infusorien. Morphol. Jahro., pp. 573-635, 1876. Geddes and Thompson. — The Evolution of Sex. London, 1889. Geddes and Thompson. — The Evolution of Sex. London, 1889. Hartog, M. M.— Some Problems of Reproduction, etc. Q. /. Hartog, M. M.— Some Problems of Reproduction, etc. Q. /. AZ. S., XXXIII.,
pp. 1-81, 1891. ;
Hertwig, R. — Ueber Kerntheilung, Richtungskérperbildung und Befruchtung von Hertwig, R. — Ueber Kerntheilung, Richtungskérperbildung und Befruchtung von
Actinospherium Eich. <Adh. d. K. bay. Akad. d. Wiss. Miinchen, 11. Kl. XIX., 1898. ;
Hertwig,
R.— Mit welchem Recht unterscheidet man geschlechtliche und unge- Hertwig,
R.— Mit welchem Recht unterscheidet man geschlechtliche und unge-
schlechtliche Fortpflanzung ? S7tz. Ber. d. Ges. f. Morph. u. Physiol. Miinchen,
II., 1899. ;
Maupas, M. — Le réjeunissement karyogamique chez les cilies. Arch. d. zool. expér. Maupas, M. — Le réjeunissement karyogamique chez les cilies. Arch. d. zool. expér. et génér., (2) VII., pp. 149-517, 1889. Minot, C. S. — Growth asa Function of Cells. Proc. Bost. Soc. Nat. Hist., XX., 1879. ot, C. S. — Growth asa Function of Cells. Proc. Bost. Soc. Nat. Hist., XX., 1879. audinn, F. — Ueber den Zeugungkreis von Paramceba Eilhardi. Siz. Ber. Akad. Minot, C. S. — Growth asa Function of Cells. Proc. Bost. Soc. Nat. Hist., XX., 1879. Schaudinn, F. — Ueber den Zeugungkreis von Paramceba Eilhardi. Siz. Ber. Akad. Wiss. Berlin, 1896. Siedlecki,
M.— Ueber
die geschlechtliche Vermehrung der Monocystis ascidiz Schaudinn, F. — Ueber den Zeugungkreis von Paramceba Eilhardi. Siz. Ber. Akad. Wiss. Berlin, 1896. Siedlecki,
M.— Ueber
die geschlechtliche Vermehrung der Monocystis ascidiz Siedlecki,
M.— Ueber
die geschlechtliche Vermehrung der Monocystis ascidiz
R. Lan. Bull. d. l’Acad. d. Sci. a. Cracovie, December, 1899. Wilson, E. B.— The Cell in Development and Inheritance. 2d ed. Mew York, Wilson, E. B.— The Cell in Development and Inheritance. 2d ed. Mew York,
1900. 1 loc. city p. 97. SPECIAL MORPHOLOGY OF THE FROTOZOAN NUCLEUS 245 245 46
THE PRO 246 THE PROTOZOA An ordinary cell in Metazoa and the higher plants consists of pro-
toplasm, which is typically differentiated into ce//-body or cytoplasm,
and nucleus. These areas of protoplasm differ considerably in their
chemical composition,
—the former being rich in proteid, of which
albumins play the most important part, and poor in phosphorus; the
latter, on the other hand, being rich in phosphorus, which is bound up
in a substance called zzclezz, and poor in albumins. Both nucleus and cytoplasm, as seen under the high power of a Both nucleus and cytoplasm, as seen under the high power of a
microscope, have a complicated structure. In both there appears to
be a general ground substance, the cytolymph in the cell-body, and
the £aryolymph in the nucleus. Throughout this ground substance,
in both the cell-body and the nucleus, extends an alveolar meshwork,
the intra-nuclear portion being known as the /inzn reticiilum (Fig. 133). The cytoplasm frequently contains other structures, such as
plastids, vacuoles, metaplasmic bodies, crystals of various kinds, etc.,
which may or may not be permanent in the cell. A more important
structure is the ceztrosome, which is usually present in the Metazoa
and which has been generally considered as playing a leading réle
in cell-division. This body, which is extremely minute, is, as a rule,
surrounded by a granular area of cytoplasm known as the a@ttraction-
sphere, centrosphere, or simply as the sphere. The nucleus, in addition
to the linin reticulum and the karyolymph, contains chromatin, a sub-
stance which, more
than anything else, distinguishes the nucleus
from the cytoplasm. As a rule, chromatin appears in the form of
small granules distributed through the linin network. These granules
in the resting nucleus may be close enough together to give the ap-
pearance of a chromatin reticulum, while during division of the nucleus
they become fused, forming structures known as chromosomes. The
latter, save in certain stages in the formation of the sex-cells, are of
definite number and appearance for the same species. The chromatin
network is frequently thickened to form relatively large masses of
chromatin known as zet-kno/s, or, together with the plasmosomes, as
nucleon. The plasmosomes, or true nucleoli, are comparatively large,
spherical masses suspended in the karyolymph and are of unknown
function. The nucleus, finally, is inclosed within a definite membrane
which usually disappears during mitosis. SPECIAL MORPHOLOGY OF THE FROTOZOAN NUCLEUS “ By cell-division, accordingly, the hereditary substance is split off from the parent-body;
and by cell-division, again, this substance is handed on by the fertilized egg-cell or odsperm
toevery part of the body arising from it. Cell-division is, therefore, one of the central
facts of development and inheritance.’
— WiLson.1 Ir has been shown in the foregoing chapters that local: modifica-
tions of the general protoplasmic basis of protozoan cells give rise to
cell-structures of considerable complexity, and, when compared with
cells in the tissues of Metazoa, these complex forms often appear to
be
the
more
highly differentiated. These
differentiations
have
mainly arisen by modifications of the cortical plasm, in response, prob-
ably, to the action of the environment and the mode of life. The
differentiations of the inner cell-plasm of the Protozoa, on the other
hand, are apparently much less complex than in Metazoa, and the
nucleus, especially, appears to be structurally much more simple in
the Protozoa. In the division of protozoan cells, structures occur
which are undoubtedly similar to those of tissue-cells, but are, on the
whole, of a simpler and more generalized character. For this reason
the careful study of these more primitive organs cannot fail to throw
light upon morphological problems which, in Metazoa and the higher
plants, are more difficult of solution. The changes which the nuclei undergo during division of the cell The changes which the nuclei undergo during division of the cell
have received
little
attention from the phylogenetic
standpoint,
although the similarity of these changes, even in the most diverse
tissues of unrelated animals and plants, bespeaks a common origin. If the structures involved in these changes can be traced back to
more generalized organs in the Protozoa, a key may perhaps be found,
— not, indeed, to ancestry of the Metazoa, as some writers have main-
tained,
— but to some of the problems of higher cell-differentiations. It is my purpose in the present chapter, therefore, to give somewhat
in detail a comparison of the various structures found in protozoan
cells with those of the cells in differentiated tissues of Metazoa and
Metaphyta, and so far as our present knowledge permits, to point out
the possible origin of different cell-organs in higher types, from the
more primitive structures as they appear in Protozoa. 1The Cell, p. 63. 245 1The Cell, p. 63. 1See Wilson, The Cell, Chap. II. SPECIAL MORPHOLOGY OF THE FROTOZOAN NUCLEUS Although the main features of indirect nuclear division, or szzfosis, Although the main features of indirect nuclear division, or szzfosis,
in animals and plants are so similar that a common type of mitotic or
division-figure can be described, there are, nevertheless, confusing
variations and differences, especially in the so-called “achromatic” por-
tions of the mitotic figure! Broadly speaking, these variations in
mitotic figures may be reduced to three main types: (1) Forms with SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 247 centrosome and spindle-figure in which central spindle, mantle-fibres,
and astral rays can be distinguished. (2) Forms with centrosome and
spindle-figure in which astral fibres are present, but no distinction
between central spindle and mantle-fibres can be seen. (3) Forms
without centrosome, but witha spindle-figure which cannot be resolved
into mantle-fibres and central spindle. In all of these cases the granular chromatin of the resting nucleus In all of these cases the granular chromatin of the resting nucleus
is arranged in the form of a reticulum upon a linin network, and, with
few exceptions, the prophases of division
are similar, in that the
granules are brought together and concentrated in the form of deeply Attraction-sphere enclosing two centrosomes. Plastids lying in the
Piasmosome or
\ en
cytoplasm
true
nucleolus
Chromatin-
network
Nucleus
4 _ |. Linin-network
Karyosome,
net-knot, or
chromatin-
L
nucleolus
Vacuole
Passive bodies (meta-
plasm or paraplasmy
suspended in the cy-
toplasmic meshwork
Fig. 133. — Diagram of acell. Its basis consists of a meshwork containing numerous minute
granules (¢crosomes) and traversing a transparent ground-substance,
[WILSON] Fig. 133. — Diagram of acell. Its basis consists of a meshwork containing numerous minute
granules (¢crosomes) and traversing a transparent ground-substance,
[WILSON] staining rods, the chromosomes, which are of definite number, shape,
and size for each species. Finally, the nuclear membrane disappears
and the chromosomes are left naked in the cytoplasm and connected
by spindle-fibres with the two poles of.the mitotic figure. As the variations in the three types have mainly to do with the As the variations in the three types have mainly to do with the
achromatic structures, further mention of the chromatin changes may
be omitted. In the first type considered, the approach of division is
signalized by the division
of the centrosome into two daughter-
centrosomes connected by fibres which form a small spindle called
by Hermann (’91) the cextral spindle. SPECIAL MORPHOLOGY OF THE FROTOZOAN NUCLEUS This continually enlarges as
the centrosomes diverge, other fibres (mantle-fibres) meantime grow- 248 THE PROTOZOA ing from the centrosome and pushing in the nuclear membrane, which
finally disappears, leaving the chromosomes in the cytoplasm. The
mantle-fibres then become attached
to the chromosomes, and the
latter, finally, surround the central spindle like a ring. The origin of
these fibres is variously interpreted. The mantle-fibres according to
some observers arise from cytoplasmic material, according to others,
from linin substance
in the nucleus. The origin of the central
spindle-fibres is also in dispute. Some observers believe that two
opposing groups of fibres starting from the centrosomes meet to form
the spindle (Driiner, ’95 ; MacFarland, ’97; etc.); others believe that
the diverging centrosomes are connected from the first by the cen-
tral spindle-fibres (Hermann, ’91; Flemming, ’91; Heidenhain, ’94;
Kostanecki, ’97; etc.), and Boveri (88) and Heidenhain maintain
that the fibres are formed from the substance surrounding the cen-
trosome (archoplasm of Boveri; substance of the ‘“centrodesmus” of
Heidenhain). Several transitional stages between the first and the second types ing from the centrosome and pushing in the nuclear membrane, which
finally disappears, leaving the chromosomes in the cytoplasm. The
mantle-fibres then become attached
to the chromosomes, and the
latter, finally, surround the central spindle like a ring. The origin of
these fibres is variously interpreted. The mantle-fibres according to
some observers arise from cytoplasmic material, according to others,
from linin substance
in the nucleus. The origin of the central
spindle-fibres is also in dispute. Some observers believe that two
opposing groups of fibres starting from the centrosomes meet to form
the spindle (Driiner, ’95 ; MacFarland, ’97; etc.); others believe that
the diverging centrosomes are connected from the first by the cen-
tral spindle-fibres (Hermann, ’91; Flemming, ’91; Heidenhain, ’94;
Kostanecki, ’97; etc.), and Boveri (88) and Heidenhain maintain
that the fibres are formed from the substance surrounding the cen-
trosome (archoplasm of Boveri; substance of the ‘“centrodesmus” of
Heidenhain). Several transitional stages between the first and the second types Several transitional stages between the first and the second types
have been described. In some of these (many egg-cells), the central
spindle appears
first as very delicate fibres between the dividing
centrosomes, but these break later, and no connection remains. SPECIAL MORPHOLOGY OF THE FROTOZOAN NUCLEUS The
complete spindle in such a case consists of fibres which apparently
pass from pole to pole, with chromosomes strung upon them, while
central spindle-fibres, if present, must be newly formed and inter-
mingled with the other fibres, and both sets must have the same origin.1
Finally, the third type of mitosis differs from the first in the ab- Several transitional stages between the first and the second types
have been described. In some of these (many egg-cells), the central
spindle appears
first as very delicate fibres between the dividing
centrosomes, but these break later, and no connection remains. The
complete spindle in such a case consists of fibres which apparently
pass from pole to pole, with chromosomes strung upon them, while
central spindle-fibres, if present, must be newly formed and inter-
mingled with the other fibres, and both sets must have the same origin.1
Finally, the third type of mitosis differs from the first in the ab-
sence of central spindle and centrosomes, and from the second by the
apparent absence of centrosomes. While great difference of opinion
exists as to the presence or absence of centrosomes in plants higher
than the Fungi, some observers denying, others affirming, its presence
in the same species, the balance of opinion at present appears to be
toward the negative side, and evidence is-certainly accumulating in
support of this view. According to Strasburger, Mottier, Osterhout,
etc., the spindle in plant mitoses arises by the gradual convergence
of rays which make their appearance tangential to the nuclear mem-
brane. Arising, as it were, from the substance of the cytoplasm, and
converging to form a bi-polar mitotic figure, the spindle-fibres are sup-
posed by Strasburger and his followers to be formed from a definite
and distinct substance to which he gave the name Kinoplasma. The
nuclear membrane, as in the other types, always disappears before the
nuclear plate is formed, and nuclear division proceeds in the usual way. Turning now tothe Protozoa, the heterogeneity in form and struc-
ture of the nuclei is particularly suggestive. All of the several parts Finally, the third type of mitosis differs from the first in the ab-
sence of central spindle and centrosomes, and from the second by the
apparent absence of centrosomes. 1Cf. Wilson (’96), 7vxopneustes. SPECIAL MORPHOLOGY OF THE FROTOZOAN NUCLEUS While great difference of opinion
exists as to the presence or absence of centrosomes in plants higher
than the Fungi, some observers denying, others affirming, its presence
in the same species, the balance of opinion at present appears to be
toward the negative side, and evidence is-certainly accumulating in
support of this view. According to Strasburger, Mottier, Osterhout,
etc., the spindle in plant mitoses arises by the gradual convergence
of rays which make their appearance tangential to the nuclear mem-
brane. Arising, as it were, from the substance of the cytoplasm, and
converging to form a bi-polar mitotic figure, the spindle-fibres are sup-
posed by Strasburger and his followers to be formed from a definite
and distinct substance to which he gave the name Kinoplasma. The
nuclear membrane, as in the other types, always disappears before the
nuclear plate is formed, and nuclear division proceeds in the usual way. Turning now tothe Protozoa, the heterogeneity in form and struc- Turning now tothe Protozoa, the heterogeneity in form and struc-
ture of the nuclei is particularly suggestive. All of the several parts SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 249 which characterize the typical nucleus of Metazoa are rarely present
here in one and the same nucleus. The nuclear membrane in some
cases is absent, in other cases well defined and persistent throughout
active as well as resting stages. The linin reticulum, with its inclosed
chromatin granules, is frequently absent, while the ground substance
of the nuclei, or the karyolymph, although it has not been critically
examined, is undoubtedly present in the majority of cases. Nucleoli
or plasmosomes have apparently been’ found in but one instance
(Actinospherium, Hertwig, ’98). The nuclei of Protozoa are, as a
rule, further distinguished from those of the Metazoa by the presence
of another intra-nuclear body, which apparently corresponds to the
sphere and centrosome of the Metazoa, and, as in the latter, plays a
prominent part in division. Boveri (’01) has recently proposed the
name Cenztronucleus for nuclei with these division centres. We can
distinguish, then, in the various nuclei of Protozoa, (1)a nuclear mem-
brane; (2) a nuclear reticulum
or linin network; (3) nucleoli, or
plasmosomes (in rare cases); (4) chromatin, occasionally in the form
of granules strung upon a linin reticulum; (5) spheres or division-
centres analogous to the extra-nuclear kinetic centres in Metazoa, but
for the most part intra-nuclear in the Protozoa. In the Cystoflagellate Moctzluca miliaris, one part of the membrane A. THE NucLEAR MEMBRANE The usual definition of a nucleus includes the membrane as an
integral part, sharply separating cytoplasmic and nuclear structures. In a few Protozoa there is no indication of
a membrane, and the
usual constituents of the nucleus are distributed throughout the cell. In others, notably in the majority
of the Sporozoa, the nuclear
membrane,
as
in most
Metazoa,
disappears during mitosis. In
all other cases, however, the nuclear membrane persists, in part at
least, throughout active as well as resting phases. It frequently ap-
pears as a very faint structure scarcely to be distinguished from the
cytoplasmic reticulum (many Flagellidia), although increasing grades
of density may be obtained, which culminate
in thick resisting
membranes
like those
of Moctzluca, Ameba proteus,
and
Ciliata.. The membrane of an isolated macronucleus of the Ciliata becomes
separated from the nuclear contents and ultimately dissolves in the
water (Biitschli). The great rigidity of this membrane and its per-
sistence during division of the nucleus led to the view that it is not
identical with a nuclear membrane of a higher cell (Biitschli, 76, 83;
Hertwig, ’76), and some observers even regarded it as highly differ-
entiated protoplasm in the form of chitin (Stein, ’54, Operczlaria), or
cellulose (Brandt, ’82, Amewéa). In the Cystoflagellate Moctzluca miliaris, one part of the membrane In the Cystoflagellate Moctzluca miliaris, one part of the membrane THE PROTOZOA 250 which is ordinarily thick and resisting disappears during division, and
the edges which are left gradually fade away into the cytoplasmic
reticulum, where they cannot be distinguished from the outer network. In this case apparently, and possibly in other Protozoa, the membranes
arise from the substance
of the cytoplasmic reticulum. Another
interpretation,
however,
is possible. Thus
in
some
Coccidiida
(Klossia), Labbé ('96) and Siedlecki (’98) described the membrane as
very delicate and staining in the same manner as the chromatin net-
work, and Siedleckiadds: ‘In fact,it represents only a more condensed
part of this network.”1! Hertwig (’98) held a similar view of the
membrane in the case of Actznospherium, regarding which he says:
“Tn well-preserved specimens the nuclear reticulum and membrane
stand in very close connection, and frequently the two cannot be dis-
tinguished.”2
The best evidence, however, of the nuclear origin of the
membrane is given by Schaudinn (’94) in the developing nuclei of Ca/
cituba polymorpha, a rhizopod belonging to the family Miliolidze (Fig. 134, 4). MSiedlecki (’98), p. 806. ° Loc. ctt., p. 635.
3 Cf. Siedlecki, ’99. MSiedlecki (’98), p. 806.
° Loc. ctt., p. 635.
3 Cf. Siedlecki, ’99. Fig. 134.— Types of nuclei.
[4. Calcituba polymorpha Roboz, from SCHAUDINN, B. Colpidium
colpoda, from a preparation,
C. Euglena virtdis Env. trom a preparation.
D, Zetramutus chilomo-
nas,n.sp.
£. Noctiluca miliaris Sur., from a preparation.]
A single karyosome (1) becomes vesicular, and ultimately gives rise to several daughter-karvyo-
somes (so-called ‘fragmentation’ Schaudinn),
Several karyosomes in ANocteduca (£) hold the
chromatin, the rest of the nucleus is filled with “achromatic” granules.
In Vetramitus chilomonas
() the chromatin is scattered throughout the cell; the lighter-colored body in the centre of the
cell is the homologue of the deeply stained central body in Euglena (C). A. THE NucLEAR MEMBRANE Here the young nucleus appears as a solid homogeneous
sphere of chromatin which gathers fluid from the surrounding plasm
and forms peripheral vacuoles. These vacuoles then pass to the inside,
and soon the entire nucleus appears vacuolated, a mere network of
chromatin. The chromatin then segregates into a central mass con-
nected by fibres with a peripheral Jayer of chromatin which forms the
nuclear membrane. The further history consists of the separation of
bits of the central mass, which pass along the radial lines to the mem-
brane, where they ultimately form a layer of chromatin similar to the
original. By the bursting of the membrane these are liberated and
recommence the cycle. In other forms, notably in Sporozoa, the nuclear membrane is
apparently of minor importance in the cell. Wolters (’91) described
a delicate membrane in some forms of Jonocystis (e.g. M. agilis);
other forms, as Monocystis ascidieg, have a thick membrane com-
posed of deeply staining fibrils,? while others of the same genus
have none
at
all. Various
other forms
of Protozoa
also have
no nuclear membranes, the chromatin in such cases being distributed
throughout the cell. Examples of this type of nucleus are found in
all classes; among the Sarcodina, Gruber (’84) and Frenzel (91),
among the Mastigophora, Biitschli (’96) and Calkins (98), among
the
Ciliata, Balbiani (60), Griiber (’84), and Bergh (’89) have de-
scribed them. In these various descriptions it is not always made
clear whether the distributed chromatin is merely a diffused nucleus
or whether each of the parts is not a single small nucleus which
divides by itself, as in the dividing granules described by Schewiakoff SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 251 (93) in Achromatium. A careful distinction should be made be-
tween these types. In Loxophyllum (Balbiani, 60), Urostyla (Bergh,
89), and Tetramitus (Calkins, ’98), the chromatin granules come
together and form a single nucleus prior to division. (93) in Achromatium. A careful distinction should be made be-
tween these types. In Loxophyllum (Balbiani, 60), Urostyla (Bergh,
89), and Tetramitus (Calkins, ’98), the chromatin granules come
together and form a single nucleus prior to division. ogether and form a single nucleus prior to division. Fig. 134.— Types of nuclei. [4. Calcituba polymorpha Roboz, from SCHAUDINN, B. Colpidium
poda, from a preparation,
C. Euglena virtdis Env. trom a preparation. D, Zetramutus chilomo-
,n.sp. £. A. THE NucLEAR MEMBRANE Noctiluca miliaris Sur., from a preparation.]
A single karyosome (1) becomes vesicular, and ultimately gives rise to several daughter-karvyo-
es (so-called ‘fragmentation’ Schaudinn),
Several karyosomes in ANocteduca (£) hold the
omatin, the rest of the nucleus is filled with “achromatic” granules. In Vetramitus chilomonas
the chromatin is scattered throughout the cell; the lighter-colored body in the centre of the
is the homologue of the deeply stained central body in Euglena (C). Fig. 134.— Types of nuclei. [4. Calcituba polymorpha Roboz, from SCHAUDINN, B. Colpidium
colpoda, from a preparation,
C. Euglena virtdis Env. trom a preparation. D, Zetramutus chilomo-
nas,n.sp. £. Noctiluca miliaris Sur., from a preparation.]
A single karyosome (1) becomes vesicular, and ultimately gives rise to several daughter-karvyo- A single karyosome (1) becomes vesicular, and ultimately gives rise to several daughter-karvyo-
somes (so-called ‘fragmentation’ Schaudinn),
Several karyosomes in ANocteduca (£) hold the
chromatin, the rest of the nucleus is filled with “achromatic” granules. In Vetramitus chilomonas
() the chromatin is scattered throughout the cell; the lighter-colored body in the centre of the
cell is the homologue of the deeply stained central body in Euglena (C). 52
THE PRO THE PROTOZOA 252 It appears, therefore, that the nuclear membrane in Protozoa can
have no'great significance, since it may be absent altogether (dis-
tributed chromatin), present only during resting phases (Sporozoa),
or persistent throughout all changes of the cell. When present, it
may be formed, apparently, from the cytoplasmic reticulum (WVoctiluca),
or from the nuclear reticulum (Actcnospherium, Reticulariida, Cocci-
diida), and these observations are in line with the results obtained by
numerous workers upon the nuclei in Metazoa. | B. Tue Linin Network Many protozoan nuclei are permanently in the condition
repre-
sented by Schaudinn’s first stage of the nucleus of Cadcztuba (Fig. 134),
and like this possess neither membrane nor linin reticulum, consist-
ing throughout of chromatin (many phytoflagellates). When, how-
ever,
a membrane is present, there is usually a clear area with little
or no trace of structure (some Heliozoa, Pénard, ’90), or else a well-
defined reticulum between the membrane and the chromatin. The
reticulum has been observed in nuclei of every class of Protozoa, as a
fine meshwork
similar
to the cytoplasmic
network,
and
to the
linin reticulum
of tissue
nuclei. In many cases,
however, the
chromatin granules are
not embedded
in such a reticulum, but
form a central mass in the nucleus. In other cases, and frequently
during preparation for division, the granules are dispersed throughout
the reticulum, which then appears like a typical nucleus (e.g. Actzno-
spherium,
Fig. 140). In some
forms
the
linin-threads
are
so
extremely minute, and the chromatin granules so large, that it appears
incredible that the latter should be inclosed in the former, as is gen-
erally believed to be the case in the nuclei of Metazoa. In many
macronuclei, the structure of the linin and chromatin, on the other
hand, is like that of tissue-cells. In nuclei with a firm and
re-
sisting membrane,
as in Amaeba proteus, there is little room
for
belief that the cytoplasmic and nuclear contents are connected, and
the two
parts appear
to be quite different
in structure. This
nucleus and a few others show no
trace of the linin reticulum,
but contain at least two kinds of granules which make up the bulk of
the chromatin and ground substance
or karyolymph. Voct2/uca
miliarts (Fig. 141, &) has a nucleus of this type with large granules
which stain intensely with acid dyes (Ishikawa, ’94; Calkins, ’98). The granules may possibly represent the cedematin-granules which
Reinke (’94) distinguished in the nuclei of Metazoa, or possibly they
represent a diffuse or distributed nucleolus, as Balbiani (’90) assumed
in regard to the nucleus of Lovophyllum meleagris. Doflein (00)
maintained that this nucleus possesses a distinct linin network which SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 253 stains with Berlin blue; in poorly preserved material the outlines of
the granules appear to form a reticulum. C. Tur NucLeoius A distinct plasmosome or true nucleolus comparable to the analo-
gous structure in Metazoa apparently exists in no case save possibly
in Actinospherium, and even here it is limited to a passing phase during
mitosis (Hertwig, '98). It is probable that the structures which have
been almost universally but erroneously called nucleoli, do not belong
at all to this category of nuclear elements, but represent either the
functional chromatin which is aggregated into a central mass (£aryo-
some) during the quiescent or vegetative period of cell-life, or the intra-
nuclear division centre. D. THE CHROMATIN The form which the chromatin assumes in Protozoa gives rise to a
great variety of nuclei which, at first sight, appear to have little in
common with each other or with nuclei of tissue-cells. There
is,
however,
,a certain relationship between
the different
types, from
extremely simple conditions, to structures as complex as in Metazoa,
and through them there is a possibility of ultimately explaining the
chromatin-changes in Metazoa. Five types of nuclei, based upon the disposition of the chromatin, Five types of nuclei, based upon the disposition of the chromatin,
can be distinguished. Of these the most primitive is, (1) the solid
sphere or karyosome (Bizznenkorper Rhumbler), which has neither linin
reticulum nor membrane (e.g. Calcituba). An advance is shown in
(2) nuclei having one such karyosome surrounded by karyolymph, the
whole inclosed within a membrane (vesicular nuclet, Gruber, ’84),
while still higher types are: (3) nuclei with several karyosomes (two
to thirteen or fourteen), with membrane, karyolymph, and with or
without a nuclear reticulum (¢.¢. Mocteluca); (4) nuclei with a large
number of smaller masses of chromatin inclosed in a definite mem-
brane with or without a linin reticulum (e.g. Amwba proteus); (5) nuclei
consisting of granules of chromatin unconfined by a nuclear membrane
and spread over the entire cell (distributed nucleus), or aggregated
about
a central
body (“intermediate”
type;
Calkins, ’98; eg. Tetramitus). Many Phytoflagellida, Choanoflagellida, some Sarcodina, and sporo- Many Phytoflagellida, Choanoflagellida, some Sarcodina, and sporo-
zoites, among the Sporozoa, have chromatin in the form of a single
homogeneous karyosome. They are usually very minute, and little is
known about them save that they are undifferentiated, and that they
divide by a simple constriction into two equal parts. A transition to the second type of nucleus is shown in Calettuba A transition to the second type of nucleus is shown in Calettuba THE PROTOZOA 254 and other marine Rhizopoda (Fig. 134). Here the result of nuclear
activity is the formation of a nuclear membrane and a nuclear reticu-
lum, the meshes of which are filled with karyolymph apparently de-
rived from the cytoplasm. Many of the stages which Schaudinn
described had already been observed in various Sarcodina by Hertwig,
F. E. Schultze, Biitschli, Gruber, Rhumbler, Hofer, and others, but the
consecutive stages in the nuclear disruption had not been seen. 1 Chromatospherite of Schneider, Binnenkorper of Rhumbler and Schaudinn, Aforulit
of Frenzel, Chromatin reservoir of Calkins, Karyosome of Siedlecki, Wucleolus of many
authors, 1The stages which Siedlecki has described may be briefly outlined as follows: The
chromatin network reappears in the vicinity of the membrane, and constantly increases by
the addition of the smaller karyosomes.
It is then drawn out into long threads and at
the same time the nuclear membrane disappears.
The karyosomes are almost entirely
used up in the formation of these elements, although a small portion remains as the charac-
teristic residual mass.
The chromatin then collects at the periphery of the cell, where little
nuclei are formed, and each nucleus becomes the chief part,of a filamentous microgamete
(Fig. 127). D. THE CHROMATIN As the karyosome SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 255 condenses, there is left a small bud, usually atta
peduncle, and this bud, which Siedlecki calls the secon
seems to be the seat of the subsequent changes in
the nucleus. The secondary karyosome enlarges as
amount of chromatin had reached it through the p
it has reached the volume of the first karyosome,
in structure. A third karyosome is then formed i
and so on until in some cases more than twenty
oped. The chromatin of the original body breaks in
in this state penetrates the connecting thread into the
somes, formed by budding. It is rather difficult t
function of this peculiar process, unless with Schn
Mingazzini (’92) we assume that it is an antecedent
phase of spore-formation. Labbé, on the other ha
karyosome as a reserve of chromatin which at first
chromatin of the
nucleus,
and
which
increases
constantly by the
addition of other
nuclear
particles
er ar
Na
a
a
1
dk
-
’
1g. 135- beans
e nucieus an
aryosome
Labbé,
’96), the
(SIEDLECKE]
increase
going
into the formation of secondary karyosomes. He
phenomena only stages in the “purification” (/’¢pur
cleus, but Siedlecki returns to the view of Schneider
and shows that they are but preparatory phases of
fact the beginning of division of the nucleus, leading
of microgametes or male reproductive cells. condenses, there is left a small bud, usually attached by a short
peduncle, and this bud, which Siedlecki calls the secondary karyosome,
seems to be the seat of the subsequent changes in the disruption of
the nucleus. The secondary karyosome enlarges as though a certain
amount of chromatin had reached it through the peduncle. When
it has reached the volume of the first karyosome, it is also similar
in structure. A third karyosome is then formed in the same way,
and so on until in some cases more than twenty have been devel-
oped. The chromatin of the original body breaks into granules, and
in this state penetrates the connecting thread into the daughter-karyo-
somes, formed by budding. It is rather difficult to determine the
function of this peculiar process, unless with Schneider (’83) and
Mingazzini (’92) we assume that it is an antecedent or preliminary
phase of spore-formation. D. THE CHROMATIN The second type of nucleus is particularly well marked in the group The second type of nucleus is particularly well marked in the group
of Sporozoa. Schneider as early as 1881 described the nucleus of
Klossia eberthi, one of the Coccidiida, as spherical, filled with “nuclear
sap” and inclosing a great ‘nucleolus,” formed of a dense cortical
layer and a central alveolar portion. He observed no reticulum, but
recent observers, Mingazzini (’92), Labbé (’96), Siedlecki (’98), de-
scribed the nucleus of Coccidiida as containing a distinct reticulum, in
which granules of chromatin and achromatin (oxychromatin in various
stages of development) can be made out. Similar nuclei are found
among the Gregarinida (Wolters, '’91; Marshall, ’93; Siedlecki, ’99). The chief interest of these nuclei, however, centres in the chro-
matin mass, the “karyosome”’ of Labbé (96), which, as in similar
nuclei among the Sarcodina, has been described under several names.! It colors so strongly with nuclear dyes that it often appears dense and
homogeneous (Fig. 135), but Schneider (’83), Labbé (96), and Sied-
lecki (98) agree that it is composed of at least two parts, an outer
cortical portion, thick and resisting in texture, and an inner granular
part. The cortical portion consists of chromatin with an exceed-
ingly fine alveolar structure, the thick walls of the alveoli being fre-
quently pressed so closely together that the striated appearance, early
described by Schneider, results. The internal granules, on the other
hand, take an intense acid stain, and are identified by Labbé as
oxychromatin granules. The history of this karyosome is strikingly similar to that of the The history of this karyosome is strikingly similar to that of the
isolated chromatin mass in Calcituba. The observations of Siedlecki
and Labbé agree on this point, and a basis is thus secured for the com-
parison of these primitive nuclear changes. As in Calcituba, the
young sporozoite (4) has an homogeneous nucleus, z.¢e. a naked karyo-
some, but, in the epithelial cell of its host, it very quickly assumes
the adult structure (Siedlecki, ’98). The intermediate stage between
the homogeneous
nucleus and the formation of the intra-nuclear
karyosome has not been observed, but, as in Ca/eztuba, the chromatin
reticulum presses more and more toward the periphery, where it is
finally condensed into the nuclear membrane. D. THE CHROMATIN Labbé, on the other hand, regarded the
karyosome as a reserve of chromatin which at first contains all the a
a
1
dk
-
in
Klossi
Fann
1g. 135- beans
e nucieus an
aryosome In
LZ0SSta C0ertht,
(SIEDLECKE] In other groups of the Protozoa, nuclei with one karyosome are
somewhat more complicated and obviously of a higher type. The
Heliozoa, for example, furnish nuclear types which, at first sight, ap-
parently agree with the above forms. The agreement is, however,
only superficial; for although similar in their general features to the
nuclei of Sporozoa, they contain achromatic structures lacking in
the latter, while their development is altogether different. A distinct 256 THE PROTOZOA linin reticulum occurs around the central karyosome, the history of
which has been recorded by Schaudinn (’96) in Actinophrys and
Acanthocystis, and by Brauer, Gruber, and Hertwig in Actznospherium. In the latter form, which may well serve as a type, the karyosome in
nuclei of ordinary vegetative forms is distinctly granular, the chromatin
granules being grouped in a variety of ways. There is a constant
tendency, however, for the granules to extend outward along the
course
of the linin reticulum, and toward the nuclear membrane,
while during mitosis the granules are grouped together in the centre
of the nucleus and along parallel lines. linin reticulum occurs around the central karyosome, the history of
which has been recorded by Schaudinn (’96) in Actinophrys and
Acanthocystis, and by Brauer, Gruber, and Hertwig in Actznospherium. In the latter form, which may well serve as a type, the karyosome in
nuclei of ordinary vegetative forms is distinctly granular, the chromatin
granules being grouped in a variety of ways. There is a constant
tendency, however, for the granules to extend outward along the
course
of the linin reticulum, and toward the nuclear membrane,
while during mitosis the granules are grouped together in the centre
of the nucleus and along parallel lines. In nuclei with from ten to twelve karyosomes, the history is prac-
tically the same as in the simpler cases with one. Here also each
karyosome breaks down into numerous granules, but there is no linin
reticulum, and the granules unite in lines to form the chromosomes
(Noctiluca, Figs. 138, 141). The formation of the numerous granules
takes place by division of the karyosomes instead of by budding as in
the Sporozoa. 1Cf. Gruber (’84). D. THE CHROMATIN Another type of nucleus is characterized by small-sized chromatin Another type of nucleus is characterized by small-sized chromatin
granules, which may or may not be strung upon linin threads. Familiar examples are seen in Euglena viridis and the majority of
the flagellates. Here the chromatin is in the form of small rod-like
granules connected by linin threads and surrounding a central division
centre, all being inclosed within a nuclear
membrane (Fig. 136). Upon division, the rods
of chromatin
separate
into two groups. Neither the observations of Blochmann nor of Keuten show any indi-
cation of splitting of the granules. In allied forms the nuclear mem-
brane may be either present or absent; when absent, the granules
of chromatin may permanently surround a central body (Chz/lomonas,
some trachelomonads),
or may be scattered throughout
the cell,
collecting only for division (Zetramitus, Urostyla). In Tetramuttus
the granules collect, without fusing, about such a centre, but in Uvo-
styla the numerous portions of the macronucleus fuse into a single
nucleus for division (Balbiani, 60; R. S. Bergh, ’89). Among the Protozoa as a whole, there is no general agreement in Among the Protozoa as a whole, there is no general agreement in
the chromatin changes preliminary to division. The object of such
changes, as Roux first pointed out, is apparently to get the chromatin
in the best position for equal division,! and the formation of granules
from the larger masses is a widely spread if not universal phenomenon
leading to this end. Three well-marked types of granule formation
have been described (1) by Schewiakoff (Euglypha); (2) by Gruber,
Brauer, and Hertwig (Actinospherium) ; and (3) by Calkins (Voeteluca). In the first type the process is described by Schewiakoff (’88) as con-
forming to the metazoan type; the granules form a distinct spireme 1Cf. Gruber (’84). SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 257 which, as in some Metazoa, splits lengthwise before it is broken into
chromosomes (Fig. 23, p. 55). A distinct spireme has also been de-
scribed in micronuclei of the Infusoria by Biitschli, Pfitzner, Maupas,
Hoyer, and Bergh, and in Heliozoa by Schaudinn, while some macro-
nuclei, as in Stylonychia and Loxophyllum, are apparently in a perma-
nent
spireme
stage (Fig. 104). 1 Doflein (’00) has recently given a very different account of chromosome-formation in
Noctiluca, based upon incomplete observations.
s E. Kinetic STRUCTURES The confusion which has arisen concerning the relation of the
centrosome to the surrounding structures in Metazoa, and the uncer-
tainty which arises from an ambiguous terminology, are magnified
many times when we come to consider analogous structures in Proto-
zoa. It is well to say at the outset that in the Protozoa there
are but few structures that can be compared with the complex astral
systems such as those in the leucocyte, or at the spindle-poles of
dividing animal cells. If, however, we regard the centrosome (in-
cluding the centriole) and attraction sphere as a unit, and as represent-
ing a structure concerned
in cell-division, we have
a basis for
comparison with structures in Protozoa which play a corresponding
réle. In the present chapter, I shall, for the sake of brevity, speak
of these structures in Protozoa
as the “division-centres.”
They
are not always of definite form and size, although consisting prob-
ably of an analogous substance or substances. In some cases the
material or substance which corresponds
to that in a more
defi-
nitely formed division-centre, is apparently spread throughout the
nucleus, and in other cases even into the cytoplasm. It will be
convenient, therefore, to speak of the “division-centre”’ not only as
a body, but also as a substance which is intimately connected with
mitosis. In some cases the division-centre is intra-nuclear, in others extra-
nuclear, and in still others it may be sometimes one, sometimes the
other. In some of the more primitive Protozoa, notably in some of
the marine Rhizopoda, and in a few Sporozoa, there appears to be no
such element present during the resting periods of the cell. Dur-
ing division,
however, there appears to be a substance which
is
derived from the chromatin and which functions as a division-centre. This is particularly interesting in the case of Monocystts ascidia,
recently described by Siedlecki (99). Here, during the formation of
the conjugating gametes or sexual reproductive bodies, there appears
in the nucleus a distinct, deeply staining granule which divides while
against the inner nuclear membrane, to form two division-centres. These become the poles of the division-figure, and during division,
there is a connecting strand of deeply staining material. There
are other
cases,
also, especially among
the
Sporozoa,
in which
a division-centre appears during nuclear division, although it cannot
be made out in the cells when at rest. D. THE CHROMATIN The latter case
is particularly
interesting, for, as in the nuclei from the salivary glands of the C/z-
vonomus larva (Balbiani), the spireme consists of alternating disks of
chromatin and a non-staining substance which Balbiani (’go) consid-
ered achromatin with the characteristics of plasmosomes. In Actinospherium and in Spirochona, Kentrochona, etc., the mass In Actinospherium and in Spirochona, Kentrochona, etc., the mass
of chromatin breaks down into granules which collect in lines across
the nucleus to form primitive chromosomes (Fig. 139). Noctiluca
is apparently
about
midway in complexity between Noctiluca
is apparently
about
midway in complexity between
Actinospherium and Euglypha. The ten or eleven karyosomes, as in
Sporozoa and Rhizopoda, break down into an immense number of
minute chromatin granules, which collect, at first, in groups in the
region of the reservoirs from which they were derived (Fig. 141);
but they are later distributed about the nucleus. The granules are
then collected in lines which radiate inward from one side of the
nucleus. By the constant addition of granules, thick chromosomes are
formed, which split down the centre. Again there is no evidence to
indicate division of the granules or the presence of a definite spireme
stage.! From the foregoing review, the facts at present appear to indicate From the foregoing review, the facts at present appear to indicate
that the most primitive nucleus is the single mass of chromatin with-
out membrane or reticulum (Coccidiida, Reticulariida). The simplest
nuclear membranes are formed directly from this chromatin (Schau-
dinn in Calcztuba), as is one type at least of the nuclear reticulum
(Reticulariida and Sporozoa). The primitive nuclear mass breaks down
into granules in preparation for reproduction,
a phenomenon which is
almost universal in Metazoa and Protozoa. In the latter group, the es-
sential feature, apparently, is the division of the chromatin mass, rather
than of the chromatin granules, as seen by the collection of chromatin
granules of distributed types (e.g. Zetramztus) into one group which
is halved, or in the formation of primitive chromosomes as in Acézno-
spherium or Noctiluca. The division of the chromatin granule is
apparently not necessary, as shown by the thick chromatin agere-
gates in Noctzluca and Actinospherium, and in the peculiar relations
in some Ciliata (Urostyla), where the long lines of chromatin are
divided into separate segments, each of which forms a nucleus. s THE PROTOZOA 258 1The “centrosomes” which Labbé (’96) described in AZossta eberthi, Bananella lacazei,
and Pfeifferia gigantea must be regarded with doubt until their relation to the nucleus
in division is made out. E. Kinetic STRUCTURES Thus, I have seen a faintly
striated spindle in the dividing nucleus of a polycystid Gregarine
of the leech Clepsine (Fig. 138), while the resting nucleus shows no
trace of a substance similar to that forming the spindle, unless, indeed, SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 259 it be a portion of the karyosome, which, in several Sporozoa, has been
shown to contain two differently staining substances.}
In still other Sporozoa there are distinct spindle-fibres, which in In still other Sporozoa there are distinct spindle-fibres, which in
some cases (¢.g. Monocystis agilis, Wolters, ’91) consist of two sets,
and their presence in resting nuclei cannot be made out. In all Spo-
rozoa, however, the origin of the spindle-substance and the division-
centre is difficult to make out because of the disappearance of the
nuclear membrane during division. In the case cited by Siedlecki,
however, there appears to be no doubt that the division-centres, with
their spindle-fibres, arise within the nucleus. In other forms of Protozoa there is usually some evidence of the In other forms of Protozoa there is usually some evidence of the
division-centre in the resting cell as well as in the mitotic figure. The
substance
of such centres
differs
from
the chromatin
of
the nucleus in its different staining reactions, and from the sub-
stances in the cytoplasm by its more intense coloration. In some
cases, such centres
are found within
the nuclear membrane
dur-
ing all phasés of cell-life (the majority of Mastigophora and Infusoria
and many Rhizopoda). In other cases they are permanently outside
of the nuclear membrane (WNoctzluca, Parameba, Heterophrys, Sphe-
rastrum). Again, they may be intranuclear during some phases and
extra-nuclear during others (TZetramitus, Actinospherium, Acantho-
cystis). 1. Lntra-nuclear Division-centres. 1.
Lntra-nuclear Division-centres. The least-differentiated division-centres are those which are per-
manently within the nucleus. They are found in all classes of the
Protozoa, and have been variously interpreted. For a long period
they were erroneously regarded as nucleoli, but since their true func-
tion was first suggested by Keuten (’95) they have been variously
regarded as “nucleolus-centrosomes ” (Keuten), centrosomes (Hert-
wig, 96), and spheres, equivalent to centrosome
plus attraction-
sphere (Calkins, ’98). In the majority of the Flagellidia the division-centre
is clearly In the majority of the Flagellidia the division-centre
is clearly
defined and distinct from all other parts of the cell. Its changes in
form, which were first made out in Euglena by Blochmann (’94) and
by Keuten (95), can be easily followed in almost any species of Lug/ena. It lies in the centre of a spherical group of chromatin granules which
are connected with one another by a linin reticulum, the whole being
inclosed within a firm nuclear membrane (Fig. 136). During resting
stages of the cell it is globular or ellipsoidal in form, but during THE PROTOZOA 260 division, it elongates to form a dumb-bell-shaped body, the two ends
remaining connected until the end of division, by a strand. After
division the daughter-centre rounds out and resumes its customary
form and position within the group of chromatin granules. An essentially similar process was described by Schaudinn (’94) in An essentially similar process was described by Schaudinn (’94) in
the division of the rhizopod Amwba crystalligera, and although he
recognized that the intra-nuclear body plays the chief réle in division,
he regarded it as the nucleolus (Fig. 137). It has since been found
in the majority of Mastigophora, in many Rhizopoda, Heliozoa, and in B
Fig. 136. — Mitosis in Euglena. [WILSON after KEUTEN.]
A. Preparing for division; the nucleus contains a “sphere” or division-centre, surrounded by
a group of chromosomes. JB. Division of the sphere to form an intra-nuclear spindle. C. Later
stage. JD. The division completed. B Fig. 136. — Mitosis in Euglena. [WILSON after KEUTEN.]
A. Preparing for division; the nucleus contains a “sphere” or division-centre, surrounded by
a group of chromosomes. JB. Division of the sphere to form an intra-nuclear spindle. C. Later
stage. JD. The division completed. Fig. 136. — Mitosis in Euglena. [WILSON after KEUTEN.]
A. Preparing for division; the nucleus contains a “sphere” or division-centre, surroun
a group of chromosomes. JB. SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 261 The micronuclei of Infusoria were first observed in division by Bal-
biani ('58, 59), but were incorrectly interpreted as bundles of sperma-
tozoa, an
error which Biitschli ('76) was
the first to point out. Biitschli showed that these supposed bundles are nothing more than
the micronucleus in division, and he correctly interpreted the micro-
nucleus as analogous to the nuclei of tissue-cells. In the figures
which were made at this time, there is a distinction between chro-
matin and achromatin (eg. Paramecium), and a mass of ‘achro-
matic’”’ substance is pictured at each pole (cf. Fig. 138, 4). Subsequent Pty. Fig. 137. — Division in Amada crystalligera Schaud. [SCHAUDINN.]
5, division-centre. Fig. 137. — Division in Amada crystalligera Schaud. [SCHAUDINN.]
5, division-centre. observers have obtained similar results. Maupas (’89) showed that
the spindle figure is made up of two distinct parts, and Hertwig (’89,
’95) made out the granular character of the chromatin, and in his
later work (’95) showed that the resting nucleus as well as the divid-
ing nucleus has an achromatic body. The relation of the intra-
nuclear body in the resting nucleus and in dividing forms was not
made out, although Hertwig assumed that the spindle in the latter is
derived from the division-centre. The later stages of the dividing
nucleus show the chromatin massed at the two ends in front of a
cap (pole-plate) of achromatic material, while a strand similar to that
in Euglena, connects the two ends (Fig. 139, D-//). It was Hertwig ('77), also, who gave the first account of the origin observers have obtained similar results. Maupas (’89) showed that
the spindle figure is made up of two distinct parts, and Hertwig (’89,
’95) made out the granular character of the chromatin, and in his
later work (’95) showed that the resting nucleus as well as the divid-
ing nucleus has an achromatic body. The relation of the intra-
nuclear body in the resting nucleus and in dividing forms was not
made out, although Hertwig assumed that the spindle in the latter is
derived from the division-centre. The later stages of the dividing
nucleus show the chromatin massed at the two ends in front of a
cap (pole-plate) of achromatic material, while a strand similar to that
in Euglena, connects the two ends (Fig. 139, D-//). 1.
Lntra-nuclear Division-centres. Division of the sphere to form an intra-nuclear spindle. C
stage. JD. The division completed. Infusoria. In many cases it loses its distinct outline and becomes
more or less indistinct, although reappearing during cell-division as
the division-centre. In the dinoflagellate
Ceratiuim hirundiuella,
Lauterborn (’95) described it as indistinct and apparently without the
usual function. The intra-nuclear division-centre becomes difficult to see, at least
in the resting phases, in micronuclei of the Infusoria, in the rhizopod
Euglypha, in the heliozoén Actinospherium, and in the ciliate Spiro-
chona, forms which may be selected as showing the variations in the
intra-nuclear division-centre. SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS It was Hertwig ('77), also, who gave the first account of the origin It was Hertwig ('77), also, who gave the first account of the origin
of the achromatic spindle-figure in the division of the macronucleus
of the peritrichous ciliate Spzrochona gemmipara. In this nucleus THE PROTOZOA 262 he described two distinct parts, one homogeneous, and with the
exception of a central granule, which he called the “nucleolus,” with-
out structure; the other, chromatin in the form of granules, which
surround the homogeneous portion. In preparation for budding, the
granule within the homogeneous portion swells, sends out pseudopodial
processes, and at the same time becomes more indistinct, until, finally,
the substance of the so-called nucleolus is lost in the surrounding
granules of chromatin. Shortly
afterward, two heaps of homo-
geneous substance (pole/flates)
appear at the ends or poles of
the
division-figure,
and
the
chromatin granules are arranged
in lines between these masses. Even at this early date Hertwig he described two distinct parts, one homogeneous, and with the
exception of a central granule, which he called the “nucleolus,” with-
out structure; the other, chromatin in the form of granules, which
surround the homogeneous portion. In preparation for budding, the
granule within the homogeneous portion swells, sends out pseudopodial
processes, and at the same time becomes more indistinct, until, finally,
the substance of the so-called nucleolus is lost in the surrounding
granules of chromatin. Shortly
afterward, two heaps of homo-
geneous substance (pole/flates)
appear at the ends or poles of
the
division-figure,
and
the
chromatin granules are arranged
in lines between these masses. Even at this early date Hertwig
compared
the
pole-plates
or
“ end-plates,” as he called them,
with the Polkérperchen (centro-
somes) in the mitotic figures of
Metazoa (Fig. 139, A-C), and
concluded that they are derived
from the original ‘ nucleolus.” he described two distinct parts, one homogeneous, and with the
exception of a central granule, which he called the “nucleolus,” with-
out structure; the other, chromatin in the form of granules, which
surround the homogeneous portion. In preparation for budding, the
granule within the homogeneous portion swells, sends out pseudopodial
processes, and at the same time becomes more indistinct, until, finally,
the substance of the so-called nucleolus is lost in the surrounding
granules of chromatin. Shortly Vist)
Fig. 138.— a. Macronucleus and micronucleus
of Stylonychia in division. 4. Micronucleus of
Paramecium aurelia in division. c. SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS The nucleus
of Chilodon cucullulus. [BUTSCHLI.]
d. Dividing
nucleus of Clepsidrina sp, Spirochona
has
been
re-
peatedly examined since 1877,
and Hertwig’s main conclusions
are confirmed. There is a dif-
ference of opinion, however, in
regard
to
the
origin
of the
“nucleolus” or division-centre,
aS We May be justified in calling
jt,
Plate (86) thought that the
corpuscle is formed anew after
division, by the accumulation of
chromatin which penetrates the
solution. He also distinguished
homogeneous portion, and main-
ed from its substance. Balbiani
mewhat similar conclusion. He
part is made up of short and fine
e chromatin dyes, and are, there-
structures. He maintained that
aggregation of several granules
of division. sent in the nucleus of Spzrechona, Spirochona
has
been
re-
peatedly examined since 1877,
and Hertwig’s main conclusions
are confirmed. There is a dif-
ference of opinion, however, in
regard
to
the
origin
of the
Vist)
“nucleolus” or division-centre,
Fig. 138.— a. Macronucleus and micronucleus
aS We May be justified in calling
Stylonychia in division. 4. Micronucleus of
jt,
Plate (86) thought that the
aramecium aurelia in division. c. The nucleus
Chilodon cucullulus. [BUTSCHLI.]
d. Dividing
corpuscle is formed anew after
cleus of Clepsidrina sp,
division, by the accumulation of
chromatin which penetrates the
omogeneous portion in a state of solution. He also distinguished
n inner structure in the supposed homogeneous portion, and main-
ained that the pole-plates are formed from its substance. Balbiani
95) more recently came to a somewhat similar conclusion. He
ound that the so-called homogeneous part is made up of short and fine
brillae which do not stain with the chromatin dyes, and are, there-
re, to be classed as “achromatic” structures. He maintained that
he “nucleolus” is formed by the aggregation of several granules
f chromatin during the later stages of division. Two substances, therefore, are present in the nucleus of Spzrechona, Fig. 138.— a. Macronucleus and micronucleus
of Stylonychia in division. 4. Micronucleus of
Paramecium aurelia in division. c. The nucleus
of Chilodon cucullulus. [BUTSCHLI.]
d. Dividing
nucleus of Clepsidrina sp, Two substances, therefore, are present in the nucleus of Spzrechona, SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 263 which play some réle in the process of division. SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS The apparent rela-
tion of these substances to one another is strikingly similar to the
relations between the centrosome and the attraction sphere in Metozoa,
but so many points remain obscure that no safe conclusions can be
drawn. In one other form, Chzlodon cucullulus, there is an intra-
nuclear structure which recalls that of Spzrochona, but nothing is
known about its division-phases (Fig. 138, C). In all other forms with well-developed “pole-plates” there is a In all other forms with well-developed “pole-plates” there is a
similar mystery regarding the material which enters into their forma-
tion. Despite the numerous observations, there is no case on record Fig. 139. — Mitotic division in the Infusoria. [From WILSON after R. HERTWIG.]
A-C. Macronucleus of Spzrochona, showing pole-plates. D-A. Successive stages in the divi-
sion of the micronucleus of Paramecium,
LD. ‘The earliest stage, showing reticulum. G. Follow-
ing stage (‘‘sickle-form") with nucleus. £, Chromosomes and pole-plates. 7. Late anaphase. #f, Final phase. Fig. 139. — Mitotic division in the Infusoria. [From WILSON after R. HERTWIG.]
A-C. Macronucleus of Spzrochona, showing pole-plates. D-A. Successive stages in the divi-
sion of the micronucleus of Paramecium,
LD. ‘The earliest stage, showing reticulum. G. Follow-
ing stage (‘‘sickle-form") with nucleus. £, Chromosomes and pole-plates. 7. Late anaphase. #f, Final phase. in which the origin of the pole-plates has been definitely made out. In all cases, however, it is assumed that the so-called nucleolus
or
“achromatic body,” which is generally present in the resting cell-
nucleus, becomes modified in some manner (Hertwig, ’95, assumed
by the addition of water or other fluid substance) until it is much en-
larged, when it forms the pole-plates. This general view is based
upon the supposition that, like a centrosome, the achromatic body
divides, half going to one pole and half to the other. In Sarcodina, as in Infusoria, there is no direct evidence to de-
termine the history of the “achromatic” body within the nucleus, THE PROTOZOA 264 although two classical objects, Actinospherium and Euglypha, have
been repeatedly examined. Nuclear division in the former was first
described by Gruber (’83), then by Hertwig (’84), by Brauer (’94),
and, finally, reéxamined in great detail by Hertwig (’98). SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS All agree
as to the general features of division, but disagree widely in details,
In some stages (before the “ primary mitosis,” Hertwig) the chromatin
is in a single large karyosome which incloses a faintly staining
achromatic mass (Gruber, Hertwig). In addition to these there is Fig. 140.— Nuclear division in Actinospherium. [HERTWIG.]
A, B. Ordinary vegetative nuclei. C. Prophase of division. D. Ordinary mitosis. &, Trans-
fusion of intra-nuclear substance (x) to the outside. , G, H#. ‘‘ Maturation-mitoses.”
#, pole
plates; c, centrosome. Fig. 140.— Nuclear division in Actinospherium. [HERTWIG.]
A, B. Ordinary vegetative nuclei. C. Prophase of division. D. Ordinary mitosis. &, Trans-
fusion of intra-nuclear substance (x) to the outside. , G, H#. ‘‘ Maturation-mitoses.”
#, pole
plates; c, centrosome. Fig. 140.— Nuclear division in Actinospherium. [HERTWIG.] a conspicuous linin network (Brauer, Hertwig), which Brauer and
Hertwig regard as forming the pole-plates during division. During
mitosis peculiar and as yet unexplained masses of protoplasm (achro-
matic) are formed on the outside of the nucleus (Protoplasmakegel). Brauer and Hertwig agree that these masses are nuclear in origin,
but neither gives a satisfactory explanation of their function in divi-
sion (Fig. 140). The pole-plates are connected by fibres of ‘achro- a conspicuous linin network (Brauer, Hertwig), which Brauer and
Hertwig regard as forming the pole-plates during division. During
mitosis peculiar and as yet unexplained masses of protoplasm (achro-
matic) are formed on the outside of the nucleus (Protoplasmakegel). Brauer and Hertwig agree that these masses are nuclear in origin,
but neither gives a satisfactory explanation of their function in divi-
sion (Fig. 140). The pole-plates are connected by fibres of ‘achro- SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 265 matic” substance, which are formed from the linin reticulum (Hertwig)
and upon which the chromatin granules are strung. At a “certain
period in mitosis” (Brauer), a period which Hertwig identified as
“maturation
mitosis,” minute
bodies, analogous
to centrosomes,
emerge from the nucleus and take a position in this extra-nuclear
mass of achromatic material (Fig. 140, #). At this stage of division,
therefore, there are structures which, more than anything else in the
Protozoa, resemble the astral system in the cytoplasm of many meta-
zoan cells. They occur, however, only at certain periods and are
not characteristic of ordinary vegetative mitosis. SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS Nuclear division in Lug/ypha as described by Schewiakoff (’88) is Nuclear division in Lug/ypha as described by Schewiakoff (’88) is
characterized by stages which are remarkably similar to those in
‘Metazoa. The chromatin passes through a spireme stage and breaks
into chromosomes by transverse division of this spireme; a spindle is
formed from “achromatic
material,’ and Polkorperchen, which are
strikingly suggestive of centrosomes, form the poles of the division-
figure (Fig. 23, p. §5). The origin, again, of the spindle-fibres and of
the pole-bodies was not determined. Schewiakoff assumed that the
latter are derived from the cytoplasm. In all of these cases, with the exception of certain maturation In all of these cases, with the exception of certain maturation
phases of Actzxospherium, the entire division-figure remains inside of
the nuclear membrane. The substance of the spindle, therefore, must
arise from within the nucleus, and although it has not been definitely
proved, there is good reason to believe that this substance is con-
tained, during the resting phases, in the so-called “nucleolus”
or
intra-nuclear sphere (division-centre). There is no doubt upon this
point
in regard
to
the
division-figure
of the simple
flagellates
(Euglena, etc.), for the clearly defined achromatic body can be traced
throughout all stages. The connecting strand in Euglena is not
fibrillated, and therefore a true spindle is lacking, but there seems
little room to question the analogy between such a strand and the
fibres in forms like Actinospherium, Spirochona,
etc. Indeed,
the
relation of the single strand to the fibrillated spindle is apparently
well marked by an intermediate form in Paramecium (Hertwig, 96),
where the central portion of the division-figure is a single strand
which
widens
and
becomes
fibrillated
at
the
ends
(cf. Fig. 139, /7). 2. Extra-nuclear Division-centres.
We now pass to a considerati We now pass to a consideration of more complicated types of
protozoan nuclei, in which, during the resting phases, the kinetic
structures are outside of the nuclear membrane. The Centralkorn in Heliozoa, with its radiating fibres which form The Centralkorn in Heliozoa, with its radiating fibres which form
the axial filaments of the pseudopodia (cf. p. 82), have often, and THE PROTOZOA 266 probably justly, been compared with the centrosome and astral rays
of Metazoa. This central granule, first observed by Grenacher (’69),
was shown
to be connected with the pseudopodia by Greeff in
the same year. Subsequent observations by F. E. Schultze (’74),
Hertwig (’77), and many others, have confirmed these results, and
demonstrated the widespread occurrence of the central granule among
the Heliozoa. Biitschli(’92) was the first to suggest the similarity to
the centrosome with its radiating fibres, a view which O. Hertwig (’93)
and Schaudinn (’96), and with them the majority of cytologists, have
accepted. The most complete observations have been made upon
species of Acanthocystis and Spherastrum by Schaudinn (’96). In
the latter form (Fig. 144, 4) the corpuscle is distinctly granular, and in. fixed preparations has a definite alveolar structure. The beginning
of division is signalized by the withdrawal of the pseudopodia; the
central granule then divides, and with it the entire astral system.! The daughter-centres
are joined together by a connecting strand
which Schaudinn regarded as a possible central-spindle (Fig. 144, C). The extra-nuclear central granule in these Heliozoa thus acts like The extra-nuclear central granule in these Heliozoa thus acts like
the intra-nuclear division-centre of Ezg/ena and other flagellates. The
extra-nuclear division-centres in the rhizopod Parame@ba and in the
cystoflagellate /Voctz/uca, on the other hand, are much more like the
centrosphere in Metazoa; and in MWoctiluca especially, the mitotic
figure, while of the protozoan type, is more like that of the Metazoa
than of any other known single-celled form. On the outside of the nucleus in WVoctzluca, in the cytoplasm, and On the outside of the nucleus in WVoctzluca, in the cytoplasm, and
close against the nuclear membrane, is a large, faintly staining spher-
ical mass, which acts as a division-centre. During the. early stages
of nuclear activity, the sphere divides into two similar halves, con-
nected by a strand composed of fibres which are formed from the
substance of the sphere. 1 The division of the central granule was first observed by Sassaki (’93) in the marine
form, Gymnosphara,
2 Cf, Calkins (’98), p. 15; Ishikawa (’99), p. 244. 2. Extra-nuclear Division-centres.
We now pass to a considerati These fibres compose the central spindle,
and are homologous in every way with the central-spindle fibres of
the usual
type of mitosis
in Metazoa
(Fig. 141, CE). The
nucleus then elongates in a direction at right angles to the central
spindle, and at the same time it bends in the centre in such a way
that the central spindle sinks into a depression in the nucleus, which
surrounds it upon three sides. In this way the nuclear plate is finally
wrapped about the central spindle in the form of an incomplete ring,
a condition which obtains in all higher mitotic figures where the cen-
tral spindle is present. The nuclear membrane then disappears? in that part of the nucleus which is turned toward the central spin-
dle, while it is retained unbroken in all other parts of the nucleus 1 Loe, cit., p. 53, SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS
267 ECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS
267 267 Fig. 141, £, F). Thus the chromosomes, as in the higher types, are
brought in contact with the central-spindle fibres. They then split
longitudinally, and through the agency of mantle-fibres are separated
into two equal groups, each group drawn toward its own daughter-
sphere. Within the sphere the fibres are focussed in a centrosome, ee ie
La
NE
8
Fig. 141. — Mitosis in Noctiluca miliaris. s, Sphere; ¢, centrosome;
ch, chromosomes. which, at this period, can be demonstrated with the greatest ease. Fig. 141. — Mitosis in Noctiluca miliaris. s, Sphere; ¢, centrosome;
ch, chromosomes. which, at this period, can be demonstrated with the greatest ease. The division is finally completed by the separation of the remainder
of the nucleus and the re-formation of the daughter-nuclei, while the
chromosomes disintegrate into granules, which again form the large
chromatin reservoirs characteristic of /Vocteluca (Fig. 134, £). As Ishikawa (94) first pointed out, the centrosome within the As Ishikawa (94) first pointed out, the centrosome within the
sphere, the mantle-fibres and their insertion in the chromosomes, 268 THE PROTOZOA the origin of the central spindle from the substance of the sphere,
are all features obviously common to the analogous structures in
Metazoa. An extra-nuclear body, somewhat similar to the sphere of octiluca, An extra-nuclear body, somewhat similar to the sphere of octiluca,
has been described by Schaudinn (’96) in the rhizopod Parameba. Parameba reproduces by swarm-spores (cf. p. 93), which are formed
by the spontaneous division of the parent organism into a large num-
ber of small parts. Before this multiple division, the extra-nuclear
achromatic mass (division-centre) divides into a number of parts equal
to the number of spores to be formed, and after the several portions
are distributed about the cell, the nucleus divides into as many parts
as there are portions of the achromatic mass. It is unfortunate that
the minutize of division are not given, and until future observation
confirms Schaudinn’s interpretation, his results must be received with
some scepticism. The swarm-spores themselves reproduce by longi-
tudinal division, and the nuclear processes involved are extremely
suggestive of the relations of the extra-nuclear to the intra-nuclear
division-centres. 3. The Relation of Extra-nuclear to Intra-nuclear Division-centres. 3. The Relation of Extra-nuclear to Intra-nuclear Division-centres. In view of the fact that the division-centres are in some cases
extra-nuclear and in others intra-nuclear, the question-naturally sug-
gests itself as to the connection, if any, between them. No one
conversant with the facts will doubt that they are analogous struc-
tures, but that one has been derived from the other is not so obvious. Hertwig (’95) held that “these centrosomes of the egg-cell (Echi-
noderms) are not specific cell-organs, but portions of the nucleus
which have become freed from the chromatic nuclear substance.” ?! This view of the origin of extra-nuclear kinetic substance in Metazoa
is difficult to accept, and in forming it, Hertwig passed over too many
questionable intermediate stages. There is considerable evidence,
however, among the Protozoa, to indicate that Hertwig’s conception
has a basis of fact, and that the extra-nuclear division-centres arose
from the intra-nuclear forms. As indirect evidence of such an origin of the extra-nuclear division- As indirect evidence of such an origin of the extra-nuclear division-
centres, it might be pointed out that in all mitotic figures in which
there is a central spindle, a portion, at least, of the spindle substance
is surrounded by chromatin, and may be said to be intra-nuclear in
position. This is certainly the case in all Protozoa, and the relations
of the extra-nuclear centres to the chromatin
is particularly sug-
gestive in the dividing swarm-spores of Parameba, in Noctiluca, in
Tetramitus, and in Actinospherium, while, according to Schaudinn's 1 Loe, cit., p. 53, SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 269 observations, in two cases at least, there is positive evidence that
the extra-nuclear centres
originate in the nucleus (Acanthocystis,
Oxyrrhis). Fig. 142. — Purumeba eilhardi Schaud. [SCHAUDINN.]
A. Section. ZB. Sporulation,
C-H. The flagellated swarm-spores in process of division. &,
the Nedenkirper ; a, the nucleus which wraps itself around the division-centre (edenkérper). Fig. 142. — Purumeba eilhardi Schaud. [SCHAUDINN.]
ZB. Sporulation,
C-H. The flagellated swarm-spores in process of division. &,
; a, the nucleus which wraps itself around the division-centre (edenkérper). In Parameba, although the division-centre (ebenkorper) appar-
ently plays no part in the nuclear division of the mother-animal, its
daughter-parts play the same réle in division of the swarm-spores as THE PROTOZOA 270 that of the intranuclear division-centre in Euglena (Fig. 142). 1A significant fact is that in Actinophrys the radiating axial filaments
centre
in the
nucleus, while in other Heliozoa with a ‘“Centradkorn” the radiating axial filaments centre
in this extra-nuclear body.
This indicates that in Acéénophrys the attraction-centre is within
the nuclear substance and presumably in the “ achromatic substance.” 3. The Relation of Extra-nuclear to Intra-nuclear Division-centres. In
Noctiluca the central spindle, which is derived from the extra-nuclear
division-centre, takes a similar intra-nuclear position during division
of the nucleus, and when the nuclear membrane disappears in the ad-
jacent portions of the nucleus, the kinetic substance is again intra-
nuclear, although the centres of attraction are outside (Fig. 141, D, £). In Actinospherium, both Brauer (’94) and Hertwig (’98) explained the
cytoplasmic accumulations (Protoplasmakegel) as coming from the
nucleus, and Hertwig pictured the outer mass and the inner achro-
matic substance as connected through openings in the membrane
(Fig. 140). Confirmatory evidence is also shown in other Heliozoa. In Actinophrys there is no extra-nuclear division-centre, and Schau- Fig. 143. — Mitosis in Tetramitus chilomonas. A, Ordinary form with distributed chromatin (c) and division-centre (s). 2B. The chromatin
granules are collected prior to division. C. The division-centre has divided. D, Later stage in. division; each daughter-nucleus is surrounded by a group of chromatin granules. Fig. 143. — Mitosis in Tetramitus chilomonas. Ordinary form with distributed chromatin (c) and division-centre (s). 2B. The chromatin
s are collected prior to division. C. The division-centre has divided. D, Later stage in. ; each daughter-nucleus is surrounded by a group of chromatin granules. Fig. 143. — Mitosis in Tetramitus chilomonas. Ordinary form with distributed chromatin (c) and division-centre (s). 2B. The chromatin
s are collected prior to division. C. The division-centre has divided. D, Later stage in. ; each daughter-nucleus is surrounded by a group of chromatin granules. dinn (’96) interprets the achromatic spindle figure as nuclear in origin
(Fig. 130, p. 236). The flagellate Zetramitus shows an apparently similar division- The flagellate Zetramitus shows an apparently similar division-
centre. During the resting phases, the chromatin is distributed
throughout the cell, while an indefinite “achromatic mass” appears
to be in direct connection with the cytoplasmic reticulum. Imme-
diately before division, however, the chromatin granules collect about
this body, and then, save for the absence of a membrane, the aggre-
gate resembles the nucleus of Euglena. Division takes place as SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 271 in Euglena, the intranuclear division-centre
dividing first. After
division the chromatin granules again disperse and the division-centre
becomes again cytoplasmic (Fig. 143). Still more convincing evidence is shown by the history of the Still more convincing evidence is shown by the history of the
division-centres of Acanthocystis and the flagellate Oxyrrhis marina
(Schaudinn,’96). 1An intermediate stage between this condition and the condition in Zuglena is shown by
some species of Chilomonas and Trachelomonas, in which there is no nuclear membrane,
but in which the chromatin remains permanently aggregated about the division-centre
(Calkins, ’98). 3. The Relation of Extra-nuclear to Intra-nuclear Division-centres. Acanthocystis has a permanent extra-nuclear division-
centre which divides and forms a complete spindle (Fig. 144, A-D). Fig. 144. Nuclear division and spore-formation in Heliozoa. [SCHAUDINN.]
A, A vegetative cell of Spherastrum with the axial filaments focussed
in a central-granule
(division-centre). B-D. Division of the nucleus in Acanthocystis,
E, F. Flagellated and amoe-
boid swarm-spores formed by budding. G. Exit of the division-centre from the nucleus. Fig. 144. Nuclear division and spore-formation in Heliozoa. [SCHAUDINN.] Fig. 144. Nuclear division and spore-formation in Heliozoa. [SCHAUDINN.]
A, A vegetative cell of Spherastrum with the axial filaments focussed
in a central-granule
(division-centre). B-D. Division of the nucleus in Acanthocystis,
E, F. Flagellated and amoe-
boid swarm-spores formed by budding. G. Exit of the division-centre from the nucleus. Fig. 144. Nuclear division and spore-formation in Heliozoa. [SCHAUDINN.]
A, A vegetative cell of Spherastrum with the axial filaments focussed
in a central-granule
(division-centre). B-D. Division of the nucleus in Acanthocystis,
E, F. Flagellated and amoe-
boid swarm-spores formed by budding. G. Exit of the division-centre from the nucleus. Like Parameba this heliozoo6n reproduces by swarm-spores;
the
division-centre, however, takes no part in their formation, but remains
intact while the nucleus divides without mitosis. The buds, therefore,
contain no portion of the original material of the division-centre, nor is
there any evidence in them of such a centre until about the fifth day. At this time Schaudinn found a “ Centralkorn” within the nucleus of
each swarm-spore, which passed later through the nuclear membrane
and into the cytoplasm, where it developed into the well-known divi-
sion-centre of the adult Acanthocystis (G). The extrusion of the kinetic substance of the nucleus which thus Like Parameba this heliozoo6n reproduces by swarm-spores;
the
division-centre, however, takes no part in their formation, but remains
intact while the nucleus divides without mitosis. The buds, therefore,
contain no portion of the original material of the division-centre, nor is
there any evidence in them of such a centre until about the fifth day. At this time Schaudinn found a “ Centralkorn” within the nucleus of
each swarm-spore, which passed later through the nuclear membrane
and into the cytoplasm, where it developed into the well-known divi-
sion-centre of the adult Acanthocystis (G). 3. The Relation of Extra-nuclear to Intra-nuclear Division-centres. The extrusion of the kinetic substance of the nucleus which thus The extrusion of the kinetic substance of the nucleus which thus THE PROTOZOA 272 takes place under normal conditions in Acanthocystis can be brought
about in Oxyrrhis marina, as Schaudinn has demonstrated, by placing
the flagellate in an abnormal medium. Ordinarily this form has an
intra-nuclear division-centre like that of Aug/exa, but if it is trans-
ferred to a more dilute salt solution, the substance of the division-
centre is forced out of the nucleus and into the cytoplasm, where it
swells to many times its usual size, and may even divide outside of
the nucleus, forming a large spindle in the cytoplasm (Schaudinn, ’96). F.
GENERAL CONSIDERATIONS The mitotic figure in plants, higher animals, and in Protozoa is
composed of two parts, of which one, the chromatin, is widely held
to be the primary agent in heredity, while the other, forming the
‘achromatic structures,” is generally regarded as the agent by means
of which the chromatin is divided equally between the two daughter-
cells. The two parts have more
or less independent antecedent
phases, and it is possible, therefore, to conceive the two portions
unequally developed. In Protozoa both portions are relatively simple
in structure, although complex chromosomes may accompany rela-
tively simple achromatic specializations, and vice versa. The stages in chromosome formation in different types of Piotocds The stages in chromosome formation in different types of Piotocds
may be briefly summarized as follows: (1) The most primitive nucleus
is, apparently, in the form of a compact sphere of chromatin, the
multiple division of which is the prelude to reproduction of the cell. (2) A higher type comprises nuclei with membranes, and with chro-
matin in one (Sporozoa) or in many (Noctiluca, etc.) karyosomes,
which break up by multiple division into granules. The granules
thus formed unite secondarily into lines forming primitive chromo-
somes. (3) In still higher forms the granules do not return to the
karyosome stage, but are widely distributed over the linin reticulum
(Flagellidia, Acrianplog, some stages of Actinospherium, Metazoa). (4) The highest type contains chromatin granules embedded in a
linin
reticulum, or aggregated to form net knots or karyosomes analogous
to the more primitive chromatin spheres. Like the primitive karyo-
somes, these net knots break up into granules which come together
in lines for division (spireme stage of some Protozoa, of plants, and
Metazoa), and these lines segment into chromosomes
of definite
number and size (Metazoa and Metaphyta). In some Protozoa the nuclei remain permanently in one or the other In some Protozoa the nuclei remain permanently in one or the other
stage described. Thus in many of the Phytoflagellida they are per-
manently in stage 1; in the simple Monadida they are typically in
stage 3, in the Rhizopoda in stage 2, while the higher types of nuclei
pass through nearly all of these stages during preparation for division. SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS
2 273 4. The so-called “ Centrosomes” in Protosoa. 4. The so-called “ Centrosomes” in Protosoa. In addition to the division-centres
or spheres described above,
there are occasional centres
in Protozoa resembling centrosomes
of the Metazoa. So little positive knowledge is at hand, however,
that the centrosome question in Protozoa must
rest in abeyance. A number of division-centres have been described as centrosomes,
but in almost all cases these are more like the spheres described above,
than the centrosomes of Metazoa. Ina number of instances, however,
the primitive division-centres contain central, deeply staining granules
which form the foci of the spindle fibres, and these would seem to be
analogous to the centrosomes of higher forms. Such granules are
found in Spzrochona, Kentrochona, Actinosphertum, and Noctiluca, and
probably they exist in other forms as well; these, however, are the
only forms that have been critically examined. In Spirochona and Kentrochona, the granules in question appear only In Spirochona and Kentrochona, the granules in question appear only
at a late stage in division (Balbiani, 95 ; Doflein,’96), and they remain
in evidence until the next division period. In Actznospherium similar
granules appear during the maturation mitosis, but are not found at
other times (Hertwig). In Woc¢zluca they appear at each mitosis and
form the insertion points for the mantle-fibres (Fig. 141, /). The origin of these so-called centrosomes still remains obscure, The origin of these so-called centrosomes still remains obscure,
although in each case it has been maintained that they arise from the
chromatin. Plate (’86) and Biitschli (’88) assumed that in the case
of Spirochona gemmipara they are formed from chromatin in solution,
while Balbiani (’95) maintained that they are actually granules of chro-
matin broken off from the polar ends of the chromosomes. Hertwig
(98) described a similar origin in the case of Actinospherium, where the
granules are supposed to arise by budding of the chromatin (Fig. 140). Brauer (’94), on the other hand, held that the centrosomes come from
the pole-plates. In Moctz/uca, finally, Ishikawa (’94, '99) and Calkins
(98) concluded that the centrosomes were nuclear in origin, while the
spheres are extra-nuclear at all times. The conclusion, however, could
not be supported by positive evidence and, at best, has only the value
of an assumption. F.
GENERAL CONSIDERATIONS He
held that in these cells the protoplasm is composed of two essential
substances, one of which, the ¢vophoplasm, is alveolar in structure and
is especially concerned with the processes of nutrition; the other,
kinoplasm, is fibrillar in structure and is devoted to the formation of
the active portions of the cell, spindle fibres, cilia, flagella, and outer
cell-covering (Hautschicht). The facts in Protozoa fit in best with the archoplasm hypothesis. matic material,” regarding which wide difference of opinion prevails. If, with van Beneden (’83), Biitschli(’92), and many others, we assume
that the spindle in Protozoa arises by the local modification of the
protoplasmic network, we leave unexplained
all of those division-
centres in Protozoa which are unmistakably permanent throughout
resting and active phases of the cell, and from which spindles are
formed (flagellates, Moctzluca, etc.). Nor can the interesting hypoth-
esis of Rabl (’89), even when supported by the evidence which
Heidenhain (’94), Biihler (’95), and Kostanecki (’97) have added, ex-
plain the facts in Protozoa, unless, indeed, it be assumed with Rabl
that the spindle fibres, never losing their identity, but merely modified
to form portions of the protoplasmic network, remain in Protozoa, in
the form of a compact and definite division-centre. Could Rabl’s
suggestion be thus adapted, the result would be an hypothesis which
agrees essentially with the archoplasm theory of Boveri (’88). Boveri
maintained that the kinetic structures of the cell are derived from a
specific substance which he called archoplasm. At first (’88) he held
that the archoplasm, in the form of granules, is a permanent substance,
but in a subsequent paper (’95) he modified the theory by the sug-
gestion that the archoplasm might be distributed about the cell in the
form of a homogeneous material which cannot be readily demonstrated,
and which under the influence of the centrosome may be crystal-
lized out from the protoplasm. An essentially similar hypothesis was
formulated by Strasburger (’92) in connection with plant-cells. He
held that in these cells the protoplasm is composed of two essential
substances, one of which, the ¢vophoplasm, is alveolar in structure and
is especially concerned with the processes of nutrition; the other,
kinoplasm, is fibrillar in structure and is devoted to the formation of
the active portions of the cell, spindle fibres, cilia, flagella, and outer
cell-covering (Hautschicht). The facts in Protozoa fit in best with the archoplasm hypothesis. F.
GENERAL CONSIDERATIONS The net knots
thus show a return to the primitive condition, the
chromatin granules to the permanent granular state in Flagellidia, or
to the disruption of the karyosomes of type 2; the fusion of the
granules into spiremes, to the primitive chromosome formation in
Noctiluca. The definite chromosomes, finally, represent the highest
grade of chromatin specialization. While it is quite probable that the chromatin of plant nuclei, of While it is quite probable that the chromatin of plant nuclei, of
metazoan nuclei, and of protozoan nuclei is everywhere essentially the
same substance, the similarity is not so obvious in case of the “achro-
T T THE PROTOZOA 274 matic material,” regarding which wide difference of opinion prevails. If, with van Beneden (’83), Biitschli(’92), and many others, we assume
that the spindle in Protozoa arises by the local modification of the
protoplasmic network, we leave unexplained
all of those division-
centres in Protozoa which are unmistakably permanent throughout
resting and active phases of the cell, and from which spindles are
formed (flagellates, Moctzluca, etc.). Nor can the interesting hypoth-
esis of Rabl (’89), even when supported by the evidence which
Heidenhain (’94), Biihler (’95), and Kostanecki (’97) have added, ex-
plain the facts in Protozoa, unless, indeed, it be assumed with Rabl
that the spindle fibres, never losing their identity, but merely modified
to form portions of the protoplasmic network, remain in Protozoa, in
the form of a compact and definite division-centre. Could Rabl’s
suggestion be thus adapted, the result would be an hypothesis which
agrees essentially with the archoplasm theory of Boveri (’88). Boveri
maintained that the kinetic structures of the cell are derived from a
specific substance which he called archoplasm. At first (’88) he held
that the archoplasm, in the form of granules, is a permanent substance,
but in a subsequent paper (’95) he modified the theory by the sug-
gestion that the archoplasm might be distributed about the cell in the
form of a homogeneous material which cannot be readily demonstrated,
and which under the influence of the centrosome may be crystal-
lized out from the protoplasm. An essentially similar hypothesis was
formulated by Strasburger (’92) in connection with plant-cells. 1 Loc. cit, p. 59. F.
GENERAL CONSIDERATIONS Here, in a great many forms, is a definite structure composed of a
specific substance which, during cell-division, forms the spindle-figure. Hertwig (’96) favored the view that the cytoplasmic and nuclear retic-
ula in the various phases which this portion of the protoplasm can
assume, are sufficient to explain the several division-centres, without
calling upon a special kinoplasm or archoplasm. He did not, how-
ever, in my opinion, give sufficient weight to the simpler division-
centres in Protozoa, but based his views upon the relatively complex
phenomena of division in Actzxospherium. He believed that in this
form the spindle fibres and the pole-plates (which he homologized with
centrosomes of the Metazoa) are derived from the reticulum of the
nucleus! No satisfactory explanation was given of the “ Plastinge- 1 Loc. cit, p. 59. SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 275 vist,’ which he described in the vegetative nucleus, although he recog-
nized a distinction between it and both the linin reticulum and the
chromatin. The achromatic figure in Actinospherium is so large as
compared with the nucleus of resting cells, that it is difficult to accept
Hertwig’s view, that it all comes from the linin of the resting nucleus
(cf. Fig. 140, A, B, D). The writer, in a recent publication (’99), pro-. posed the application of Boveri's theory of archoplasm to the division-
centres of Protozoa; and, applicable, apparently, to all types of the
division-figure in Protozoa, it may serve for the time as a working
hypothesis. Briefly stating this hypothesis, it was held that the
division-centre, consisting of archoplasm, retains its definite form
and size in the nucleus of many of the primitive forms (flagellates),
but under certain conditions may become enlarged or diffuse. Thus,
in Yetramitus, the division-centre is much more diffuse during the
resting phases of the cell than during division (cf. Figs. 134, 143);
and in Oxyrrhis marina Schaudinn (’96) found that the division-centre
becomes much enlarged when the cells are transferred to a medium
of less density, and conversely, when placed in a denser medium, the
structures become reduced in size and more definite. It was held
that the intra-nuclear
division-centre
becomes
similarly
diffused. 1 Loc. cit, p. 224. F.
GENERAL CONSIDERATIONS In its latest
form, Hertwig’s theory may be summarized
as follows: (1) The
achromatic substance is at first uniformly distributed in the resting
nucleus, but appears during division as pole-plates, the analogue of
centrosomes ; (2) the achromatic substance becomes permanently an
intra-nuclear centrosome ; (3) it is extruded from the nucleus to form
an extra-nuclear centrosome. Still another point of view was suggested by Schaudinn (’96) after improbability of this theory, from the phylogenetic standpoint, was con-
vincingly shown by Boveri (’95); and Lauterborn (’96), admitting
this difficulty, proposed the theory in a slightly modified form. He
assumed that micronucleus and centrosome may have had a common
ancestor in some primitive bi- nucleated protozoon, such as Ame@ba
binucleata (Schaudinn), and that intermediate stages may be seen in
certain existing Protozoa, such as Parameba etthardi (Schaudinn)
and Noctiluca miliaris. He considered the micronucleus to have
arisen from one of these primitive nuclei, but by a differentiation
quite different from that which gave rise to a centrosome. This
theory, also, has been shown to be untenable by Boveri (or). A
different theory elaborately worked out has appeared in several of
the recent publications of R. Hertwig (’95, ’96, ’98). In its latest
form, Hertwig’s theory may be summarized
as follows: (1) The
achromatic substance is at first uniformly distributed in the resting
nucleus, but appears during division as pole-plates, the analogue of
centrosomes ; (2) the achromatic substance becomes permanently an
intra-nuclear centrosome ; (3) it is extruded from the nucleus to form
an extra-nuclear centrosome. Still another point of view was suggested by Schaudinn (’96) after Still another point of view was suggested by Schaudinn (’96) after
his very remarkable observations and experiments on Acanthocystis
and Oxyrrhis marina. He suggested the following two possibilities:
Either the division-centre (his centrosome) is a structure formed within
the nucleus, becoming subsequently cytoplasmic in position, as it does
in the buds of Acanthocystis, or as the nucleolus-centrosome does in
the nucleus of Oxyrrhis when immersed in dilute sea-water ; or else
it is normally cytoplasmic in position, and becomes secondarily intra-
nuclear. Not only in Protozoa, but also in Metazoa, the sphere and centro-
some have been described, in some cases, as coming from the nucleus. 1Cf. Calkins (’98, ’99). F.
GENERAL CONSIDERATIONS The nucleus of Ameba proteus, for example, contains chromatin in the
form of minute granules, which are arranged about the periphery of
the nucleus, while the central portion is occupied by a large homo-
geneous mass, which can be explained as an enlarged or diffuse intra-
nuclear division-centre. The pole-plates,
also, which
are
widely
distributed throughout the Protozoa, may be explained as a temporary
accumulation of this ordinarily diffuse archoplasmic substance, and thus
homologous with the “nucleolus-centrosome”
or division-centre of
Euglena, and with the centrosphere of Metazoa1
The substance
which may thus become diffused through the nucleus may also pene-
trate the nuclear membrane, until accumulations on the outside of the
nucleus result. Hertwig described such transfusion of achromatic
material from the nucleus of Act‘nospherium to the aggregates (Piv-
toplasmakegel) on the outside (Fig. 140, £). Finally, just as it be-
comes diffused throughout the nucleus in Protozoa and Metazoa, so
it may become diffused throughout the cytoplasm in Metazoa, as
postulated by Boveri. A number of ingenious theories have been made to account for the
origin of the division-centres of the Metazoa. Biitschli (’91) was the
first to suggest that the micronucleus of Infusoria might be the pro-
tozoan analogue of the metazoan centrosome. Hertwig (’92) and
Heidenhain (’94) accepted the suggestion, and the latter, in partic-
ular, worked out a complicated theory of phylogeny upon it. The 1 Loc. cit, p. 224. 276 THE PROTOZOA improbability of this theory, from the phylogenetic standpoint, was con-
vincingly shown by Boveri (’95); and Lauterborn (’96), admitting
this difficulty, proposed the theory in a slightly modified form. He
assumed that micronucleus and centrosome may have had a common
ancestor in some primitive bi- nucleated protozoon, such as Ame@ba
binucleata (Schaudinn), and that intermediate stages may be seen in
certain existing Protozoa, such as Parameba etthardi (Schaudinn)
and Noctiluca miliaris. He considered the micronucleus to have
arisen from one of these primitive nuclei, but by a differentiation
quite different from that which gave rise to a centrosome. This
theory, also, has been shown to be untenable by Boveri (or). A
different theory elaborately worked out has appeared in several of
the recent publications of R. Hertwig (’95, ’96, ’98). F.
GENERAL CONSIDERATIONS The most noteworthy of these is Brauer’s description of Ascaris mega-
locephala univalens (’93) and Riickert’s (’94) description of its nuclear
origin from the germinal vesicle of a copepod. In these two cases, however, it seems obvious that such an origin
among higher metazoan cells can have little value in problems relat-
ing to the historic origin of the centrosomes ; for here differentiation
is fully as complete as in any other cells, and the origin of such centro-
somes can give no light on the problem. To a certain extent, Hert-
wig’s view, also, is open to the same criticism. Schaudinn’s other alternative has opened the way for another view,} Schaudinn’s other alternative has opened the way for another view,}
which, based upon the lower Flagellidia and Rhizopoda, explains
more primitive conditions in more primitive organisms. It is not SPECIAL MORPHOLOGY OF THE PROTOZOAN NUCLEUS 277 improbable that the condition of the distributed nucleus, as found in
Tetramitus, may have been widely spread among the lower forms of
life. In Yetramdtus the division-centre becomes more compact and
distinct during the preparatory division stages, while the chromatin
granules collect in a small aggregate in its immediate vicinity. The
sphere then divides and the chromatin aggregate separates into two
portions. This stage of the division probably corresponds with the
stage described by Schaudinn
in Parameba, where the chromatin
granules form a ring about the divided sphere. The swarm-spores
increase by simple division, as in 7etramitus, and a dumb-bell struc-
ture results. The nucleus, which is a well-defined body, then moves
around the connecting strand of the daughter-centres until it sur-
rounds it as the nucleus of MVocéz/uca surrounds the central spindle
(Fig. 142, %, G). The connecting strand of the daughter-centres of
Parameba is analogous, therefore, to the central spindle of Mocti/uca
and of the Metazoa. It is a temporary stage like this in Tetramitus or Parameba, that It is a temporary stage like this in Tetramitus or Parameba, that
lends plausibility to Schaudinn’s other alternative,
although
the
reverse view may also be conceived, the diffusion of the chromatin
granules taking place by reason of the disappearance through gradual
degeneration of the nuclear membrane. _ If the latter view of the origin
of diffused chromatin be accepted, then the theory of the original
intra-nuclear position of the sphere, for which there are certainly a
great many supporting facts, is warranted. F.
GENERAL CONSIDERATIONS There is considerable
evidence, on
the other hand, to support
the argument
that the
division-centre was originally a distinct cytoplasmic structure, which
only secondarily became connected with the nucleus (e.g. Tetramitus
and Parameba). Intermediate stages between the temporary stage in
Tetramitus and Parameba, and the permanent intra-nuclear division-
centre, may be seen in numerous Flagellidia, such as Chz/omonas and
some species of 7rachelomonas, where no nuclear membrane surrounds
the chromatin granules. Even in these low forms, the sphere appar-
ently exerts some force of attraction, perhaps chemotactic, upon the
chromatin, and this may or may not be strong enough to keep the
granules permanently aggregated. If not, the distributed nucleus may
result ; if so, the intra-nuclear condition of the sphere is the outcome. In Parameba, Noctiluca, in diatoms, and in the majority of Metazoa
and plants, a nuclear membrane is formed and the sphere remains in
its cytoplasmic or extra-nuclear condition, and, as a cytoplasmic body,
or as a kinetic substance (archoplasm or kinoplasm), may undergo fur-
ther differentiations leading to the complicated mitotic figures of
higher animals and plants. In the majority of cases the nuclear
membrane disappears during mitosis, and the primitive conditions are
thus repeated. By rupture of the membrane, the substance of the 278 THE PROTOZOA division-centre comes into direct contact with the chromatin, and is,
in part at least, surrounded by it. The facts point toward the conclusion that the centre of activity in The facts point toward the conclusion that the centre of activity in
the division of the protozoan cell, as in Metazoa, resides in a special
structure, which, to avoid confusion in terminology, has been called
the division-centre. In some cases this structure resembles the astral
system of Metazoa, in consisting of an outer spherical mass with radi-
ating processes (astrosphere), and an inner focal granule or granules
(centrosome). The evidence further tends to show that the division-
centre in Protozoa consists of a specific substance different from the
chromatin and from the cytoplasm, and possessing above all other
portions of the cell an active réle in division. No conclusive evidence
is forthcoming to show whether this substance is permanent in all
cells, or whether it was originally nuclear or cytoplasmic in origin,
although the widespread intra-nuclear condition favors the view that
it originated there. F.
GENERAL CONSIDERATIONS The origin of the central granule (centrosome or centriole) within The origin of the central granule (centrosome or centriole) within
the division-centre
is even more uncertain;
the few data at hand
lead us provisionally to believe with Balbiani and Hertwig that it is
derived from the chromatin. The observations, however, upon which
this conclusion
is based are scanty, and further research must be
undertaken before we can hope for a sufficient basis of facts upon
which to generalize. SPECIAL BIBLIOGRAPHY VIII Boveri, T. — Zellenstudien. /ezazsche Zeitschrift, XXI1.and XXIV., 1888 and 120%
and Part IV., Igor. Brauer,
Aug.—Ueber die Encystierung von Actinospherium
Eich. 2. w. Z., Brauer,
Aug.—Ueber die Encystierung von Actinospherium
Eich. 2. w. Z.,
LVIII., 1894. Biitschli,
0.— Ueber die so-genannten Central-korper der Zelle und ihre Bedeu- schli,
0.— Ueber die so-genannten Central-korper der Zelle und ihre Bedeu-
tung. Vehr.d. Nat. Med. Ver. z. Heidelberg, N. F.,1V., pp. 535-538, 1892. ins, G. N. — The Phylogenetic Significance of Certain Protozoan Nuclei. i. Biitschli,
0.— Ueber die so-genannten Central-korper der Zelle und ihre Bedeu-
tung. Vehr.d. Nat. Med. Ver. z. Heidelberg, N. F.,1V., pp. 535-538, 1892. Calkins, G. N. — The Phylogenetic Significance of Certain Protozoan Nuclei. i. Biitschli,
0.— Ueber die so-genannten Central-korper der Zelle und ihre Bedeu-
tung. Vehr.d. Nat. Med. Ver. z. Heidelberg, N. F.,1V., pp. 535-538, 1892. Calkins, G. N. — The Phylogenetic Significance of Certain Protozoan Nuclei. i. tung. Vehr.d. Nat. Med. Ver. z. Heidelberg, N. F.,1V., pp. 535-538, 1892. Calkins, G. N. — The Phylogenetic Significance of Certain Protozoan Nuclei. i. MV. Y. Acad. Sciences, X1., 1898. ins, G. N. — The Phylogenetic Significance of Certain Protozoan Nuclei. i. MV. Y. Acad. Sciences, X1., 1898. wig, R.— Ueber Kern‘heilung, Richtungskérperbildung und Befruchtung von Hertwig, R.— Ueber Kern‘heilung, Richtungskérperbildung und Befruchtung von
Actinospherium
Eich. <Adh. d. K. bay. Akad. d. Wiss. Aliinchen, I. 1. XIX., 1898. Ishikawa, C. — Ueber die Kerntheilung bei Noctiluca miliaris. Ber. Maturf. Ges- Ishikawa, C. — Ueber die Kerntheilung bei Noctiluca miliaris. Ber. Maturf. Ges-
fretburg, VAIl., 1893. Schaudinn, F. — Ueber die Centralkorn der Heliozoén, eine Beitrag zur Centrosomen- Schaudinn, F. — Ueber die Centralkorn der Heliozoén, eine Beitrag zur Centrosomen-
frage. Verh. d. deutsch. Zool. Gesell., 1896. Strasburger, E.-— Histologische Beitrage. 1V., Jena, 1892. Strasburger, E.-— Histologische Beitrage. 1V., Jena, 1892. Wilson, E. B. — The Cell in Inheritance and Development. Wilson, E. B. — The Cell in Inheritance and Development. 2d Ewition, New York,
1900. 2 Lee’s Verworn, p. 46. 1 Psycho-Protisten Studien, p. 211.
2 SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA “Die psychischen Vorgange im Protistenreich sind daher die Briicke, welche die che-
mischen Processe in der unorganischen Natur mit dem Seelenleben der hédchsten Thiere
verbindet.” — VERWORN.} - ALL animals are subject to disintegration and waste of substance
through oxidation, and to reintegration and renewal of substance
through the addition of new materials. Waste and renewal are
usually spoken of together under the head of metabolism, and
together they constitute one of the essential properties by which
living matter is distinguished from non-living. When renewal of
substance exceeds its waste, the phenomenon of growth results, and
growth leads to reproduction. In the higher animals these various
functions are distributed among many organs, but in the Protozoa
they are all performed by the single cell. The simplest of living
forms, these organisms naturally invite a comparison of their functions,
on the one hand, with those of the higher animals, and, on the other
hand, with the physical and chemical operations of inorganic nature. A modern attitude on the second of these comparisons is taken by
Verworn, an ardent opponent of the old conception of a specific vital
force, different from the forces of the inorganic world. “An ex-
planatory principle,” says Verworn, “can never hold good in physi-
ology, in reference to the physical phenomena of life, that is not also
applicable in chemistry and physics to lifeless nature. The assump-
tion of a specific vital force in every form is not only wholly super-
fluous, but inadmissible.” 2? This extreme
reaction from the old
vitalistic point of view appears to be somewhat premature; for, as
Driesch, Whitman, Wilson, and many others have suggested, there
exists in every organism a power of adaptation and certain codrdinat-
ing factors by which the organism acts as an individual or a unit,
notwithstanding the fact that its body is composed of a great number
of chemically different substances. At the present time these coérdi-
nating factors and the power of adaptation transcend physical or
chemical analysis, and
raise the lowest protozoon immeasurably
above inanimate objects, and perhaps justify, in a modern sense, the
much-abused term
‘‘ vitalism.” 1 Psycho-Protisten Studien, p. 211. 27 279 279 PROTOZOA 280 Among the most interesting problems suggested by the Protozoa
are those relating to their apparently conscious activities. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA _Conscious-
ness has often been ascribed to the Protozoa in order to account for
certain actions which appear to be voluntary. Thus they are often de-
scribed as “selecting” their food, of ‘‘ choosing” building material
for their shells and tests, or of “voluntarily? moving around an
object,
etc. Dujardin
was
one
of the
first
to
discredit
con-
sciousness in the Protozoa, and, with a truly modern point of view,
he wrote
as follows:
‘If one
invokes
the faculty which
the
Protozoa have of directing themselves in the liquid, and of wilfully
pursuing their prey, at least will it be necessary first to verify the
reality of this faculty which I believe as fabulous as everything else
reported as instinct on the part of these animalcula.”! Neverthe-
less, it is generally recognized that consciousness, like life itself,
could not have arisen
at once
in the higher animals, but must
have developed by a slow process of evolution from some property of
protoplasm, which, if the principle of genetic continuity involved in
the doctrine of evolution holds good for the lowest forms, must be
present in some form in all Protozoa. Of the many functions which
make up the vital activities of Protozoa, that of irritability or response
to external stimuli undoubtedly stands nearest to the basis of con-
sciousness of the higher animals, and it may be expressed either by
the general protoplasm
or by specialized “sensory” ectoplasmic
modifications. The other functions of Protozoa, notably those of nutrition and
excretion, can be treated with greater assurance, and can be more
readily compared with similar functions in higher animals. In the
present chapter we may first inquire how closely these more ele-
mentary functions agree with those of the higher forms, and then
consider some of the evidence upon which consciousness has been
attributed to these primitive forms. 1 (41), p. III. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
28 SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
28 Only those Protozoa which take in solid food have been satisfacto-
rily studied in this connection. The processes of digestion and assimi-
lation in parasitic, holophytic, and saprophytic forms are in the main
unknown. Among the carnivorous forms, however, some advance
has been made, although it cannot be said that the digestive processes
even here are fully understood. The classic objects for research in
this direction have been the large predatory ciliates (S¢plonychia,
Prorodon, Climacostomum, Cyrtostomum, Stentor, etc.), the Heliozoa
(Actinophrys, Actinospherium),
and
the rhizopods (Amaeba, Poly-
stomella, and Pelomyxa). The first observations upon intra-cellular
digestion in these forms were made in the last century (Corti, 1774;
Goeze, 1777), when it was seen that living ciliates or flagellates, when
taken into the cell-body of another protozoén, soon cease their strug-
gling and die. The details of the process and the causes of death
have been made out in the last quarter-century, and it is now safely
determined that an acid secretion plays the most important part in
the killing and subsequent digestion of the captives. The length of time which an ingested protozo6n can live in a gastric
vacuole of another form varies, according to the organisms, from five
or ten minutes to several hours. After the prey has become quiet,
the digestive processes, indicated by the disruption of the body of
the captive and gradual absorption of its digestible parts, go on more
or less rapidly. The indigestible parts, in the form of a granular
residue, are voided to the outside. It has been determined upon pretty safe evidence that the chief It has been determined upon pretty safe evidence that the chief
and probably the main source of nutriment of the Protozoa consists
of the proteid substances of the organisms taken in as food, while,
with a few exceptions, carbohydrates and fats are not assimilated. Carbohydrates,
in
the
form
of ordinary
starch, appear
to
be
untouched by the body fluids of many Rhizopoda (Greenwood, ’86 ;
Meissner, ’88; Fabre-Dumergue, ’88); although the recent observations
of Stole (’0o) indicate that in one form, at least (Pelomyxa palustris
Greeff), starch grains
are
easily corroded and
at least partially
digested. Certain kinds of starch are more easily digested than
others;
rice starch
is much more soluble than potato starch
in
the digestive fluids of Pelomyxa. 1 “Tn man, according to recent investigations, starch is said to be broken up by diastase into
five successive hydrolytic cleavage products, as follows:
(1) Amylodextrin (Co2H20O10
)s4, A. INTRA-CELLULAR DIGESTION IN PROTOZOA A distinction must be made at the outset between the digestive
processes of most Metazoa and of Protozoa. In the former, with
some
exceptions,
the digestive
fluids
are poured
out
from
the
epithelial cells which line the digestive tract, into the lumen of that
tract, and the food is digested in the stomach or intestine. In the
Protozoa and in some Metazoa (e.g. the Coelenterata), on the other
hand, the food is taken directly into the cells and there digested. The former method of digestion is said to be inter-cellular, the latter
intra-cellular. a substance giving a deep blue color with iodine.
This is next changed to (2) Erythro-
dextrin (Cy2H20010)18 + H,0, or (Cy2gH 20010) 17- (Cy2H22011), which
is readily soluble in
water, and gives with iodine a reddish brown color.
Erythrodextrin is converted into (3)
Achroédextrin (Cy2H29O10)6 + H20, or (CyzH20010)5.
(C1gH22011), which is likewise very
soluble, tastes slightly sweet, but gives no coloration with iodine.
Achroddextrin now
breaks up into (4) Isomaltose, which through change in configuration is transformed to its
isomere (5) maltose.”
From Howell (’97), p. 1007.
1Cf. Butschli, p. 1802. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
28 The Infusoria seem to have a much
more developed power of starch dissolution, for the grains of potato
starch are all more or less disfigured (Fig. 145), resembling in this
respect the partial digestion of the starch grains in the higher animals
(Meissner, Fabre-Dumergue, Greenwood). The starch after solution
forms a dextrin (Meissner) or an erythrodextrin (Fabre-Dumergue),
but the transformation of these into glucose has not been made out.! THE PROTOZOA 282 The emulsification of fats has never been observed, and opinions
differ as toits possibility. Meissner (’88)and Greenwood (87) asserted
upon empirical grounds that it does not take place in Rhizopoda,
Heliozoa, or Infusoria. Their evidence is mainly based upon the
observations that the fat particles which had been taken in were
thrown out unaltered after a number of days. Fabre-Dumergue (’88)
and Biitschli (’83) take a less positive view, saying that the possibility
of emulsification is certainly not excluded, and that probably a certain
amount of the ingested fat is digested. Chitin, cellulose, and the shells, tests, etc., of other Protozoa pass Chitin, cellulose, and the shells, tests, etc., of other Protozoa pass
through the body plasm with little or no change. Chlorophyl also
may be placed in the same category (Meissner, Le Dantec,
’92),
although a few well-authenticated observations
show that in some
I GW? OB I GW? OB
QeQ0
MF
Fig. 145. — Starch grains in Ciliata, after partial digestion. [MEISSNER.] Fig. 145. — Starch grains in Ciliata, after partial digestion. [MEISSNER.] cases, at least, chlorophyl becomes red, yellow, brown, and finally
black (Perty, ’52), thus indicating some change in its constitution. There is, however, no evidence to show that it becomes dissolved,! while the careful observations of Le Dantec (’92) show that in some
cases, far from being injured by contact with the plasm, the plant-cells
containing chlorophyl may actually thrive in it. Single cells of the
alga which Beyerinck (’90) identified as Chlorella vulgaris, belonging
to the order Protococcacez, when taken into the protoplasm of Pava-
mecium bursarta, are surrounded by a vacuole like any food particle. Soon, however, the vacuole disappears and the plant-cell is left in direct
contact with the plasm, where it divides to form a layer of symbiotic
algee characteristic of this species of Paramcecium. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
28 Thus, in this case SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
283 283 at least, symbiotic forms arise through the inability of the animal pro-
toplasm to digest the plant; either can live equally well without the
other, although it is probable that there is some mutually beneficial
action between the two when together. Beyond the fact of the indi-
gestible cellulose coating, there is nothing to show why the plant-cell
is not acted upon by the dissolving fluids of the body, as is the case
when the same plant-cells are taken in by other Protozoa, and the
subject approaches very near to the time-honored but yet unsolved
problem why the stomach does not digest itself. Considerable interest attaches to the nature
of the fluid which Considerable interest attaches to the nature
of the fluid which
causes the disruption and digestion of the proteids in living proto-
plasm of plants and animals serving as food. It has been asserted
(Maupas, ’83) that the protoplasm of the prey becomes a part of
the protoplasm of the captor without further change, the implica-
tion being that there is no especial fluid created by the carnivorous
organism, as in higher animals, to digest the food. A consider-
able body of evidence has grown up, however, showing that this is
not true, and that a definite acid is formed, by means of which the
solid food particles are disintegrated and dissolved. The method of
procedure in determining this point is based upon the same principle
of differential staining as that employed by cytologists in working out
the chemical nature of the various parts of the cell in higher animals. The application, however, is very much more limited, for only those
chemical substances can be used which by zzéra-vitam application
have no deleterious effects upon the organisms. As far back as 1879,
Engelmann, observing that blue litmus granules turn red and remain
so in the protoplasm of some rhizopods and ciliates (Ameba proteus,
Paramecium aurelia, Stylonychia mytilus, and S. pustulata), cred-
ited the color-change
to an
acid in the cytoplasm. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
28 Subsequent
work by numerous physiologists has fully substantiated the results
obtained by Engelmann, and similar but more refined methods in the
hands of Meissner, Fabre-Dumergue, Metschnikoff, Greenwood, and
Le Dantec have given a firm basis for the belief that digestion of
proteids in Protozoa is similar to that in the Metazoa, but beyond the
fact that the dissolving fluid is a mineral acid, nothing further con-
cerning it is known. The bare statement of Engelmann’s view of an acid cytoplasm is The bare statement of Engelmann’s view of an acid cytoplasm is
subject to misinterpretation. While it is undoubtedly true that an
acid is present in the cytoplasm, it by no means follows that the cyto-
plasm itself is acid. On the contrary, it was soon demonstrated that
in some forms
at least (ciliates, Metschnikoff,
’88, and Meissner,
88, as well as Ameba and Actinospherium, Meissner), the cytoplasm
has a decidedly alkaline reaction while the acid is confined to the fluid
in the gastric vacuoles. Dujardin ('41) long before had noted that a THE PROTOZOA 284 considerable quantity of water accompanies the food particles, what-
ever they might be, into the protoplasm, and forms the fluid of the
gastric vacuole, and later physiologists have found that this water be-
comes acid by gradual secretion from the surrounding protoplasm. (Fabre-Dumergue, Meissner, Metschnikoff,
’89; Le Dantec, ’go).+
It thus appears, if these observations be complete, that in these cases
at least the food particles never come in direct contact with the pro- OS
PDT
AE
Te
CLO
DOE
a
a
a
Fearn
aa
RS
=U
ice
pore
BW
ESET. 28g
we
Sa
<a
Yo: Yom
ees
o
. 23238. Fd
ofe =~
;
Fig. 146. — Digestion in Reticulariida. [VERWORN.]
A, B, C, D, E, successive stages in the disintegration of a ciliate (Co/poda) (c), in a pseudo-
podium of Lieberkihnia, Fig. Fig. 146. — Digestion in Reticulariida. [VERWORN.]
A, B, C, D, E, successive stages in the disintegration of a ciliate (Co/poda) (c), in a pseudo-
podium of Lieberkihnia, toplasm, but are always suspended in the liquid of the vacuole. There
are,
however,
certain
exceptions
to
this
rule,
and
with
these in mind,
it is not yet possible to regard the subject
as
definitely established. 1Cf., however, Greenwood (’94). 1J.e Dantec (’95) does not believe that the vacuole disappears during the so-called quies-
cent phase, but is invisible merely because at this time it has the same index of refraction as
the surrounding plasm, becoming visible again after diffusion of the digested parts.
The
strength of Le Dantec’s criticism is taken away by his own observations upon Gromia fluvi-
atilis, where the ingesta is digested without the formation of a vacuole. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
28 Food-taking in certain
marine
Rhizopoda
is apparently accomplished without the formation of a gastric vacuole,
and the prey, possibly a small ciliate, disintegrates while in contact
with the plasm, and the disintegrated parts move about in cyclosis
with the endoplasmic granules (Liedberkiihnia, Gromia fluviatilis,
Fig. 146). In these cases the digestive fluids must be in any and all toplasm, but are always suspended in the liquid of the vacuole. There
are,
however,
certain
exceptions
to
this
rule,
and
with
these in mind,
it is not yet possible to regard the subject
as
definitely established. Food-taking in certain
marine
Rhizopoda
is apparently accomplished without the formation of a gastric vacuole,
and the prey, possibly a small ciliate, disintegrates while in contact
with the plasm, and the disintegrated parts move about in cyclosis
with the endoplasmic granules (Liedberkiihnia, Gromia fluviatilis,
Fig. 146). In these cases the digestive fluids must be in any and all 1Cf., however, Greenwood (’94). SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA 285 parts of the protoplasm, to be called out, perhaps, by the stimulus of
the ingested substances, and the conclusion
is obvious
that the
vacuole or improvised stomach in the various forms is not absolutely
essential. Greenwood (’94) has given a very careful description of the gastric Greenwood (’94) has given a very careful description
vacuoles in the ciliate Carchestum polypinum, one of th
Here the gastric vacuoles, after leaving the mouth
downward
into an
area
bounded
by the horse-
shoe - shaped
macronu-
cleus (Fig. 147), where
they pass into a state of
“storage” (characterized
by the loss of the water
taken in with the food),
and the food particles are
thus
left,
for the time
being, in direct contact
with
the
protoplasm. This
stage, which
may
last from one to twenty
hours, is eventually ended
by the formation
of a
vacuole again, about the
ingesta.! Preceding the
period of vacuole forma-
tion
there
is a sudden
concentration of the pe-
ripheral food particles of
the ingesta into a central,
solid
ball. This
condi-
tion—Greenwood calls it
the “aggregation” stage,
Fig. 147. — Digestion in Carchesium. aj
|
The
path which the food takes is
is
brought about,
she
P
beli
:
lottine
a (circular round marks) represents
CNEVES,
as
10
c otting,
age; 6 (crosses) represents the position
by the accumulation and _ region of the later changes. concentration of some re-
tractile substance. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
28 The fluid freshly secreted to form
vacuole has a decidedly acid reaction, and a powerful Greenwood (’94) has giv
vacuoles in the ciliate Carc
Here the gastric vacuoles,
downward
into an
area
bounded
by the horse-
shoe - shaped
macronu-
cleus (Fig. 147), where
they pass into a state of
“storage” (characterized
by the loss of the water
taken in with the food),
and the food particles are
thus
left,
for the time
being, in direct contact
with
the
protoplasm. This
stage, which
may
last from one to twenty
hours, is eventually ended
by the formation
of a
vacuole again, about the
ingesta.! Preceding the
period of vacuole forma-
tion
there
is a sudden
concentration of the pe-
ripheral food particles of
the ingesta into a central,
solid
ball. This
condi-
tion—Greenwood calls it
the “aggregation” stage,
aj
|
is
brought about,
she
beli
:
lottine
CNEVES,
as
10
c otting,
by the accumulation and _
concentration of some re-
tractile substance. The flu
vacuole has a decidedly ac Fig. 147. — Digestion in Carchesium. [GREENWOOD.]
The
path which the food takes is represented by dots;
P
a (circular round marks) represents
the position of stor-
age; 6 (crosses) represents the position of rest; ¢ (dots), the
_ region of the later changes. Fig. 147. — Digestion in Carchesium. [GREENWOOD.]
The
path which the food takes is represented by dots;
P
a (circular round marks) represents
the position of stor-
age; 6 (crosses) represents the position of rest; ¢ (dots), the
region of the later changes. THE PROTOZOA 286 upon proteids, so that in this secondary vacuole the real disinte-
gration of the food substance takes place. According to Greenwood’s observations, therefore, it appears that According to Greenwood’s observations, therefore, it appears that
the original vacuole does not become the digestive vacuole, but that
the food particles first become free in the protoplasm, to be re-collected
and digested in an acid-holding gastric vacuole. There are no observations to indicate the changes which take place There are no observations to indicate the changes which take place
in the disintegrated food particles from the time they leave the gas-
tric vacuole until the absorption, by intussusception, of the nutritive
parts contained in them. Most observers, however, are agreed that
there must be a sort of chyme formed which mixes with the proto-
plasmic fluids and is absorbed by them. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
287 crystals in Metazoa (Stein, 59; Wrzesniowski,’79; Entz,’79; Maupas,
83; Rhumbler, 88), while from the form of the crystals and chemical
reactions, Biitschli! at first regarded them as crystals of oxalic acid,
but later 2 was content to regard them as the main end product of the
metabolism of proteids in the body. While numerous observers have experimented in various ways to crystals in Metazoa (Stein, 59; Wrzesniowski,’79; Entz,’79; Maupas,
83; Rhumbler, 88), while from the form of the crystals and chemical
reactions, Biitschli! at first regarded them as crystals of oxalic acid,
but later 2 was content to regard them as the main end product of the
metabolism of proteids in the body. While numerous observers have experimented in various ways to While numerous observers have experimented in various ways to
determine the chemical nature of these questionable bodies, the most
convincing results have been obtained by Schewiakoff (’94). After
repeated experiments, many of which only confirmed the earlier con- Bog
we x
I:
f
KY *
Fig. 148.— Excretory granules in Paramacium,
[SCHEWIAKOFF.]
C. The isolated crystals. —>
—— Fig. 148.— Excretory granules in Paramacium,
[SCHEWIAKOFF.]
C. The isolated crystals. 1 Protozoa, p. 103. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
28 Closely connected with the metabolism of the protozoon cell, but Closely connected with the metabolism of the protozoon cell, but
as yet of unknown origin, are the so-called “excretory granules”
(Assimilationskorperchen
of Rhumbler,
Lxcretkorner
of Biitschli,
Schewiakoff, corpuscules biréfringents of Maupas, etc.). The wide
distribution of these granules, or crystals, their formation and disap-
pearance under varying conditions of the organisms, make it probable
that they play an important part in the physiology of the Protozoa. Whether, however, they represent a final stage in the processes of
digestion, or represent the products of katabolic metabolism, has not
been satisfactorily made out. The granules in question, with or without a definite crystalline The granules in question, with or without a definite crystalline
form, have been described in almost every class of Protozoa. In
shelled and in naked fresh-water rhizopods (Auerbach, ’55; Carter,
64; Lankester,’79; F. E. Schultze,’75; Maupas,’83; Schewiakoff,’88),
in Heliozoa (Hertwig & Lesser,’79; Maupas,’83), in Flagellidia (Biit-
schli, 78, ’83), and in Infusoria (Maupas, ’83; Stein, ’59; Wrzesniowski,
*79; Rhumbler, 88; Schewiakoff ’94), they occur in varying numbers
and positions. In the Ciliata, where they have been most thoroughly
studied, they may lie well distributed about the endoplasm (Fig. 148),
or may be concentrated
at the two ends of the animal in the
vicinity of the contractile vacuoles (Paramecium;
cf. Schewiakoff,
94). When distributed about the body, they lie in vacuoles which
move freely with the protoplasmic flow. They vary considerably in
form
(C), but, as
a rule, have
a crystalline appearance,
while
the larger granules are striated by radial or parallel lines, indicating,
Schewiakoff believes, the coalescence of needle-form crystals. They
vary in size according to the degree of aggregation, but measure, on
the average, from 0.003 to 0.014 mm. in length (Schewiakoff). Numerous suggestions as to the significance of these crystals, anda Numerous suggestions as to the significance of these crystals, anda
few valuable experiments to determine their chemical composition,
have been made. From analogy with other animals it was early sup-
posed that they represent concretions which correspond to uric acid SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
287 2 Loc. cit., p. 1484 (788). 1“ Calcium is by far the most abundant metallic element in the body. ...
It is found
in all the cells and fluids of the body, probably loosely combined with proteid.” — Howell, doc.
cit. p. 967. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
287 clusions of others, he obtained the following results: (1) the crys-
tals are insoluble, in the ordinary sense, in water; (2) slightly soluble
in concentrated acetic acid and in dilute ammonia ; (3) more soluble
in solutions
of different
salts, weak acetic
acid, and ammonia;
(4) easily soluble in mineral acids and alkalies ; (5) insoluble in alcohol,
ether, and carbon bisulphate ; (6) negative results with staining agents
showed that they can be neither albuminate nor carbohydrate in na-
ture; (7) reactions to osmic acid, alcohol, and ether excluded the
possibility of fats; (8) delicate tests showed that the crystals were
composed of an unorganized substance; (9) final tests showed this clusions of others, he obtained the following results: (1) the crys-
tals are insoluble, in the ordinary sense, in water; (2) slightly soluble
in concentrated acetic acid and in dilute ammonia ; (3) more soluble
in solutions
of different
salts, weak acetic
acid, and ammonia;
(4) easily soluble in mineral acids and alkalies ; (5) insoluble in alcohol,
ether, and carbon bisulphate ; (6) negative results with staining agents
showed that they can be neither albuminate nor carbohydrate in na-
ture; (7) reactions to osmic acid, alcohol, and ether excluded the
possibility of fats; (8) delicate tests showed that the crystals were
composed of an unorganized substance; (9) final tests showed this 2 Loc. cit., p. 1484 (788). 288 THE PROTOZOA to be a calcium salt, which was determined as calcium orthophos-
phate (Ca,H,(PO4),).? While there are many chances for error in Schewiakoff’s work, it While there are many chances for error in Schewiakoff’s work, it
is probable that he has come very near to the correct interpretation
of these bodies. Their origin, however, as well as their significance,
remains in doubt. Their disposal also has not been satisfactorily ac-
counted for. Stein reported their defecation with the undigested
remains through the anus, but Entz, Maupas, and Schewiakoff believe,
apparently on justifiable grounds, that they are dissolved and pass to
the outside through the contractile vacuole. B. RESPIRATION Until Leydig (’57) demonstrated the excretory function of the water
vascular system, it was supposed that the flatworms obtained oxygen
from a stream of water taken in by the vascular system from the out-
side, in a manner analogous to the air supply from the trachea of
insects, and Schmidt (’67), following Dujardin, and followed by Bal-
biani (’60, ’61) and Maupas (’79), attempted to explain respiration in
Protozoa in the same manner. At the present time, while the prob-
ability is very strong that the contractile vacuole expels water in which
the oxygen has been replaced by carbon dioxid, there have been singu-
larly few actual observations to confirm it. Certes’s (’85) experiments
show that some alteration takes place in the water after its entrance
into the protoplasm. This was demonstrated by placing Infusoria in
water colored by dissolved aniline dyes; the water of. the contractile
vacuole
remained
clear
and
colorless,
although the
surrounding
medium was intensely colored. The only direct observations of the
presence of carbon dioxid was made by Brandt (’81) upon Amedéa. Placing these organisms in a medium colored by dilute haematoxylin,
he found that the water of the vacuole became yellow and then red,
thus showing the characteristic
reaction of hamatoxylin
in the
presence of an acid. The observation is not conclusive, however,
for the presence of uric acid might also give this reaction. Numerous attempts have been made to describe the series of events Numerous attempts have been made to describe the series of events
which lead up to, and cause, the contraction of the vacuole. Being
entirely hypothetical, they may be dismissed with a brief mention. Schwalbe, Rossbach, Engelmann, Maupas, and many others explained
the bursting as due to the contractility of the protoplasm. Schwalbe
attempted
to trace the impulse or stimulus of contraction
to the
products
of destructive
metabolism,
which
become
stored
up in
the vacuole
so that the latter, when full, presses upon the pro-
toplasm and causes it to contract. Rossbach (’72) more obscurely
attempted
to trace the stimulus
to the chemical
change which
takes place at the moment of oxidation. Each oxidation forms an
oxidation product which, as soon
as formed, incites the stimulus. B. RESPIRATION The assumption that Protozoa take in oxygen and liberate carbon
dioxid rests almost entirely upon indirect evidence, which, however, is
so strong as to leave little reason to doubt the validity of the assump-
tion. An infusorian, for example, moving rapidly day and night
during its entire life, and eating constantly throughout this period,
must undergo continual waste in the liberation of energy by com-
bustion. A constant supply of oxygen and a constant excretion of
the waste products of combustion appear to be equally necessary for
the continuance of this activity. With remarkable intuition, Spallan-
zani (1776) suggested the contractile vacuole as the organ of respira-
tion by means of which the waste matters are thrown out, and later
observers have offered no evidence of value to disprove the suggestion. In a ciliate, for example, the volume of a contractile vacuole at com-
plete diastole is about one-tenth of the volume of the animal itself,
and, contracting every two or three minutes, the vacuole must in half
an hour expel to the outside a volume of water equal to that of the
entire animal. The oxygen-laden water which is thus expelled must
have entered the body through the mouth opening or by osmosis
through the body walls. The important réle which respiration plays
in the physiology of the Protozoa, and the agency of the contractile
vacuole in this process, was first clearly recognized by Schwalbe ('66)
and Zenker ('66), then by Wrzesniowski (’69), and later by Ross-
bach (72), Biitschli (’77), Limbach (’80), Maupas (’83), and Fiszer
(85). Before the period of Schwalbe and Zenker, the contractile
vacuole had been interpreted as a heart and the centre of a circu-
latory system (Corti, 1774; Gleichen, 1778; Wiegmann, ’35 ; Siebold,
’48), etc., and Pouchet (’64) went so far as to describe colored blood
pumped by the vacuole throughout the body. This view, which now SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
28G has but an historic interest, has recently been rather feebly advocated
by Greeff (’91) and Pénard (’90), who adopted it without the supposed
justification which the older naturalists had in comparing the contractile
vacuole of Protozoa with the water vascular system of the flatworms. B. RESPIRATION Zenker (’66) was more clear in describing the process as due to the
attraction of protoplasm for oxygen-holding water, and repulsion for
water without oxygen, the result being that when the oxygen is re-
moved from the imbibed water, the latter is expelled from the body. Rhumbler (’98) gave a similar interpretation and adduced experi-
ments
with
inorganic
fluids
simulating
the contractile
vacuole. Biitschli (’83) regarded the systole as
due to a simple physical
- - THE PROTOZOA 290 force, —surface tension. The liquid of the vacuole mixes quickly
with the surrounding water, as a small drop fuses with a larger mass,
as soon as the intervening layer of protoplasm gives way before the
pressure of the growing vacuole. Delage and Hérouard (’96), on
the other hand, held that contractility of the protoplasm brings about
the contraction just as it causes the expulsion of undigested food
matters. When present, carbon dioxid is probably dissolved in the water of
the contractile vacuole, although, in some cases, especially among the
Sarcodina, gas vacuoles containing, probably, carbon dioxid have been
repeatedly observed, first by Perty (’49) in Avce//a, and subsequently
by Biitschli (’74), Engelmann (’78), Entz (’78), and others, not only
in Avcella, but in other rhizopods as well. According to Engelmann,
the gas is secreted very rapidly, but at irregular intervals, and he, with
other modern observers, accepted and confirmed the suggestion made
by Perty, that the gas vacuoles serve as an hydrostatic apparatus, by
means of which the organisms can raise and lower themselves in the
water. Although the contractile vacuole appears to play an important réle Although the contractile vacuole appears to play an important réle
in respiration, it is not absolutely necessary for the performance of
this function, for a great many forms have no such organ. The ma-
rine rhizopods and Radiolaria, and some of the fresh-water forms of
Rhizopoda (¢.g. Pelomyxa), have no contractile vacuoles. Respira-
tion in such cases must take place by osmosis. An interesting series
of forms are the Opalinidz, parasitic ciliates of which some genera
have a contractile vacuole with numerous feeding canals (Azoplophrya,
Hloplitophrya), while others have no contractile vacuole, although the
canals are present (Ofalina, according to Fabre-Dumergue). In
none of these forms is there a mouth, and respiration must take place
by osmosis. B. RESPIRATION There is, on the whole, very little direct evidence to support the There is, on the whole, very little direct evidence to support the
conclusion which on @ przore grounds appears indisputable, that, like
other organisms, the Protozoa take in oxygen and give off carbon
dioxid. The fragmentary evidence which we have tends to the con-
clusion that, when present, the contractile vacuole, probably in addi-
tion to other excretory functions, is the active agent in the disposal of
carbon dioxid, while the income of oxygen-holding water takes place
by osmosis through the body walls, by ingulfing through the mouth,
or by both methods. C. SECRETION AND EXCRETION
s but one of the waste matters form Carbon dioxid is but one of the waste matters formed by the decom-
position of proteids in vital activities. In the higher animals the final SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
291 products are carbon dioxid and urea (CO(NH,),), and it has long been
assumed upon @ priovz grounds that a similar result follows combus-
tion in Protozoa. Again, there have been but few observations to
confirm this supposition. The contractile vacuole was regarded as an
excretory vesicle (Urizdlase) by Boeck ('47), Rood (’53), Stein (56),
Leydig (57), Kolliker (64), and more recently as an excretory organ
by Maupas (’83), Rhumbler (’88), Griffiths (89), Schewiakoff (’94),
Delage and Hérouard (’96), besides many others. Biitschli! believed
that it is a pure hypothesis to assume that the vacuole has an excre-
tory function other than that of respiration, but Maupas (’83) insisted
upon the physiological necessity of such an excretory organ, and cited
as an argument the presence of contractile vacuoles in vegetable
zoéspores, which, having chlorophyl, can presumably make use of all
the carbon dioxid formed, and which, therefore, probably make use of
the vacuole for secretion. Maupas’s argument is offset by the fact of
numerous Protozoa which have no contractile vacuoles, and it follows
that if these can get rid of their waste organic matters by osmosis, it
is quite possible that forms with vacuoles candothe same. Entz(’88)
held that the crystals occasionally found in the vacuoles and reser-
voirs of different forms are uric acid (arnconcremente), a supposition
which was supported with direct evidence by Griffiths (89). The lat-
ter determined the presence of uric acid in several different types of
Protozoa, including the rhizopod Amada and the ciliates Paramecium
and Vorticella. A number of animals were placed on a slide under a
cover-glass, and killed with alcohol followed by nitric acid. The
slide was then gently warmed, and ammonia was introduced. When
the experiments were successful,
a number of purple prismatic crys-
tals of murexide appeared in the contractile vacuoles, showing that
uric acid had been present. These results were repeatedly obtained,
although the experiments were not always successful, showing, Grif-
fiths says, that the vacuole may have some other functions besides
secretion. Until this interesting series of experiments is confirmed,
however, Griffiths’s results must be inconclusive. 1°88), p. 1452. 2 Loc, cil. p. 135+ C. SECRETION AND EXCRETION
s but one of the waste matters form The materials
thus secreted vary in nature from purely inorganic solids, like calcium
carbonate, silica, etc., to chitin, cellulose, fats, and jelly-like protoplas-
mic products. The simplest cases of secretion are seen in those
Mastigophora and Sarcodina where the outer protoplasm becomes
gelatinous, to form the jelly-like mantles of different types (many
Flagellidia, Heliozoa, Radiolaria). In Ameéa there is a secretion of
such a substance which aids the animal in securing food by sticking
it to larger objects, as well as by ensnaring the prey (Rhumbler, Ver-
worn, Hofer). In Luglena, according to Klebs (’86), the protoplasm
throws out a slimy mantle when the surrounding conditions are un-
suitable. This mantle at first is not homogeneous, but is in the form
of minute gelatinous threads which arise beneath the cuticle from the
outer protoplasmic layer of the body. A network is then formed be-
tween the threads, which finally unite to form a homogeneous man-
tle about the animal. An identical process has been described by
Schewiakoff (’94) and by Siedlecki (’99), in the movements of certain
gregarines,! where a gelatinous layer beneath the membrane secretes
filaments of slime-like material which harden outside the body. In other instances the secreted material is in the form of granules In other instances the secreted material is in the form of granules
which unite outside the body to form stalks or houses, or even shells. In many such cases the granules have not been a part of the body
protoplasm, although they may have been created there. Such, for
example, are the lime shells of the’ Reticulariida, or the silicious and
acanthin skeletons of the Radiolaria. In other cases they appear to
be a growth product of the organism, as in the branched stalks of
many Flagellidia, although even here foreign particles may be used
for this end. Thus, in freshly formed Azthophysa stalks, Kent
and Biitschli observed that the excreted material was granular, and
Ehrenberg had already noticed that, if Azthophysa colonies are fed
with indigo, the colored particles collect at the base of the animal,
while Kent (’81), repeating the experiment, observed that the gran-
ules were actually deposited to form a part of the new stalk material. The materials for the shells of Rhizopoda may be foreign particles
analogous to the indigo granules, or they may be the result of chem-
ical activity of the protoplasm. 1Cf p. 149. C. SECRETION AND EXCRETION
s but one of the waste matters form If they are confirmed,
on the other hand, the following reflection is warranted, and has a sin-
gular interest in the present-day problems of biology: “ Through all
the multitudinous changes,” says Griffiths, “that have taken place dur-
ing the lapse of ages in the development of the mammalian kidney, we
find that the physiological functions are the same as occur in its origi-
nal or primitive form, as represented in the Protozoa.” ? The secretion from the protoplasmic body of definite particles of
matter, which may or may not have been at some time a part of the
animal protoplasm, and which play some further part in the life activi- The secretion from the protoplasmic body of definite particles of
matter, which may or may not have been at some time a part of the
animal protoplasm, and which play some further part in the life activi- 2 Loc, cil. p. 135+ THE PROTOZOA 292 ties, is definitely established in a number of cases. The materials
thus secreted vary in nature from purely inorganic solids, like calcium
carbonate, silica, etc., to chitin, cellulose, fats, and jelly-like protoplas-
mic products. The simplest cases of secretion are seen in those
Mastigophora and Sarcodina where the outer protoplasm becomes
gelatinous, to form the jelly-like mantles of different types (many
Flagellidia, Heliozoa, Radiolaria). In Ameéa there is a secretion of
such a substance which aids the animal in securing food by sticking
it to larger objects, as well as by ensnaring the prey (Rhumbler, Ver-
worn, Hofer). In Luglena, according to Klebs (’86), the protoplasm
throws out a slimy mantle when the surrounding conditions are un-
suitable. This mantle at first is not homogeneous, but is in the form
of minute gelatinous threads which arise beneath the cuticle from the
outer protoplasmic layer of the body. A network is then formed be-
tween the threads, which finally unite to form a homogeneous man-
tle about the animal. An identical process has been described by
Schewiakoff (’94) and by Siedlecki (’99), in the movements of certain
gregarines,! where a gelatinous layer beneath the membrane secretes
filaments of slime-like material which harden outside the body. In other instances the secreted material is in the form of granules ties, is definitely established in a number of cases. C. SECRETION AND EXCRETION
s but one of the waste matters form The various shells of Diflugia, Cen-
tropyxts, Cyphoderia, Lithicolla, etc., are examples of the first type,
while Euglypha, Quadrula, most Heliozoa, Reticulariida, and Radio-
laria are examples of the second. In the Sarcodina, where the shells, as in Reticulariida, are formed
by deposition of calcium carbonate, it has been shown by Carpenter,
Kolliker, Wallich, and especially by Dreyer (’92), that the material 1Cf p. 149. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
293 PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA 293 is deposited from a well-defined portio
Wallich), and not upon the outside o
chitinous
lamellze
situated
in the
ectoplasm (Fig. 149). The manufac-
ture
of the shell
material
of the
Mollusca
was
explained
by Stein-
mann as a purely chemical phenome-
non, and not as an expression of vital
activity. The early experiments of
Harting (’73) in allowing carbonic
acid alkalies to act upon albumin or
other nitrogenous substances, there-
by obtaining a precipitate of calcium
carbonate in the form of granules
similar to Coccoliths, gave Steinmann
the clue to his theory that calcium
chloride and other salts acting upon
albumin in animal protoplasm give a
similar
precipitate. Applying
this
theory
to
the
marine
rhizopods,
Dreyer argued that the protoplasmic
body is saturated with dissolved cal-
cium salts, and that albuminoid stuffs
secreted by the living animal undergo
fermentation through the agency of
bacteria, and ammonium carbonate is
formed, wha acting i eage Epe eal
cium salts, results
in the formation
of calcium
carbonate! <A similar
explanation
can
be given
for the
diverse skeletons of the Radiolaria, the
the interstices of the protoplasmic al
the four-rayed type of spicule, and t
(Fig. 41, p. 77):
Among the most interesting materi
are those which give rise, through rapid Fig. 149. — Shell formation in Gromia
fluviatilis Daj. [DREYER,]
J, the deposit between two lamella. Fig. 149. — Shell formation in Gromia
fluviatilis Daj. [DREYER,]
J, the deposit between two lamella. Among the most interesting materials secreted by the Protozoa
are those which give rise, through rapid oxidation, to phosphorescence,
A great many forms have this power, especially among the Dino-
flagellidia and the Cystoflagellidia; and although the cause is not fully
established, yet it is generally agreed, at the present time, that the
light is due to the rapid oxidation of certain products of metabolism. 1Cf. Dreyer, p. 224. C. SECRETION AND EXCRETION
s but one of the waste matters form One of the most noteworthy illustrations of phosphorescence is shown
by Noctiluca miliaris, in which Quatrefages (’50) found that the light, THE PROTOZOA 204 which appears homogeneous under low magnification, is, in reality,
caused by a vast number of minute light centres. These are closely
grouped together in the centre of the light area, but are easily distin-
guished at the edges (Fig. 150). Radziszewski (’80) found that a
number of different substances which are present in living organisms
have this power of phosphorescence when in an alkaline medium. which appears homogeneous under low magnification, is, in reality,
caused by a vast number of minute light centres. These are closely
grouped together in the centre of the light area, but are easily distin-
guished at the edges (Fig. 150). Radziszewski (’80) found that a
number of different substances which are present in living organisms
have this power of phosphorescence when in an alkaline medium. Fig. 150. — Phosphorescence in Noctiluca miliaris Sur. [QUATREFAGES.]
A portion of the body is represented with numerous scintillating dots. Fig. 150. — Phosphorescence in Noctiluca miliaris Sur. [QUATREFAGES.]
A portion of the body is represented with numerous scintillating dots. Amongst them are fat, lecithin, cholestearin, certain oils, grape sugar,
etc. Watasé (’98) regarded the protrusion or secretion of such sub-
stances as due to protoplasmic contraction induced by any external
stimulus, such as a blow or shock of any kind. D. IRRITABILITY When, by reason of an external stimulus, any normal movement of
a protozodn is interrupted, or changed to another type of motion
(which may be definite or indefinite in regard to the source of the
stimulus), the change is brought about by protoplasmic reaction,
which is expressed by the general term z777tadbz/ity. The effect of heat upon the activities of Protozoa was early recog- The effect of heat upon the activities of Protozoa was early recog-
nized by the students of the group. Spallanzani, for example, noted
that increased warmth brought about increased activity in the con-
tractile vacuole, as well as in movements of the animal. Subsequent
observers have established the fact that movement is possible only
within certain temperatures, and, further, that this limit in either
direction varies with the organism considered. Between such limits,
the amoeboid movements of Rhizopoda and the vibratile movements. of Flagellidia and Ciliata increase to a certain optimum, which again
varies with the organism, but after this optimum is passed, movement
gradually decreases until, at the point of maximum temperature, heat
rigor sets in; and this temperature, if passed, results in death. On
the other hand, with cooling, the movements become slower and
slower, until a minimum temperature is reached and all motion ceases, SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
295 after which, with further decrease in temperature, cold rigor and
death ensue. In spite of these limitations, however, the Protozoa
have the power of adaptation to high temperatures on the one hand,
and low temperatures on the other. Ehrenberg cites the presence of
Nassula, Enchelys, and Amphileptus in the waters of the Ischian hot
springs, which reach a temperature far above the normal for most
Ciliata and Flagellidia, while the presence of Hematococcus on high
mountains and in polar regions, or of various Flagellidia in frozen
pools, is common knowledge. The maximum temperature for com-
mon Flagellidia varies usually between 40° and 50° C. (Schultze, 63 ;. Strasburger, 78; Klebs, ’83), although Dallinger cites a number of
common forms which live until 60° is reached, while the spores of
these forms withstand temperatures far beyond the boiling point. With Rhizopoda, the maximum temperature seems to be somewhat
less than with Flagellidia. Kihne (’64) found the maximum for
Ameba diffiuens and Actinospherium at 45° C., and similar results. were obtained by Schultze (63) and Verworn (’89). D. IRRITABILITY The Ciliata also havea generally lower maximum temperature than The Ciliata also havea generally lower maximum temperature than
do the Flagellidia, although important exceptions are found where, by
adaptation to new conditions, the organisms are able to withstand a
considerable temperature. The common forms stand a temperature
of from 38° to 42° C., and the activity of the cilia resembles that of
ciliated
epithelium described by Engelmann
('79). According
to:
Rossbach’s description of the increase of heat upon the activity of the
ciliate Stylonychia, 4° marks the lower limit of motion when the animal
is nearly at rest, above 25° the movements become violent, although
still spontaneous and normal, while from
30° to 35° the motion
becomes uncertain and the animals lose their power of direction, a
condition which gives place to a violent rotation about the axis until
death finally ensues. In connection with the general stimulation of
the body by heat, there is frequently a definite movement of the
organism toward or away from the source of the stimulus (¢hermo-
taxis),
This seems to have been first observed by Stahl (’84) in con-
nection with the plasmodia of Athalium septicum. He placeda
strip
of filter paper, upon which a plasmodium had flowed, between two
beakers in such a way that one end of the plasmodium lay in cold
water (7°), while the other lay in warm water (30°). The streaming
of the plasm began at once toward the warmer water until the plas-
modium was completely immersed, although previously the movement
had been in the opposite direction. Verworn ('89) observed a similar
thermotactic phenomenon
in Amaba imax, while, more recently,
Mendelssohn (95) has shown that, in the Ciliata, Paramecium seeks
warmer water in temperatures below 24° to 28°, while in temperatures. above this the reverse reaction takes place. 2096 THE PROTOZOA Light as well as heat rays frequently have a similar directive
effect upon Protozoa, a phenomenon called phototaxts by Strasburger,
which is explained as follows by Verworn: “A ray of light extends
through space from a source of light, in a straight direction, and
diminishes in intensity with the distance, hence any two points in
the line of the ray possess different intensities; the point that is
nearer the source has the greater, that which is farther away has the
less, intensity. 1 Lee, 1900, p. 447.
2 The slight effect of light upon Ciliata is shown by the following table from Maupas
(788).
The organisms were kept for one month in the light and one month in darkness,
and during that time they increased by division as fullows: —
Colpidium colpoda
in darkness 48 times;
in light 46
Glaucoma scintillans
se
es
98
«
&
99
Parameecium bursaria
“
“
9
«
“6
9
Stylonychia pustulata
“
“
48
«
“
«59 D. IRRITABILITY A ray of light, therefore, fulfils very completely the
conditions that are necessary to the appearance of unilateral stimula-
tion; in fact, it is extremely difficult to establish conditions under
which an organism is stimulated by light uniformly on all sides. As
a result of this, stimulation by light calls out very pronounced direc-
tive effects.” 1! A difference of opinion exists, however, as to the
general effect of light upon organisms; Klebs (’85), on the one hand,
asserted that all protoplasm is sensitive to light, while Verworn, after
trying in vain to get reactions on the part of certain organisms, con-
cluded that such is not the case. It appears from numerous investi-
gations that the most phototactic forms are the flagellated
cells,
Strasburger (’78) observing that swarm-spores of different plants are
attracted toward a light of a certain intensity, and move away from
alight of greater intensity, while he and Cohn (’64) determined the
fact that rays having a short wave-length, especially the blue and the
violet, are more effective than those of the longer wave-length, such
as the red. Engelmann (’82) saw Euglena gather in heaps along the
region of the line F of a microspectrum, and he also established
the fact that colorless forms, such as Chilomonas paramecium, or the
colorless swarm-spores of the Chytridize, are positively phototactic in
weak, and negatively phototactic in strong, light. Among the Infusoria, light reactions are much less marked than Among the Infusoria, light reactions are much less marked than
among the Mastigophora, and light seems to be quite unnecessary for
their existence.? Nevertheless, in one form, at least, positive reactions
have been demonstrated by Verworn (’89), who showed that Plew-
ronema chrysalis reacts vigorously to light stimuli through cobalt
glass, while light through red glass gives no effects, an experiment
which shows that it is not the longer but the shorter light vibrations
that are effective. The results with Rhizopoda have been somewhat more definite The results with Rhizopoda have been somewhat more definite 1 Lee, 1900, p. 447. 2 The slight effect of light upon Ciliata is shown by the following table from Maupas
8). D. IRRITABILITY The organisms were kept for one month in the light and one month in darkness,
during that time they increased by division as fullows: —
Colpidium colpoda
in darkness 48 times;
in light 46
Glaucoma scintillans
se
es
98
«
&
99
Parameecium bursaria
“
“
9
«
“6
9
Stylonychia pustulata
“
“
48
«
“
«59 SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
297 than with the Ciliata. Pelomyxa palustris, according to Engelmann
(79), moves energetically in darkness, but if a strong light be sud-
denly thrown upon it, a sudden contraction results and the organism
rounds out into a spherical mass; but if light gradually increases,
there is no response. Rhumbler (’98) came to a somewhat similar
conclusion in regard to Ameba proteus, holding that food-taking is
more energetic in the night than during the day, and is not frequently
observed because the intense light of the microscope mirror causes
sluggishness. The experiments of Leaming and Harrington (’99)
upon the effect of light of different colors shows that Amada proteus
reacts vigorously in red lights, but protoplasmic movements almost
cease in rays from the violet end of the spectrum. Living protoplasm can also, than with the Ciliata. Pelomyxa palustris, according to Engelmann
(79), moves energetically in darkness, but if a strong light be sud-
denly thrown upon it, a sudden contraction results and the organism
rounds out into a spherical mass; but if light gradually increases,
there is no response. Rhumbler (’98) came to a somewhat similar
conclusion in regard to Ameba proteus, holding that food-taking is
more energetic in the night than during the day, and is not frequently
observed because the intense light of the microscope mirror causes
sluggishness. The experiments of Leaming and Harrington (’99)
upon the effect of light of different colors shows that Amada proteus
reacts vigorously in red lights, but protoplasmic movements almost
cease in rays from the violet end of the spectrum. Living protoplasm can also, within Living protoplasm can also, within a certain limit, accommodate
itself to chemical changes in the surrounding medium. Thus, Am@ebe
may become accustomed to a 4 per cent solution of common salt,
if it be slowly added, while a 1 per cent solution, if added suddenly,
will kill them. Heliozoa can be transferred from salt water to fresh,
and vice versa. D. IRRITABILITY Both organisms collect in a ring around a bubble
of air, the ciliate nearer the source, the bacillus away from it. Carbon
dioxid is said to give the same reaction as dilute acids, causing the
aggregation of Paramecium about it (Jennings), although other ob-
servers have been unable, thus far, to confirm these experiments. The singularly interesting investigations which Pfeffer (84, ’88) The singularly interesting investigations which Pfeffer (84, ’88)
made upon the chemotaxis of reproductive elements have thrown no
little light upon the problems of fertilization, and have shown at the
same time how minute a quantity of the stimulant is needed to give
the required effect, too much: causing a negative result. He found
that antherozoids of the fern are attracted to the capillary tube, which
holds a slight trace of malic acid, while a larger quantity turns them
away. Carrying the experiment to other forms of life, it is probable
that analogies to this may be found in almost all kinds of organisms,
both animal and plant, and that positive chemotaxis is a necessary
condition of the fertilization of many eggs by spermatocytes. Jennings’s (’99) interesting experiments and observations upon Jennings’s (’99) interesting experiments and observations upon
various forms of Infusoria and Mastigophora show that many hith-
erto supposedly directive chemical stimuli are effective, not because of
their unilateral stimulation through the differences in density, but
because they induce a natural motor response on the part of the organ-
ism. To illustrate with the case of Paramecium, which affords the
most satisfactory basis for this view, Jennings maintains that these
organisms, which for a long time have been known to have an appar-
ent affinity for weak acids, are not attracted by a drop of acid placed
in the vessel which contains them, but wander into it in their aimless
movements. The usual motor response of Paramecium is to swim
backward from the object struck, turn toward its side containing the
peristome, and swim forward again. By this manceuvre an ordinary
obstacle is avoided. The acid does not cause this reaction, but when
the organism attempts to leave the drop, contact with the surrounding
walls brings about the motor response, and the organism thus remains
entrapped (Fig. 151). D. IRRITABILITY dthalium septicum will live in a 2 per cent solution
of sugar if slowly added, and many other instances might be given. Such adaptation cn the part of the organism signifies a gradual change
in the chemical or physical make-up of its own substance, and in a
variety of cases the rapidity of the change varies inversely as the
distance from the source, hence as the intensity, of the stimulus. If
a stimulus, therefore, comes from a certain direction, the effect, like
that of a light stimulus, is a unilateral reaction, to which the general
term chemotaxis
has been given (Verworn). The greatest variety
of substances may give these reactions, water, air, and other gases,
alkalies, and acids of various kinds. Pfeffer (’88) found that, in
various Flagellidia, including Cry/fomonas, several species of Bodo,
Monas guttula, Trepomonas, Polytoma, Euglena, etc., different sub-
stances cause very different reactions, —substances which cause posi-
tive reactions in some forms causing negative reactions in others. He
also obtained the interesting result that substances which in weak
solutions cause a positive reaction, in strong solutions cause a nega-
tive one, so that, as in light stimuli,
a chemotactic optimum exists
toward
which
the
organism
constantly
strives. In Rhizopoda,
although fewer instances of chemotaxis are known, the same general
results have been obtained. Stahl’s often cited experiments on the
mycetozoan .2thaltum septicum showed that this organism is posi-
tively chemotactic toward both harmful and beneficial stuffs. In all
cases of harmful action upon the Rhizopoda, the reaction is expressed
by the withdrawal of the pseudopodia, rounding out of the body, and
final disintegration. With the Ciliata, according to Pfeffer, chemo-
tactic reactions appear to be less delicate than with the Mastigophora. 298 THE PROTOZOA Oxygen, as with most organisms, exercises
a chemotactic effect,
although the positive are much more rare than negative effects
(Verworn and Jennings). Jennings, in a series of very careful ex-
periments, found that alkalies give clearly marked negative chemo-
taxis, while substances
“in which Paramecia
collect,
giving the
motor reaction only when they attempt to pass out of them, are
substances having a weak acid reaction” (’99). The relative effect
of the same stimulus upon different organisms is prettily shown
by Massart’s (’91) experiments upon Spiri//um and Anophrys, in the
presence of oxygen. D. IRRITABILITY Rosenhof (1755) observed
that Ameba becomes globular upon being shaken, an observation
which was followed by numerous experiments by De Bary on Myce- THE PROTOZOA 300 tozoa, by Haeckel (’70) on Rhizopoda and Radiolaria, and by Verworn
('89), who demonstrated on a number of Rhizopoda that stimuli of
different grades of intensity cause different degrees of reaction, and
that a stimulus applied at the end of a pseudopodium causes only
a local irritation expressed by contraction
at that point, and he
expressed the belief that a regular graduated scale of irritability
could be established for this group. Many forms show almost no
reaction to slight stimuli, others a distinct contraction. Among the Mastigophora and Infusoria, not only is the body more Among the Mastigophora and Infusoria, not only is the body more
easily affected by mechanical stimuli, but transmission
of the stimulus
is far more rapid than in the Sarcodina. Impact of a ciliate with any
object, possibly an organism much smaller than itself, causes a vig-
orous reaction expressed by sudden change of motion (Flagellidia,
hypotrichous and holotrichous Ciliata), by contraction of myonemes
(Stentor,
Vorticellide,
etc.),
or
by body contractions
(£zg/ena,
Spirostomum, etc.), all of which may be expressed by Jennings’s term
motor response. A general result of mechanical stimulation is a motor response fol- A general result of mechanical stimulation is a motor response fol-
lowed by the tendency to turn away from the source, and the general
reaction, whether positive or negative, since it deals with the question
of pressure in some form or other, is called davotaxis (Verworn). In all forms of barotaxis, e.g. in ¢higmotaxis, the reactions may be In all forms of barotaxis, e.g. in ¢higmotaxis, the reactions may be
either positive or negative. Negative thigmotaxis, the ordinary reac-
tion to mechanical stimuli, such as pricking or crushing a
local area, is
expressed by movement away from the source of irritation. Positive
thigmotaxis
is expressed by movement toward the source of the
stimulus. The latter is commonly seen in the attachment of pseudo-
podia of Asda and other fresh-water Rhizopoda to solid bodies, and
it affords an explanation of the ingulfing of solid bodies (¢,g. food par-
ticles). Dewitz (’86) found positive thigmotaxis on the part of sper-
matozoa of the cockroach, a discovery of the greatest interest in view,
again, of the question of fertilization. D. IRRITABILITY While this ingenious view is acceptable in the
case of Paramecium, it by no means follows that all so-called chemo-
tropic or chemotactic
effects can be similarly explained, and that SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
299 chance brings the organism in the sphere of influence of the stimulant. On the other hand, it is equally probable that many of the so-called
directive effects may be explained by this assumption, and extreme
caution is needed in interpreting all such reactions. Garry (’00)
has applied the distinction, first suggested by Loeb ('93), to be used
in interpreting the responses of organisms to stimuli; if a directive
effect is produced (that is, if the organism is distinctly oriented in
respect to the centre of the stimulus), the effect is said to be chemo-
tropic, but if an effect is produced which is not directive, it is said to
be chemokinetic (Uuderschiedsempfindlich, Loeb). According to Jen-
nings, the reaction of Paramecium would be neither chemokinetic nor
equivalent to the unilateral stimuli induced by light or heat (photo-
taxis and thermotaxis). Fig. 151.— Motor response in Paramecium. [JENNINGS.]
a-f. Successive positions after meeting with an obstruction 4. Param Fig. 151.— Motor response in Paramecium. [JENNINGS.]
a-f. Successive positions after meeting with an obstruction 4. The various instances which have been described of positive and
negative chemotaxis must be reinvestigated in the light of Jennings’s
conclusions, for it is obvious that the mere collection of organisms in
a substance does not necessarily indicate the directive effect of that
substance. Nevertheless, in some cases such an effect appears to be
definitely established. Garry showed that certain organic acids have
a positive directive effect upon the flagellate Chz/omonas paramecium,
while various other acids, alkalies, and salts have merely a chemo-
kinetic effect. He also obtained results which seem to indicate that
it is the ions dissociated in certain solutions which are capable of
stimulating the organism. These observations open out
an
unex-
plored
field
for investigation
and promise extremely interesting
results. Irritability on the part of the Protozoa, in response to mechanical Irritability on the part of the Protozoa, in response to mechanical
Stimuli, has long been known. Résel v. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA 301 surface toward the posterior end.!_
In some forms, ¢.¢. Paramecium
and the majority of Ciliata, and different species of Ameba, mak-
ing the current results in active and direct movements toward the
kathodic electrode ; in others, e.g. the flagellate Polytoma, the reverse
reaction takes place, the organisms swimming vigorously toward the
anode, and still a third reaction may be observed in Spirostomum
ambiguum, which turns at right angles to the direction of the current
and remains so (Verworn, ’89). From the various experiments which have been made upon Proto- surface toward the posterior end.!_
In some forms, ¢.¢. Paramecium
and the majority of Ciliata, and different species of Ameba, mak-
ing the current results in active and direct movements toward the
kathodic electrode ; in others, e.g. the flagellate Polytoma, the reverse
reaction takes place, the organisms swimming vigorously toward the
anode, and still a third reaction may be observed in Spirostomum
ambiguum, which turns at right angles to the direction of the current
and remains so (Verworn, ’89). From the various experiments which have been made From the various experiments which have been made upon Proto-
zoa,
it appears
that more or less similar motor reactions follow
different kinds of stimuli, and that the organism reacts similarly to
the same stimulus. With each type of stimulus there is, in most
cases, an optimum of intensity below which a positive reaction is
observed, but which, if exceeded, is followed by a negative reaction,
while over-stimulation results in no response or in death. Further-
more, the experiments show that each type of organism behaves in
its own way, a given stimulus violently affecting some species, while
others are unaffected, although different individuals
of the same
species always react
similarly. The directive
effects
of certain
stimuli through operation in certain lines or on certain sides giving
rise to unequal stimulation of the protoplasm, induce reactions which
again are invariable. The thermotactic response of Amebda, or the
phototactic reactions of Eug/ena, can be predicted with almost
as
much
certainty
as the reaction
of mercury
to
heat. In some
instances, notably in the more complicated forms, it appears that the
organism as a whole is endowed with a set of motor responses which
might be identified as instinctive. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA Stimulation at one point induces
not a local response, as in Rhizopoda, but a reaction of the entire
organism, which is poorly explained by the assumption of a machine-
like organization of the cell, or by the statement that these responses
are merely the expression of chemical and physical forces. 1 Loc. cit., p. 123. D. IRRITABILITY The reactions to galvanic currents are, as a rule, quite definite, and The reactions to galvanic currents are, as a rule, quite definite, and
as in iron
filings over a magnet, there is usually a definite polar
arrangement of the single-celled organisms, a reaction expressed by
the term ga/vanotaxis. Pearl (’00), examining the reactions to gal-
vanic stimuli in several ciliates and in the flagellate Chzlomonas para-
mecium in the light of Jennings’s theory of motor responses, came to
the conclusion that the usual reaction is distinctly affected by the
current so that a “forced movement” in many cases is superimposed
upon the normal motor response. The forced movement in ciliates,
he states, is due to the action of certain cilia which are constrained
by the current to lie in certain definite positions, —those on the
cathode surface pointing toward the anterior end, those on the anode E. GENERAL CONSIDERATIONS All of the normal functions of the protozoan cell show themselves
second in complexity only to the analogous processes which take
place in the Metazoa, and, as with the latter, many of them
still
remain unexplained. One point, nevertheless, has been very thor-
oughly demonstrated, vzz. the importance of the nucleus in the main-
tenance of many of these functions. Brandt (’77) was the first to
observe that non-nucleated fragments of Actznospherium
will die,
while nucleated parts will live, and his observations were made the 1 Loc. cit., p. 123. THE PROTOZOA 302 basis of a long series of experiments by Nussbaum (’84, ’85), Gruber
(86), Balbiani (’88), Hofer (89), and Verworn (’88, ’89, ’91), which
have led to fruitful results. Amongst these, the most striking and
suggestive fact is, that without the nucleus, the process of digestion
cannot take place in any form of Protozoa. The beautiful and clear-
cut experiments of Hofer (’89) and Verworn (’91) have demonstrated
beyond a doubt that the digestive fluid is not prepared in the cyto-
plasm when the nucleus is absent. Hofer demonstrated that the
slimy secretion which Amada throws out to anchor itself ‘before food-
taking is never formed by the enucleated portions, and Verworn (’88)
proved that enucleated pieces of Polystome//a could not repair or regen-
erate the lost shell, while nucleated pieces quickly repaired it. Ver-. worn came to the conclusion, which seems to be demonstrated, that
enucleated protoplasmic masses cease entirely those chemical pro-
cesses by which products of the normal cell are used or formed. In-
deed, the generalization may now be made that no secretion takes
place in enucleated fragments. On the other hand, the nucleus by
itself, ze. separated from the cytoplasm, has no longer the power to
regenerate the lost parts, and like the enucleated cytoplasm, soon dies. “The nucleus needs the plasm, the plasm the nucleus,” says Biit-
schli, ‘the activities of both are reciprocal, and one without the other
cannot live,’ a generalization confirmed by Verworn’s conclusive
experiments (’91). The processes of secretion, therefore, whether for
the purpose of digestion or for any other purpose in the life of the
unicellular organism, are expressed by the constant chemical inter-
change which goes on between the cytoplasm and the nucleus. 1(’88), p. 1642. E. GENERAL CONSIDERATIONS Outside of secretion and reproduction, however, the usual functions Outside of secretion and reproduction, however, the usual functions
of motion, and those of the contractile vacuole, are performed nearly
as well by the enucleated as the nucleated fragments. Balbiani (’88)
observed that non-nucleated pieces of infusoria, while unable to regen-
erate the lost parts, were nevertheless capable of limited motion, and of
living and swimming about actively for several days, while the contractile
vacuole continued its rhythmic pulsations. Hofer (’89) also observed
that non-nucleated pieces of Amba proteus will form pseudopodia
and live for some time after the operation in active motion, a point
which Verworn established in a striking manner in the case of Tha-
lasstcolla nucleata. ere the central capsule, after extirpation of the
nuclei, forms a spherical mass which soon begins to assume the form
of a typical radiolarian. The vacuolated portion, however, only begins
to appear when degeneration sets in and the animal dies (Fig. 152). That the enucleated portions require oxygen and can use oxygen is
shown by Verworn’s negative experiments with enucleated Infusoria in SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA 303 an oxygen-free medium. Enucleated pieces in the ordinary medium
will live, but those in the oxygen-free medium quickly die, showing
that oxygen-taking is possible without the nucleus. So, too, the con-
tractile vacuole continues to pulsate in Ameéa even nine days after
the operation which separated it from the nucleus (Hofer). The nucleus, furthermore, appears to have nothing to do directly
with the functions of irritability, for non-nucleated fragments respond
as readily to stimuli as do the nucleated parts. They show the same
chemotactic, thermotactic, and galvanotactic reactions as the complete
organisms, and Eimer’s view that the nucleus is the seat of psychic
activity as expressed by motion, is flatly contradicted (Verworn, ’89). Perty (52), as well as Dujardin (’41), long since questioned the intel- A
Fig. 152. — Isolated nucleus of Thadassicolla nucleata Hux. [VERWORN.]
A, B, C, D. Regenerative changes. £, F, G, A. Degenerative changes. Fig. 152. — Isolated nucleus of Thadassicolla nucleata Hux. [VERWORN.]
A, B, C, D. Regenerative changes. £, F, G, A. Degenerative changes. ligent power of the Infusoria in their reactions to stimuli, and more
modern observers have generally accepted the view that the proto-
plasm of these animals responds automatically, in greater or less
degree, to stimuli of various kinds. E. GENERAL CONSIDERATIONS In the Sarcodina, the entire cell
is equally irritable ; but higher in the scale, the outer plasm becomes
more and more sensitive in relation to the entire body until, in the
membranes of Infusoria, a generally sensitive skin is found which is
sometimes drawn out into so-called
sensory processes (feelers and
tasters of hypotrichous forms), while in the Mastigophora special
sensory or receptive spots are frequently present (“‘eye-spots””). This
tendency to localization of the sensitive parts of the protoplasm is
only in accord with our a@ priori conceptions of differentiation brought
about by reaction to the environment or adaptation, and is strictly ligent power of the Infusoria in their reactions to stimuli, and more
modern observers have generally accepted the view that the proto-
plasm of these animals responds automatically, in greater or less
degree, to stimuli of various kinds. In the Sarcodina, the entire cell
is equally irritable ; but higher in the scale, the outer plasm becomes
more and more sensitive in relation to the entire body until, in the
membranes of Infusoria, a generally sensitive skin is found which is
sometimes drawn out into so-called
sensory processes (feelers and
tasters of hypotrichous forms), while in the Mastigophora special
sensory or receptive spots are frequently present (“‘eye-spots””). This
tendency to localization of the sensitive parts of the protoplasm is
only in accord with our a@ priori conceptions of differentiation brought
about by reaction to the environment or adaptation, and is strictly 304. THE PROTOZOA comparable to the differentiation of the diffuse ectodermal nervous
system of the Coelenterata, or to the ectodermal origin of the nervous
system in all higher Metazoa. Movement and reaction to stimuli have been the grounds usually Movement and reaction to stimuli have been the grounds usually
adopted in support of the view that Protozoa are endowed with intel-
ligence, and Eimer’s view (’88) may be given as an example of this
type of reasoning. He says: “The ciliated Infusoria behave so in
regard to the outer world that one must ascribe to them the presence
of a will. The simple consideration of their movement from place to
place shows this. Change of position is entirely voluntary and is
effected through the greatest variety of motions; sometimes all of the
cilia, sometimes this one or that one, move more slowly or more rapidly
or are kept quiet. The hypotrichously ciliated Infusoria, e.g. 1 Loc, cit, p. 340. E. GENERAL CONSIDERATIONS In the preceding chapters it has been shown that in many forms isolated muscle of a frog, and the question of consciousness resolves
itself into the query, Does the single-celled organism compare with
the isolated muscle or with the individual frog of which that muscle
forms a part ? In the preceding chapters it has been shown that in many forms
certain parts of the cell become differentiated for special functions,
some of which are sensory, and in the whole group of Protozoa it is
possible to arrange a scale of forms in which sensory differentiations
of the plasm become more and more complex, z.e. certain regions of
the cell become more irritable than others. The reactions are very
much the same, however, whether external stimuli be applied to the
least differentiated or the most complex of Protozoa. Verworn has
further shown (89) that enucleated and minute parts of different
kinds of Protozoa also react to stimuli in exactly the same way as
does the entire animal, observations which confirm Gruber’s earlier
view, that each protoplasmic element has its own “will expression ”
(86), and justify Verworn’s assertion that each protoplasmic part is
the independent centre and source of its own movement, while the
movement of a single-celled organism is only the synchronous move-
ment of its many parts. Responses to stimuli are “reflex,” there-
fore, and the conclusion forces itself that the protozoan organism has
no more “consciousness” than the isolated muscle of the frog,
In addition
to movement, however, other vital phenomena have In addition
to movement, however, other vital phenomena have
been cited as evidence of intelligent action. Two of the most strik-
ing phenomena among the Protozoa are the apparent choice of food
and the selection of certain materials for building the shell. Chlamy-
dodontidze live almost exclusively on diatoms and Oscz//arza, although
other food is abundant; the ciliates Exchelys, Spathidium, Chenta,
Amphileptus, Lionotus, Dilepius, and Didinium feed on ciliates alone
(Biitschli), Actznobolus on Halteria, while numerous other forms of
ciliates may be limited
in other ways,
or may be omnivorous. Among Mastigophora Cienkowsky (’65) observed Colpodella pugnax
feeding exclusively on Chlamydomonas, while the rhizopod Vampy-
vella spirogyr@ is limited to the cells of the alga Spzrogyra. E. GENERAL CONSIDERATIONS Euplotes
charon, sometimes paddle rapidly through the water by means of all
the cilia, sometimes run upon immersed objects, using their cilia
as legs, with a motion like that of an Asellus,” etc. If Eimer
could explain how he knows
that
such movement
is voluntary
or involuntary, the conclusions concerning intelligence would have
some basis, but, as Haeckel says, the difference between voluntary and
involuntary movement is as difficult to define as it is to fix the boundary
between sensation (Empfindung) and irritability (Redsbarkeit), while
the latter shows no more trace of intelligence than do the very sensi-
tive reactions of Mzmosas, Dionea muscipula, and other higher plants. If by voluntary action is understood a movement brought about by an
impulse or impulses, engendered more or less directly by external
stimuli, or by changes in the inner condition of the organism, then,
as Biitschli remarks, there is no reason for opposing the assumption ;
but if by voluntary action is meant that each stimulus or change in
outer conditions induces a response brought about by the intelligent
act of a will, then we are entirely without basis for the assumption of
such action in the Protozoa. While it is impossible
to prove
that movement is involuntary,
observations upon living organisms under different conditions, such,
for example, as the reactions of entire cells or the parts of cells to
artificial stimuli of various kinds, make it reasonably certain. Observations and experiments upon entire individuals are not con- Observations and experiments upon entire individuals are not con-
clusive, and the possibility of consciousness
in perception and in
reaction is not excluded. It might indeed be argued that acids
“taste good” to Paramecium, or that antherozoids of the fern “like”
malic acid in small quantities as man likes alcohol, or that various
Protozoa “know enough” to turn away from harmful substances,
having acquired these characteristics through long ages of natural
selection. But similar responses to similar stimuli are shown by the 1 Loc, cit, p. 340. 1 Loc, cit, p. 340. SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA 305 isolated muscle of a frog, and the question of consciousness resolves
itself into the query, Does the single-celled organism compare with
the isolated muscle or with the individual frog of which that muscle
forms a part ? E. GENERAL CONSIDERATIONS The
latter, suspecting
that
in
such
cases
it is only
the mechanical irritation
of moving organisms which causes the
pseudopodial reaction, experimented with inorganic objects, such as
a needle-point, a bit of paper, etc., and found that Actznospherium
among
Heliozoa,
and Polystomella
among marine rhizopods, will
absorb a moving needle-point or a bit of paper as readily as a strug-
gling ciliate, and he concluded that the direct cause of food absorption,
in these cases at least, was the mechanical stimulus. Again, in forms
which draw food to the mouth opening by means of a vortex current
due to flagella or cilia, Entz (’88) assumed a selective action from the
fact that certain objects are passively thrown out of the current and
do not reach the mouth opening at all. Stein accounted for the same
occurrence by attributing a function of taste to certain of the adoral
cilia. But Verworn called attention to the old experiments of Gleichen
and Ehrenberg in feeding ciliates with powdered carmine and indigo,
which was quickly ingested, while he also observed that digestible as
well as indigestible particles are thrown out of the vortex current. Selection in such cases is not subjective, but depends upon the physi-
cal or chemical nature of the substances; the animal itself does not
distinguish between good and bad. Even more interesting in this respect are the observations and ex- based upon a comparison with our own consciousness or upon physical
phenomena in the non-living world, must be considered only tenta-
tive. If protoplasm be regarded merely as a fluid substance, or
better, a mixture of various, different fluids, forming an exceedingly
complex chemical substance distinct in its individuality, and possess-
ing certain recognizable properties, then it must be subject to the
same laws which govern all fluids, and many of the so-called vital
phenomena can be reduced to processes which in the inorganic world
are familiar to physicists and chemists. Haeckel (’66) pointed out in
connection with the supposed selective power of Protozoa, that an
alum crystal selects only alum molecules from a-mother liquid holding
numerous salts in solution, and Berthold (’86) suggested that lifeless
fluids will not absorb all kinds of stuff, but only such as have a certain
chemical composition. Verworn (’89), experimenting with Am@ba
and Pelomyxa, found no selective action with the objects used; every-
thing was surrounded by the pseudopodia and drawn into the body. E. GENERAL CONSIDERATIONS Carpen-
ter, Romanes, and Brady (’84) all remarked upon the selective power
of the marine rhizopods in building their shells, and the latter in par-
ticular ascribed a power of intelligent action on the part of certain
forms, eg. Truncatulina lobatula, which
“ protect themselves under
certain circumstances with a covering of sand.”
While these observations undoubtedly suggest willed acts on the While these observations undoubtedly suggest willed acts on the
part of the Protozoa in question, there is nevertheless room for an
explanation along quite different lines. It is ever necessary to bear
in mind, however, that
all vital phenomena are exceedingly com-
plex, even in the simplest of forms, and any explanation, whether x THE PROTOZOA 306 based upon a comparison with our own consciousness or upon physical
phenomena in the non-living world, must be considered only tenta-
tive. If protoplasm be regarded merely as a fluid substance, or
better, a mixture of various, different fluids, forming an exceedingly
complex chemical substance distinct in its individuality, and possess-
ing certain recognizable properties, then it must be subject to the
same laws which govern all fluids, and many of the so-called vital
phenomena can be reduced to processes which in the inorganic world
are familiar to physicists and chemists. Haeckel (’66) pointed out in
connection with the supposed selective power of Protozoa, that an
alum crystal selects only alum molecules from a-mother liquid holding
numerous salts in solution, and Berthold (’86) suggested that lifeless
fluids will not absorb all kinds of stuff, but only such as have a certain
chemical composition. Verworn (’89), experimenting with Am@ba
and Pelomyxa, found no selective action with the objects used; every-
thing was surrounded by the pseudopodia and drawn into the body. On the other hand, certain Rhizopoda (as in the case of Vampyrella
spirogyr@, which feeds solely on Spzvogyra) eat only certain kinds of
food, while Heliozoa, according to Meissner (’88), and Reticulariida,
according to Verworn (’89), normally take in only living organisms as
food. E. GENERAL CONSIDERATIONS On the other hand, certain Rhizopoda (as in the case of Vampyrella
spirogyr@, which feeds solely on Spzvogyra) eat only certain kinds of
food, while Heliozoa, according to Meissner (’88), and Reticulariida,
according to Verworn (’89), normally take in only living organisms as
food. The
latter, suspecting
that
in
such
cases
it is only
the mechanical irritation
of moving organisms which causes the
pseudopodial reaction, experimented with inorganic objects, such as
a needle-point, a bit of paper, etc., and found that Actznospherium
among
Heliozoa,
and Polystomella
among marine rhizopods, will
absorb a moving needle-point or a bit of paper as readily as a strug-
gling ciliate, and he concluded that the direct cause of food absorption,
in these cases at least, was the mechanical stimulus. Again, in forms
which draw food to the mouth opening by means of a vortex current
due to flagella or cilia, Entz (’88) assumed a selective action from the
fact that certain objects are passively thrown out of the current and
do not reach the mouth opening at all. Stein accounted for the same
occurrence by attributing a function of taste to certain of the adoral
cilia. But Verworn called attention to the old experiments of Gleichen
and Ehrenberg in feeding ciliates with powdered carmine and indigo,
which was quickly ingested, while he also observed that digestible as
well as indigestible particles are thrown out of the vortex current. Selection in such cases is not subjective, but depends upon the physi-
cal or chemical nature of the substances; the animal itself does not
distinguish between good and bad. Even more interesting in this respect are the observations and ex- Even more interesting in this respect are the observations and ex-
periments of Rhumbler (’98) with organic and inorganic fluids. While
corroborating the observations of Hofer and others, that objects are
ingulfed at the posterior end of Ammqda, he found that the ectoplasm SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
307 is constantly moving inward to become changed again into endo-
plasm. The food particles accompany this inturning, and a filament is constantly moving inward to become changed again into endo-
plasm. The food particles accompany this inturning, and a filament
Fig. 153. — Reactions of Amaba verrucosa and of fluid substances. [RHUMBLER.]
A. Ameba verrucosa with a filament of Oscilaria. B,C. Chloroform drops with a filamen
of shellac. a, 4, ¢, d, e. E. GENERAL CONSIDERATIONS successive stages in the rolling of a filament of Oscil/aria,
B, C. Simila
rolling of a shellac filament. of Oscillaria, if pulled out after being partly ingested, leaves a channel
in which it had lain (Fig. 153, a-g). After being drawn in, the filament Fig. 153. — Reactions of Amaba verrucosa and of fluid substances. [RHUMBLER.]
A. Ameba verrucosa with a filament of Oscilaria. B,C. Chloroform drops with a filament
of shellac. a, 4, ¢, d, e. successive stages in the rolling of a filament of Oscil/aria,
B, C. Similar
rolling of a shellac filament. Fig. 153. — Reactions of Amaba verrucosa and of fluid substances. [RHUMBLER.]
A. Ameba verrucosa with a filament of Oscilaria. B,C. Chloroform drops with a filament
of shellac. a, 4, ¢, d, e. successive stages in the rolling of a filament of Oscil/aria,
B, C. Similar
rolling of a shellac filament. of Oscillaria, if pulled out after being partly ingested, leaves a channel
in which it had lain (Fig. 153, a-g). After being drawn in, the filament 308 THE PROTOZOA of Oscillavia is rolled up into a small coil, and considerable space is
thus saved. An Ameba, 90 » in length, absorbed and coiled up a
filament of Osctllaria 540 w long. Quite similarly, a drop of water
quickly draws into its substance a minute splinter of wood or glass,
while all fluids show the same power in respect to certain substances. A drop of chloroform will draw in a shellac thread from the surround-
ing water, and will roll it up within its substance in exactly the same
way that an Ameéa rolls up a filament of Oscel/laria (Pigs 153; J, C),
Egg albumin and gum arabic in solution show the same phenomenon,
the rapidity of ingestion depending upon the density of the medium. To a physicist such a process is explained by the phenomena of
cohesion and adhesion; the coefficient of adhesion between the chloro-
form and the shellac filament is greater than the coefficient between
shellac and water. But if a splinter of glass be mechanically inserted
in a drop of chloroform suspended in water, the splinter will quickly
leave the chloroform and seek the water. In this case the coefficient
between glass and water is greater than that between glass and chlo-
roform. E. GENERAL CONSIDERATIONS The stimuli necessary to
bring about protoplasmic reaction may be so delicate that we cannot
perceive them, and we are thus led to assign some mystic cause under The naked Am@ba might be considered a complex chemical com-
pound, endowed, like all compounds, with special properties
— in this
case with the power of motion, of irritability, of metabolism, and of
growth and reproduction. Like many chemical compounds it is, dur-
ing the living state, of unstable equilibrium, which involves a constant
change in chemical composition. These
several
properties with
which it is endowed, however, are not confined exclusively to the liv-
ing organisms, for many inorganic compounds possess one or more of
them. Thus, a drop of oil quite spontaneously assumes forms which
simulate different species of Amada, while a mixture of sugar or salt
and olive oil simulates not only the movements, but even the struc-
ture, of living protoplasm (Biitschli). In these cases the motion is satis-
factorily explained by the laws of surface tension, although the move-
ments are almost as remarkable as those of a rhizopod. The motion
of the protoplasm of a plant-cell is explained as the resultant of the
chemical changes taking place within the body of the plant, and simi-
larly the motion of Amaéa has recently been interpreted by Berthold,
Verworn, Biitschli, and Rhumbler, as a series of responses to changes
in the chemical composition, with corresponding changes in density
within the organism. The movements which engender the phenom-
ena of phototaxis and thermotaxis, of chemotaxis and barotaxis, are also
duplicated by inorganic substances. Thus the impact of ether waves
upon various
bodies brings about
a rearrangement
of molecules. Affinity, in chemistry, is the elective property by which one substance
seeks another, and the mutual action is analogous to, if not the same
as, chemotaxis; while the phenomena of cohesion and of adhesion
come under the head of barotaxis. Ina similar way it can be shown
that irritability, or response to stimuli, has its analogue in the inorganic
world; any compound substance in a state of unstable equilibrium, as
in explosive compounds, will react to stimuli of various kinds, while
even metabolism is simulated in inorganic objects, as shown in the
excellent illustration cited by Verworn (’94) of nitric acid in the pres-
ence of sulphurous anhydride. E. GENERAL CONSIDERATIONS Rhumbler tried the ingenious experiment of coating a glass
splinter with a layer of shellac; when one end was placed against the
chloroform drop, the splinter and shellac were quickly drawn into the
drop. Here the shellac was soon dissolved by the chloroform, and
the splinter was gradually left naked, whereupon it soon left the drop,
being drawn into the surrounding water by reason of the greater
coefficient of adhesion between glass and water. Here, according to
Rhumbler, is an analogue of the process of feeding on the part of
Rhizopoda. Bodies are ingested into the plasm because of the greater
attraction to the fluid protoplasm than to water, then, through the
chemical changes between protoplasm and the digestible parts of the
foreign substances, the constituents of the foreign body are changed,
and a corresponding change is wrought in the attractive force which
keeps them together, that is, in the coefficient of adhesion, and defe-
cation results. Similarly with shell-formation,
it has been shown by Verworn,
Dreyer, Rhumbler, and others, that Rhizopoda pick up all sorts of
foreign particles and excrete them at certain times upon the outside. Rhumbler found that the same process may be repeated by inorganic
liquids, some of which (e.g. chloroform) show a selective tendency
in picking up some objects and leaving others, while Verworn has
shown that Amada will not pick up certain objects unless the latter
be made to irritate it so that a slimy secretion is poured out. In those
forms of Rhizopoda which appear tq select their building material,
the selection is often due to the character of the material at their
disposal, and partly to purely physical conditions, such as the ex-
clusion of large sand grains because of the small mouth opening, or SOME PROBLEMS IN THE PHYSIOLOGY OF THE PROTOZOA
309 the inability
of the protoplasm
to hold objects above
a certain
weight. In other cases where the shell material is deposited, such as the the inability
of the protoplasm
to hold objects above
a certain
weight. In other cases where the shell material is deposited, such as the
lime or silicious shells of Sarcodina, the substance itself is built up by
a chemical process within the protoplasm, and the deposition may
take place periodically or continuously. The naked Am@ba might be considered a complex chemical com- take place periodically or continuously. E. GENERAL CONSIDERATIONS The naked Am@ba might be considered a complex chemical com-
pound, endowed, like all compounds, with special properties
— in this
case with the power of motion, of irritability, of metabolism, and of
growth and reproduction. Like many chemical compounds it is, dur-
ing the living state, of unstable equilibrium, which involves a constant
change in chemical composition. These
several
properties with
which it is endowed, however, are not confined exclusively to the liv-
ing organisms, for many inorganic compounds possess one or more of
them. Thus, a drop of oil quite spontaneously assumes forms which
simulate different species of Amada, while a mixture of sugar or salt
and olive oil simulates not only the movements, but even the struc-
ture, of living protoplasm (Biitschli). In these cases the motion is satis-
factorily explained by the laws of surface tension, although the move-
ments are almost as remarkable as those of a rhizopod. The motion
of the protoplasm of a plant-cell is explained as the resultant of the
chemical changes taking place within the body of the plant, and simi-
larly the motion of Amaéa has recently been interpreted by Berthold,
Verworn, Biitschli, and Rhumbler, as a series of responses to changes
in the chemical composition, with corresponding changes in density
within the organism. The movements which engender the phenom-
ena of phototaxis and thermotaxis, of chemotaxis and barotaxis, are also
duplicated by inorganic substances. Thus the impact of ether waves
upon various
bodies brings about
a rearrangement
of molecules. Affinity, in chemistry, is the elective property by which one substance
seeks another, and the mutual action is analogous to, if not the same
as, chemotaxis; while the phenomena of cohesion and of adhesion
come under the head of barotaxis. Ina similar way it can be shown
that irritability, or response to stimuli, has its analogue in the inorganic
world; any compound substance in a state of unstable equilibrium, as
in explosive compounds, will react to stimuli of various kinds, while
even metabolism is simulated in inorganic objects, as shown in the
excellent illustration cited by Verworn (’94) of nitric acid in the pres-
ence of sulphurous anhydride. Growth, too, is not confined to organic
substances, as shown by the continual growth by accretion of various
crystals or solids, while growth by intussusception takes place when-
ever a solid becomes dissolved in a liquid. E. GENERAL CONSIDERATIONS Growth, too, is not confined to organic
substances, as shown by the continual growth by accretion of various
crystals or solids, while growth by intussusception takes place when-
ever a solid becomes dissolved in a liquid. The stimuli necessary to
bring about protoplasmic reaction may be so delicate that we cannot
perceive them, and we are thus led to assign some mystic cause under THE PROTOZOA 310 a term like “vital phenomena.”
Unexplained phenomena are daily
seen in the laboratory, but the explanations are not regarded as
hopeless; the sudden stoppage of a piece of camphor moving on
the surface of pure water, by the introduction of the end of a finger,
which gives to the water an exceedingly minute quantity of fatty
matter, yet enough to equalize the surface tension,
is understood
through the laws of physics, although causes cannot be seen. At the
present time, we can no more hope to understand the properties
which we call “life” than we understand the ultimate causes of surface
tension, of aquosity, or universal gravity; yet its manifestations can
be examined and measured, and reduced to laws which are already
applied in the domain of physical science. The Protozoa, finally, must be regarded as forms in which the
organism is less developed than in any other animal. In other words,
the coédrdinating factors by means of which each protozo6n acts as a
unit are less complex than in other representatives of the animal
kingdom, and this fact justifies the hope which lies at the bottom
of modern “vitalism,” that some day we may interpret these unknown
factors in chemical and physical terms. BIBLIOGRAPHY The titles included here are arranged in alphabetical order according to the system adopted in
Minot's Human Embryology, in Wilson's The Cell in Inheritance and Development, and in other
recent works. Each author's name is followed by the year of publication (abbreviated to the last
two digits in all years between 1802-1901 inclusive);
and where two or more publications ap-
peared in the same year, they are referred to in the proper order, Thus, BLOCHMANN, F., '94, 2,
refers to Blochmann’s second paper in 1894. SPECIAL BIBLIOGRAPHY IX Balbiani, G.— Recherches expérimentales sur la metotomie des Infusoires ciliés. Recueil zool. Suisse, V., 1888. Berthold, G. — Studien iiber Protoplasmamechanik. Lezpzig, 1886. Berthold, G. — Studien iiber Protoplasmamechanik. Lezpzig, 1886. Biitschli, 0. — Untersuchungen iiber mikroskopische Schaume und d Biitschli, 0. — Untersuchungen iiber mikroskopische Schaume und das Protoplasma. Leipzig, 1892. Greenwood, M.— On the Digestive Process in Some Rhizopoda. Jour. of Physiol. Greenwood, M.— On the Digestive Process in Some Rhizopoda. Jour. of Physiol. (English), VII. and VIII., 1886 and 1887. Griffiths. — A Method of demonstrating the Presence of Uric Acid in the Contractile Griffiths. — A Method of demonstrating the Presence of Uric Acid in the Contractile
Vacuoles of Some Lower Organisms. Proc. Roy. Soc. Edinburgh, XV1., 1889. Hofer, B. —Expérimentelle Untersuchungen iiber den Einfluss des Kerns auf das Hofer, B. —Expérimentelle Untersuchungen iiber den Einfluss des Kerns auf das
Protoplasma. /en. Zezt., 1889. Jennings, H. S. — Studies on Reactions to Stimuli in Unicellular Organisms. Amer. Jennings, H. S. — Studies on Reactions to Stimuli in Unicellular Organisms. Amer. Jour. Phys., I1., 1899. Le Dantec, F. — Recherches sur la digestion intra-cellulaire chez les protozoaires. Dantec, F. — Recherches sur la digestion intra-cellulaire chez les protozoaires. Ann. ad. Lust. Pasteur, 1V., 1890. ssner,
M. — Beitrdge zur Ernahrungsphysiologie
der FProtozoen. Z. w. Z., Le Dantec, F. — Recherches sur la digestion intra-cellulaire chez les protozoaires. Ann. ad. Lust. Pasteur, 1V., 1890. Meissner,
M. — Beitrdge zur Ernahrungsphysiologie
der FProtozoen. Z. w. Z., ssner,
M. — Beitrdge zur Ernahrungsphysiologie
der FProtozoen. Z. w. Z.,
XLVI., 1888. mbler, L. — Physikalische Analyse von Lebenserscheinungen der Zelle. Arch. f Rhumbler, L. — Physikalische Analyse von Lebenserscheinungen der Zelle. Arch. f-
Entwickelungsmechanik, V11., 1898. aa:
Verworn, M. — Psychophysiologische Protistenstudien. Jena, 1889. Verworn, M. — Psychophysiologische Protistenstudien. Jena, 1889. Id. — Die physiologische Bedeutung des Zellkerns. Arch. f. ad. G Id. — Die physiologische Bedeutung des Zellkerns. Arch. f. ad. Ges. Physiologie,
LI., 1891. Id. — Allgemeine Physiologie. Lefzzg, 1894. Translated by F. S. Lee, New Id. — Allgemeine Physiologie. Lefzzg, 1894. Translated by F. S. Lee, New
York, 1899. ABBREVIATIONS Anatomischer Anzeiger. Archives de Biologie. Archives de Biologie. Archiv fiir Anatomie u Archiv fiir Anatomie und Physiologie. Annals and Magazine of Natural History Annals and Magazine of Natural History. Archiv fiir mikroskopische Anatomie. m Archiv fiir mikroskopische Anatomie. Archiv fiir Naturgeschichte. Archiv fiir Naturgeschichte. Archiv fiir Entwicklungsgesch Archiv fiir Entwicklungsgeschichte. Archiv de zoologie expérimentale et
a>
ada Archiv de zoologie expérimentale et générale. Biologisches Centralblatt. a>
ada Archiv de zoologie expérim
Biologisches Centralblatt. a>
ada Biologisches Centralblatt. Comptes Rendus de l’Acad Comptes Rendus de l’Académie de Sciences. Comptes Rendus de la Société de Biologie. Ee Comptes Rendus de l’Acad
Comptes Rendus de la Soci
e Comptes Rendus de la Société de Biologie. Journal of Morphology. y Ee Journal of Morphology. Jenaische Zeitschrift. Jenaische Zeitschrift. Miiller’s Archiv. Miiller’s Archiv. Morphologisches Morphologisches Jahrbuch. Quarterly Journal of Microscopical Science. Quarterly Journal of Microscopical Science. Zoologischer Anzeiger. Zoologischer Anzeiger. Zeitschrift fiir wissenscha
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N AGARDG, ’28. Icones Algar. Europ. — Agassiz, L., 57. Contribution to the
Natural History of the United States of America. 1st Monograph: Essay on Classi-
fication, Boston. — Alder, J., "51. An Account of Three New Species of Animal-
cules: 4. M. MV. A. (2), VII, p. 426.— Allman, G. J., 72. Note on Noctiluca:
Q. J. XII (N. S.), pp. 326-32. — Altmann, R., ’94. Die Elementarorganismen
und ihre Beziehungen zu den Zellen: 2d Zdition.— Archer, W.,’69. On Some
Fresh-water Rhizopoda, new or little known:
Q. /. (N. S.), [X.—Id., °70. On
Some Fresh-water Rhizopoda, new or little known: Q. /. (N. S.), IX and X.—I4d.,
°75. On Chlamydomyxa labyrinthuloides,
nov. gen. et sp., a New Fresh-water
Sarcodic Organism:
Q. /., XV (N. S.), p. 107.—Id., "76. Résumé of Recent
Contributions to our Knowledge of Fresh-water Rhizopoda:
Q. /. (N.S.), XVI,
p- 347-— Auerbach, L., 55. Ueber die Einzelligkeit der Amoeben:
Z. w. Z,,
VII, pp. 365-430. BALBIANI, G., 58. Note relative 4 existence d’une génération sexuelle chez AGARDG, ’28. Icones Algar. Europ. — Agassiz, L., 57. Contribution to the
Natural History of the United States of America. 1st Monograph: Essay on Classi-
fication, Boston. — Alder, J., "51. An Account of Three New Species of Animal-
cules: 4. M. MV. A. ABBREVIATIONS (2), VII, p. 426.— Allman, G. J., 72. Note on Noctiluca:
Q. J. XII (N. S.), pp. 326-32. — Altmann, R., ’94. Die Elementarorganismen
und ihre Beziehungen zu den Zellen: 2d Zdition.— Archer, W.,’69. On Some
Fresh-water Rhizopoda, new or little known:
Q. /. (N. S.), [X.—Id., °70. On
Some Fresh-water Rhizopoda, new or little known: Q. /. (N. S.), IX and X.—I4d.,
°75. On Chlamydomyxa labyrinthuloides,
nov. gen. et sp., a New Fresh-water
Sarcodic Organism:
Q. /., XV (N. S.), p. 107.—Id., "76. Résumé of Recent
Contributions to our Knowledge of Fresh-water Rhizopoda:
Q. /. (N.S.), XVI,
p- 347-— Auerbach, L., 55. Ueber die Einzelligkeit der Amoeben:
Z. w. Z,,
VII, pp. 365-430. BALBIANI, G., 58. Note relative 4 existence d’une génération sexuelle chez BALBIANI, G., 58. Note relative 4 existence d’une génération sexuelle chez
les Infusoires: Jozwr. de la Physiol., 1, pp. 347-52. —1d., 59. Du réle des organes
générateurs dans la division spontanée des Infusoires ciliés: C. &., XLVIII, pp. 266-9. —Id., 60. Note sur un cas de parasitisme improprement pris pour un mode
de reproduction des Infusoires ciliés: C. 2., LI, pp. 319-22. — Id., °61. Recherches
sur les phénoménes sexuelles des Infusoires: Jour. de la Physiol., 1V, pp. 102-30,
311 311 THE PROTOZOA 312 194-220, 431-48, 465-520.—Id., 63. Sur l’organisation et la nature des Psoro-
spermies: C. #., LVII, pp. 157-61.—Id., 82. Les Protozoaires: jour. de Micro-
graphie, V (1881), p. 63, VI (1882), p. 9. —Id., "88. Recherches expérimentales
sur la mérotomie des Infusoires ciliés: Recueil Zoolog. Siisse, V, pp. 1-72. —1d., 89. Sur trois entophytes nouveaux du tube digestif des Myriapodes: Jour. de l’ Anatom. et de la Physiolog., XXV, pp. 5-45.—Id., 90. Sur la structure intime du noyau du
Loxophyllum meleagris: Z. A., XIII, pp. 110-15 and 132-6.—Id.,’95. Sur la
structure et la division du noyau chez le Spirochona gemmipara: Aun. de Microg.,
VII, p. 289. Baranetski, J., °75.— Influence de la lumiére sur les plasmodia des
Myxomycétes: Jem. d. 1. Soc. Nat. d. Cherbourg, XIX, pp. 321-60. Barbagello,
P. (with Cassagrandi,O.),’95. Ricerche biologiche e clinic sull’ Amoeba coli (Lésch.) :
Bull. della sedute della Accademia Gioenia di Sct. Nat. in Catania, XLI, pp. 7-19. —De Bary, 64. Die Mycetozoen:
2d Ed. Leifzig.—Id.,
’°87. Comparative
Morphology and Biology of the Fungi, Mycetozoa, and Bacteria. Translated from
German Edition of 1884: Oxford. — Behla, Robt.,’98. ABBREVIATIONS Die Amében insbesondere
vom parasitaren und culturellen Standpunkt : Berdiz.— Van Beneden, E., ’83. Recherches sur la maturation de l’ceuf et la fécondation: A. B., IV, pp. 265-637. — Van Beneden et Neyt,’87. Nouvelle recherches sur la fécondation et la division
mitosique chez l’Ascaris megalocephalus: Budi. Acad. Roy. d. Belgique, XIV, pp. 215-95. — Bergh, R. S.,°79. Tiarina fusus Cl. and Lach.:
Vidensk. Meddel. fr. d. Naturh. Foren. t. Kjobenhavn, 1879-80, pp. 265-70. —Id., °81. Der Organismus
der Cilioflagellaten: AZ. /., VII, pp. 177-288. —Id.,’89. Recherches sur les noyaux
de l'Urostyla grandis et de l'Urostyla intermedia n. sp.: 4. 2., 1X, pp. 497-513. Berthold, G.,’86. Studien tiber Protoplasmamechanik: Lezfzzg, 1886. — Beyerinck,
—,’90. Culturversuche mit Zoochlorellen, Lichengonidien und andere Algen : Lot. Zeit. — Biainville, H. de, ’22. Article ‘“Infusoire,” in Dzctzonnatre des Sciences
Naturelles, T. 23, pp. 416-20.— Blochmann, F., ’88. Zur Kenntniss der Fort-
pflanzung von Euglypha alveolata: MZ. /., XIII, pp. 173-83.—Id., 94, I. Ueber
die Kerntheilung bei Euglena: &. C., XIV, pp. 194-7. Id., '94, 2. Zur Kennt-
niss von Dimorpha nutans (Gruber): B. C., XIV, pp. 197-201. — Boeck, C. P. B.,
47. Nogle Forhold af Bygningen og Udviklingem af Polygastrica: Forh. Skandin. Naturfor. Christiania, p. 270.— Borgert, Adolf, ’91. Ueber die Dictyochiden,
insbesondere iiber Distephanus speculum; so wie Studien an Phaeodarien: Z. w. Z.,
LI, pp. 629-76. —Id., 86. Fortpflanzungsverhaltnisse bei tripyleen Radiolarien
(Phaeodarien):
Verh. d. Deutsch. Zool. Ges. — Bory de Saint Vincent,
'24. Classification des animaux domestiques: Evcycl. Méthode, 11, Paris. — Bose, F. J.,
98. Le Cancer maladie infectieuse a Sporozoaires: Arch. de Phys. norm. et path.,
Paris, XXX, pp. 458-71 and 484-93. — Bourne, A. G.,’91. On Pelomyxa viridis
sp. n. and on the vesicular nature of Protoplasm: Q. //. (N.S), XXXII, pp. 357-74. — Boveri, Th., ’88. Zellenstudien
I, I]: /. Z., XXII.—Id., 90. Zellenstudien,
Ill, e¢c.: J. Z., XXIV., pp. 314-401. —Id., 95. Ueber das Verhalten der Centro-
somen bei der Befruchtung des Seeigeleies, nebst allgem. Bemerkungen tiber Cen- 98. Le Cancer maladie infectieuse a Sporozoaires: Arch. de Phys. norm. et path.,
Paris, XXX, pp. 458-71 and 484-93. — Bourne, A. G.,’91. On Pelomyxa viridis
sp. n. and on the vesicular nature of Protoplasm: Q. //. (N.S), XXXII, pp. 357-74. — Boveri, Th., ’88. Zellenstudien
I, I]: /. Z., XXII.—Id., 90. Zellenstudien,
Ill, e¢c.: J. Z., XXIV., pp. 314-401. —Id., 95. ABBREVIATIONS 381-407.— Bruch, C., 50. Einige Bemerkungen iiber die
Gregarinen: Z. w. Z., II, pp. 110-14. — Buck, B.,°77. Einige Rhizopodenstudien :
Z. Ww. Z, XXX, pp. I-49. — Buehler, A.,’95. Protoplasma-Structur in Vorder-
hirnzellen der Eidechsen:
Verh. Phys. Med. Ges. Wiirzburg
(2), XX1X, pp. 209-52. — Buffon, G. L. de, 1749. Histoire Naturelle générale et particulier:
Z. 1-111, des Golfes von Neapel:
Fauna and Flora, G.v. N. — Brauer, Aug., 93. Zur
Kenntniss d. Spermatogenese v. Ascaris megalocephalus: 4. #. 4., XLII, p- 153. —Id.,’94. Ueber die Encystierung von Actinosphaerium Eich: 2. w. Z., LVILI, pp-
189-221. — Briicke, E., 61. Die Elementarorganismen:
I Zener Sttzungsber. XLIV, Abth. 11, pp. 381-407.— Bruch, C., 50. Einige Bemerkungen iiber die
Gregarinen: Z. w. Z., II, pp. 110-14. — Buck, B.,°77. Einige Rhizopodenstudien :
Z. Ww. Z, XXX, pp. I-49. — Buehler, A.,’95. Protoplasma-Structur in Vorder-
hirnzellen der Eidechsen:
Verh. Phys. Med. Ges. Wiirzburg
(2), XX1X, pp. 209-52. — Buffon, G. L. de, 1749. Histoire Naturelle générale et particulier:
Z. 1-111,
Edition 1812.— Burnett, W. J.,'54. On the zodlogical nature of Infusoria: Proc. Boston Soc. Nat. Hiest.,1V, pp. 331-5. — Biitschli, O., °73. Einiges iiber Infusorien:
A. m. A., 1X, pp. 657-78. —Id., 74. Zur Kenntniss der Fortpflanzung bei Arcella
vulgaris Ehr., 4. #. A., XI, pp. 460-6. —Id., 76. Studien iiber d. erst. Entwick-
lungsvorg. d. Eizelle, d. Zelltheilung u. die Conjugation der Infusorien: Adhand. a. Senkenberg. naturfor. Gesell. Fr. a. Al., X, pp. 213-452. (Preliminary in Z.w.Z.,
XXV, 1875, p. 426.) —Id., 77. Ueber den Dendrocometes paradoxus Stein, nebst
einigen Bemerkungen, efc.: Z. w. Z., XXVIII, pp. 49-67. —Id., °78. Beitrige
zur Kenniniss der Flagellaten und einiger verwandten Organismen: Z. w. Z., XXX,
pp. 205-81.—Id., ’82. Beitrage zur Kenntniss der Fischpsorospermien: Z. w. Z., XXV, pp. 629-51. —Id., 83. Protozoa in Bronn’s Klassen und Ordnungen
des Thierreichs, 1883-88. —Id., 85. Einige Bermerkungen tiber gewisse Orga-
nisationverhaltnisse der Cilioflagellaten und der Noctiluca:
47. /., X, pp. 529-77-
—Id.,’92,1. Ueber die so-genannten Centralkérper der Zelle und ihre Bedeutung :
Verh. d. Nat. Med. Verh. 2. Heidelberg (N. F.), IV, pp. 535-8. —Id., "92, 2. Untersuchungen iiber mikroskopische Schaume und das Protoplasma: Le/zig, 1892. (Translated by E. A. Minchin, Zoxdon, 94.) —1d., 96. Weitere Ausfiihrungen
iiber den Bau der Cyanophyceen und Bakterien: Lezpzig. CALENDRUCCIO,
S., ’90. Animali parasiti dell’ uomo in Sicilia:
Azz. d. CALENDRUCCIO,
S., ’90. Animali parasiti dell’ uomo in Sicilia:
Azz. ABBREVIATIONS Ueber das Verhalten der Centro-
somen bei der Befruchtung des Seeigeleies, nebst allgem. Bemerkungen tiber Cen-
trosomen u. Verwandtes:
Verh. d. Phys. Med. Ges. z. Wiirsburg (N. F.), XXIX,1,
p- 41.—Id., ’01. Zellenstudien, IV, /exa, 1901.— Brady, H. B.,’79. Notes on
some of the reticularian Rhizopoda of the “ Challenger” Expedition. 1. On new or
little-known Arenaceous types: Q. /., XIX, p. 28.—Id., ’82,’84. Report on the
Foraminifera dredged by H. M.S. “Challenger” during the years 1873-6: Challenger
Reports Zoology, 1X.—Id., °84. On Keramosphera, a new type of Porcellanous
Foraminifera:
A. A/. MN. A. (5) X, pp. 242-5.— Brandt, H., °77. Ueber
Actinosphaerium Eich: Dessertation, Halle. — Brandt, K.,’81. Farbung lebender
einzelligen Organismen: &.C., I, pp. 202-5. —Id., 82. Ueber die morphologische
und physiologische Bedeutung des Chlorophyls bei Thieren: <lrch. f. Anat. u. Phys. (Piys. Abth.),
pp. 125-51.—1d.,’85. Koloniebildenden Radiolarien (Sphaerozoeén) 98. Le Cancer maladie infectieuse a Sporozoaires: Arch. de Phys. norm. et path.,
Paris, XXX, pp. 458-71 and 484-93. — Bourne, A. G.,’91. On Pelomyxa viridis
sp. n. and on the vesicular nature of Protoplasm: Q. //. (N.S), XXXII, pp. 357-74. — Boveri, Th., ’88. Zellenstudien
I, I]: /. Z., XXII.—Id., 90. Zellenstudien,
Ill, e¢c.: J. Z., XXIV., pp. 314-401. —Id., 95. Ueber das Verhalten der Centro-
somen bei der Befruchtung des Seeigeleies, nebst allgem. Bemerkungen tiber Cen-
trosomen u. Verwandtes:
Verh. d. Phys. Med. Ges. z. Wiirsburg (N. F.), XXIX,1,
p- 41.—Id., ’01. Zellenstudien, IV, /exa, 1901.— Brady, H. B.,’79. Notes on
some of the reticularian Rhizopoda of the “ Challenger” Expedition. 1. On new or
little-known Arenaceous types: Q. /., XIX, p. 28.—Id., ’82,’84. Report on the
Foraminifera dredged by H. M.S. “Challenger” during the years 1873-6: Challenger
Reports Zoology, 1X.—Id., °84. On Keramosphera, a new type of Porcellanous
Foraminifera:
A. A/. MN. A. (5) X, pp. 242-5.— Brandt, H., °77. Ueber
Actinosphaerium Eich: Dessertation, Halle. — Brandt, K.,’81. Farbung lebender
einzelligen Organismen: &.C., I, pp. 202-5. —Id., 82. Ueber die morphologische
und physiologische Bedeutung des Chlorophyls bei Thieren: <lrch. f. Anat. u. Phys. (Piys. Abth.),
pp. 125-51.—1d.,’85. Koloniebildenden Radiolarien (Sphaerozoeén) BIBLIOGRAPHY 313 des Golfes von Neapel:
Fauna and Flora, G.v. N. — Brauer, Aug., 93. Zur
Kenntniss d. Spermatogenese v. Ascaris megalocephalus: 4. #. 4., XLII, p- 153. —Id.,’94. Ueber die Encystierung von Actinosphaerium Eich: 2. w. Z., LVILI, pp-
189-221. — Briicke, E., 61. Die Elementarorganismen:
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den Bau der Bursaria truncatella; mit besonderer Beriicksichtigung der protoplas-
matischen Strukturen: JZ. /., XII, pp. 333-63. —Id., 88. Die Parasitischen Am6-
ben des menschlichen Darmes:
Cent. f. Bakt. und Par., XIII, pp. 598-609. —14.,
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Rhizopodenstudien
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Lezfz7g.— Schwalbe, G., 66. Ueber die Contractilen
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skopische Untersuchungen iiber die Uebereinstimmung
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Bull. d. Acad. d. Sci. ad. Cracovie, 1899, Dec. — Siedlecki und
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C. #. S. B. (10), IV, pp. 425-8.—Id.,
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Anz. d. l’fust. Past., 1], pp. 545-80.—Sorokin, N., °78. Ueber Gloidium quodrifidum, e¢c.:
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New
Infusoria from American
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A. AZ. NM. H. (5), XVII.—
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Guerin,
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Bull. d. Acad. d. Sci. ad. Cracovie, 1899, Dec. — Siedlecki und
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C. #. S. B. (10), IV, pp. 425-8.—Id.,
'97, 2. L’Evolution des Sporozoaires der
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509.—Id., 54. Die Infusionsthiere
auf ihre Entwicklungsgesch. untersucht:
Leipzig.—Id., 56. In Tageblatt der 32 Versammlung deutscher Naturforscher
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Svtz. Ber. d. K. bohm. Ak. W.,
X, pp. 41-3. —Id., 59. Der Organismus der Infusionsthiere,
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gend bekannte Infusorienformen aus der Ostsee bei Wismar:
Amtl. Ber. a. Vers. deutsch. Naturf. u. Aerzte, Karlsbad, 1862, p. 165.—Id., 67. Der Organismus
der Infusionsthiere,
Bd. I]:
Lecpzig, 1867.—Id.,
"78. Der Organismus
der
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Z. w. Z., XXXI, pp. 29-58. —Stéhr, P., 80. Die Radiolarienfauna der Tripoli
von Grotte, etc.: Paleontographica, XXVI, p. 121. — Stokes, A. C.,’°84. Notices
of Some New Parasitic Infusoria: Amer. Mat., XVIII, pp. 1081-6. —1d.,’86. Some
New
Infusoria from American
Fresh Waters:
A. AZ. NM. H. (5), XVII.—
Id., 93. The Contractile Vacuole: Amer. Mon. Mic. Jour., XVI, pp. 182-8. —
Stolc, A., 00. Beobachtungen und Versuche iiber die Verdauung und Bildung
der Kohlenhydrate bei einem Am6benartigen Organismus
Pelomyxa
palustris
Greeff: Z. w. Z., LXVIII, pp. 625-68.— Strasburger, E., “78. Wirkung des
Lichtes und der Warme auf Schwarmsporen: /. Z., XII, pp. 551-625. —Id., °92. Histologische Beitrage, IV: /ena.— Suriray, ‘36. Recherches sur la cause ordi-
naire de la phosphorescence marine et description du Noctiluca miliaris:
Guerin,
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Coregonus:
Cent. Bakt., XXIII, pp. 602-76, 46-55, and 699-703. Carpenter, classification, 18, 107, 108, 109;
types of shells, 73; food-selection, 305.
Carter, 109; conjugation in Audorina, 134; Calkins, odors due to Protozoa,62; Uroglena,
130; Lymphosporidium, 169; mitosis in
Mastigophora, 250, 252; historic origin of
mitotic figure, 274.
Carpenter, classification, 18, 107, 108, 109; Carus, J. V., classification, Sporozoa, 168.
Cassagrandi, dysentery, 64. Carus, C. G., 10;
spontaneous generation,
25.
Carus, J. V., classification, Sporozoa, 168. INDEX OF AUTHORS Agardh, 139. Agassiz, L., 12. Alder, 12. Allman, Woctiluca, 136. Altmann, structure of protoplasm, 35. Archer, classification, 106, 109. Aristotle, animals and plants, 23, 25. Auerbach, excretory granules, 286. Agardh, 139. Agassiz, L., 12. Alder, 12. Allman, Woctiluca, 136. Altmann, structure of protoplasm, 35. Archer, classification, 106, 109. Aristotle, animals and plants, 23, 25. Auerbach, excretory granules, 286. Bruch, Sporozoa, relationships, 166. Briicke, 2; pseudopodia-formation, 83. Buck, swarm-spores in Arcella, 95. Buffon, 13; animals and plants, 23; sp Bruch, Sporozoa, relationships, 166. Briicke, 2; pseudopodia-formation, 83. Buck, swarm-spores in Arcella, 95. Buffon, 13; animals and plants, 23; sp Buck, swarm-spores in Arcella, 95. Buffon, 13; animals and plants, 23; Allman, Woctiluca, 136. Altmann, structure of pr Buffon, 13; animals and plants, 23; spon-
taneous generation, 26; theory of genera-
tion, 27. Biihler, origin of spindle, 274. Burnet, 12. Biitschli, 2, 12, 14; animals and Aristotle, animals and plants, 23, 25. Auerbach, excretory granules, 286. Biihler, origin of spindle, 274. Burnet, 12. Auerbach, excretory granules, 286. Burnet, 12. Biitschli, 2, 1 Biitschli, 2, 12, 14; animals and plants, 25;
structure of protuplasm, 35, 79; odors, 62;
pseudopodia-formation, 84; swarm-spores
in Arcella, 95;
conjugation, 60, 96, 193,
228;
classification, 20-22, 99, 101, 169,
206, 208, 209, 210; shells of Dinoflagel-
lidia, 117; flagella-motion, 121; nutrition
in Mastigophora,
126;
catenation, 131;
inter-relationships of Mastigophora, 135;
spore-formation
in Sporozoa, 152, 154;
Sporozoa, inter-relationships, 166; mem-
brane of ciliates, 176, 178;
contraction
of the vacuole, 188; tentacles of suctoria,
195; inter-relationships of the Infusoria,
199-200;
mouth-shilting in ciliata, 185;
rejuvenescence, 213, 220; conjugation in
Mastigophora,
228, 241;
nucleus,
249,
250;
mitosis, 264; division-centre, 266;
digestion of fat, 282; function of vacuole,
289; gas vacuoles, 290. Balbiani,
2, 13;
conjugation,
15;
spore-
formation in Sporozoa,
154;
classifica-
tion, 167; nucleus of Loxophyllum, 189,
190; nucleus, 250, 252; mitosis in micro-
nuclei, 261; in Sfzrochona, 262; function
of vacuole, 289; merotomy, 302. Balbiani,
2, 13;
conjugation,
15;
spore-
formation in Sporozoa,
154;
classifica-
tion, 167; nucleus of Loxophyllum, 189,
190; nucleus, 250, 252; mitosis in micro-
nuclei, 261; in Sfzrochona, 262; function
of vacuole, 289; merotomy, 302. Barbagello, dysentery, 64. Barbagello, dysentery, 64. Barry, nucleus, 2; Protozo Barry, nucleus, 2; Protozoa as single cells,
Il. De Bary, 18; pseudopodia in mycetozoa,
84; irritability, 299. Van Beneden, maturation, 233. Bergh, 21; animals and plants, 25; cuticula, Barry, nucleus, 2; Protozoa as single cells,
Il. Calendruccio, dysentery, 64.
Calkins, odors due to Protozoa, Cattaneo, swarm-spores in .47‘ced/a, 95.
9 Cassagrandi, dysentery, 64.
Cattaneo, swarm-spores in .4 INDEX OF AUTHORS Barry, nucleus, 2; Protozoa as single cells,
Il. De Bary, 18; pseudopodia in mycetozoa,
84; irritability, 299. Van Beneden, maturation, 233. Bergh, 21; animals and plants, 25; cuticula, Il. De Bary, 18; pseudopodia in mycetozoa,
84; irritability, 299. 84; irritability, 299. Van Beneden, maturation, 233. Bergh, 21; animals and plants, Bergh, 21; animals and plants, 25; cuticula,
38;
coloring matter
in Dinoflagellidia,
117; feeding in Dinoflagellidia, 126; in-
ter-relations, 135; classification, 140, 206;
nuclei, 250, 251. Berthold, explanation of pseudopodia-forma- Bergh, 21; animals and plants, 25; cuticula,
38;
coloring matter
in Dinoflagellidia,
117; feeding in Dinoflagellidia, 126; in-
ter-relations, 135; classification, 140, 206;
nuclei, 250, 251. Berthold, explanation of pseudopodia-forma- Berthold, explanation of pseudopodia-forma-
tion, 84. Bichat, 2. Berthold, explanation of pseudopodia-forma-
tion, 84. Bichat, 2. Calendruccio, dysentery, 64. Calkins, odors due to Protozoa, Blainville, spontaneous generation, 28. Blochmann, conjugation in Fuglypha, 2 Blochmann, conjugation in Fuglypha, 219,
234; division of Zuglena, 256, 259. Carpenter, classification, 18, 107, 108, 109;
types of shells, 73; food-selection, 305. Carter, 109; conjugation in Audorina, 134; Bourne, bacteria in Protozoa, 83; nuclei of
Pelomyxa, 87. Boveri, origin of the mitotic spindle, 248; of Carter, 109; conjugation in Audorina, 134;
classification, 138; conjugation in Masti-
gophora, 224, 228, 232. Carus, C. G., 10;
spontaneous generation, Boveri, origin of the mitotic spindle, 248; of
the centrosome, 276. Brady, classification, 107, 108, 109; shell, 305. Brady, classification, 107, 108, 109; shell, 305. Brandt, symbiosis in Radiolaria, 71; conju- Carus, C. G., 10;
spontaneous generation,
25. Carus, J. V., classification, Sporozoa, 168. Brandt, symbiosis in Radiolaria, 71; conju-
gation, 95;
nucleus,
249;
function
of
vacuole, 289; merotomy, 301. Brauer, encystment of Actinospherium, 90; Carus, J. V., classification, Sporozoa, 168. Cassagrandi, dysentery, 64. Brauer, encystment of Actinospherium, 90;
maturation, 235; mitosis, 264. "3 Cassagrandi, dysentery, 64. Cattaneo, swarm-spores in Cattaneo, swarm-spores in .47‘ced/a, 95. 9 "329 INDEX OF AUTHORS 330 23, 33, 106, 107, 137, 138, 139, 206, 207,
208, 209;
sarcode,
34;
immortality
of
Protozoa, 60; pseudopodia and’ flagella,
44;
gastric vacuoles
in rhizopods,
91;
consciousness
in Protozoa,
280,
303;
function of the vacuole, 289. Dunal, odors due to Protozoa, 62. Celli, malaria, 163. Celli and Fiocca, dysentery, 64. Certes, respiration, 289. Celli, malaria, 163. Celli and Fiocca, dysentery, 64. Certes, respiration, 289. Celli, malaria, 163. Celli and Fiocca, dysentery, 64. Certes, respiration, 289. INDEX OF AUTHORS Cienkowsky, 13;
animals Cienkowsky, 13;
animals and plants, 23;
symbiosis in Radiolaria, 71; pseudopodia-
formation, 83; budding Clathrulina, 96;
classification,
106, 109,
137,
138, 139;
conjugation of monads, 132; A/udlticilia,
200; conjugation in Voctiluca, 219; food-
selection, 305. Claparéde, 12; classification, 18, 21, 22; gas Dunal, odors due to Protozoa, 62. Ecker, pseudopodia-furmation, 83. Ehrenberg, 9, 13, 15; classification, Ecker, pseudopodia-furmation, 83. Ehrenberg, 9, 13, 15; classification, 15, 18,
20, 33, 106, -137, 138-40, 206, 207, 208,
210; immortality of Protozoa, 60; odors
due
to
Protozoa,
62;
Cvroglena,
130;
markings on
ciliates,
177;
conjugation,
222; function of vacuole, 292. Claparéde, 12; classification, 18, 21, 22; gas
vacuoles in rhizopods, 89. Claparéde and Lachmann, classification, 106, Claparéde and Lachmann, classification, 106,
140; myonemes, 180; classification, 206,
207, 208, 209, 210; conjugation, 217. Clark-James,
feeding
in Choanoflagellida, Clark-James,
feeding
in Choanoflagellida,
127; classification, 137. Claude-Bernard, 29. Eimer, consciousness in Protozoa, 304. Eismond, suction in the tentacles of Suctor Eismond, suction in the tentacles of Suctoria,
196. —
Ellis, discovery of trichocysts, II. Claude-Bernard, 29. Cohn, F., 23; conju Ellis, discovery of trichocysts, II. Engelmann,
12;
conjugation,
14, Cohn, F., 23; conjugation of Volvox, 134;
of Gontum, 226; irritability, 296. Cohn, L., dysentery, 64; mesoplasm, 144; Engelmann,
12;
conjugation,
14, 61, 193,
212,
213,
220, 222, 241;
stigmata,
37,
118; inotogmata and pseudopodia forma-
tion, 83; gas vacuoles in rhizopods, 89;
chlorophyl
in
Vorticella,
174;
nemato-
cysts in Vorticella, 176; myonemes, 180;
classification,
207, 209;
digestion, 283;
gas vacuoles, 2y0; irritability, 296, 297. Cohn, L., dysentery, 64; mesoplasm, 144;
myonemes of Vorticella, 180, 207, 209. Corti, 7; digestion, 281; respiration, 288. myonemes of Vorticella, 180, 207, 209. Corti, 7; digestion, 281; respiration, 288. Councilman, dysentery, 64. Cunningham, dysentery, 64. Corti, 7; digestion, 281; respiration, 288. Councilman, dysentery, 64. Cunningham, dysentery, 64. Cunningham, dysentery, 64. Dallinger and Drysdale, spontaneous genera-
tion, life-history of monads, 28, 132;
fla-
gellum-formation,
120;
conjugation
of
flagellates, 219, 221. Dangeard, feeding in Dinoflagellidia, 126; Entz, Actinobolus, 50; symbiosis in Radio-
laria, 71; gas vacuoles, 89; classification,
106, 109, 206, 207;
mouth of Choano-
flagellida, 123; nutrition in Mastigophora,
126, 127; myonemes in ciliates, 178, 180;
inter-relations of Infusoria, 201; tentacles
of Actinobolus,
183; gas vacuoles, 290;
function of vacuole, 291. Dangeard, feeding in Dinoflagellidia, 126;
in Suctoria, 196; conjugation, 219. Danilewsky, Polymitus form, 162; classifica- : Danilewsky, Polymitus form, 162; classifica- :
tion, 168. Gabriel, Sporozoa as plants, 166, INDEX OF AUTHORS 95; budding in Clathrulina, 96;
origin
of tentacles, 197; sex, 211; maturation,
235, 237, 238; nucleus, 249, 261; mitosis
in Actinospharium,
261, 266; origin of
centrosome, 276. Gasser, dysentery,*64. Geddes and Thompson, origin of sex, 212. Giard, classification Sporozua, 168. Gleichen, 7; function of the vacuole, 288. Goeze, digestion, 281. Hertwig and Lesser, 109; excretory granules,
286. Goldfuss, spontaneous generation, 28. Goroschankin, conjugation of Mastigoph Goroschankin, conjugation of Mastigophora,
224, 228. Hieronymous, conjugation in Gontum, 227. Hill, classification, 206. Hofer, 2; enucleated Amada, 86, 302; se- Gould, Bacteria in Rhizopoda, 83. Grassi,
dysentery,
64; classificati Hofer, 2; enucleated Amada, 86, 302; se-
cretion, 292. Hofmeister, pseudopodia-formation, 84. Grassi,
dysentery,
64; classification,
138,
168; malaria, 163, 164. Hofmeister, pseudopodia-formation, 84. Holman, conjugation in Amedla, 97, 217. Gray, classification, 109. Greeff, 33; contractile fib Holman, conjugation in Amedla, 97, 217. Hooke, 6. Greeff, 33; contractile fibres in Amada, 83;
budding in Clathrulina, 96; membrane in
Actinospharium, 105; classification, 106, ©
109; conjugation in Zoothamnium, 222;
division-centre in Heliozoa, 266. Greenwood,
digestion
in
rhizopods, Howell, starch, 281; calcium in man, 288. Hoyer, conjugation, 194. Hoyer, conjugation, 194. Huxley, classification, 18, Huxley, classification, 18, 207; animals and
plants, 23; spontaneous generation, 29;
epidemic among silkworms, 165. Greenwood,
digestion
in
rhizopods,
280; in Ciliata, 285. 92, Ishikawa, tentacles of Suctoria, 197; conju-
gation in Mocliluca, 219; nucleus, 252;
mitosis in Mocti/uca, 267. Israel, bacteria in Rhizopoda, 83. Grenacher, division-centre in Heliozoa, 266. Griffiths, excretion of urea, 291. Gruber, 2; conjugation, 15,97; Actinophrys,
69; classification,
106,
206, 208, 210;
inter-relations of Sarcudina, 101; matura-
tion, 235, 241; nucleus, 250; mitosis, 4ctz-
nospherium, 264; merotomy, 302, 305. Gruby, classification, 137. Israel, bacteria in Rhizopoda, 83. Jackson, classification, 209. Jennings, irritability in Ciliata, 298. Jensen, lifting power of Paramecium, 181,
Jickeli, 15; conjugation in Rhizopoda, 97,
219. Joblot, 7; spontaneous generation, 26. Gruby, classification, 137. Gruithuisen, I1. Gruithuisen, I1. Gurley, Myxosporidiida, reproduction, 154; Gruithuisen, I1. Gurley, Myxospori Jickeli, 15; conjugation in Rhizopoda, 97,
219. Joblot, 7; spontaneous generation, 26. Gurley, Myxosporidiida, reproduction, 154;
classification, 169. Johnson, division of Stentor, 192; pseudo-
podia of Stentor, 183; plastogamy in Ac-
tinospherium, 218. Joly, odors due to Protozoa, 62. Jones, Rupert, classification, 108. Joukowsky, conjugation in Cihata, 220. Julin, function of the macronucleus, 188. INDEX OF AUTHORS Le Dantec, symbiosis in Radiolaria, 71; in Le Dantec, symbiosis in Radiolaria, 71; in
Infusoria, 174; digestion, 282, 284. Dawson, Eozo6én, 29. Dawson, Eozo6én, 29. Defrance, classification, Dawson, Eozo6én, 29. Defrance, classification, 107, 108. Fabre-Dumergue,
contractile
vacuole,
54,
187; classification, 209; digestion, 281, 284. Feletti, malaria, 163. Fisch, classification, 138. Fischer, classification, 109; structure of fla-
gella, 121. Fiszer, function of the vacuole, 288. Flemming, origin of the mitotic spindle, 248. Focke, 11. :
Feettinger, classification, 207. Fol, classification, 208. Foulke, Actinospherium, 97. Francé, collar of Choanoflagellida, 123, 127. Frenzel, classification, 106, 137,
138, 167,
210; pyxinine granules, 143; nucleus, 250. Fresenius, classification, 138, 207. Fromentel, classification, 139. Gabriel, Sporozoa as plants, 166, Defrance, classification, 107, 108. Delage and Hérouard,
18; ani Delage and Hérouard,
18; animals
and
plants, 22, 25; flagella~-motion, 121; con-
tractile vacuole of ciliates, 188; tentacles
of Suctoria, 195; nervous organ of lorti-
cella, 183; function of the vacuole, 290. Dewitz, thigmotaxis, 300. Fisch, classification, 138. Fischer, classification, 109; Fischer, classification, 109; structure of fla-
gella, 121. Fiszer, function of the vacuole, 288. Fischer, classification, 109; structure of fla-
gella, 121. Fiszer, function of the vacuole, 288. Fiszer, function of the vacuole, 288. Flemming, origin of the mitotic spind Dewitz, thigmotaxis, 300. Diesing, 20; animals and plant Dewitz, thigmotaxis, 300. Diesing, 20; animals and plants, 23; classi- Diesing, 20; animals and plants, 23; classi-
fication, 139, 140. Doflein, structure
of Sporozoa, 142, 144; Doflein, structure
of Sporozoa, 142, 144;
classification, 209; centrosomes in Ciliata,
272. Donné, classification, 138. Fol, classification, 208. Foulke, Actinospherium, 97. Foulke, Actinospherium, 97. Francé, collar of Choanoflagell Francé, collar of Choanoflagellida, 123, 127. Frenzel, classification, 106, 137,
138, 167, Donné, classification, 138. Dreyer, skeleton-formation, Frenzel, classification, 106, 137,
138, 167,
210; pyxinine granules, 143; nucleus, 250. Fresenius, classification, 138, 207. Frenzel, classification, 106, 137,
138, 167,
210; pyxinine granules, 143; nucleus, 250. Fresenius, classification, 138, 207. Dreyer, skeleton-formation, Radiolaria, 77;
secretion, 292, 308. Driiner, origin of the spindle, 248. Driiner, origin of the spindle, 248. Fromentel, classification, 139. Fromentel, classification, 139. Dufour, classification, 21, 167. Dujardin, classification, 6, 10, 1 Gabriel, Sporozoa as plants, 166, Dujardin, classification, 6, 10, 15, 16, 18, 20, INDEX OF ’
AUTHORS 331 Garry, irritability, 299. Gasser, dysentery,*64. 95; budding in Clathrulina, 96;
origin
of tentacles, 197; sex, 211; maturation,
235, 237, 238; nucleus, 249, 261; mitosis
in Actinospharium,
261, 266; origin of
centrosome, 276. INDEX OF AUTHORS Haeckel, 12; classification, 17, 21, 106, 109,
110, 208; animals and plants, 23; struc-
ture of Radiolaria, 69, 76; conjugation in
Radiolaria, 95; development
of Radio-
laria, 95; origin of Sporozoa, 167; phy-
logeny of Infusoria, 205, irritability, 300,
304, 306. Hallez, classification, 106. Haeckel, 12; classification, 17, 21, 106, 109,
110, 208; animals and plants, 23; struc-
ture of Radiolaria, 69, 76; conjugation in
Radiolaria, 95; development
of Radio-
laria, 95; origin of Sporozoa, 167; phy-
logeny of Infusoria, 205, irritability, 300,
304, 306. Hallez, classification, 106. Hammerschmitt, classification, 168. Harrington and Leaming; see Leaming. Harting, shell-formation, 293. Hartog, polar bodies, 238. Harvey, 5. Heidenhain, origin of spindle, 248. Heitzmann, pseudopodia-formation, 83. Henle, Sporozoa as worms, 166. Henneguy, classification, 169. Hermann, origin of spindle, 247, 248. Hertwig, O., rejuvenation, 212. Hertwig, R., 2, 12;
conjugation,
15, 211;
classification, 21, 106, 109, 140;
structure
of Radiolaria, 69; encystment of -fctinos-
pherium, 90; swarm-spores in aArcella, Kartulis, dysentery, 64. Kent, 21; nutrition in Mastigophora, 126;
Uroglena, 130;
classification,
137,
138,
139, 208,
209, 210;
conjugation,
217;
secretion, 292. Keuten, division of Euglena, 256. King and Rowney, Eozoén, 29. Kirschner, conjugation in Voliox, 232. Klebs, inter-relations of the Sarcodina, 99; Kartulis, dysentery, 64. Kent, 21; nutrition in Mastigophora, 126;
Uroglena, 130;
classification,
137,
138,
139, 208,
209, 210;
conjugation,
217;
secretion, 292. Keuten, division of Euglena, 256. King and Rowney, Eozoén, 29. Kirschner, conjugation in Voliox, 232. Klebs, inter-relations of the Sarcodina, 9 Klebs, inter-relations of the Sarcodina, 99;
of Mastigophora, 135; ectoplasmic modi-
fications, 113, 117; classification, 137, 138,
139; irritability, 296. Kofoid, classification, 140. Klebs, inter-relations of the Sarcodina, 99;
of Mastigophora, 135; ectoplasmic modi-
fications, 113, 117; classification, 137, 138,
139; irritability, 296. Kofoid, classification, 140. K6lliker,
12;
classification, 21; Sporozoa,
166; function of vacuole, 291. Korotneff, budding in Heliozoa, 95. Hermann, origin of spindle, 247, 248. Hertwig, O., rejuvenation, 212. Hertwig, R., 2, 12;
conjugation,
15, 211;
classification, 21, 106, 109, 140;
structure
of Radiolaria, 69; encystment of -fctinos-
pherium, 90; swarm-spores in aArcella, K6lliker,
12;
classification, 21; Sporozoa,
166; function of vacuole, 291. Korotneff, budding in Heliozoa, 95. Korotneff, budding in Heliozoa, 95. INDEX OF AUTHORS 332 Kostanecki, origin of spindle, 248, 274. Kowalewsky, classification, 209. Krassilstschik, conjugation in Polytoma, 221. Kruse, classification, 168. Kiihne, conjugation of 4mada, 218. Kostanecki, origin of spindle, 248, 274. Manson, malaria, 163-165. Marchiafava and Celli, /a&sm Kowalewsky, classification, 209. INDEX OF AUTHORS Marchiafava and Celli, /a&smodium malaria,
168. Krassilstschik, conjugation in Polytoma, 221. Kruse, classification, 168 Mark, polar bodies, 238. Massart, irritability, 298. Kruse, classification, 168. Kiihne, conjugation of 4mada, 218. Kiihne, conjugation of 4mada, 218. Massart, irritability, 298. Maupas, conjugation, 15, Maupas, conjugation, 15, 193, 207, 234, 2373
food-taking, 48; senescence, 60; preda-
tory ciliates, 175, 199 ; inter-relationships
of Infusoria, 201, 204 ; mitosis in micro-
nuclei, 261;
digestion, 283;
excretory
granules, 286;
function of the vacuole,
288, 291; rejuvenescence, 212, 220. Meissner, digestion in Rhizopoda, 92, 280, Labbé, 22; protoplasmic structure of Sporo-
zoa, 142, 144; division in Sporozoa, 149 ;
spore-furmation, 151; malaria, 163; clas-
sification, 167-169; nucleus, 250, 254. Lachmann,
myonemes
in Vorticella,
178;
classification,
209. See Claparéde and
Lachmann. Meissner, digestion in Rhizopoda, 92, 280,
284. Mereschowsky,
Meissner, digestion in Rhizopoda, 92, 280,
284. Mendelssohn, irritability, 295. classification,
109,
1383, Lamarck, 9; spontaneous generation, 28;
classification, 107, 108, 109, 208. Lambl, dysentery, 64. Mereschowsky,
Mendelssohn, irritability, 295. classification, Lambl, dysentery, 64. Mereschowsky,
classification,
phylogeny of Ciliata, 205. Mesnil, conjugation in Sporozoa, 225. 109,
1383, Lameere, 12. Langmann, Myxosporidiida
in reptiles and Lameere, 12. Langmann, Myxo Lameere, 12. Langmann, Myxosporidiida
in repti Mesnil, conjugation in Sporozoa, 225. Metschnikoff, 13; gastric vacuoles in R Langmann, Myxosporidiida
in reptiles and
amphibia, 165. Lankester, classification,
18, 169; flagella- Metschnikoff, 13; gastric vacuoles in Rhizo-
poda, 91; malaria,
163; myonemes
of
Vorticella, 180; digestion, 283, 284. Meyen, II. Lankester, classification,
18, 169; flagella-
motion, 121,
Lauterborn, classification, 137, 139; division Meyen, II. Meyer, nutrition in Mastigophora, 126. Lauterborn, classification, 137, 139; division
of Ceratium, 260; historic origin of cen-
trosome, 276. Laveran,
dysentery,
643
Sporozoa,
159; Meyer, nutrition in Mastigophora, 126. Milne-Edwards, spontaneous generation, 29. Meyer, nutrition in Mastigophora, 126. Milne-Edwards, spontaneous generation, 29. Meyer, nutrition in Mastigophora, 126. Milne-Edwards, spontaneous generation, 29. Mingazzini,
life-cycle
of Coccidiida,
158; Milne-Edwards, spontaneous generation, 29. Mingazzini,
life-cycle
of Coccidiida,
158; Laveran,
dysentery,
643
malaria, 162-165. Leaming and Harrington, effect of light on
Sporozoa,
159; Mingazzini,
life-cycle
of Coccidiida,
158;
classification, 168, 169; nuclei, 254. Mingazzini,
life-cycle
of Coccidiida,
158
classification, 168, 169; nuclei, 254. Minot, rejuvenescence, 212. Miller, classification, 109. Montfort, classification, 107. Moore, nerve-organ of J orticella, 183. Mottier, origin of spindle, 248. Leaming and Harrington, effect of light on
Ameba, 297. Le Blanc, swarm-spores in Diflugia, 95. Minot, rejuvenescence, 212. Miller, classification, 109. Montfort, classification, 107 Le Blanc, swarm-spores in Diflugia, 95. INDEX OF AUTHORS Roux, cytotropy, 218. Riickert, origin of the Riickert, origin of the centrosome, 276. Perty, 12, 13, 22; animals and plants, 25;
nutrition in Mastigophora, 126; classifi-
cation, 138, 139, 207, 208; digestion, 282;
gas vacuoles, 290; consciousness, 303. Petridis, dysentery, 64. Sacharoff, malaria, 163. St. Vincent, classification, 137, 140. Sandahl, classification, 107. Sanfelice, malaria, 163. Sars, classification, 107. Sassaki, division-centre in Heliozoa, 266. Schaffhausen, Vorticella movement, 180. Schaudinn, 2; axial filaments, 79, 82, 10 Sacharoff, malaria, 163. St. Vincent, classification, 137, 140. Sandahl, classification, 107. Sandahl, classification, 107. Sanfelice, malaria, 163. Petridis, dysentery, 64. Pfeffer, chemotaxis in reproductive element Sanfelice, malaria, 163. Sars, classification, 107. Pfeffer, chemotaxis in reproductive elements,
297. Sars, classification, 107. Sassaki, division-centre in Heliozoa, 266. Pfeiffer, L., classification, 169. Pfeiffer,
R., life-cycle
of Cocc Schaffhausen, Vorticella movement, 180. Pfeiffer,
R., life-cycle
of Coccidium, 158;
Serumsporidium, 165. Schaudinn, 2; axial filaments, 79, 82, 101;
Parameba,
93;
conjugation
in Reticu-
lariida, 95, 99;
in Actinophrys, 97;
in
Sporozoa,
156, 158; classification,
109;
conjugation of Rhizopoda, 218, 219; nu-
cleus, 250; division of Ameba crystaili-
gera, 260; mitosis in Heliozoa, 266; in
Parameba,
268;
origin of centrosome,
276. Phillips, classification, 207. Pineau, 12. Pineau, 12. Plate, mitosis in Spzrochona, 262. Podwyssozki, microsporozoite
an Podwyssozki, microsporozoite
and macro-
sporozoite, 158. Pouchet, stigmata, 119;
catenation,
131; Pouchet, stigmata, 119;
catenation,
131;
respiration, 288,
Pringsheim, conjugation of Pandorina, 132, Pringsheim, conjugation of Pandorina, 132,
228. Schewiakoff,
structure
of Luglypha,
68;
membrane of gregarine, 145; movement
of gregarines, 149; protoplasm of ciliates,
174, 178; myonemes, 180; classification,
208, 209; nucleus, 250; division of Zugly-
pha, 265; excretory granules, 286; secre-
tion, 292. Prowazek, conjugation in Luglypha, 234. Przesmycki, excretory granules, 287. Przesmycki, excretory granules, 287. Purkinje and Valentin, classification, 20 Purkinje and Valentin, classification, 207. Purkinje and Valentin, classification, 207. Quatrefages,
spontaneous
generation,
29;
phosphorescence in Noctiluca, 293. Quennerstedt, classification, 206. Schilling, feeding in Dinoflagellidia, 126. Schleiden, 10. Quennerstedt, classification, 206. Schleiden, 10. Schlumberger, dimorphism in Reticulariida, Schlumberger, dimorphism in Reticulariida,
95; classification, 106. Schmarda, feeding in Dinoflagellidia, 126; Rabl, origin of the spindle, 274. Racovitza and Labbé, classification, 168. Radziszewski, phosphorescence, 294. Rabl, origin of the spindle, 274. Racovitza and Labbé, classification, 168. Radziszewski, phosphorescence, 294. Schmarda, feeding in Dinoflagellidia, 126;
classification, 139. ? Schmidt, 11; function of vacuole, 289. Radziszewski, phosphorescence, 294. Reaumur, spontaneous generation, 26. Reaumur, spontaneous generation, 26. Redi, Sporozoa, 21. Schmidt, 11; function of vacuole, 289. INDEX OF AUTHORS Schneider, classification, 106, 109, 167- Redi, Sporozoa, 21. Reinhardt, conjugatio Redi, Sporozoa, 21. Reinhardt, conjugation in flagellates, 224. Schneider, classification, 106, 109, 167-169;
carminophilous granules, 143;
nuclei of
Sporozoa, 146; spore-formation in Sporo-
zoa, 152; nucleus, 254. Schrank, classification, 140, 206. Reinhardt, conjugation in flagellates, 224. Reuss, classification, 107, 108. Reuss, classification, 107, 108. Rhumbler, structure of Zzglypha Rhumbler, structure of Zzglypha, 68; pseu-
dopodia-formation, 84;
ecto- and endo-
- plasmic interchange, 85; gas vacuoles, 89;
axial filaments, 101; contractile vacuoles
in ciliates, 188; conjugation in Rhizopoda,
217; action of vacuole, 289; function, 291;
secretion, 292;
irritability, 297;
experi-
ments with inorganic fluids, 306-309. Risso, classification, 109. Robin, classification, 136. Roboz, classification, 107. Rémer, classification, 109. Rémpel, classification, 209. Rood, function of the vacuole, 291. Rosenhof, irritability, 299. Ross, malaria, 164. Rossbach, function of the vacuole, 288, 289. Rhumbler, structure of Zzglypha, 68; pseu-
dopodia-formation, 84;
ecto- and endo-
- plasmic interchange, 85; gas vacuoles, 89;
axial filaments, 101; contractile vacuoles
in ciliates, 188; conjugation in Rhizopoda,
217; action of vacuole, 289; function, 291;
secretion, 292;
irritability, 297;
experi-
ments with inorganic fluids, 306-309. Risso, classification, 109. Rhumbler, structure of Zzglypha, 68; pseu-
dopodia-formation, 84;
ecto- and endo-
- plasmic interchange, 85; gas vacuoles, 89;
axial filaments, 101; contractile vacuoles
in ciliates, 188; conjugation in Rhizopoda,
217; action of vacuole, 289; function, 291;
secretion, 292;
irritability, 297;
experi-
ments with inorganic fluids, 306-309. Risso, classification, 109. Robin, classification, 136. Roboz, classification, 107. Rémer, classification, 109. Rémpel, classification, 209. Rood, function of the vacuole, 291. Rosenhof, irritability, 299. Ross, malaria, 164. Rossbach, function of the vacuole, 288, 289. Schrank, classification, 140, 206. Schuberg, dysentery, 64; life-cyc Schuberg, dysentery, 64; life-cycle of Coc-
cidiida, 158; membrane of ciliates, 176;
classification, 206, 207, 208. Schultze, F. E., inter-relations of the Sarco- Schultze, F. E., inter-relations of the Sarco-
dina, 103;
classification,
106, 109, 137;
conjugation of Rhizopoda, 217; division-
centre in Heliozoa, 266; excretory gran-
ules, 286; irritability, 295. Schultze, M., protoplasm and sarcode, 12; Risso, classification, 109. Robin, classification, 136. Schultze, M., protoplasm and sarcode, 12;
‘axial
filaments,
79,
101;
classification,
107. Schiitt, coloring matter in Dinoflagellidia, Roboz, classification, 107. Rémer, classification, 109. Rémer, classification, 109. Rémpel, classification, 209. Rémpel, classification, 209. Rood, function of the vacuol Schiitt, coloring matter in Dinoflagellidia,
117; classification, 140. Schwalbe, function of the vacuole, 288. Rood, function of the vacuole, 291. Rosenhof, irritability, 299. INDEX OF AUTHORS Le Clerc, classification, 106. Montfort, classification, 107. Moore, nerve-organ of J ortic Le Clerc, classification, 106. Moore, nerve-organ of J orticella, 183. Mottier, origin of spindle, 248. Ledermiiller, 8, 22. Mottier, origin of spindle, 248. Miiller, Johannes,
13; classificat Leeuwenhoek, 5; spontaneous
generation,
26; classification, 140. Léger, structure of gregarines, 144; micro- Miiller, Johannes,
13; classification, 18, 21;
conjugation of Gozzum, 226. Miiller, O. F., 8, 13; classification, 15, 208 ; Léger, structure of gregarines, 144; micro-
sporozoites, 159; classification, 167. Miiller, O. F., 8, 13; classification, 15, 208 ;
spontaneous generation, 28. Munier-Chalmas
et Schlumberger,
dimor- Leidy, classification, 106, 138, 208. Leuckart, 21 ; animals and plants, 2 Munier-Chalmas
et Schlumberger,
dimor-
phism in Reticulariida, 95. Leuckart, 21 ; animals and plants, 23; life-
cycle of Cocetdium, 158; Sporozoa rela-
tionships, 166. Leydig,
12;
Sporozoa
inter-relationships, Nageli, spontaneous generation, 29, 169. Needham, theory of generation, 27. Nitsch, 25; classification, 138, 206, 207. Nussbaum, merotomy, 302. Leydig,
12;
Sporozoa
inter-relationships,
166; respiration, 289;
function of vacu-
ole, 291. Limbach, function of the vacuole, 288. Leydig,
12;
Sporozoa
inter-relationships,
166; respiration, 289;
function of vacu-
ole, 291. Limbach, function of the vacuole, 288. Limbach, function of the vacuole, 288. Linneus, 5. Limbach, function of the vacuole, 288. Linneus, 5. Oersted, animals and plants, 24. Oken, 9, 12; spontaneous generation, 28;
classification, 208,
WOrbigny, classification, 18, 107, 108, 109. Osterhout, origin of spindle, 248. Owen, II. Lister, alternation of generations in Reticu-
lariida, 74, 95. Loeb, irritability, 299. Loeb, irritability, 299. Loeb and Hardesty, ma Loeb, irritability, 299. Loeb and Hardesty, mac Loeb and Hardesty, macronucleus, 188,
Loesch, dysentery, 64. Loesch, dysentery, 64. Logan, Eozoén, 29. Logan, Eozoén, 29. Lutz, classification, 1 Parker, classification, 107, 108. Pasteur, spontaneous generation, 29. Pearl, irritability, 300. Pénard, structure of Pelomyxa, 38; contrac-
tile vacuole, 54; structure of Luglypha, Lutz, classification, 169. Maccallum, malaria, 164. Maclarland, origin of spind Maclarland, origin of spindle, 248. Mannaberg, dysentery, 64. Mannaberg, dysentery, 64. INDEX OF AUTHORS 333 68 ; food-taking in Heliozoa, QI; conju-
gation in Heliozoa, 97, 217, 219;
classifi-
cation, 106, 109;
phylogeny of Ciliata,
204. 68 ; food-taking in Heliozoa, QI; conju-
gation in Heliozoa, 97, 217, 219;
classifi-
cation, 106, 109;
phylogeny of Ciliata,
204. Rostafinski, conjugation in Gondtzm, 227. Rouget, Vorticella myonemes, 180. Roux, cytotropy, 218. Riickert, origin of the centrosome, 276. Rostafinski, conjugation in Gondtzm, 227. Rouget, Vorticella myonemes, 180. Roux, cytotropy, 218. INDEX OF AUTHORS Schwalbe, function of the vacuole, 288. Schwann, 2. Schwalbe, function of the vacuole, 288. Schwann, 2. Schwann, 2. ossbach, function of the vacuole, 288, 289. INDEX OF AUTHORS 334 Thélohan, protoplasmic structure of Sporo-
z0a, 142; reproduction in Sporozoa, 154;
classification, 169. Thompson, classification, 108. Thompson, classification, 108. Topsent, classification, 106. Siebold, V., 11; classification, 16; animals
and
plants,
22;
function
of vacuole,
289. Topsent, classification, 106. Trembley, 7; conjugation of Zoothamnium,
222. Siedlecki,
movement
of gregarines,
149;
conjugation, 156, 159, 225, 232; nucleus,
250;
division-centre
in Sporozoa,
258;
secretion, 292. Treviranus, 9, 28. Tyndall, spontaneou Tyndall, spontaneous generation, 29. Tyndall, spontaneous generation, 29. Vejdowsky, classification, 207. Verworn, 2, 32; isolated central Simond, conjugation in Coccidiida, 159. Sjébring, Sporozoa, 159. Sjébring, Sporozoa, 159. Sorokin, classification, 106 Verworn, 2, 32; isolated central capsule, 69;
enucleated Protozoa, 86; cytotropy, 218;
secretion, 292; irritability, 295, 297, 300,
302, 305, 306, 308, 309. Sorokin, classification, 106,
Spallanzani, 7, 11; spontane Spallanzani, 7, 11; spontaneous generation,
27; respiration, 288; irritability, 294. 27; respiration, 288; irritability, 294. Spencer, growth and reproduction, 212. Stache, classification, 107. Stahl, irritability, 295. Steenstrup, alternation of generations, 12. Stein,
12,
13, 22;
animals
and_
plants, 302, 305, 306, 308, 309. Villot, 12. Villot, 12. Walker and Boys, classification, 108. Wallich, pseudopodia-formation, 84; Wallich, pseudopodia-formation, 84; chito-
sarc, 293. Wasielewsky,
granules in Sporozoa,
143; Stein,
12,
13, 22;
animals
and_
plants,
24; classification, 15, 21, 106, 107, 109,
137, 138, 139, 167, 168, 206-210; shells
of
Dinoflagellidia,
117;
nutrition
in
Mastigophora, 126; inter-relationships of
Mastigophora, 135; Symphyta, 166; myon-
emes,
177,
178;
phylogeny
of Ciliata,
204; conjugation of Codosiga, 222; Chloro-
ONIUM, 225; Gontum, 226; Volvox, 232;
excretory granules, 286; function of vacu-
ole, 291. :
Steinhaus, classification, Sporozoa, 168. Wasielewsky,
granules in Sporozoa,
143;
conjugation, 159; origin of Sporozoa, 167. Watasé, sex, 243. Weber, pseudopodia-formation, 84. Wasielewsky,
granules in Sporozoa,
143;
conjugation, 159; origin of Sporozoa, 167. Watasé, sex, 243. Watasé, sex, 243. Weber, pseudopodia-forma Watasé, sex, 243. Weber, pseudopodia Weber, pseudopodia-formation, 84. Weismann, origin of death, 60; mat Weismann, origin of death, 60; maturation,
233. Wiegmann, function of the vacuole, 288, 289. Williamson, classification, 109. Wilson,
maturation,
233;
origin
of the
spindle, 248; vital phenomena, 279. Wolters, nuclei of Sporozoa, 146; conjuga- Wolters, nuclei of Sporozoa, 146; conjuga-
tion, 156, 225; maturation, 235; nucleus,
250. Wright, classification, 107, 210. Wrzesniowsky, classification, 206, 209. Wysotzki, classification, 139. Steinhaus, classification, Sporozoa, 168. INDEX OF AUTHORS Steinmann, shell-formation, 293. Wolters, nuclei of Sporozoa, 146; conjuga-
tion, 156, 225; maturation, 235; nucleus,
250. Steinmann, shell-formation, 293. Sterki, classification, 209. Steinmann, shell-formation, 293. Sterki, classification, 209. Steinmann, shell-formation, 293. Sterki, classification, 209. Sterki, classification, 209. Stohr, classification, 140. Stokes, contractile vacuole of Amada, 88; Sterki, classification, 209. Stohr, classification, 140. Wright, classification, 107, 210. Wrzesniowsky, classification, 206, 2 Stohr, classification, 140. Stokes, contractile vacuol Stokes, contractile vacuole of Amada, 88;
classification, 206, 207. Stolc, starch
digestion in Rhizopoda,
92, Wrzesniowsky, classification, 206, 209. Wysotzki, classification, 139. Wysotzki, classification, 139. Stolc, starch
digestion in Rhizopoda,
92,
284. Strasburger, origin of the spindle, 248; irri- Zacharias,
function
of pseudopodia, 90;
Uroglena, 130, 131; classification, 210. Zenker, classification, 210;
function of the Strasburger, origin of the spindle, 248; irri-
tability, 295, 297. tability, 295, 297. Suriray, classification, Zenker, classification, 210;
function of the
vacuole, 288. :
Zschokke, spore-cysts of AZyxobolus, 155. Suriray, classification, 140. Suriray, classification, 140. Zschokke, spore-cysts of AZyxobolus, 155. Zschokke, spore-cysts of AZyxobolus, 155. INDEX OF SUBJECTS Acantharia, 70, IIo. Acanthocystis,
skeleto Acantharia, 70, IIo. Acanthocystis,
skeleto Alveolininz, 107. Ammodiscus, 107. Alveolininz, 107. Ammodiscus, 107. Acantharia, 70, IIo. Acanthocystis,
skeleton,
75, 82; budding,
95;
axial filaments,
101; . classification,
109; centrosomes, 266,
Acanthonida, 110. Acanthospora,168. Acanthosporide, 168. Acephalina, 168. Acineta, budding, 199; classification, 210. Acineta-theory, Stein, 13. Acinetidz, 210. Actinelida, 110. Actinobolus,
food-taking,
50;
fig. p. 51;
tentacles, 183; classification, 206. Actinocephalidz, 167. Actinocephalus, 168. Actinolophus, skeleton, 74 ; pseudopodia, 82 ;
classification, 109 ; phylogeny, 204. Actinomonas,
103;
fig. p. 103;
classifica-
tion, 137. Actinophrys, 109;
fig. p. 16; pseudopodia,
82; contractile vacuole, 87; conjugation,
97, 218; axial filaments, 101. Actinospherium, alveoli in protoplasm, 35;
skeleton, 36;
encystment, go ;
artificial
reproduction, 97;
axial
filaments,
Io1 ;
membrane, 105 ; classification, 109 ;_ con-
jugation, 218;
mitosis, 264; maturation,
236; irritability, 295, 301. Actinotricha, 209. Actipylea, 70, 110. Actissa, fig. p. 17. Adelea, conjugation,
158, 229;
tion, 168 ; maturation, 237. Adinida, 5, 140. adoral zone, 6, 171. ligyria, 207. Agegregata, 167. Aggregatidee, 167. Allomorphina, 108. alternation
of generations, 12;
in Reticu-
lariida, 74, 95. alveolar structure of protoplasm, 35. Alveolina, 107. classifica- Acanthocystis,
skeleton,
75, 82; budding,
95;
axial filaments,
101; . classification,
109; centrosomes, 266, Ammodiscus, 107. Ameba coli, fig. p. Ameba coli, fig. p. 63. Amcebea, 18, 109; centrosomes, 266,
Acanthonida, 110. Amcebea, 18,
Amaba proteu . Amaba proteus,
ectoplasm,
fig. p. 36;
pseudopodia, 81; nucleus, 87; contractile
vacuole, 54, 88;
fig. p. 89; conjugation,
97, 105 ; classification, 105;
conjugation,
218;
nucleus, 252, 276;
secretion, 291,
292; irritability, 295, 297, 301. Amoebida, classification, 105. Ameebide, classification, 105. Ameebosporidia, 169. Amphidinium, 140. Amphidoma, 140. Amphileptus, 206;
irritability, 295;
food-
selection, 305. Ampbhilonchide, r1o. Amphinionas, 137. Amphisia, 209. Amphisolenia, 140. Amphistegina, 109. Amphistomine, 106. Amphitholus, 140. Amphitrema, 106. Ainphizsonella, 106. Amphoroides, 168. anal tube, in Infusoria, 186. Ancistrum, 207. Ancyromonas, 137. Ancyrophora, 168. androgonidia, in Volvox, 134. Androspyride, 110. Anentera, 9. Angiosporea, 167. Animalcula, 7. animals and plants, 22. anisogamy, 224. Antsonema, 139. anisospore, 95. Anomalina, 109. Anoplophrya,
207. Anthocyrtide, 111. Anthophysa, 137; secretion, 292. Acanthonida, 110. Acanthospora,168. Acanthospora,168. Acanthosporide, 16 Acanthosporide, 168. Acephalina, 168. Acephalina, 168. Acineta, budding, 199; classification, 210. Acineta-theory, Stein, 13. Acineta-theory, Stein, 13. Acinetidz, 210. 292; irritability, 295, 297, 301. Amoebida, classification, 105. Acinetidz, 210. Amoebida, classification, 105. Ameebide, classification, 105. Actinelida, 110. Actinobolus,
food Ameebide, classification, 105. Ameebosporidia, 169. Ameebide, classification, 105. Ameebosporidia, 169. Actinobolus,
food-taking,
50;
fig. Adelea, conjugation,
158, 229;
tion, 168 ; maturation, 237.
Adinida, 5, 140.
classifica- INDEX OF SUBJECTS p. 51;
tentacles, 183; classification, 206. Actinocephalidz, 167. Actinocephalidz, 167. Actinocephalus, 168. Actinocephalidz, 167. Actinocephalus, 168. Actinolophus, skeleton, Actinocephalus, 168. Actinolophus, skeleton, Actinospherium, alveoli in protoplasm, 35;
skeleton, 36;
encystment, go ;
artificial
reproduction, 97;
axial
filaments,
Io1 ;
membrane, 105 ; classification, 109 ;_ con-
jugation, 218;
mitosis, 264; maturation,
236; irritability, 295, 301. Actinotricha, 209. Actipylea, 70, 110. Actipylea, 70, 110. Actissa, fig. p. 17. Adelea, conjugation,
158, 229;
tion, 168 ; maturation, 237. Adinida, 5, 140. classifica- adoral zone, 6, 171. ligyria, 207. 335 INDEX OF SUBJECTS 336 Bifarina, 108. Bigenerina, 108. Biloculina, fig. p. 74; classification, 107
Blanchardina, 169. Blepharacysta, 140. Blepharisma, 208,
Bodo,
138; conjugation,
2173;
irritab
297. Bodonide, 138. Bolivina, 108. Botellina, 107. Bothriopsis, 168. Bradyina, 108. budding (see Reproduction), in Sarcod
95-
Bulimina, 108. Buliminine, 108. Bullaria, obsolete, 15. Bursaria, sphincter, 181; classification,
Bursariidz, 208. Bittschlia, 206. Cenomorpha, 208. Calcarina, 73, 109. Calcituba, classification, 107; nucleus,
chromosomes, 253, 254. calymma, 23, 69. Calyptotricha, 207. Campanella, adoral zone, 202. Campascus, 106. Camptonema, pseudopodia, 82. Candeina, 108. Cannobotryide, 110. Cannopylea, 70, III. Cannoraphide, I11. Cannospheeridee, 111. capitate tentacles, fig. p. 196; function,
carbohydrates in digestion, 282. carbon
dioxid
in contractile
vacuole,
288, 289. Carchesium, 209; digestion in, 285. carminophilous granules, 143. Carpenteria, 109. Carteria, 139. Carterina, 107. Caryolysus, 168. Caryophagus, 168. Cassidulina, 108. Cassiduline, 108. Castanellide, 111. catenoid colony, 57. cell-structure, 246. Bifarina, 108. Bigenerina, 108. Biloculina, fig. p. 74; classification, 107. Blanchardina, 169. Blepharacysta, 140. Blepharisma, 208,
Bodo,
138; conjugation,
2173;
irritability
297. Bodonide, 138. Bolivina, 108. Botellina, 107. Bothriopsis, 168. Bradyina, 108. budding (see Reproduction), in Sarcodina,
95-
Bulimina, 108. Buliminine, 108. Bullaria, obsolete, 15. Bursaria, sphincter, 181; classification, 208
Bursariidz, 208. Bittschlia, 206. Aphrothoracida, 109. arboroid colony, 57. arboroid colony, 57. Arcella, pseudopodia, Arcella, pseudopodia, 86; contractile vacu-
ole, 87; budding, 95; classification, 106;
conjugation, 218; gas vacuoles, 290. Arcellidz, 106. Arcellidz, 106. Archeodiscus, 109. archigony, 29. archispore, I51. archispore, I51. archoplasm, 274. archoplasm, 274. Arcuothrix, 106. Arcuothrix, 106. Articulina, 107. Artiscide, 110. Artiscide, 110. Artodiscus, cause Artodiscus, cause of motion, IOI. Aschemonella, 107. Aschemonella, 107. Ascoglena, 138. Buliminine, 108. Bullaria, obsolete, 15. Bursaria, sphincter, 181; classificatio
Bursariidz, 208. Bittschlia, 206. Cenomorpha, 208. Calcarina, 73, 109. Calcituba, classification, 107; nucleus
chromosomes, 253, 254. calymma, 23, 69. Calyptotricha, 207. Campanella, adoral zone, 202. Campascus, 106. Camptonema, pseudopodia, 82. Candeina, 108. Cannobotryide, 110. Cannopylea, 70, III. Cannoraphide, I11. Cannospheeridee, 111. capitate tentacles, fig. p. INDEX OF SUBJECTS 19; nutrition, double
nature, 25, 126; classification, 139. Chrysalidina, 108. Chrysameba, 139. Chrysococcus, 139. Chrysomonadide, 139. Chrysopyxis, 139. Chrysospherella, 139. chyle, 92. cilia, structure, 181. Ciliata, classification, 206,
Cilio-flagellata, 121. Ciliophrys, 103, 137. Cinetochilum, 207. cirri, structure, 182. Citharistes, 139. Cladomonas, 137. classification, history of, 15. Clathrulina,
skeleton,
75;
96; classification, 109. Clavulina, 108. Climacostomum, vacuole, 187; classification,
208. Cnemidiophora, 167. Coccidiida, 21, 168. Cocetdium,
spores,
classification, 168. Coccodiscide, 110. Coccomonas, 139. Cochliopodium, 106. Codonella, 208. swarm-spores,
153;
life-cycle,
158; chromatoid granules, 144. chromatophore, 37. chromatospherite, 254. Chromomonadina, 139. chromosomes, 247. E
Chromutina,
fig. p. 19; nutrition, double
nature, 25, 126; classification, 139. Chrysalidina, 108. Chrysameba, 139. Chrysococcus, 139. Chrysomonadide, 139. Chrysopyxis, 139. Chrysospherella, 139. chyle, 92. cilia, structure, 181. Ciliata, classification, 206,
Cilio-flagellata, 121. Ciliophrys, 103, 137. Cinetochilum, 207. cirri, structure, 182. Citharistes, 139. Cladomonas, 137. classification, history of, 15. Clathrulina,
skeleton,
75;
96; classification, 109. Clavulina, 108. Climacostomum, vacuole, 187; classification,
208. Cnemidiophora, 167. Coccidiida, 21, 168. Cocetdium,
spores,
classification, 168. Coccodiscide, 110. Coccomonas, 139. Cochliopodium, 106. Codonella, 208. Codonocladium,
137. Codoneca, gelatinous cup, 114; classification,
137. Codoncecidee, 137. Codosiga, fig. p. 57; colony-form, 131; classi-
fication, 137; conjugation, 222. Coelodendride, 111. Coelographidee, 111. Calomonas, 139. Colacium, 138. Coleorhynchus, 168. Coleps, covering, 176; fig. p. 176; classifica-
tion, 206. collar, in Choanoflagellida, 122. Collodictyon, 138. Colloidida, 110. Collospheeridze, 110. swarm-spores,
153;
life-cycle,
158;
stalk,
131;
classification, centrosome; see division-centre,
centrosphere, 246. centrosphere, 246. Cephalina, 167. cephalont, 145. Chromomonadina, 139. cephalont, 145. Cephalothamnium chromosomes, 247. Cephalothamnium, 137. Chromutina,
fig. p. 19; nutrition, double
nature, 25, 126; classification, 139. Chrysalidina, 108. Ceratium, 41; shell, 116; catenation, i303
classification, 140; mitosis, 260. Ceratocorys, 136, 140. Ceratomyxa, 169. Ceratomyxa, 169. Ceratospora, 168. Ceratospora, 168. Cercomonadidee, 13 Cercomonadidee, 137. Cercomonas, reproduct Cercomonas, reproduction,
1323
classifica-
tion, 137; conjugation, 214,
Cercoporide, 111. Cercoporide, 111. Certesia, 209. Chetoproteus, Chetoproteus, 106. Chalarothoracida, 10 Chalarothoracida, 109,
Challengeride, 111. Challengeride, 111. Chasmostoma, 207. chemotaxis, 297. chemotaxis, 297. Chiastolide, 110. Chiastolide, 110. Chiliferidee, 207. Chiliferidee, 207. ‘Chilodon, 207; nu ‘Chilodon, 207; nucleus, 263. Chilodontinze, 207. Chilodontinze, 207. Chilomonas, fig. p. Chilomonas, fig. p. 36; protoplasmic struc-
ture,
113;
starch,
118;
nucleus,
124;
classification, 139; irritability, 296, 300. Chilostomella, 108. Chilostomella, 108. Chilostomellide, 108. chitin-shell, 39. chitin-shell, 39. chitosarc, 293. Cocetdium,
spores,
classification, 168. Coccodiscide, 110. 153;
life-cycle,
158; chitosarc, 293. Chlamydodon, Chlamydodon, 207. Chlamydodontide, 2 Coccodiscide, 110. Coccomonas, 139. Chlamydodontide, 207. Chlamydomonadide, 139. Chlamydomonadina, 139. Chlamydomonadina, 139. INDEX OF SUBJECTS 196; functio
carbohydrates in digestion, 282. carbon
dioxid
in contractile
vacuol
288, 289. Carchesium, 209; digestion in, 285. carminophilous granules, 143. Carpenteria, 109. Carteria, 139. Carterina, 107. Caryolysus, 168. Caryophagus, 168. Cassidulina, 108. Cassiduline, 108. Castanellide, 111. catenoid colony, 57. cell-structure, 246. cellulose shells, 39. Cenodiscide, 110. central capsule, 17, 67. Ascoglena, 138. asexual reproduct asexual reproduction; see Reproduction. Aspidisca, 209. Aspidisca, 209. Astasia, membran Astasia, membrane, 114; classification, 138. Astasiida, 138. Cenomorpha, 208. Calcarina, 73, 109. Calcituba, classification, 107; nucleus,
chromosomes, 253, 254. calymma, 23, 69. Calyptotricha, 207. Campanella, adoral zone, 202. Campascus, 106. Camptonema, pseudopodia, 82. Candeina, 108. Cannobotryide, 110. Cannopylea, 70, III. Cannoraphide, I11. Cannospheeridee, 111. capitate tentacles, fig. p. 196; function,
carbohydrates in digestion, 282. carbon
dioxid
in contractile
vacuole,
288, 289. Astasiida, 138. Cenomorpha, 208. Calcarina, 73, 109. Asterophora, 168. Astomea, 138. Calcarina, 73, 109. Calcituba, classificati Astomea, 138. astral granule ( astral granule (Centralkorn), 18, 82. Astrodisculus, 109. Calcituba, classification, 107; nucleus, 250;
chromosomes, 253, 254. calymma, 23, 69. Astrodisculus, 109. Astrolonchide, Ir10. calymma, 23, 69. Calyptotricha, 207. Calyptotricha, 207. Campanella, adoral Astrolophide, I10. Astropyle, 70. Campanella, adoral zone, 202. Campascus, 106. Astropyle, 70. Astrorhiza, 107. Astropyle, 70. Astrorhiza, 107. Astrorhizide, 107. Astrorhizinz, 107. Astrospheeride, 110. Astylozoén, 209. Atractonema, 138. attraction sphere, 246. Aulocanthide, III. Aulospheeride, 111,
auto-infection, 158. axial filament, 81, 265. axoneme, in Vorticella, 179. axopodia
(pseudopodia
with
axial
fila-
ments), Dimorpha, 101. Campascus, 106. Camptonema, pse Astrorhiza, 107. Astrorhizide, 107 Camptonema, pseudopodia, 82. Candeina, 108. Astrorhizide, 107. Astrorhizinz, 107. Candeina, 108. Cannobotryide, Astrorhizinz, 107. Astrospheeride, 110. Cannobotryide, 110. Cannopylea, 70, III. Astrospheeride, 110. Astylozoén, 209. Cannopylea, 70, III. Cannoraphide, I11. Astylozoén, 209. Atractonema, 138 Cannoraphide, I11. Cannospheeridee, 111. attraction sphere, 246. Aulocanthide, III. capitate tentacles, fig. p. 196; function, 195.. carbohydrates in digestion, 282. carbon
dioxid
in contractile
vacuole, 54, Aulocanthide, III. Aulospheeride, 111, carbohydrates in digestion, 282. carbon
dioxid
in contractile
v carbon
dioxid
in contractile
vacuole, 54,
288, 289. Carchesium, 209; digestion in, 285. axoneme, in Vorticella, 179. axopodia
(pseudopodia
wi carminophilous granules, 143. Carpenteria, 109. axopodia
(pseudopodia
with
axial
fila-
ments), Dimorpha, 101. Balantidiopsis, 208. Balantidium, fig. p. 63; classification, 208. Balladina, 209. barotaxis, 300. Barrouxta, 168. Bathysiphon, 107. Bdelloidina, 107. Belloides, 168. Benedenia, 168. Biccecidee, 137. Bicoseca, 137+ INDEX OF SUBJECTS 337 Centralkorn , see central granule; division-
centre. chromatin, 40, 253. chromatoid granules, 144. chromatophore, 37. chromatospherite, 254. Chromomonadina, 139. chromosomes, 247. E
Chromutina,
fig. p. Dactylophoride, 167.
Dactylophorus, 167.
Dactylosphara, 106.
Datlasia, 207.
Dallingeria, 138. INDEX OF SUBJECTS Chlamydomonas, odor in Codonella, 208. Codonocladium, 127;
reproduction,
131;
classification,
139; conjugation, 224. Chloromonadina, 139. Chloromyxidz, 169. Chloromyxum, spore-capsules, 156;
classi- Chloromyxidz, 169. Chloromyxum, spore- Chloromyxum, spore-capsules, 156;
classi-
fication, 169. chlorophyl,
distinguishing
animals
and Coleps, covering, 176; fig. p. 176; classifica-
tion, 206. collar, in Choanoflagellida, 122. chlorophyl,
distinguishing
animals
and
plants, 23; in Paramecium, 282. chlorophyllin, in Dinoflagellidia, 118. collar, in Choanoflagellida, 122. Collodictyon, 138. chlorophyllin, in Dinoflagellidia, 118. Choanoflagellida, 137. Collodictyon, 138. Colloidida, 110. Colloidida, 110. Collospheeridze, 1 Collospheeridze, 110. Chromatella, 106. Z INDEX OF SUBJECTS 338 Dasytricha, cortical plasm, 175; classi
tion, 207; cilia, 177. Dendrocometes, 210. Dendrometidz, 210. Dendromonas, stalk,
131; classification,
Dendrophrya, 107. Dendrosoma, 197, 210. Dendrosomide, 210. Desmothoracida, 109. deutomerite, 144. Diaspora, 168. diastole, 288. diatomin, 37. Dictyocysta, 208. dictyotic moment, 51, 76. Didinium, phylogeny, 206;
classificat
206; food-selection, 305. Didymophyes, protoplasmic structure,
classification, 167. Didymophyidz, 167. Diffiugia, pseudopodia, 86; budding,
conjugation, 97, 106, 218. digestion, 280. :
Dileptus, form, 172; trichocysts, 175; cla
fication, 206; food-selection, 305. Dimorpha, pseudopodia,. 82; fig. p. 1
classification,
137. dimorphism, in Reticulariida, 94. Dinema, 138. Diniferidee, 140. Dinobryon, 57;
fig. p. 115; classificat
139. Dinoflagellidia, 140. Dinophrya, 206. Diophrys, 209. Dinophyside, 140. Dinophysis, 140. Diploconide, 110. Diplocystis, skeleton, 75 ; classification,
Diplomita, 137. Diplophrys, 106,
Diplopsalis, 140. Diplosiga, collar, 123; classification, 137. Diplospora, 168. Discophrya,
vacuole,
207. Discorbina, 109; conjugation, 219. Discorhynchus, 168. Disporocystide, 168. Distephanus,
skeleton, 114;
classificat
140. Distomea, 138. Ditrema, 106. division-centre, 261, 266, 268, 272. dizoic cyst or spore, 153. 187;
classificat Collozoida, 110. colony-formation, varieties of, 57. color and odors in drinking water, 62. Colpidium, 207. Colpoda,
27;
cyclosis,
173;
classification,
207. ‘Colponema, 138. Cometoides, 168. Concharidee, III. Conchopthir
us, 208. Condylostomum, 208. conjugation, 41, 60; in ciliata, 193; general,
214. consciousness in Protozoa, 280, 304. contractile vacuole, 52; function, 187. Cornuspira, 107. Coronidz, 110. cortical plasm, 174. Coskinolina, 107. Cothurnia, 209. Craspedomonadide, 137. Cryptoglena, 138. Cryptomonadidee, 139. Cryptomonas, 139; irritability, 297. Crystallispora, 168. Cubospheeridee, 110. Cuneolina, 108. Cyathomonas, 139. Cyclammina, 108. Cyclidium, 207. Cyclocheta, 209. Cycloclypeina, 109. Cycloclypeus, 109. Cyclospora, 168. Cyphinidze, 110. Cyphoderia, protoplasmic structure, 68, 106,
Cyrtoidida, 111. cyst, varieties, fig. p. 47. Cystobia, 168. Cystocalpidee, 111. Cystocephalus, 168. Cystodiscus, 169. Cystoflagellidia, 140. Cytameba, 168. cytolymph, 246. cytophan, 179. cytoplasm, 246. Cytosporidia, 142. cytotropy, 217. Dactylophoride, 167. Dactylophorus, 167. Dactylosphara, 106. Datlasia, 207. Dallingeria, 138. Dasytricha, cortical plasm, 175; classi
tion, 207; cilia, 177. Dendrocometes, 210. Dendrometidz, 210. INDEX OF SUBJECTS Dendromonas, stalk,
131; classification,
Dendrophrya, 107. Dendrosoma, 197, 210. Dendrosomide, 210. Desmothoracida, 109. deutomerite, 144. Diaspora, 168. diastole, 288. diatomin, 37. Dictyocysta, 208. dictyotic moment, 51, 76. Didinium, phylogeny, 206;
classificat
206; food-selection, 305. Didymophyes, protoplasmic structure,
1
classification, 167. Didymophyidz, 167. Diffiugia, pseudopodia, 86; budding,
conjugation, 97, 106, 218. digestion, 280. :
Dileptus, form, 172; trichocysts, 175; cla
fication, 206; food-selection, 305. Dimorpha, pseudopodia,. 82; fig. p. 1
classification,
137. dimorphism, in Reticulariida, 94. Dinema, 138. Diniferidee, 140. Dinobryon, 57;
fig. p. 115; classificat
139. Dinoflagellidia, 140. Dinophrya, 206. Diophrys, 209. Dinophyside, 140. Dinophysis, 140. Diploconide, 110. Diplocystis, skeleton, 75 ; classification,
Diplomita, 137. Diplophrys, 106,
Diplopsalis, 140. Diplosiga, collar, 123; classification, 137. Diplospora, 168. Discophrya,
vacuole,
207. Discorbina, 109; conjugation, 219. Discorhynchus, 168. Disporocystide, 168. Distephanus,
skeleton, 114;
classificat
140. Distomea, 138. Ditrema, 106. division-centre, 261, 266, 268, 272. dizoic cyst or spore, 153. 187;
classificat Collozoida, 110. colony-formation, varieties of, 57. color and odors in drinking water, 62. Colpidium, 207. Colpoda,
27;
cyclosis,
173;
classification,
207. ‘Colponema, 138. Cometoides, 168. Concharidee, III. Conchopthir
us, 208. Condylostomum, 208. conjugation, 41, 60; in ciliata, 193; general,
214. consciousness in Protozoa, 280, 304. contractile vacuole, 52; function, 187. Cornuspira, 107. Coronidz, 110. cortical plasm, 174. Coskinolina, 107. Cothurnia, 209. Craspedomonadide, 137. Cryptoglena, 138. Cryptomonadidee, 139. Cryptomonas, 139; irritability, 297. Crystallispora, 168. Cubospheeridee, 110. Cuneolina, 108. Cyathomonas, 139. Cyclammina, 108. Cyclidium, 207. Cyclocheta, 209. Cycloclypeina, 109. Cycloclypeus, 109. Cyclospora, 168. Cyphinidze, 110. Cyphoderia, protoplasmic structure, 68, 106,
Cyrtoidida, 111. cyst, varieties, fig. p. 47. Cystobia, 168. Cystocalpidee, 111. Cystocephalus, 168. Cystodiscus, 169. Cystoflagellidia, 140. Cytameba, 168. cytolymph, 246. cytophan, 179. cytoplasm, 246. Cytosporidia, 142. cytotropy, 217. Dasytricha, cortical plasm, 175; classific
tion, 207; cilia, 177. Dendrocometes, 210. Dendrometidz, 210. Dendrosoma, 197, 210. Dendrosomide, 210. Desmothoracida, 109. deutomerite, 144. deutomerite, 144. Diaspora, 168. consciousness in Protozoa, 280, 304. contractile vacuole, 52; function, 187 contractile vacuole, 52; function, 187. Cornuspira, 107. Diffiugia, pseudopodia, 86; budding, 95
conjugation, 97, 106, 218. digestion, 280. : Cryptoglena, 138. Cryptomonadidee, 1 Cryptomonadidee, 139. Cryptomonas, 139; irrit Cryptomonas, 139; irritability, 297. Crystallispora, 168. Dileptus, form, 172; trichocysts, 175; classi
fication, 206; food-selection, 305. Dimorpha, pseudopodia,. 82; fig. p. 100 Crystallispora, 168. Cubospheeridee, 110. Cubospheeridee, 110. Cuneolina, 108. Dimorpha, pseudopodia,. 82; fig. p. 100
classification,
137. dimorphism, in Reticulariida, 94. dimorphism, in Reticulariida, 94. Dinema, 138. INDEX OF SUBJECTS 339 Doliocystide, 168,
Doliocystis, 168. Dorataspide, 110. Drepanomonas, 207. Luglypha, protoplasmic structure, 38, 68;
shell, 40;
encystment, fig. p. 41;
bud-
ding, fig. p. 55, 93; contractile vacuole,
87; encystment, 90;
classification, 106 ;
conjugation, 219 ; mitosis, 264. INDEX OF SUBJECTS Druppulidee, 110. dysentery, 64. Dysteria, 207. LEchinomera, 167. Ectoplasm,
34;
interchange
with
endo-
plasm, 85. ;
Ehrenbergina, 108. Llermocystis, fig. p. 58. Eikenia, 106. Eimeria, conjugation, 229. Ellipsidze, 110. Lllipsoidina, 108. Enchelinide, 206. Enchelys, 206 ; irritability, 295; food selec-
tion, 305. encystment, 46, 193. endogenous budding, 198. endogenous sporozoite-formation, 158. endoplasm,
34;
interchange
with
ecto-
plasm, 85. LEndosphera, 210. endospore, 151. Endothyra, 108,
Endothyrinz, 108. Enterodela, 9. Entodinium, 208. Entosiphon, 139. Lozoin, 29. Ephelota, fig. p. 58; classification, 210. Epiclintes, 209. epimerite, 144. Epipyxis, division, 128; classification, 137. epispore, 151. Lpistylis,
nematocysts,
209. Erviliinee, 207. Eudorina, colony-formation,
130;
classifi-
cation, 140; conjugation, 227. Euglena, fig. p. 37, 62; membrane, 114;
classification,
373 Euglyphidz, 106. Euplotes, fig. p. 54; classification, 209. Euplotidz, 209. Euspora, 167. Eutreptia, 138. excretion, 52. excretory granules, 286,
exogenous budding, 198. extra-capsular protoplasm, 69. extra-nuclear division-centre, 265. Lxuviella, inter-relations,
136;
classifica-
tion, 140. eye-spot;
see stigma. Euglyphidz, 106. Euplotes, fig. p. 54; classification, 209. Euplotidz, 209. Euspora, 167. Eutreptia, 138. excretion, 52. excretory granules, 286,
exogenous budding, 198. extra-capsular protoplasm, 69. extra-nuclear division-centre, 265. Lxuviella, inter-relations,
136;
classifica-
tion, 140. eye-spot;
see stigma. eye-spot;
see stigma. eye-spot;
see stigma. Fabularia, 107. fertilization, in Coccidiida, 159, 229. Flagellidia, 20;
classification, 137. flagellum, 60;
structure, absorption,
etc.,
119. flagellum fissure, 117. folliculina,
covering,
176;
classification,
208. food-selection, 305, 309. food-taking,
in
Mastigophora,
125;
in
Sporozoa, 146. Foraminifera, 18. frondicularia, 108. Frontonia, trichocysts, 175 ; vacuoles, 187;
classification, 207. Fusulina, 109. Fusulininz, 109. galvanotaxis, 300. Gamocystis, 167. gastric vacuole, in Sarcodina, 91;
in In-
fusoria, 185. Gastrostyla, 50, 209. gas vacuole, 290. gemmation;
see reproduction. generation de novo, 26. Gerda, degeneration of peristome, 204; clas-
sification, 209. Glaucoma, 207. Glenodinium, color in water, 62; shell, 1163
food-taking, 126; classification, 140. Clobigerina, 108. Globigerina ooze, 32. Globigerinidze, 108. Gloidium, 106. Glossatella, 140, 209. Glugea, spore-capsules, 156; silk-worm epi-
demic, 165 ; classification, 169. Gontodoma, 140. Gonium, colony-formation,
129;
classifica-
tion, 140; conjugation, 226. Fabularia, 107. fertilization, in Coccidiida, 159, 229. Flagellidia, 20;
classification, 137. flagellum, 60;
structure, absorption,
etc.,
119. flagellum fissure, 117. folliculina,
covering,
176;
classification,
208. food-selection, 305, 309. food-taking,
in
Mastigophora,
125;
in
Sporozoa, 146. Foraminifera, 18. frondicularia, 108. Frontonia, trichocysts, 175 ; vacuoles, 187;
classification, 207. Fusulina, 109. Flagellidia, 20;
classification, 137. flagellum, 60;
structure, absorption food-taking,
in
Mastigophora,
125;
in
Sporozoa, 146. Foraminifera, 18. LEndosphera, 210. endospore, 151. Foraminifera, 18. endospore, 151. Glugea, spore-capsules, 156; silk-worm epi-
demic, 165 ; classification, 169. INDEX OF SUBJECTS Gonyaulax, 140. Gonyostomum,
tr Hexamitus, 138. Gonyostomum,
trichocysts,
119;
classifica-
tion, 209. Hippocrepina, 107. flirmidium, 137. flirmidium, 137. Hirmocystis, 167. Goussia, 168. Hirmocystis, 167. Hlistioneis, 140. Grassia, 138;
relation to Ciliata, 200. Hlistioneis, 140. flolophrya, cilia, gregaloid colony, 57. Gregarina, 167. flolophrya, cilia, 177; classification, 206. holophytic nutrition, 24. Gregarina, 167. holophytic nutrition, 24. Ftolosticha, 209. Gregarinida, 167. Gregarinidx, 167. Gregarinidx, 167. Gringa, 105. Holotrichida, 171, 206. holozoic nutrition, 24. holozoic nutrition, 24. Hoplitophrya,
vacuole, Gromia, food-taking, fig. p. 91; classifica-
tion, 106; digestion, 285. Hoplitophrya,
vacuole,
187; classification,
207. Hoplitophrya,
vacuole,
187; classification,
207. LHoplorhynchus, 168. Groemide, 106. Gymnameebina, classification, 105. Gymnodinium
furrow,
116; stigm Gymnodinium
furrow,
116; stigma,
119;
food-taking, 126; primitive, 136. Gymnophrys, 106. Gymnosphera, pseu Hyaloklossia, 168. Hyalopus, conjugati Gymnosphera, pseudopodia, 82. Gymnostomata, 22. Hyalopus, conjugation, 99, 225. Hyalosphenia, 106. Gymnostomata, 22. Gymnostomina, 206. Gymnospora, 167. Hyalospora, 167. Hymenomonas, 139 Gymnospora, 167. gynogonidia, in Vol Hymenomonas, 139. Hypocoma, cilia, 195; gynogonidia, in Volvox, 134. Gypsina, 109. gynogonidia, in Volvox, 134. Gypsina, 109. Hypocoma, cilia, 195; classification, 210. Hypocomide, 210. Gypsina, 109. Gyrocorys, 208. Gyrocorys, 208. Hypotrichida, 171;
peristomial
structures,
181; classification, 208. Hypotrichida, 171;
peristomial
structures,
181; classification, 208. hzematochrome, in Mastigophora, 118. Huematococcus,
odor
in water,
62;
fo INDEX OF SUBJECTS karyosome, 42, 87, 146. 973;
general, . 0. trichocysts,
119;
classifica-
relation to Ciliata, 200. y, 57. 7. 67. 67. taking, fig. p. 91; classifica-
gestion, 285. ;
classification, 105. furrow,
116; stigma,
119;
126; primitive, 136. 106. pseudopodia, 82. 22. 206. 167. n Volvox, 134. . in Mastigophora, 118. odor
in water,
62;
;
classification,
139;
, 168. 168. 22. a, 168. 107. of Actinobolus, 50; classifica-
168. 8. um, 107. 07. 08. . 07. 109. shell and furrows, 116; classi-
69. 137. a, 137. onjugation, 221. de, 137. 138. 3. 109. food-
irrita-
INDEX OF SUBJECTS
Heterotrichida, 171, 208. Hexalaspidze, 110. Hexamitus, 138. Hippocrepina, 107. flirmidium, 137. Hirmocystis, 167. Hlistioneis, 140. flolophrya, cilia, 177; classification, 206. holophytic nutrition, 24. Ftolosticha, 209. Holotrichida, 171, 206. holozoic nutrition, 24. Hoplitophrya,
vacuole,
187; classification,
207. LHoplorhynchus, 168. flormosina, 107. LHyalobryon, 139. Hyalodiscus, 106. Hyaloklossia, 168. Hyalopus, conjugation, 99, 225. Hyalosphenia, 106. Hyalospora, 167. Hymenomonas, 139. Hypocoma, cilia, 195; classification, 210. Hypocomide, 210. Hypotrichida, 171;
peristomial
structures,
181; classification, 208. Lleonema,
tentacles,
classification,
206. Imperforina, 106. Infusoria, 15; protoplasmic structure, 173;
mouth-shifting, 185; vacuoles,
186; nu-
cleus, 188; reproduction, 192; inter-rela-
tions, 199; classification, 206. inorganic fluids simulating Protozoa, 306. Inotogmata, 83. inter-alveolar substance, 35. intermediate skeleton, 73. intra-capsular protoplasm, 69. irritability, 294. isogamy, 214. Ssospora, 168. isospore, 95. Lsotricha, 207. Isotrichidz, 207. 183;
Jaculella, 107. Jenia, 138. karyogamy,
Sarcodina,
90,
220. karyolymph, 246. karyophan, 179. karyosome, 42, 87, 146. 973;
general INDEX OF SUBJECTS 340 Heterotrichida, 171, 208. Hexalaspidze, 110. Hexamitus, 138. Hippocrepina, 107. flirmidium, 137. Hirmocystis, 167. Hlistioneis, 140. flolophrya, cilia, 177; classification, 206. holophytic nutrition, 24. Ftolosticha, 209. Holotrichida, 171, 206. holozoic nutrition, 24. Hoplitophrya,
vacuole,
187; classification,
207. LHoplorhynchus, 168. flormosina, 107. LHyalobryon, 139. Hyalodiscus, 106. Hyaloklossia, 168. Hyalopus, conjugation, 99, 225. Hyalosphenia, 106. Hyalospora, 167. Hymenomonas, 139. Hypocoma, cilia, 195; classification, 210. Hypocomide, 210. Hypotrichida, 171;
peristomial
structures,
181; classification, 208. Gonospora, 168. Gonyaulax, 140. Gonyostomum,
trichocysts,
119;
classifica-
tion, 209. Goussia, 168. Grassia, 138;
relation to Ciliata, 200. gregaloid colony, 57. Gregarina, 167. Gregarinida, 167. Gregarinidx, 167. Gringa, 105. Gromia, food-taking, fig. p. 91; classifica-
tion, 106; digestion, 285. Groemide, 106. ;
Gymnameebina, classification, 105. Gymnodinium
furrow,
116; stigma,
119;
food-taking, 126; primitive, 136. Gymnophrys, 106. Gymnosphera, pseudopodia, 82. Gymnostomata, 22. Gymnostomina, 206. Gymnospora, 167. gynogonidia, in Volvox, 134. Gypsina, 109. Gyrocorys, 208. Heterotrichida, 171, 208. Hexalaspidze, 110. Gonospora, 168. Gonyaulax, 140. Hexalaspidze, 110. Hexamitus, 138. INDEX OF SUBJECTS Endothyra, 108, frondicularia, 108. Frontonia, trichocysts Frontonia, trichocysts, 175 ; vacuoles, 187;
classification, 207. Fusulina, 109. Endothyrinz, 108. Enterodela, 9. Enterodela, 9. Entodinium, 208 Fusulina, 109. Fusulininz, 109. Entodinium, 208. Entosiphon, 139. Fusulininz, 109. Entosiphon, 139. Lozoin, 29. galvanotaxis, 300. Gamocystis, 167. gastric vacuole, in Sarcodina, 91;
in In-
fusoria, 185. Gastrostyla, 50, 209. gas vacuole, 290. gemmation;
see reproduction. generation de novo, 26. Gerda, degeneration of peristome, 204; clas-
sification, 209. Glaucoma, 207. Glenodinium, color in water, 62; shell, 1163
food-taking, 126; classification, 140. Clobigerina, 108. Globigerina ooze, 32. Globigerinidze, 108. Gloidium, 106. Glossatella, 140, 209. Glugea, spore-capsules, 156; silk-worm epi-
demic, 165 ; classification, 169. Gontodoma, 140. Gonium, colony-formation,
129;
classifica-
tion, 140; conjugation, 226. Ephelota, fig. p. 58; classification, 210. Epiclintes, 209. Ephelota, fig. p. 58; classification, 210. Epiclintes, 209. Epiclintes, 209. epimerite, 144. Epiclintes, 209. epimerite, 144. epimerite, 144. Epipyxis, divisio Lpistylis,
nematocysts,
209. Erviliinee, 207. classification,
373 Erviliinee, 207. Eudorina, colo Eudorina, colony-formation,
130;
classifi-
cation, 140; conjugation, 227. Euglena, fig. p. 37, 62; membrane, 114; Euglena, fig. p. 37, 62; membrane, 114;
pyrenoids, 118; stigmata, 118 ;- classifica-
tion, 138; chromosomes, 256;
secretion,
292; irritability, 296. food-taking, 126; classification, 140. Clobigerina, 108. Globigerina ooze, 32. Globigerinidze, 108. Euglenida, 138. Euglenide, 138. Luglypha, protoplasmic structure, 38, 68;
shell, 40;
encystment, fig. p. 41;
bud-
ding, fig. p. 55, 93; contractile vacuole,
87; encystment, 90;
classification, 106 ;
conjugation, 219 ; mitosis, 264. Glugea, spore-capsules, 156; silk-worm epi-
demic, 165 ; classification, 169. Gonium, colony-formation,
129;
classifica-
tion, 140; conjugation, 226. -
;
-
-
-
-
OF SUBJECTS
Heterotrichida, 171, 208. Hexalaspidze, 110. Hexamitus, 138. Hippocrepina, 107. flirmidium, 137. Hirmocystis, 167. Hlistioneis, 140. flolophrya, cilia, 177; classification, 206. holophytic nutrition, 24. Ftolosticha, 209. Holotrichida, 171, 206. holozoic nutrition, 24. Hoplitophrya,
vacuole,
187; classification,
207. LHoplorhynchus, 168. flormosina, 107. LHyalobryon, 139. Hyalodiscus, 106. Hyaloklossia, 168. Hyalopus, conjugation, 99, 225. Hyalosphenia, 106. Hyalospora, 167. Hymenomonas, 139. Hypocoma, cilia, 195; classification, 210. Hypocomide, 210. Hypotrichida, 171;
peristomial
structures,
181; classification, 208. Lleonema,
tentacles,
classification,
206. Imperforina, 106. Infusoria, 15; protoplasmic structure, 173;
mouth-shifting, 185; vacuoles,
186; nu-
cleus, 188; reproduction, 192; inter-rela-
tions, 199; classification, 206. inorganic fluids simulating Protozoa, 306. Inotogmata, 83. inter-alveolar substance, 35. intermediate skeleton, 73. intra-capsular protoplasm, 69. irritability, 294. isogamy, 214. Ssospora, 168. isospore, 95. Lsotricha, 207. Isotrichidz, 207. 183;
Jaculella, 107. Jenia, 138. karyogamy,
Sarcodina,
90,
220. karyolymph, 246. karyophan, 179. hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f 181; classification, 208. hzematochrome, in Mastigophora, 118. Huematococcus,
odor
in water,
62;
taking,
125;
classification,
139;
bility, 295. Hemogregarina, 168. Hlemoproteus, 168. Hzmosporidia, 22. Hemosporidiida, 168. Haliphysema, 107. Hlalteria, food of Actinobolus, 50; classifica-
tion, 208. Halteridium, 168. Halteriide, 208. Haplophragmium, 107. Haplostiche, 107. Hastigerina, 108. Hlauerina, 107. Hauerinine, 107. Heliozoa, 17, 109. LHemidinium, shell and furrows, 116; classi-
fication, 140. Henneguya, 169. LHerpetomonas, 137. Heteromastigida, 137. Fleteromita, conjugation, 221. Heteromonadide, 137. Heteronema, 138. STeterophrys, 93. LTelterostegina, 109. food-
irrita- Huematococcus,
odor
in water,
62;
taking,
125;
classification,
139;
bility, 295. Hemogregarina, 168. food-
irrita- Lleonema,
tentacles,
classification,
206. Imperforina, 106. 183; Lleonema,
tentacles,
classification,
206. Imperforina, 106. 183; Lleonema,
tentacles,
classification,
206. Imperforina, 106. Infusoria, 15; protoplasmic structure, 173;
mouth-shifting, 185; vacuoles,
186; nu-
cleus, 188; reproduction, 192; inter-rela-
tions, 199; classification, 206. inorganic fluids simulating Protozoa, 306. Inotogmata, 83. inter-alveolar substance, 35. intermediate skeleton, 73. intra-capsular protoplasm, 69. irritability, 294. isogamy, 214. Ssospora, 168. isospore, 95. Lsotricha, 207. Isotrichidz, 207. 183;
Jaculella, 107. Jenia, 138. karyogamy,
Sarcodina,
90,
220. karyolymph, 246. karyophan, 179. karyosome, 42, 87, 146. 973;
general, Imperforina, 106. Infusoria, 15; prot Hlemoproteus, 168. Hzmosporidia, 22. Infusoria, 15; protoplasmic structure, 173;
mouth-shifting, 185; vacuoles,
186; nu-
cleus, 188; reproduction, 192; inter-rela-
tions, 199; classification, 206. inorganic fluids simulating Protozoa, 306. Hzmosporidia, 22. Hemosporidiida, 168 inorganic fluids simulating Protozoa, 306. Inotogmata, 83. Hlauerina, 107. Hauerinine, 107. Hauerinine, 107. Heliozoa, 17, 109. Heliozoa, 17, 109. LHemidinium, shell Henneguya, 169. LHerpetomonas, 137 LHerpetomonas, 137. Heteromastigida, 137 Heteromastigida, 137. Fleteromita, conjugation karyogamy,
Sarcodina,
90,
220. karyolymph, 246. karyophan, 179. karyosome, 42, 87, 146. 973;
general, Fleteromita, conjugation, 221. Heteromonadide, 137. Heteromonadide, 137. Heteronema, 138. INDEX OF SUBJECTS 341 Lophomonas, 138; relation to Ciliata, 200. lorication moment, 76. Lophomonas, 138; relation to Ciliata, 200. lorication moment, 76. Kentrochonopsis, 209. Keramosphera, 107. Kentrochonopsis, 209. Keramosphera, 107. lorication moment, 76. Loxocephalus, 207. Keramosphera, 107. Keramospherinz, 107. Loxocephalus, 207. Keramospherinz, 107. Kerona, 209. Loxodes, 206. Loxophyllum, Kerona, 209. Loxophyllum, trichocysts, 175 ; nucleus, 189,
250, 257; classification, 206. Lymphosporidium,
fig. p. 363 kinetic centre; see division-centre. kinoplasm, 248, 274. kinetic centre; see division-centre. kinoplasm, 248, 274. 250, 257; classification, 206. 250, 257; classification, 206. Lymphosporidium,
fig. p. 363
147; classification, 169. \
life-cycle,
macrogamete, Vordicella, 195. macronucleus, 14, 188. macrospore, 151. macrosporozoite, 158. Magosphera, 26, 57, 130. Malaria, 65, 160. Mallomonas, 139. Marginulina, 108. Marsipella, 107. Maryna, 208. hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f Mastigameba, 99; protoplasm, 113; classifi-
cation, 137. Mastigophora, 15; structure,
113; flagella,
119; nucleus, 124; vacuoles, 127; inter-
relations,
135;
classification,
136. Mastigophrys, 103, 137. maturation, 233. megalosphzeric chamber, 74, 95. Megastoma,
fig. p. 63, 126;
classification,
138. melanin granules, 144. membrane, structure, 182. membranelle, 171; structure, 182. Menoidium, 138. Menospora, 168. Menosporide, 168. merozoite, 157, 229. Mesodinium, tentacles, 183; phylogeny, 206;
classification, 206. mesoplasm, 144. Mesostigma, 139. Metacineta, 210. Metacinetidz, 210. metasitism, 30. Metopus, 208. micelle, 29. microgamete, 195. Microglena, 139. Microgromia,
fig. p. 56; in division, 93;
classification, 106. micronucleus, 14, 188. micropyle, 229. microspheric chamber, 74. microspore, 151. Microsporidiina, 169. microsporozoite, 158. kinoplasm, 248, 274. Klossia,
168;
conjug Lymphosporidium,
fig. p. 363
147; classification, 169. \
life-cycle, Klossia,
168;
conjugation,
229; nucleus,
250, 254. 147; classification, 169. \ macrogamete, Vordicella, 195. macronucleus, 14, 188. Lacrymaria,
cilia, 177;
phylogeny, 206;
classification, 206,
Lagena, 108. macronucleus, 14, 188. macrospore, 151. macrospore, 151. macrosporozoite, Lagena, 108. Lagenidze, 108. Lagena, 108. Lagenidze, 108. macrosporozoite, 158. Magosphera, 26, 57, 13 Lagenidze, 108. Lageninz, 108. Magosphera, 26, 57, 130. Malaria, 65, 160. Lageninz, 108. Lagenophrys, 209 Malaria, 65, 160. Lagenophrys, 209. Lankesterella, 168. Mallomonas, 139. Marginulina, 108. Lankesterella, 168. Marginulina, 108. Marsipella, 107. Larcaridz, 110. Larcoidida, 110, Marsipella, 107. Maryna, 208. Larcoidida, 110,
Larnacide, 110. Maryna, 208. Mastigameba, Larnacide, 110. Laverania, 168. Mastigameba, 99; protoplasm, 113; classifi-
cation, 137. Mastigophora, 15; structure,
113; flagella, Laverania, 168. Lecguereusia, 106,
Légeria, 168. Mastigophora, 15; structure,
113; flagella,
119; nucleus, 124; vacuoles, 127; inter-
relations,
135;
classification,
136. Mastigophrys, 103, 137. Légeria, 168. Leidyonella, 13 Leidyonella, 138 ; relation to Ciliata, 200. Lembadion, cilia, 177 ; vacuole, 187; classi- Leidyonella, 138 ; relation to Ciliata, 200. Lembadion, cilia, 177 ; vacuole, 187; class Lembadion, cilia, 177 ; vacuole, 187; classi-
fication, 207. : Mastigophrys, 103, 137. maturation, 233. maturation, 233. megalosphzeric chamber, 74, 95. Megastoma,
fig. p. 63, 126;
cl Lembus, 207. Leptocinclis, 138 Leptocinclis, 138. Leptodiscus, 21, 1 Megastoma,
fig. p. 63, 126;
classification,
138. melanin granules, 144. Leptodiscus, 21, 112;
origin, 136; classifi-
cation, 140. Leptotheca, 169. melanin granules, 144. membrane, structure, 18 Leptotheca, 169. Leucophrys, 207; membrane, structure, 182. membranelle, 171; structure membranelle, 171; structure, 182. Menoidium, 138. Leucophrys, 207; conditions of conjugation,
220. Menoidium, 138. Menospora, 168. Lichnaspis, fig. p. 78. Lichnophora, phylogeny Menospora, 168. Menosporide, 168 Menosporide, 168. merozoite, 157, 229. Lichnophora, phylogeny, 203; classification,
209. Lichnophoridze, 209. merozoite, 157, 229. hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f Myxobolidze, 169. Myxobolus, fig. p. 39; spore-formation, 154;
classification, 169. Afyxodictyum, contractile vacuole, 87; classi-
fication, 106. Myxomycetes, 18. Myxosoma, 169. Myxosporidia, 21. Myxosporidiida, 169. Nadinella, 106,
Nasselaria, 70; classification, 110,
Nasselidae, 109. Nassoidida, 109. Miliolinge, 107. mitosis, 246. mitotic figure, 246. Monadida, 137. Monas, 104, 137; Nephroseluris, 139. Noctiluca, 21, 112; alveoli, 35; feeding, 50,
127; budding, 59; color in water, 62; ori-
gin, 136; classification, 140; conjugation,
219; nucleus, 249, 252; mitosis, 256, 257;
phosphorescence, 293. Nodosaria, fig. p. 72; classification, 108. Nodosarina, fig. p. 73. ‘Nodosinella, 108. Nonionina, 109. Nosema, 169. Nosematide, 169. Nubecularia, 107. Nubecularinze, 107. Nuclearia, shell, 74; pseudopodia, 82; divi-
sion, 93; fig. p. 102; classification, 109. nuclein, 246. nucleolus, 253. nucleus, types in Protozoa, 40; in Sarcodina,
86; in Mastigophora, 124; in Sporozoa,
146; in Infusoria, 188; general, 246, 301. Nuda, 106. Nummulinide, 109. Nummulites 109. Nummulitine, 109. nutrition, 24, 280. Nyctotherus, 208. Nephroseluris, 139. Noctiluca, 21, 112; alveoli, 35; feeding, 50,
127; budding, 59; color in water, 62; ori-
gin, 136; classification, 140; conjugation,
219; nucleus, 249, 252; mitosis, 256, 257;
phosphorescence, 293. Nodosaria, fig. p. 72; classification, 108. Nodosarina, fig. p. 73. ‘Nodosinella, 108. Nonionina, 109. Nosema, 169. Nosematide, 169. Nubecularia, 107. Nubecularinze, 107. Nuclearia, shell, 74; pseudopodia, 82; divi-
sion, 93; fig. p. 102; classification, 109. nuclein, 246. nucleolus, 253. nucleus, types in Protozoa, 40; in Sarcodina,
86; in Mastigophora, 124; in Sporozoa,
146; in Infusoria, 188; general, 246, 301. Nuda, 106. Nummulinide, 109. Nummulites 109. Nummulitine, 109. nutrition, 24, 280. Nyctotherus, 208. Ochromonas, food-taking, 126; classification,
139. odors caused by Protozoa, 62. Oikomonas,
fig. p. 49;
food-taking,
1255
classification, 137. old age, 60. Oligotrichina, 208. Onychodactylus, 207. Onychodromus, degeneration, 60; classifica-
tion, 209; conjugation, 220, 234. Obcephalus, 168. Opalina, contractile vacuole, 187; nucleus,
190; classification, 207. Opalinidze, 207. Opalinopsis, 207. Opercularia, fig. p. 73; classification, 209. Ophrydium,
covering,
176; vacuole, 187;
classification, 209. Ophryocystis, 169. Monas, 104, 137; irritability, 297. Monaster, 140. Monaster, 140. monaxonic forms Noctiluca, 21, 112; alveoli, 35; feeding, 50,
127; budding, 59; color in water, 62; ori-
gin, 136; classification, 140; conjugation,
219; nucleus, 249, 252; mitosis, 256, 257;
phosphorescence, 293. Noctiluca, 21, 112; alveoli, 35; feeding, 50,
127; budding, 59; color in water, 62; ori-
gin, 136; classification, 140; conjugation,
219; nucleus, 249, 252; mitosis, 256, 257;
phosphorescence, 293. Nodosaria, fig. p. 72; classification, 108. monaxonic forms, 34. Monera, 30. Monobia, 109. Monocercomonas, 138. hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f Mesodinium, tentacles Lichnophoridze, 209. Lieberkithnia, 106; d Mesodinium, tentacles, 183; phylogeny, 206;
classification, 206. mesoplasm, 144. Lieberkithnia, 106; digestion, 284. Lieberkiihnide, 208. Lieberkithnia, 106; digestion, 284. Lieberkiihnide, 208. Lieberkiihnide, 208. Lingulina, 108. mesoplasm, 144. Mesostigma, 139. Mesostigma, 139. Metacineta, 210. Lingulina, 108. linin-reticulum, 246, Metacineta, 210. Metacinetidz, 210. linin-reticulum, 246, 252. Lionotus, form, 172; tricho Metacinetidz, 210. metasitism, 30. Lionotus, form, 172; trichocysts, 175; classi-
fication, 206; food-selection, 305. Liosphzeridee, 110. Liosphzeridee, 110. Lithelidz, 110. Lithelidz, 110. Lithobotryidze, 11 Lithobotryidze, 110. Lithocampide, 110. Lithocampide, 110. Lithocystis, 168. Lithocystis, 168. Litholophidze, 110 Litholophidze, 110. Lituola, 107. microspore, 151. Microsporidiina, Microsporidiina, 169. microsporozoite, 158. INDEX OF SUBJECTS 342 Microthoracidee, 207, Nassula, trichocysts, 175; classification, 207;
irritability, 295. Nassulinz, 207. Nebenkern (micronucleus), 14, 188. Nebenkirper, 93. nematocysts, 37; in Mastigophora, 119; in
Vorticella, 176. Neosporidia, 168. Nephroseluris, 139. Noctiluca, 21, 112; alveoli, 35; feeding, 50,
127; budding, 59; color in water, 62; ori-
gin, 136; classification, 140; conjugation,
219; nucleus, 249, 252; mitosis, 256, 257;
phosphorescence, 293. Nodosaria, fig. p. 72; classification, 108. Nodosarina, fig. p. 73. ‘Nodosinella, 108. Nonionina, 109. Nosema, 169. Nosematide, 169. Nubecularia, 107. Nubecularinze, 107. Nuclearia, shell, 74; pseudopodia, 82; divi-
sion, 93; fig. p. 102; classification, 109. nuclein, 246. nucleolus, 253. nucleus, types in Protozoa, 40; in Sarcodina,
86; in Mastigophora, 124; in Sporozoa,
146; in Infusoria, 188; general, 246, 301. Nuda, 106. Nummulinide, 109. Nummulites 109. Nummulitine, 109. nutrition, 24, 280. Nyctotherus, 208. Ochromonas, food-taking, 126; classification,
139. odors caused by Protozoa, 62. Oikomonas,
fig. p. 49;
food-taking,
1255
classification, 137. old age, 60. Oligotrichina, 208. Onychodactylus, 207. Onychodromus, degeneration, 60; classifica-
tion, 209; conjugation, 220, 234. Obcephalus, 168. Microthorax, 207. Miliolidz, 107. Miliolidz, 107. Miliolina, 107. Miliolinge, 107. Nebenkern (micronucleus), 14, 188. Nebenkirper, 93. Miliolina, 107. Miliolinge, 107. mitosis, 246. mitotic figure, 246. Monadida, 137. Monas, 104, 137; irritability, 297. Monaster, 140. monaxonic forms, 34. Monera, 30. Monobia, 109. Monocercomonas, 138. Monocystis,
fig. p. 63; pseudopodia,
144;
life-cycle,
151;
conjugation,
157,
225;
classification, 168; nucleus, 250. Monodontophrya, vacuole, 187; classification,
208. monophagous Sporozoa, 142. Monopylea, 70, 110. Monosiga, 137. Monostomea, 138. Monostomine, 106. monozoic spore, 153. morulit, 254. mosquitoes and malaria, 164. motor response, 298. mouth-shifting, 178. movement, Sporozoa, 147. Miillerian law, 77. Alulticilia, flagella, 121; classification, 138;
relation to Ciliata, 200. Mycetozoa, 18. Mycetozoida, 18. myoneme, 38; in Sporozoa, 145; in Infu-
soria, 178. Alyxastrum, 109. Myxidiide, 169. Myxidium, 169. Myxinia, fig. p. 20. hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f Monocystis,
fig. p. 63; Nodosaria, fig. p. 72; classification, 108. Nodosarina, fig. p. 73. Nodosaria, fig. p. 72; classification, 108. Nodosarina, fig. p. 73. Monocystis,
fig. p. 63; pseudopodia,
144;
life-cycle,
151;
conjugation,
157,
225;
classification, 168; nucleus, 250. Monodontophrya, vacuole, 187; classification, Nodosarina, fig. p. 73. ‘Nodosinella, 108. ‘Nodosinella, 108. Monodontophrya, vacuole, 187; classification,
208. monophagous Sporozoa, 142. Nonionina, 109. Nosema, 169. monophagous Sporozoa, 142. Monopylea, 70, 110. monophagous Sporozoa, 142. Monopylea, 70, 110. Monopylea, 70, 110. Monosiga, 137. Monosiga, 137. Monostomea, 138 Monostomea, 138. Monostomine, 106. Monostomine, 106. monozoic spore, 153. monozoic spore, 153. morulit, 254. morulit, 254. mosquitoes an mosquitoes and malaria, 164. motor response, 298. motor response, 298. mouth-shifting, 178. mouth-shifting, 178. movement, Sporozoa, movement, Sporozoa, 147. Miillerian law, 77. Miillerian law, 77. Alulticilia, flagella, Nummulites 109. Nummulitine, 109. Alulticilia, flagella, 121; classification, 138;
relation to Ciliata, 200. Mycetozoa, 18. Nummulitine, 109. nutrition, 24, 280. Mycetozoa, 18. Mycetozoida, 18. nutrition, 24, 280. Nyctotherus, 208. nutrition, 24, 280. Nyctotherus, 208. Nyctotherus, 208. myoneme, 38; in Sporozoa, 145; in Infu-
soria, 178. Alyxastrum, 109. myoneme, 38; in Sporozoa, 145; in Infu-
soria, 178. Alyxastrum, 109. Ochromonas, food-taking, 126; classification,
139. odors caused by Protozoa, 62. Oikomonas,
fig. p. 49;
food-taking,
1255
classification, 137. old age, 60. Oligotrichina, 208. Onychodactylus, 207. Onychodromus, degeneration, 60; classifica-
tion, 209; conjugation, 220, 234. Obcephalus, 168. Opalina, contractile vacuole, 187; nucleus,
190; classification, 207. Opalinidze, 207. Opalinopsis, 207. Opercularia, fig. p. 73; classification, 209. Ophrydium,
covering,
176; vacuole, 187;
classification, 209. Ophryocystis, 169. Afyxodictyum, contractile vacuole, 87; classi-
fication, 106. Myxomycetes, 18. Myxosoma, 169. Myxosporidia, 21. Myxosporidiida, 16 Myxosporidiida, 169. Nasselaria, 70; classification, 110,
Nasselidae, 109. Nasselidae, 109. Nassoidida, 109. Nassoidida, 109. Ophryocystis, 169. INDEX OF SUBJECTS 343 SUBJECTS
343
Peridinium, fig. p. 20; color, 62; shell, 1163
pseudonoctiluca, 136; classification, 140. periplast, 113. Peripylea, 69, 110.°
peristome, 184. Peritrichida, 171, 209. Peritromidz, 208. Peritromus,
cilia,
classification, 208. Petalomonas, 138. Phacodiscide, 110. Phacotide, 139. Phacotus,
shell,
114;
conjugation, 224. Phacus, fig. p. 19; classification, 138. Phzenocystina, 169. Pheeoconchida, 111. Pheeocystinida, 111. Pheeodaria, 111. Pheeodinidee, 111. phzeodium, III. Phzeogromida, 111. Phzeospherida, 111. Phalacroma, 140. Phalansteridz, 137. 181;
phylogeny,
202;
classification,
1393
Phalansterium,
tig. p. 122; classification, 137. Phascolodon, 207. Phialoides, 168. Phormocampide, I11. Phormocyrtide, 111. Phormospyride, 110. Phorticidee, 110. phosphorescence, 62, 293. phototaxis, 296. Phractopeltidze, 110. phycopyrrin, in Dinoflagellidia, 118. Phyllomitus, 138. Phyllomonas, 137. Phytoflagellida, 19, 25, 139. Phytomastigoda, 25. Pileocephalus, 168. Pilulina, 107. hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f Porodiscide, 110. Porospora, 167. Porosporide, 167. Poteriodendron, 137. primite, 156. 198;
classifica-
regeneration,
302;
Prorodon,
trichocysts,
175;
classification,
206; myonemes, 177. Protameba, contractile vacuole, 87, 105. protein, Sporozoa, 143. Proterospongia, fig. p. 113; classification, 137. Protista, 25. Protoceratium, 140. Protogenes, contractile vacuole, 87, 106. protomerite, 144. Protomonas, 103. Protomyxa, 106,
protoplasm, of Sarcodina, 67; Mastigoph-
ora, 113; Sporozoa, 142; Ciliata, 173. Protozoa, name, 28; mode
of life, 32; old
age and immortality,
60; economic as-
pects, 62; cancer, malaria, etc., 65; sex,
211; physiology, 279. Prunoidida, 110. Prunophractida, 110. Psammosphera, 107. Pseudochlamys, 40, 106. pseudoconjugation, 156. pseudocyst, 151. Pseudodiffiugia, 106. Pseudonavicella, 151. Pseudopodia,
17, 79-86;
movement, 83. Pseudospora, 103. Pseudospore, 103. Psilotricha, 209. psorosperm, 21. Plerocephalus, 167. Pterospora, 168. Ltychodiscus, 140. Ptychostomum, 207. Pullenia, 108. Pylobotryide, 110. Pylodiscidee, 110. Pylonide, 110. Pyramimonas, 139. pyrenoid, 117. Pyrophacus, 140. Pyrsonympha, 138. Pyxidicula, 106,
Pyxinia, fig. p. 33 143; classification, 168. pyxinine, 143. explanation
of
Quadrilonchide, 110. Quadrula, budding, 93; classification, 106. Radiolaria, 17; central capsule, 67, 70; pro-
toplasmic structure, 69; modifications of plasmosome, 246. plastic granules in Sporozoa, 144. plastogamy, in Sarcodina, 90, 97; general, 218. Platoum, 106. Platydorina, 140. Platytheca, 137. Plectanide, 110,
Plectoidida, 110. Plectophrys, 106. Pleuronema, 207; irritability, 296. Pleuronemide, 207. Pleurotricha, 209. LPlistophora, 169. Podocampide, III. podoconus, 70. Podocyathus, 210. Podocyrtidee, 111. Podolampas, 140. Podophrya,
reproduction,
tion, 210. Podophryide, 210. Podostoma, 106. polar bodies, 237. polar capsule, 154. pole-plates, 262. Polydinida, 140. Polygastrica, 9. Polykrikos, 37, 41;
trichocysts, 119; food-
taking, 126. )
Polymastigida, 138. Polymitus form, 162. Polymorphina, 108. Polymorphininz, 108. Polyweca, 137. polyphagous Sporozoa, 142. Polyphragma, 108. Polysporocystidze, 168. Polystomella,
72, 109;
irritability, 306. Polystomellinze, 109. Polytoma, starch,
118; reproduction,
131;
classification, 139; irritability, 297, 301. Polytrema, 109. Polytrichina, 208. polyzoic spore, 153. Ponpholyxophrys, 109. Pontomyxa, 106. Porodiscide, 110. Porospora, 167. Porosporide, 167. Poteriodendron, 137. primite, 156. 198;
classifica-
regeneration,
302; Platytheca, 137. Plectanide, 110, Protoceratium, 140. Protogenes, contractile Plectanide, 110,
Plectoidida, 110. Plectoidida, 110. Plectophrys, 106. Pleuronema, 207; protomerite, 144. Protomonas, 103. Pleuronema, 207; irritability, 296. Pleuronemide, 207. Protomonas, 103. Protomyxa, 106, Pleuronemide, 207. Protomyxa, 106,
protoplasm, of Sa Pleurotricha, 209. LPlistophora, 169. protoplasm, of Sarcodina, 67; Mastigoph-
ora, 113; Sporozoa, 142; Ciliata, 173. Protozoa, name, 28; mode
of life, 32; old protoplasm, of Sarcodina, 67; Mastigoph-
ora, 113; Sporozoa, 142; Ciliata, 173. Protozoa, name, 28; mode
of life, 32; old LPlistophora, 169. Podocampide, III. Podocampide, III. hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f protein, Sporozoa, 143. Proterospongia, fig. p. 113; classification, 137. Protista, 25. Protoceratium, 140. Protogenes, contractile vacuole, 87, 106. protomerite, 144. Protomonas, 103. Protomyxa, 106,
protoplasm, of Sarcodina, 67; Mastigoph-
ora, 113; Sporozoa, 142; Ciliata, 173. Protozoa, name, 28; mode
of life, 32; old
age and immortality,
60; economic as-
pects, 62; cancer, malaria, etc., 65; sex,
211; physiology, 279. Prunoidida, 110. Prunophractida, 110. Psammosphera, 107. Pseudochlamys, 40, 106. pseudoconjugation, 156. pseudocyst, 151. Pseudodiffiugia, 106. Pseudonavicella, 151. Pseudopodia,
17, 79-86;
movement, 83. Pseudospora, 103. Pseudospore, 103. Psilotricha, 209. psorosperm, 21. Plerocephalus, 167. Pterospora, 168. Ltychodiscus, 140. Ptychostomum, 207. Pullenia, 108. Pylobotryide, 110. Pylodiscidee, 110. Pylonide, 110. Pyramimonas, 139. pyrenoid, 117. Pyrophacus, 140. Pyrsonympha, 138. Pyxidicula, 106,
Pyxinia, fig. p. 33 143; classification, 168. pyxinine, 143. explanation
of
Quadrilonchide, 110. Quadrula, budding, 93; classification, 106. Prorodon,
trichocysts,
175;
classification,
206; myonemes, 177. Protameba, contractile vacuole, 87, 105. protein, Sporozoa, 143. Proterospongia, fig. p. 113; classification, 137. Protista, 25. Protoceratium, 140. Protogenes, contractile vacuole, 87, 106. protomerite, 144. Protomonas, 103. Protomyxa, 106,
protoplasm, of Sarcodina, 67; Mastigoph-
ora, 113; Sporozoa, 142; Ciliata, 173. Protozoa, name, 28; mode
of life, 32; old
age and immortality,
60; economic as-
pects, 62; cancer, malaria, etc., 65; sex,
211; physiology, 279. Prunoidida, 110. Prunophractida, 110. Psammosphera, 107. Pseudochlamys, 40, 106. pseudoconjugation, 156. pseudocyst, 151. Pseudodiffiugia, 106. Pseudonavicella, 151. Pseudopodia,
17, 79-86;
movement, 83. Pseudospora, 103. Pseudospore, 103. Psilotricha, 209. psorosperm, 21. Plerocephalus, 167. Pterospora, 168. Ltychodiscus, 140. Ptychostomum, 207. Pullenia, 108. Pylobotryide, 110. Pylodiscidee, 110. Pylonide, 110. Pyramimonas, 139. pyrenoid, 117. Pyrophacus, 140. Pyrsonympha, 138. Pyxidicula, 106,
Pyxinia, fig. p. 33 143; classification, 168. pyxinine, 143. explanation
of
Quadrilonchide, 110. Quadrula, budding, 93; classification, 106. plasmosome, 246. plastic granules in Sporozoa, 144. plastogamy, in Sarcodina, 90, 97; general, 218. Platoum, 106. Platydorina, 140. Platytheca, 137. Plectanide, 110,
Plectoidida, 110. Plectophrys, 106. Pleuronema, 207; irritability, 296. Pleuronemide, 207. Pleurotricha, 209. LPlistophora, 169. Podocampide, III. podoconus, 70. Podocyathus, 210. Podocyrtidee, 111. Podolampas, 140. Podophrya,
reproduction,
tion, 210. Podophryide, 210. Podostoma, 106. polar bodies, 237. polar capsule, 154. pole-plates, 262. Polydinida, 140. Polygastrica, 9. Polykrikos, 37, 41;
trichocysts, 119; food-
taking, 126. )
Polymastigida, 138. Polymitus form, 162. Polymorphina, 108. Polymorphininz, 108. Polyweca, 137. polyphagous Sporozoa, 142. Polyphragma, 108. Polysporocystidze, 168. Polystomella,
72, 109;
irritability, 306. Polystomellinze, 109. Polytoma, starch,
118; reproduction,
131;
classification, 139; irritability, 297, 301. Polytrema, 109. Polytrichina, 208. polyzoic spore, 153. Ponpholyxophrys, 109. Pontomyxa, 106. hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f Pilulininz, 107. Pinaciophora,
skeleton,
75;
classification,
109. Pinacocystts, 109. Pleodorina, 140. Plagiopogon, 206. Plagiotoma, 208. Plagiotomidee, 208. Plagonide, 110. Plakopus, 106. Planorbulina, 109. Plasmodium malaria, 65; conjugation and
life-cycle,
160, Ophryodendridz, 210,
Ophryodendron, budding Ophryodendron, budding, 199; classification,
210. Ophryoglena, 207. Ophryoglena, 207. Ophryoscolecide, 2 Ophryoscolecide, 208. Ophryoscolex, 208. Peritrichida, 171, 209. Peritromidz, 208. Opisthodvn, 207. Peritromidz, 208. Orbiculina, 107. Peritromus,
cilia,
classification, 208. Petalomonas, 138. 181;
phylogeny,
202; Orbiloides, 109. Petalomonas, 138. Orbitolites, 73, 107. Orbulinella, 109. Orbulinella, 109. Phacodiscide, 110. Phacotide, 139. Ornithocercus, 140. Phacotide, 139. Phacotus,
shell, Orosphveridee, 111. Orthodon, 207. Phacotus,
shell,
114;
conjugation, 224. Phacus, fig. p. 19; classification, 138. classification,
1393 Orthodon, 207. Oxyrrhis, divisi Phacus, fig. p. 19; classification, 138. Phzenocystina, 169. Oxyrrhis, division, 128; classification, 138;
nucleus and division-centre, 271. Oxytoxunt, 140. Phzenocystina, 169. Pheeoconchida, 111. Oxytoxunt, 140. Oxytricha,
phylo Pheeoconchida, 111. Pheeocystinida, 111. Pheeocystinida, 111. Pheeodaria, 111. Oxytricha,
phylogeny,
202;
209. Oxytrichidze, 209. classification, Oxytricha,
phylogeny,
202;
209. Oxytrichidze, 209. classification, Oxytrichidze, 209. Phzeospherida, 111. Phalacroma, 140. Phalacroma, 140. Phalansteridz, 137. Pandorina,
fig. p. 37;
colony-formation,
129;
reproduction,
132;
classification,
140; conjugation, 227. pan-sporoblast, 155. pan-sporoblast, 155. paraglycogen, 143. Parameba, reproduct Phialoides, 168. Phormocampide, Phormocampide, I11. Phormocyrtide, 111. Parameba, reproduction, 59; spore-forma-
tion, 93; classification, 105; conjugation,
218; chromosomes, 233; reduction, 234;
division-centre, 276. Paramcecidz, 207. Phormocyrtide, 111. Phormospyride, 110. Phormospyride, 110. Phorticidee, 110. Phorticidee, 110. phosphorescence, phosphorescence, 62, 293. phototaxis, 296. Paramcecidz, 207. Paramecium, trich Paramecium, trichocysts, 37, 175; cyclosis,
173; strength of, 181; classification, 207;
digestion, 282; urea, 291; motor response. 298; irritability, 301. paramylum, in Mastigophora, 117. Paramecium, trichocysts, 37, 175; cyclosis,
173; strength of, 181; classification, 207;
digestion, 282; urea, 291; motor response. 298; irritability, 301. paramylum, in Mastigophora, 117. parasitic Protozoa, 63. Parkeria, 108. parthenogonidia, in Volvox, 134, 232. Patellina, 109; conjugation, 219. Pavonina, 108. pellicula, 38, 114, 176. Pelomyxa, 106; protoplasmic structure, 35,
38; nuclei, 87; vacuole, 87; budding, 95;
starch digestion, 281; irritability, 297. Pelosina, 107. Peneroplidinee, 107. Peneroplis, 107. Peranema, motion, 121; classification, 138. Peranemide, 138. Perforina, 108. Peridinide, 140. peridinin, in Dinoflagellidia, 118. Phractopeltidze, 110. phycopyrrin, in Dinofl phycopyrrin, in Dinoflagellidia, 118. Phyllomitus, 138. phycopyrrin, in Dinoflagellidia, 118. Phyllomitus, 138. paramylum, in Mastigophora, 117. parasitic Protozoa, 63. parthenogonidia, in Volvox, 134, 232. Patellina, 109; conjugation, 219. Patellina, 109; conjugation, 219. Pavonina, 108. peridinin, in Dinoflagellidia, 118. INDEX OF SUBJECTS 344 Prorodon,
trichocysts,
175;
classification,
206; myonemes, 177. Protameba, contractile vacuole, 87, 105. Radiolaria, 17; central capsule, 67, 70; pro-
toplasmic structure, 69; modifications of
skeleton, 77; spore-formation, 95;
con-
jugation, 96; development, 105; classifi-
cation, TIO, hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f Protozoa, name, 28; mode
of life, 32; old
age and immortality,
60; economic as-
pects, 62; cancer, malaria, etc., 65; sex,
211; physiology, 279. Protozoa, name, 28; mode
of life, 32; old
age and immortality,
60; economic as-
pects, 62; cancer, malaria, etc., 65; sex,
211; physiology, 279. podoconus, 70. Podocyathus, 210 Podocyathus, 210. Podocyrtidee, 111. Podocyrtidee, 111. Podolampas, 140. Podolampas, 140. Podophrya,
reprod Podophrya,
reproduction,
tion, 210. 198;
classifica- Podophrya,
reproduction,
tion, 210. Podophryide, 210. 198;
classifica- Podophrya,
reproduction,
tion, 210. Podophryide, 210. Podostoma, 106. polar bodies, 237. polar capsule, 154. pole-plates, 262. Polydinida, 140. Polygastrica, 9. Polykrikos, 37, 41;
trichocysts, 119; food-
taking, 126. )
Polymastigida, 138. Polymitus form, 162. Polymorphina, 108. Polymorphininz, 108. Polyweca, 137. polyphagous Sporozoa, 142. Polyphragma, 108. Polysporocystidze, 168. Polystomella,
72, 109;
irritability, 306. Polystomellinze, 109. Polytoma, starch,
118; reproduction,
131;
classification, 139; irritability, 297, 301. Polytrema, 109. Polytrichina, 208. polyzoic spore, 153. Ponpholyxophrys, 109. Pontomyxa, 106. Porodiscide, 110. Porospora, 167. Porosporide, 167. Poteriodendron, 137. primite, 156. Prorocentridz, 140. Prorocentrum,
color,
62;
flagella,
121;
inter-relations, 136; classification, 140. 198;
classifica-
regeneration,
302; Podophryide, 210. Podostoma, 106. Podostoma, 106. polar bodies, 237. polar bodies, 237. polar capsule, 154. Polydinida, 140. Polygastrica, 9. Polygastrica, 9. Polykrikos, 37, Pseudospora, 103. Pseudospore, 103. Polykrikos, 37, 41;
trichocysts, 119; food-
taking, 126. )
Polymastigida, 138. Polymastigida, 138. Polymitus form, 162. Polymitus form, 162. Polymorphina, 108. Polymorphininz, 108. Polymorphininz, 108. Polyweca, 137. polyphagous Sporozoa, 142. Polyphragma, 108. Polyphragma, 108. Polysporocystidze, 16 Polystomella,
72, 109;
irritability, 306. Polystomellinze, 109. regeneration,
302; Polystomella,
72, 109;
irritability, 306. Polystomellinze, 109. regeneration,
302; Polystomellinze, 109. Polytoma, starch,
11 Polytoma, starch,
118; reproduction,
131;
classification, 139; irritability, 297, 301. Polytrema, 109. Polytoma, starch,
118; reproduction,
131;
classification, 139; irritability, 297, 301. Polytrema, 109. INDEX OF SUBJECTS 345 . 156. p. 49; skeleton, 75, 93. rejuvenescence. 61, 213. rejuvenescence. vision, 55; spore-forma-
59; in Sporozoa, 149;
, 106. 7. ell,
114;
classification,
137. n, 24. shells and tests, 71; di-
pseudopodia, 82; nuclei,
cuole, 87; nutrition, 90;
reproduction, 92; classifi-
. n malaria organism, 161;
Sciadiophora, 168. Scyphidia, 209. Scytomonas, 138. secretion and excretion, 290. selection of food, 48. Semantidze, 110. senile degeneration, 220. sensory organs, 61. Serumsporidia, 169. Serumsporidium, 165; classification, 169. Sethocyrtidae, 111. sex-differentiation, definition, 213. shells and tests, Reticulariida, 71. Shepheardella, 106. Silicoflagellida, 140. Solenophrya, 210. Soreumide, 110,
Sorosphara, 107. Sparotricha, 209. spasmoneme, in Vordtcella, 179. Spathidium, 206. hzematochrome, in Mastigophora, 118.
Huematococcus,
odor
in water,
62;
f Spherastrum, pseudopodia, 82, 93; classifi-
cation, 109; division-centre, 266,
Spheerocapsida, 110. Spherocystis, 167. Sphareca, 137. sphzeroid colony, 57. Spheeroidida, 110. Spheroidina, 108. Spheromyxa, 169. Sphzerophractida, 110. Spherophrya, 210. Spharorhynchus, 168. Spherospora, 169. Spheerozoidze, 110. Sphenomonas, 138. Spirillina, 109. Spirilling, 109. Spirochona, mitosis, 191, 261; classification,
209. Spirochonidz, 209. Spiroloculina, 107. Spironema, 138. spironeme, 179. Spirostomum,
vacuole,
187;
nuclei,
190;
classification, 208; irritability, 301. Spondylomorum, 139. Spongodiscide, 110. « Spongomionas, 137. Sponguride, 110. spontaneous division, 59. spontaneous generation, 26. spore-formation, 58. sporoblast, 151, 225. sporocyst, I51. Sciadiophora, 168. Scyphidia, 209. Scytomonas, 138. secretion and excretion, 290. selection of food, 48. Semantidze, 110. senile degeneration, 220. sensory organs, 61. Serumsporidia, 169. Serumsporidium, 165; classification, 169. Sethocyrtidae, 111. sex-differentiation, definition, 213. shells and tests, Reticulariida, 71. Shepheardella, 106. Silicoflagellida, 140. Solenophrya, 210. Soreumide, 110,
Sorosphara, 107. Sparotricha, 209. spasmoneme, in Vordtcella, 179. Spathidium, 206. Spherastrum, pseudopodia, 82, 93; classifi-
cation, 109; division-centre, 266,
Spheerocapsida, 110. Spherocystis, 167. Sphareca, 137. sphzeroid colony, 57. Spheeroidida, 110. Spheroidina, 108. Spheromyxa, 169. Sphzerophractida, 110. Spherophrya, 210. Spharorhynchus, 168. Spherospora, 169. Spheerozoidze, 110. Sphenomonas, 138. Spirillina, 109. Spirilling, 109. Spirochona, mitosis, 191, 261; classification,
209. Spirochonidz, 209. Spiroloculina, 107. Spironema, 138. spironeme, 179. Spirostomum,
vacuole,
187;
nuclei,
190;
classification, 208; irritability, 301. Spondylomorum, 139. Spongodiscide, 110. « Spongomionas, 137. Sponguride, 110. spontaneous division, 59. spontaneous generation, 26. spore-formation, 58. Sciadiophora, 168. Scyphidia, 209. Rainey’s corpuscles, 156. Rainey’s tubes, 145. Rainey’s tubes, 145. Ramutlina, 108. Ramulinine, 108. selection of food, 48. Kaphidiophrys, fig. p. 49; skeleton, 75, 93. réjuentssement,; see rejuvenescence. Semantidze, 110. senile degeneration réjuentssement,; see rejuvenescence. senile degeneration, 220. sensory organs, 61. rejuvenescence, 14, 61, 213. sensory organs, 61. reorganization ; see rejuvenescence. reproduction, 54; division, 55; spore- Serumsporidia, 169. Serumsporidium, 16 reproduction, 54; division, 55; spore-forma-
tion, 58; budding, 59; in Sporozoa, 149;
in Infusoria, 192. sex-differentiation, definition, 213. shells and tests, Reticulariida, 71. respiration, 288. shells and tests, Reticulariida, 71. Shepheardella, 106. Reticulariida, 18, 67, 106. Rhabdammina, 107. Reticulariida, 18, 67, 106. Rhabdammina, 107. Rhabdammina, 107. Silicoflagellida, 140. Solenophrya, 210. Rhabdamminine, 107. Rhabdomonas, 138. Solenophrya, 210. Rhabdomonas, 138. Rhabdostyla, 209. Soreumide, 110,
Sorosphara, 107. Rhabdostyla, 209. Rhipidodendron,
s Sorosphara, 107. Sparotricha, 209. Rhipidodendron,
shell,
114;
classification,
137. Rhizammina, 109. Rhipidodendron,
shell,
114;
classification,
137. Sparotricha, 209. spasmoneme, in V spasmoneme, in Vordtcella, 179. Spathidium, 206. 137.
hizamm Rhizammina, 109. Rhizomastigidz, 99, Rhizomastigidz, 99, 137. Rhizopoda, 105. Rhizopoda, 105. Rhopalonia, 167. cation, 109; division-centre, 266,
Spheerocapsida, 110. Spheerocapsida, 110. Spherocystis, 167. Rhopalonia, 167. khyncheta, 210. khyncheta, 210. Rimulina, 108, Rimulina, 108,
Rotalia, 109. Rimulina, 108,
Rotalia, 109. Rotalia, 109. Rotalide, 108, Rotalia, 109. Rotalide, 108, Rotalide, 108,
Rotalinz, 109. Rotalide, 108,
Rotalinz, 109. Rotalinz, 109. Rotalinz, 109. Saccammina, 107. Saccamminine, 107. Saccamminine, 107. Sagenella, 107. Sagospheeridee, Sagospheeridee, 111. Sagrina, 108. Sagrina, 108. Salpingeca, 13 Salpingeca, 137. Saltonella, 106. Saltonella, 106. saprophytic nutrit saprophytic nutrition, 24. Sarcocystis, 169. Spirochona, mitosis, 191, 261; classification,
209. Sarcodina, 15, 67; shells and tests, 71; di-
morphism, 74; pseudopodia, 82; nuclei,
86; contractile vacuole, 87; nutrition, 90;
encystment, 90; reproduction, 92; classifi-
cation, 105. sarcomatrix, 69. Sarcosporidiida, 169. satellite, 156. Schaumplasma, 35. schizogony, 159; in sporocyst, I51. INDEX OF SUBJECTS 346 Telosporidia, 167. tentacles, 52, 196. Testacea, 18. Tetramitus, flagella,
cleus, 124, 251;
jugation, 214. Tetrasporocystide, 1
Tetrastyla, 209. Tetratoma, 139. Textularia, 108. Textularide, 108. Textularine, 108. Thalamophora, 18. Thalamopora, 109. Thalassicolide, 18, 1
Thalassicolla, 110;
sule, 302. Thalassospheerida, 11
Thaumatomastix, 13
Thecameebina, 106. Thélohania, 169. Theocyrtide, 111. thermotaxis, 295. thigmotaxis, 300. Tholonide, I10. Tholospyridze, 110. Thurammina, 107. Thylakidium, 208. Tiarina, 206. Tinoporinze, 109. Tinoporus, 109. Tintinnide, 208. Tintinnidium, 208. Tintinnopsis, 208. Tintinnus, 208,
Tokophrya, 210. Trachelinidze, 206. Trachelius, trichocys
206. Trachelocerca, 206. Trachelomonas, shel
classification, 138. Trepomonas, 138; ir
trichocysts, in Masti
soria, 175. Trichodina, phyloge
209. Trichodinopsis, 209. Trichogaster, 209. Trichomonas, 138. Trichonympha, 138;
Trichonymphinea, 13
Trichophrya, 210. Trichorhynchus, 167. Trichospherium, 106 Telosporidia, 167. tentacles, 52, 196. tentacles, 52, 196. Testacea, 18. Testacea, 18. Tetramitus, f Sporozoa,
15;
structure,
142, 146;
food-
taking, 146; motion, 148; reproduction,
149;
inter-relationships,
166;
classifica-
tion, 167. Tetramitus, flagella,
121;
distributed
nu-
cleus, 124, 251; classification, 133; con-
jugation, 214. Tetrasporocystide, 168. sporozoite, 141. Tetrasporocystide, 168. Tetrastyla, 209. Tetrasporocystide, 168. Tetrastyla, 209. Tetrastyla, 209. Tetratoma, 139. Spumellaria, 70, 110. Spyroidida, 110. Tetrastyla, 209. Tetratoma, 139. Spyroidida, 110. Squamulina, 107. Tetratoma, 139. Textularia, 108. Tetratoma, 139. Textularia, 108. Textularia, 108. Textularide, 108. Squamulina, 107. Stacheia, 108, Stacheia, 108, Textularide, 108. Staurophrya, 210. Textularine, 108. Thalamophora, 18. Staurophryidze, 210. Thalamophora, 18. Thalamopora, 109. Staurospheeride, 110. Thalamopora, 109. Thalassicolide, 18, Steiniella, 140. Stenophora, 167. Thalassicolide, 18, 110. Thalassicolla, 110; enuc Stenophora, 167. Stentor, pseudopod Thalassicolla, 110; enucleated central cap-
sule, 302. Thalassospheerida, 110. Stenophora, 167. Stentor, pseudopodia, 175, 183; nuclei, 190;
division, 192; classification, 208. Stentoridze, 208. 137.
hizamm Stephanidee, 110. Stephanophora, 168. Stephanopogon, phylogeny,
201;
classifica-
tion, 206. Stephanosphara, 140. Stephoidida, 110. Stichotricha, 209. Stictospora, 168. stigma (eye-spot), fig. p. 37; 61; in Infusoria,
174. Streblonide, 110. Strombidium, trichocysts, 175; classification,
208. Strongylidium, 209. Stylameba, 106. styliform tentacles, 195. Stylochrysalis,
division, 128;
classification,
139. Stylocometes, 210. ,
Stylonychia, 209; conjugation, 220; nucleus,
257; irritability, 295. Stylorhynchide, 168. Stylorhynchus, 168. Stylosphzeridee, 110. Sucto-ciliata, 205. Suctorella, 210. Suctoria, 18, 22, 195 ; food-taking, 52; ten-
tacles, fig. p. 52; reproduction, 198; clas-
sification, 209. symbiosis, in Radiolaria, 70;
in Parame-
cium, 282. Symphyta, 166. Syncrypta, 139. Syncystis, 168. Synura, odor, 62; classification, 139. Syringammina, 107. Stentor, pseudopodia, 175, 183; nuclei, 190;
division, 192; classification, 208. Stentor, pseudopodia, 175, 183; nuclei, 190;
division, 192; classification, 208. division, 192; classification, 208. Stentoridze, 208. Thalassospheerida, 110. Thaumatomastix, 139. Stentoridze, 208. Thaumatomastix, 139. Stephanidee, 110. Stephanophora, 16 Thecameebina, 106. Thélohania, 169. Stephanophora, 168. Stephanopogon, phylo Thélohania, 169. Theocyrtide, 111. Stephanopogon, phylogeny,
201;
classifica-
tion, 206. Stephanopogon, phylogeny,
201;
classifica-
tion, 206. Theocyrtide, 111. thermotaxis, 295. thigmotaxis, 300. Stephanosphara, 140. Stephoidida, 110. thigmotaxis, 300. Tholonide, I10. Stephoidida, 110. Stichotricha, 209. Tholonide, I10. Tholospyridze, 110. Thurammina, 107. Stichotricha, 209. Stictospora, 168. Thurammina, 107. Thylakidium, 208. Stictospora, 168. stigma (eye-spot), Thylakidium, 208. Tiarina, 206. stigma (eye-spot), fig. p. 37; 61; in Infusoria,
174. Tiarina, 206. Tinoporinze, 109 Tinoporinze, 109. Tinoporus, 109. Tinoporus, 109. Tintinnide, 208. Tintinnide, 208. Tintinnidium, 208. Tintinnopsis, 208. Tintinnopsis, 208. Tintinnus, 208, Tintinnus, 208,
Tokophrya, 210. Tokophrya, 210. Trachelinidze, 206. Trachelinidze, 206. Trachelius, trichocy Trachelius, trichocysts, 175;
classification,
206. Trachelocerca, 206. Symphyta, 166. Syncrypta, 139. Syncrypta, 139. Syncystis, 168. Syncystis, 168. Synura, odor, 6 Trichorhynchus, 167. Trichospherium, 106. Syringammina, 107. INDEX OF SUBJECTS 347 Trichostomata, 22. Trichostomina, 207. Trigonomenas, 138,
Trimastigide, 138. Trimastix, 138. Trinema, 106. Tripocalpidae, 111,
Tripocyrtida, 111. Tritaxia, 108. Trochammina,
107. Trochammininz, 107. Trochilia, 207. trophoplasm, 274. Tropidoscyphus, 139. Truncatulina, 109; shell, 305. Trypanosoma, 137. Tubulata, 18. Tuscaroride, 111. Tympanide, 110. Urceolarinz, 209. Urceolus, 138. urea, 54, 291. Urnula, 210. Urnulide, 210. Urocentridz, 207. Urocentrum,
caudal
cilia,
182;
187; classification, 207. Uroglena, 25; odor, 62; fig. p. 57; colony-
formation,
129;
division
of the colony,
131; classification, 139. Uroleptus, 209. Uronema, 207. Uronychia, 209. Urophagus, 138. Urosoma, 209. Urospora, 168. Urostyla, 209; nucleus, 251. Urotricha, 206. Urozona, 207. vacuoles, Urthiere, 28. Uvigerina, 108. Vacuolaria, 139. vacuoles, in Sarcodina, 87; in Mastigophora,
127; in Infusoria, function, 291. Vaginicola, 209. Vaginulina, 108. OPINIONS OF THE PRESS. “Professor Henry Fairfield Osborn has rendered
an important service by the preparation of a concise
history of the growth of the idea of Evolution. The
chief contributions of the
different thinkers from
Thales to Darwin are brought into clear perspective,
and a
just estimate of the methods and results of each
one is reached. ‘lhe work is extremely well done,
and it has an added value of great importance in the
fact that the author is a trained biologist. Dr. Os-
born is himself one of the authorities in the science
of Evolution, to which he has made important con-
tributions. He is therefore in a position to estimate
the value of scientific theories more justly than would
be possible to one who approached the subject from
the standpoint of metaphysics or that of literature.”
—President Davip Starr JorDAN, widely read, not only by science teachers, by biologi-
cal students, but we hope that historians, students of
social science, and theologians will acquaint them-
selves with this clear, candid, and catholic statement
of the origin and early history of a theory, which not
only explains the origin of life-forms, but has trans-
formed the methods of the historian, placed philoso-
phy on a higher plane, and immeasurably widened
our views of nature and of the Infinite Power work-
ing in and through the universe.” — Professor A. S. Packarn,
in Sczence, New Y in Sczence, New York. “‘This is an attempt to determine the history of
Evolution, its development and that of its elements,
and the indebtedness of modern to earlier investi-
gators. The book is a valuable contribution;
it will
do a great deal of good in disseminating more accu-
rate ideas of the accomplishments of the present as
compared with the past, and in broadening the views. of such as have confined themselves too closely to
the recent or to specialties. ... As a whole the
book is admirable. The author has been more im-
partial than any of those who have in part anticipated
him in the same line of work.”
— The Nation, —President Davip Starr JorDAN, —President Davip Starr JorDAN, in The Dial, Chicago. in The Dial, Chicago. 137.
hizamm Valviulina, 108. Vampyrella, pseudopodia, 82; encystment,
90; division, 92;
conjugation, 98, 104;
classification, 109; food-selection, 305. Verjiingung ,; see rejuvenescence. Verneutlina, 108. Virgulina, 108. Volvocina, 139. Volvox, 26, 57;
colony-formation, 129; re-
production, 134;
classification, 140;
con-
jugation, 232. Vorticella, chlorophyl, 174;
origin of, 202;
nematocysts, 176; myonemes, 178; clas-
sification, 209; urea, 291."
Vorticellide, origin of, 202;
209. Vorticellidinze, 209. classification,
Wagnerella, 109. Webbina, 107. Wimperring, 179. Zonaridz, 110. Zodglea, 28. zoéphyte, 25. Zoothamnium, conjugation, 195, 222; clas-
sification, 209. Zygartidee, 110. Zygocystis, 168. Zygoselmis, 138. zygospore, in Mastigophora, 133. Zygospyride, 110. Trichostomata, 22. Trichostomina, 207. Trigonomenas, 138,
Trimastigide, 138. Trimastix, 138. Trinema, 106. Tripocalpidae, 111,
Tripocyrtida, 111. Tritaxia, 108. Trochammina,
107. Trochammininz, 107. Trochilia, 207. trophoplasm, 274. Tropidoscyphus, 139. Truncatulina, 109; shell, 305. Trypanosoma, 137. Tubulata, 18. Tuscaroride, 111. Tympanide, 110. Vampyrella, pseudopodia, 82; encystment,
90; division, 92;
conjugation, 98, 104;
classification, 109; food-selection, 305. Verjiingung ,; see rejuvenescence. Verneutlina, 108. Tubulata, 18. Tuscaroride, 1 Tuscaroride, 111. Tympanide, 110. Tympanide, 110. Vorticella, chlorophyl, 174;
origin of, 202;
nematocysts, 176; myonemes, 178; clas-
sification, 209; urea, 291."
Vorticellide, origin of, 202;
classification, Urceolarinz, 209. Urceolus, 138. urea, 54, 291. Urnula, 210. Urnulide, 210. Urocentridz, 207. Urocentrum,
caudal
cilia,
182;
187; classification, 207. Uroglena, 25; odor, 62; fig. p. 57; colony-
formation,
129;
division
of the colony,
131; classification, 139. Uroleptus, 209. Uronema, 207. Uronychia, 209. Urophagus, 138. Urosoma, 209. Urospora, 168. Urostyla, 209; nucleus, 251. Urotricha, 206. Urozona, 207. vacuoles, Vorticellide, origin of, 202;
209. Vorticellidinze, 209. classification, Vorticellidinze, 209. Wimperring, 179. Uroleptus, 209. Uronychia, 209. Urophagus, 138. Urophagus, 138. Zygartidee, 110. Zygocystis, 168. Urosoma, 209. Urospora, 168. Zygocystis, 168. Zygoselmis, 138. Urospora, 168. Urostyla, 209; Zygoselmis, 138. zygospore, in Mas Urostyla, 209; nucleus, 251. Urotricha, 206. Urotricha, 206. Urozona, 207. Zygospyride, 110. Urozona, 207. By HENRY FAIRFIELD OSBORN, Sc.D. Cloth. 8vo. 259 pages. Illustrated. Price, $2.00. 316 pages.
135 Illustrations.
Price, $2.50. “‘This important monograph will be welcomed by
all students of zodlogy as a valuable accession to the
literature of the theory of descent. More than this,
the volume bears internal evidence throughout of
painstaking care in bringing together, in exceedingly
readable form, all the essential details of the structure
and metamorphosis of Amphioxus as worked out by
anatomists and embryologists since the time of Pallas,
its discoverer. The interesting history of the changes
it undergoes during metamorphosis, especially its sin-
gular symmetry, is clearly described and ingenious
explanations of the phenomena are suggested. Most
important, perhaps, are the carefully suggested homol-
ogies of the organs of A mphzo.cus
with those of the
embryos of the Vertebrates above it in rank, especiall
those of the Marsipobranchs and Selachians. Thoug
the comparisons with the organisms next below Am-
phioxus, such as Asczdzans, Balanoglossus, Cepha-
lodiscus,
Rhabdopleura, and
the Echinoderms,
will be found no less interesting. In short, the book
may be commended
to students already somewhat
familiar with zodlogical facts and principles, as an
important one to read. They may thus be brought
to appreciate to what an extent the theory of descent
is indebted to the patient labors of the zodlogists of the last forty years fora secure foundation in observed
facts, seen in their correlations, according to the com-
parative method. ... The present work contains
everything that should be known about Amphioxus,
besides
a great deal that is advantageous to know
about the Tunicata, Balanoglossus, and some other
types which come into structural relations with Am-
phioxus.”
— Professor Joun A. RypEr, the last forty years fora secure foundation in observed
facts, seen in their correlations, according to the com-
parative method. ... The present work contains
everything that should be known about Amphioxus, everything that should be known about Amphioxus,
besides
a great deal that is advantageous to know
about the Tunicata, Balanoglossus, and some other
types which come into structural relations with Am-
phioxus.”
— Professor Joun A. RypEr, types which come into structural relations with Am-
phioxus.”
— Professor Joun A. RypEr, — Professor Joun A. RypEr,
in The
American Naturali in The
American Naturalist, Philadelphia. “‘ The observations on Amphioxus made before the
second half of the present century, amongst which. 316 pages.
135 Illustrations.
Price, $2.50. those of Johannes Miiller take a foremost place, showed
that this remarkable animal bears certain resemblances
to Vertebrates; and since then its interest in this re-
spect has gradually become more apparent.... A
consecutive history of the more recent observations
was, therefore, greatly needed by those whose oppor-
tunities did not permit them to follow out the matter
for themselves, and who will welcome a book written
in an extremely lucid style by a naturalist who can
speak with authority
on the subject.”
— Professor W. NEwTon PARKER, “‘ The observations on Amphioxus made before the
second half of the present century, amongst which. those of Johannes Miiller take a foremost place, showed
that this remarkable animal bears certain resemblances
to Vertebrates; and since then its interest in this re-
spect has gradually become more apparent.... A
consecutive history of the more recent observations — Professor W. NEwTon PARKER,
in Nature, Londo — Professor W. NEwTon PARKER,
in Nature, Londo in Nature, London. “The length to which this review has extended
must be evidence of the importance of Dr. Dean's By BASHFORD DEAN, Ph.D., Adjunct Professor of Zotlogy, Columbia Univer
300 pages.
344 Illustrations.
Price, $2.50. 300 pages. 344 Illustrations. Price, $2.50. This work has been prepared to meet the need of the general student for a concise knowledge of the living
and extinct Fishes. Itcovers the recent advances in the comparative anatomy, embryology, and paleontology
of the five larger groups of Lampreys, Sharks, Chimzroids, Teleostomes, and Dipnoans—the aim being to
furnish a well-marked ground plan of Ichthyology. The figures are mainly original and designed to aid in prac-
tical work as well as to illustrate the contrasts in the development of the principal organs through the five groups. “‘The
intense
specialization which
prevails
in
zoGlogy at the present day can lead to no other result
than this, that a well-educated zodlogist who becomes
a student of one group is in a few years quite left
behind by the student
of other groups. Books,
therefore, like those of Mr. Dean are necessary for
zovlogists at large.”
— The Atheneum, London. work. The suggestions here offered may be of use
for another edition. That another may be called for,
we may hope. For the work as it is, and for the care
and thought bestowed on it, our thanks are due.”
— THEODORE GILL, — THEODORE GILL,
in Sczence,
New — THEODORE GILL,
in Sczence,
New in Sczence,
New York. in Sczence,
New York. — The Atheneum, London. in Natural Science, London. — The Atheneum, London. ‘*L’ouvrage de M. Bashford Dean nous parait fait
avec soin;
les illustrations sont excellentes et trés
nombreuses, et i] mérite le meilleur accueil de la part
des zoologistes.”
:
— Cu. Brononiart, “‘Dr. Bashford Dean is known to zodlogists, first,
as the author of exhaustive and critical articles in the
publications of the United States Fish Commission,
on the systems of oyster culture pursued in Europe,
and, secondly, as an embryologist who has lately been
doing good work on the development of various Ga-
noid fishes and the comparison that may be instituted
with Teleostei. His recent addition to the well-known
“Columbia University Biological Series,’ now being
brought out by The Macmillan Company, under the
editorship of Professor H. F. Osborn, is an interesting
volume upon fishes, in which considerable prominence
is given to the fossil forms, and the whole subject is
presented to us from the point of view of the evolu-
tionist. This is the characteristic feature of the book. From the very first page of the introduction to the
last page in the volume, preceding the index, which
is a table of the supposed descent of the groups of
fishes, the book is full of the spirit and the language
of evolution.”
— Professor W. A. HerpMan,
in Nature, London. OPINIONS OF THE PRESS. “A somewhat new and very interesting field of in-
quiry is opened in this work, which is devoted to
demonstrating that the doctrine of Evolution, far
from being a child of the middle of the nineteenth
century, of sudden birth and phenomenally rapid
growth, as it is by many supposed to be, has really
been in men’s minds for ages. It appears in the
germ in the earliest Greek philosophy; in vigorous
childhood in the works of Aristotle ; in adolescence
at the closing period of the last century; and reaches
full-grown manhood in our own age of scientific
thought and indefatigable research.”
— New Science Review. “But whether the thread be broken or continuous,
the history of thought upon this all-important subject
is of the deepest interest, and Professor Osborn’s
work will be welcomed by all who take an intelligent
interest in Evolution. Up to the present, the pre-
Darwinian evolutionists have been for the most part
considered singly, the claims of particular naturalists
being urged often with too warm an enthusiasm. Professor Osborn has undertaken a more compre-
hensive
work,
and with well-balanced judgment
assigns a place to each writer.”
— Professor Epwarp B. Poutton, — New Science Review. “This is a timely book. For it is time that both
the special student and general public should know
that the doctrine of Evolution has cropped out of the
surface of human thought from the period of the
Greek philosophers, and that it did not originate
with Darwin, and that natural selection is not
a
synonym of Evolution... . The book should be — Professor Epwarp B. Poutton,
in Nature, Lon — Professor Epwarp B. Poutton,
in Nature, Lond in Nature, London, VOL. Ill. FISHES, LIVING AND FOSSIL.
AN INTRODUCTORY STUDY. By BASHFORD DEAN, Ph.D., Adjunct Professor of Zotlogy, Columbia Unive By EDMUND B. WILSON, Ph.D.,
of Invertebrate Zoblogy, Columbia Un 371 pages. 142 Illustrations. Price, $3.00. VOL. V.
THE FOUNDATIONS OF ZOOLOGY.
By WILLIAM KEITH BROOKS, Professor of Zodlogy, Johns Hopkins University. 8vo. Cloth. Pp. viii+ 339. Price, $2.50 ver. “A book that will live as a permanent addition to the common sense of science. It belongs to literature as
well as to science. It belongs to philosophy as much as to either, for it is full of that fundamental wisdom
about realities which alone is worthy of the name of philosophy.”
Sczence. — Cu. Brononiart,
in Le Revue Sci — Cu. Brononiart,
in Le Revue Sci — Cu. Brononiart,
in Le Revue Sci in Le Revue Scientifique, Paris. ‘For the first time in the history of Ichthyology,
students are now provided with an elementary
hand-
book affording a general view of the whole subject... .. The last sixty pages of the volume are devoted to
a list of derivations of proper names, a copious bibli-
ography, and a series of illustrated tabular statements
of the anatomical characters of the great groups of
fishes. These sections bear signs of having been
prepared most carefully and laboriously, and form an
admirable appendix for purposes of reference. There
will be much difference of opinion among specialists
as to the value of some of the tables and the judgment
pronounced by the author; but we have detected a
very small proportion of errors for so bold an enter-
rise, and students of the lower Vertebrata are much
indebted to Dr. Dean for an invaluable compendium.”
— ARTHUR SMITH Woopwarp,
in Natural Science, London. in Nature, London. in Nature, London. “The length to which this review has extended
must be evidence of the importance of Dr. Dean's in Natural Science, London. [EXTRACTS FROM PREFACE AND INTRODUCTORY CHAPTER.] “This volume is the outcome of a course of lec-
tures, delivered at Columbia University in the winter
of 1892-1893, in which I endeavored to give to an
audience of general university students some account
of recent advances in cellular biology, and more espe-
cially to trace the steps by which the problems of
evolution have been reduced to problems of the cell. It was my first intention to publish these lectures in
a simple and general form, in the hope of showing to
wider circles how the varied and apparently hetero-
geneous cell-researches of the past twenty years have
grown together in a coherent group, at the heart of
which are a few elementary phenomena, and how
these phenomena, easily intelligible to those having
no special knowledge of the subject, are related to
the problems of development. ... The rapid ad-
vance of discovery in the meantime has made it
seem desirable to amplify the original plan of the
work, in order to render it useful to students as well
as to more general readers... . This book does
not, however, aim to be a treatise on general his-
tology.” ““The theory of evolution originally grew out of
the study of natural history, and it took definite
shape long before the ultimate structure of living
bodies was in any degree comprehended. ... The
study of microscopical anatomy, in which the cell-
theory was based, lay in a different field... . Only
within a few years has the ground been cleared for
that close alliance of the evolutionists and the cytolo-
gists which forms so striking a feature of the contem-
porary biology. . . .” “‘The opening chapter is devoted
to a general
sketch of cell-structure, and the second to the phe-
nomena of cell-division. The following three chap-
ters deal with the germ-cells,
— the third with their
structure and mode of origin, the fourth with their
union in fertilization, the fifth with the phenomena
of maturation. . . The sixth chapter contains a
critical discussion of cell-organization,
... In the critical discussion of cell-organization,
... In the
seventh chapter the cell is considered with reference
to its more fundamental chemical and physiological
properties. . . .” VOL. VI.
THE PROTOZOA.
By GARY N. CALKINS, Ph.D. The object of this volume is to set forth the main characteristics of the Protozoa without undertaking an
exhaustive description. It is intended for students and for general readers who wish to know what the Pro-
tozoa are, and what their relations are to current biological problems. In the first few chapters of the book
the Protozoa are treated as a phylum of the animal kingdom. A short historical sketch leading up to the
present systems of classification is followed by a general description of the group, touching upon some of the
more special subjects, such as mode of life, motion, excretion, respiration, reproduction, colony-formation,
encystment, etc., and this is followed by more general subjects dealing with the Protozoa in relation to man
and other animals; e.g. their sanitary aspects, parasitism, symbiosis, etc. :
In the final chapter the Protozoa are dealt with from the standpoint of phylogeny. Theories as to the In the final chapter the Protozoa are dealt with from the standpoint of phylogeny. Theories as to the
origin of life, spontaneous generation, and the relations
of the classes of Protozoa to one another are con-
sidered, and the volume ends with a discussion of the various views regarding the origin of the Metazoa from
the Protozoa.
|
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Formation of Oil-Particle-Aggregates: Numerical Model Formulation and Calibration
|
Frontiers in marine science
| 2,021
|
cc-by
| 11,877
|
ORIGINAL RESEARCH
published: 13 May 2021
doi: 10.3389/fmars.2021.629476 Formation of
Oil-Particle-Aggregates: Numerical
Model Formulation and Calibration
Linlin Cui1*, Courtney K. Harris1* and Danielle R. N. Tarpley1,2
1 Virginia Institute of Marine Science, William & Mary, Gloucester Point, VA, United States, 2 Coastal and Hydraulics
Laboratory, United States Army Corps of Engineers, Vicksburg, MS, United States When oil spills occur in turbid waters, the oil droplets and mineral grains can combine
to form oil-particle aggregates (OPAs). The formation of OPAs impacts the vertical
transport of both the oil and the mineral grains; especially increasing deposition of oil
to the seabed. Though the coastal oceans can be very turbid, to date, few numerical
ocean models have accounted for aggregation processes that form OPAs. However,
interactions between oil and mineral aggregates may be represented using techniques
developed to account for sediment aggregation. As part of Consortium for Simulation of
Oil Microbial Interactions in the Ocean (CSOMIO), we modified an existing, population
dynamics-based sediment flocculation model to develop OPAMOD, a module that
accounts for the formation of OPAs. A zero-dimensional model using OPAMOD is
shown to be capable of reproducing the size distribution of aggregates from existing
laboratory experimental results. Also using the zero-dimensional model, sensitivity tests
were performed on two model parameters, the fractal dimension and collision efficiency. Results showed that fractal dimension played a role in the OPA size distribution by
influencing the effective particle density, which modified the number concentration of
flocs for a given mass concentration. However, the modeled particle characteristics
and oil sequestration were relatively insensitive to collision efficiency. To explore OPA
formation for an outer continental shelf site, two simulations were conducted using
a one-dimensional (vertical) implementation of the model. One scenario had high
sediment concentration near the seabed to mimic storm-induced resuspension. The
other scenario represented river plume sediment delivery by having high sediment
concentration in surface waters. Results showed that OPA formation was sensitive to
the vertical distribution of suspended sediment, with the river plume scenario creating
more OPA, and sequestering more oil within OPA than the storm resuspension scenario. OPAMOD was developed within the Coupled Ocean-Atmosphere-Wave-and-Sediment
Transport (COAWST) modeling system, therefore the methods and parameterizations
from this study are transferrable to a three-dimensional coupled oil-sediment-microbial
model developed by CSOMIO within the COAWST framework. Keywords: oil-particle aggregates, flocculation, fractal dimension, numerical modeling, oil spill Edited by:
Andrew James Manning,
HR Wallingford, United Kingdom Reviewed by:
Byung Joon Lee,
Kyungpook National University,
South Korea
Junhong Liang,
Louisiana State University,
United States *Correspondence:
Linlin Cui
lcui@vims.edu
Courtney K. Harris
ckharris@vims.edu Specialty section:
This article was submitted to
Marine Pollution,
a section of the journal
Frontiers in Marine Science
Received: 14 November 2020
Accepted: 14 April 2021
Published: 13 May 2021
Citation:
Cui L, Harris CK and Tarpley DRN
(2021) Formation
of Oil-Particle-Aggregates: Numerical
Model Formulation and Calibration. Front. Mar. Sci. 8:629476. doi: 10.3389/fmars.2021.629476 Specialty section:
This article was submitted to
Marine Pollution,
a section of the journal
Frontiers in Marine Science Specialty section:
This article was submitted to
Marine Pollution,
a section of the journal
Frontiers in Marine Science
Received: 14 November 2020
Accepted: 14 April 2021
Published: 13 May 2021 Received: 14 November 2020
Accepted: 14 April 2021
Published: 13 May 2021 INTRODUCTION Oil spills have detrimental impacts on coastal ecosystems and
marine life (Nixon et al., 2016; Bam et al., 2018; Robinson
and Rabalais, 2019; Martin et al., 2020). As a result, oil
models have been developed to predict the transport and
fate of oil, often by employing Lagrangian particle tracking
(Zhu et al., 2018). However, predicting the fate of oil is
difficult as it is influenced by a variety of processes, such as
spreading, evaporation, and degradation for surface slicks; and
emulsification, degradation, and dissolution for oil droplets in
the subsurface. In addition, aggregation of oil and suspended
particulate material has been considered an important process to
remove free oil (Lee, 2002; Payne et al., 2003). It was reported
that significant oil sedimentation can occur when suspended
particulate concentrations reach 0.01 kg m−3, and large
deposition events were associated with sediment concentrations
of at least 0.1 kg m−3 (Boehm, 1987). The Mississippi River
mouth delivers significant quantities of both freshwater and
sediment into the northern Gulf of Mexico. Suspended sediment
concentrations vary seasonally. The Mississippi River discharge
peak occurs during the spring, when it is about twice as high as
the low flow in the fall (Thorne et al., 2008). A previous study
showed that the formation of OPAs in the Mississippi River varied
depending on the time of year with a higher probability during
winter and spring when suspended sediment concentrations were
high, ∼0.35 kg m−3 (Danchuk and Willson, 2011). With an
aggregation kinetic model, Sterling et al. (2004) found that the
timescale for aggregation of oils and clays was within an order
of magnitude for the timescale of clay settling, thus, aggregation
provided a mechanism for oil sedimentation. Nevertheless,
numerical models for oil-sediment interactions are limited,
especially in three-dimensional oil models for the coastal ocean. Numerical
flocculation
models
that
represent
particle
aggregation and breakup have a long history within sediment-
transport (Lick et al., 1992; Winterwerp, 1998), and can provide
a method to represent OPA formation. There are two main
approaches to representing particle sizes in these sediment
flocculation models. The first type are distribution-based models,
in which floc size distribution is fixed but the modeled sizes
change in response to turbulence, concentration, etc. (Khelifa
and Hill, 2006; Winterwerp et al., 2006). The other method uses
size-class-based models that calculate the particle population
by distributing mass among a discrete number of fixed size
classes (Verney et al., 2011). INTRODUCTION Unlike distribution-based models,
size-class-based models can provide detailed information about
floc populations. Recently, a size-class-based flocculation model
(FLOCMOD) (Verney et al., 2011) has been implemented
within
the
COAWST
(Coupled-Ocean-Atmosphere-Wave-
Sediment Transport) model (Sherwood et al., 2018) and tested
in an idealized, partially mixed estuary to investigate cohesive
sediment distribution over a tidal cycle (Tarpley et al., 2019). To date, however, these types of flocculation models have solely
been used to characterize the fate and aggregation of sediment. We expand upon these by using the FLOCMOD numerical
framework to account for the formation of OPA that incorporate
oil, sediment, and water within aggregates. When oil interacts with organic matter, such as bacteria,
phytoplankton, or detritus; they can form a type of aggregate
called Marine Oil Snow (MOS). Marine Oil Snow Sedimentation
and Flocculent Accumulation (MOSSFA) is a significant pathway
for the fate of oil. Studies showed that ∼4–31% of the Deepwater
Horizon oil settled on the seafloor via MOSSFA (Valentine
et al., 2014). These aggregates are more common in areas
where primary productivity is high and suspended sediment
concentrations are relatively low (<0.01 kg m−3), such as the
open ocean (Daly et al., 2016). Compared to OPA, the MOS
tend to reach larger diameters (Stoffyn-Egli and Lee, 2002). Simulation of MOS has obtained substantial interest since the
Deepwater Horizon oil spill. For example, Dissanayake et al. (2018) developed a one-dimensional model to simulate MOS
formation and settling in the ocean. This provided valuable
insight into the vertical processes that govern MOSSFA formation
and transport, but to date these processes have not been included
in a three-dimensional model capable of estimating dispersal. An important parameter in representing the formation and
evolution of aggregates of particles is the fractal dimension,
which generally represents the shape and packing of an aggregate
(Wiesner, 1992; Kranenburg, 1994). Fractal dimension has been
used to represent sediment flocs, marine snow, and oil-mineral
aggregates, among other aggregate types. Fractal dimensions
can range from 1 to 3, with a value of three representing
a solid particle, such as sand grains. Natural sediment flocs
typically have a fractal dimension close to 2.1 (Tambo and
Hozumi, 1979; Kranenburg, 1994), with broadly reported fractal
dimensions with the range of 1.7 ∼2.85 (Wiesner, 1992). Citation: Cui L, Harris CK and Tarpley DRN
(2021) Formation
of Oil-Particle-Aggregates: Numerical
Model Formulation and Calibration. Front. Mar. Sci. 8:629476. doi: 10.3389/fmars.2021.629476 May 2021 | Volume 8 | Article 629476 1 Frontiers in Marine Science | www.frontiersin.org Modeling of Oil-Particle Aggregates Cui et al. been preferred to describe microscopic aggregates. In terms of
structure, three types of OPAs were identified from microscopy
imageries: droplet OPAs, solid OPAs, and flake OPAs (Stoffyn-
Egli and Lee, 2002). Droplet OPAs are single or multiple oil
droplets coated by sediment particles; solid OPAs are a mixture
of oil and sediment particles where clear oil droplets cannot be
seen; flaked OPAs have a membrane-like shape, are fragile and
break apart in high shear conditions, thus they are rarely observed
in nature (Stoffyn-Egli and Lee, 2002). When oil is delivered to
areas of high turbidity, such as surf zones and river plumes, OPAs
provide a means of delivering oil to the seafloor (Daly et al.,
2016). In general, OPAs observed in situ are small (<50 micron–
1 mm) (Stoffyn-Egli and Lee, 2002). Sediment mineralogy can
affect the formation and shape of OPAs. A recent laboratory study
investigated the formation of OPAs with crude oil and two types
of cohesive minerals: kaolinite and bentonite clay (Ye et al., 2020). They showed that bentonite clay had a higher stickiness than
kaolinite, and tended to produce solid, higher-settling velocity
OPAs. Based on these results, along with the fact that bentonite
is more like the clay observed near Mississippi River Delta than
kaolinite (Jaisi et al., 2008), solid OPAs formed from oil and
bentonite clay were considered in this study. Frontiers in Marine Science | www.frontiersin.org Existing COAWST System Including
FLOCMOD The COAWST modeling system was developed to couple
the ROMS hydrodynamic model with Weather Research and
Forecasting Model (WRF) and Simulating Waves Nearshore
(SWAN) to provide two-way links between the hydrodynamic
(ROMS), atmospheric (WRF), and wave (SWAN) models
(Warner et al., 2010). ROMS is capable of solving for the
transport of tracer fields, including salinity, temperature,
sediment, etc. (Haidvogel et al., 2008). The Community Sediment
Transport
Modeling
System
(CSTMS)
was
implemented
in ROMS for sediment transport and evolution of bottom
morphology (Warner et al., 2008). Further model development
added
processes
of
sediment
flocculation
(FLOCMOD)
including aggregation and disaggregation (Sherwood et al.,
2018). FLOCMOD is a population-balance module using a
finite number of floc size classes based on Verney et al. (2011). Each floc class is assumed to have the same fractal dimension,
but assigned a different floc diameter, floc density, and setting
velocity. FLOCMOD has been tested in one-dimensional
simulations to reproduce a tidal cycle variation of sediment flocs
from a laboratory experiment (Sherwood et al., 2018), and two-
dimensional simulation for an idealized, muddy estuary (Tarpley
et al., 2019). Within this paper we implemented COAWST
to use ROMS, CSTMS, and the newly developed OPAMOD
in zero-dimensional and one-dimensional configurations, as
described below. An objective of our study was to develop a module capable
of simulating oil-particle aggregation that could be coupled
to other components of the COAWST modeling system. To accomplish this, we developed OPAMOD, a population-
balance module based on FLOCMOD, but modified to account
for the formation of OPAs. Further motivation was to
facilitate the use of OPAMOD within a three-dimensional
implementation of COAWST that coupled hydrodynamic,
sediment, microbial, and oil components (Dukhovskoy et al.,
2021). This coupled model included hydrodynamics, sediment
transport, and biogeochemical modules available from ROMS
(Shchepetkin and McWilliams, 2005; Fennel et al., 2006;
Haidvogel et al., 2008; Warner et al., 2008). To these modules,
Dukhovskoy et al. (2021) added OPAMOD, OWTM (the Oil
Weathering and Transport Module), and a module to account
for biological weathering of oil (Coles et al., 2020). They
then used the fully coupled model to estimate oil dispersal
for situation similar to the Deepwater Horizon event for a
timescale of several days. To be useful within the fully coupled
model required that OPAMOD carry a moderate computational
expense. MATERIALS AND METHODS This section describes the modeling framework and assumptions
used to represent the formation of OPAs. The new module,
OPAMOD, was implemented within the hydrodynamic model,
ROMS, which is the ocean model component of COAWST. INTRODUCTION Different terminologies have been used to describe aggregates
that incorporate oil along with sediment, such as oil-mineral
aggregate (OMA) (Lee and Stoffyn-Egli, 2001; Ye et al., 2020),
oil-sediment aggregate (OSA) (Bandara et al., 2011), and oil-
particle aggregate (OPA) (Fitzpatrick et al., 2015; Zhao et al.,
2016). We use the term OPA in this study, because this has May 2021 | Volume 8 | Article 629476 Frontiers in Marine Science | www.frontiersin.org 2 Modeling of Oil-Particle Aggregates Cui et al. Cui et al. However, for sediment flocs, some studies have reported higher
fractal dimensions, between 2.6 and 3.0 (Lee et al., 2002). Fractal dimensions have also been reported for marine snow,
falling in the range of 1.3 ∼2.3 (Burd and Jackson, 2009). The main factors that can affect the fractal dimension include
turbulence (Logan and Kilps, 1995; Dyer and Manning, 1999),
the shape (e.g., ellipsoidal and spherical) of the primary particles
(Perry et al., 2012), and the makeup of the components (e.g.,
mineral types, organic matters, and oil) (Lee et al., 2002;
Jarvis et al., 2005; Ye et al., 2020). In the laboratory, fractal
dimensions can be indirectly estimated from properties related
to aggregate size, such as settling velocity. Based on this method,
Ye et al. (2020) reported that fractal dimension of kaolinite-oil
aggregates varied from 2.4 to 2.6, while fractal dimension of
bentonite-oil aggregates was in the range of 2.2–2.4. Flocculation
and aggregation models often assume a single average fractal
dimension throughout all size ranges. For instance, Dissanayake
et al. (2018) chose a fractal dimension of 2.2 to represent the
MOS aggregates in their one-dimensional model. FLOCMOD
also assumes a constant fractal dimension (Verney et al., 2011;
Sherwood et al., 2018). For example, Tarpley et al. (2019) used
a fractal dimension of 2.4 for FLOCMOD in a two-dimensional
sediment transport model. Model Implementation and Results”). Next, a one-dimensional
model was used to represent an oil spill on a continental
slope setting to evaluate the relative importance of OPA
formation under storm, and freshwater plume scenarios (Section
“Scenarios:
Implementation
of
One-Dimensional
Model”). Conclusions and discussion are given in Section “Conclusion
and Discussion.” Frontiers in Marine Science | www.frontiersin.org Existing COAWST System Including
FLOCMOD Additionally, the application focused on transport of
oil droplets over the continental slope and outer continental
shelf, where sediment concentrations and turbulence levels are
relatively low. This meant that our formulation could focus on
the aggregate formation process to account for settling of oil
via OPA, and neglect potential breakup of OPA once formed
as has been done by similar models (Bandara et al., 2011;
Zhao et al., 2016). OPA Module Development To account for OPA, the tracer fields in ROMS were expanded
to include sediment flocs, oil droplets, and OPAs. These tracers
for flocs, oil droplets, and OPAs were represented using a finite
number of classes; each with a prescribed diameter, settling
velocity, and density. Assumptions The rest of this paper describes the development of
OPAMOD
and
two
stand-alone
applications. Specifically,
Section “Materials and Methods” describes model development. A zero-dimensional model implementation was then used to
validate model performance based on available laboratory data,
and test its sensitivity to parameters (Section “Zero-Dimensional Our approach used a population-balance model for OPA
formation and growth, specifically representing interactions
between three types of tracers: oil droplets, sediment flocs, and
OPAs (Figure 1). In nature, the formation, aggregation, and
disaggregation of an OPA is complicated. Interactions include May 2021 | Volume 8 | Article 629476 3 Cui et al. Modeling of Oil-Particle Aggregates FIGURE 1 | Schematic of modeled formation processes for OPA. Mopa,k is the mass of newly formed OPA of class k, which is the mass addition of two collision
components (e.g., collision between the ith – class oil Moil,i and the jth – class sediment Mp,j). Assuming that Mopa,k falls between two OPA classes, then this mass
is mass-linearly distributed into the two neighboring classes [i.e. the nth and (n+1)th OPA classes]. FIGURE 1 | Schematic of modeled formation processes for OPA. Mopa,k is the mass of newly formed OPA of class k, which is the mass addition of two collision
components (e.g., collision between the ith – class oil Moil,i and the jth – class sediment Mp,j). Assuming that Mopa,k falls between two OPA classes, then this mass
is mass-linearly distributed into the two neighboring classes [i.e. the nth and (n+1)th OPA classes]. (4) The fractal behavior of OPAs was assumed to be the
same as that of sediment flocs, which is consistent with
laboratory observations of Ye et al. (2020). Therefore,
the effective density was related to the primary particle
diameter and density through fractal dimension in the
following relationship (Kranenburg, 1994): collisions between particles and droplets that can form larger
particles or droplets; or can result in the breakup into smaller
particles and droplets (Lee et al., 1997). An oil droplet can
coalesce with other oil droplets to form a larger oil droplet;
or with sediment flocs to create OPAs; also, an oil droplet can
interact with OPAs. Likewise, a sediment floc can combine with
other sediment flocs to form larger flocs; or with oil droplets
to form OPAs; or interact with existing OPAs. Finally, OPAs
can interact with other OPAs. Assumptions The simulation was conducted
with concentration thresholds for both sediment and oil; i.e., the
OPA aggregation process was not invoked when concentrations
were below 0.001 kg m−3. To reduce model complexity while
addressing the primary goal of providing estimates of OPA
formation and sinking for use on continental shelves, the
following assumptions were made: ρe = (ρs −ρw)( D
Dp
)
nf −3
(1) (1) where ρe is the effective density of the floc or OPA, ρs
is the density of the particle, and ρw is the density of
water. D is the diameter; and Dp is the diameter of the
primary particle, i.e. the smallest floc particle. The fractal
dimension is nf . f
(5) Each sediment class and OPA class was assigned a diameter,
density, and settling velocity as model input parameters. f
(5) Each sediment class and OPA class was assigned a diameter,
density, and settling velocity as model input parameters. (1) Including all potential aggregation and disaggregation
processes in the model would require a large number of
parameters and high degree of complexity. As noted above,
a variety of aggregation processes can occur in coastal
systems that include sediment and oil droplets. To reduce
the complexity and increase computational efficiency, the
implementation of this model only considered interactions
between oil droplets and sediment flocs, oil droplets
and OPAs, and sediment flocs and OPAs (Figure 1),
following Zhao et al. (2016). (6) Formed OPAs are considered to be stable, i.e., the model
neglected OPA breakup processes. This assumption is
consistent with previous models (Bandara et al., 2011;
Zhao et al., 2016), and seemed reasonable for meeting
the primary objective of accounting for OPA formation,
settling and subsequent removal of oil from the water
column. It effectively considers the OPA to be resilient to
breakup and avoids an added complexity in the model of
specifying the particle makeup that would be produced
via breakup. (6) Formed OPAs are considered to be stable, i.e., the model
neglected OPA breakup processes. This assumption is
consistent with previous models (Bandara et al., 2011;
Zhao et al., 2016), and seemed reasonable for meeting
the primary objective of accounting for OPA formation,
settling and subsequent removal of oil from the water
column. It effectively considers the OPA to be resilient to
breakup and avoids an added complexity in the model of
specifying the particle makeup that would be produced
via breakup. Assumptions (2) Collision between two entities can result from turbulent
motions, differential settling, and Brownian motion. Collisions due to Brownian motion are unimportant in
ocean systems and shear-induced collisions are dominant
in turbulent environments (Sherwood et al., 2018),
therefore, we only implemented collisions due to fluid
shear in the model. ZERO-DIMENSIONAL MODEL
IMPLEMENTATION AND RESULTS (2) OPAMOD was implemented within a quasi-zero-dimensional
framework,
as
described
below. This
was
first
used
to
demonstrate
that
the
model
results
compared
favorably
to laboratory observations of OPA properties, and to aid with
selection of model parameters (Section “Model Comparison With
Laboratory Experimental Data”). Next, the zero-dimensional
implementation was used to evaluate the sensitivity of OPA
properties to the choice of the collision efficiency and fractal
dimension (Section “Sensitivity Tests”). where α represents collision efficiency. Note that this formulation
used a single value for α regardless of the types of particles
involved in a collision. Zhao et al. (2016) developed an
estimate for coagulation efficiency applied on the conceptual
basis of sediment particles coating an oil droplet. Dissanayake
et al. (2018) cited values of alpha that ranged from 0.08 to
0.8, depending on the makeup of the particle (i.e., organic
material, mineral content, oil content). However, these were
not directly applicable to the solid OPAs conceptualized
for this study, nor could they be directly implemented
within the full coupled model. Additionally, our sensitivity
study below indicated that the model was not particularly
sensitive to α. For these reasons we used a constant value
for α, though it would be relatively straightforward to
use particle-type dependent values provided guidance for its
parameterization. Finally, the collision probability, A
i, j
is
given by: Governing Equations of OPA Formation Process Governing Equations of OPA Formation Process
At model implementation, the input files specify the number
of tracer classes to be included; specifically, the value of nsed
represents the number of sediment floc classes, noil represents the
number of oil droplet classes, and nopa represents the number
of OPA classes. Changes to the number concentration (i.e. NOIL,i, NP,j, and NOPA,i) due to mass exchanges among classes
were simulated to represent aggregation between two different (3) Sediment flocs, oil droplets, and OPAs were assumed
to be spherical. May 2021 | Volume 8 | Article 629476 Frontiers in Marine Science | www.frontiersin.org 4 Modeling of Oil-Particle Aggregates Cui et al. Finally, the time variation of the number concentrations of oil
droplets in the ith class (NOIL,i) is calculated as: Finally, the time variation of the number concentrations of oil
droplets in the ith class (NOIL,i) is calculated as: class types. The model tracks the time variation of the number
concentration of OPA in the mth class (NOPA,m) as the sum of
gain and loss terms: dNOIL,i
dt
= −NOIL,i
nsed
X
j=1
αA
i, j
NP,j
|
{z
}
oil−sediment loss
−NOIL,i
nopa
X
j=1
αA
i, j
NOPA,j
|
{z
}
oil−opa loss
(5) dNOPA,m
dt
=
noil
X
i=1
nsed
X
j=1
αA
i, j
NOIL,iNP,j
|
{z
}
oil−sediment gain
+
nopa
X
i=1
nsed
X
j=1
αA
i, j
NOPA,iNP,j
|
{z
}
opa−sediment gain
+
noil
X
i=1
nopa
X
j=1
αA
i, j
NOIL,iNOPA,j
|
{z
}
oil−opa gain
−NOPA,m
nsed
X
j=1
αA
m, j
NP,j
|
{z
}
opa−sediment loss
−NOPA,m
noil
X
j=1
αA
i, j
NOIL,j
|
{z
}
oil−opa loss
(2) {z
oil−opa loss {z
oil−sediment loss (5) (5)
OPAMOD classifies OPAs by density (ρi,opa), vertical terminal
velocity (wi,opa), and median diameter (Di,opa), for OPA sizes
i=1, . . . nopa. Since OPAMOD tracks a finite number of OPA
classes, the mass of each newly formed OPA is mass-linearly
distributed into two neighboring classes (Bandara et al., 2011;
Verney et al., 2011) rather than assigning the total mass to one
class (Figure 1). {z
oil−sediment gain Model Comparison With Laboratory
Experimental Data The first set of model results were compared with experimental
data from Ye et al. (2020) who conducted six laboratory
experiments to investigate characteristics (e.g., settling velocity,
effective density, and fractional dimension) of OPAs formed
from kaolinite or bentonite clays mixed with crude oil. Previous
studies have shown that when the ratio of oil to sediment is
close to two, it can reach a maximum OPA formation efficiency
(Guyomarch et al., 2002; Khelifa et al., 2008). Ye et al. (2020)
therefore generated OPAs by adding 0.5 g of clay and 1.0 g of
Texas crude oil into a jar containing 1.0 L of artificial seawater. The mixture was stirred at 490 rpm, which gave an estimated
turbulence dissipation rate ε about 0.02 m2s−3. The turbulence
was maintained for up to 2 h, and then flocs and OPAs were
collected and analyzed from materials after they settled overnight. Statistical analysis of these experimental samples showed that
the floc and OPA sizes varied from 20 to 700 µm. Bentonite
clays tended to generate compact, negatively buoyant OPAs
(Ye et al., 2020). A
i, j
= 1
6G(Di + Dj)3
(3) (3) where Di and Dj are diameters of two colliding entities; G is the
shear rate, which can be expressed by energy dissipation rate ε
and kinematic viscosity ν, as G= √ε/ν. The time variation of the number concentration of sediment
particles in the ith class (NP,i) is calculated as: To
compare
with
these
laboratory
measurements,
we
configured a quasi-zero-dimensional model in ROMS to
simulate the scenario where OPAs formed from oil and bentonite
clay. The shear rate G was specified as 20 s−1 over the simulation
time of 2 h, replicating the laboratory experiment. In actuality,
the ROMS implementation was a small three-dimensional
grid, but the model was effectively zero-dimensional because dNP,i
dt
= −NP,i
noil
X
j=1
αA
i, j
NOIL,j
|
}
−NP,i
nopa
X
j=1
αA
i, j
NOPA,j
|
} {z
oil−sediment loss {z
oil−sediment loss (4) May 2021 | Volume 8 | Article 629476 Frontiers in Marine Science | www.frontiersin.org Frontiers in Marine Science | www.frontiersin.org Modeling of Oil-Particle Aggregates Cui et al. conditions were uniform, the horizontal boundary conditions
were periodic, and settling velocities were set to zero to maintain
a vertically uniform suspension. Model Comparison With Laboratory
Experimental Data The quasi-zero-dimensional
simulation therefore operated as uniform in all three directions,
so that the only mechanism for changes to the distributions of
sediment, oil droplets, and OPA were via the OPAMOD terms. The model used five sediment classes, twelve OPA classes, and six
oil classes (Table 1). The initial concentrations of sediment and
oil, shown in Figure 2, were 0.5 and 1.0 kg m−3, respectively, to
match laboratory conditions (Ye et al., 2020). The sediment mass
concentration was evenly distributed among the five sediment
classes (Figure 2A), and the initial oil distribution was as shown
(Figure 2B). In this simulation, the collision efficiency was 0.35,
and fractal dimension was 2.3, which were obtained using model
calibration simulations to match the laboratory results. The
primary particle diameter was 4 µm, which matched the value
used by Ye et al. (2020). experimental OPA size peaked in class 5 (D = 180 µm) with a
number fraction of ∼20% (Figure 3A). Both the modeled and
experimental OPAs were mainly composed of small and medium
aggregates (>95% of the population, Figure 3B). However,
the number fraction of modeled small OPAs (class 2 and 3,
D = 60 and 100 µm) slightly exceeded that of experimental
ones (Figure 3B). Sensitivity Tests (A) Histogram of OPA sizes, (B) Cumulative number percentage of OPAs. FIGURE 3 | Comparison of the number fraction of oil-bentonite aggregates between the lab experiments (Ye et al., 2020) and 0-D model that included OPAMOD
after a simulation time of 2 h. (A) Histogram of OPA sizes, (B) Cumulative number percentage of OPAs. distribution shifted from coarse toward fine aggregates. For the
sensitivity test that used a fractal dimension of 2.5, the finer
classes (D ≤140 µm) accounted for almost 65% of the number
concentration (Figure 5A). For the simulation that used a fractal
dimension of 2.0, finer classes made up less than 10% of the
total (Figure 5A). For simulations with intermediate fractal
dimensions (2.2 and 2.3), the intermediate classes (D from
180 to 360 µm) contained greater than 60% of the number
concentrations. In terms of mass concentrations, as the fractal formation, with collision efficiencies that ranged from 0.2 to
0.7. For these simulations, the fractal dimension (nf) was
held constant at 2.3. The final OPA size distributions for
these simulations are shown in Figure 4. The case that
had a higher collision efficiency had slightly higher OPA
concentrations in the middle-sized classes (180 µm < D <
360 µm), and concentrations were slightly shifted to the larger
OPA classes for the case having lower collision efficiency. The modeled OPA size distributions were fairly insensitive to
collision efficiency. y
Next, to estimate the relative influence of fractal dimension
on the size distribution of OPAs, six numerical experiments
were run where fractal dimension was varied from 2.0
to 2.5. For these simulations, collision efficiency was held
at 0.3. Note that because fractal dimension represents the
shape and packing of aggregates, particles with higher fractal
dimensions are denser than particles of the same diameter
with lower fractal dimension (Equation 1). Each of these
sensitivity tests had the same initial sediment concentration
by mass (0.1 kg m−3, matching Ye et al., 2020). Because
they had different fractal dimensions, however they were
each initialized with different number concentrations (NP,i). As the fractal dimension for the test increased, the initial
number of sediment particles decreased. Because the population
balance equations are based on number concentrations, this
had an impact on the frequency of collisions and thus
likelihood of formation of OPAs. Sensitivity Tests Next, sensitivity tests were run to evaluate the impact of
uncertainties in model parameters on the resulting size
distributions of OPAs. The primary model parameters, fractal
dimension and collision efficiency (nf in Equation 1; and α in
Equations 2, 4, 5), were each varied for a series of model runs, and
the resultant effect on the size distribution of OPAs was assessed. The final size distribution of OPAs was seen to be quite sensitive
to the fractal dimension, and insensitive to collision efficiency. Within the model, the mixtures of sediment and oil quickly
combined to form OPAs, and the OPA concentrations and size
distributions reached a steady state within the first few minutes
of the simulation. Figure 3 compares the number fraction of
modeled and experimental OPAs. The size distribution produced
by the model had good agreement with experimental data. The
modeled OPA size peaked in class 6 (D = 220 µm), which
accounted for about 18% of the total number of OPA, while the Collision efficiency (α) represents the probability with
which colliding constituents (sediment, oil droplets, or OPA)
combine to form larger OPA. Values reported in the literature
include 0.4 for heterogeneous collision efficiency (e.g., oil-
clay), and 0.7 for homogeneous collision efficiency (e.g.,
sediment-sediment), respectively (Sterling et al., 2004; Bandara
et al., 2011). Six numerical experiments were run to test
how variations in collision efficiency influenced modeled OPA TABLE 1 | Diameters (D, µm) for sediment (MUD, five size classes), OPA (twelve size classes), and oil droplets (six size classes). 1
2
3
4
5
6
7
8
9
10
11
12
MUD
30
60
100
140
180
OPA
30
60
100
140
180
220
280
360
440
520
600
680
Oil
40
60
80
100
120
140
FIGURE 2 | Initial size mass concentration distributions for OPAMOD simulation of (A) sediment particles, and (B) oil droplets. Oil
40
60
80
100
120
140
G
|
O
O
( )
( ) GURE 2 | Initial size mass concentration distributions for OPAMOD simulation of (A) sediment particles, and (B) oil droplets. May 2021 | Volume 8 | Article 629476 Frontiers in Marine Science | www.frontiersin.org 6 Modeling of Oil-Particle Aggregates Cui et al. FIGURE 3 | Comparison of the number fraction of oil-bentonite aggregates between the lab experiments (Ye et al., 2020) and 0-D model that included OPAMOD
after a simulation time of 2 h. Frontiers in Marine Science | www.frontiersin.org Sensitivity Tests The results of the fractal
dimension sensitivity tests are illustrated as the final OPA
size distributions by number of particles (Figure 5A), and
the final size distribution by mass concentration (Figure 5B). As the assumed fractal dimension increased, the OPA size FIGURE 4 | Size distribution by mass concentration of OPA at the end of 2 h
simulation calculated using different collision efficiencies (see legend). A fractal
dimension of nf = 2.3 was used for these simulations. FIGURE 4 | Size distribution by mass concentration of OPA at the end of 2 h
simulation calculated using different collision efficiencies (see legend). A fractal
dimension of nf = 2.3 was used for these simulations. May 2021 | Volume 8 | Article 629476 Frontiers in Marine Science | www.frontiersin.org Frontiers in Marine Science | www.frontiersin.org 7 Modeling of Oil-Particle Aggregates Cui et al. FIGURE 5 | Size distributions of OPA calculated at the end of 2 h simulation using different fractal dimensions (see legend). A collision efficiency of 0.3 was used for
these simulations. (A) The cumulative distribution by % of the number of particles. (B) Size distribution by mass concentration. FIGURE 5 | Size distributions of OPA calculated at the end of 2 h simulation using different fractal dimensions (see legend). A collision efficiency of 0.3 was used for
these simulations. (A) The cumulative distribution by % of the number of particles. (B) Size distribution by mass concentration. Sediment from the Mississippi River is widely dispersed
within 20–40 km from the bird-foot delta (Xu et al., 2011). Located within 16 km of the South Pass of the Mississippi
River, the MC20 site is therefore often influenced by the
Mississippi River freshwater and sediment plume. Additionally,
during large storms, this location may be impacted a bottom
nepheloid layer sourced from sediment resuspended from
the adjacent continental shelf by energetic waves. However,
it is difficult to quantify potential interactions between oil
released at this site, and sediments in either the surface
freshwater plume, or in a bottom nepheloid layer. Therefore,
the MC20 scenario provides a good example to implement
one-dimensional (vertical) model to explore the potential for
sediment / oil interactions to impact the dispersal of oil in
similar settings. dimension decreased, the OPA shifted toward the larger diameter
particles (Figure 5B). SCENARIOS: IMPLEMENTATION OF
ONE-DIMENSIONAL MODEL Next, the formation of OPA was examined in the context of
an oceanographic water column, using a quasi-one-dimensional
approach. These scenarios were motivated by consideration of a
contamination event at the Taylor Energy Company’s Mississippi
Canyon Block 20 platform (MC20), and used oceanographic
fields (SSC, velocity) from a three-dimensional numerical model
as described below. Background of MC20 Site In September of 2004, Hurricane Ivan passed through the
northern Gulf of Mexico as a category 3 storm generating
22 m waves, and destroying seven drilling platforms (Mason,
2019). The Taylor Energy Mississippi Canyon 20 (MC20) oil and
gas drilling platform, which is located about 16 km southeast
of the mouth of the South Pass of the Mississippi River,
was toppled by storm-triggered submarine mudslides. On 17
September 2004, 1 day after the passage of Ivan, surface oil
slicks were found around the MC20 site. About 10 days later,
satellite images showed oil floating over the sediment-laden
coastal waters offshore of the Mississippi delta (Mason, 2019). Since then, oil plumes and surface oil slicks have been observed
at the MC20 site from ships and satellite remote sensing
(Mason, 2019). Estimates of the oil flux rate span from roughly
9–108 barrels of oil per day, based on acoustic surveys and
bubblometer surveys (MacDonald et al., 2019). The MC20
site is located at a water depth of approximately 140 m,
where the fine sediment (silt and clay) percentage exceeds 90%
(Mason, 2019). Configuration of One-Dimensional Model TABLE 3 | Effective density (ρe, kg m−3) and settling velocity (ws, mm s−1) for
OPA size classes. The fractal dimension was 2.3 in the calculation. interactions between oil contamination and a bottom nepheloid
layer created by resuspended sediment during a tropical storm. The model results from Harris et al. (2020) estimated that about
30% of the suspended sediment delivered to the continental
slope of the northern Gulf of Mexico for the 1-year simulation Configuration of One-Dimensional Model As done for
the zero-dimensional model (Section “Zero-Dimensional Model
Implementation and Results”), the one-dimensional sediment-
OPAMOD model used five cohesive sediment classes, twelve
OPA classes, and six oil droplet classes (Table 1). The effective
density (Equation 1) and settling velocity for sediment flocs
and OPAs are shown in Tables 2, 3, respectively. Here settling
velocity are obtained from laboratory experiment (Ye et al.,
2020). To avoid complexity in the suspended concentrations,
and because little is known about either the erosion thresholds
for OPA or the characteristics of eroded OPA; the critical shear
stress was defined a high value (10 Pa) to prevent erosion from
occurring. Rather, the initial suspended sediment concentrations
were derived from the larger scale ROMS model of Harris et al. (2020), which did include both river plume sedimentation and
seabed resuspension. flow condition; matching values of velocity, temperature, salinity,
and suspended sediment concentrations extracted from a larger-
scale ROMS simulation (Harris et al., 2020). After a 2 h spin-up
time to allow for the flow to accelerate and reach steady state,
OPAMOD was activated. The fractal dimension and collision
efficiency were set to 2.3 and 0.35, respectively. As done for
the zero-dimensional model (Section “Zero-Dimensional Model
Implementation and Results”), the one-dimensional sediment-
OPAMOD model used five cohesive sediment classes, twelve
OPA classes, and six oil droplet classes (Table 1). The effective
density (Equation 1) and settling velocity for sediment flocs
and OPAs are shown in Tables 2, 3, respectively. Here settling
velocity are obtained from laboratory experiment (Ye et al.,
2020). To avoid complexity in the suspended concentrations,
and because little is known about either the erosion thresholds
for OPA or the characteristics of eroded OPA; the critical shear
stress was defined a high value (10 Pa) to prevent erosion from
occurring. Rather, the initial suspended sediment concentrations
were derived from the larger scale ROMS model of Harris et al. (2020), which did include both river plume sedimentation and
seabed resuspension. TABLE 3 | Effective density (ρe, kg m−3) and settling velocity (ws, mm s−1) for
OPA size classes. 1
2
3
4
5
6
ρe
396.56
244.11
170.72
134.90
113.14
98.31
ws
0.433
0.597
0.725
1.459
2.186
2.873
7
8
9
10
11
12
ρe
83.04
69.64
60.51
53.84
48.71
44.62
ws
3.673
5.497
7.087
8.348
13.696
11.003
The fractal dimension was 2.3 in the calculation. Configuration of One-Dimensional Model Configuration of One-Dimensional Model
The quasi-one-dimensional model was configured to generally
represent steady conditions for the MC20 site. The one-
dimensional model was actually a small three-dimensional
implementation, with 5×6 horizontal grid cells, and 50 vertical
grid cells. Lateral periodic boundary conditions were applied
meaning that anything exiting the domain horizontally reentered
on the upstream side, creating a uniform flow field in the
horizontal. The oil concentration was initialized to be vertically
uniform and set to 0.2 kg m−3, based on estimates from the
BP/Deepwater Horizon Oil Spill (Sammarco et al., 2013). The size
distribution of oil was assumed to be the same mass percentage as
in the 0D simulation (Figure 2B). Hydrodynamic and suspended
sediment conditions were obtained from a 1-year long ROMS
simulation (1 October, 2007 through 30 September, 2008) of
the northern Gulf of Mexico that included times of large river
discharge, and two hurricanes (Harris et al., 2020). The one-
dimensional model started from rest and was nudged to a steady May 2021 | Volume 8 | Article 629476 Frontiers in Marine Science | www.frontiersin.org 8 Modeling of Oil-Particle Aggregates Cui et al. TABLE 2 | Effective density (ρe, kg m−3) and settling velocity (ws, mm s−1) for
sediment size classes. 1
2
3
4
5
ρe
396.56
244.11
170.72
134.90
113.14
ws
0.135
0.480
1.105
1.480
1.886
The fractal dimension was 2.3 in the calculation. TABLE 3 | Effective density (ρe, kg m−3) and settling velocity (ws, mm s−1) for
OPA size classes. 1
2
3
4
5
6
ρe
396.56
244.11
170.72
134.90
113.14
98.31
ws
0.433
0.597
0.725
1.459
2.186
2.873
7
8
9
10
11
12
ρe
83.04
69.64
60.51
53.84
48.71
44.62
ws
3.673
5.497
7.087
8.348
13.696
11.003
The fractal dimension was 2.3 in the calculation. TABLE 2 | Effective density (ρe, kg m−3) and settling velocity (ws, mm s−1) for
sediment size classes. flow condition; matching values of velocity, temperature, salinity,
and suspended sediment concentrations extracted from a larger-
scale ROMS simulation (Harris et al., 2020). After a 2 h spin-up
time to allow for the flow to accelerate and reach steady state,
OPAMOD was activated. The fractal dimension and collision
efficiency were set to 2.3 and 0.35, respectively. Hurricane Scenario First, the one-dimensional model was implemented to study
oil-sediment interaction under energetic waves to explore FIGURE 6 | Profiles of (A) velocity and salinity, (B) initial suspended sediment mass concentration, and (C) shear rate for the hurricane scenario (A,B are based on
model output from Harris et al., 2020. C is from 1D model output). FIGURE 6 | Profiles of (A) velocity and salinity, (B) initial suspended sediment mass concentration, and (C) shear rate for the hurricane scenario (A,B are based on
model output from Harris et al., 2020. C is from 1D model output). May 2021 | Volume 8 | Article 629476 Frontiers in Marine Science | www.frontiersin.org 9 Modeling of Oil-Particle Aggregates Cui et al. period occurred during hurricanes Gustav and Ike. For this
scenario we therefore used Hurricane Gustav, the second most
catastrophic hurricane of the year 2008 (Brown et al., 2010). Gustav made landfall in southeast Louisiana on 1 September
2008 as category 2 hurricane. Model results indicated that wave-
induced bottom orbital velocities exceeded 4 m s−1 in coastal
areas and wave-induced resuspensions occurred offshore to
water depth of 189 m (Harris et al., 2020). Figure 6A shows
the vertical profiles of velocity and salinity used as forcing,
which were extracted from the three-dimensional model output
from Harris et al. (2020). The velocity exceeded 1.0 m s−1
at mid-depth. The salinity profile showed an upper mixed
layer extending to about 15 m depth. Fresher water in the mixed layer indicated the influence of the Mississippi River
plume. The vertical profile of initial sediment concentration
included a ∼40 m thick bottom nepheloid layer where
concentrations increased to about 0.01 kg m−3 near the seafloor
(Figure 6B). The sediment concentration was assumed to be
evenly distributed among five sediment classes at each vertical
level. The simulation period was 12 h, which allowed OPAMOD
to operate for 10 h. Figures 7A,B show the depth-integrated OPA and suspended
sediment concentrations, respectively. After the initial 2-h
hydrodynamic spin-up time, OPAMOD was activated in the
model and the mixture of oil and sediment began to form OPA. The depth-integrated OPA mass concentration peaked around FIGURE 7 | Time-series for the hurricane scenario of depth-integrated (A) OPA mass concentration and (B) SSC mass concentration. Profiles of OPA mass
concentration at (C) 10 min, (D) 25 min, and (E) 360 min after activating OPAMOD, respectively. Hurricane Scenario Lines in panels (C–E) share the same legend shown in panel (A),
with dotted gray line representing the total OPA size classes, black line representing the OPA size classes from 1 to 4, blue line representing the OPA size classes
from 5 to 8, and red line representing the OPA size classes from 9 to 12, respectively. Frontiers in Marine Science | www frontiersin org
10
May 2021 | Volume 8 | Article 629476 FIGURE 7 | Time-series for the hurricane scenario of depth-integrated (A) OPA mass concentration and (B) SSC mass concentration. Profiles of OPA mass
concentration at (C) 10 min, (D) 25 min, and (E) 360 min after activating OPAMOD, respectively. Lines in panels (C–E) share the same legend shown in panel (A),
with dotted gray line representing the total OPA size classes, black line representing the OPA size classes from 1 to 4, blue line representing the OPA size classes
from 5 to 8, and red line representing the OPA size classes from 9 to 12, respectively. May 2021 | Volume 8 | Article 629476 Frontiers in Marine Science | www.frontiersin.org 10 Modeling of Oil-Particle Aggregates Cui et al. 45 min after the aggregation process commenced, and increased
to about 0.3 kg m−2. During this time, depth-integrated sediment
concentrations saw a rapid decrease (from 0.25 to < 0.1 kg m−2),
as suspended sediment was converted to OPA. After the peak,
OPA concentrations decreased over the next 8 h as the OPAs
settled to the seafloor (Figure 7A). The size distribution of OPAs
mainly fell in the medium size range (classes 5–8, having D =
180 −360 µm ), whose concentration was about 10 times greater
than that of small and large size ranges. River Plume Scenario Next, the one-dimensional model was applied to examine
how sediment from river discharge can affect OPA formation. The velocity and salinity forcing came from the same model
simulation as in hurricane scenario (i.e., Harris et al., 2020), but
conditions including velocities, salinity, and suspended sediment
concentrations were extracted for a period in March of 2008
that had elevated river discharge. The maximum velocity was
∼0.8 m s−1 in the surface layer (Figure 8A). The vertical
salinity profile reflected the influence of the Mississippi River
plume in this locality, with a salinity difference greater than 10
PSU in the upper 20 m (Figure 8A). The suspended sediment
concentration peaked near 30 m water depth, reaching ∼0.017 kg
m−3 (Figure 8B). The initial oil concentration was the same as
used in the hurricane scenario, again initialized to be vertically
uniform at 0.2 kg m−3, based on Sammarco et al. (2013). The
simulation period was 48 h, with the OPAMOD being activated
after the first 2 h to allow for spin up of the hydrodynamic model. g
g
Vertical profiles of the OPA concentrations show that the
aggregation process was evident in the bottom 40 m of
water (Figures 7C–E). Directly after OPAMOD was initiated,
OPA concentrations were especially high in the bottom few
meters of the water column (Figure 7C) where sediment
concentration was high (e.g., see Figure 6B) and turbulence
was energetic (Figure 6C). The smaller classes of OPA
(D < 180 µm) were fairly well-mixed throughout the bottom
boundary layer (black lines in Figures 7A,C–E), but did not
reach high mass concentrations. The medium size classes of
OPA (D = 180 −360 µm) dominated the presence of OPA,
and were distributed through the bottom boundary layer
(depths of ∼130–140 m in Figures 7C–E). The larger OPA
(red lines in Figures 7A,C–E, D > 360 µm) settled to the
seafloor quickly. Figures 9A,B show the depth-integrated OPA and suspended
sediment mass concentrations, respectively. It took about 4 h for
the mass concentration of suspended OPA to reach a maximum
(Figure 9A, the first and second vertical gray lines). Suspended
sediment mass concentrations began decreasing when OPAMOD
was initiated (time = 2 h, Figure 9B), as sediment became Figures 9A,B show the depth-integrated OPA and suspended
sediment mass concentrations, respectively. River Plume Scenario It took about 4 h for
the mass concentration of suspended OPA to reach a maximum
(Figure 9A, the first and second vertical gray lines). Suspended
sediment mass concentrations began decreasing when OPAMOD
was initiated (time = 2 h, Figure 9B), as sediment became FIGURE 8 | Profiles of (A) velocity and salinity, (B) initial suspended sediment mass concentration, and (C) shear rate for the river plume scenario (A,B are based on
model output from Harris et al., 2020. C is from 1D model output). FIGURE 8 | Profiles of (A) velocity and salinity, (B) initial suspended sediment mass concentration, and (C) shear rate for the river plume scenario (A,B are based on
model output from Harris et al., 2020. C is from 1D model output). May 2021 | Volume 8 | Article 629476 11 Frontiers in Marine Science | www.frontiersin.org Modeling of Oil-Particle Aggregates Cui et al. FIGURE 9 | Time-series for the river plume scenario of depth-integrated (A) OPA mass concentration and (B) SSC mass concentration. Profiles of OPA
oncentration at (C) hour 4, (D) hour 8, and (E) hour 28 after activating OPAMOD, respectively. Lines in panels (C–E) share the same legend shown in panel (A), with
otted gray line representing the total OPA size classes, black line representing the OPA size classes from 1 to 4, blue line representing the OPA size classes from 5
o 8, and red line representing the OPA size classes from 9 to 12, respectively. FIGURE 9 | Time-series for the river plume scenario of depth-integrated (A) OPA mass concentration and (B) SSC mass concentration. Profiles of OPA
concentration at (C) hour 4, (D) hour 8, and (E) hour 28 after activating OPAMOD, respectively. Lines in panels (C–E) share the same legend shown in panel (A), with
dotted gray line representing the total OPA size classes, black line representing the OPA size classes from 1 to 4, blue line representing the OPA size classes from 5
to 8, and red line representing the OPA size classes from 9 to 12, respectively. incorporated into OPA. Then, for the next ∼30 h, the suspended
sediment and OPA mass concentrations both decreased as
sediment continued to be incorporated into OPA, and both
OPA and sediment settled to the seafloor (Figure 9A). River Plume Scenario For the
river plume case, the OPA mass concentrations were mainly
distributed within the medium (D = 180 −360 µm), and larger
(D > 360 µm) sizes (red and blue lines in Figure 9A), though the
medium sizes had a longer residence time in the water column. were present in the freshwater plume, they did not reach large
concentrations (Figures 9C–E), but instead served mainly to
“seed” the growth of the larger sized OPAs. The medium and
larger sizes reached notably high concentrations in the water
column, and because they have relatively high settling velocities,
provided a downward flux that resulted in elevated suspended
OPA concentrations in the mid-water column (Figure 9D). After approximately 28 h, OPAs completely settled to the
seafloor (Figure 9E). The vertical concentration profiles show that OPA were
present throughout the water column (Figures 9C–E). Initially,
elevated suspended OPA concentrations were formed in the
upper water column, where suspended sediment was available,
so that OPA appeared in the top 80 m of the water column
(Figure 9C). The higher settling velocities of the OPA, relative
to the suspended sediment, caused the OPA to sink out of surface
waters, but they continued to grow by scavenging the residual oil
in the water column. While the smaller OPA sizes (D < 180 µm) Frontiers in Marine Science | www.frontiersin.org CONCLUSION AND DISCUSSION An
oil-particle-aggregate
module
was
developed
and
implemented in ROMS within the COAWST framework. Implementation
of
a
quasi-zero-dimensional
simulation
demonstrated the ability to predict OPA formation and May 2021 | Volume 8 | Article 629476 12 Modeling of Oil-Particle Aggregates Cui et al. within a three-dimensional, fully coupled model which included
OPAMOD, developed by the Consortium for Simulation of Oil-
Microbial Interactions in the Ocean (CSOMIO) (Dukhovskoy
et al., 2021). Implemented to represent the transport and fate
of oil sourced from an oil spill in the northern Gulf of Mexico,
this three-dimensional model represented oil transformations
and transport, biological processes and feedbacks with oil (Coles
et al., 2020) and formation of OPA through OPAMOD. The full
three-dimensional coupled model therefore accounts for water
column transport of hydrocarbons, as well as biodegradation, and
delivery to the seabed via OPAs (Dukhovskoy et al., 2021). For the
case modeled, the source of oil was in very deep waters and the
results showed that the feedbacks between microbial processes
and oil were more significant than OPA formation (Dukhovskoy
et al., 2021). We postulate that the OPA formation would be
significant for cases where the source of oil was near a turbid river
plume, as shown for our one-dimensional river plume scenario;
or in shallower water where resuspension creates higher turbidity. reproduce the particle size distribution observed in a laboratory
study. Both the modeled and laboratory results showed that
OPAs were dominated by small and medium sized aggregates
(D ≤360 µm). Comparison with laboratory results showed that
our implementation of OPAMOD in ROMS behaved reasonably,
indicating that the model can be a useful tool in representing
the removal of oil via OPA formation. Sensitivity tests were
conducted to examine how the model responded to two
parameters: fractal dimension and collision efficiency. Results
showed that the modeled size distributions were much more
sensitive to fractal dimension than collision efficiency. According
to Equation 1, floc density varies with fractal dimension; when
a smaller fractal dimension was assumed, the initial number
concentrations of sediment increased, which encouraged the
formation of more and larger OPA. Simulations of OPA formation impacted by a surface river
plume and a hurricane event were conducted by configuring
a quasi-one-dimensional model using conditions based on
a 140-m deep site near Mississippi River Delta. CONCLUSION AND DISCUSSION Including OPA breakup
would be more difficult, because the mass balance equations
would need to specify the products formed by the disaggregation
of large OPA. Within FLOCMOD, the fragmentation distribution
function (FDBS) specifies how sediment mass from a larger floc
is distributed into smaller flocs upon breakup (Verney et al.,
2011; Sherwood et al., 2018). Similarly, inclusion of OPA breakup
would require specification of how to partition the oil and
sediment mass into smaller OPA, and / or sediment or oil classes. The current formulation of OPAMOD also does not account
for interactions between the biogenic detritus, oil, and sediment,
which have been shown to form MOSSFA (Vonk et al., 2015;
Quigg et al., 2020). Additionally, recent laboratory studies have
shown that the collision efficiency for OPA formation may be
sensitive to organic content of the particles (Ye et al., 2021). Adding coupling between the sediment, OPA, and biological
modules within the CSOMIO model (Dukhovskoy et al.,
2021) would be relatively straightforward because all of these
components have been coded within the ROMS and COAWST
frameworks. This model could become an important tool in
determining the fate of oil and prediction of the impact of
future oil spills. The current study is the first attempt of integrating an
OPA module within a coupled hydrodynamic and sediment
transport model. The one-dimensional model was run for
scenarios representative of conditions that could lead to OPA
formation over the Gulf of Mexico continental slope. In
order to fully represent the formation and dispersal of OPA,
however, OPAMOD can be implemented within a model that
also accounts for horizontal transport. This has been done Frontiers in Marine Science | www.frontiersin.org CONCLUSION AND DISCUSSION These two
scenarios allowed us to compare the relative importance of
surface suspended sediments associated with a freshwater plume
to bottom nepheloid layer suspended sediments associated
with a resuspension event. Model results showed that the
plume conditions were more effective at forming OPAs and
creating a deposition event of OPAs. The main difference
between these two scenarios was the vertical distributions of
suspended sediment. In the hurricane scenario, high sediment
concentrations were located in a bottom nepheloid layer. Thus,
newly formed OPAs quickly settled out of the water column. In comparison, suspended sediment concentration was high in
the surface layer for the river plume scenario. OPAs formed
in surface waters, which gave OPAs longer residence times
in the water column. During settling, OPAs scavenged more
residual oil and sediment in the water column. For comparison,
the depth-integrated suspended sediment mass concentration
(∼0.5 kg m−2, Figure 9B) in the river plume scenario was
about double that of the hurricane scenario (Figure 9A), but
the maximum depth-integrated OPA mass concentration (∼5 kg
m−2, Figure 9B) in the river plume scenario was about 15
times greater than that in the hurricane scenario (Figure 7A). Additionally, in the river plume scenario, the OPA remained in
the water column for a longer time period, because the settling
distance was so much greater. This implies that OPA formation
via river plume sediment would lead to a more widely dispersed
depositional footprint, compared to OPA formation via storm
resuspension. Additionally, because river plume generated OPAs
had longer residence times in the water column compared to
resuspension generated OPAs, they would have more opportunity
for degradation via water column weathering and consumption
prior to deposition. p
g
y
However, application of OPAMOD to scenarios that have
the potential for resuspension would require further model
development to account for the resuspension of OPA and
potential breakup of OPA under energetic conditions. For the
current implementations, we set the erosion parameters for
OPA to be very large so that resuspension would not occur. However, previously deposited OPA might be resuspended later,
under more energetic conditions, thus providing a source of
contamination some time after the initial contamination event. The model is equipped to account for resuspension, but would
require proper parameterization of critical shear stress and the
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and effective density of flocs, and their fractal dimensions. J. Sea Res. 41, 87–95. DATA AVAILABILITY STATEMENT The datasets presented in this study can be found on an online
repository. The name of the repository/repositories and accession May 2021 | Volume 8 | Article 629476 13 Modeling of Oil-Particle Aggregates Cui et al. Simulation of Oil-Microbial Interactions in the Ocean) (GoMRI
Award R01983). numbers can be found below: https://data.gulfresearchinitiative. org/. The dataset page is https://data.gulfresearchinitiative.org/
data/R6.x803.000:0009. AUTHOR CONTRIBUTIONS Leiping Ye (Sun Yat-sen University) provided laboratory data
from his publications. The authors appreciate input from
the CSOMIO group during model development, including
Eric Chassignet, Dmitry Dukhovskoy, Tian-Jian Hsu, Andrew
Manning, and Steve Morey. We appreciate input from the
two reviewers, which greatly helped us to improve the
manuscript. The authors acknowledge William and Mary
Research Computing (https://www.wm.edu/it/rc) for providing
computational resources that have contributed to the results
reported within this paper. This paper is Contribution No. 4007 of the Virginia Institute of Marine Science, William &
Mary. LC, CH, and DT: conceptualization and writing-review and
editing. LC and CH: methodology. LC: model development,
implementation, analysis, and writing-original draft preparation. CH: supervision and funding acquisition. All authors have read
and agreed to the published version of the manuscript. FUNDING This research was made possible by a grant from the Gulf of
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00128-7 Copyright © 2021 Cui, Harris and Tarpley. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Marine Science | www.frontiersin.org REFERENCES The
use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with
these terms. Copyright © 2021 Cui, Harris and Tarpley. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The
use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
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English
| null |
TKTL1 Knockdown Impairs Hypoxia-Induced Glucose-6-phosphate Dehydrogenase and Glyceraldehyde-3-phosphate Dehydrogenase Overexpression
|
International journal of molecular sciences
| 2,022
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cc-by
| 17,078
|
Inês Baptista 1,2,3,†
, Effrosyni Karakitsou 1,2,3,4,†, Jean-Baptiste Cazier 3,4, Ulrich L. Günther 5,
Silvia Marin 1,2,6,*,‡
and Marta Cascante 1,2,6,*,‡ Inês Baptista 1,2,3,†
, Effrosyni Karakitsou 1,2,3,4,†, Jean-Baptiste Cazier 3,4, Ulrich L. Günther 5,
Silvia Marin 1,2,6,*,‡
and Marta Cascante 1,2,6,*,‡ 1
Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, Universitat de Barcelona (UB),
08028 Barcelona, Spain; inesvbaptista@gmail.com (I.B.); e.karakitsou@ub.edu (E.K.) p
p
g
(
)
(
)
2
Institute of Biomedicine of University of Barcelona (IBUB), University of Barcelona (UB), 08028 Barcelona,
Spain p
p
g
2
Institute of Biomedicine of University of Barcelona (IBUB), University of Barcelona (UB), 08028 Barcelona,
Spain p
3
Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK;
j.cazier@bham.ac.uk 3
Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
j.cazier@bham.ac.uk j
4
Centre for Computational Biology, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
5
Institute of Chemistry and Metabolomics University of Lübeck 23562 Lübeck Germany; j
4
Centre for Computational Biology, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 4
Centre for Computational Biology, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
5
Institute of Chemistry and Metabolomics, University of Lübeck, 23562 Lübeck, Germany;
ulrich.guenther@uni-luebeck.de 5
Institute of Chemistry and Metabolomics, University of Lübeck, 23562 Lübeck, Germany;
ulrich.guenther@uni-luebeck.de g
6
CIBER of Hepatic and Digestive Diseases (CIBEREHD), Institute of Health Carlos III (ISCIII), 28029 Madrid,
Spain p
*
Correspondence: silviamarin@ub.edu (S.M.); martacascante@ub.edu (M.C.) †
These authors contributed equally to this work. ‡
These authors contributed equally to this work. Abstract: Increased expression of transketolase (TKT) and its isoform transketolase-like-1 (TKTL1)
has been related to the malignant leukemia phenotype through promoting an increase in the non-
oxidative branch of the pentose phosphate pathway (PPP). Recently, it has also been described
that TKTL1 can have a role in survival under hypoxic conditions and in the acquisition of radio
resistance. However, TKTL1’s role in triggering metabolic reprogramming under hypoxia in leukemia
cells has never been characterized. Using THP-1 AML cells, and by combining metabolomics and
transcriptomics techniques, we characterized the impact of TKTL1 knockdown on the metabolic
reprogramming triggered by hypoxia. Results demonstrated that TKTL1 knockdown results in
a decrease in TKT, glucose-6-phosphate dehydrogenase (G6PD) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) activities and impairs the hypoxia-induced overexpression of G6PD and
GAPDH, all having significant impacts on the redox capacity of NADPH- and NADH-related
cells. International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences
Citation: Baptista, I.; Karakitsou, E.;
Cazier, J.-B.; Günther, U.L.; Marin, S.;
Cascante, M. TKTL1 Knockdown
Impairs Hypoxia-Induced
Glucose-6-Phosphate Dehydrogenase
and Glyceraldehyde-3-Phosphate
Dehydrogenase Overexpression. Int. J. Mol. Sci. 2022, 23, 3574. https://
doi.org/10.3390/ijms23073574
Academic Editor: Kwang Dong Kim
Received: 1 February 2022
Accepted: 23 March 2022
Published: 25 March 2022 Citation: Baptista, I.; Karakitsou, E.;
Cazier, J.-B.; Günther, U.L.; Marin, S.;
Cascante, M. TKTL1 Knockdown
Impairs Hypoxia-Induced
Glucose-6-Phosphate Dehydrogenase
and Glyceraldehyde-3-Phosphate
Dehydrogenase Overexpression. Int. J. Mol. Sci. 2022, 23, 3574. https://
doi.org/10.3390/ijms23073574
Academic Editor: Kwang Dong Kim
Received: 1 February 2022
Accepted: 23 March 2022
Published: 25 March 2022 Citation: Baptista, I.; Karakitsou, E.;
Cazier, J.-B.; Günther, U.L.; Marin, S.;
Cascante, M. TKTL1 Knockdown
Impairs Hypoxia-Induced
Glucose-6-Phosphate Dehydrogenase
and Glyceraldehyde-3-Phosphate
Dehydrogenase Overexpression. Int. J. Mol. Sci. 2022, 23, 3574. https://
doi.org/10.3390/ijms23073574 Received: 1 February 2022
Accepted: 23 March 2022
Published: 25 March 2022 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Keywords: metabolism; hypoxia; transketolase-like 1; pentose phosphate pathway; transcriptomics;
leukemia; AML; glucose-6-phosphate dehydrogenase; glyceraldehyde-3-phosphate dehydrogenase Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Inês Baptista 1,2,3,†
, Effrosyni Karakitsou 1,2,3,4,†, Jean-Baptiste Cazier 3,4, Ulrich L. Günther 5,
Silvia Marin 1,2,6,*,‡
and Marta Cascante 1,2,6,*,‡ Moreover, TKTL1 knockdown impedes hypoxia-induced transcription of genes encoding key
enzymes and transporters involved in glucose, PPP and amino acid metabolism, rendering cells
unable to switch to enhanced glycolysis under hypoxia. Altogether, our results show that TKTL1
plays a key role in the metabolic adaptation to hypoxia in THP-1 AML cells through modulation of
G6PD and GAPDH activities, both regulating glucose/glutamine consumption and the transcriptomic
overexpression of key players of PPP, glucose and amino acids metabolism. 1. Introduction Synthesis of ribose-5-phosphate (R5P) is crucial to ensure the production of nucleotide
building blocks for DNA duplication. R5P can be synthesized through both branches of
the pentose phosphate pathway (PPP), mediated by glucose-6-phosphate dehydrogenase
(G6PD) on the oxidative side with the additional production of nicotinamide-adenine https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 3574. https://doi.org/10.3390/ijms23073574 Int. J. Mol. Sci. 2022, 23, 3574 2 of 20 dinucleotide phosphate (NADPH) and controlled by TKT on the non-oxidative side, which
links glycolysis to the production of R5P [1,2]. dinucleotide phosphate (NADPH) and controlled by TKT on the non-oxidative side, which
links glycolysis to the production of R5P [1,2]. The TKTL1 isoenzyme is missing 38 amino acids in the active site [3] and increased ex-
pression of TKT and its isoform TKTL1 has been reported in different types of cancers [4–7],
and is related to malignant transformation [8] and poor prognosis [9–12]. Evidence across
several cancer tissues up until now have highlighted that TKTL1 is tightly linked to adap-
tation to hypoxia and its overexpression has been correlated with resistance to ionizing
radiation and chemotherapy [13] as well as to the “Warburg effect” [14]. For example,
in glioma cells, it has been demonstrated that hypoxia led to TKTL1 induction and that
its knockdown promoted cell death [13]. Induction of TKTL1 under hypoxia has also
been observed in different cell lines of colorectal cancer (CRC) and upregulation has been
demonstrated for a subset of CRC patients [10]. It has also been reported that hypoxia
induced major metabolic changes in AML cell lines, including adaptation of both lipid
and glycolytic metabolism [15]. In addition, increased levels of TKTL1 have been reported
in response to decitabine (DAC) treatment in primary acute myeloid leukemia (AML)
samples [16] and correlated to the acquisition of tyrosine kinase inhibitor resistance in
chronic myeloid leukemia (CML) [17]. y
However, to date, there has been little research regarding the importance of TKTL1
for the metabolic response triggered by hypoxia in leukemia and its specific role during
metabolic reprogramming of AML cells towards hypoxia remains unknown. One of the objectives of this work is to untangle the functional relationship between
TKTL1 expression and metabolic adaptation of AML cells to hypoxia. 1. Introduction For this purpose, we
performed a comparative analysis of hypoxia-induced changes using the THP1 cell line, a
monocytic leukemic cell line established in 1980 from the blood of an AML child patient
with an MLL fusion [18]. Using THP1 transduced with a lentiviral vector expressing a
TKTL1-specific shRNA (TKTL1KD) and a wild-type control transduced with an empty vec-
tor (THP-1WT), we attested the differences between the two cell lines at the transcriptional
and metabolic levels. Together, our findings highlight that in response to hypoxia, THP-1
cells deeply rewire central metabolism at the transcriptomic level to sustain an enhanced
consumption of glucose and glutamine, and that this response is severely impaired in
the cells lacking TKTL1 that are not able to increase the activity of key players in redox
homeostasis such as GAPDH and G6PD. 2. Results Enzy-
matic activity assays performed through spectrophotometry in normoxia and hypoxia (1% O2); for
(B) transketolase (TKT), (C) glucose-6-phosphate dehydrogenase (G6PD) and (D) 6-phospo-gluconate
dehydrogenase (6PGD). Data represented as mean ± SD (n = 2). Statistically significant differences in
all panels were determined by two-tailed independent sample Student’s t-test: p < 0.05 (*). p < 0.01
(**). p < 0.001 (***). Through transcriptomic analysis, we verified the following differences in activity origi-
nating from differential expression. The THP-1WT and THP-1KD changes in gene expression
levels in response to hypoxia are listed in Tables A1 and A2, respectively, in Appendix A. Consistent with the hypoxia-induced decrease in proliferation rates of these cells, we
observed that G6PD expression in hypoxia (quantified as Log2-fold-change in hypoxia
versus normoxia transcript levels (Log2FC)) was decreased significantly for THP-1WT
(Log2FC = −0.72; padj < 0.05) as well as for TKTL1KD (Log2FC = −1.24; padj < 0.05) (Figure
2). Considering that G6PD is regulated by post-translational modifications (PTMs), the
fact that G6PD activity did not change could be due to PTM enzymatic activation. In fact,
Table 1. Duplication times of THP-1WT and THP-1KD under normoxia and hypoxia. Duplication Time (hours)
% Increment Versus WT
THP-1WT Normoxia
38.4 ± 2.24
–
THP-1KD Normoxia
49.4 ± 2.48 **
25.1
THP-1WT Hypoxia
113.5 ± 41.81
–
THP-1KD Hypoxia
151.3 ± 19.38
28.6
Data represented as mean ± SD (n = 3). Statistically significant differences between THP-1WT and THP-1KD in
normoxia or in hypoxia were determined by two-tailed independent sample Student’s t-test: p < 0.01 (**). Through transcriptomic analysis, we verified the following differences in a
ati
f o
diffe e tial e
e io
The THP 1WT a d THP 1KD ha
e i
e
Table 1. Duplication times of THP-1WT and THP-1KD under normoxia and hypoxia. y
y
g
y
[
]
transcriptomic results show that under hypoxic conditions, SIRT5 levels increased in both
THP-1WT (Log2FC = 0.78; padj < 0.05) and THP-1KD (Log2FC = 1.08; padj < 0.05) (Figure 2). Since it is thought that the synthesis of R5P and other pentoses formed in both branches
of the PPP mainly contribute to proliferation, we determined the effect of TKTL1 knock-
down on the enzyme activity of TKT, G6PD and 6-phosphogluconate dehydrogenase
(6PGD) using spectrophotometric methods (see Section 4.3). 2. Results 2.1. TKTL1 Contributes to Cell Growth and Pentose Phosphate Pathway in Normoxia and Hypoxia 2.1. TKTL1 Contributes to Cell Growth and Pentose Phosphate Pathway in Normoxia and Hypoxia
To characterize the role of TKTL1 for the metabolic response of AML cells to hy-
poxia, we initially evaluated the effect of the shRNA-mediated silencing of TKTL1 on the
expression of TKTL1 and TKT genes by RT-PCR. We obtained a knockdown efficiency
of 63.3% ± 0.03 for TKTL1 expression, whereupon TKT expression was not significantly
affected (Figure 1A). Next, we evaluated the impact of TKTL1 knockdown on cell proliferation during
hypoxia. At 96 h of cell incubation, an increase in duplication time of TKTL1KD cells
compared to THP-1WT of around 25% and 29% for normoxia (O2 at 21%) or hypoxia (O2 at
1%), respectively, was observed (Table 1). When compared to normoxia, hypoxia induced
a 3-fold increase in duplication time of both THP-1WT and THP-1KD cells. These results
reinforce the notion that TKTL1 is a necessary enzyme for proliferation, as reported in
many previous studies, highlighting that its function is essential under normal oxygen and
hypoxic conditions. Int. J. Mol. Sci. 2022, 23, 3574 3 of 20
6PGD Figure 1. Effects of TKTL1 knockdown on TKTL1 and TKT gene expression and the pentose phos-
phate pathway. (A) Effects of TKTL1 silencing on TKTL1 and TKT gene expression. RT-PCR per-
formed using PPA1 as a housekeeping gene for data normalization. Decrease in TKTL1 expression
determined as 63.3% ± 0.03. Data shown as mean ± SD (n = 3) of normalized values. Enzymatic
activity assays performed through spectrophotometry in normoxia and hypoxia (1% O2); for (B)
transketolase (TKT), (C) glucose-6-phosphate dehydrogenase (G6PD) and (D) 6-phospo-gluconate
dehydrogenase (6PGD). Data represented as mean ± SD (n = 2). Statistically significant differences
in all panels were determined by two-tailed independent sample Student’s t-test: p < 0.05 (*). P <
0.01 (**). p < 0.001 (***). Figure 1. Effects of TKTL1 knockdown on TKTL1 and TKT gene expression and the pentose phos-
phate pathway. (A) Effects of TKTL1 silencing on TKTL1 and TKT gene expression. RT-PCR per-
formed using PPA1 as a housekeeping gene for data normalization. Decrease in TKTL1 expression
determined as 63.3% ± 0.03. Data shown as mean ± SD (n = 3) of normalized values. 2. Results TKTL1 knockdown resulted
in a decrease in transketolase activity of around 45% in normoxia and 49% in hypoxia
(Figure 1B) and a decrease in G6PD activity of around 65% and 75% for normoxia and
hypoxia, respectively (Figure 1C). By contrast, the knockdown did not alter 6PGD activity
in normoxia nor in hypoxia (Figure 1D). yp
g
Through transcriptomic analysis, we verified the following differences in activity
originating from differential expression. The THP-1WT and THP-1KD changes in gene
expression levels in response to hypoxia are listed in Tables A1 and A2, respectively, in
Appendix A. Consistent with the hypoxia-induced decrease in proliferation rates of these cells, we
observed that G6PD expression in hypoxia (quantified as Log2-fold-change in hypoxia
versus normoxia transcript levels (Log2FC)) was decreased significantly for THP-1WT
(Log2FC = −0.72; padj < 0.05) as well as for TKTL1KD (Log2FC = −1.24; padj < 0.05)
(Figure 2). Considering that G6PD is regulated by post-translational modifications (PTMs),
the fact that G6PD activity did not change could be due to PTM enzymatic activation. In
fact, it has been described that G6PD is activated by SIRT5-catalyzed deglutarylation [19]. The transcriptomic results show that under hypoxic conditions, SIRT5 levels increased
in both THP-1WT (Log2FC = 0.78; padj < 0.05) and THP-1KD (Log2FC = 1.08; padj < 0.05)
(Figure 2). With respect to the TKT and TKTL1 genes, there was a very slight increase Int. J. Mol. Sci. 2022, 23, 3574
Int. J. Mol. Sci. 2022, 23, x FOR 4 of 20
4 of 21 in gene expression induced by hypoxia, yet with no change in transketolase activity (see
Figure 1B). With respect to the TKT and TKTL1 genes, there was a very slight increase in gene expres-
sion induced by hypoxia, yet with no change in transketolase activity (see Figure 1B). in gene expression induced by hypoxia, yet with no change in transketolase activity (s
Figure 1B). With respect to the TKT and TKTL1 genes, there was a very slight increase in gene expre
sion induced by hypoxia, yet with no change in transketolase activity (see Figure 1B). Figure 2. Gene expression changes of key enzymes and transporters between THP-1WT and THP-1
in hypoxia. Significantly changing genes due to TKTL1 highlighted in pink. Expression changes
normoxia versus hypoxia, indicated in log2-fold-change of n = 3 samples per condition. 2. Results Abbrevi
tions: G6PD—glucose-6-phosphate dehydrogenase, GAPDH—glyceraldehyde-3-phosphate deh
drogenase, GLUT3—glucose transporter 3, HK1–3—hexokinase 1 to 3, LDHD—lactate dehydroge
ase D, MCT4—monocarboxylate transporter 4, PDK1–4—pyruvate dehydrogenase kinase 1 to
SIRT5—sirtuin 5, SLC—solute carrier family, TKT—transketolase, TKTL1—transketolase-like
Created using BioRender.com (accessed date: 22 May 2022). 2.2. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes for the Fate of Glucose in THP-1
Figure 2. Gene expression changes of key enzymes and transporters between THP-1WT and THP-1K
in hypoxia. Significantly changing genes due to TKTL1 highlighted in pink. Expression changes
normoxia versus hypoxia, indicated in log2-fold-change of n = 3 samples per condition. Abbreviatio
G6PD—glucose-6-phosphate dehydrogenase, GAPDH—glyceraldehyde-3-phosphate dehydrogena
GLUT3—glucose transporter 3, HK1–3—hexokinase 1 to 3, LDHD—lactate dehydrogenase D, MCT4
monocarboxylate transporter 4, PDK1–4—pyruvate dehydrogenase kinase 1 to 4, SIRT5—sirtu
5, SLC—solute carrier family, TKT—transketolase, TKTL1—transketolase-like 1. Created usin
BioRender.com (accessed date: 31 January 2022). Figure 2. Gene expression changes of key enzymes and transporters between THP-1WT and THP-1KD
in hypoxia. Significantly changing genes due to TKTL1 highlighted in pink. Expression changes in
normoxia versus hypoxia, indicated in log2-fold-change of n = 3 samples per condition. Abbrevia-
tions: G6PD—glucose-6-phosphate dehydrogenase, GAPDH—glyceraldehyde-3-phosphate dehy-
drogenase, GLUT3—glucose transporter 3, HK1–3—hexokinase 1 to 3, LDHD—lactate dehydrogen-
ase D, MCT4—monocarboxylate transporter 4, PDK1–4—pyruvate dehydrogenase kinase 1 to 4,
SIRT5—sirtuin 5, SLC—solute carrier family, TKT—transketolase, TKTL1—transketolase-like 1. Created using BioRender.com (accessed date: 22 May 2022). 2 2 TKTL1 I E
ti l t T i
H
i I d
d Ch
f
th F t
f Gl
i
THP 1
Figure 2. Gene expression changes of key enzymes and transporters between THP-1WT and THP-1KD
in hypoxia. Significantly changing genes due to TKTL1 highlighted in pink. Expression changes in
normoxia versus hypoxia, indicated in log2-fold-change of n = 3 samples per condition. Abbreviations:
G6PD—glucose-6-phosphate dehydrogenase, GAPDH—glyceraldehyde-3-phosphate dehydrogenase,
GLUT3—glucose transporter 3, HK1–3—hexokinase 1 to 3, LDHD—lactate dehydrogenase D, MCT4—
monocarboxylate transporter 4, PDK1–4—pyruvate dehydrogenase kinase 1 to 4, SIRT5—sirtuin
5, SLC—solute carrier family, TKT—transketolase, TKTL1—transketolase-like 1. Created using
BioRender.com (accessed date: 31 January 2022). Int. J. Mol. Sci. 2022, 23, 3574 5 of 20 5 of 20 . TKTL1 Is Essential to Trigger Hypoxia-Induced Changes for the Fate of Glucose in THP-1 Cells A common behavior of most cancer cells under hypoxic conditions is the increase
in the glycolytic pathway flux as a metabolic adaptation to their environment. 2. Results To see
how this feature is dependent on TKTL1, we measured the changes in glucose and lactate
concentrations in cell culture media of THP-1WT and THP-1KD cells incubated for 48 h
under normoxic and hypoxic conditions. In normoxia, THP-1WT glycolytic parameters
(changes in glucose and in lactate concentrations) were around 2-fold higher than in THP-
1KD cells (Figure 3A–C). The ratio of increased lactate per decreased glucose concentrations
in cell culture media was significantly different in WT vs. KD cells in normoxia but not in
hypoxia (Figure 3C). Nevertheless, the increased values of around 2 for both cell lines in
hypoxia indicate that under O2 restriction, glucose is readily oxidized to lactate. R REVIEW
6 of 21 Figure 3. Loss of TKTL1 impairs hypoxia response. Changes in concentration of glucose (A) and
lactate (B) in incubation medium, normalized by cell number and incubation time. Glucose and
lactate concentrations were measured in incubation medium of THP-1WT and THP-1KD cells incu-
bated for 48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in lactate per change
in glucose concentration. Enzymatic activity assays performed through spectrophotometry in
normoxia and hypoxia for enzymes (D) hexokinase (HK), (E) glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) and (F) lactate dehydrogenase (LDH). Data represented are mean ± SD, with n =
3 in panels A, B and C, and n = 2 in panels D, E and F. Statistically significant differences in all panels
were determined by two-tailed independent sample Student’s t-test: p < 0.05 (*). p < 0.01 (**). p <
0.001 (***). Figure 3. Loss of TKTL1 impairs hypoxia response. Changes in concentration of glucose (A) and
lactate (B) in incubation medium, normalized by cell number and incubation time. Glucose and lactate
concentrations were measured in incubation medium of THP-1WT and THP-1KD cells incubated for
48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in lactate per change in glucose
concentration. Enzymatic activity assays performed through spectrophotometry in normoxia and
hypoxia for enzymes (D) hexokinase (HK), (E) glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
and (F) lactate dehydrogenase (LDH). Data represented are mean ± SD, with n = 3 in panels A, B and
C, and n = 2 in panels D, E and F. Statistically significant differences in all panels were determined by
two-tailed independent sample Student’s t-test: p < 0.05 (*). p < 0.01 (**). p < 0.001 (***). Figure 3. 2. Results 2022, 23, 3574 6 of 20 It has been well-documented that the induction of hypoxia responses in induction
of glycolysis occurs as a consequence of increased gene expression of enzymes and trans-
porters (identified in Figure 2) [20–29]. We investigated whether the impaired enhancement
of glycolysis in response to hypoxia observed in THP-1KD was caused by a failure to
upregulate gene expression. Results showed that the expression of the isoforms of hexokinase that bind to mito-
chondria (HK1 and HK2) and the pyruvate dehydrogenase kinase isoenzymes (PDK1, 3
and 4) were strongly up-regulated in THP-1WT in response to hypoxia. On the contrary, in
THP-1KD, hypoxic conditions induced a decrease in the expression of PDK3 and PDK4 but
did not alter the expression of HK1 nor PDK1. It is worth noting that the hypoxia-induced
increase in gene expression of the hexokinase isoenzyme HK3, that binds to the nucleus, is
2.5-fold higher in THP-1KD than in THP-1WT, suggesting a TKTL1-independent mechanism
of regulation of the expression of this gene in hypoxia. In fact, HK3 has been described
to have a role in decreasing reactive oxygen species (ROS) and to have a perinuclear
localization [30]. Other glycolytic enzymes and transporters that are reported to be overexpressed in hy-
poxia, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose transporter
SLC2A3 (GLUT3), lactate dehydrogenase D (LDHD) and lactate transporter SLC16A3
(also known as monocarboxylate transporter MCT4) were found to be upregulated under
hypoxic conditions in both THP-1WT and to a lesser extent also in THP-1KD. yp
Measurement of total HK and GAPDH activity in THP-1WT and THP-1KD cells
(Figure 3D,E) showed that the activity was higher in THP-1WT than in THP-1KD in both nor-
moxic and hypoxic conditions. On the contrary, we observed that the knockdown of TKTL1
did not affect total LDH activity and that its activity was increased in both THP-1WT and
THP-1KD cells under hypoxia (Figure 3F). The obtained results are consistent with the lower
Km for glucose reported for HK3 isoenzyme, with respect to HK1 and HK2 [31]. LDHD has
been reported to function as a “metabolite repair enzyme” that controls metabolic damage
in glycolysis by contributing to the elimination of methylglyoxal formed from another
glycolytic enzyme, namely the triose-phosphate isomerase (TPI) [32]. Total GAPDH activity
(Figure 3E) as well as GAPDH gene expression (Figure 2) increased in both cell lines in
response to hypoxia, favoring the THP-1WT. 2. Results Loss of TKTL1 impairs hypoxia response. Changes in concentration of glucose (A) and
lactate (B) in incubation medium, normalized by cell number and incubation time. Glucose and
lactate concentrations were measured in incubation medium of THP-1WT and THP-1KD cells incu-
bated for 48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in lactate per change
in glucose concentration. Enzymatic activity assays performed through spectrophotometry in
normoxia and hypoxia for enzymes (D) hexokinase (HK), (E) glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) and (F) lactate dehydrogenase (LDH). Data represented are mean ± SD, with n =
3 in panels A, B and C, and n = 2 in panels D, E and F. Statistically significant differences in all panels
were determined by two-tailed independent sample Student’s t-test: p < 0.05 (*). p < 0.01 (**). p <
0.001 (***). Figure 3. Loss of TKTL1 impairs hypoxia response. Changes in concentration of glucose (A) and
lactate (B) in incubation medium, normalized by cell number and incubation time. Glucose and lactate
concentrations were measured in incubation medium of THP-1WT and THP-1KD cells incubated for
48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in lactate per change in glucose
concentration. Enzymatic activity assays performed through spectrophotometry in normoxia and
hypoxia for enzymes (D) hexokinase (HK), (E) glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
and (F) lactate dehydrogenase (LDH). Data represented are mean ± SD, with n = 3 in panels A, B and
C, and n = 2 in panels D, E and F. Statistically significant differences in all panels were determined by
two-tailed independent sample Student’s t-test: p < 0.05 (*). p < 0.01 (**). p < 0.001 (***). 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1
Cells
Second to glucose, glutamine is a major carbon source for energy production and
anabolic processes [34]. It has been described that hypoxia enhances glutamine synthesis
and uptake in cancer cells by increasing glutamine synthetase (GS, also known as GLUL)
Hypoxia triggered a metabolic switch, as evidenced by a 2.2-fold increase in the change
in glucose and lactate concentrations in the incubation medium of THP-1WT cells, whereas
in THP-1KD, these glycolytic parameters were not statistically different in hypoxia vs. normoxia (Figure 3A,B). These results unveil a key role of TKTL1 in the adaptive metabolic
switch to glycolysis that THP-1WT cells undergo under hypoxia. Int. J. Mol. Sci. 2. Results Altogether, the gene expression profile observed provides strong evidence that the
knockdown of TKTL1 affects the metabolic switch to glycolysis in response to hypoxia
through impaired upregulation of gene expression for HK and PDKs isoforms. Our ob-
servations demonstrated that hypoxia triggers a dramatic increase in Estrogen-Related
Receptor b (ESRRb) (log2FC = 4.6; padj < 0.05) in THP-1WT, whereas ESRRb levels remain
constant in THP-1KD. The family of Estrogen-Related Receptors (ESRRs) has been described
to mediate PDKs gene overexpression in hypoxia [26,33], allowing us to hypothesize that
TKTL1 plays a key role in the switch from OXPHOS to glycolysis by induction of PDKs
that result in pyruvate dehydrogenase (PDH) inhibition in an ESRRb-mediated way. 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells Second to glucose, glutamine is a major carbon source for energy production and
anabolic processes [34]. It has been described that hypoxia enhances glutamine synthesis
and uptake in cancer cells by increasing glutamine synthetase (GS, also known as GLUL)
and the number of glutamine transporters such as SLC38A2 [34]. To attest how TKTL1
could impact glutamine metabolism, we measured the changes in glutamine and glutamate
concentrations in incubation medium of THP-1WT and THP-1KD cells incubated for 48 h
under normoxic and hypoxic conditions. We found that TKTL1 knockdown resulted in a
20–25% decrease in the change in glutamine and glutamate concentrations under normoxia
and in a 70–75% decrease under hypoxia (Figure 4A,B). When comparing the effect of
this knockdown on the ratio of change in glucose concentration vs. change in glutamine
concentration (Figure 4C), we observed a decrease of around 20% in normoxia and of 27%
in hypoxia, which indicates that the overall impact of silencing TKTL1 is more pronounced Int. J. Mol. Sci. 2022, 23, 3574 7 of 20
trans- 7 of 20
trans- in hypoxia. In fact, the hypoxic metabolic switch triggered an increase of around 20%
for glutamine and glutamate concentrations in THP-1WT cells, while the THP-1KD cells in
hypoxia had around a 50–60% decrease in these metabolites’ levels (Figure 4A,B). y
g
,
p
y
g
,
changes at the transcriptomic level relating to glutamine/glutamate intake and metabo-
lism correlate with an impairment of the metabolic response induced by hypoxia as a con-
sequence of TKTL1 knockdown. Figure 4. Loss of TKTL1 impairs hypoxia response. Changes in glutamate (A) and in glutamine (B)
concentrations in incubation medium, normalized by cell number and incubation time. Glutamate
and glutamine concentrations were measured in incubation medium of THP-1WT and THP-1 KD cells
incubated for 48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in glucose per
change in glutamine concentration. Data represented are mean ± SD (n = 3). Statistically significant
differences in all panels were determined by two-tailed independent sample Student’s t-test: p < 0.05
(*). p < 0.01 (**). p < 0.001 (***). Figure 4. Loss of TKTL1 impairs hypoxia response. Changes in glutamate (A) and in glutamine
(B) concentrations in incubation medium, normalized by cell number and incubation time. 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells Glutamate
and glutamine concentrations were measured in incubation medium of THP-1WT and THP-1 KD cells
incubated for 48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in glucose per
change in glutamine concentration. Data represented are mean ± SD (n = 3). Statistically significant
differences in all panels were determined by two-tailed independent sample Student’s t-test: p < 0.05
(*). p < 0.01 (**). p < 0.001 (***). Figure 4. Loss of TKTL1 impairs hypoxia response. Changes in glutamate (A) and in glutamine (B)
concentrations in incubation medium, normalized by cell number and incubation time. Glutamate
and glutamine concentrations were measured in incubation medium of THP-1WT and THP-1 KD cells
incubated for 48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in glucose per
change in glutamine concentration. Data represented are mean ± SD (n = 3). Statistically significant
differences in all panels were determined by two-tailed independent sample Student’s t-test: p < 0.05
(*). p < 0.01 (**). p < 0.001 (***). Figure 4. Loss of TKTL1 impairs hypoxia response. Changes in glutamate (A) and in glutamine
(B) concentrations in incubation medium, normalized by cell number and incubation time. Glutamate
and glutamine concentrations were measured in incubation medium of THP-1WT and THP-1 KD cells
incubated for 48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in glucose per
change in glutamine concentration. Data represented are mean ± SD (n = 3). Statistically significant
differences in all panels were determined by two-tailed independent sample Student’s t-test: p < 0.05
(*). p < 0.01 (**). p < 0.001 (***). Figure 4. Loss of TKTL1 impairs hypoxia response. Changes in glutamate (A) and in glutamine (B)
concentrations in incubation medium, normalized by cell number and incubation time. Glutamate
and glutamine concentrations were measured in incubation medium of THP-1WT and THP-1 KD cells
incubated for 48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in glucose per
change in glutamine concentration. Data represented are mean ± SD (n = 3). Statistically significant
differences in all panels were determined by two-tailed independent sample Student’s t-test: p < 0.05
(*). p < 0.01 (**). p < 0.001 (***). Figure 4. Loss of TKTL1 impairs hypoxia response. Changes in glutamate (A) and in glutamine
(B) concentrations in incubation medium, normalized by cell number and incubation time. 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells Expression changes in normoxia versus hypoxia, indicated in log2-fold-change for
n = 3 samples per condition. Abbreviations: ALDH—aldehyde dehydrogenase family, ALDH4A1—
pyrroline-5-carboxylate dehydrogenase. mitochondrial, ALDH18A1—pyrroline-5-carboxylate syn-
thase, ARG1—arginase 1, ASL—argininosuccinate lyase, ASS1—argininosuccinate synthase 1, GLS—
glutaminase, GS—glutamine synthetase, OAT—ornithine aminotransferase, PRODH—proline de-
hydrogenase, PYCR2 and 3—pyrroline-5-carboxylate reductase 2 and 3, SLC—solute carrier family. Created with BioRender.com (accessed on 31 January 2022). Figure 5. Gene expression alterations of key players in glutamine and glutamate metabolism and
transport between THP-1WT and THP-1KD in hypoxia. Significant changing genes due to TKTL1 high-
lighted in pink. Expression changes in normoxia versus hypoxia, indicated in log2-fold-change for
n = 3 samples per condition. Abbreviations: ALDH—aldehyde dehydrogenase family, ALDH4A1—
pyrroline-5-carboxylate dehydrogenase. mitochondrial, ALDH18A1—pyrroline-5-carboxylate syn-
thase, ARG1—arginase 1, ASL—argininosuccinate lyase, ASS1—argininosuccinate synthase 1,
GLS—glutaminase, GS—glutamine synthetase, OAT—ornithine aminotransferase, PRODH—pro-
line dehydrogenase, PYCR2 and 3—pyrroline-5-carboxylate reductase 2 and 3, SLC—solute carrier
family. Created with BioRender.com (accessed on 22 May 2022). Figure 5. Gene expression alterations of key players in glutamine and glutamate metabolism and
transport between THP-1WT and THP-1KD in hypoxia. Significant changing genes due to TKTL1 high-
lighted in pink. Expression changes in normoxia versus hypoxia, indicated in log2-fold-change for
n = 3 samples per condition. Abbreviations: ALDH—aldehyde dehydrogenase family, ALDH4A1—
pyrroline-5-carboxylate dehydrogenase. mitochondrial, ALDH18A1—pyrroline-5-carboxylate syn-
thase, ARG1—arginase 1, ASL—argininosuccinate lyase, ASS1—argininosuccinate synthase 1, GLS—
glutaminase, GS—glutamine synthetase, OAT—ornithine aminotransferase, PRODH—proline de-
hydrogenase, PYCR2 and 3—pyrroline-5-carboxylate reductase 2 and 3, SLC—solute carrier family. Created with BioRender.com (accessed on 31 January 2022). 2.4. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes in the Fate of Amino Acids in
2.4. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes in the Fate of Amino Acids in THP-1 Cells 2.4. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes in the Fate of Amino Acids in
2.4. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes in the Fate of Amino Acids in THP-1 Cells gg
yp
g
f
THP-1 Cells
Besides glutamine and glutamate, we also analyzed and compared the changes in
concentrations of amino acids in the incubation medium from THP-1WT and THP-1KD cells
incubated in normoxic and hypoxic conditions. Concentrations in the medium of proline
and ornithine increased more in THP-1WT than THP-1KD in response to hypoxia (Figure
6B). 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells Glutamate
and glutamine concentrations were measured in incubation medium of THP-1WT and THP-1 KD cells
incubated for 48 h, in both normoxic and hypoxic conditions. (C) Ratio of change in glucose per
change in glutamine concentration. Data represented are mean ± SD (n = 3). Statistically significant
differences in all panels were determined by two-tailed independent sample Student’s t-test: p < 0.05
(*). p < 0.01 (**). p < 0.001 (***). Another key characteristic adaptation to hypoxia in cancer cells is observed in altered
expression of key glutamine metabolism genes and transporters [34]. We investigated
whether the contrary response seen in hypoxia in our results was due to a failure in the
upregulation of gene expression (Figure 5). We observed that GS gene expression was
significantly upregulated (log2FC = 1.3; padj < 0.05) and also SLC38A2 was slightly upreg-
ulated in THP-1WT, whereas glutaminase (GLS) expression did not change. By contrast,
GLS and SLC38A2 were downregulated in THP-1KD and the observed overexpression of
GS (log2FC = 0.60; padj < 0.05) was 50% lower than that observed in the control cells. In
addition, SLC1A3 (glutamate–aspartate transporter) and SLC17A7 (glutamate transporter)
were strongly overexpressed under hypoxia conditions in THP-1WT, whereas in THP-1KD
they were downregulated or unaltered, respectively. Altogether, the observed changes at
the transcriptomic level relating to glutamine/glutamate intake and metabolism correlate
with an impairment of the metabolic response induced by hypoxia as a consequence of
TKTL1 knockdown. Int. J. Mol. Sci. 2022, 23, 3574 8 of 20 8 of 20 Figure 5. Gene expression alterations of key players in glutamine and glutamate metabolism an Figure 5. Gene expression alterations of key players in glutamine and glutamate metabolism and
transport between THP-1WT and THP-1KD in hypoxia. Significant changing genes due to TKTL1 high-
lighted in pink. Expression changes in normoxia versus hypoxia, indicated in log2-fold-change for
n = 3 samples per condition. Abbreviations: ALDH—aldehyde dehydrogenase family, ALDH4A1—
pyrroline-5-carboxylate dehydrogenase. mitochondrial, ALDH18A1—pyrroline-5-carboxylate syn-
thase, ARG1—arginase 1, ASL—argininosuccinate lyase, ASS1—argininosuccinate synthase 1,
GLS—glutaminase, GS—glutamine synthetase, OAT—ornithine aminotransferase, PRODH—pro-
line dehydrogenase, PYCR2 and 3—pyrroline-5-carboxylate reductase 2 and 3, SLC—solute carrier
family. Created with BioRender.com (accessed on 22 May 2022). Figure 5. Gene expression alterations of key players in glutamine and glutamate metabolism and
transport between THP-1WT and THP-1KD in hypoxia. Significant changing genes due to TKTL1 high-
lighted in pink. 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells p < 0.01
(**) and p < 0.001 (***). Figure 6. Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a
decrease in concentration during incubation time, and a positive value denotes an increase in con-
centration during incubation time. (A) Alanine and serine. (B) Branched-chain amino acids isoleu-
cine (Ile), leucine (Leu) and valine (Val). (C) Histidine (His), lysine (Lys), methionine (Met), orni-
thine (Orn), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Tryp) and tyrosine
(Tyr). (D) Acetylornithine (AcOrn), alpha-aminoadipic acid (αAAA), kynurenine and total dime-
thylarginine (total DMA). Data represented are mean ± SD (n = 3). Statistically significant differences
Figure 6. Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a
decrease in concentration during incubation time, and a positive value denotes an increase in concen-
tration during incubation time. (A) Alanine and serine. (B) Branched-chain amino acids isoleucine
(Ile), leucine (Leu) and valine (Val). (C) Histidine (His), lysine (Lys), methionine (Met), ornithine
(Orn), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Tryp) and tyrosine (Tyr). (D)
Acetylornithine (AcOrn), alpha-aminoadipic acid (αAAA), kynurenine and total dimethylarginine
(total DMA). Data represented are mean ± SD (n = 3). Statistically significant differences were
determined by two-tailed independent sample Student’s t-test with p-values of p < 0.05 (*). p < 0.01
(**) and p < 0.001 (***). Figure 6. Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a
decrease in concentration during incubation time, and a positive value denotes an increase in con-
centration during incubation time. (A) Alanine and serine. (B) Branched-chain amino acids isoleu-
cine (Ile), leucine (Leu) and valine (Val). (C) Histidine (His), lysine (Lys), methionine (Met), orni-
thine (Orn), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Tryp) and tyrosine
(Tyr). (D) Acetylornithine (AcOrn), alpha-aminoadipic acid (αAAA), kynurenine and total dime-
thylarginine (total DMA). 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells The inhibition on the expression levels of these enzymes was around 1.6-
fold higher in THP-1KD than in THP-1WT (Figure 7), which is consistent with the observed
higher decrease in Ile and Leu consumption induced by hypoxia in THP-1KD. Figure 6. Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a
Figure 6. Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a Figure 6. Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a
decrease in concentration during incubation time, and a positive value denotes an increase in con-
centration during incubation time. (A) Alanine and serine. (B) Branched-chain amino acids isoleu-
cine (Ile), leucine (Leu) and valine (Val). (C) Histidine (His), lysine (Lys), methionine (Met), orni-
thine (Orn), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Tryp) and tyrosine
(Tyr). (D) Acetylornithine (AcOrn), alpha-aminoadipic acid (αAAA), kynurenine and total dime-
thylarginine (total DMA). Data represented are mean ± SD (n = 3). Statistically significant differences
Figure 6. Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a
decrease in concentration during incubation time, and a positive value denotes an increase in concen-
tration during incubation time. (A) Alanine and serine. (B) Branched-chain amino acids isoleucine
(Ile), leucine (Leu) and valine (Val). (C) Histidine (His), lysine (Lys), methionine (Met), ornithine
(Orn), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Tryp) and tyrosine (Tyr). (D)
Acetylornithine (AcOrn), alpha-aminoadipic acid (αAAA), kynurenine and total dimethylarginine
(total DMA). Data represented are mean ± SD (n = 3). Statistically significant differences were
determined by two-tailed independent sample Student’s t-test with p-values of p < 0.05 (*). 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells The observed major increase in gene expression of proline dehydrogenase (PRODH),
that converts proline in pyrroline-5-carboxylate (5PC), and of arginase 1 (ARG1), that con-
verts arginine into ornithine, is in accordance with the observed increase in metabolite
concentration in incubation medium between THP-1WT and THP-1KD (Figure 5). Moreover,
in THP-1WT, we observed for hypoxia a lower increase in the concentration of acetylorni-
thine in incubation medium (Figure 6D), the latter being an intermediate metabolite be-
tween glutamate and ornithine [35]. This finding is consistent with a more active produc-
tion of ornithine in the wild-type cells under hypoxic conditions. Interestingly, we also
observed a significant increase in the excretion of total dimethylarginines (total DMA) in
THP-1KD cells (Figure 6D), which is consistent with a significant overexpression of argi-
nine methyltransferase 8 (PRMT8) (log2FC = 5.7; padj < 0.05). It is worth noting that
Besides glutamine and glutamate, we also analyzed and compared the changes in
concentrations of amino acids in the incubation medium from THP-1WT and THP-1KD cells
incubated in normoxic and hypoxic conditions. Concentrations in the medium of proline
and ornithine increased more in THP-1WT than THP-1KD in response to hypoxia (Figure 6B). The observed major increase in gene expression of proline dehydrogenase (PRODH), that
converts proline in pyrroline-5-carboxylate (5PC), and of arginase 1 (ARG1), that converts
arginine into ornithine, is in accordance with the observed increase in metabolite concen-
tration in incubation medium between THP-1WT and THP-1KD (Figure 5). Moreover, in
THP-1WT, we observed for hypoxia a lower increase in the concentration of acetylornithine
in incubation medium (Figure 6D), the latter being an intermediate metabolite between
glutamate and ornithine [35]. This finding is consistent with a more active production of
ornithine in the wild-type cells under hypoxic conditions. Interestingly, we also observed
a significant increase in the excretion of total dimethylarginines (total DMA) in THP-1KD
cells (Figure 6D), which is consistent with a significant overexpression of arginine methyl-
transferase 8 (PRMT8) (log2FC = 5.7; padj < 0.05). It is worth noting that PRMP8 has been
recently described as a key player in maintaining stress tolerance by ensuring proper DMA
levels [36]. Int. J. Mol. Sci. 2022, 23, 3574 9 of 20
but the
ecrease in consumption of histidine induced by hypoxia was significant only for THP-1WT. Figure 6. 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a
decrease in concentration during incubation time, and a positive value denotes an increase in con-
centration during incubation time. (A) Alanine and serine. (B) Branched-chain amino acids isoleu-
cine (Ile), leucine (Leu) and valine (Val). (C) Histidine (His), lysine (Lys), methionine (Met), orni-
thine (Orn), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Tryp) and tyrosine
(Tyr). (D) Acetylornithine (AcOrn), alpha-aminoadipic acid (αAAA), kynurenine and total dime-
thylarginine (total DMA). Data represented are mean ± SD (n = 3). Statistically significant differences
Figure 6. Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a
decrease in concentration during incubation time, and a positive value denotes an increase in concen-
tration during incubation time. (A) Alanine and serine. (B) Branched-chain amino acids isoleucine
(Ile), leucine (Leu) and valine (Val). (C) Histidine (His), lysine (Lys), methionine (Met), ornithine
(Orn), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Tryp) and tyrosine (Tyr). (D)
Acetylornithine (AcOrn), alpha-aminoadipic acid (αAAA), kynurenine and total dimethylarginine
(total DMA). Data represented are mean ± SD (n = 3). Statistically significant differences were
determined by two-tailed independent sample Student’s t-test with p-values of p < 0.05 (*). p < 0.01
(**) and p < 0.001 (***). Regarding branched-chain amino acid (BCAA) metabolism, we observed, for hypoxia,
a significantly reduced consumption of isoleucine (Ile) and leucine (Leu), at similar ex-
tents, for both THP-1WT and THP-1KD cells. With respect to valine (Val), we observed
that the decrease in the change in valine concentration in incubation medium was only
statistically significant in THP-1WT cells (Figure 6C). To complete the characterization of
BCAA metabolism, we further looked at the gene expression of key enzymes in their
catabolism and found that the expression of branched-chain aminotransferase 1 (BCAT1)
and branched-chain keto acid dehydrogenase B (BCKDHB) genes decreased under hypoxia
in both cell lines. 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells Data represented are mean ± SD (n = 3). Statistically significant differences
Figure 6. Changes in concentration of specific metabolites measured in incubation medium of THP-
1WT and THP-1 KD cells incubated for 48 h, for both normoxic and hypoxic conditions. All changes
in concentrations were normalized by cell number and incubation time. Negative values denote a
decrease in concentration during incubation time, and a positive value denotes an increase in concen-
tration during incubation time. (A) Alanine and serine. (B) Branched-chain amino acids isoleucine
(Ile), leucine (Leu) and valine (Val). (C) Histidine (His), lysine (Lys), methionine (Met), ornithine
(Orn), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Tryp) and tyrosine (Tyr). (D)
Acetylornithine (AcOrn), alpha-aminoadipic acid (αAAA), kynurenine and total dimethylarginine
(total DMA). Data represented are mean ± SD (n = 3). Statistically significant differences were
determined by two-tailed independent sample Student’s t-test with p-values of p < 0.05 (*). p < 0.01
(**) and p < 0.001 (***). Regarding branched-chain amino acid (BCAA) metabolism, we observed, for hypoxia,
a significantly reduced consumption of isoleucine (Ile) and leucine (Leu), at similar ex-
tents, for both THP-1WT and THP-1KD cells. With respect to valine (Val), we observed
that the decrease in the change in valine concentration in incubation medium was only
statistically significant in THP-1WT cells (Figure 6C). To complete the characterization of
BCAA metabolism, we further looked at the gene expression of key enzymes in their
catabolism and found that the expression of branched-chain aminotransferase 1 (BCAT1)
and branched-chain keto acid dehydrogenase B (BCKDHB) genes decreased under hypoxia
in both cell lines. The inhibition on the expression levels of these enzymes was around 1.6-
fold higher in THP-1KD than in THP-1WT (Figure 7), which is consistent with the observed
higher decrease in Ile and Leu consumption induced by hypoxia in THP-1KD. 10 of 20
11 of 21 Int. J. Mol. Sci. 2022, 23, 3574
Int. J. Mol. Sci. 2022, 23, x FO 10 of 20
11 of 21 Figure 7. Gene expression changes in amino acid transport and catabolism between THP-1WT and
THP-1KD in hypoxia. Significant changing genes due to TKTL1 highlighted in pink. Expression
changes in normoxia versus hypoxia, indicated in log2-fold-change of n = 3 samples per condition. Abbreviations: BCAT1—branched-chain amino acid transaminase 1, BCKDHB—branched-chain
keto acid dehydrogenase B, IDO2—indoleamine 2,3 dioxygenase 2, PCK1—phosphoenolpyruvate
carboxykinase 1, PHGDH—phosphoglycerate dehydrogenase, SHMT2—serine hydroxymethyl-
transferase 2, SLC—solute carrier family, TPH2—tryptophan hydroxylase 2. 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells Created with BioRen-
der.com (accessed on 22 May 2022). 3. Discussion
Figure 7. Gene expression changes in amino acid transport and catabolism between THP-1WT and
THP-1KD in hypoxia. Significant changing genes due to TKTL1 highlighted in pink. Expression
changes in normoxia versus hypoxia, indicated in log2-fold-change of n = 3 samples per condition. Abbreviations: BCAT1—branched-chain amino acid transaminase 1, BCKDHB—branched-chain
keto acid dehydrogenase B, IDO2—indoleamine 2,3 dioxygenase 2, PCK1—phosphoenolpyruvate
carboxykinase 1, PHGDH—phosphoglycerate dehydrogenase, SHMT2—serine hydroxymethyltrans-
ferase 2, SLC—solute carrier family, TPH2—tryptophan hydroxylase 2. Created with BioRender.com
(accessed on 31 January 2022). Figure 7. Gene expression changes in amino acid transport and catabolism between THP-1WT and
THP-1KD in hypoxia. Significant changing genes due to TKTL1 highlighted in pink. Expression
changes in normoxia versus hypoxia, indicated in log2-fold-change of n = 3 samples per condition. Abbreviations: BCAT1—branched-chain amino acid transaminase 1, BCKDHB—branched-chain
keto acid dehydrogenase B, IDO2—indoleamine 2,3 dioxygenase 2, PCK1—phosphoenolpyruvate
carboxykinase 1, PHGDH—phosphoglycerate dehydrogenase, SHMT2—serine hydroxymethyl-
transferase 2, SLC—solute carrier family, TPH2—tryptophan hydroxylase 2. Created with BioRen-
der.com (accessed on 22 May 2022). 3 Di
i
Figure 7. Gene expression changes in amino acid transport and catabolism between THP-1WT and
THP-1KD in hypoxia. Significant changing genes due to TKTL1 highlighted in pink. Expression
changes in normoxia versus hypoxia, indicated in log2-fold-change of n = 3 samples per condition. Abbreviations: BCAT1—branched-chain amino acid transaminase 1, BCKDHB—branched-chain
keto acid dehydrogenase B, IDO2—indoleamine 2,3 dioxygenase 2, PCK1—phosphoenolpyruvate
carboxykinase 1, PHGDH—phosphoglycerate dehydrogenase, SHMT2—serine hydroxymethyltrans-
ferase 2, SLC—solute carrier family, TPH2—tryptophan hydroxylase 2. Created with BioRender.com
(accessed on 31 January 2022). Leukemia is a type of cancer characterized by a rapid expansion of immature hema-
topoietic cells [41]. This uncontrolled proliferation can arise from many distinct conjuga-
tions of mutations, whose compiled synergy drives tumorigenesis [42–46]. The resulting
phenotype presents a different metabolism from the surrounding normal cells, granting
it advantages in proliferation and survival in the hypoxic conditions of the hematopoietic
SLC7A8, an antiport transporter that intakes BCAAs as well as other neutral amino
acids (tyrosine, phenylalanine, tryptophan, methionine and glutamine) through obligatory
exchange mechanisms, as well as SLC7A7 [37], another antiport transporter that similarly
exchanges neutral for cationic amino acids, were dramatically overexpressed in response to
hypoxia only in THP-1WT (Figure 7). it advantages in proliferation and survival in the hypoxic conditions of the hematopoietic
niche [47–51]. 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells On the contrary, kynurenine net consumption was strongly enhanced
under hypoxia in THP-1KD cells, which is consistent with the inability of the KD cells to
enhance IDO2 in response to hypoxia. Serine net consumption was also slightly enhanced in hypoxia in both cell lines, but
the increase was only statistically significant in THP-1WT, in accordance with the observed
greater increase in phosphoglycerate dehydrogenase (PHGDH) and serine hydroxymethyl-
transferase 2 (SHMT2) gene expression in THP-1WT (Figure 7). Secretion of alanine to the medium was also enhanced under hypoxia, but only for
THP-1WT (Figure 6A), which is consistent with the enhanced change in glucose and glu-
tamine concentrations measured in incubation medium of THP-1WT previously observed
with respect to THP-1KD. We also measured intracellular amino acid content to see whether the pattern of
alterations in THP-1KD incubation medium under hypoxic conditions is consistent with net-
total amino acids’ intracellular content. The THP-1WT cells had three times less aspartate
and five times more serine in hypoxia, whereas these metabolite levels did not significantly
change intracellularly under hypoxia in THP-1KD cells (Table A3 in Appendix B). The
observed THP-1WT decrease in aspartate intracellularly under hypoxia is consistent with
reported evidence across different tumors of a negative correlation between hypoxia and
intracellular aspartate content [38]. p
The rise in intracellular serine content triggered by hypoxia in THP-1WT together with
the previously observed increase in serine net consumption (Figure 6A) could be related to
the use of this amino acid for pyrimidine synthesis and cell proliferation under hypoxia. In
fact, it has been described that serine synthesis through PHGDH is crucial for the mainte-
nance of nucleotide levels [39], and a similar effect was reported for phosphoenolpyruvate
carboxykinase 1 (PCK1), which is a key player that drives pyrimidine biosynthesis under
hypoxia [40]. It is worth noting that PCK1 gene expression was significantly increased
(log2FC = 5.0; padj < 0.05) under hypoxia in THP-1WT, whereas hypoxia did not trigger
any statistically significant changes in THP-1KD (Figure 7). The fact that we did not observe these changes in intracellular amino acid content
in hypoxia in THP-1KD corroborates the importance of TKTL1 in triggering the switch to
enhanced glycolysis and the consequent adaptation of amino acid metabolism to support
such a response. 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells Transketolase-like 1 is a key enzyme of metabolism, linking glycolysis to the produc-
tion of nucleotides a necessary component for de novo synthesis of RNA and DNA es-
yp
y
g
Methionine and tyrosine consumption by cells decreased under hypoxia, but the
changes were statistically significant only in THP-1KD cells. On the contrary, the decrease
in consumption of histidine induced by hypoxia was significant only for THP-1WT. tion of nucleotides, a necessary component for de novo synthesis of RNA and DNA, es-
pecially in rapidly proliferating cells [52–54]. In fact, expression of TKTL1 is low in normal
tissues except for testis [55,56]. Although the full scope of its functions is still unknown to
this day, a clear correlation of its expression with cancer malignancy and poor prognosis
has been established across many types of cancer [5–7,57,58]. One such type is AML,
where the putative role of TKTL1 overexpression remains to be properly pursued [16,59]. This study combined targeted metabolomics and transcriptomics profiling for a com-
parative analysis of a stable acute monocytic leukemia cell line with a TKTL1 knockdown
Net lysine, threonine and phenylalanine consumptions decreased significantly under
hypoxia in both cell lines, but the changes in concentration in incubation medium observed
for lysine and threonine were much larger in THP-1KD (Figure 6B). With respect to pheny-
lalanine, the observed changes were larger in THP-1WT (Figure 6B). It is worth noting that
the net consumption of α-aminoadipic acid (αAAA), a biogenic amine generated in the
intermediate steps of the catabolism of lysine, decreased under hypoxia in both cell lines
(Figure 6D). However, the observed decrease was lower for THP-1KD, with cells consuming Int. J. Mol. Sci. 2022, 23, 3574 11 of 20 11 of 20 significantly more αAAA in hypoxia compared to THP-1WT. In normoxic conditions, there
were no significant differences in αAAA net consumption between the cell lines. g
p
Tryptophan uptake was slightly decreased in both cell lines under hypoxia (Figure 6B). Tryptophan can be metabolized to kynurenine with indoleamine 2.3-dioxygenase 2 (IDO2),
the latter being the rate-limiting step of this pathway, or to serotonin through TPH1 and
TPH2. Both pathways produce biogenic amines and other active metabolic intermediates
with signaling functions. Gene expression of IDO2 was strongly enhanced under hypoxia
only in THP-1WT, which is consistent with a decreased net consumption of kynurenine in
these cells (Figure 6B). 2.3. TKTL1 Is Essential to Trigger Hypoxia-Induced Changes to the Fate of Glutamine in THP-1 Cells Overall, this new role of TKTL1, beyond its enzymatic function on the
non-oxidative branch of the PPP unveiled in this study, opens new opportunities for the
design of combined therapies targeting TKTL1 and for exploiting the impaired adaptation
of these cells to hypoxic stress. 3. Discussion Leukemia is a type of cancer characterized by a rapid expansion of immature hematopoi-
etic cells [41]. This uncontrolled proliferation can arise from many distinct conjugations of
mutations, whose compiled synergy drives tumorigenesis [42–46]. The resulting phenotype
presents a different metabolism from the surrounding normal cells, granting it advantages
in proliferation and survival in the hypoxic conditions of the hematopoietic niche [47–51]. Transketolase-like 1 is a key enzyme of metabolism, linking glycolysis to the pro-
duction of nucleotides, a necessary component for de novo synthesis of RNA and DNA,
especially in rapidly proliferating cells [52–54]. In fact, expression of TKTL1 is low in nor-
mal tissues except for testis [55,56]. Although the full scope of its functions is still unknown Int. J. Mol. Sci. 2022, 23, 3574 12 of 20 12 of 20 to this day, a clear correlation of its expression with cancer malignancy and poor prognosis
has been established across many types of cancer [5–7,57,58]. One such type is AML, where
the putative role of TKTL1 overexpression remains to be properly pursued [16,59]. p
p
p
p
y p
This study combined targeted metabolomics and transcriptomics profiling for a com-
parative analysis of a stable, acute, monocytic leukemia cell line with a TKTL1 knockdown
and a wild-type counterpart, in order to discern TKTL1’s role in metabolic adaptation
under the state of hypoxia. yp
The results obtained unveiled that the main metabolic changes triggered by hypoxic
conditions, such as increased glucose and glutamine consumptions and increased lactate
production, are impaired by TKTL1 knockdown. These differences occur as a consequence
of changes at the level of gene expression, enzyme activities, metabolite exchange with
extracellular media and intracellular metabolite homoeostasis. In fact, we have observed impaired gene expression and/or enzyme activity of
GAPDH, a glycolytic protein transcriptionally upregulated in hypoxia with moonlighting
activities [60], and other key metabolic players of glucose metabolism reported in the
literature to be induced under hypoxia [20–25], such as lactate and glucose transporters,
PDKs, HKs, PCK1, and LDHs. Moreover, TKTL1 knockdown results in a decrease in
G6PD activity, an enzyme whose main function is to maintain redox homeostasis [61], thus
expanding the metabolic impact on pentose-phosphate and NADPH synthesis capabilities
of the TKTL1-knockdown cells. 3. Discussion Worth noting that the impaired increase in GAPDH and
G6PD, the key enzymes of the upper part of glycolysis and the oxidative branch of the
PPP pathway, can indirectly contribute to the observed decrease in TKT activity to main-
tain metabolic homeostasis. Moreover, the decrease in reducing power associated with
decreases in GAPDH and G6PD, two critical mediators of the cellular response to oxidative
stress, will impact every aspect of energetic metabolism. At the level of glutamine–glutamate transport and metabolism, we also observed
that the expression of key genes and transporters, reported in the literature to be induced
by hypoxia [34], such as SLC1A3, SLC17A7, SLC38A2 and glutamine synthase, was also
impaired in TKTL1 knockdown cells when submitted to the hypoxia challenge. It is worth noting that loss of TKTL1 also resulted in a reduction in the hypoxia-
triggered switch to proline production when compared with wild-type cells, and that
the expression pattern of key players in proline metabolism (PYCRs and PRODH) is also
altered. Taking into account that TKTL1 knockdown results in a decrease in G6PD activity
and that proline synthesis requires NADPH, these results could be explained by a decreased
NADPH pool triggered by G6PD activity deficiency. In fact, there is increasing evidence
that proline synthesis plays a key role in the regulation of cellular redox homeostasis
and that it is synthesized beyond the cell proliferative need as part of a “redox valve”
mechanism [62]. Another consequence of impaired GAPDH activity induced by TKTL1 is the possible
effects in serine synthesis. It has been described that GAPDH controls D-serine synthesis in
astrocytes [63]. The impaired expression in hypoxia of GAPDH, together with PCK1 and
key cationic and essential amino acids transporters (SLC7A7 and SLC7A8), as well as of
the attenuated expression of key players in serine synthesis, could be related to an altered
pyrimidine nucleotide synthesis owing to TKTL1 knockdown. In fact, it has been described
that PCK1 and PHGDH are key players in maintaining appropriate nucleotide synthesis
under hypoxia [39,40]. yp
The fact that TKTL1 knockdown results in downregulation of so many metabolic
genes indicates that TKTL1 could be a “moonlight protein” and may well also act by other
mechanisms not involving enzyme activity. Further studies are required to investigate
whether TKTL1 catalyzes a mono-substrate reaction or an unsuspected reaction that could
induce transcription factors. 4. Materials and Methods
4.1. Cell Culture The THP-1 cell line is a human monocytic cell line derived from an acute monocytic
leukemia patient, and is widely used as a model for AML in drug screenings. THP-1
cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). THP-1WT and THP-1KD cells were generated by Sirion Biotech GmbH (Munich, Germany). THP-1 cells were grown in suspension in RPMI-1640 culture medium of 10 mM glucose,
2 mM glutamine and supplemented with 10% heat-inactivated fetal calf serum (FCS) and
1% penicillin/streptomycin at 37 ◦C and 5% CO2. In hypoxia settings, cells were cultured
for 5 days for chronic hypoxic adaptation in Hypoxystation H35 (Don Whitley Scientific
Limited, Bingley, UK) prior to any experiment. 3. Discussion p
Of note, the cells in hypoxia initiated a compensatory mechanism in order to sur-
vive in response to TKTL1 knockdown. Increased gene expression of SIRT5 stimulates
the regulation of G6PD through post-translational modifications in order to maintain its Int. J. Mol. Sci. 2022, 23, 3574 13 of 20 13 of 20 activity in hypoxia. Increased use of the isoenzyme HK3 and upregulation of PRMT8 are
clear responses to ROS increase, since both have been reported to mediate mitochondrial
biogenesis and stress tolerance. g
While there is previous evidence demonstrating that TKTL1 expression is triggered
in hypoxia [13,14], our study goes beyond the state of the art, unveiling the metabolic
role of TKTL1 triggering the switch to enhanced glycolysis and glutamine consumption
at the molecular level. In fact, our results provide evidence that TKTL1 plays a key role
in facilitating the overexpression of key proteins necessary for the switch to enhanced
glycolysis and glutamine consumption modes characteristic of hypoxia adaptation. Therefore, our results reveal that the role of TKTL1 in the adaptation to hypoxia is
essential for the coordination of central metabolism to trigger metabolic reprogramming,
both regulating glucose/glutamine consumption and also contributing to oxidative stress
regulation through the overexpression of GAPDH and G6PD. Further studies should be carried out to better understand the mechanisms involved
in this new facet of TKTL1 function in metabolism. Additional investigations into the
changes identified here on other AML cell lines and primary patient-derived cells could
reveal new approaches for therapies that combine targeting TKTL1 and the “weaknesses”
that arise from its knockdown. 4.2. Cell Viability Assays Cell proliferation curves were performed by cell counting using a ScepterTM Hand-
held Automated Cell Counter (Merck Millipore, Burlington, MA, USA) and Countess II
Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA). THP-1 cell lines
were seeded at 3 × 105 cells per 25 cm2 flasks in normoxia and at 4 × 105 cells in hypoxia. Cell counting was performed at 24 h timepoints up until a total of 96 h. After counting,
THP-1 cells were collected in Eppendorf tubes from seeding flasks, with volumes of 500 µL,
centrifuged at 350× g for 5 min, supernatant was removed and resuspended in 1 mL PBS
solution (phosphate-buffered saline). 4.3. Measurement of Enzyme Activities, Concentration of Metabolites in Incubation Medium, and
Intracellular Concentrations of Glucose, Lactate, Amino Acids, and Biogenic Amines Specific enzyme activities of G6PD, GAPDH, HK, LDH, PK, 6PGD and TKT were
determined by using NAD(P)H-coupled enzymatic reactions [64]. For measurement of consumption and production rates of metabolites and measure-
ment of intracellular concentrations, THP-1 cell lines were seeded at 4 × 105 cells/mL
for both normoxia and hypoxia. After 48 h incubation, extracts were collected from
5 × 106 cells each. Media and cell pellets were frozen until analysis. Changes in metabolite concentrations in incubation media were determined by measur-
ing metabolite concentration at the beginning and end of incubation time and normalizing
the difference between these two concentrations by time and cell number. Exponential
cell growth was considered during the entire incubation time [65]. Glucose, lactate, glu-
tamate and glutamine concentrations in incubation media aliquots were measured using Int. J. Mol. Sci. 2022, 23, 3574 14 of 20 14 of 20 NAD(P)H-coupled enzymatic reactions in a COBAS Mira Plus spectrophotometer (Horiba
ABX, Kyoto, Japan) [64]. NAD(P)H-coupled enzymatic reactions in a COBAS Mira Plus spectrophotometer (Horiba
ABX, Kyoto, Japan) [64]. The concentration of amino acids and biogenic amines in incubation media was
determined using the AbsoluteIDQ™p180 Kit (Biocrates Life Sciences, Innsbruck, Austria)
and the AB Sciex 4000 QTRAP MS/MS mass spectrometer coupled to an Agilent HPLC
1200, according to manufacturer’s instructions. Next, 10 µL of media were plated in each
well of the kit. Analyst and the MetIDQ™software packages were used to analyze the
obtained data and calculate metabolite concentrations. 4.2. Cell Viability Assays Amino acids’ and biogenic amines’ intracellular concentrations were determined from
cell lysates using the AbsoluteIDQ™p180 Kit (Biocrates Life Sciences, Innsbruck, Austria)
and the AB Sciex 4000 QTRAP MS/MS mass spectrometer coupled to an Agilent HPLC
1200 (AB Sciex LLC, Framingham, MA, USA; Agilent Technologies, Santa Clara, CA, USA). Cell pellets containing ca. 5 × 106 cells were resuspended in 70 µL of EtOH:PBS 85:15. Suspensions were treated twice as follows: suspensions were sonicated using titanium
probe (3 × 15 s; output 25, tune 50), then submerged in liquid N2 for 30 s and thawed at
95 ◦C in a dry bath. Subsequently, suspensions were centrifuged at 20,000× g for 5 min at
4 ◦C, and supernatants were collected. Then, 30–50 µL of supernatant was plated in each
well of the kit. Analyst and the MetIDQ™software packages were used to analyze the
obtained data and calculate metabolite concentrations. Intracellular concentrations were
corrected by protein content in cell lysates, measured using bicinchoninic acid (BCA) assay. 4.4. Real-Time PCR RNA was extracted from THP-1 cells, both fresh and frozen extracts using Trizol
(Sigma-Aldrich Co LLC, Saint Louis, MO, USA) according to the manufacturer’s protocol. Chloroform was added to the mixture and centrifuged to generate aqueous and organic
phases. The aqueous phase was collected, added to cold isopropanol and incubated
overnight at 4 ◦C to precipitate the RNA. Samples were then centrifuged at 14,000× g
at 4 ◦C for 15 min. RNA was purified with several washing steps using 75% ethanol
and resuspended in RNAse-free water. Purified RNA was quantified using Nanodrop
Spectrophotometer (ND 1000 V3.1.0. Thermo Fisher Scientific Inc., Waltham, MA, USA). p
p
The Reverse Transcription reaction (converting RNA into DNA) was performed at
37 ◦C using 1 µg of RNA added to a mixture containing 5× Buffer (Invitrogen, Waltham,
MA, USA), 0.1 M dithiothreitol (DTT) (Invitrogen, Waltham, MA, USA), Random Hexam-
ers (Roche, Basilea, Switzerland), 40 U·µL−1 RNAse inhibitor (Promega, Fitchburg, WI,
USA), 40 mM dNTPs (Bioline, London, UK), 200 U·µL−1 M-MLV-RT (Invitrogen, Waltham,
MA, USA). Gene-expression analysis was performed with a RT-PCR system (Applied Biosystems®
7500 Real Time PCR, Applied Biosystems, Waltham, MA, USA) using the manufacturer’s
standard protocol employing TaqMan® (Applied Biosystems, Waltham, MA, USA) and
gene-specific probes for TKT (Hs00169074_m1),
TKTL1 (Hs00202061_m1),
GLS
(Hs01014019_m1). Reactions were performed in a volume of 20 µL containing 9 µL of cDNA
and 11 µL of TaqMan Master Mix (Applied Biosystems, Waltham, MA, USA). RT-PCR pro-
gram set-up parameters were: (1) initial incubation at 50 ◦C for 2 min, (2) denaturalization
at 95 ◦C for 10 min, (3) amplification of 40 cycles alternating between 95 ◦C for 15 s and
60 ◦C for 1 min. The house-keeping gene used as reference was PP1A (Hs99999904_m1,
Applied Biosystems) and expression levels were quantified using the ∆∆Ct method. 4.5.3. Analysis of the Raw RNA Sequencing Data The quality of the raw RNA sequencing reads was assessed by FastQC version 0.11.3,
a computational quality-control tool for high-throughput sequence data in Java developed
by Simon Andrews and the Babraham Bioinformatics group (http://www.bioinformatics. babraham.ac.uk/projects/fastqc/ accessed on 31 January 2022, BaBraham Institute, Cam-
bridge, UK). The alignment of the reads was performed using STAR version 2.5.2a open-
source software and the FASTA sequences were generated using Homo sapiens high-
coverage GRCh37.75.dna.primary_assembly. Gene-level count tables were obtained using
the count script of the HTSeq python library version 0.6.1p1 [66] with default options. Only
reads with unique mappings were considered. For performing dimensionality reduction
by principal component analysis (PCA) and hierarchical clustering, normalized transcript
counts were utilized after transformation with the “rlog” function of the Bioconductor pack-
age DESeq2 version 1.12.4 [67]. R version 3.3.1 [68] was used for conducting biostatistical
analyses. 4.5.4. Differential Expression Analysis The statistical analysis to identify differentially expressed genes was performed using
the Bioconductor package DESeq2 version 1.12.4 [67]. Size-factor-based normalization
was performed to control for batch effects and inter-sample variability. Genes with less
than 10 counts across all samples were filtered out. Differential expression analysis was
performed using package defaults for dispersion estimation and the function “Deseq”,
to cover independent filtering, cooks cutoff [69] for outlier detection and a Wald test. Adjusted p-values (padj) were computed from the DESeq2-calculated p-values by applying
a Bonferroni correction for multiple testing. Genes with a Padj-value < 0.05 were considered
as statistically significantly differentially expressed. All aforementioned biostatistical
analyses were performed using R version 3.3.1 [68]. 4.5.1. RNA Extraction Cell pellets of THP-1 cell lines were collected and frozen. Total RNA from lysates
of the THP1WT and THP1KD cell lines in triplicates (n = 3) under normoxic and hypoxic
conditions, and with 10 × 106 cells each, were extracted using the RNeasy Mini Kit (Qiagen,
Hilden, Germany). RNA integrity was further tested using lab-on-a-chip technology on the
BioAnalyzer 2100. Int. J. Mol. Sci. 2022, 23, 3574 15 of 20 15 of 20 4.5.2. RNA-Seq Library Preparation, Sequencing and Generation of FastQ Files High-quality RNA-seq (transcriptome) was performed in the CNIC (National Centre
for Cardiovascular Diseases, Madrid, Spain) genomic unit using the Illumina HiSeq 2500
sequencer. There, 200 ng of total RNA was used to generate barcoded RNA-seq libraries
using the NEBNext Ultra RNA Library preparation kit (New England Biolabs, Ipswich,
MA, USA). Briefly, poly A+ RNA was purified using poly-T oligo-attached magnetic beads
followed by fragmentation and then first and second cDNA strand synthesis. Next, cDNA
ends were repaired and adenylated. The NEBNext adaptor was then ligated, followed by
uracil excision from the adaptor and PCR amplification. Finally, the size of the libraries
was checked using the Agilent 2100 Bioanalyzer DNA 1000 chip and their concentration
was determined using the Qubit® fluorometer (Life Technologies, Carlsbad, CA, USA). Libraries were sequenced on a HiSeq2500 (Illumina, San Diego, CA, USA) to generate
61-base single reads. Finally, FastQ files for each sample were obtained using CASAVA v1.8
software (Illumina, San Diego, CA, USA). Author Contributions: I.B. and S.M. performed the experiments. E.K. and M.C. performed the
transcriptomics data curation and analysis. M.C. and S.M. performed the conceptualization. M.C., I.B.
and E.K. wrote the first draft of the manuscript. I.B., E.K., S.M. and M.C. analyzed and interpreted
data. S.M. and M.C. designed and supervised experiments. I.B., E.K., J.-B.C., U.L.G., S.M. and M.C.
discussed results and participated in writing and revising the manuscript. U.L.G., S.M., J.-B.C. and 4.6. Statistical Analysis Experiments were performed in triplicate for cell lines in at least three independent
experiments. Statistical analyses were performed using the parametric unpaired, two-tailed
independent sample Student’s t-test, assuming normality of distribution and homogeneity
of variance based on past experience in similar measurements. p < 0.05 (*) was considered
statistically significant. Author Contributions: I.B. and S.M. performed the experiments. E.K. and M.C. performed the
transcriptomics data curation and analysis. M.C. and S.M. performed the conceptualization. M.C., I.B. and E.K. wrote the first draft of the manuscript. I.B., E.K., S.M. and M.C. analyzed and interpreted
data. S.M. and M.C. designed and supervised experiments. I.B., E.K., J.-B.C., U.L.G., S.M. and M.C. discussed results and participated in writing and revising the manuscript. U.L.G., S.M., J.-B.C. and Int. J. Mol. Sci. 2022, 23, 3574 16 of 20 M.C. performed overall supervision and funding acquisition. All authors have read and agreed to
the published version of the manuscript. Funding: I. Baptista and E. Karakitsou were funded by the EU grant HaemMetabolome H2020-MSCA-
ITN-2015-675790. J.B. Cazier, U.L. Günther, S. Marin and M. Cascante acknowledged the funding from
the European Commission (HaemMetabolome [EC-675790]). M. Cascante and S. Marin acknowledge
grant PID2020-115051RB-I00 funded by MCIN/AEI/ 10.13039/501100011033, the Agència de Gestió
d’Ajuts Universitaris i de Recerca (AGAUR) Generalitat de Catalunya (2017SGR1033) and CIBERehd
(CB17/04/00023) (ISCIII. Spain). M. Cascante also received support through the prize “ICREA
Academia” for excellence in research, funded by the ICREA foundation–Generalitat de Catalunya. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript. Acknowledgments: We would like to thank the CCiTUB (Scientific and Technological Centers of the
UB) of the Universitat de Barcelona (Barcelona, Spain) for their expert and technical help using the
LC-MS/MS equipment. Conflicts of Interest: The authors declare no conflict of interest. Appendix A
Table A1. Differential gene expression comparing THP1WT cells in hypoxia (n = 3) versus normoxia
(n = 3). Gene Symbol
Description
Log2 Fold Change
lfcSE 1
p-Value
p-adj. 1 lfcSE is the log fold change Standard Error, 2 p-adj. is adjusted p-value. 1 lfcSE is the log fold change Standard Error, 2 p-adj. is adjusted p-value. 4.6. Statistical Analysis Hyp)
Alanine
402.30 ± 143.45
473.60 ± 144.63
346.52 ± 5.56
279.01 ± 79.01
0.5378
0.1103
Arginine
273.40 ± 77.02
272.04 ± 150.85
292.70 ± 144.44
111.34 ± 86.22
0.8482
0.1844
Asparagine
771.52 ± 228.94
563.26 ± 54.48
474.11 ± 39.58
702.79 ± 395.83
0.0909
0.5779
Aspartate
118.19 ± 37.52
136.50 ± 46.37
36.98 ± 10.07
55.27 ± 44.06
0.0223
0.0927
Citrulline
0.24 ± 0.23
3.20 ± 4.66
2.54 ± 2.93
0.52 ± 0.89
0.2464
0.3824
Glutamine
1636.46 ± 205.69
1760.44 ± 510.50
1377.17 ± 295.79
1100.21 ± 204.71
0.2806
0.1061
Glutamate
855.50 ± 1481.77
1089.97 ± 943.94
0.00 ± 0.00
1609.70 ± 571.89
0.3739
0.4605
Glycine
915.60 ± 375.13
1075.90 ± 575.77
583.59 ± 285.89
778.96 ± 222.69
0.1685
0.4516
Histidine
72.78 ± 13.73
46.93 ± 17.33
58.96 ± 8.22
36.64 ± 16.40
0.2093
0.4967
Isoleucine
185.40 ± 38.58
200.03 ± 38.97
210.65 ± 76.16
153.94 ± 44.02
0.6355
0.2461
Leucine
236.18± 55.20
248.53 ± 52.65
249.76 ± 63.02
168.81 ± 66.49
0.7928
0.1789
Lysine
40.25 ± 17.59
53.42 ± 8.72
63.64 ± 23.21
38.99 ± 25.69
0.2368
0.4090
Methionine
71.61 ± 20.62
66.44 ± 25.40
63.10 ± 21.25
41.63 ± 22.51
0.6446
0.2741
Ornithine
22.30 ± 10.41
21.32 ± 9.00
35.94 ± 6.38
29.14 ± 16.43
0.1253
0.5096
Phenylalanine
42.47 ± 7.37
39.58 ± 11.15
59.42 ± 22.21
34.28 ± 9.91
0.2781
0.5709
Proline
705.55 ± 139.47
743.98 ± 116.06
249.71 ± 37.70
191.38 ± 38.52
0.0055
0.0014
Serine
70.15 ± 23.94
129.16 ± 90.81
353.88 ± 116.37
186.89 ± 34.27
0.0144
0.3612
Threonine
173.83 ± 92.88
142.23 ± 28.16
200.71 ± 89.78
111.91 ± 40.27
0.7368
0.3455
Tryptophan
11.02 ± 3.72
12.83 ± 1.75
16.12 ± 6.45
9.69 ± 1.04
0.3014
0.0555
Tyrosine
76.14 ± 26.81
91.19 ± 14.14
89.76 ± 32.79
82.07 ± 19.26
0.6074
0.5448
Valine
54.40 ± 19.08
40.16 ± 5.70
60.33 ± 7.25
51.91 ± 20.82
0.6410
0.3992
Acetylornithine
2.44 ± 2.34
1.18 ± 1.91
3.27 ± 1.82
2.57 ± 3.02
0.6537
0.5368
ADMA
2.17 ± 1.48
1.57 ± 0.75
1.31 ± 0.38
0.74 ± 0.69
0.3849
0.2322
α-AAA
7.09 ± 2.44
6.27 ± 2.80
5.35 ± 5.40
4.62 ± 0.51
0.6387
0.3711
Carnosine
0.84 ± 0.55
0.51 ± 0.19
0.38 ± 0.17
0.21 ± 0.09
0.2395
0.0732
Creatinine
16.70 ± 6.86
15.20 ± 2.31
10.38 ± 2.29
11.00 ± 1.91
0.2047
0.0722
Histamine
0.32 ± 0.29
0.23 ± 0.40
0.00 ± 0.00
0.25 ± 0.44
0.1252
0.9480 Table A2. 4.6. Statistical Analysis Differential gene expression comparing THP1KD cells in hypoxia (n = 3) versus normoxia
(n = 3). Gene Symbol
Description
Log2 Fold Change
lfcSE 1
p-Value
p-adj. 2
G6PD
glucose-6-phosphate dehydrogenase
−1.24
0.10
3.15 × 10−37
1.51 × 10−35
SIRT5
sirtuin 5
1.08
0.12
2.92 × 10−19
3.50 × 10−18
TKTL1
transketolase-like 1
0.90
0.12
2.14 × 10−14
1.66 × 10−13
TKT
transketolase
0.24
0.07
5.9 × 10−4
1.5 × 10−3
HK2
hexokinase 2
0.67
0.17
7.82 × 10−5
2.2 × 10−4
HK3
hexokinase 3
2.73
0.17
7.21 × 10−57
1.17 × 10−54
PDK3
pyruvate dehydrogenase kinase 3
−0.61
0.21
3.9 × 10−3
8.9 × 10−3
PDK4
pyruvate dehydrogenase kinase 4
−2.20
0.53
3.53 × 10−5
1.1 × 10−4
GAPDH
glyceraldehyde-3-phosphate
dehydrogenase
0.57
0.11
1.86 × 10−7
7.30 × 10−7
LDHD
lactate dehydrogenase D
1.34
0.14
1.53 × 10−20
2.03 × 10−19
SLC2A3
solute carrier family 2 member 3
1.81
0.32
2.11 × 10−8
9.12 × 10−8
SLC16A3
solute carrier family 16 member 3
0.48
0.12
7.73 × 10−5
2.2 × 10−4
ESRRB
estrogen related receptor beta
–
–
–
–
GLUL
glutamate-ammonia ligase
0.60
0.12
2.07 × 10−7
8.09 × 10−7
GLS
glutaminase
−0.88
0.13
1.67 × 10−11
9.95 × 10−11
SLC1A3
solute carrier family 1 member 3
−0.89
0.32
5.4 × 10−3
1.1 × 10−2
SLC38A2
solute carrier family 38 member 2
−0.38
0.10
2.8 × 10−4
7.1 × 10−4
IDO2
indoleamine 2,3-dioxygenase 2
–
–
–
–
PHGDH
phosphoglycerate dehydrogenase
0.46
0.09
1.10 × 10−7
4.42 × 10−7
SHMT2
serine hydroxymethyltransferase 2
0.37
0.05
1.81 × 10−15
1.53 × 10−14
TPH2
tryptophan hydroxylase 2
6.93
1.40
6.88 × 10−7
2.53 × 10−6
BCAT1
branched chain amino acid
transaminase 1
−2.41
0.14
9.87 × 10−66
2.61 × 10−63
1 lfcSE is the log fold change Standard Error, 2 p-adj. is adjusted p-value. Table A3. Intracellular concentrations of metabolites in THP-1WT and THP-1KD cell lines. Table A3. Intracellular concentrations of metabolites in THP-1WT and THP-1KD cell lines. Metabolite
THP-1WT
Normoxia
THP-1KD
Normoxia
THP-1WT
Hypoxia
THP-1KD
Hypoxia
p-Value
THP-1WT
(Nor vs. Hyp)
p-Value
THP-1KD
(Nor vs. 4.6. Statistical Analysis 2
G6PD
glucose-6-phosphate dehydrogenase
−0.72
0.09
1.45 × 10−15
1.41 × 10−14
SIRT5
sirtuin 5
0.78
1.10
7.01 × 10−13
5.32 × 10−12
TKTL1
transketolase-like 1
0.34
0.11
2.4 × 10−3
5.8 × 10−3
TKT
transketolase
0.20
0.07
2.7 × 10−3
6.5 × 10−3
HK1
hexokinase 1
0.73
0.12
1.49 × 10−9
8.00 × 10−9
HK2
hexokinase 2
1.82
0.17
2.71 × 10−27
6.56 × 10−26
HK3
hexokinase 3
1.09
0.17
2.27 × 10−10
1.32 × 10−9
PDK1
pyruvate dehydrogenase kinase 1
0.70
0.21
7.6 × 10−4
2.0 × 10−3
PDK3
pyruvate dehydrogenase kinase 3
0.51
0.21
1.6 × 10−2
3.1 × 10−2
PDK4
pyruvate dehydrogenase kinase 4
1.42
0.49
4.0 × 10−3
9.1 × 10−3
GAPDH
glyceraldehyde-3-phosphate
dehydrogenase
1.05
0.11
9.71 × 10−22
1.59 × 10−20
LDHD
lactate dehydrogenase D
2.18
0.25
7.55 × 10−18
8.89 × 10−17
SLC2A3
solute carrier family 2 member 3
0.67
0.32
3.11 × 10−8
1.44 × 10−7
SLC16A3
solute carrier family 16 member 3
3.60
0.12
6.36 × 10−30
1.81 × 10−28
ESRRB
estrogen related receptor beta
4.64
1.4
1.2 × 10−3
3.0 × 10−3
GLUL
glutamate-ammonia ligase
1.38
0.12
2.16 × 10−30
6.37 × 10−29
SLC1A3
solute carrier family 1 member 3
0.80
0.29
5.9 × 10−3
1.3 × 10−2
SLC17A7
solute carrier family 17 member 7
3.31
0.61
5.31 × 10−8
2.40 × 10−7
SLC38A2
solute carrier family 38 member 2
0.28
0.10
2.6 × 10−2
4.9 × 10−2
IDO2
indoleamine 2,3-dioxygenase 2
4.55
1.75
9.4 × 10−3
2.0 × 10−2
PHGDH
phosphoglycerate dehydrogenase
0.77
0.09
3.11 × 10−19
4.15 × 10−18
SHMT2
serine hydroxymethyltransferase 2
0.75
0.04
7.34 × 10−65
1.46 × 10−6
PCK1
phosphoenolpyruvate carboxykinase 1
5.03
1.51
8.8 × 10−4
2.3 × 10−3
TPH2
tryptophan hydroxylase 2
–
–
–
–
BCAT1
Branched chain amino acid
transaminase 1
−1.56
0.14
9.14 × 10−31
2.74 × 10−29
BCKDH
Branched chain keto acid
dehydrogenase E1 subunit beta
−0.38
0.10
2.3 × 10−4
6.7 × 10−4
SLC7A7
solute carrier family 7 member 7
2.28
0.16
6.47 × 10−46
4.86 × 10−44
SLC7A8
solute carrier family 7 member 8
3.21
0.85
0.00015
0.00044
1 lf SE i
h l
f ld h
S
d
d E
2
dj i
dj
d
l Table A1. Differential gene expression comparing THP1WT cells in hypoxia (n = 3) versus normoxia
(n = 3). Int. J. Mol. Sci. 4.6. Statistical Analysis 2022, 23, 3574 17 of 20 17 of 20 Int. J. Mol. Sci. 2022, 23, 3574
17 of 20
Table A2. Differential gene expression comparing THP1KD cells in hypoxia (n = 3) versus normoxia
(n = 3). Gene Symbol
Description
Log2 Fold Change
lfcSE 1
p-Value
p-adj. 2
G6PD
glucose-6-phosphate dehydrogenase
−1.24
0.10
3.15 × 10−37
1.51 × 10−35
SIRT5
sirtuin 5
1.08
0.12
2.92 × 10−19
3.50 × 10−18
TKTL1
transketolase-like 1
0.90
0.12
2.14 × 10−14
1.66 × 10−13
TKT
transketolase
0.24
0.07
5.9 × 10−4
1.5 × 10−3
HK2
hexokinase 2
0.67
0.17
7.82 × 10−5
2.2 × 10−4
HK3
hexokinase 3
2.73
0.17
7.21 × 10−57
1.17 × 10−54
PDK3
pyruvate dehydrogenase kinase 3
−0.61
0.21
3.9 × 10−3
8.9 × 10−3
PDK4
pyruvate dehydrogenase kinase 4
−2.20
0.53
3.53 × 10−5
1.1 × 10−4
GAPDH
glyceraldehyde-3-phosphate
dehydrogenase
0.57
0.11
1.86 × 10−7
7.30 × 10−7
LDHD
lactate dehydrogenase D
1.34
0.14
1.53 × 10−20
2.03 × 10−19
SLC2A3
solute carrier family 2 member 3
1.81
0.32
2.11 × 10−8
9.12 × 10−8
SLC16A3
solute carrier family 16 member 3
0.48
0.12
7.73 × 10−5
2.2 × 10−4
ESRRB
estrogen related receptor beta
–
–
–
–
GLUL
glutamate-ammonia ligase
0.60
0.12
2.07 × 10−7
8.09 × 10−7
GLS
glutaminase
−0.88
0.13
1.67 × 10−11
9.95 × 10−11
SLC1A3
solute carrier family 1 member 3
−0.89
0.32
5.4 × 10−3
1.1 × 10−2
SLC38A2
solute carrier family 38 member 2
−0.38
0.10
2.8 × 10−4
7.1 × 10−4
IDO2
indoleamine 2,3-dioxygenase 2
–
–
–
–
PHGDH
phosphoglycerate dehydrogenase
0.46
0.09
1.10 × 10−7
4.42 × 10−7
SHMT2
serine hydroxymethyltransferase 2
0.37
0.05
1.81 × 10−15
1.53 × 10−14
TPH2
tryptophan hydroxylase 2
6.93
1.40
6.88 × 10−7
2.53 × 10−6
BCAT1
branched chain amino acid
transaminase 1
−2.41
0.14
9.87 × 10−66
2.61 × 10−63
1 lfcSE is the log fold change Standard Error, 2 p-adj. is adjusted p-value. Appendix B
Table A3. Intracellular concentrations of metabolites in THP-1WT and THP-1KD cell lines. Metabolite
THP-1WT
Normoxia
THP-1KD
Normoxia
THP-1WT
Hypoxia
THP-1KD
Hypoxia
p-Value
THP-1WT
(Nor vs. Hyp)
p-Value
THP-1KD
(Nor vs. 4.6. Statistical Analysis Hyp)
Alanine
402.30 ± 143.45
473.60 ± 144.63
346.52 ± 5.56
279.01 ± 79.01
0.5378
0.1103
Arginine
273.40 ± 77.02
272.04 ± 150.85
292.70 ± 144.44
111.34 ± 86.22
0.8482
0.1844
Asparagine
771.52 ± 228.94
563.26 ± 54.48
474.11 ± 39.58
702.79 ± 395.83
0.0909
0.5779
Aspartate
118.19 ± 37.52
136.50 ± 46.37
36.98 ± 10.07
55.27 ± 44.06
0.0223
0.0927
Citrulline
0.24 ± 0.23
3.20 ± 4.66
2.54 ± 2.93
0.52 ± 0.89
0.2464
0.3824
Glutamine
1636.46 ± 205.69
1760.44 ± 510.50
1377.17 ± 295.79
1100.21 ± 204.71
0.2806
0.1061
Glutamate
855.50 ± 1481.77
1089.97 ± 943.94
0.00 ± 0.00
1609.70 ± 571.89
0.3739
0.4605
Glycine
915.60 ± 375.13
1075.90 ± 575.77
583.59 ± 285.89
778.96 ± 222.69
0.1685
0.4516
Histidine
72.78 ± 13.73
46.93 ± 17.33
58.96 ± 8.22
36.64 ± 16.40
0.2093
0.4967
Isoleucine
185.40 ± 38.58
200.03 ± 38.97
210.65 ± 76.16
153.94 ± 44.02
0.6355
0.2461
Leucine
236.18± 55.20
248.53 ± 52.65
249.76 ± 63.02
168.81 ± 66.49
0.7928
0.1789
Lysine
40.25 ± 17.59
53.42 ± 8.72
63.64 ± 23.21
38.99 ± 25.69
0.2368
0.4090
Methionine
71.61 ± 20.62
66.44 ± 25.40
63.10 ± 21.25
41.63 ± 22.51
0.6446
0.2741
Ornithine
22.30 ± 10.41
21.32 ± 9.00
35.94 ± 6.38
29.14 ± 16.43
0.1253
0.5096
Phenylalanine
42.47 ± 7.37
39.58 ± 11.15
59.42 ± 22.21
34.28 ± 9.91
0.2781
0.5709
Proline
705.55 ± 139.47
743.98 ± 116.06
249.71 ± 37.70
191.38 ± 38.52
0.0055
0.0014
Serine
70.15 ± 23.94
129.16 ± 90.81
353.88 ± 116.37
186.89 ± 34.27
0.0144
0.3612
Threonine
173.83 ± 92.88
142.23 ± 28.16
200.71 ± 89.78
111.91 ± 40.27
0.7368
0.3455
Tryptophan
11.02 ± 3.72
12.83 ± 1.75
16.12 ± 6.45
9.69 ± 1.04
0.3014
0.0555
Tyrosine
76.14 ± 26.81
91.19 ± 14.14
89.76 ± 32.79
82.07 ± 19.26
0.6074
0.5448
Valine
54.40 ± 19.08
40.16 ± 5.70
60.33 ± 7.25
51.91 ± 20.82
0.6410
0.3992
Acetylornithine
2.44 ± 2.34
1.18 ± 1.91
3.27 ± 1.82
2.57 ± 3.02
0.6537
0.5368
ADMA
2.17 ± 1.48
1.57 ± 0.75
1.31 ± 0.38
0.74 ± 0.69
0.3849
0.2322
α-AAA
7.09 ± 2.44
6.27 ± 2.80
5.35 ± 5.40
4.62 ± 0.51
0.6387
0.3711
Carnosine
0.84 ± 0.55
0.51 ± 0.19
0.38 ± 0.17
0.21 ± 0.09
0.2395
0.0732
Creatinine
16.70 ± 6.86
15.20 ± 2.31
10.38 ± 2.29
11.00 ± 1.91
0.2047
0.0722
Histamine
0.32 ± 0.29
0.23 ± 0.40
0.00 ± 0.00
0.25 ± 0.44
0.1252
0.9480 Int. 4.6. Statistical Analysis J. Mol. Sci. 2022, 23, 3574 18 of 20 Table A3. Cont. Metabolite
THP-1WT
Normoxia
THP-1KD
Normoxia
THP-1WT
Hypoxia
THP-1KD
Hypoxia
p-Value
THP-1WT
(Nor vs. Hyp)
p-Value
THP-1KD
(Nor vs. Hyp)
Kynurenine
0.38 ± 0.17
0.43 ± 0.09
0.39 ± 0.17
0.45 ± 0.16
0.9685
0.9024
Met-SO
1.88 ± 0.58
1.98 ± 0.95
2.81 ± 1.47
2.79 ± 1.40
0.3630
0.4562
Putrescine
2.96 ± 1.32
3.59 ± 1.84
3.00 ± 0.89
3.22 ± 0.51
0.9657
0.7542
Spermidine
45.35 ± 16.23
36.51 ± 5.33
23.81 ± 9.00
26.50 ± 7.43
0.1148
0.1306
Spermine
20.86 ± 11.70
14.61 ± 2.37
13.77 ± 7.30
9.65 ± 4.51
0.4232
0.1667
T4-OH-Pro
221.31 ± 22.58
202.10 ± 40.27
130.55 ± 4.57
130.59 ± 58.46
0.0024
0.1559
Taurine
469.08 ± 82.46
464.12 ± 361.99
196.01 ± 188.34
219.15 ± 200.75
0.0829
0.3633
Total DMA
0.12 ± 0.12
0.05 ± 0.05
0.12 ± 0.12
0.20 ± 0.16
0.9458
0.2015
The intracellular content of these metabolites was obtained using the Biocrates Absolute IDQTM p180 kit (Biocrates
Life Sciences AG, Austria) after 48 h incubation with RPMI 1640 10 mM Glc and 4 mM Gln, 5% S/P and 1% FBS,
in both normoxia and hypoxia conditions. Data is represented as mean ± SD (n = 3). Table A3. Cont. The intracellular content of these metabolites was obtained using the Biocrates Absolute IDQTM p180 kit (Biocrates
Life Sciences AG, Austria) after 48 h incubation with RPMI 1640 10 mM Glc and 4 mM Gln, 5% S/P and 1% FBS,
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Carbon storage potential and its distributions in the particle size fractions in Harran Plain, Turkey
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ABSTRACT that the particle size distribution in the surface layer varied in the
following order depending on soil weight: 850-250> 2000-850> 250-
150> 150-75> 75 µm. The organic C content of the soils is low due to the
semi-arid climate conditions. Fraction-based soil SOC distribution was in
the following order: 11% at 2000-850 µm, 15% at 850-250 µm, 21% at
250-150 µm, 23% at 150-75 µm and <75 µm 30%. Organic matter
fractions differed according to the particle size distribution and the
applicable soil management system. Stable organic matter content was
significantly related to clay content and greatly influenced by the type of
soil management used. that the particle size distribution in the surface layer varied in the
following order depending on soil weight: 850-250> 2000-850> 250-
150> 150-75> 75 µm. The organic C content of the soils is low due to the
semi-arid climate conditions. Fraction-based soil SOC distribution was in
the following order: 11% at 2000-850 µm, 15% at 850-250 µm, 21% at
250-150 µm, 23% at 150-75 µm and <75 µm 30%. Organic matter
fractions differed according to the particle size distribution and the
applicable soil management system. Stable organic matter content was
significantly related to clay content and greatly influenced by the type of
soil management used. In recent years, there has been increasing international interest in
increasing and sustainably managing soil C stocks to contribute to
combating climate change and support food security. In this context,
determining the C storage capacity of soils and examining the distribution
of soil C based on fractions is of great importance for a better
understanding of C dynamics. The present study investigated the storage
potential of soil organic carbon (SOC), inorganic carbon (SIC) and total
carbon (TC) in 16 selected profiles, and SOC and SIC distribution in five
different particle size fractions (2000-425µm, 425-150 µm, 150-106 µm,
106-75 µm, <75 µm) of the Harran plain in Turkey. The results revealed Keywords: Soil organic carbon, Soil inorganic carbon, Carbon storage, Particle size, Harran plain Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) 2022, 28 (3) : 501 – 510 DOI: 10.15832/ankutbd.907173
Journal of Agricultural Sciences
(Tarim Bilimleri Dergisi)
J Agr Sci-Tarim Bili
e-ISSN: 2148-9297
jas.ankara.edu.tr
Carbon Storage Potential and its Distributions in the Particle Size Fractions in
Harran Plain, Turkey
İbrahim Halil YANARDAĞa*
, Asuman BÜYÜKKILIÇ YANARDAĞa
, Ahmet R. MERMUTb,c
,
Ángel FAZ CANOd
aSoil Science and Plant Nutrition Department, Agriculture Faculty, Malatya Turgut Özal University, Battalgazi, Malatya, TURKEY
bSoil Science Department, Agriculture Faculty, Harran University, Şanlıurfa, TURKEY
cDepartment of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, CANADA
dSustainable Use, Management, and Reclamation of Soil and Water Research Group, Agrarian Science and Technology Department, Technical University of Cartagena,
Cartagena, Murcia, SPAIN Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) 2022, 28 (3) : 501 – 510 DOI: 10.15832/ankutbd.907173
Journal of Agricultural Sciences
(Tarim Bilimleri Dergisi)
J Agr Sci-Tarim Bili
e-ISSN: 2148-9297
jas.ankara.edu.tr
Carbon Storage Potential and its Distributions in the Particle Size Fractions in
Harran Plain, Turkey
İbrahim Halil YANARDAĞa*
, Asuman BÜYÜKKILIÇ YANARDAĞa
, Ahmet R. MERMUTb,c
,
Ángel FAZ CANOd
aSoil Science and Plant Nutrition Department, Agriculture Faculty, Malatya Turgut Özal University, Battalgazi, Malatya, TURKEY
bSoil Science Department, Agriculture Faculty, Harran University, Şanlıurfa, TURKEY
cDepartment of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, CANADA
dSustainable Use, Management, and Reclamation of Soil and Water Research Group, Agrarian Science and Technology Department, Technical University of Cartagena,
Cartagena, Murcia, SPAIN Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) 2022, 28 (3) : 501 – 510 DOI: 10.15832/ankutbd.907173
Journal of Agricultural Sciences
(Tarim Bilimleri Dergisi)
J Agr Sci-Tarim Bili
e-ISSN: 2148-9297
jas.ankara.edu.tr
Carbon Storage Potential and its Distributions in the Particle Size Fractions in
Harran Plain, Turkey
İbrahim Halil YANARDAĞa*
, Asuman BÜYÜKKILIÇ YANARDAĞa
, Ahmet R. MERMUTb,c
,
Ángel FAZ CANOd
aSoil Science and Plant Nutrition Department, Agriculture Faculty, Malatya Turgut Özal University, Battalgazi, Malatya, TURKEY
bSoil Science Department, Agriculture Faculty, Harran University, Şanlıurfa, TURKEY
cDepartment of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, CANADA
dSustainable Use, Management, and Reclamation of Soil and Water Research Group, Agrarian Science and Technology Department, Technical University of Cartagena,
Cartagena, Murcia, SPAIN 2022, 28 (3) : 501 – 510
Journal of Agricultural Sciences
(Tarim Bilimleri Dergisi) Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) DOI: 10.15832/ankutbd.907173 ARTICLE INFO Research Article
Corresponding Author: İbrahim Halil YANARDAĞ, E-mail: ibrahim.yanardag@ozal.edu.tr
Received: 1 April 2021 / Revised: 8 October 2021 / Accepted: 9 October 2021 / Online: 01 Se Research Article
Corresponding Author: İbrahim Halil YANARDAĞ, E-mail: ibrahim.yanardag@ozal.edu.tr Corresponding Author: İbrahim Halil YANARDAĞ, E-mail: ibrahim.yanardag@ozal.edu.tr
Received: 1 April 2021 / Revised: 8 October 2021 / Accepted: 9 October 2021 / Online: 01 September 2022 Cite this article
YANARDAĞ İ H, BÜYÜKKILIÇ YANARDAĞ A, MERMUT A R, FAZ CANO A (2022). Carbon Storage Potential and its Distributions in the Particle Size Fractions in Harran
Plain, Turkey. Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 28(3):501-510. DOI: 10.15832/ankutbd.907173 1. Introduction Soil carbon (C) is the largest terrestrial carbon reserve and contains about 1.5 Eg (ie 1.5 × 1018 g) of carbon, of which 0.68 Eg is
organic carbon (Zhang et al. 2020). Soil organic matter (SOM) positively affects the physical, chemical and biological properties
of the soil (Sakin & Yanardağ 2019). It is also one of the most important components in the soil due to its capacity to affect plant
growth and yield (Bongiovanni & Labartini 2006). In C stabilization processes, the structure of organic compounds in SOM,
their true resistance to weathering and their interactions with the soil mineral fraction are closely related (Marinari et al. 2010). The new origin of SOM is closely related to biological activity in the soil, while new and middle-aged organic matter can
contribute to the improvement of the physical structure of the soil (Wander 2004). However, different soil types may also react
differently to the stabilization and sequestration of C (Yanardağ et al. 2015). Measuring the potential of agricultural soils to soil organic carbon (SOC) dynamics will help evaluate the contribution of
cultivated soils to the global C balance as a carbon source or sink. However, there are many uncertainties about the impact of
SOC dynamics on the soil system (Mermut et al. 2000; Li 2002), and the ultimate potential for C stabilization in soils is unknown
(Smith 2004). The quality of SOM depends on its distribution between unstable and stable organic components. Stable organic
compounds in the soil include humus materials and other macromolecules or are physically preserved by their adsorption on
mineral surfaces or bound in aggregates (Tobiasova et al. 2012). One of the key elements to reliably evaluate SOM dynamics is
the experimental identification of SOM pools associated with stabilization mechanisms (Abdul Kader 2006). Soil organic matter can be analyzed based on different fractions and basic information about the processes and products of
soil formation can be provided by soil particle size distributions (Gunal et al. 2011). Interactions of physical, chemical and
biological processes in the soil affect aggregate formation and stabilization (Kocyigit & Demirci 2010). These materials with a Yanardağ et al. - Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 2022, 28(3): 501-510 particle size of <2 µm, mostly silt and clay, have slow cycle times and stabilize in the primary organo-mineral structure by
interacting mainly with minerals (Chenu & Plante 2006). 1. Introduction Here, the clay-sized OM usually accounts for more than 50% of the
SOM, and the sum of clay and silt (<20 μm) can be over 90% (Christensen 1996). In addition, changes in organic matter levels
caused by land use can be better understood by determining the distribution of fractions (Figueiredo et al. 2010). Dalal & Mayer
(1986) reported that the change of some soil properties depends on which fractions of SOM are accumulated rather than the total
amount of SOM. particle size of <2 µm, mostly silt and clay, have slow cycle times and stabilize in the primary organo-mineral structure by
interacting mainly with minerals (Chenu & Plante 2006). Here, the clay-sized OM usually accounts for more than 50% of the
SOM, and the sum of clay and silt (<20 μm) can be over 90% (Christensen 1996). In addition, changes in organic matter levels
caused by land use can be better understood by determining the distribution of fractions (Figueiredo et al. 2010). Dalal & Mayer
(1986) reported that the change of some soil properties depends on which fractions of SOM are accumulated rather than the total
amount of SOM. The objectives of this study are (1) to determine the soil organic carbon, inorganic carbon, and total carbon storage potential
of the Harran plain in 16 selected profiles, and (2) to determine the SOC and SIC distribution in five different particle size
fractions (2000-425µm, 425-150 µm, 150-106 µm, 106-75 µm, <75 µm). 2. Material and Methods 2. Material and Methods 2.1. Description of the study area 2.1. Description of the study area The soil locations and taxonomies are Kısas 1 (P1) (36°58'00.3"N, 38°55'42.3"E);
Typic Torrert, Çekçek (P2) (36°56'49.9"N, 38°54'05.8"E); Typic Torrifluvent, Harran 1 (P3) (37°00.0'06.2"N, 38°58'57.3"E);
Vertic Camborthid, Kısas 2 (P4) (36°58 '05.6"N, 39°01'09.5"E); Typic Torrert, Bellitaş (P5) (37°01'09.6 "N, 39°09'13.2"E);
Typic Torrifluvent, Gürgelen 1 (P6) (37°00'0.504"N, 39°09'0.632"); Typic Calciorthid, İkizce (P7) (37°01'0.501"N,
39°07'0.299"E); Vertic Torrifluvent, Sırrın (P8) (37°05'0.461"N, 39°02'0.144"E); Vertic Camborthid, İrice (P9) (37°06'0.062"N, 502 Yanardağ et al. - Journal of Agricultural Sciences (Tarım Bilimleri Dergisi), 2021, 28(3):501-510 39°03'0.766"E); Typic Calciorthid, Beğdeş (P10) (36°50'0.491"N, 38°54'0.169"e); Typic Torrert, Harran 2 (P11)
(36°53'0.034"N, 38°57'0.567"E); Vertic Camborthid, Gürgelen 2 (P12) (36°47'0.136"N, 39°05'0.258"E); Typic Calciorthid,
Akören (P13) (36°46'0.353"N, 39°04'0.529"E); Typic Calciorthid, Ekinyazı (P14) (36°43'0.374"N, 39°06'0.811"E); Typic
Calciorthid, Akçakale (P15) (36°45'0.370"N, 39°58'0.538"E); Typic Torrert, Gürgelen 3 (P16) (36°44'0.370"N, 38°52'0.343");
Typic Calciorthid. The soils of the plain are clayey with a slightly alkaline pH (pHH2O 7.5–8.0). The minimum permeability
values of the soils are between 0.22 and 3.51 m day-1 (GDSHW 2003). The majority of soils in the plain are classified as Vertisol
according to Soil Survey Staff (2006). The dominant silicate clay minerals are smectite, and palygorskite, however, illite and
kaolinite are also found in the mixture. The soils are generally well developed with ABC horizons and although lime content is
high, soil organic matter is usually around 1.0% (GDSHW 2003). The selected physical-chemical soil characteristics were given in Table I. The soil textures were generally clayey and the
amount of clay tended to increase towards the center of the study region. The southern sample sites had higher salinity (15.62
dS m-1). Due to excessive and uncontrolled irrigation and fertilization, in addition to poor natural drainage, soil salinity was very
high. The soils were alkaline and the pH was ranged between 7.37 and 8.40 (Table 1). The studied area had high carbonate contents (mean 26.60%). The organic matter concentrations were between 0.87 and
2.12%. The studied area had high amount of clay, which were ranged between 30 and 60%. The dominant silicate clay minerals
were smectite therefore, cation exchange capacities were high and ranged between 28.61 and 48.12 cmol+ kg-1. The bulk density
of 16 studied soil profiles was changed between 1.25 and 1.35 g cm-3 (Table I). 2.2. Soil sampling and analysis 2.2. Soil sampling and analysis Soil samples were taken from 16 selected profiles to determine soil characteristics and especially carbon contents. The samples
were air-dried, sieved to pass a 2-mm mesh, and stored in plastic bags for analysis of selected physical and chemical soil
properties. Soil samples for particle size analysis were taken from each of the surface soils (0-20 cm). The following soil analysis was carried out: pH measured in a 1:1 water soil ratio mixture according to Peech's method
(1965); soluble salts according to Bower & Wilcox (1965) method; CaCO3 by the Bernard calcimeter method (Vatan 1967);
organic carbon according to Duchaufour (1970), cation exchange capacity (CEC) following the method of Chapman (1965). SOC, SIC, and TC in 16 profiles and each particle size fractions were measured by TOC analyzer (TOC – V-CSH Shimadzu
(Kyoto-Japan)). Particle size analysis was carried out by using the FAO-ISRIC system (1990) after the combination of the pipette Robinson
and sieving. The fraction of bulk soils into five particle size fractions was conducted using sieves with the following sizes: 2000,
425, 150, 106, and 75 µm. These fractions were studied in the 20 cm surface soil from the studied area. For the quantification of
soil organic carbon in each fraction, a subset of each particle size fraction was ground and determined according to Duchaufour
(1970). 2.1. Description of the study area Harran Plain is located in the south-eastern part of Şanlıurfa province, Turkey, which is in the center of Turkey's major irrigation
and development project (Southeastern Anatolian Project, GAP). It lies between the longitudes of 38°39'-39°30' E and the
latitudes of 36°43'-37°11' N and spans an area of 225 000 ha. The elevation ranges between 350 and 450 m a.s.l. and it increases from the south to the north. The plain has a semi-arid
climate with limited precipitation between June and September. The long-term mean annual temperature is about 18 °C, the
highest annual mean temperature is 31.4 °C in July, and the lowest annual mean temperature is 5.8 °C in January. The annual
mean relative humidity and precipitation are 57% and 284.2 mm, relatively (Yesilnacar & Güllüoğlu 2007). The dominant crops
in the area are cotton, corn, and wheat. Figure 1- Distribution of 16 selected soil profiles in the studied area
samples were taken from 16 series of genetic horizons in 3 replications in wheat fields in Harran Plain. In the study, soil
differ between 1.4 m and 3.6 m were dug up until reaching the main material. The distribution of 16 selected soil profiles
e studied area is presented in Figure 1. The soil locations and taxonomies are Kısas 1 (P1) (36°58'00.3"N, 38°55'42.3"E);
orrert, Çekçek (P2) (36°56'49.9"N, 38°54'05.8"E); Typic Torrifluvent, Harran 1 (P3) (37°00.0'06.2"N, 38°58'57.3"E);
Camborthid, Kısas 2 (P4) (36°58 '05.6"N, 39°01'09.5"E); Typic Torrert, Bellitaş (P5) (37°01'09.6 "N, 39°09'13.2"E);
Torrifluvent, Gürgelen 1 (P6) (37°00'0.504"N, 39°09'0.632"); Typic Calciorthid, İkizce (P7) (37°01'0.501"N,
299"E); Vertic Torrifluvent Sırrın (P8) (37°05'0 461"N 39°02'0 144"E); Vertic Camborthid İrice (P9) (37°06'0 062"N Figure 1- Distribution of 16 selected soil profiles in the studied area Soil samples were taken from 16 series of genetic horizons in 3 replications in wheat fields in Harran Plain. In the study, soil
profiles differ between 1.4 m and 3.6 m were dug up until reaching the main material. The distribution of 16 selected soil profiles
(P) in the studied area is presented in Figure 1. 2.1. Description of the study area Table 1- Selected physical-chemical characteristics of the soil study Table 1- Selected physical-chemical characteristics of the soil study
SP*
pH
EC
dS m-1
OM
%
CaCO3
%
CEC
cmol+ kg-1
BD
g cm-3
Mean
7.98
2.42
1.41
26.60
38.08
1.31
Std Dev
0.34
4.25
0.37
5.75
5.23
0.03
Min
7.37
0.48
0.87
14.81
28.61
1.25
Max
8.40
15.62
2.12
37.02
48.12
1.35
SP*
Sand
%
Silt
%
Clay
%
Mg++
mg kg-1
K+
mg kg-1
Na+
mg kg-1
Mean
17.06
34.25
48.44
170.8
449.7
480.7
Std Dev
7.08
4.52
7.63
45.2
78.2
151.8
Min
5.00
24.00
30.00
100.1
328.5
303.6
Max
31.00
39.00
60.00
234.3
664.7
943.0
* Statistical Properties of Level EC: Electric conductivity, CaCO3: Lime Content, OM: Organic Matter,
CEC: Cation Exchange Capacity, BD: Bulk Density * Statistical Properties of Level EC: Electric conductivity, CaCO3: Lime Content, OM: Organic Matter,
CEC: Cation Exchange Capacity, BD: Bulk Density 3.1. Organic, Inorganic, and Total carbon storage of the soils Storage of carbon (OC, IC, and TC) in the studied soil profiles were given in Figure 2. The total amount of SOC within soil
profiles varied significantly among the selected areas (P<0.05). The SOC concentration were 7.60 g kg-1 in P1, 7.96 g kg-1 in P2,
11.55 g kg-1 in P3, 10.84 g kg-1 in P4, 8.31 g kg-1 in P5, 13.94 g kg-1 in P6, 9.22 g kg-1 in P7, 12.81 g kg-1 in P8, 12.39 g kg-1 in
P9, 9.65 g kg-1 in P10, 8.45 g kg-1 in P11, 6.76 g kg-1 in P12, 9.93 g kg-1 in P13, 14.93 g kg-1 in P14, 6.13 g kg-1 in P15, and 7.89
g kg-1 in P16, respectively. The SOC contents were low due to low precipitation and high temperature. Microbial activity in soil
is highly affected by soil moisture and soil temperature, which has played an important role in CO2 emissions and 65-85% of
this emission is due to the soil temperature (Büyükkılıç Yanardağ et al. 2004). In addition, the CO2 emission of the soils is very
effective on the SOC and TN reserves. Furthermore, researchers indicated that the variability of soil C depends on soil texture,
climate, vegetation, and land use and management, as well as differences in species and plant density (Yoo et al. 2006). Hontoria
et al. (1999) stated that 45% of the variability in SOC in the Spanish peninsula can be explained by the annual precipitation,
annual average temperature, and altitude parameters. In addition to the climate, the clay content and type of the soil in the region
also affected the organic matter content. Also, the oxidation of organic matter was faster due to the calcareous soil character
(Homann et al. 1995). It means that the presence of CaCO3 in the soil might also contribute to the stabilization of poorly
crystallized Fe forms; this will contribute to SOC retention, likely regarding triple Fe-Ca-SOC complexes (Sowers et al. 2018). The highest SOC content was found in P14 while, lowest in P15. The elevation and climate conditions of these soils were
approximately similar, whereas SOC ratios were varied due to different soil management practices. Soil conditions in turn were
most influenced especially in our region by the temperature and soil moisture regimes, although the mineralogical and biological
regimes were also important (Buringh 1984). Yanardağ et al. - Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 2022, 28(3): 501-510 Yanardağ et al. - Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 2022, 28(3): 501-510 3. Results and Discussion 3. Results and Discussion 3.1. Organic, Inorganic, and Total carbon storage of the soils The inorganic C concentration of the soils ranged; 3.93 in P1, 3.51 in P2, 2.57 in P3, 4.44 in P4, 4.02 in P5, 3.04 in P6, 3.27
in P7, 3.41 in P8, 2.81 in P9, 2.99 in P10, 3.09 in P11, 2.76 in P12, 2.95 in P13, 2.43 in P14, 1.78 in P16, and 4.07% in P16,
respectively. The plain soil had a high carbonate ratio, also pedogenic carbonate was predominant due to alkaline soils features. Khademi & Mermut (1998) stated that if there is sufficient calcium in the soil solution, that is when the pH range is between 7.3
and 8.5, secondary carbonate accumulations start, and calcite is formed in the soil. Therefore, the inorganic carbon concentration
of the soils was high. The vertical distribution of SOC, SIC, TC are given in Figure 2. The results showed that SOC concentrations were decreased
with increasing soil depth. However, SOC levels were distributed along with the whole soil profile. Batjes (1996) observed that
the highest SOC concentration was accumulated in upper soil layers, but a large amount was also stored between 1 and 2-m
depth. Furthermore, the storage of OC in soil depended on the balance between the addition of organic material (freshly dead
plants and animal waste) mixing into the soil and losses of C through decomposition (Sollins et al. 1996). The degradation or
decomposition of the organic matter here depends on its chemical composition and physical relationship with other soil
components (Rovira et al. 2008). Inorganic C concentration of the selected soils was high and increased with soil depth. Primary carbonates dissolve under
arid to semi-humid climatic conditions and combine with the CO2 present in the soil solution and recrystallize and a significant
amount of pedogenic (secondary) carbonates accumulate in the soil. In these soil horizons, carbonate accumulation is generally
observed depending on the characteristics of the parent material and climatic conditions (Gocke et al. 2012). 3.2. Soil particle sizes distribution 2.3. Statistical methods For the statistical analysis, multiple linear tests were performed using analysis of variance (ANOVA) to determine whether there
are any statistically significant differences between the means of the independent groups. Relationships among properties were
studied using Pearson correlations. Soil chemical properties related to carbon content and pools were subjected to principal
components analysis (PCA) to elucidate major variation patterns in terms of C pools. All statistical tests were performed with
SPSS V26.0 and differences were considered significant when P<0.05. 503 3.2. Soil particle sizes distribution The distribution of particle size fractions in the surface soil from study areas was given in Figure 3. The soil fraction distribution
in the size of 2000-850 µm varied between 15.3% and 42.3%, in the size of 850-250 µm varied between 34.6% and 51.5%, in
the size of 250-150 µm varied between 8.8 and 17.2%, in the size of 150-75 µm varied between 7.2 and 17.7%, and <75 µm in
size varied between 1.8 and 13.0%. The results showed that the dominant size fractions were the largest (850-250 and 850-2000
µm). Although the Harran Plain is Tethys Sea of sedimentary origin (Şengör et al. 1988), it can be easily observed that different
land use has affected the soil particle size. Different soil management systems including cultivation and irrigation can modify
soil particle distribution throughout the soil profile (Jaiyeoba 2003). The high content of smectite in the soils and especially the transformation of palygorskite into this mineral enabled the plain
soils to enrich with smectite. Soils dense as smectite are saturated with Ca++ ions (Seyrek et al. 2005). When we evaluated the
clay results (between 30 and 60%), we thought that the reason for having the lowest percentage in <75 µm would be that the
high Ca++ ions in the soil bind the clay particles tightly together. 504 Yanardağ et al. - Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 2022, 28(3): 501-510 Yanardağ et al. - Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 2022, 28(3): 501-510 Yanardağ et al. - Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 2022, 28(3): 501-510
Figure 2- Carbon percentage concentration in 16 profiles (IC: Inorganic Carbon; OC: Organic Carbon; TC: Total Carbon, n=3) Figure 2- Carbon percentage concentration in 16 profiles (IC: Inorganic Carbon; OC: Organic Carbon; TC: Total Carbon, n=3) Figure 3- Distribution of particle size fractions in surface soil horizon
3.3. Soil organic and inorganic carbon distribution in the different particle sizes
Stable organic matter content at a particle size of <50 μm was significantly related to clay content, while coarse organic matter
with a size of >100 μm was greatly affected by soil management systems (Quiroga et al. 1996). 3.2. Soil particle sizes distribution The highest SOC accumulation was observed between 250-150 um fractions. Accoe et al. (2002) reported that SOC concentration was largest in small size fraction in a sandy loam textured soil. Carter
et al. (2003) found that clay plus silt content (lower than 40%) consisted of 60% of total organic carbon in 15 different sandy
loam to heavy clay textured soil. Zhang et al. (2006) reported that the highest organic carbon accumulated in the clay size fraction
in silty textured soil in Loess Plateau, Gansu- China. Chenu et al. (2000) found a significant relationship between organic matter
and particle size fractions in varied humic loamy soils. Chen & Chiu (2003), found a similar relationship in subalpine areas in
central Taiwan. Contrarily, some researchers examined that the SOM level could reflect the intensity of soil use (Puget et al. 1995; Schulten et al. 1993). Figure 4- Distribution of soil organic carbon (% SOC) in the five particle size fractions analyzed in the surface soil
samples
-
0.50
1.00
1.50
2.00
2.50
3.00
3.50
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16
2000-850 µm
850-250 µm
250-150 µm
150-75 µm
75> µm -
0.50
1.00
1.50
2.00
2.50
3.00
3.50
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16 P1 P3 P4 P5 P9 2000-850 µm
850-250 µm
250-150 µm
150-75 µm
75> µm Figure 4- Distribution of soil organic carbon (% SOC) in the five particle size fractions analyzed in the surface soil
samples In general, the quantity and nature of the clay affected the amount of C stabilized in the soil. Clay size fraction contributed
most of the organic matter both for organic carbon and for nitrogen, with considerably smaller contributions from a silt-and sand-
sized fraction (Schmidt & Kögel-Knabber 2002). The clay content of the soil is one of the most important factors affecting SOC
accumulation. If the clay content is high, it may have higher SOC under similar climatic conditions and land use compared to
soils with low clay content. Also, here the climate factor is the main determinant of the decomposition rate and time of C in the
soil, thus directly influencing the amount of SOC (Milne 2008). 3.2. Soil particle sizes distribution Distributions of SOC content in
33.1
23.3
24.6
42.3
21.0
16.9
21.2
26.6
26.7
12.0
16.4
30.2
28.7
15.3
29.1
16.1
43.8
34.6
42.5
36.9
47.5
45.3
40.8
51.5
45.8
42.4
40.4
40.5
43.0
43.5
48.3
40.1
10.3
11.4
12.4
8.8
13.5
15.5
16.5
12.0
11.0
17.1
17.2
11.5
11.5
17.1
11.3
15.5
8.0
17.7
12.0
7.9
11.8
14.5
16.3
8.0
9.2
16.2
15.7
10.1
10.1
14.8
7.2
15.7
4.8
13.0
8.5
4.1
6.1
7.7
5.1
1.8
7.3
12.2
10.3
7.7
6.7
9.4
4.1
12.6
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
2000-850 µm. 850-250 µm. 250-150 µm. 150-75 µm. 75> µm. Profiles
Particle Size (%) 33.1
23.3
24.6
42.3
21.0
16.9
21.2
26.6
26.7
12.0
16.4
30.2
28.7
15.3
29.1
16.1
43.8
34.6
42.5
36.9
47.5
45.3
40.8
51.5
45.8
42.4
40.4
40.5
43.0
43.5
48.3
40.1
10.3
11.4
12.4
8.8
13.5
15.5
16.5
12.0
11.0
17.1
17.2
11.5
11.5
17.1
11.3
15.5
8.0
17.7
12.0
7.9
11.8
14.5
16.3
8.0
9.2
16.2
15.7
10.1
10.1
14.8
7.2
15.7
4.8
13.0
8.5
4.1
6.1
7.7
5.1
1.8
7.3
12.2
10.3
7.7
6.7
9.4
4.1
12.6
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Profiles
Particle Size (%) Figure 3- Distribution of particle size fractions in surface soil horizon
2000-850 µm. 850-250 µm. 250-150 µm. 150-75 µm. 75> µm. Profiles Figure 3- Distribution of particle size fractions in surface soil horizon 3.3. Soil organic and inorganic carbon distribution in the different particle sizes Stable organic matter content at a particle size of <50 μm was significantly related to clay content, while coarse organic matter
with a size of >100 μm was greatly affected by soil management systems (Quiroga et al. 1996). Distributions of SOC content in 3 Yanardağ et al. - Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 2022, 28(3): 501-510 five particle size fractions in the surface horizon were given in Figure 4. Soil organic C content was increased with small size
fractions. The highest SOC accumulation was observed between 250-150 um fractions. five particle size fractions in the surface horizon were given in Figure 4. Soil organic C content was increased with small size
fractions. 3.2. Soil particle sizes distribution The SIC ranged between 1.29% (in P11) and 6.49% (in P16) in the particle sizes of 850-2000 µm, ranged between 0.48% (in
P6) and 5.12% (in P16) in the particle sizes of 250-850 µm, ranged between 0.71% (in P12) and 4.90% (in P4) in the particle
sizes of 150-250 µm, ranged between 0.31% (in P10) and 5.39% (in P4) in the particle sizes of 75-150 µm, and ranged between
0.07% (in P10) and 4.62% (P1) in the particle sizes of <75 µm, respectively (Figure 5). Contrary to the SOC, the SIC results
were more recent and there were no major differences between the results. It can be said here that SIC is more stable than SOC
and is more resistant to weathering. Also, Dong et al. (2017) reported that carbonates have different stabilities in different particle
fractions, and this stability is more intense in clay than in sand and silt (Loeppert & Suarez 1996). 506 Yanardağ et al. - Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 2022, 28(3): 501-510 Figure 5- Distribution of soil inorganic carbon (% SIC) in five particle size fractions of the surface soils
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16
2000-850 µm. 850-250 µm
250-150 µm
150-75 µm
75> µm 0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16
2000-850 µm. 850-250 µm
250-150 µm
150-75 µm
75> µm P5 P9 Figure 5- Distribution of soil inorganic carbon (% SIC) in five particle size fractions of the surface soi As a result of the correlation analysis between SOC and SIC in the 5 different particles fractions analysed on the 16 soil
profiles, it was observed that these two parameters did not have a statistically significant relationship with each other (p > 0.05)
(Table 2). Dong et al. (2017) found a strong positive correlation between the SOC stock and SIC stock in selected 25 sites (3 in
desert land, 9 in shrubland, and 13 in cropland) in soil from the semi-arid region in Yanqi Basin, China. 3.2. Soil particle sizes distribution With the breakdown of
organic matter, there is an increase in the amount of CO2 in the environment, where CO2 combines with H2O to form HCO3- and
in the next phase, HCO3- combines with Ca++ ions found in large calcareous soils to form CaCO3 (Wang et al. 2015). Therefore,
a positive relationship is expected between SOC and SIC. However, if the organic matter of these soils is very limited, this
relationship is likely not to be significant. Table 2- The correlation analysis between SOC and SIC in the 5 different soil particles fractions in 16 soil profiles
SIC
SOC
Particle size
850-250
µm
250-150
µm
150-75
µm
<75 µm 2000-850
µm
850-250
µm
250-150
µm
150-75
µm
<75 µm
SIC
2000-850 µm
0.836**
0.718**
0.441
0.429
-0.179
-0.319
-0.013
-0.215
-0.385
850-250 µm
0.778**
0.341
0.419
-0.039
-0.238
-0.031
-0.260
-0.298
250-150 µm
0.796**
0.734**
0.109
-0.087
0.175
-0.314
-0.323
150-75 µm
0.822**
0.197
0.006
0.062
-0.330
-0.303
<75 µm
-0.038
-0.175
0.020
-0.217
-0.142
SOC
2000-850 µm
0.771**
0.304
-0.101
-0.082
850-250 µm
0.632**
0.301
0.143
250-150 µm
0.450
0.456
150-75 µm
0.714**
**, Significant at P<0.01. correlation analysis between SOC and SIC in the 5 different soil particles fractions in 16 soil profiles Table 2- The correlation analysis between SOC and SIC in the 5 different soil particles fractions in 1 3 Yanardağ et al. - Journal of Agricultural Sciences (Tarim Bilimleri Dergisi), 2022, 28(3): 501-510 In addition, a similarity was observed in the relationship between SIC and SOC fraction distributions. Accordingly, when the
fractions were close to each other, the significance increased, and no relationship was observed between the largest and smallest
fractions. It can be said that this result is expected. Because it is quite common for particle sizes to show similar distribution
between fractions. In addition, a similarity was observed in the relationship between SIC and SOC fraction distributions. Accordingly, when the
fractions were close to each other, the significance increased, and no relationship was observed between the largest and smallest
fractions. It can be said that this result is expected. Because it is quite common for particle sizes to show similar distribution
between fractions. Accoe F, Boeckx P, Van Cleemput O, Hofman, G X, Hui H Bin & Guanxiong C (2002). Characterization of soil Organic Matter Fraction from
Grassland and Cultivated Soils via C Content and δ13C Signature. Rapid Commun Mass Spectrom 16: 2157-2164.
https://doi.org/10.1002/rcm.827
Batjes N H (1996). Total carbon and nitrogen in the soils of the world. European Journal of Soil Science 47: 151-163.
https://doi.org/10.1111/j.1365-2389.1996.tb01386.x 3.2. Soil particle sizes distribution In Axis 1, SOC, IC, fraction 2000-850 µm and fraction 850-250 µm showed a
positive distribution, whereas pH and CEC showed a negative distribution. In Axis 2, the 250-150 µm fraction and CaCO3
showed a negative distribution, no remarkable distribution was observed in other parameters. According to the PCA analysis
results, it showed a distribution of 19.35% on axis 1 and 18.89% on axis 2 between inorganic C fractions and some soil properties
in 16 profiles. Similar to the SOC fraction in Axis 1, SOC, IC, fraction 2000-850 µm and fraction 850-250 µm showed a positive
distribution, whereas pH and CEC showed a negative distribution. In Axis 2, all IC fractions, IC, and CaCO3 showed a positive
distribution, a slightly negative distribution was observed for pH, SOC, and Clay. 4. Conclusions Semiarid climate conditions affected organic matter with the high temperature and deficiency of organic material affected the
accumulation of organic carbon in the soil. Despite the high clay content of the soil, usually low organic matter and high lime
contents depended on the structure was not develop the well. Particle-size distribution of soils was very important to understand
their properties especially in terms of soil classification. Also, the features of clay minerals were another important factor for the
distribution of particle sizes. In addition to soil, cultivation was of other importance for the accumulation of organic matter. The
particle size distribution of surface soil samples was founded 850-250 > 2000-850 > 250-150 > 150-75 > 75 µm. respectively. Distribution of SOC in particle size fractions were respectively 11% in 2000-850 µm., 15% in 850-250 µm., 21% in 250-150
µm., 23% in 150-75 µm. and 30% in <75 µm. The particle size distribution influenced t h e quantity as well as the quality
of soil organic matter. The high quantity of SOC accumulated in the below of 75 μm fraction and it was concluded that the
most important fraction in storing SOC was clay, followed by silt. When the vertical distribution of SOC accumulation was
evaluated, it was found that SOC did not differ significantly between soil depths in any of the 16 evaluated soils, but increased
strongly in the surface soil layer due to root density. However, IC distribution among soil layers varied, possibly due to different
land uses. This study demonstrated that there was an inverse relation between SOC and particle size in Vertisol soils. Organic
matter fractions differed according to particle size distribution and the prevailing soil management system. Stable organic matter
content was significantly related to clay content and was strongly influenced by the type of soil management utilized. 3.2. Soil particle sizes distribution Figure 6- Biplot of principal component analysis (PCA) performed with SOC fractions (a) SIC fractions (b) and soil
properties in 16 surface soil layers
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16
CEC
SOC
pH
IC
Clay
CaCO3
150-75
<75
250-…
2000-850
850-250
-3.0
-2.0
-1.0
0.0
1.0
2.0
3.0
-2.0
-1.0
0.0
1.0
2.0
Axis 2 (14.66%)
Axis 1 (19.43%)
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16
SOC
pH
CEC
IC
150-75
250-150
<75
2000-850
850-…
CaCO3
Clay
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
-2.0
-1.0
0.0
1.0
2.0
Axis 2 (18.89%)
Axis 1 (19.35%)
a. b. P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16
SOC
pH
CEC
IC
150-75
250-150
<75
2000-850
850-…
CaCO3
Clay
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
-2.0
-1.0
0.0
1.0
2.0
Axis 2 (18.89%)
Axis 1 (19.35%)
b. Figure 6- Biplot of principal component analysis (PCA) performed with SOC fractions (a) SIC fractions (b) and soil
properties in 16 surface soil layers The results of PCA analysis showed a distribution of 19.43% on axis 1 and 14.66% on axis 2 between SOC fractions and
some soil properties in 16 profiles (Figure 6). In Axis 1, SOC, IC, fraction 2000-850 µm and fraction 850-250 µm showed a
positive distribution, whereas pH and CEC showed a negative distribution. In Axis 2, the 250-150 µm fraction and CaCO3
showed a negative distribution, no remarkable distribution was observed in other parameters. According to the PCA analysis
results, it showed a distribution of 19.35% on axis 1 and 18.89% on axis 2 between inorganic C fractions and some soil properties
in 16 profiles. Similar to the SOC fraction in Axis 1, SOC, IC, fraction 2000-850 µm and fraction 850-250 µm showed a positive
distribution, whereas pH and CEC showed a negative distribution. In Axis 2, all IC fractions, IC, and CaCO3 showed a positive
distribution, a slightly negative distribution was observed for pH, SOC, and Clay. The results of PCA analysis showed a distribution of 19.43% on axis 1 and 14.66% on axis 2 between SOC fractions and
some soil properties in 16 profiles (Figure 6). References https://doi.org/10.1016/j.soilbio.2007.07.021 p
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g
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ermogravimetry and scanning calorimetry. Soil Biol Biochem 40: 172–185. https://doi.org/10.1016/j.soilbio.2007.07.0 Schmidt M W I & Kögel-Knabber I (2002). Organic Matter in Particle Size Fraction from A and B Horizons of a Haplic Alfisol. European
Journal of Soil Science 53: 383-391. https://doi.org/10.1046/j.1365-2389.2002.00460.x Schulten H R, Leinweber P & Sorge C (1993). Composition of organic matter in particle-size fractions of an agricultural soil. Journal of Soil
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biological C cycle. Geoderma 130: 47–65. https://doi.org/10.1016/j.geoderma.2005.01.008 Yoo K, Amundson R, Heimsath A M & Dietrich W E (2006). Spatial patterns of soil organic carbon on hillslopes: integrating geomorphic
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Zhang J H, Quine T A, Ni S J & Ge F L (2006). Stocks and dynamics of SOC in relation to soil redistribution by water and tillage erosion. © 2022 by the author(s). Published by Ankara University, Faculty of Agriculture, Ankara, Turkey. This is an
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and reproduction in any medium, provided the original work is properly cited. 510
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The off-line effect of affective touch on multisensory integration and tactile perceptual accuracy during the somatic signal detection task
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PLOS ONE
RESEARCH ARTICLE
The off-line effect of affective touch on
multisensory integration and tactile
perceptual accuracy during the somatic signal
detection task
Sofia Sacchetti ID*, Francis McGlone, Valentina Cazzato ID‡, Laura Mirams‡
School of Psychology, Liverpool John Moores University, Liverpool, United Kingdom
‡ VC and LM have shared the senior authorship on this work.
* S.V.Sacchetti@2017.ljmu.ac.uk
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Sacchetti S, McGlone F, Cazzato V,
Mirams L (2021) The off-line effect of affective
touch on multisensory integration and tactile
perceptual accuracy during the somatic signal
detection task. PLoS ONE 16(12): e0261060.
https://doi.org/10.1371/journal.pone.0261060
Editor: Marcello Costantini, University G
d’Annunzio, ITALY
Received: February 1, 2021
Accepted: November 23, 2021
Published: December 31, 2021
Copyright: © 2021 Sacchetti et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Abstract
Affective touch refers to the emotional and motivational facets of tactile sensation and has
been linked to the activation of a specialised system of mechanosensory afferents (the CT
system), that respond optimally to slow caress-like touch. Affective touch has been shown
to play an important role in the building of the bodily self: the multisensory integrated global
awareness of one’s own body. Here we investigated the effects of affective touch on subsequent tactile awareness and multisensory integration using the Somatic Signal Detection
Task (SSDT). During the SSDT, participants were required to detect near-threshold tactile
stimulation on their cheek, in the presence/absence of a concomitant light. Participants
repeated the SSDT twice, before and after receiving a touch manipulation. Participants
were divided into two groups: one received affective touch (CT optimal; n = 32), and the second received non-affective touch (non-CT optimal; n = 34). Levels of arousal (skin conductance levels, SCLs) and mood changes after the touch manipulation were also measured.
Affective touch led to an increase in tactile accuracy, as indicated by less false reports of
touch and a trend towards higher tactile sensitivity during the subsequent SSDT. Conversely, non-affective touch was found to induce a partial decrease in the correct detection
of touch possibly due to a desensitization of skin mechanoreceptors. Both affective and
non-affective touch induced a more positive mood and higher SCLs in participants. The
increase in SCLs was greater after affective touch. We conclude that receiving affective
touch enhances the sense of bodily self therefore increasing perceptual accuracy and
awareness. Higher SCLs are suggested to be a possible mediator linking affective touch to
a greater tactile accuracy. Clinical implications are discussed.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This work was supported by an Early
Career Researcher PhD Studentship bursary from
Liverpool John Moores University.
1 Introduction
Competing interests: The authors declare that they
have no competing interests exist.
Affective touch, or tactile processing with a hedonic or emotional component [1], is understood to be coded by specialised, unmyelinated mechanosensitive slow-conducting, peripheral
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Affective touch and perceptual accuracy in a multisensory context
nerves, known as C-tactile afferents (CT-afferents; [2, 3]). CT-afferents are mainly found in
the hairy skin of the body [4, 5, but see [6] for recent evidence of CT in glabrous skin] and
respond optimally to gentle stroking touch (1–10 cm/s stroking velocity; [7]. Specifically,
responses of CT-afferents during single unit recordings with microneurography in response to
different stroking velocities show that their spike discharge follows an inverted U-shaped pattern, with the greatest response at ~3 cm/s, and weaker responses at slower (0.1 cm/s) and
faster velocities (30 cm/s; [7, 8]). Importantly, activation of CT-afferents correlates with the
subjective rating of pleasantness, indicating that CT-afferents may constitute the peripheral
physiological substrate for pleasant tactile information [7, 9, 10].
Because of their role in contributing to the hedonic value of social physical interactions,
CT-afferents have been proposed to play a pivotal part in fostering affiliative behaviours and
proximity seeking. Accordingly, different streams of research converging in the so called
‘Social Touch Hypothesis’ have recently underlined the importance of CT-afferents in promoting social bonding and attachment [1, 11]. Supporting this hypothesis, it has been shown that
CT-afferents respond optimally to touch delivered at skin temperature [8]. Moreover, when
asked to stroke babies or their partners, people spontaneously deliver touch at CT optimal
velocities [12].
CT-afferents ascend via spinothalamic pathways to the posterior insula, a limbic area that is
understood to support an early convergence of affective and sensory signals from the body [2,
13, 14]. In turn, remapping of information from the posterior to the anterior insula is thought
to allow the integration of sensory signals with other bodily information as well as with other
cognitive and social factors, ultimately serving body awareness and body self-consciousness
[15, 16]. Indeed, this ascending neural path is hypothesised to be involved in the construction
and maintenance of the so called ‘bodily self’, that is defined as a global, multimodal awareness
of one’s own body, arising from the integration of information coming from different sensory
modalities [17, 18].
Following this line of reasoning, researchers have investigated the role of affective touch in
tasks assessing the integration of competing multisensory information such as the Rubber
Hand Illusion (RHI; [19–22]). During the RHI, an experimenter strokes a visible rubber hand,
that is placed in front of the participant, simultaneously to the participant’s own hand, that
conversely is hidden from sight. The integration of contrasting visuo-tactile information
induces many participants to perceive the rubber hand as their own (embodiment) and to
mis-locate the position of their own hand as closer to the rubber hand (proprioceptive drift).
Of relevance here is the finding that the illusion is strongest when the stroking touch is applied
at CT-afferent preferred velocities (1–10 cm/s) vs non-affective touch stimuli, in terms of a
higher embodiment (the misperception of the rubber hand as participants’ own hand [20, 22])
or in terms of a stronger proprioceptive drift (the mis-location of participants’ hand as closer
to the rubber hand; [21]). The authors concluded that affective touch may have a unique contribution to the sense of body ownership as assessed using the RHI.
Similar results were found by Panagiotopoulou and colleagues [23] who investigated the
effects of administering touch at CT optimal vs non-CT optimal velocities during the ‘enfacement illusion paradigm’. During this paradigm, participants are stroked on the cheek (a body
site densely innervated by CT-afferents) whilst watching a video of another person’s face being
stroked on the specular cheek. Subsequently, they are shown a video of the other person’s face
gradually morphing with a photograph of their own face, and they are asked to say at what
point the face looks more like them, than the other person. In agreement with previous
research using the RHI, affective touch enhanced subjective self-face recognition during this
task as indicated by participants’ self-reported experience of the illusion.
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Affective touch and perceptual accuracy in a multisensory context
Taken together results of these studies suggest that in a multisensory body-awareness context, affective touch is perceived as more meaningful compared to a non-affective touch [3],
leading participants to anchor their sense of bodily self to a greater extent to the affective tactile
information mediated by CT-afferents, rather than other sensory information. This means
that affective touch is potentially more relevant than discriminative touch information in
building a global, multimodal perceptive model of one’s own body (bodily self). During multisensory integration tasks, such as the RHI and the enfacement illusion, this translates with
affective touch enhancing the experience of body ownership [20, 23, 24].
Further support for the role of CT-afferents in generating the sense of bodily self comes
from a positron emission tomography study (PET) which found that when contrasting affective touch on the forearm (high-CT innervated hairy skin) with affective touch on the palm
(low-CT innervated glabrous skin) not only was there the previously reported activations in
dorsal posterior insular cortex and mid-anterior orbitofrontal cortex, but there was also activation in the angular gyrus in the parietal cortex [3]. The angular gyrus has been shown by
Blanke and colleagues [25] to trigger repeated out-of-body experiences when electrically stimulated in a study with an epilepsy patient. The activation in this area to CT-directed touch during the PET study poses an intriguing question as to the role of this area in coding for the
sense of a bodily self.
However, in the studies mentioned above analysing the effects of affective touch on multisensory integration (i.e., the synthesis of sensory information from two or more sensory
modalities to the building of a unitary body perception [26]), affective touch was an on-line
active component of the tasks that participants were asked to perform, in the sense that affective touch was one of the concomitant and competing sensory information that participants
were asked to elaborate and integrate in order to perform the task. Therefore, it was not possible to disentangle whether affective touch induced subsequent off-line alterations in multisensory integration. In a similar direction, a previous study by von Mohr and colleagues [27]
indicated that affective touch can induce an emotional off-line effect in the form of a subsequent reduction in feelings of social exclusion. However, no studies to date have analysed
whether a similar effect of affective touch can impact also subsequent multisensory integration.
Information on this topic would be crucial in determining the potential for affective touch to
be used as an intervention in clinical populations presenting with aberrant body perception
and multisensory integration (i.e., people with eating disorders, body dysmorphic disorders,
and medically unexplained symptoms).
In the current study, we investigated the effects of CT optimal (affective) vs CT non-optimal
(non-affective) touch on subsequent multisensory integration using the Somatic Signal Detection Task (SSDT). The SSDT is a paradigm for the assessment of tactile perception and visuotactile multisensory integration. The SSDT involves detecting near-threshold vibrotactile
pulses that occur on 50% of experimental trials, with and without a simultaneous spatially
aligned flashing LED [28–30]. During this task, the presence of the light (LED) increases
reports of feeling the touch when the tactile pulse is administered (as indicated by a significant
increase in hit rates between light and no light trials). Alongside, the presence of the light also
induces participants to erroneously report perceiving the touch when the tactile pulse was not
administered (as indicated by an increase in false alarms between light and no light trials [28]).
This effect of the light has been found to be a robust phenomenon consistent over time and it
is therefore thought not to be influenced by learning mechanisms (e.g., learned association
between the light and touch [31]).
In comparison to the RHI, and other paradigms assessing multisensory integration, the
SSDT takes advantage of the use of Signal Detection Theory, which provides a more comprehensive description of participants responses, with separate measures of sensitivity (d0 , i.e., the
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Affective touch and perceptual accuracy in a multisensory context
ability to correctly discriminate whether the tactile pulse was absent or present) and response
criterion (i.e., the propensity to report feeling the tactile pulse regardless of the type of trial;
[32]). Furthermore, it has been argued that participants’ responses during the RHI can be
driven by suggestibility (demand characteristics), since during the illusion participants are
aware of a distortion in their experience of an existing touch [33, 34]. Conversely, during the
SSDT, participants are unaware of whether or not their experience of touch is accurate or distorted, and so this paradigm may be less susceptible to suggestibility and therefore be a more
objective measure of tactile distortions due to multisensory integration. Moreover, it should be
noted that the RHI aims to assess body ownership through the manipulation of multisensory
information. Therefore, the RHI does not measure multisensory integration per se but rather
it constitutes an indirect/implicit measure of multisensory processes, whereas the SSDT is a
direct/explicit measure of multisensory integration.
As opposed to previous research in which CT optimal vs non-CT optimal touch was administered online as part of a multisensory task [20, 23], in the current study, the SSDT was performed offline: before and after receiving the touch. Indeed, previous research using the SSDT
paradigm [35] has shown that offline interventions can have a carry-over effect to subsequent
performance on the task. Specifically, focusing on interoceptive sensations (i.e., inner body signals) during a heart-beat perception task was found to increase participants’ reports of touch
in the presence and absence of the target tactile pulse; conversely, focusing on external touch
during a grating orientation task was found to decrease participants reports of touch [35].
Building on these results, in the current study, we investigated whether affective touch can also
induce an offline carry-over effect on subsequent perceptual processes during multisensory
integration measured by the SSDT.
Participants completed the SSDT twice before and after receiving either CT optimal vs
non-CT optimal touch. Throughout the experiment, Skin Conductance Levels (SCLs) were
also recorded to investigate whether the touch manipulation would induce changes in autonomic physiological arousal, and whether these changes would relate to SSDT responses. Furthermore, before and after receiving the touch manipulation participants were administered
with self-report measures of mood (with a focus on Anxiety, Calmness, Happiness, Sadness).
Due to the functional role of CT-afferents in multisensory perception and body awareness,
the main hypothesis of this study was that CT optimal touch would lead to a more accurate
perception during the subsequent SSDT. More precisely, we predicted that affective touch
would enhance perceptual awareness on the body site stimulated to a greater extent than nonaffective touch. In turn, we expected this enhancement in perceptual awareness to enhance
touch perception in that body site, leading participants to be better able to discriminate when
the tactile pulse was administered and when it was not (higher sensitivity, d’). According to
previous research that showed a unique link between affective touch and body awareness, we
expected this effect to be specific for CT optimal touch as opposed to non-CT optimal touch
[23, 24].
However, differently from previous research that assessed the effects of affective touch during the RHI, we expect CT optimal touch to enhance perceptual, tactile accuracy rather than
amplify perceptual distortions. Indeed, during the RHI tactile information contributes to eliciting the perceptual illusion. Therefore, an enhancement in the salience of the tactile information (affective touch; i.e., CT optimal touch) determined a stronger perceptual distortion [20–
22]. Conversely, during the SSDT, the presence of the touch (vibrotactile pulse) does not play a
role in eliciting the illusion but rather it represents the genuine information required to detect
in the absence of the perceptual illusion. Therefore, due to paradigm differences, in the current
study we predicted affective touch to enhance perceptual accuracy rather than increase perceptual distortions. Moreover, as affective touch should enhance multisensory integration
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processes, we expected this manipulation also to increase the effect of the light on touch
reports during the SSDT. Specifically, we expected participants receiving affective touch to be
more accurate in detecting the tactile pulse during the SSDT (d’) especially in trials during
which the light was present. Affective touch can be considered an interoceptive modality.
However, hypotheses of this study are not built upon the fact that affective touch is mainly
interoceptive vs exteroceptive but rather upon the fact that CT-afferents converge in a neural
network responsible for body awareness and multisensory integration processes. For the purposes of this study, therefore, the nature of affective touch as an interoceptive vs exteroceptive
modality has not been discussed.
Moreover, in line with previous studies that have investigated the effects of CT optimal
touch using implicit measures of emotional valence (e.g., facial electromyography and physiological responses of skin conductance and heart rate variability; [36–38], we also hypothesized
that CT optimal touch would increase positive mood (happiness and calmness) and decrease
negative mood (anxiety and sadness).
2. Materials and methods
2.1 Design
Participants were tested in a single study session. All participants performed the SSDT at the
beginning of the testing session, they then received a touch manipulation, and subsequently
performed the SSDT a second time. Participants were randomly assigned to one of two possible touch manipulations: one group received CT optimal touch (3cm/s; n = 32), and the second group received non-CT optimal touch (30cm/s; n = 34). Therefore, the study employed a
2×2×2 mixed designed with group (CT optimal touch vs non-CT optimal touch) as a between
subject variable, Light (Light vs No Light) and Time (Pre vs Post-touch) during the SSDT as
within-subject variables, and hit rate (HR), false alarm rate (FA), d’ and c as dependent
variables.
2.2 Participants
Sixty-six women between 19 and 60 years of age (M age = 32.67, SD = 15.42) were recruited
from the staff and student population at Liverpool John Moores University (LJMU) and from
the general population via advertisements placed around the university campus and on social
media. The sample size was based on a power analysis using G� Power 3.1.9.7 [39], which indicated that overall a minimum sample of n = 56 was needed to detect a medium effect (f = .20)
with 95% power, using a mixed design ANOVA (“number of groups” = 2 × “number of measurements” = 4) with alpha at .05 (two tailed). The sample size was expanded to 66 participants
to increase statistical power (CT optimal group: n = 32; non-CT optimal group: n = 34).
Any individual who self-reported to have been previously clinically diagnosed with, or
treated for body dysmorphic disorder (BDD) or eating disorders (EDs) were excluded from
the study, as well as individuals who self-reported a history of, or any current neurological
and/or psychiatric disorder. Further exclusion criteria included uncorrectable visual impairments, tactile impairments, skin conditions and pregnancy. To avoid any effect of gender differences on affective touch responses [40, 41], and consistent with previous studies by our
research group [29, 30], we recruited only participants who self-identified as women. All participants but two were right-handed as assessed using the Edinburgh Handedness Inventory
(EHI; [42]).
According to the Helsinki declaration of ethical standards, the study was approved by the
LJMU’s Research Ethics Committee. All participants gave their informed consent to take part,
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and they were compensated for their time with a £5 shopping voucher or “participation
points” for course credit for BSc Psychology students.
2.3 Material and measures
2.3.1 The Somatic Signal Detection Task (SSDT)–face version [28, 29]. Participants sat
in a light attenuated room approximately 40cm in front of a computer monitor (5:4 ratio;
270mm × 330mm). A tactor delivering vibrations (Z-Voom phones type YVE-01B-03, Yeil
Electronics, South Korea; 1.8cm diameter) was fixed to participants’ left cheeks using double
sided adhesive tape and a bandage tape to prevent movements. Tactile stimuli (20ms, 100Hz
vibrations) were produced by sending amplified sound files (.wav files, sine wave), controlled
by E-Prime software (Psychology Software Tools Inc., Pittsburgh, PA, USA), to the tactor.
A mirror-reversed photograph of the participant’s face (768 × 583 pixels in size) was presented on the computer screen during all trials of the SSDT. Instructions for participants were
presented on the top section of the computer screen in order not to hinder vision of the photograph. During the experimental phase of the SSDT, a 4mm red light emitting diode (LED) was
fixed to the computer monitor mirroring the location of the tactor on the participant’s face
(see Fig 1). The LED was therefore placed on the right cheek of the face depicted on the
monitor.
Gaze direction, and distance from the computer monitor during the task was controlled
using a chin-rest that discouraged participants from moving their head. Throughout the
Fig 1. Schematic depiction of the experimental set-up during the SSDT. The experimenter (left) delivered the touch
manipulation to the participant (right) between the first (Pre) and the second (Post) repetition of the SSDT.
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experiment, participants listened to white noise via headphones to mask any informative
sounds from the tactor. Underneath the headphones, participants wore a second pair of small
earphones which administered auditory cues for signalling the beginning of each trial of the
SSDT.
2.3.2 Touch manipulation. During the touch manipulation, tactile stimulation (brush
strokes) were delivered from a female experimenter using a cosmetic brush (No7 cosmetic
brush, Boots UK). Prior to testing, the experimenter trained on a high precision scale to deliver
strokes at a constant pressure of 220 mN (22gr/cm2; [43–45]). During the touch manipulation,
participants were instructed to look at a blank screen presented on the computer monitor in
front of them. As during the SSDT, movements of the head were prevented by the use of a
chin-rest. The CT optimal group received strokes at the velocity of 3cm/s, and the non-CT
optimal group at the velocity of 30cm/s. A visual metronome was presented on a computer
screen behind the participant to guide the experimenter in delivering the strokes at the correct
velocity (3cm/s or 30cm/s; [38, 46]). The metronome used a custom made PsychoPy script
[47], showing a 3s countdown followed by a 9cm rectangle filling at the required stroking
velocity (3s for the 3cm/s touch, and 0.3s for the 30cm/s touch).
The touch manipulation consisted of 4 blocks of 4 trials each, with each trial corresponding
to a 6s window during which participants were stroked back and forth on a 9cm segment on
their left cheek. Therefore, for the CT-optimal group (3cm/s) each trial consisted of 2 consecutive strokes administered, while for the non-CT optimal (30cm/s) each trial consisted of 20
strokes.
After each of the 4 blocks, participants were asked to rate the Pleasantness and Intensity of
the stimulation on a 15cm Visual Analogue Scale (VAS) ranging from -10 (unpleasant) to +10
(pleasant), and from 0 (least intense) to 100 (most intense). Pleasantness and Intensity VASs
were used as a manipulation check to ensure that CT optimal touch was perceived as more
pleasant and less intense compared to non-CT optimal touch in accordance with previous
research [e.g., 7, 8, 48].
Moreover, prior to, and after receiving the touch manipulation, participants were asked to
report on four 15cm VASs how calm, anxious, happy, and sad they felt from a minimum of 0
(not at all) to a maximum of 100 (very much so). Mood VASs (Calmness, Anxiety, Happiness,
Sadness) to analyse the influence of the touch manipulation on participants’ emotional state.
VASs for psychosocial measurements have been shown to be a valid and reliable instrument
[49]. In the current study, VASs were preferred to other available measures of mood because
they are easy to use, they have been shown to have a better responsiveness (i.e., ability to detect
significant changes), and validity (i.e., the ability to assess more closely the actual experience of
participants) as compared to other instruments such as Likert scales [49–51]. The experimental
set-up is illustrated in Fig 1.
2.3.3 Physiological arousal. Electrodermal Activity (EDA) signals (i.e., the electrical
activity of the skin resulting from changes in sweating) were recorded using the Biopac MP150
Systems (Version 4.2, Biopac Systems Inc., CA, USA), in three time windows: during the first
repetition of the SSDT (Pre), during the touch manipulation, and during the second repetition
of the SSDT (Post). The three recordings were interspersed with 90s breaks. Two electrodes
were placed on the index and the middle finger of the left hand. The electrodes were connected
to the Biopac MP150 Systems and the Biopac Student Lab Pro 3.7 software, which was programmed to filter in real time EDA data with a band-pass of 0–35Hz. The sampling rate for
data acquisition was set at 1000Hz.
EDA data were then used for calculating Skin Conductance Levels (SCLs), a measure of the
background tonic EDA that is commonly used as an index of psychological arousal. Particularly, SCLs are deemed to reflect slow changes in the autonomic sympathetic nervous system
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(SNS) in response to stressors or emotional stimuli [52]. Higher SCLs indicate a stronger activation of the SNS and therefore a higher level of arousal [36]. In the current study, SCLs were
preferred over cardiac measures of physiological arousal, in accordance with previous research
that suggested EDA to be a better index for detecting arousal changes when investigating
responses that are predominantly of lower arousal [36, 52]. However, tactile stimuli have been
previously reported to affect also other autonomic and cardiac measures (e.g., [53–55]). SCLs
were recorded to investigate changes in arousal due to the touch manipulation, and to analyse
possible influences of arousal levels on subsequent SSDT responses.
2.4 Self-report questionnaires
Self-report measures were used to control the sample for possible confounding variables that
have been shown to impact touch perception during the SSDT, that are eating disorder symptoms, body dysmorphic disorder symptoms and individual differences in body awareness [29,
56, 57]. Pre-existing abnormalities and between groups differences in these variables could
indeed bias interpretation of results. Scores were compared to normative data to ensure comparability with the general population, and were compared statistically between the two groups
to ensure homogeneity of participants.
2.4.1 Eating Disorder Inventory-3 (EDI-3; [58]). The EDI-3 is 91-item self-report questionnaire used to assess disordered eating symptomatology. Participants are asked to rate how
frequently they do what is reported in each item on a 6-point Likert scale ranging from
“never” to “always”. Example items are: “I eat when I’m upset”, “I stuff myself with food”, “I
think about dieting” and “I think my hips are too big”. The EDI-3 comprises 12 subscales
assessing different domains defining, and/or associated with EDs: Drive for Thinness, Bulimia,
Body Dissatisfaction, Low Self-Esteem, Personal Alienation, Interpersonal Insecurity, Interpersonal Alienation, Interoceptive Deficits, Emotional Dysregulation, Perfectionism, Asceticism, and Maturity Fears. In the current study, we focused on the ED Risk Composite which
constitutes an index of the risk to develop an ED and correspond to the sum of scores on the
Drive for Thinness, Bulimia and Body Dissatisfaction subscales. Clinical samples have been
found to score a mean value of 61 on the ED Risk Composite (between a minimum of 51 and a
maximum of 75; [59]). The EDI-3 has been found to have an excellent specificity and sensitivity, and a good internal consistency (α = between .75 and .92; [59]).
2.4.2 Dysmorphic Concerns Questionnaire (DCQ; [60]). The DCQ is a 7-item selfreport scale investigating body image preoccupation and dysmorphic concerns. It includes
items such as: “Have you ever been very concerned about some aspect of your appearance?”
and “Have you ever spent a lot of time worrying about a defect in your appearance or bodily
functioning?”. Items are rated on 4-point Likert scale from a minimum of 0 (“not at all”) to a
maximum of 4 (“much more than most people”). Previous research indicated that total scores
above 9 are indicative of clinical concern [61]. The DCQ was shown to have a good internal
consistency with α = .80 [62].
2.4.3 Body Perception Questionnaire-Very Short Form (BPQ-VSF; [63]). The BPQ is a
12-item self-report questionnaire of body awareness and autonomic reactivity. Participants are
asked to state how often they consider themselves to be aware of different bodily signals such
as: “muscle tension”, “goose bumps”, “digestive problems”, and “heart-beating”. Items are
rated on a 5-point Likert scale ranging from 1 (“Never”) to 5 (“Always”). Total scores range
between 12 and 60, with higher values reflecting a hypersensitivity to bodily sensations, lower
values a hyposensitivity, and average scores a normal sensitivity. The BPQ has been shown to
have an excellent test-retest reliability and a good internal consistency (α = between .83 and
.91; [64]).
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Fig 2. Timeline representation of the different steps of the study procedure.
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2.5 Procedure
2.5.1 General procedure. At the beginning of each testing session, the experimenter took
a photograph of the participant’s face using a Nikon D50 digital SLR camera. Participants were
standing against a plain-coloured grey background, and they were photographed with a neutral facial expression, without hair covering their face. Each participants’ original photograph
was centred and cropped to adjust to the computer screen, and it was flipped horizontally as
seen in a mirror using Microsoft Picture Manager 2013. The photograph of the participant was
presented on the computer monitor during all trials of SSDT as part of the set-up (see above).
Subsequently, electrodes were placed on participants’ hand as previously explained for recording EDA.
Participants were then asked to complete the first repetition of the SSDT protocol (Pre),
which consisted of a thresholding procedure and a testing phase (see below). After completing
the SSDT, participants were asked to fill in the mood VASs (Pre), they were administered with
the touch manipulation according to their group assignment (CT optimal vs non-CT optimal),
and they were then asked to fill in the mood VASs a second time (Post). After the touch
manipulation, participants repeated the testing phase of the SSDT a second time (Post). They
were then administered each of the self-report questionnaires: the EHI, the EDI-3, the DCQ
and the BPQ. Lastly, height and weight were measured with a stadiometer and a scale for calculating the Body Mass Index (BMI; kg/m2) using the NHS online calculator. The testing procedure lasted approximately 90 minutes. A schematic representation of the general procedure
is presented in Fig 2.
2.5.2 SSDT thresholding procedure. Replicating the methods used by Mirams and colleagues [65], before the beginning of the testing phase, participants completed a thresholding
procedure to individually calibrate the strength (amplitude) of the tactile pulse used during the
subsequent testing phase. A threshold was found for each participant using the Parameter Estimation by Sequential Testing (PEST; [66]) algorithm. Participants were presented with a series
of pairs of trials (1,020ms each), during which either in the first or in the second trial a 20ms
tactile pulse (Touch) was delivered with a delay of 500ms on either side (500ms–Touch–
500ms). The other trial consisted of an empty 1,020ms period during which no touch occurred
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(No Touch). The beginning of each trial was signalled by a 250ms beep sound administered
through a pair of earphones. Participants were then asked to decide whether they felt a pulse
during the first or second trail in a two alternative forced choice design (i.e., “Did you feel the
vibration during the first or second trial?”) by pressing “1” or “2” on the computer keyboard.
The PEST procedure was set to identify the intensity necessary for participants to detect the
pulse in 75% of trials (75% threshold; [65]). The procedure began by presenting the same
above threshold tactile pulse to all participants. If participants responded correctly on a series
of trials (> 75% correct responses), the programme automatically reduced the strength of the
tactile pulse. Conversely, if they began to respond incorrectly (< 75% correct), the programme
automatically increased the strength of the pulse. A Wald [67] sequential likelihood-ratio test
was used to determine when to change the strength of the stimulus. The thresholding procedure took approximately 15 minutes. If the minimum step size was not reached after 120 trials,
the strength of the tactile pulse was set to the average stimulus strength over the last 50 thresholding trials. Immediately after completing the thresholding procedure, participants started
the experimental phase of the SSDT.
The thresholding procedure was performed only once during the first repetition of the
SSDT. The strength of the tactile pulse was then set at the same threshold level during the first
and the second repetitions of the testing phase of the SSDT.
2.5.3 SSDT testing phase. At the beginning of the testing phase, the experimenter placed
the LED on the computer monitor on the right cheek of the photograph presented, therefore
mirroring the position of the tactor on participants’ face. The testing phase of the SSDT
employed a repeated-measures design with tactile pulse (Touch/No Touch) and light (Light/
No Light) as within-subjects factors. The tactile pulse was administered in 50% of trials. Simultaneously, the LED flashed in 50% of trials, giving the following four trial types: touch only
(No Light/Touch); light only (Light/No Touch); light and touch (Light/Touch); and catch (No
Light/No Touch). Each trial type was presented 20 times in a random order, giving a total
number of 80 trials. As in the thresholding procedure, the beginning of each trial was signalled
by an auditory cue followed by a 1,020ms stimulus window. As during the thresholding procedure, in touch only trials, a 20ms tactile pulse was administered alone preceded and followed
by a 500ms delay. In light and touch trials, the LED flashed for 20ms synchronously with the
tactile pulse. In light only trials, the LED flashed for 20ms alone. In catch trials, no stimulation
was administered. At the end of each trial, participants were asked to report whether or not
they felt a touch (i.e., “Did you feel a vibration?”), and they were given four possible answers:
‘definitely yes’ (keyboard button ‘1’), ‘maybe yes’ (keyboard button ‘2’), ‘maybe no’ (keyboard
button ‘3’), or ‘definitely no’ (keyboard button ‘4’; [68]). For the purposes of this study and
consistent with previous studies using the SSDT, ‘definitely’ and ‘maybe’ responses were combined in a yes/no binary coding [29, 30, 68].
3. Results
3.1 Data processing
3.1.1 SSDT outcomes. Participants’ responses on the SSDT were classified as hits (reports
of feeling the touch on touch-present trials), false alarms (erroneous reports of feeling the
touch on touch-absent trials), misses (reports of not feeling the touch on touch-present trials),
or correct rejections (reports of not feeling the touch on touch-absent trials; [28]). According
to the log-linear correction, hit rates (HR) were calculated using the formula: [hits + .5/(hits
+ misses +1)], and false alarm rates (FA) using the formula: [false alarms + .5/(false alarms
+ correct rejections +1)] [69]. HR and FA were then used to calculate the signal detection theory test statistics d0 and c, where d0 (i.e., sensitivity) indicates participants’ ability to
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Table 1. Descriptive statistics for Age, SSDT threshold levels, mood at baseline and questionnaire scores in each group.
CT optimal
M (sd)
t
df
sig
d
32.09 (15.32)
31.97 (12.75)
.12
64
.90
.03
-1067.31 (390.86)
-1085 (394.27)
.18
64
.85
.04
Age
Threshold
non-CT optimal
M (sd)
BMI
26.37 (5.81)
25.18 (5.56)
.85
64
.40
.21
Calmness_Pre
79.82 (19.66)
72.94 (20.55)
1.39
64
.17
.34
Anxiety_Pre
24.89 (24.09)
25.55 (23.48)
-.11
64
.91
.03
Happiness_Pre
70.49 (16.53)
72.62 (16.07)
-.53
64
.60
.13
Sadness_Pre
17.23 (19.22)
15.97 (19.47)
.28
64
.78
.06
EDI-3
32.19 (18.25)
29.47 (21.31)
.55
64
.58
.14
DCQ
6.56 (3.93)
6.12 (4.14)
.44
64
.66
.11
BPQ
35.94 (12.35)
36.50 (11.69)
-.190
64
.85
.05
https://doi.org/10.1371/journal.pone.0261060.t001
discriminate between touch-present and touch-absent trials [zHR − zFA], and c (response criterion) indicates participants’ tendency to report stimuli as present regardless of the type of
trail [−.5� zHR + zFA] [70]. Lower scores on c (c < 0) indicate a higher tendency to report
touch (answer “yes”) across trials.
3.1.2 SCLs data extrapolation. EDA data were recorded continuously during three time
windows: the first repetition of the SSDT (SSDT Pre), the touch manipulation, and the second
repetition of the SSDT (SSDT Post), resulting on a total of 3 recordings per participant (each
lasting approximately 15 minutes; see Fig 2). Each recording was visually inspected for artefacts which were manually removed using Biopac (MP150) Systems. Three SCLs per participant were then extrapolated averaging across the EDA signal in each recording.
3.2 Demographics and self-reports analyses
Statistical analyses were performed using SPSS (SPPS Inc., Chicago, IL). All data are reported
as Mean (M) and Standard Deviation (SD). A significance threshold of p < .05 was set for all
effects, and effect sizes were estimated using Cohen’s d and partial eta square (η2).
A series of t-tests was performed to analyse group differences in demographics, baseline
mood, and self-report personality traits. As reported in Table 1, the two groups (CT vs nonCT) were found not to differ in Age, SSDT threshold levels, or Body Mass Index (BMI). No
significant between-groups differences were found in mood prior to the touch manipulation,
nor in eating disorder symptoms (EDI), body dysmorphic symptoms (DCQ), or body awareness (BPQ). The two groups were therefore comparable in all measures that were identified as
possible confounding variables. Moreover, mean scores for the EDI, DCQ, and BPQ of our
two groups were consistent with mean scores of the normative healthy samples, indicating our
sample to be representative of the general population [59, 61, 64].
3.3 SSDT analyses
Descriptive statistics for HR, FA, d0 and c in each Light and Time condition of the SSDT, in
each group are presented in Table 2. Before performing the analyses, these SSDT outcomes
were tested for normality. HR, d0 and c were normally distributed, therefore parametric analyses were conducted. Based on the main hypothesis of the study that the touch manipulation
would increase d’ in the CT optimal group only, independent sample t test were planned a-priori to compare d’ in the CT vs non-CT optimal group before and after the touch manipulation.
Three repeated-measures mixed design ANOVAs were then performed with Group as the
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Table 2. Descriptive statistics for hit rate, false alarm rate, d’ and c in each Light (Light and No Light) and Time (Pre and Post) condition during the SSDT, in each
group (CT and non-CT).
CT optimal
Pre
Post
HR (%)
Post
d’
c
M (SD)
M (SD)
M (SD)
M (SD)
No Light
47.69 (21.45)
10.55 (6.97)
1.41 (.77)
.78 (.43)
Light
59.53 (25.28)
8.43 (7.09)
1.58 (.91)
.45 (.43)
No Light
44.69 (27.94)
7.74 (7.45)
1.41 (1.03)
.92 (.50)
Light
53.34 (26.97)
6.46 (6.55)
1.66 (1.03)
.73 (.53)
HR (%)
FA (%)
d’
c
Non-CT optimal
Pre
FA (%)
M (SD)
M (SD)
M (SD)
M (SD)
No Light
44.30 (26.68)
9.44 (7.10)
1.35 (.90)
.89 (.52)
Light
46.91 (25.24)
7.74 (6.93)
1.30 (.82)
.77 (.48)
No Light
33.50 (23.14)
9.21 (7.21)
.91 (.69)
.97 (.56)
Light
45.82 (30.14)
8.59 (6.44)
1.19 (.93)
.83 (.57)
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between-subject factor, and Light and Time as within-subject factors, using HR, d’ and c as
dependent variables. Paired and independent-sample t tests were performed to follow up significant interactions. Bonferroni correction was used to correct for multiple comparisons.
Conversely, FA in all experimental conditions were not normally distributed, with a significant positive skewness. As FA remained not normal after attempts to transform the data, nonparametric analyses were conducted. As there is no non-parametric test equivalent to a mixed
design ANOVA, two Freidman’s ANOVAs, for the CT optimal and the non-CT optimal
groups, were used to investigate within-subjects effects, with Light and Time as within-subject
factors. Wilcoxon tests were used to follow up significant results. Mann-Whitney U tests were
used to investigate between-subject effects.
3.3.1 Hit Rate (HR). There was a significant main effect of the Light (F(1,64) = 33.48, p =
.000, η2 = .34) and a significant three way interaction Time × Light × Group (F(1,64) = 5.73, p
= .020, η2 = .08). Follow-up analyses showed higher HR in Light compared to No Light trials
in the CT optimal group (F(1,31) = 19.98, p = .000, η2 = .39; Light: M = 56.44, SD = 21.90; No
Light: M = 46.19, SD = 21.80). Conversely, in the non-CT optimal group there was no main
effect of the Light, driven by the fact that before the touch manipulation the presence of the
light did not increase HR (Pre; t(33) = -1.05, p = .30, d = .18). This indicates that during the
first repetition of the SSDT (Pre), for the non-CT optimal group, HRs were less influenced by
the presence of the Light than the CT optimal group.
Indeed, in the non-CT optimal group the effect of the Light was present only after the touch
manipulation (Post; t(33) = -3.89, p = .000, d = .67) showing higher HR in Light (M = 45.82,
SD = 30.14) compared to No Light (M = 33.50, SD = 23.14) trials. However, this effect seemed
to be driven by the fact that HR dropped down significantly in No Light trials from before
(Pre: M = 44.30, SD = 26.68) to after the touch manipulation (Post: M = 33.50, SD = 23.14; t
(33) = 2.89, p = .007, d = .50), indicating that participants in the non-CT optimal group were
less able to perceive the touch after the touch manipulation without the prompt of the Light.
No main effect of Time and Group, and no interactions were found to be significant (all
Fs � 1.98; all ps � .16).
Analyses were repeated using the difference score in HR from the first (baseline) and the
second repetition of the SSDT to correct for between-group differences at baseline. Results
showed a significant Light × Group interaction (F(1,64) = 19.98, p = .020, η2 = .08). Post-hoc
analyses showed an effect of the Light in the non-CT optimal group only (t(33) = -.09, p =
.020, d = .23). This is consistent with previous results showing in this group an effect of the
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Fig 3. Mean HR during the SSDT in Light and No Light trials before (Pre) and after (Post) receiving the touch
manipulation in the CT optimal and non-CT optimal group. Error bars show standard deviations. For the CT
optimal group there was an effect of the light during the first (Pre) and the second repetition (Post) of the SSDT. For
the non-CT optimal group an effect of the light was found only during the second repetition (Post).
https://doi.org/10.1371/journal.pone.0261060.g003
light only after the touch manipulation. Therefore the correction for baseline scores did not
change this effect. No significant results were found in the CT optimal group resulting from
the fact that the effect of the Light before and after the touch manipulation remained fairly stable. No other significant results were found (all ts � 1.38; all ps � .17). Results are reported in
Fig 3.
3.3.2 False Alarms (FA). A Friedman’s test showed that there was a significant difference
in FA in at least one of the four experimental conditions (Light × Time) in the CT optimal
group (X2(3) = 19.32, p = .000, W = .20). Post-hoc tests using Wilcoxon signed-rank test
showed that in the CT optimal group there was a significant effect of Time with lower FA after
the touch manipulation (Post; Mdn = 4.50) as compared to before it (Pre; Mdn = 8.90; z =
-2.52, p = .012, r = .45), indicating that receiving CT optimal touch decreased subsequent false
reports of the tactile pulse during the SSDT. No other significant within-subjects effects were
found in the CT optimal group (all zs � -1.85, all ps � .064). Regarding the non-CT optimal
group, a Friedman’s test showed that there was a tendency towards a significant difference in
FA between the four experimental conditions (Light × Time; X2(3) = 7.58, p = .056, W = .07).
However, follow up analyses showed no within-subjects effects to be significant (all zs � -1.86,
all ps � .083).
Mann-Whitney tests revealed a significant between-groups difference in FA in the change
score between Pre and Post FA (before and after the touch manipulation; U = 376.50, z =
-2.18, p = .029, η2 = .07), with a stronger decrease in FA from Pre to Post in the CT optimal
group (Mdn = -2.50) as compared to the non-CT optimal group (Mdn = .00). No other significant differences between the two groups were found (U � 406; p � .062). Results are reported
in Fig 4.
3.3.3 Sensitivity (d’). Following the main hypothesis of this study, a priori analyses were
planned to assess differences in d’ between the CT optimal and the non-CT optimal groups
before (Pre) and after (Post) the touch manipulation. According to our expectations, results
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Fig 4. Median change score in FA between the first (Pre) and the second (Post) repetition of the SSDT in the CT
optimal and non-CT optimal group. Error bars show interquartile ranges (IQR). The CT optimal group showed a
stronger decrease in FA during the second repetition (Post) of the SSDT as compared to the non-CT optimal group.
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showed a significantly higher d’ in the CT optimal vs the non-CT optimal group only after the
touch manipulation (Post; t(64) = 2.36, p = .022, d = .57; CT optimal: M = 1.54, SD = .90; nonCT optimal: M = 1.05, SD = .77, see Fig 5). This difference may reflect the fact that a decrease
in FA after the touch manipulation was found in the CT optimal group only. Conversely, there
was no between-groups difference in d’ before the touch manipulation (Pre; t(64) = .84, p =
.403, d = .21).
Fig 5. Mean d’ during the SSDT before (Pre) and after (Post) receiving the touch manipulation in the CT optimal
and non-CT optimal group. Error bars show standard deviations. the CT optimal group showed a significantly higher
d’ compared to the non-CT optimal group only during the second repetition (Post) of the SSDT.
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In order to have a more thorough description of the data and consistently with the analyses
run for the other SSDT outcomes, a repeated-measures mixed design ANOVA was also performed. Results showed a main effect of Light with higher d’ in Light (M = 1.43, SD = .80) compared to No Light (M = 1.27, SD = .74) trials. Moreover, there was a tendency towards a main
effect of Group (F(1,63) = 2.97, p = .071 η2 = .04) due to a higher d’ in the CT optimal
(M = 1.51, SD = .73) compared to the non-CT optimal group (M = 1.19, SD = .71). This tendency was probably driven by the fact that the CT optimal showed a higher d’ after the touch
manipulation (Post) as explained before.
There was no significant main effect of Time, and no two or three way interactions were
significant (all Fs � 1.98; all ps � .16). Therefore, it should be noted that there was no significant Group × Time interaction to support the analyses of the a priori simple effects comparing
d’ in the CT optimal vs the non-CT optimal groups before (Pre) and after (Post) the touch
manipulation. Although, as argued by Howell [71], significant interactions in the omnibus
ANOVA are unnecessary for performing simple effects analyses when specific simple effects
are predicted a priori by the study’s hypothesis, we also believe that this non-significant interaction should be acknowledged as a limitation of current results.
3.3.4 Response criterion (c). There was a significant main effect of Time (F(1,64) = 7.71,
p = .007, η2 = .10) with higher c during the second repetition of the SSDT (Post; M = .87, SD =
.50) compared to the first repetition of the SSDT (Pre; M = .73, SD = .46), indicating that participants were overall more inclined to report feeling the tactile pulse, regardless whether it
was administered or not, before (Pre) rather than after (Post) the touch manipulation.
There was also a main effect of the Light (F(1,64) = 37.75, p = .000, η2 = .37) and a significant Light × Group interaction (F(1,64) = 4.65, p = .035, η2 = .07). Follow-up analyses showed
a lower c in Light compared to No Light trials in both the CT optimal (t(31) = 6.88, p = .000,
d = 1.21; Light: M = .59, SD = .41; No Light: M = .85, SD = .42) and the non-CT optimal group
(t(33) = 2.53, p = .016, d = .43; Light: M = .80, SD = .46; No Light: M = .93, SD = .47). Further
follow up analyses showed no other significant between-groups differences in c. However,
when looking at change scores between Light and No Light trials, the CT optimal group
showed a stronger decrease in c from No Light to Light trials (M = -.26, SD = .22) compared to
the non-CT optimal group (M = -.13, SD = .29; t(64) = -2.16, p = .035, d = .53). However this
last difference was no longer significant when using Bonferroni correction. Overall, these
results indicate that all participants had a higher tendency to report perceiving the tactile pulse
when the Light was present; however, this tendency was slightly stronger in the CT optimal
group (possibly due to the fact that the non-CT optimal group showed no effect of the Light
on HR before the touch manipulation; see below for a summary).
There was no significant main effect of Group, and no other two or three way interactions
were significant (all Fs � 1.98; all ps � .16).
3.3.5 SSDT results summary. Overall SSDT outcomes showed that in the CT optimal
group, according to previous literature, the presence of the light induced an increase in HR as
compared to trials in which the light was absent [29, 30]. Conversely, in the non-CT optimal
group the presence of the light was found to induce higher HR only after receiving the touch
manipulation. However, this effect was driven by the fact that after receiving the non-affective
touch, there was a decrease in HR in trials during which the light was absent. This suggests
that participants in the non-CT optimal group were less able to perceive the touch after the
touch manipulation when there was not the prompt of the light.
Conversely, receiving affective touch was found to decrease FA, with participants in the CT
optimal group reporting less FA after the touch manipulation. Accordingly, the change score
in FA from before to after the touch manipulation was significantly greater in the CT optimal
group as compared to the non-CT optimal group. Results in FA were partially mirrored by
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results in d’. Indeed, the CT optimal group showed a tendency toward a higher d’ after the
touch manipulation when compared to the non-CT optimal group. Taken together, results in
FA and d`suggest that affective touch induced an increase in tactile accuracy. However, analyses in d’ were exploratory and followed-up a main effect of Group that was only approaching
significance and as such they should be interpreted with cation.
Moreover, there were significant effects of the Light on d’ and c indicating that the presence
of the light induced participants to correctly report perceiving the tactile pulse more often.
This effect of the Light on c was slightly stronger in the CT optimal group, which exhibited a
higher change score between Light and No Light trials compared to the non-CT optimal
group. However, this difference in c between the two groups seemed to be driven by previously
shown results in HR, where the presence of the light was found to induce an increase in HR in
the CT optimal group only. In contrast to some previous studies, there was no effect of the
Light on FAs [28, 68].
3.4 CT vs non CT touch manipulation check
Two Independent sample t-tests were performed with Intensity and Pleasantness as dependent
variables and Group as a between-subjects factor. According to our expectations and in accordance with previous literature, the CT optimal group rated the touch manipulation as significantly more pleasant (M = 6.72, SD = 3.09; t(64) = 5.73, p = .000, d = 1.42) and less intense
(M = 33.44, SD = 23.16; t(64) = -3.46, p = .001, d = .85) compared to the non-CT optimal
group (Pleasantness: M = 1.70, SD = 3.94; Intensity: M = 51.09, SD = 18.05; [7, 8, 48].
3.5 Mood VASs
Four mixed design ANOVAs were performed with Time as within-subject factor and Group
as a between-subjects factor to assess how the touch manipulation effected mood (Pre vs Post
the touch manipulation) in the CT optimal vs non-CT optimal group. The touch manipulation
increased participants’ self-reported Calmness (F(1,64) = 4.36, p = .041, η2 = .06; Pre:
M = 76.28, SD = 20.27; Post: M = 81.01, SD = 17.76) and Happiness (F(1,64) = 7.65, p = .007,
η2 = .11; Pre: M = 71.59, SD = 16.20; Post: M = 76.17, SD = 16.19), and decreased participants’
self-reported Anxiety (F(1,64) = 12.56, p = .001, η2 = .16; Pre: M = 25.23, SD = 23.60; Post:
M = 16.74, SD = 20.06) and Sadness (F(1,63) = 5.36, p = .018, η2 = .09; Pre: M = 16.59,
SD = 19.21; Post: M = 12.96, SD = 17.05). However, contrary to our expectations, these effects
were not specific for the CT optimal group. Indeed, there were no significant main effects of
Group and no Time × Group interactions were significant (all Fs � 1.91; all ps � .17).
3.6 Physiological arousal
A mixed ANOVA was performed with Time as a within-subjects factor, Group as a betweensubjects factor and SCLs as the dependent variable, to analyse changes in physiological arousal
during testing. For this analysis three temporal windows were considered: the first repetition
of the SSDT (SSDT Pre), the touch manipulation (Touch) and the second repetition of the
SSDT (SSDT Post). Therefore, in the context of the current analyses the factor Time had three
levels. Results showed a significant main effect of Time (F(2,64) = 59.67, p = .000, η2 = .48) and
a significant Time × Group interaction (F(2,64) = 3.13, p = .047, η2 = .05). Follow-up analyses
indicated that SCLs during Touch (M = 8.87, SD = 4.79) were significantly higher compared to
both the SSDT Pre (M = 6.04, SD = 4.46; t(65) = -10.69, p = .000, d = .61) and the SSDT Post
time window (M = 7.53, SD = 5; t(65) = 5.30, p = .000, d = .27). Moreover, SCLs during the
SSDT Post time window were found to be significantly higher than SCLs during the SSDT Pre
time window (t(65) = -10.69, p = .000, d = .61). Further Independent sample t-tests indicated
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Fig 6. SCLs in the CT optimal and in the non-CT optimal groups during the first repetition of the SSDT (SSDT
Pre), the touch manipulation (Touch), and the second repetition of the SSDT (SSDT Post). SCLs increased during
the touch manipulation, especially for the CT optimal group.
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that the CT optimal group had a significantly higher change score in SCLs from SSDT Pre to
Touch (M = 3.47, SD = 2.32) as compared to the non-CT optimal group (M = 2.22, SD = 1.80; t
(64) = 2.45, p = .017, d = .60). No other between-groups comparisons were found to be significant (all ts � 1.40; all ps � .17). Overall results indicated that the touch manipulation induced
an increase in arousal that was slightly higher in the CT optimal group as compared to the
non-CT optimal group. After the touch manipulation, during the second repetition of the
SSDT, the level of arousal decreased. However, it remained significantly higher than during
the first repetition of the SSDT. SCLs data are presented in Fig 6.
3.7 Exploratory correlational analyses
No a priori hypotheses drove these analyses, they were rather run for exploratory reasons and
with the aim to inform future research in this field.
3.7.1 SSDT outcomes, mood and arousal. Exploratory Pearson’s and Spearman’s correlations were run to test whether participants’ emotional states (Mood VASs), levels of arousal
(SCLs), and perceived quality of touch (Intensity and Pleasantness) were related to performance during the SSDT (HR, FA, d’ and c mean values across all experimental conditions).
There was a relationship between the HR and d’ indices and levels of arousal (SCLs). Particularly, there was a significant positive correlation between overall HR (across all experimental
conditions) and SCLs during the touch manipulation (r = .34; p = .005) and SCLs during the
second repetition of the SSDT (Post; r = .28; p = .023). However, this last correlation did not
remain significant when using a Bonferroni correction. Alongside d’ (across all experimental
conditions) was positively correlated to SCLs in all three temporal windows: during the first
repetition of the SSDT (r = .27; p = .027; only approaching significance when using a Bonferroni correction), the touch manipulation (r = .38; p = .002) and the second repetition of the
SSDT (r = .32; p = .009). Overall, these results suggest that participants exhibiting higher levels
of arousal were also more accurate in correctly reporting the tactile pulse during the SSDT as
indicated by HR and d’. Conversely, SSDT responses were not related to participants’
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emotional states (as measured by the Mood VASs), or perceived quality of touch (ratings of
Intensity and Pleasantness; all rs � .17; all ps � .18).
3.7.2 Manipulation check, mood and arousal. Further correlational analyses were run to
analyse the relationship between participants’ perceived quality of touch during the touch
manipulation (Intensity and Pleasantness; i.e., manipulation check), and subsequent emotional states (Mood VASs_Post) and levels of arousal. Perceived Pleasantness of touch was
found to be positively correlate with subsequent Calmness (r = .35; p = .004) and negatively
correlated with subsequent Anxiety (r = .34; p = .005). Therefore, the more pleasant the touch
manipulation was perceived the calmer and less anxious participants felt after it. Moreover,
Intensity of touch was found to be positively correlated with subsequent Sadness (r = .28; p =
.020), suggesting that the more intense the touch was perceived the sadder participants were
after the touch. However, this correlation did not remain significant when using a Bonferroni
correction. There were no other significant correlations (all rs � -.17; all ps � .16).
4. Discussion
The aim of the current study was to investigate the off-line carry-over effects of CT optimal
(affective) vs non-CT optimal (non-affective) touch on subsequent visuo-tactile multisensory
integration using the Somatic Signal Detection Task (SSDT). According to previous literature,
linking CT optimal affective touch to body awareness and multisensory perception, we predicted affective touch to enhance perceptual accuracy during the subsequent SSDT as compared to non-affective touch.
Affective touch led to a decrease in false reports of feeling the tactile pulse (FA) during the
SSDT. Results in FA were partially mirrored by results in d`. There was a tendency towards a
main effect of group on d’, and exploratory analyses showed that sensitivity in detecting the
tactile pulse (d`) was significantly higher after receiving affective touch (CT optimal group) in
comparison to after receiving non-affective touch (non-CT optimal group). It should be
noted, however, that there was no significant group × time interaction for d’. Nevertheless,
these results showed that participants receiving affective touch were then less inclined to erroneously report the tactile pulse when absent (FA). Alongside, in line with significant results in
FA, the CT optimal group also showed a tendency towards a better discrimination of detecting
when the tactile pulse was administered from when it was not (d`). Therefore, according to
our expectations, affective touch was shown to induce a partial increase in tactile accuracy.
To date, this is the first study demonstrating an off-line effect of affective touch on perceptual accuracy and visuo-tactile, multisensory integration. Prior studies have indicated that in a
multisensory context (i.e., during multisensory integration tasks) affective touch was perceived
as more meaningful when compared to non-affective discriminative touch [18, 72]. During
the Rubber Hand Illusion (RHI) or the Enfacement Illusion, for example, affective touch has
been linked to a stronger experience of the illusion and an enhancement of body ownership
[20–23]. According to these results, it has been argued that CT-afferents may play a unique
role in the construction of the bodily self, that is the multimodal perceptually integrated model
of one’s own body [18, 20, 25]. However, it remained unclear whether affective touch could
induce subsequent alterations in multisensory integration and body perception. This study
aimed to fill in this gap in the current literature. Results showed that affective touch can indeed
induce a subsequent off-line effect on a multisensory integration task that is the SSDT by
increasing participants’ tactile accuracy.
In this respect, it is worth noting that from a neurophysiological point of view, CT-afferents
have been linked to the activation of brain areas that are involved in the elaboration and integration of multisensory information (e.g., the posterior insula; [13, 14]), and in the codification
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of the sense of a bodily self (e.g., the angular gyrus; [3]). Therefore, it could be the case that
receiving affective touch alters the way subsequent sensory information are coded and integrated in the building of the bodily self. Specifically, affective touch may increase awareness
about one’s own body and therefore determine a subsequent more accurate integration of multisensory information.
This effect was specific for affective touch; non-affective touch did not impact on FA and
d`. Conversely, non-affective touch induced a partial decrease in the correct detection of touch
(HR) during the SSDT. Indeed, the non-CT optimal group showed a significant effect of the
Light on HR only after receiving the touch manipulation, with higher HR in trials during
which the light was present as compared to trials in which the light was absent. However, this
effect was driven by the fact that after the touch manipulation, in the non-CT optimal group
there was a decrease in HR in trials during which the light was absent, suggesting that participants receiving non-affective touch were then less able to perceive the tactile pulse without the
prompt of the light.
It is possible that the fast touch (30 cm/s) administered in the non-CT optimal group desensitized the same tactile channels required to detect the vibrotactile stimulation administered
during the SSDT. Indeed, previous research has shown mechanoceptors to have receptive
fields that can vary and adapt according to circumstances (e.g., exposure to visual information
and/or sustained stimulation; see [73, 74]). In turn, adaptation of receptive fields has been
shown to be related to impaired performances in the detection of subthreshold stimuli due to
an increase in the tactile threshold [73–75]. In this respect, a previous psychophysiological
study indicated that adaptation to prolonged fast/vibrating tactile stimuli induced a desensitization of skin mechanoreceptors that were then less sensitive in discriminating different textures [75, 76]. Therefore, it could be hypothesised that a similar mechanism influenced
participants responses in this study. Specifically, receiving a prolonged sustained tactile stimulation (non-affective fast touch) targeting the same receptive fields involved in the detection of
subthreshold touch during the SSDT may have caused an increase in participants’ tactile
threshold and therefore a worse detection (i.e., desensitization) of the tactile pulse during the
SSDT. According to this explanation, in a previous study by Mirams and colleagues [35] participants showed decreased touch reports during the SSDT after performing a grating orientation task which involved focusing on external touch. Coherently to our reasoning, it could be
the case that also in this previous study, the tactile stimulation involved in discriminating grating orientations fatigued and desensitized skin mechanoreceptors, leading to a decrease in
touch reports during the subsequent SSDT.
Further SSDT results showed that, in line with previous literature, the presence of the light
facilitated participants perception of the tactile pulse during the task. This was reflected by
overall higher HR and d`, and lower c in trials during which the light was present. Contrary to
our expectations, the touch manipulation, in both the conditions of affective and non-affective
touch, was found not to alter this effect of the light on touch detection during the SSDT.
Although the non-CT optimal group showed an effect of the light (higher HR in light-present
vs light-absent trials) only after receiving non-affective touch, as previously discussed, this
effect seemed to be driven by a decrease in HR in trials during which the light was absent,
which can be linked to a desensitization of mechanoreceptors after receiving a fast touch.
In contrast with the original SSDT study [28] the presence of the light was not found to
increase false reports of touch. However, this is consistent with some other previous studies
which investigated the effect of the light on SSDT performance under different experimental
conditions [68, 77], overall suggesting that the presence of the light can influence both correct
and incorrect reports of touch. Further research should focus on whether participants’ states
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or personality traits play a role in determining whether the presence of the light influence
more correct or incorrect report of touch during the SSDT.
Throughout the experiment, Skin Conductance Levels (SCLs) were also recorded to analyse
whether the touch manipulation induced changes in autonomic physiological arousal. Results
indicated that the touch manipulation induced an increase in arousal that was slightly higher
for affective vs non-affective touch. After the touch manipulation, during the second repetition
of the SSDT, the level of arousal slowly decreased. However, SCLs remained significantly
higher compared to baseline levels (during the first repetition of the SSDT). A single study
only previously analysed the relationship between affective touch and SCLs with results similar
to the results of this study [36]. Indeed, Ree and colleagues [36] showed SCLs to increase after
receiving a tactile stimulation, with a greater increase for CT optimal vs non-CT optimal
touch, although this difference did not reach significance. However, it should be noted that
other studies using different indexes of arousal, such as the heart rate, the heart rate variability,
and the amplitude in skin conductance response, all suggested affective touch to be associated
with a decrease, rather than an increase in arousal [53, 55, 78]. Therefore, a comprehensive picture of how different sympathetic and parasympathetic indexes of arousal vary due to affective
touch is still to be drawn by future research.
Interestingly, SCLs were positively correlated with participants’ ability to correctly detect
the tactile pulse during the SSDT (HR), and to accurately discriminate when the tactile pulse
was administered and when it was not (d`). These results are in agreement with results of a
previous study from our research group, in which the change scores in SCLs between experimental conditions was found to be associated with a parallel increment in participants ability
to discriminate touch (d`) in light-present trials of the SSDT [29]. Taken together, these findings suggest that participants’ responses during the SSDT can be influenced by their arousal
levels, with higher SCLs predicting a more accurate tactile perception. Moreover, SCLs were
found to increase more steeply during affective touch as compared to non-affective touch.
Therefore, it could be hypothesized that SCLs may mediate the relationship between the touch
manipulation and tactile accuracy, with affective touch being associated with higher SCLs and
increased tactile accuracy. Future research should further investigate this hypothesis.
In line with our expectations, affective touch was found to induce an increase in positive
mood (Happiness and Calmness) and a decrease in negative mood (Anxiety and Sadness).
However, and in contrast to our expectation, this effect was not specific for affective touch, but
was also present after receiving non-affective touch. Mood was partially influenced by participants’ perceived quality of touch during the touch manipulation (Intensity and Pleasantness).
Indeed, participants reported feeling calmer and less anxious the more pleasant they had rated
the touch manipulation, and reported feeling sadder the more intense the touch manipulation
was perceived to feel. However, mood and perceived quality of touch were found not to influence SCLs.
To summarize, results of this study showed for the first time an off-line effect of affective
touch on visuo-tactile, multisensory integration and perceptual awareness. In the context of
the SSDT, where participants are required to detect a tactile pulse independently from the
presence/absence of a concomitant light flashing, this effect translated in a greater tactile accuracy. Moreover, our results suggested SCLs to be a possible mediating mechanism linking
affective touch to a greater tactile accuracy during the SSDT. As previously explained, from a
neurophysiological point of view, CT-afferents have been linked to the activation of brain
areas that are involved in body awareness and in the building of the sense of a bodily self
through the integration of multisensory information (e.g., the posterior insula; [13, 14] and the
angular gyrus; [3]). Therefore, it could be the case that an activation of this brain network after
receiving affective touch increases body awareness altering the way subsequent sensory
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information are coded and integrated in the building of the bodily self. Specifically, results of
this study might suggest that by enhancing the sense of a bodily self through affective touch,
body perception may benefit from a greater accuracy and awareness. In other words, affective
touch may increase awareness about one’s own body as indicated by a more accurate elaboration of multisensory information during the SSDT.
However, this study is not without limitations. For example, it could be argued that repeating the SSDT could affect participants’ performance during the task. However, previous
research has shown participants’ responses during the SSDT to be consistent over time (i.e.,
multiple repetitions) with no learning processes involved [31]. Moreover, it should be noted
that the two experimental groups of this study presented with a significant difference in the
way they were influenced by the light in the correct detection of touch during the SSDT at
baseline (before the experimental manipulation). The reason for this difference in baseline is
not clear. Although no other between-groups differences were found at baseline. To overcome
this issue, future research may benefit from the use of a within-subject design that minimizes
possible biases linked to between-subjects differences. An alternative implementation of the
current design would be to repeat this study adding an additional experimental group undergoing a no touch or a very slow touch (e.g., 0.3 cm/s) manipulation. Although in this case the
risk of between-subjects differences at baseline would increase, an additional experimental
condition would allow to control for specificity of results. For example, it would allow us to
determine whether our interpretation of results in the non-CT optimal group as being linked
to desensitization of mechanoceptors is correct. Following this stream of research, another
adjustment that could be used to control for pre-existing between-groups differences would be
to adopt a linear mixed model analysis with participants’ responses at baseline used as
covariates.
Nonetheless, conclusions of the current research promote a possible use of affective touch
as an intervention for those pathological conditions in which perception of the body is altered.
Previous studies have shown affective touch to increase the sense of body ownership in neuropsychological conditions such as asomatognosia and somatoparaphrenia [72, 79, 80]. A future
stream of research should investigate whether the use of affective touch as an intervention can
be expanded also to those psychopathological disorders characterized by alterations in multisensory integration and perceptual accuracy such eating disorders, body dysmorphic disorders, and medically unexplained symptoms.
Supporting information
S1 Dataset. Affective touch and perceptual accuracy.
(XLSX)
Acknowledgments
A special thanks goes to the MSc student Emily Hughes for helping with part of the data collection. We would like also to thank the SomAffect research group for supporting the project
with technical advice and inspiring discussion - https://somaffect.org/.
Author Contributions
Supervision: Francis McGlone, Valentina Cazzato, Laura Mirams.
Writing – original draft: Sofia Sacchetti.
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FAK auto-phosphorylation site tyrosine 397 is required for development but dispensable for normal skin homeostasis
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cc-by
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RESEARCH ARTICLE Abstract This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This research was supported by the
Mayo Clinic Lucille and Smith Gibson Research
Fellowship to JBH and SSD and also the
Foundation for the National Institutes of Health
(CA215105 to AM). The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. FAK auto-phosphorylation site tyrosine 397 is
required for development but dispensable for
normal skin homeostasis Joel B. Heim1☯¤, Cera A. McDonald1☯, Saranya P. Wyles1, Sindhuja Sominidi-Damodaran1,
Edwin J. Squirewell1, Ming Li1, Catherine Motsonelidze1, Ralph T. Bo¨ttcher2,3, Jan van
Deursen4,5,6, Alexander Meves1,4,6* 1 Department of Dermatology, Mayo Clinic, Rochester, Minnesota, United States of America, 2 Department
of Molecular Medicine, Max Planck Institute for Biochemistry, Martinsried, Germany, 3 German Center for
Cardiovascular Research-Munich Partner Site, Munich, Germany, 4 Department of Biochemistry and
Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States of America, 5 Department of Pediatric
and Adolescent Medicine, Mayo Clinic, Rochester, Minnesota, United States of America, 6 Mayo Clinic
Cancer Center, Mayo Clinic, Rochester, Minnesota, United States of America a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 ☯These authors contributed equally to this work. ¤ Current address: Department of Chemistry, University of Oslo, Oslo, Norway
* meves.alexander@mayo.edu ☯These authors contributed equally to this work. ¤ Current address: Department of Chemistry, University of Oslo, Oslo, Norway
* meves.alexander@mayo.edu ☯These authors contributed equally to this work. Abstract Citation: Heim JB, McDonald CA, Wyles SP,
Sominidi-Damodaran S, Squirewell EJ, Li M, et al. (2018) FAK auto-phosphorylation site tyrosine 397
is required for development but dispensable for
normal skin homeostasis. PLoS ONE 13(7):
e0200558. https://doi.org/10.1371/journal. pone.0200558 Citation: Heim JB, McDonald CA, Wyles SP,
Sominidi-Damodaran S, Squirewell EJ, Li M, et al. (2018) FAK auto-phosphorylation site tyrosine 397
is required for development but dispensable for
normal skin homeostasis. PLoS ONE 13(7):
e0200558. https://doi.org/10.1371/journal. pone.0200558 Focal adhesion kinase (FAK) is an intensely studied non-receptor tyrosine kinase with roles
in cancer and other common human diseases. Despite the large interest in FAK, the in vivo
contribution of FAK auto-phosphorylation site tyrosine (Y) 397 to FAK function is incom-
pletely understood. To study FAK Y397 in vivo we analyzed mice with ‘non-phosphorylata-
ble’ Y-to-phenylalanine (F) and ‘phospho-mimicking’ Y-to-glutamate (E) mutations in the
germline. We found that FAK Y397F mice die early during embryogenesis with abnormal
angiogenesis like FAK kinase-dead mice. When Y397 is mutated to a glutamate mice sur-
vive beyond mid-gestation like mice where Y397 is lost by deletion of FAK exon 15. In cul-
ture, defects in proliferation, invasion and gene expression were more severe with the FAK
Y397F than with the FAK Y397E mutation despite the inability of FAK Y397E to bind SRC. Conditional expression of FAK Y397F or Y397E in unchallenged avascular epidermis, how-
ever, resulted in no appreciable phenotype. We conclude that FAK Y397 is required for the
highly dynamic tissue remodeling during development but dispensable for normal homeo-
stasis of avascular epidermis. In contrast to the Y397F mutation, FAK Y397E retains suffi-
cient biological activity to allow for development beyond mid-gestation. Editor: Nils Cordes, Technische Universitat
Dresden, GERMANY
Received: February 20, 2018
Accepted: June 28, 2018
Published: July 12, 2018
Copyright: © 2018 Heim et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Editor: Nils Cordes, Technische Universitat
Dresden, GERMANY
Received: February 20, 2018
Accepted: June 28, 2018
Published: July 12, 2018
Copyright: © 2018 Heim et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Copyright: © 2018 Heim et al. Citation: Heim JB, McDonald CA, Wyles SP,
Sominidi-Damodaran S, Squirewell EJ, Li M, et al.
(2018) FAK auto-phosphorylation site tyrosine 397
is required for development but dispensable for
normal skin homeostasis. PLoS ONE 13(7):
e0200558. https://doi.org/10.1371/journal.
pone.0200558 Mouse strains Targeting of embryonic stem cells and mouse chimera production was through the Mayo
Clinic Transgenic Core Facility (Rochester, MN). FAK Y397F mice have been reported previ-
ously [10]. FAKfl/fl mice [17] were obtained from the Mutant Mouse Regional Resource Center
at UC Davis. Keratin 5-Cre mice were as previously described [18]. To humanely and effi-
ciently euthanize animals carbon dioxide (CO2) was used following American Veterinary
Medical Association guidelines. All animal experiments were approved by the Institutional
Animal Care and Use Committee at Mayo Clinic, Rochester, MN, and all experiments were
performed in accordance with relevant guidelines and regulations. Role of FAK tyrosine 397 in development and skin Competing interests: The authors have declared
that no competing interests exist. The structure of FAK consists of a focal adhesion targeting (FAT) domain on the C-
terminus, an adjacent catalytic kinase domain and a four-point one, ezrin, radixin, moesin
(FERM) domain on the N-terminus. The central catalytic and FERM domains are connected
by a linker region which contains the major FAK auto-phosphorylation site, tyrosine (Y)
397 [1]. When FAK is in the inactive conformation, the FERM domain auto-inhibits the
catalytic kinase domain and Y397 is nonphosphorylated [8]. Upon activation, the linker
region becomes exposed and Y397 undergoes autophosphorylation [8], providing a high-
affinity binding site [9] for proteins such as proto-oncogene tyrosine-protein kinase Src
(SRC) [10]. Competing interests: The authors have declared
that no competing interests exist. Although much is known about the mechanisms and biological activities of FAK in vitro,
the regulation of FAK in vivo is incompletely understood. FAK-null mice experience embry-
onic lethality by embryonic day (E) 8.5 [11, 12]. Deletion of FAK Y397 by linker domain trun-
cation mutation results in embryonic death between E13.5-E14.5 [13], while the substitution
of Y397 with a non-phosphorylatable phenylalanine (Y397F) results in embryonic death by
E11.5 [10]. Conditional FAK deletion has been shown to suppress tumor formation in breast
epithelium [14] and the epidermis [15]. Further, phosphorylated Y397 FAK has been localized
in the nuclei of cancerous cells, including melanoma [10] and colorectal cancer [16]. These
findings suggest that FAK Y397 plays an important role during embryonic development and
may influence cell survival and gene transcription in cancerous cells, however may not be nec-
essary for cell survival in mature differentiated cells. Here, we explored the role of FAK Y397 in embryonic development and mature differenti-
ated epidermis by comparing mice with Y397-to-phenylalanine (Y397F) and Y397-to-gluta-
mate (Y397E) germline mutations, allowing us to examine the in vivo activities of FAK lacking
a phosphorylatable tyrosine (Y397F) or harboring a constitutive negative charge at Y397
(Y397E). Introduction Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase [1] enriched in focal adhesions
[2]. FAK has been shown to play important roles in diverse events such as cell adhesion[3],
wound healing [4], and metastasis [5]. FAK has also been localized to the nucleus, where it
may influence cell survival [6] and modulate gene transcription [7]. 1 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 Timed mating and embryo isolation In all mating procedures, female mice were exposed to male mice overnight. Identification of a
vaginal plug the next morning was used to determine embryonic day (E) 0.5. Staged embryos
(E8.5-E16.5) were dissected in ice-cold PBS. Embryos were observed under an Olympus
SZX12 Microscope. Small pieces of tail or paw were digested in PBS containing proteinase K at
56 ˚C for 90 min. Proteinase K was heat-inactivated at 80 ˚C for 30 min. 2 μl of the sample
were used to set up a PCR reaction for genotyping. Primary cells and cell lines Heterozygous FAK Y397F mice were intercrossed and MEFs were isolated from E9.5 embryos,
immortalized with the SV40 large T antigen. FAK Y397F mutation was confirmed by RNA
sequencing. Primary mouse keratinocytes were isolated and cultured as previously described30. p53-/- FAK knock-out mouse embryonic fibroblasts (MEFs; CRL-2644) were purchased from
the American Type Culture Collection. Cell lines were tested for mycoplasma and viral con-
tamination by the RapidMAP™21 test (Taconic) or the Mayo IMPACT Profile (IDEXX BioRe-
search). MEFs were cultured in DMEM containing 10% FBS. Peptide pulldowns Pulldowns were performed as previously described [19]. Purified GST-tagged SRC was pur-
chased from Sigma-Aldrich (S1076). Antibodies The following antibodies were used for immunoprecipitation and/or microfluidic western
blot analysis by ProteinSimple (PS), immunohistochemistry (IHC), immunofluorescence
(IF): CD31 (553371, MEC13.3, BD Biosciences; 1:500 for IF), laminin (ab11575, Abcam;
1:400 for IF), involucrin (ab28057, Abcam; 1:100 for IHC), FAK (06–543, EMD Millipore;
1:50 for PS; 1:100 for IF), paxillin (610051, 349, BD Biosciences, 1:100 for IF), phospho-FAK
Y397 (AF4528, R&D Systems; 1:200 for PS), phospho-FAK Y397 (44-624G, Thermo Fisher
Scientific; 1:100 for IF), β-tubulin (ab15568; Abcam; 1:50 for PS), SRC (2109, 36D10, Cell Sig-
naling Technology; 1:50 for PS), phospho-SRC Y416 (2101, Cell Signaling Technology; 1:10
for PS). 2 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 Role of FAK tyrosine 397 in development and skin Whole-mount three-dimensional imaging of CD31 in embryos Whole-mount three-dimensional imaging of CD31 was performed as previously described
[18]. To allow for deep penetration of laser light and confocal sectioning, embryos were dehy-
drated by increasing methanol concentrations, cleared in benzyl alcohol/benzyl benzoate (1:2). Images were obtained with a Zeiss LSM780 confocal microscope using ZEN software (2012,
release 8.0). Transient and stable transfection/transduction To generate stable cell lines, MEFs were infected with lentivirus in 2 ml complete medium con-
taining 8 μg/ml polybrene and a virus multiplicity of infection (MOI) of 2 to 20. Selection was
by 1 to 1.5 μg per ml of puromycin. Plasmids and constructs For stable FAK variant expression in cell lines, cDNA was cloned into the lentiviral expression
vector LV022 (Applied Biological Materials). Point mutations were introduced by site-directed
mutagenesis using the Quikchange II XL kit (200521, Agilent Technologies). PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 Microfluidic western blotting Western blots were performed as Simple Western™assays using the Wes system (ProteinSim-
ple), a combination of capillary electrophoresis and immunodetection techniques, following
the manufacturer’s protocols. Quantification of chemiluminescence was based on peak height
after correction for a baseline signal. Raw data was generated by the Compass software (version
2.5.8 to 2.7.1, build ID 0201–0826). Compass is the control and data analysis application for
Simple Western instruments. 3 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 Role of FAK tyrosine 397 in development and skin Proliferation and Matrigel invasion For proliferation analysis, cells were seeded at low densities (2,500 to 5,000 cells per well). Cells
were incubated using an IncuCyte ZOOM1 (Essen Bioscience). Cell growth based on percent
confluence was determined from phase contrast images. For proliferation assays based on auto-
mated counting of fluorescent nuclei, cells were first infected with the NucLight Red reagent
(4476, Essen Bioscience) and selected using 1 μg per ml of puromycin to obtain a stable nuclear
red fluorescent label. Invasion assays were performed by seeding cells at a density of 30,000
cells/well on a thin coating of Matrigel (0.1 mg/ml, standard formulation, Corning) in 96 well
ImageLock microplates (4379, Essen BioScience). Cells were then allowed to grow confluent. Prior to scratching, cell proliferation was inhibited by 30 minutes exposure to 10 μg/ml mito-
mycin C (M4287, Sigma-Aldrich). Monolayers were scratched using the WoundMaker™pin
tool (4493, Essen BioScience). Medium was changed and cells were covered in 2 mg/ml Matri-
gel (standard formulation, Corning) diluted in complete medium. Relative wound density was
calculated using the IncuCyte™Scratch Wound Cell Migration Software Module (9600–0012). Immunocytochemistry and immunohistochemistry For immunocytochemistry, cells were cultured on LAB-TEK II chamber slides (154453,
Thermo Scientific), fixed for 10 min in 4% PFA at room temperature and stained with a
BOND-MAX autostainer (Leica Biosystems). Briefly, slides were washed with 1x Bond Wash
solution (Leica Biosystems) and cells were permeabilized for 10 min with 0.1% Triton X-100
in PBS. After washing and blocking with 3% BSA in PBS for 60 min, slides were incubated
with a 1:100 dilution of primary antibody in Bond Primary Antibody Diluent (Leica Biosys-
tems) for 90 min. Slides were washed three times for 2 min and incubated with a 1:100 dilution
of secondary antibody in BOND Primary Antibody Diluent for 60 min. After three 2 min
wash steps, DAPI staining was performed for 10 min. Images were collected at room tempera-
ture by confocal microscopy (Zeiss LSM780) with a x40/1.0 objective using ZEN software
(2012, release 8.0). For immunohistochemistry on paraffin sections using involucrin antibody,
sections were baked, dewaxed and exposed to EDTA based pH 9.0 solution (AR9640, Leica
Biosystems) using a BOND-MAX autostainer. Antibody detection was by alkaline phospha-
tase-linked polymers (DS9390, Leica Biosystems). For immunohistochemistry on frozen
murine skin sections, epitope retrieval was not required. Antibody detection was by secondary
Alexa 488-conjugated antibody (1:200 dilution). Hematoxylin and eosin-stained paraffin sec-
tions were digitalized with an Aperio ScanScope (Leica Biosystems). Adhesion turnover LentiBrite™Paxillin (Millipore)-GFP-tagged FAK Y397E and FAK Y397F cells were plated
(10,000 per well) in regular growth medium in Lab-Tek II chambered coverglass (Nunc)
coated with 10 μg/ml of fibronectin (Calbiochem). The focal adhesion turnover analysis was
performed on a custom-made system (Visitron Systems) based on an Nikon Eclipse Ti
inverted microscope, an Nikon 60x 1.40 NA objective and a ProEM EMCCD camera (Prince-
ton Instruments) in an incubation chamber (Oxolab) to control temperature, CO2 (5% pre-
mixed) and humidity. Images were acquired every three minutes for four hours using
VisiView software (Visitron Systems) and analyzed by ImageJ. Successful generation of FAK Y397 mutant mice To study the phenotype of mice expressing FAK Y397F or Y397E during development and in
adult tissue, we targeted exon 15 of the murine FAK gene by homologous recombination in
embryonic stem (ES) cells. Positive ES cell clones of each mutation were injected into blasto-
cysts to establish the mutant knock-in (KI) mouse strains (KIneo+) [10]. The Flippase Recombi-
nation Target (FRT)-flanked neomycin gene was then removed by intercrossing the mutant
mouse strains with a deleter-Flippase strain, resulting in mice carrying an intronic FRT site
and defined point mutations in exon 15 of the FAK gene (KIfrt). Heterozygous intercrosses of
FAK Y397F [10] and Y397E mice (Table 1) yielded no live homozygous offspring at birth. However, homozygous embryos could be detected by PCR genotyping on embryonic (E) day
9.5 (Fig 1A). Sanger sequencing of cDNA derived from the tails of adult heterozygous KIfrt
mice showed double peaks at amino acid codon 397, indicating base pair changes introduced
by homologous recombination (Fig 1B). Experiments on micropatterns FAK variant cells were seeded on fibronectin-coated micropatterned discs (10-900-10,
CYTOO) according to instructions and allowed to attach and spread for 1 hour. Cells were PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 4 / 14 Role of FAK tyrosine 397 in development and skin then fixed for 10 min in 4% PFA at room temperature and processed for immunocytochemis-
try. Images were collected at room temperature by confocal microscopy (Zeiss LSM780) with a
x40/1.0 objective using ZEN software (2012, release 8.0). Reference cells were created using a
CYTOO-provided image analysis macro (CYTOOL-IP-Reference cell/April 2014) in ImageJ
(release 1.44i). Gene expression by next-generation sequencing RNA was isolated using the RNeasy Plus Mini kit (74134, Qiagen). RNA sequencing was per-
formed as previously described [20]. Briefly, RNA-derived cDNA libraries were prepared
using the TruSeq RNA Library Prep Kit v2 (Illumina). Concentration and size distribution of
the resulting libraries were determined on an Agilent Bioanalyzer DNA 1000 chip and con-
firmed by Qubit fluorometry (Life Technologies). Unique indexes were incorporated at the
adaptor ligation phase for three-plex sample loading. Libraries were loaded onto paired end
flow cells to generate cluster densities of 700,000 per mm2 following Illumina’s standard proto-
col. The flow cells were sequenced as 51 paired end reads on an Illumina HiSeq 2000. The sam-
ples were processed through the Mayo RNA-Seq analysis pipeline, MAP-RSeq. Raw and
normalized (read per kilobase of gene per million mapped reads) gene expression read counts
were obtained per sample. Differential gene expression analysis was carried out using the freely
available edgeR bioconductor software package (http://bioconductor.org). Because scaling by
total lane counts can bias estimates of differential expression, edgeR uses trimmed mean nor-
malization on raw read counts to determine whether genes are differentially expressed using
the negative binomial method. The Benjamini and Hochberg correction is used to control for
multiple testing to obtain a false discovery rate. Statistics Statistical analysis was performed using the GraphPad Prism software (version 6.05, GraphPad
Software). Statistical significance was determined as indicated. Statistical analysis was performed using the GraphPad Prism software (version 6.05, GraphPad
Software). Statistical significance was determined as indicated. Role of FAK tyrosine 397 in development and skin Table 1. Genotypes of embryos obtained from crosses between FAK Y397E/+ heterozygous mice derived from ES cell clones 227 and 405. Genotype
Clone 227
Clone 405
9.5
10.5
13.5
14.5
16.5
Born (P0)
Born (P0)
+/+
5
10
12
15
12
211
13
+/Y397E
11
8
19
38
25
432
26
Y397E/Y397E
5
18
10
15
0
0
0
No. of litters
2
4
5
7
4
100
8
The total number of embryos of each genotype obtained from crosses between mice (FAK Y397E/+; ES cell clones 227 and 405) is shown at various embryonic days (E)
and at birth (P0). obtained from crosses between FAK Y397E/+ heterozygous mice derived from ES cell clones 227 and 405. ypes of embryos obtained from crosses between FAK Y397E/+ heterozygous mice derived from ES cell clones 227 and 405 The total number of embryos of each genotype obtained from crosses between mice (FAK Y397E/+; ES cell clones 227 and 405) is shown at various embryonic days (E)
and at birth (P0). The total number of embryos of each genotype obtained from crosses between mice (FAK Y397E/+; ES cell clones 227 a
and at birth (P0). embryonic development stalled at E9.5, a stage at which intersomitic vessels (ISVs) were clearly
visible between the somites of wild type (WT) but not FAK Y397F embryos (Fig 2A). Mutant
embryos demonstrated malformed hearts and an unorganized somite structure while the
allantois was unfused and enlarged [10]. Whole-mount three-dimensional (3D) imaging of
platelet endothelial cell adhesion molecule-stained (PECAM-1; CD31) Y397F FAK embryos
indicated the presence of a dorsal aorta (DA) adjacent to an incompletely formed heart, and a
lack or malformation of ISVs, as compared to the clearly stained and normally developed
heart, DA, and ISVs of the WT embryo (Fig 2B). Additionally, the FAK Y397F yolk sacs lacked
the differentiated blood vessels seen on WT yolk sacs (Fig 2C). FAK Y397E embryos in contrast were comparable in size and organ development to wild
type (WT) embryos at E10.5 with visible and well-defined ISVs (Fig 2D). Whole-mount 3D
imaging of PECAM-1-stained FAK Y397E embryos demonstrated developing vascularization
as well as an identifiable DA and ISVs (Fig 2E). The yolk sac showed differentiated blood ves-
sels (Fig 2F). At E12.5, however, embryos demonstrated edema, and by E13.5 both edema and
hemorrhage were clearly visible (Fig 2G). FAK Y397F mice die during early mesoderm development whereas FAK
Y397E mice display abnormalities after mid-gestation Timed mating revealed that FAK Y397F mice die between E9.5 and E11.5 [10] whereas FAK
Y397E mice survive until E14.5 (Table 1). No live embryos were seen at E16.5. FAK Y397F 5 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 https://doi.org/10.1371/journal.pone.0200558.t001 Role of FAK tyrosine 397 in development and skin Fig 2. Y397E FAK embryos survive longer than Y397F FAK embryos. (A) Bright-field images of E9.5 WT and Y397F FAK embryos. Arrows point to heart, ISVs,
DA, somites, and allantois. WT, wild-type; ISV, intersomitic vessels; DA, dorsal aorta. (B) Whole-mount 3D imaging of CD31-stained E9.5 WT and Y397F FAK
embryos. Arrows point to DA, ISVs, and heart. 3D, three-dimensional; CD31, platelet endothelial cell adhesion molecule (PECAM-1). (C) Bright-field images of E10.5
WT and Y397F FAK yolk sacs. (D) Bright-field images of E10.5 WT and Y397E FAK embryos. Arrows point to ISV and heart. (E) Whole-mount 3D imaging of
CD31-stained E10.5 WT and Y397E FAK embryos. Arrows point to ISV, DA, and heart. (F) Bright-field images of E10.5 WT and Y397E FAK yolk sacs. (G) Bright-
field images of E12.5-E13.5 WT and Y397E FAK embryos. Arrows point to edema and hemorrhage. (H) Survival rate of homozygous FAK mutants by embryonic day,
with class of mutant and specific mutation indicated. Scale bars, 200 μm. Fig 2. Y397E FAK embryos survive longer than Y397F FAK embryos. (A) Bright-field images of E9.5 WT and Y397F FAK embryos. Arrows point to heart, ISVs,
DA, somites, and allantois. WT, wild-type; ISV, intersomitic vessels; DA, dorsal aorta. (B) Whole-mount 3D imaging of CD31-stained E9.5 WT and Y397F FAK
embryos. Arrows point to DA, ISVs, and heart. 3D, three-dimensional; CD31, platelet endothelial cell adhesion molecule (PECAM-1). (C) Bright-field images of E10.5
WT and Y397F FAK yolk sacs. (D) Bright-field images of E10.5 WT and Y397E FAK embryos. Arrows point to ISV and heart. (E) Whole-mount 3D imaging of
CD31-stained E10.5 WT and Y397E FAK embryos. Arrows point to ISV, DA, and heart. (F) Bright-field images of E10.5 WT and Y397E FAK yolk sacs. (G) Bright-
field images of E12.5-E13.5 WT and Y397E FAK embryos. Arrows point to edema and hemorrhage. (H) Survival rate of homozygous FAK mutants by embryonic day,
with class of mutant and specific mutation indicated. Scale bars, 200 μm. https://doi.org/10.1371/journal.pone.0200558.g002 phenotype similar to FAK Δ15 mice. FAK Y397 mutants may be broadly classed into loss of
function mutations, including Y397F FAK, and those that are partially functional, including
Y397E FAK and FAK Δ15 (Fig 2H). Compared to published FAK mutant mice [11–13,
21] the in utero survival curve of FAK Y397E embryos resembled that of mice with a 19 amino
acid FAK linker deletion (FAK Δ15) (Fig 2H) [13]. In contrast, the FAK Y397F mutation was
early embryonic lethal akin to FAK-null or kinase-dead mutations (Fig 2H) with a phenotype
resembling fibronectin (FN)-deficient embryos [22]. Together, our data suggested that Y397F
sustained a non-active FAK conformation in vivo, while Y397E was partially functional with a Fig 1. Generation of FAK Y397 mutant FAK. (A) Generation of hetero- and homozygous mutants was confirmed by PCR of E9.5 embryos. Arrows
indicate bands representing mutant (Kl) or WT alleles. +, wild-type; Kl/+, heterozygous mutant; Kl/Kl, homozygous mutant; bps, base pairs. (B) Sanger
sequencing was performed with mouse tail cDNA to detect heterozygous FAKY397E/WT mutants. Arrows indicate sites of single base pair mutagenesis. https://doi.org/10.1371/journal.pone.0200558.g001 Fig 1. Generation of FAK Y397 mutant FAK. (A) Generation of hetero- and homozygous mutants was confirmed by PCR of E9.5 embryos. Arrows
indicate bands representing mutant (Kl) or WT alleles. +, wild-type; Kl/+, heterozygous mutant; Kl/Kl, homozygous mutant; bps, base pairs. (B) Sanger
sequencing was performed with mouse tail cDNA to detect heterozygous FAKY397E/WT mutants. Arrows indicate sites of single base pair mutagenesis. https://doi.org/10.1371/journal.pone.0200558.g001 https://doi.org/10.1371/journal.pone.0200558.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 6 / 14 FAK Y397E exhibits a higher degree of biological activity than FAK Y397F Phospho (p)-Y397 FAK has been shown to bind the Src-homologue-2 domain (SH2) of SRC
and form a protein complex that is implicated in integrin signaling and cell adhesion [23]. We PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 7 / 14 Role of FAK tyrosine 397 in development and skin therefore decided to investigate the binding potential and activity of FAK Y397E to determine
whether residual binding with SRC would explain the extended lifespan and advanced devel-
opment of FAK Y397E versus Y397F embryos. The affinity of recombinant full-length SRC for
synthesized peptide corresponding to the FAK linker region, including FAK proline rich
region 1 (PRR1) and amino acids (aa) 358–409 harboring modifications at aa 397 was tested in
vitro as shown. Peptides also contained FAK proline rich region 1 (aa368-375) which serves as
a binding site for SH3 domains including SRC-SH3. Desthiobiotinylated peptide was immobi-
lized to streptavidin-coated beads and incubated with glutathione S-transferase-tagged recom-
binant SRC (SRC-GST). The amount of precipitated SRC-GST was then determined by
microfluidic immunoblotting using the ProteinSimple Wes platform. We found that only the
pY397 FAK peptide but not scrambled, nonphosphorylated (FAK), Y397F, or Y397E FAK
peptides precipitated SRC-GST (Fig 3A). Quantification of the chemiluminescent signal from
pY397 FAK peptide-SRC-GST pulldowns demonstrated a large peak at 80 kDa indicating
effective binding of pY397 FAK peptide to SRC-GST. No or minimal signal was seen with
scrambled, nonphosphorylated (WT), Y397F or Y397E peptides (Fig 3B) indicating weak or
no binding of nonphosphorylated peptides to SRC-GST. These results suggested that efficient
FAK binding to full length SRC requires pY397 FAK and cannot be achieved by a negatively
charged ‘phospho-mimicking’ glutamate at amino acid 397. To determine expression and activation of FAK and SRC in culture, we stably transduced
FAK-null mouse embryonic fibroblasts (MEFs) with lentiviral constructs encoding for FAK
variants (Fig 3C). MEFs expressing FAK WT, FAK Y397F, or FAK Y397E were then seeded on
gelatin-coated surfaces where they maintained a classic fibroblast morphology (Fig 3D). Immunoblotting experiments showed that FAK was detected in all three FAK variant lysates. pY397 FAK was however only detected in the WT FAK lysate (Fig 3E) as FAK Y397E did not
bind anti-pY397 FAK antibody. SRC was expressed at a similar level in all three FAK variant
and knock out (KO) cell lines. PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 Role of FAK tyrosine 397 in development and skin Fig 3. Y397E FAK demonstrates residual activity. (A) Peptide pulldowns using bead-immobilized FERM-kinase linker domain peptides and recombinant SRC-GST. The pulldown blot indicates which samples captured SRC-GST, while the input blot indicates the amount of SRC-GST in each sample prior to experimentation. SRC-GST, SRC tagged with glutathione-s-transferase; Mr (K), weight in kilo-Daltons; aa, amino acid. (B) Chemiluminescent signal of SRC-GST bound to peptide in
each pulldown sample. (C) FAK-null MEFs were transduced with lentiviral constructs encoding for WT FAK, Y397F FAK, and Y397E FAK. KO, knock-out; WT,
wild-type. (D) Phase-contrast images of MEF lines expressing either WT, Y397F, Y397E or no FAK. KO, knock-out. (E) Western blots of FAK, pY397 FAK, and
loading control TUBB in lysate of FAK variant MEF lines and FAK-null MEFs. TUBB, tubulin β-chain. (F) Western blots of total SRC, pY416 SRC, and TUBB in
lysates of FAK variant MEF lines and FAK-null MEFs. (G) Adhesion turnover measurements of GFP-Paxillin-positive focal adhesions in WT and FAK variant MEFs
(mean ± SD, n = 180; P < 0.001, Student’s t test; n.s., not significant). (H) Averaged N confocal images of paxillin (PAX)-stained FAK variant cells (reference cells);
signal density was processed over the image stack and pseudocolored. Arrows indicate areas of PAX adhesions in low contractility areas. (I) Phase object confluence of
FAK variant MEF lines, indicating the percentage of cells covering the field of view over 72 hours. Statistically significant differences between WT FAK and Y397F Fig 3. Y397E FAK demonstrates residual activity. (A) Peptide pulldowns using bead-immobilized FERM-kinase linker domain peptides and recombinant SRC-GST. The pulldown blot indicates which samples captured SRC-GST, while the input blot indicates the amount of SRC-GST in each sample prior to experimentation. SRC-GST, SRC tagged with glutathione-s-transferase; Mr (K), weight in kilo-Daltons; aa, amino acid. (B) Chemiluminescent signal of SRC-GST bound to peptide in
each pulldown sample. (C) FAK-null MEFs were transduced with lentiviral constructs encoding for WT FAK, Y397F FAK, and Y397E FAK. KO, knock-out; WT,
wild-type. (D) Phase-contrast images of MEF lines expressing either WT, Y397F, Y397E or no FAK. KO, knock-out. (E) Western blots of FAK, pY397 FAK, and
loading control TUBB in lysate of FAK variant MEF lines and FAK-null MEFs. TUBB, tubulin β-chain. (F) Western blots of total SRC, pY416 SRC, and TUBB in
lysates of FAK variant MEF lines and FAK-null MEFs. FAK Y397E exhibits a higher degree of biological activity than FAK Y397F In contrast, active SRC as measured by pY416 was abundant in
FAK WT lysates, and almost undetectable in KO and FAK Y397F lysates but slightly more
abundant in Y397E lysates (Fig 3F). These data suggested that while SRC is present in all tested
MEF lines, its activation depends on pY397 FAK. Since FAK is involved in focal adhesion turnover, wound healing, and survival, assays com-
paring adhesion, proliferation and motility allowed us to infer the functionality of the variants. FAK variant MEFs were analyzed microscopically for their ability to affect turnover of green
fluorescent-paxillin-positive focal adhesions. We found that focal adhesion lifetime was
increased almost 2-fold in FAK Y397F MEFs whereas no significant change was detected with
FAK Y397E versus WT (Fig 3G). FAK variant fibroblasts were also seeded on fibronectin
micropatterned discs and stained for paxillin (PAX), a focal adhesion marker. N confocal
images of PAX-stained FAK variant cells were averaged and their signal density processed
over the image stack and pseudocolored. In all FAK variant cells, PAX-containing adhesions
were found in a predominantly circumferential distribution. However, in FAK Y397F cells,
PAX-containing adhesions were also found in less contractile areas towards the center of the
discs. Such aberrant formation of adhesion structures was thought to impede FAK Y397F cell
migration and invasion. We therefore analyzed FAK variant MEFs for their ability to migrate
and invade into Matrigel. Cells were imaged and analyzed for phase object confluence, which measures the percent of
the field of view covered by confluent cells. A significant reduction in confluence was seen
with FAK Y397F but not Y397E versus WT (Fig 3I). Similarly, primary FAK Y397F MEFs that
were directly derived from knock-in mice showed delayed spreading and reduced phase object
confluence after plating versus WT (S1 Fig). To more directly measure cell proliferation, FAK
variant MEFs were transduced to express a nuclear-restricted red fluorescent protein. This PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 8 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 Role of FAK tyrosine 397 in development and skin FAK confluence are indicated below relevant data points (mean ± SEM, n = 24; P < 0.05, Student’s t test; h, hours). (J) Nuclear counts of fluorescently labeled nuclei
of FAK variant MEFs are depicted as a measure of cell proliferation (mean ± SEM, n = 24; P < 0.05, Student’s t test). (K) Relative wound density of FAK variant MEF
lines indicating the percentage of cells to invade Matrigel following wound treatment. Statistically significant differences between WT and Y397F FAK lines are
indicated below relevant data points. Immunoblots are pseudoimages generated by the ProteinSimple Compass software (mean ± SEM, n = 12; P < 0.05, Student’s t
test). https://doi.org/10.1371/journal.pone.0200558.g003 https://doi.org/10.1371/journal.pone.0200558.g003 enabled real-time counting of cells. We found that the doubling time of nuclei was reduced in
Y397F but not in Y397E MEFs compared to WT (Fig 3J). Likewise, Matrigel invasion follow-
ing scratch wounding was significantly reduced with FAK Y397F but not Y397E versus WT
(Fig 3K). These data showed that FAK Y397E retains a higher degree of biological activity than
FAK Y397F. This is further demonstrated by Y397E but not Y397F FAK MEFs maintaining
normal osteopontin (SPP1) expression (S2 Fig and S1 Dataset), a downstream read-out of
FAK activity [10]. (G) Adhesion turnover measurements of GFP-Paxillin-positive focal adhesions in WT and FAK variant MEFs
(mean ± SD, n = 180; P < 0.001, Student’s t test; n.s., not significant). (H) Averaged N confocal images of paxillin (PAX)-stained FAK variant cells (reference cells);
signal density was processed over the image stack and pseudocolored. Arrows indicate areas of PAX adhesions in low contractility areas. (I) Phase object confluence of
FAK variant MEF lines, indicating the percentage of cells covering the field of view over 72 hours. Statistically significant differences between WT FAK and Y397F l activity. (A) Peptide pulldowns using bead-immobilized FERM-kinase linker domain peptides and recombinant SRC-GST. es captured SRC-GST, while the input blot indicates the amount of SRC-GST in each sample prior to experimentation. f
( )
h
k l
l
d ( )
h
l
l f
b
d
d PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 9 / 14 https://doi.org/10.1371/journal.pone.0200558.g003 Skin homeostasis unaltered in conditional Y397F and Y397E mutant mice To determine whether Y397F or Y397E FAK would present a phenotype if selectively
expressed in the epidermis, FAK mutant mice were crossed with mice expressing Cre recombi-
nase under a keratin 5 promoter (K5-Cre; Fig 4A). Conditional Y397F and Y397E FAK mice
retained a normal phenotype. On gross inspection, three week-old mice harboring Y397F or
Y397E FAK were indistinguishable from control litter-mates (Fig 4B). No hair phenotype was Fig 4. No epidermal phenotype in conditional Y397F FAK and Y397E FAK mice. (A) Heterozygous mice with one FAK allele encoding for Y397F FAK or Y397E
FAK and one allele containing floxed exon 17 were crossed with mice expressing Cre recombinase under a K5 promoter to ensure exclusive expression of Y397F FAK
or Y397E FAK in the epidermis of offspring. K5, keratin-5. (B) Images of adult WT, Y397F FAK, and Y397E FAK mice. (C) HE staining of WT, Y397F FAK, and
Y397E FAK epidermis. HE, hematoxylin and eosin. (D) Immunohistochemical staining of involucrin using an alkaline phosphatase-based red detection kit. (E)
Immunofluorescent staining of laminin-332 expression in WT, Y397F FAK, and Y397E FAK epidermis. Laminin shown in green, DAPI shown in blue. DAPI,
4’,6-Diamidine-2’-phenylindole. (F) Immunocytochemical staining of FAK variant epidermal keratinocytes. FAK staining shown in green, paxillin staining shown in
red, and DAPI staining shown in blue. PAX, paxillin. https://doi.org/10.1371/journal.pone.0200558.g004 Fig 4. No epidermal phenotype in conditional Y397F FAK and Y397E FAK mice. (A) Heterozygous mice with one FAK allele encoding for Y397F FAK or Y397E
FAK and one allele containing floxed exon 17 were crossed with mice expressing Cre recombinase under a K5 promoter to ensure exclusive expression of Y397F FAK
or Y397E FAK in the epidermis of offspring. K5, keratin-5. (B) Images of adult WT, Y397F FAK, and Y397E FAK mice. (C) HE staining of WT, Y397F FAK, and
Y397E FAK epidermis. HE, hematoxylin and eosin. (D) Immunohistochemical staining of involucrin using an alkaline phosphatase-based red detection kit. (E)
Immunofluorescent staining of laminin-332 expression in WT, Y397F FAK, and Y397E FAK epidermis. Laminin shown in green, DAPI shown in blue. DAPI,
4’,6-Diamidine-2’-phenylindole. (F) Immunocytochemical staining of FAK variant epidermal keratinocytes. FAK staining shown in green, paxillin staining shown in
red, and DAPI staining shown in blue. PAX, paxillin. Discussion In the present study, we show that phosphorylated Y397 FAK is necessary for normal embry-
onic development but dispensable for unchallenged skin homeostasis. Our data suggests that
inactivating mutations in Y397 FAK result in decreased expression of FN-like ECM and defec-
tive mesenchymal angiogenesis—a process that depends on FN-like ECM [22]–but have no
effect on normal epidermis and epidermal appendages which are avascular tissues. Substituting Y397 with a glutamate or phenylalanine residue in the germline allowed us to
investigate the role of Y397 FAK phosphorylation in vivo. Glutamate introduces a negative
charge, thus retaining FAK function compared to Y397F, including enhanced embryonic
development and reduced alteration of gene expression. However, glutamate is not truly
“phospho-mimicking” as it does not fit the binding pocket of SH2 adaptor domains [24]. Spe-
cifically, we have shown that SRC SH2 does not interact with Y397E FAK linker peptide. The
combination of a constitutive negative charge and a disrupted SH2 binding pocket ultimately
results in hemorrhage and embryonic death by E16.5. Interestingly, mice with a deletion of the
19 amino acid FERM-kinase domain linker (Δ15 FAK) which includes Y397 show a phenotype
that is similar to Y397E embryos [13]. A shortened linker would likely impair FERM-kinase
domain interaction due to structural constraints [25]. Indeed, basal FAK activity is increased
by Δ15 deletion [13]. Structural predictions lead to the assumption that the same may be true
for FAK Y397E [25]. Y397 FAK plays an important role in complex mesenchymal processes such as angiogenesis
and drives the expression of FN-type ECM. FN secretion and matrix assembly in the ECM are
necessary for angiogenesis and vasculogenesis, and FN is primarily expressed during develop-
ment, wound-healing, and tumor formation [26]. FN-integrin binding activates FAK to form
focal adhesions and initiate signals to assist in FN-based matrix assembly [27]. Y397F FAK
embryos resemble FN-deficient embryos [10], and FAK-null embryos exhibit deficient FN
matrices and patterning [28]. These findings help explain why Y397E and Y397F FAK
embryos experienced stalled development and impaired angiogenesis, namely that the activity
of Y397E and Y397F FAK was insufficient to maintain their roles in FN-based matrix assembly
prior to angiogenesis. However, adult skin only expresses FN in the basement membrane [29],
and FN-receptors like α5β1 integrin are not expressed at meaningful levels in the epidermis
[30]. Therefore, the lack of a phenotype in unchallenged adult skin of Y397E and Y397F FAK
mice is expected. Skin homeostasis unaltered in conditional Y397F and Y397E mutant mice https://doi org/10 1371/journal pone 0200558 g004 10 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 Role of FAK tyrosine 397 in development and skin encountered throughout the entire life span of FAK Y397F or Y397E mice indicating normal
hair follicle stem cell function. Histology revealed normal interfollicular epidermis without
sub-epidermal blistering or inflammatory infiltrates (Fig 4C). Immunolabeling of laminin 332,
a basement membrane marker, and involucrin, a marker of epidermal differentiation, also
demonstrated no obvious difference in epidermal or basement membrane architecture (Fig
4D and 4E). FAK mutant protein was expressed in keratinocytes and co-localized with paxillin
to focal adhesions (Fig 4F). The unaltered epidermal morphology and expected expression and
cellular localization of FAK Y397F and Y397E suggested that Y397 is not necessary for normal
epidermal function. This finding is in line with previously reported research, wherein FAK
was deleted in keratin-14-expressing basal keratinocytes, resulting in no deleterious phenotype epidermal function. This finding is in line with previously reported research, wherein FAK
was deleted in keratin-14-expressing basal keratinocytes, resulting in no deleterious phenotype
but rather tumor suppression [15]. PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 Discussion FAK has been shown to promote an immunosuppressive microenvironment that allows
cancerous cells to evade immune surveillance [7]. Conditional FAK knock-out reduces the
risk of carcinogenesis in breast and skin tissue [14, 15]. FAK Y397 drives FN-type gene expres-
sion [10], which in turn protects the stem cell niche [31]. Cancer stem cells overexpress FAK, PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 11 / 14 Role of FAK tyrosine 397 in development and skin where it induces stem cell self-renewal and metastasis [32]. FAK likely promotes ECM remod-
eling and contributes to the pathogenesis of cancer and other prevalent diseases. Future
research must provide additional detail on the role of FAK in subcellular compartments that
sustain the FAK-induced ECM remodeling response such as the nucleus. S1 Dataset. List of genes induced by FAK variants as determined by total RNA sequencing.
(XLSX) S1 Dataset. List of genes induced by FAK variants as determined by total RNA sequencing. (XLSX) Supporting information S1 Fig. Reduced cell spreading in primary FAK Y397F MEFs. (A) Wild-type and FAK
Y397F mutant MEFs were plated on plastic and monitored by live cell imaging over time. Mutant MEFs were delayed in their ability to spread after attachment. (B-C) Automated quan-
tification of phase object confluence (B) and average phase object area (C) based on a pre-
defined confluence mask. Both outcome measures confirm the delayed spreading and phase
confluence as observed on images (mean ± SD, n = 24; P < 0.001, Student’s t test; n.s., not sig-
nificant; h, hours). (T
) S2 Fig. Y397E FAK MEFs exhibit gene expression closer to WT MEFs. (A) Logarithmic
change in gene expression of KO, Y397F FAK, and Y397E FAK embryos normalized to wild-
type expression. KO/WT expression shown in blue, Y397F FAK/WT expression shown in red,
and Y397E/WT expression shown in green. KO, knock-out; WT, wild-type. (B) Expression of
FN-associated genes in KO, Y397F FAK, and Y397E FAK embryos normalized to WT expres-
sion, with blue indicating decreased expression and red indicating increased expression. FAK
Y397F but not Y397E embryos resembled that of fibronectin (FN)-deficient embryos10,18. We therefore hypothesized that FAK Y397F may impact FN-type gene expression to a greater
extent than FAK Y397E. FC, fold change. (TIF) References 1. Parsons JT. Focal adhesion kinase: the first ten years. J Cell Sci. 2003; 116(8):1409–16. https://doi.org/
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acts with FAK proline-rich region 1 to induce fibronectin-type matrix. Proc Natl Acad Sci U S A. 2017;
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enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature. 1995; 377
(6549):539–44. https://doi.org/10.1038/377539a0 PMID: 7566154 12. Shen T-L, Park AY-J, Alcaraz A, Peng X, Jang I, Koni P, et al. Conditional knockout of focal adhesion
kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogen-
esis. J Cell Biol. 2005; 169(6):941–52. https://doi.org/10.1083/jcb.200411155 PMID: 15967814 13. Corsi JM, Houbron C, Billuart P, Brunet I, Bouvree K, Eichmann A, et al. Autophosphorylation-indepen-
dent and -dependent functions of focal adhesion kinase during development. J Biol Chem. 2009; 284
(50):34769–76. https://doi.org/10.1074/jbc.M109.067280 PMID: 19776009 14. Author Contributions Conceptualization: Ralph T. Bo¨ttcher, Alexander Meves. Conceptualization: Ralph T. Bo¨ttcher, Alexander Meves. Data curation: Joel B. Heim, Cera A. McDonald, Saranya P. Wyles, Sindhuja Sominidi-Damo-
daran, Edwin J. Squirewell, Ming Li, Catherine Motsonelidze, Ralph T. Bo¨ttcher, Alexander
Meves. Formal analysis: Joel B. Heim, Cera A. McDonald, Ming Li, Ralph T. Bo¨ttcher, Alexander
Meves. Funding acquisition: Alexander Meves. Investigation: Joel B. Heim, Cera A. McDonald, Saranya P. Wyles, Sindhuja Sominidi-Damo-
daran, Edwin J. Squirewell, Ming Li, Catherine Motsonelidze, Ralph T. Bo¨ttcher, Alexander
Meves. Methodology: Joel B. Heim, Cera A. McDonald, Ming Li, Ralph T. Bo¨ttcher, Alexander
Meves. Project administration: Alexander Meves. Resources: Ralph T. Bo¨ttcher, Jan van Deursen, Alexander Meves. Supervision: Alexander Meves. 12 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0200558
July 12, 2018 Role of FAK tyrosine 397 in development and skin Visualization: Alexander Meves. Writing – original draft: Joel B. Heim, Cera A. McDonald, Ralph T. Bo¨ttcher, Alexander
Meves. Writing – review & editing: Joel B. Heim, Cera A. McDonald, Ralph T. Bo¨ttcher, Alexander
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adhesion kinase suppresses tumor formation and blocks malignant progression. Genes Dev. 2004;
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appropriate for tissue specific or generalized cre mediated recombination. Genesis. 2004; 39(1):52–7. https://doi.org/10.1002/gene.20025 PMID: 15124227 18. Yokomizo T, Yamada-Inagawa T, Yzaguirre AD, Chen MJ, Speck NA, Dzierzak E. Whole-mount three-
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promote skin tumorigenesis independent of their phosphorylation. Proc Natl Acad Sci U S A. 2011;
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a Risk Factor for Sentinel Lymph Node Metastasis in Primary Cutaneous Melanoma. J Clin Oncol. 2015; 33(23):2509–15. https://doi.org/10.1200/JCO.2014.60.7002 PMID: 26150443 21. Lim ST, Chen XL, Tomar A, Miller NLG, Yoo J, Schlaepfer DD. Knock-in mutation reveals an essential
role for focal adhesion kinase activity in blood vessel morphogenesis and cell motility-polarity but not
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FAK and Src adhesion signaling occurs independently of adhesion complex composition. J Cell Biol. 2016; 212(3):349–64. https://doi.org/10.1083/jcb.201508080 PMID: 26833789 24. Dephoure N, Gould KL, Gygi SP, Kellogg DR. Mapping and analysis of phosphorylation sites: a quick
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fibronectin matrix and fibrillar adhesions. J Cell Sci. 2004; 117(2):177–87. 29. Coolen NA, Schouten KC, Middelkoop E, Ulrich MM. Comparison between human fetal and adult skin. Arch Dermatol Res. 2010; 302(1):47–55. https://doi.org/10.1007/s00403-009-0989-8 PMID: 19701759 30. Lowell CA, Mayadas TN. Overview: studying integrins in vivo. Integrin and Cell Adhesion Molecules:
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English
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Telomere protein Rap1 is a charge resistant scaffolding protein in chromosomal bouquet formation
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BMC biology
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cc-by
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© 2015 Amelina et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Chromosomes reorganize in early meiotic prophase to form the so-called telomere bouquet. In fission
yeast, telomeres localize to the nuclear periphery via interaction of the telomeric protein Rap1 with the membrane
protein Bqt4. During meiotic prophase, the meiotic proteins Bqt1-2 bind Rap1 and tether to the spindle pole body
to form the bouquet. Although it is known that this polarized chromosomal arrangement plays a crucial role in
meiotic progression, the molecular mechanisms of telomere bouquet regulation are poorly understood. Results: Here, we detected high levels of Rap1 phospho-modification throughout meiotic prophase, and identified
a maximum of 35 phosphorylation sites. Concomitant phosphomimetic mutation of the modification sites suggests
that Rap1 hyper-phosphorylation does not directly regulate telomere bouquet formation or dissociation. Despite
the negative charge conferred by its highly phosphorylated state, Rap1 maintains interactions with its binding
partners. Interestingly, mutations that change the charge of negatively charged residues within the Bqt1-2 binding
site of Rap1 abolished the affinity to the Bqt1-2 complex, suggesting that the intrinsic negative charge of Rap1 is
crucial for telomere bouquet formation. Conclusions: Whereas Rap1 hyper-phosphorylation observed in meiotic prophase does not have an apparent role
in bouquet formation, the intrinsic negative charge of Rap1 is important for forming interactions with its binding
partners. Thus, Rap1 is able to retain bouquet formation under heavily phosphorylated status. Keywords: Telomere, Shelterin complex, Schizosaccharomyces pombe, Phosphorylation, Meiosis studies suggest that the bouquet also plays a crucial role
in meiotic spindle formation [5]. Amelina et al. BMC Biology (2015) 13:37
DOI 10.1186/s12915-015-0149-x Amelina et al. BMC Biology (2015) 13:37
DOI 10.1186/s12915-015-0149-x Open Access Telomere protein Rap1 is a charge resistant
scaffolding protein in chromosomal
bouquet formation Hanna Amelina, Shaan Subramaniam, Vera Moiseeva, Christine Anne Armstrong, Siân Rosanna Pearson
and Kazunori Tomita* a Amelina, Shaan Subramaniam, Vera Moiseeva, Christine Anne Armstrong, Siân Rosanna Pears
Kazunori Tomita* * Correspondence: k.tomita@ucl.ac.uk
Chromosome Maintenance Group, UCL Cancer Institute, University College
London, London WC1E 6DD, UK Background Telomeres are specialized nucleoprotein structures that
form the natural ends of linear chromosomes. While
telomeres are mostly known for their essential function
in chromosome maintenance, they also play an important
role in meiotic progression [1]. During meiotic prophase,
the position of chromosomes within the nucleus is dra-
matically reorganized and telomeres cluster within a lim-
ited area of the nuclear envelope to form the so-called
telomere bouquet [2, 3]. This conserved reorganization of
chromosomes during meiotic prophase has been observed
in most eukaryotic organisms and is shown to promote
homolog pairing and meiotic recombination [4]. Recent In fission yeast Schizosaccharomyces pombe, the telo-
mere bouquet is observed throughout meiotic prophase. This period is also known as the ‘horsetail nucleus’ stage,
during which the nucleus elongates and oscillates back
and forth between the cell poles, following the spindle
pole body or SPB (the yeast equivalent of the centro-
some) driven by cytoplasmic microtubules [6]. Bouquet
formation is achieved by expression of a pair of meiosis-
specific proteins, Bqt1 and Bqt2, which bridge the telo-
meric proteins Taz1 and Rap1 to the SPB component
Sad1 [7]. To ensure telomere attachment to the SPB, telo-
meres must be tethered to the nuclear envelope via the
interaction between Rap1 and the inner nuclear mem-
brane complex Bqt3 and Bqt4 [8]. Disruption of any of * Correspondence: k.tomita@ucl.ac.uk
Chromosome Maintenance Group, UCL Cancer Institute, University College
London, London WC1E 6DD, UK Amelina et al. BMC Biology (2015) 13:37 Page 2 of 9 during meiosis, Rap1 was endogenously tagged with PK
(V5) epitope peptide and detected by anti-V5 anti-
bodies. Western blotting analysis of synchronous cul-
ture extracts showed that Rap1 protein is rather stably
expressed during meiosis, although lower molecular
weight, potentially truncated forms of Rap1, were ob-
served at the end of meiosis (Fig. 1c, top panel). these components leads to failure of telomere clustering
and defective spore formation in meiosis [7–11]. Sporula-
tion defects in the bouquet mutants occur mainly due to
impaired spindle formation and partly due to compro-
mised meiotic centromere assembly, followed by chromo-
some segregation defects [5, 12]. Recent studies suggest
that recruitment of a single telomere tract or centromere
to the SPB is sufficient to confer functional spindles
[13, 14]. Background Hence, the telomere bouquet does not only func-
tion in alignment of chromosomes but is also crucial for
the recruitment of a chromosome to the SPB to create a
functional meiotic spindle. Interestingly, a number of distinctly shifted bands of
Rap1 were detected during meiotic prophase. Similar
shifted bands of Rap1 have also been recently reported
[18]. To determine if Rap1 is phosphorylated during
meiosis, cell extracts were further analyzed using Phos-
tag™SDS-PAGE [19]. Phos-tag™gel analysis revealed that
the Rap1 protein is highly phosphorylated during mei-
osis. Strikingly, the maximum level of phosphorylation
was observed at 4.5–5 hr, when almost none of the fast-
migrating forms of Rap1 were detected (Fig. 1d). Phos-
phatase treatment confirmed that the shifted bands ob-
served at 4.5–5 hr represented phosphorylated forms of
Rap1 (Fig. 1e). Thus, our data indicates that Rap1 phos-
phorylation accumulates as meiotic prophase progresses,
and Rap1 becomes hyper-phosphorylated at the onset of
meiosis I, when the bouquet stage ends [5]. p
Although the bouquet composition and its function
are becoming better understood, the molecular mecha-
nisms that govern dissociation of telomeres from the
SPB remain elusive. Interestingly, in fission yeast, polar-
ized bouquet configuration is maintained until the end
of meiotic prophase, and upon entry into the first mei-
otic division, telomeres dissociate from the SPB in a con-
certed manner, dubbed ‘telomere fireworks’ [5]. Another
interesting observation is that Bqt1 and Bqt2 do not
localize to telomeres at the onset of the first meiotic div-
ision [7]. Moreover, Rap1 is highly phosphorylated in mi-
totic cells [15]. We therefore hypothesized that disruption
of the interaction between Rap1 and the Bqt1-2 complex,
potentially by means of post-translational modifications of
one or both interacting partners, may be responsible for
telomere dissociation from the SPB. In this report, we in-
vestigated whether Rap1 is involved in the termination of
the telomere bouquet. Comprehensive phosphoproteomic
analysis of the meiotic Rap1 protein revealed that it is pro-
gressively phosphorylated throughout meiotic prophase
and the number of phosphosites peaks after completion of
the bouquet stage. Surprisingly, this massive phosphoryl-
ation of Rap1 is dispensable for telomere bouquet dissoci-
ation, as indicated by our live microscopy analysis and
protein interaction studies of the phospho-mutants. Our
study illuminates that Rap1 is a protein resistant to nega-
tive charge and functions as a ‘scaffolding’ protein in the
telomere bouquet. Mass spectrometry analysis of Rap1 reveals an increasing
number of phosphosites detected upon completion of
the bouquet stage To determine the location of phosphorylation sites in
Rap1, meiotic Rap1 was purified from fractions of the
synchronized culture at 3.5 hr and 4.5 hr, and was
subjected to mass spectrometry analysis. Using trypsin
digestion, we covered 70–75% of the Rap1 protein se-
quence at 95% peptide threshold, and identified 19 and
35 phosphorylation sites from 3.5 hr and 4.5 hr, respect-
ively (Additional file 1). Notably, all phosphorylated sites
identified at 3.5 hr were also detected at 4.5 hr, suggest-
ing that Rap1 phosphorylation accumulates with pro-
gression of meiotic prophase. Our analysis revealed
several meiosis-specific phosphorylation sites in addition
to those detected in mitosis-arrested cells [15]. With
respect to known protein binding domains of Rap1
[15, 20], the phosphorylated sites at 3.5 hr (early prophase)
fell into three clusters, whereas at 4.5 hr phosphosites
were fairly evenly distributed across Rap1 (Fig. 2). Interest-
ingly, two and six phosphosites were identified in the
Bqt1-2 binding area (311–370 amino acids) at 3.5 hr and
4.5 hr, respectively. Notably, although phosphorylations
were detected within the Bqt4 and Poz1 binding regions,
we did not detect any phosphorylation within known
structural domains of Rap1. Altogether, our mass spec-
trometry data suggest that the number of phosphorylated
residues of Rap1 increases with the progression of meiosis,
which is in agreement with our Phos-tag™gel analysis
(Fig. 1d). Rap1 is hyper-phosphorylated in meiosis
h
b l
f h e Treatment of Rap1-3xPK from the 4.5
hr fraction with lambda-phosphatase and/or phosphatase inhibitors as a control. Note that fast-migrating bands of Rap1 are observed in this case
due to the presence of endogenous phosphatases Fig. 1 Rap1 is hyper-phosphorylated in meiosis. a Schematic diagram of meiotic culture synchronization using homozygous diploid cells carrying
the temperature-sensitive pat1-114 mutation and the mat-Pc cassette. b Distribution graph of the number of nuclei in meiocytes through meiosis
(top left) and images of DAPI-stained cells from indicated fractions (bottom left). FACS analysis shows DNA duplication from 2C to 4C (right). c,d Western blot analysis of Rap1-3xPK from mitotic cycling cells (mit), G1 arrested cells (time 0) and meiotic cell fractions at indicated times. Anti-Cdc2 (CDK) and anti-Cdc13 (Cyclin B) antibodies were used as a loading control and meiosis synchronicity marker, respectively. c Separation
of cell extracts on a standard gradient gel. d Separation of phosphorylated Rap1-3xPK on a Phos-tag gel. e Treatment of Rap1-3xPK from the 4.5
hr fraction with lambda-phosphatase and/or phosphatase inhibitors as a control. Note that fast-migrating bands of Rap1 are observed in this case
due to the presence of endogenous phosphatases Fig. 1 Rap1 is hyper-phosphorylated in meiosis. a Schematic diagram of meiotic culture synchronization using homozygous diploid cells carrying
the temperature-sensitive pat1-114 mutation and the mat-Pc cassette. b Distribution graph of the number of nuclei in meiocytes through meiosis
(top left) and images of DAPI-stained cells from indicated fractions (bottom left). FACS analysis shows DNA duplication from 2C to 4C (right). c,d Western blot analysis of Rap1-3xPK from mitotic cycling cells (mit), G1 arrested cells (time 0) and meiotic cell fractions at indicated times. Anti-Cdc2 (CDK) and anti-Cdc13 (Cyclin B) antibodies were used as a loading control and meiosis synchronicity marker, respectively. c Separation
of cell extracts on a standard gradient gel. d Separation of phosphorylated Rap1-3xPK on a Phos-tag gel. e Treatment of Rap1-3xPK from the 4.5
hr fraction with lambda-phosphatase and/or phosphatase inhibitors as a control. Note that fast-migrating bands of Rap1 are observed in this case
due to the presence of endogenous phosphatases Rap1 is hyper-phosphorylated in meiosis
h
b l
f h To investigate the stability of the Rap1 protein through-
out meiosis, a homozygous diploid (h−/h−) temperature-
sensitive pat1-114 strain carrying a mat-Pc cassette
was utilized to synchronize meiosis [16, 17]. Meiosis
was induced after nitrogen starvation, followed by a
temperature shift from permissive (26°C) to restrictive
conditions (34°C) (Fig. 1a). Progression of meiosis was
monitored by assessing the number of nuclei and DNA
content per cell from fractions collected at 30-minute
or 1-hour intervals during the synchronization proced-
ure (Fig. 1b). In order to assess Rap1 protein stability Page 3 of 9 Amelina et al. BMC Biology (2015) 13:37 C
A
E
D
B
Fig. 1 Rap1 is hyper-phosphorylated in meiosis. a Schematic diagram of meiotic culture synchronization using homozygous diploid cells carrying
the temperature-sensitive pat1-114 mutation and the mat-Pc cassette. b Distribution graph of the number of nuclei in meiocytes through meiosis
(top left) and images of DAPI-stained cells from indicated fractions (bottom left). FACS analysis shows DNA duplication from 2C to 4C (right). c,d Western blot analysis of Rap1-3xPK from mitotic cycling cells (mit), G1 arrested cells (time 0) and meiotic cell fractions at indicated times. Anti-Cdc2 (CDK) and anti-Cdc13 (Cyclin B) antibodies were used as a loading control and meiosis synchronicity marker, respectively. c Separation
of cell extracts on a standard gradient gel. d Separation of phosphorylated Rap1-3xPK on a Phos-tag gel. e Treatment of Rap1-3xPK from the 4.5
hr fraction with lambda-phosphatase and/or phosphatase inhibitors as a control. Note that fast-migrating bands of Rap1 are observed in this case
due to the presence of endogenous phosphatases C A C E
D B B E
D D E Fig. 1 Rap1 is hyper-phosphorylated in meiosis. a Schematic diagram of meiotic culture synchronization using homozygous diploid cells carrying
the temperature-sensitive pat1-114 mutation and the mat-Pc cassette. b Distribution graph of the number of nuclei in meiocytes through meiosis
(top left) and images of DAPI-stained cells from indicated fractions (bottom left). FACS analysis shows DNA duplication from 2C to 4C (right). c,d Western blot analysis of Rap1-3xPK from mitotic cycling cells (mit), G1 arrested cells (time 0) and meiotic cell fractions at indicated times. Anti-Cdc2 (CDK) and anti-Cdc13 (Cyclin B) antibodies were used as a loading control and meiosis synchronicity marker, respectively. c Separation
of cell extracts on a standard gradient gel. d Separation of phosphorylated Rap1-3xPK on a Phos-tag gel. Hyper-phosphorylation of Rap1 in meiosis is dispensable
for telomere bouquet formation and dissociation BRCT, BRCA1 C-terminus domain; Myb domain, Myb-like domain; RCT,
Rap1 C-terminus domain Fig. 2 Domain organization and schematic of phosphorylation sites of Rap1 protein detected at 3.5 hr and 4.5 hr into meiosis. Phosphorylation
sites are highlighted as bars with a colour code (yellow, less than 10%; orange, 10–50%; and red, over 50%). Protein interaction domains are
indicated above and the structural domains are shown at the bottom. BRCT, BRCA1 C-terminus domain; Myb domain, Myb-like domain; RCT,
Rap1 C-terminus domain was impaired in the rap1 phospho-mutants. Since C-
terminus tagging of Rap1 impaired telomere length
homeostasis (Fig. 3e), the PK epitope tag was fused to the
N-terminus. Although phosphomimetic forms of the Rap1
protein (Rap1-32E and 17E) migrate slower than wild-type
Rap1, none of the cluster mutations affected protein
stability (Fig. 3f). The strains carrying mutant Rap1 main-
tained their telomere length comparable to that of wild-
type (Fig. 3e). Accordingly, all mutants retained their
ability to interact with Poz1 by the yeast two-hybrid assay
(Fig. 3g). Additionally, both 32A and 32E mutant forms of
Rap1 retained the ability to interact with Bqt4 (Fig. 3h). Indeed, telomere localization to the nuclear periphery in
interphase was not impaired in rap1-32A and 32E mu-
tants (Additional file 3). Thus, hyper-phosphorylation of
Rap1 observed in meiosis does not appear to have a role
in telomere bouquet regulation. Furthermore, our muta-
genesis analysis suggests that Rap1 is able to resist high
negative charge changes without affecting its function in
meiosis or telomere length homeostasis. its ability to interact with Bqt4 and Poz1 in yeast two-
hybrid assay (Fig. 3d,g,h). To study the function of Rap1-DD337AA, endogenous
rap1 was mutated and fused to YFP. Accordingly, rap1-
DD337AA mutants were defective in sporulation (Figs. 3c
and 4b). Live cell imaging of the mutant showed that
Rap1-DD337AA localized to telomeres, as determined
by co-localization to Taz1 (Fig. 4c), but did not cluster at
the SPB in meiotic prophase (Fig. 4d). Furthermore, in
many cases the SPB was destabilized and detached from
the nucleus and, as a consequence, aberrant chromo-
some segregation was observed (Fig. 4d). These meiotic
phenotypes are characteristic of rap1Δ mutants as well
as the bouquet-defective mutants [5]. However, Rap1-
DD337AA was stably expressed and telomere length of
the rap1-DD337AA mutant was the same as that of
wild-type (Fig. 3e,f). Hyper-phosphorylation of Rap1 in meiosis is dispensable
for telomere bouquet formation and dissociation Hyper-phosphorylation of Rap1 in meiosis is dispensable
for telomere bouquet formation and dissociation extracts confirmed that the mutant forms of Rap1 were
stably expressed, and the phospho-modification of Rap1-
32A was significantly reduced (Additional file 2). Finally,
our yeast two-hybrid assay confirmed that the Bqt1/2 bind-
ing domain of Rap1 falls within 216–388 amino acids, and
introduced cluster mutations did not affect its interaction
with the Bqt1-2 complex (Fig. 3d). Since we observed that Rap1 phosphorylation peaks at
meiosis I, we speculated that the resulting highly nega-
tive charge of Rap1 is responsible for the change in its
affinity to the Bqt1-2 complex. In order to mimic hyper-
phosphorylated Rap1, all validated phosphorylation sites
from S-212 to S-562 were substituted with negatively
charged glutamate residues (rap1-32E) (Fig. 3a). To moni-
tor telomeres and the SPB through meiosis, endogenous
Taz1 and Sid4 were tagged with YFP and mCherry, respect-
ively. To our surprise, the phosphomimetic rap1-32E mu-
tants did not exhibit any detectable meiotic defects and
their telomeres clustered and dissociated from the SPB in a
timely manner very similar to that of the wild-type (Fig. 3b). Accordingly, rap1-32E mutants exhibited no sporulation
defects (Fig. 3c). The corresponding non-phosphorylatable
mutant form of Rap1 (rap1-32A) also did not cause defects
in meiotic progression and telomere bouquet behaviour
(Fig. 3a,b,c). Western blot analysis from meiotic cell Suspecting that some phospho-modifications might
remain unidentified in our study, five additional serine
and threonine residues (S-317, T-321, S-322, T-328 and
S-364), along with 12 detected phosphosites within and
adjacent to the Bqt1/2 binding domain, were all substituted
to glutamate (rap1-17E) or alanine (rap1-17A) (Fig. 3a). However, these mutations also did not cause any defects in
meiosis (Fig. 3b,c). Thus, we conclude that accumulation
of negative charge at the Bqt1-2 binding domain of Rap1
does not affect its ability to form the bouquet. Because rap1-32A and rap1-32E bear mutations within
the binding domain of the telomerase negative regulator
Poz1 [21], we checked whether telomere length regulation Amelina et al. BMC Biology (2015) 13:37 Page 4 of 9 Fig. 2 Domain organization and schematic of phosphorylation sites of Rap1 protein detected at 3.5 hr and 4.5 hr into meiosis. Phosphorylation
sites are highlighted as bars with a colour code (yellow, less than 10%; orange, 10–50%; and red, over 50%). Protein interaction domains are
indicated above and the structural domains are shown at the bottom. Hyper-phosphorylation of Rap1 in meiosis is dispensable
for telomere bouquet formation and dissociation Additionally, telomeres of the mutant
cells were retained at the nuclear periphery in interphase
(Additional file 3). Thus, rap1-DD337AA is a meiosis-
specific loss-of-function mutation, and negatively charged
aspartates at positions 337 and 338 are crucial for bouquet
formation. Discussion 3 Rap1 hyper-phosphorylation in meiosis is dispensable for telomere bouquet clustering/dissociation. a Schematic of phosphomimetic and
unphosphorylatable cluster mutants of Rap1 created and analyzed in this study. Additional mutation sites introduced in 17E/A (see text) are
highlighted in red. b Series of frames from films of meiosis. The SPB and telomeres were observed via endogenously tagged Sid4-mCherry and
Taz1-YFP, respectively. Time count starts from the beginning of filming. Scale bar equals 2 μm. Example of defective meiotic SPB is shown in
rap1Δ. None of the rap1 cluster mutants exhibit defective SPB (examined cell number of indicated strains is more than 20). c Frequency of normal
four-spore asci in rap1 phosphomutants. Zygotic asci generated from the indicated genotypes in an h90 (homothallic) background were scored
by light microscopy. Two hundred asci per genotype were counted in each experiment. Data represent the average of three experiments. Error
bars indicate standard deviations. d,g,h Yeast two-hybrid analysis of the interaction between mutant Rap1 and the (d) Bqt1-2 fusion protein,
(g) Poz1 and (h) Bqt4. e Telomere lengths of the rap1 phosphomutants. Telomere Southern blot of genomic DNA digested with EcoRI and
hybridized with a telomeric probe. A fragment of the SafeView Nucleic Acid Stain stained gel image at 2.5 kb is shown below the blots as a
loading control. f Protein expression levels of the N-terminal PK-tagged mutant Rap1. SPB, spindle pole body chromosomal bouquet configuration, the telomere-bound
proteins are likely to be exposed to these kinases. Additionally, DNA damage checkpoint kinases are acti-
vated during meiotic recombination [26]. In fact, some
phosphorylations originate from telomere-associated ki-
nases as indicated by reduced shifted bands of Rap1 at
4–4.5 hr in the absence of Taz1 (Additional file 4). Never-
theless, our study suggests that the hyper-phosphorylation
of Rap1 observed during meiosis is not directly involved
in the regulation of the bouquet. Rap1 can withstand sig-
nificant charge changes that do not affect interactions
with its binding partners and its function in meiosis. Hence, rather than a functional regulatory protein, meiotic
Rap1 appears to be a ‘scaffolding’ protein, that is targeted
by multiple kinases. Interestingly, while mutating 32 phosphorylation sites did not alter Rap1 function, muta-
tion of only two highly conserved residues (D-337 and
D-338) disrupted its ability to bind to the Bqt1-2 complex,
causing pronounced defects in telomere clustering and
chromosome segregation. Discussion Rap1 protein is negatively charged, and the Bqt1/2
binding region is particularly rich in hydrophobic and
negatively charged amino acid residues. Some of these
negatively charged residues (D-335, D-337, D-338 and
E-342) are well-conserved among fission yeast species
(Fig. 4a). Importantly, mutation analysis indicated that
Rap1-DD337AA (D337A and D338A mutations) no
longer interacts with the Bqt1-2 complex, but retains In this study we have shown that the level of phosphor-
ylation of Rap1 gradually increases during the course of
meiotic prophase, peaking in meiosis I. The SPB is
known to recruit a number of kinases and phosphatases
that modify its subunits, and these modifications play a
critical role in regulating mitotic commitment and meiotic
progression [22–25]. Since telomere heterochromatin
comes into close contact with the meiotic SPB during Page 5 of 9 Page 5 of 9 Amelina et al. BMC Biology (2015) 13:37 B
C
A
G
H
D
F
E
Fig. 3 Rap1 hyper-phosphorylation in meiosis is dispensable for telomere bouquet clustering/dissociation. a Schematic of phosphomimetic and
unphosphorylatable cluster mutants of Rap1 created and analyzed in this study. Additional mutation sites introduced in 17E/A (see text) are
highlighted in red. b Series of frames from films of meiosis. The SPB and telomeres were observed via endogenously tagged Sid4-mCherry and
Taz1-YFP, respectively. Time count starts from the beginning of filming. Scale bar equals 2 μm. Example of defective meiotic SPB is shown in
rap1Δ. None of the rap1 cluster mutants exhibit defective SPB (examined cell number of indicated strains is more than 20). c Frequency of normal
four-spore asci in rap1 phosphomutants. Zygotic asci generated from the indicated genotypes in an h90 (homothallic) background were scored
by light microscopy. Two hundred asci per genotype were counted in each experiment. Data represent the average of three experiments. Error
bars indicate standard deviations. d,g,h Yeast two-hybrid analysis of the interaction between mutant Rap1 and the (d) Bqt1-2 fusion protein,
(g) Poz1 and (h) Bqt4. e Telomere lengths of the rap1 phosphomutants. Telomere Southern blot of genomic DNA digested with EcoRI and
hybridized with a telomeric probe. A fragment of the SafeView Nucleic Acid Stain stained gel image at 2.5 kb is shown below the blots as a
loading control. f Protein expression levels of the N-terminal PK-tagged mutant Rap1. SPB, spindle pole body E B
C
A D D E A B G G H F H C C F Fig. Discussion In contrast, the binding part-
ners Bqt1 and Bqt2 are positively charged, suggesting that
Rap1 binds to the Bqt1-2 complex through hydrogen
bonding interactions. Thus, we speculate that negatively
charged residues at Rap1’s interaction surfaces are evolu-
tionarily conserved in order to retain affinity under shifts
in charge occurring throughout meiotic prophase. Rap1 is also highly phosphorylated in the mitotic cell
cycle, particularly in M-phase. Among the phosphoryl-
ation sites reported for mitotic Rap1, five phosphoryl-
ation sites (S-213, T-378, S-422, S-456 and S-513) were Page 6 of 9 Amelina et al. BMC Biology (2015) 13:37 A
B
C
D
Fig. 4 Negative charge of Rap1 Bqt1/2 binding domain is important for functional telomere bouquet. a Alignment of Rap1 protein sequences
from different fission yeast species, highlighting a highly conserved area within Rap1 (performed using Clustal Omega; EMBL-EBI, Cambridge,
UK). Sp, Schizosaccharomyces pombe; Scr, S. cryophilus; So, S. octosporus; Sj, S. japonicus. b Morphology of zygotic asci of rap1-DD337AA mutant
compared to cells expressing wild-type Rap1. Frequency of normal four-spore asci is shown in Fig. 3b. c Mitotic cells expressing Rap1-YFP or
Rap1(DD337AA)-YFP (green in the merged pictures) and Taz1-mCherry (red in the merged pictures). Both wild-type Rap1 and Rap1-DD337AA
co-localize with Taz1-mCherry. Scale bar equals 5 μm. d Series of frames from films of meiosis. The SPB, telomeres and chromosomes were
observed via endogenously tagged Sid4-mCherry, Rap1-YFP and Hht1-Cerulean, respectively. Time count starts from the beginning of filming. Scale bar equals 2 μm. Top image, wild-type cells; and bottom four images, examples of defective meiosis in rap1-DD337AA. Note that rap1-DD337AA
meiosis is reminiscent of rap1Δ meiosis shown in Fig. 3b A
D D A B B C Fig. 4 Negative charge of Rap1 Bqt1/2 binding domain is important for functional telomere bouquet. a Alignment of Rap1 protein sequences
from different fission yeast species, highlighting a highly conserved area within Rap1 (performed using Clustal Omega; EMBL-EBI, Cambridge,
UK). Sp, Schizosaccharomyces pombe; Scr, S. cryophilus; So, S. octosporus; Sj, S. japonicus. b Morphology of zygotic asci of rap1-DD337AA mutant
compared to cells expressing wild-type Rap1. Frequency of normal four-spore asci is shown in Fig. 3b. c Mitotic cells expressing Rap1-YFP or
Rap1(DD337AA)-YFP (green in the merged pictures) and Taz1-mCherry (red in the merged pictures). Both wild-type Rap1 and Rap1-DD337AA
co-localize with Taz1-mCherry. Scale bar equals 5 μm. d Series of frames from films of meiosis. Telomere Southern blotting Southern blotting was performed as described previously
[27]. Equal amounts of EcoRI-digested DNA fragments
were separated on a 1% agarose gel and subjected to
Southern blotting with a telomere probe. Discussion BMC Biology (2015) 13:37 Page 7 of 9 phosphorylations among them) counteracts Cdc2 kinase
action to preserve affinity to Bqt4, and thereby maintain
telomere localization to the nuclear membrane and bou-
quet configuration. media containing a nitrogen source and incubated for
24 hr until mid-log phase (OD = 0.5–0.7). The cells were
next transferred to EMM media without a nitrogen
source (EMM-N) by filtering, and cells were incubated
for another 15–16 hr to arrest cells at G1 phase. To in-
activate the pat1 kinase gene and induce meiosis, the
temperature was shifted up to 34°C, cultures were sup-
plemented with one-fifth volume of EMM media pre-
warmed to 34°C, and meiotic fractions were collected at
the required time point. Western blotting and Phos-tag gel g
g g
Whole-cell protein extracts prepared using a trichloroacetic
acid method were separated by SDS-PAGE using 10%
acrylamide gels. Western blotting was performed with anti-
V5 peptide (Bio-Rad, Hercules, CA, USA), anti-Cdc2 (Santa
Cruz Biotechnology, Dallas, TX, USA) and anti-Cdc13
(Santa Cruz Biotechnology) antibodies following a stand-
ard protocol. For detection of phosphorylated Rap1 forms,
7.5% acrylamide gels were supplemented with 25 μM
PhosTag ligand (AAL-107; NARD Institute, Amagasaki,
Japan) and 50 μM MnCl2, according to the protocol [19]. A Phos-tag gel was treated with 1 mM EDTA prior to
transfer. Two technical replicates of the Western blotting
image in Fig. 1c are shown in Additional file 6. Phosphatase treatment of native cell extracts Native whole-cell extracts were prepared using modi-
fied HB buffer (50 mM HEPES-KOH, pH 7.5; 140 mM
NaCl, 0.1% NP-40; 1 mM MnCl2). Then 20 μl of lysate
was treated with 4 μl of lambda-phosphatase; 10 μl of Preparation of cell extracts and immunoprecipitation for
mass spectrometry Six litres of synchronous meiotic cells expressing Rap1-
3xPK were harvested at 3.5 hr (early prophase) or 4.5 hr
(late prophase) following the pat1-114 synchronization
protocol as described above. Rap1-3xPK was immuno-
precipitated from native cell extracts in RIPA buffer
(50 mM Tris–HCl, pH 8; 150 mM NaCl; 1% NP-40;
0.5% sodium deoxycholate, and 0.1% SDS), supple-
mented with 1X PhosSTOP (Roche, Basel, Switzerland);
1× cOmplete EDTA-free protease inhibitor (Roche); 1 mM
PMSF, 1 mM DTT, 0.1 ng/mL MG132 (Sigma-Aldrich, St
Louis, MO); and 10 U/mL TURBO DNase (Ambion, Life
Technologies, Carlsbad, CA, USA), using Dynabeads M-
270 epoxy (Life Technologies) pre-coated with anti-V5
peptide antibodies. Yeast genetics and plasmids The genotypes of the strains used for this study are listed
in Additional file 5. All media and supplements were pur-
chased from Formedium™(Hunstanton, UK). Fission yeast
was grown at 32°C in standard rich media (YES) unless in-
dicated. Epitope tag insertion at the C-terminus was de-
scribed previously [27]. A plasmid for N-tagged rap1 was
constructed by cloning the rap1 gene, including 1,360
bases of the upstream and 1,500 bases of the downstream
regions, with a primer set including a KpnI site. The start
codon of rap1+ was replaced with a single V5 (PK) se-
quence. A kanMX6 cassette was inserted 700 bases up-
stream of the gene. The resulting plasmid pRap1a-nPK
was digested with KpnI and replaced the ura4+ cassette at
the rap1 gene locus in rap1Δ cells. The transformants
were backcrossed with a wild-type strain and cultured for
2 weeks before analysis of telomere length. To generate
rap1 mutants, cluster-mutated rap1 gBlocks were synthe-
sized (Integrated DNA Technologies, Coralville, IA, USA)
and replaced wild-type rap1+ in the pRap1a-nPK plasmid
and the yeast two-hybrid pGAD and pGBK plasmids. Yeast two-hybrid assays The assay was conducted according to the Matchmaker
Gold
Yeast
Two-Hybrid
System
manual
(Clontech,
Mountain View, CA, USA). Expression vectors for the
GAL binding domain (BD) fused proteins and the GAL
activation domain (AD) fused proteins were generated
by subcloning of the indicated cDNAs into pGBK and
pGAD, respectively. Expression of BD and AD fused
proteins were confirmed by Western blotting using anti-
myc and anti-HA antibodies, respectively. To express
Bqt1 and Bqt2 together in the yeast two- hybrid plas-
mids, bqt2+ cDNA was inserted at the start codon of the
bqt1+ cDNA sequence; the resulting plasmid was named
pGBK-Bqt2-1. Conclusions Rap1 hyper-phosphorylation observed during meiotic
prophase does not have a direct role in telomere bou-
quet regulation. Rap1 uses its negatively charged amino
acid residues to bind the Bqt1-2 complex. Therefore the
interaction is not affected by changes in net charge caused
through progressive hyper-phosphorylation. Discussion The SPB, telomeres and chromosomes were
observed via endogenously tagged Sid4-mCherry, Rap1-YFP and Hht1-Cerulean, respectively. Time count starts from the beginning of filming. Scale bar equals 2 μm. Top image, wild-type cells; and bottom four images, examples of defective meiosis in rap1-DD337AA. Note that rap1-DD337AA
meiosis is reminiscent of rap1Δ meiosis shown in Fig. 3b Fig. 4 Negative charge of Rap1 Bqt1/2 binding domain is important for functional telomere bouquet. a Alignment of Rap1 protein sequences
from different fission yeast species, highlighting a highly conserved area within Rap1 (performed using Clustal Omega; EMBL-EBI, Cambridge,
UK). Sp, Schizosaccharomyces pombe; Scr, S. cryophilus; So, S. octosporus; Sj, S. japonicus. b Morphology of zygotic asci of rap1-DD337AA mutant
compared to cells expressing wild-type Rap1. Frequency of normal four-spore asci is shown in Fig. 3b. c Mitotic cells expressing Rap1-YFP or
Rap1(DD337AA)-YFP (green in the merged pictures) and Taz1-mCherry (red in the merged pictures). Both wild-type Rap1 and Rap1-DD337AA
co-localize with Taz1-mCherry. Scale bar equals 5 μm. d Series of frames from films of meiosis. The SPB, telomeres and chromosomes were
observed via endogenously tagged Sid4-mCherry, Rap1-YFP and Hht1-Cerulean, respectively. Time count starts from the beginning of filming. Scale bar equals 2 μm. Top image, wild-type cells; and bottom four images, examples of defective meiosis in rap1-DD337AA. Note that rap1-DD337AA
meiosis is reminiscent of rap1Δ meiosis shown in Fig. 3b and raises the possibility that telomeres remain associ-
ated with Bqt4 and the SPB via different mechanisms. Notably, four residues of Rap1 including Cdc2 targets
were also found to be phosphorylated throughout mei-
otic prophase in our study (except for S-456, detected
only at 4.5 hr) (Additional file 1). Unlike the Rap1-5E
mutant protein, our phosphomimetic cluster mutant
Rap1-32E, which includes 5E mutation (Fig. 3a), was
able to interact with Bqt4. Thus, we predict that pre-
meiotic hyper-phosphorylation of Rap1 (or particular shown to have an inhibitory effect on Rap1-Bqt4 inter-
action, which was demonstrated by the phosphomimetic
rap1-5D/5E mutants [15]. In mitosis, Cdc2 phosphory-
lates three of these sites in order to temporarily release
telomeres from the nuclear envelope. This mechanism
assists faithful chromosome segregation in anaphase. However, rap1-5D and rap1-5E mutants do not have any
sporulation defects [15], which suggests that the bouquet
is intact. This is surprising since the Rap1-Bqt4 inter-
action is required for telomere clustering in meiosis [8], Page 7 of 9 Amelina et al. References 1. Jain D, Cooper JP. Telomeric strategies: means to an end. Annu Rev Genet. 2010;44:243–69. 1. Jain D, Cooper JP. Telomeric strategies: means to an end. Annu Rev Genet. 2010;44:243–69. pat1-114 synchronization Cells were first cultured in YE media at 26°C overnight
until late stationery phase, then transferred to EMM Page 8 of 9 Page 8 of 9 Amelina et al. BMC Biology (2015) 13:37 Page 8 of 9 phosphatase inhibitors; both phosphatase and inhibi-
tors; or water as a control. The samples were incubated
at 30°C for 1 hour before being separated on SDS-
PAGE gel. Authors’ contributions HA carried out most of the experiments. HA and KT designed the study and
wrote manuscript. SS performed yeast two-hybrid analysis. SRP assisted with
yeast two-hybrid assays. VM assisted with live cell imaging. CAA supported
yeast genetics analysis. HA, KT, SS and VM generated strains and plasmids. Al
authors read and approved the final manuscript. HA carried out most of the experiments. HA and KT designed the study and
wrote manuscript. SS performed yeast two-hybrid analysis. SRP assisted with yeast two-hybrid assays. VM assisted with live cell imaging. CAA supported
yeast genetics analysis. HA, KT, SS and VM generated strains and plasmids. All
authors read and approved the final manuscript. Further details about microscopy and mass spectrom-
etry methods can be found in Additional file 7. A list of
phosphopeptides detected by mass spectrometry is shown
in Additional file 8. Acknowledgments We thank the Proteomics and Metabolomics Core Facility at the Institute for
Cancer Research (ICR) for mass spectrometry analysis of the samples and
technical advice on the sample preparation. We thank Junko Kanoh (Osaka
University, Japan) for critical reading before submission. This work is
supported mainly by the European Research Council (281722-HRMCB) and
partly by Cancer Research UK (C36439/A12097) and the Cancer Research
UK - UCL Centre. Open access of data partly by Cancer Research UK (C36439/A12097) and the Cancer Research
UK - UCL Centre. The mass spectrometry proteomics data have been
deposited to the ProteomeXchange consortium [1] via
the PRIDE partner repository with the dataset identifier
PXD001841. Received: 5 March 2015 Accepted: 4 June 2015 Received: 5 March 2015 Accepted: 4 June 2015 Additional files 2. Harper L, Golubovskaya I, Cande WZ. A bouquet of chromosomes. J Cell Sci. 2004;117:4025–32. Additional file 1: List of Rap1 phosphorylation sites identified in
the study. 3. Scherthan H. A bouquet makes ends meet. Nat Rev Mol Cell Biol. 2001;2:621–7. Additional file 2: Western blot images of TCA-extracted Rap1
proteins from meiotic cells. Indicated cells were induced to undergo
meiosis using the pat1-114 mutation, and were collected from the
4.5 hr fraction. Cell extracts were separated in regular gradient (left)
and Phos-tag SDS-PAGE (right), and subjected to Western blot using
anti-PK(V5) antibody. Unlike wild-type meiotic Rap1 that shows multiple
bands, Rap1-32A exhibits a single band in the Phos-tag gel, suggesting
the phospho-modifications were diminished in this mutant form. 4. Niwa O, Shimanuki M, Miki F. Telomere-led bouquet formation facilitates
homologous chromosome pairing and restricts ectopic interaction in fission
yeast meiosis. EMBO J. 2000;19:3831–40. y
5. Tomita K, Cooper JP. The telomere bouquet controls the meiotic spindle. Cell. 2007;130:113–26. 6. Chikashige Y, Ding DQ, Funabiki H, Haraguchi T, Mashiko S, Yanagida M,
et al. Telomere-led premeiotic chromosome movement in fission yeast. Science. 1994;264:270–3. 7. Chikashige Y, Tsutsumi C, Yamane M, Okamasa K, Haraguchi T, Hiraoka Y. Meiotic proteins bqt1 and bqt2 tether telomeres to form the bouquet
arrangement of chromosomes. Cell. 2006;125:59–69. Additional file 3: Localization of telomeres in mitotic interphase. (A)
Projected images of cells in mitotic interphase. Taz1-YFP, Sid4-mCherry and
Bqt3-Cerulean were used to visualize telomeres, spindle pole body and the
membrane, respectively. White and grey arrows indicate examples of
telomeres localized at the nuclear periphery and away from the nuclear
envelope, respectively. Scale bar equals 5 μm. (B) Percentages of telomeres
localized at the nuclear periphery in interphase cells. Cells containing one
nucleus and one Sid4-mCherry focus (not dissociated) were selected as
mitotic interphase cells. Telomeres were scored as localized at the nuclear
periphery when the distance between the brightest Taz1-YFP focus and
Bqt3-Cerulean-labeled nuclear membrane was less than 0.4 μm using
SoftWoRks (Applied Precision; GE Healthcare, Chalfont St Giles, UK). (C)
Middle z-section image of the cells in mitotic interphase. Example of
telomeres detached from the nuclear envelope (left) and telomeres
localized at the nuclear periphery (right). Scale bar equals 5 μm. g
8. Chikashige Y, Yamane M, Okamasa K, Tsutsumi C, Kojidani T, Sato M, et al. Membrane proteins Bqt3 and −4 anchor telomeres to the nuclear envelope
to ensure chromosomal bouquet formation. Additional files Abbreviations
AD: Activation domain; BD: Binding domain; SPB: Spindle pole body. Competing interests
The authors declare that they have no competing interests. 19. Kinoshita E, Kinoshita-Kikuta E, Takiyama K, Koike T. Phosphate-binding tag,
a new tool to visualize phosphorylated proteins. Mol Cell Proteomics. 2006;5:749–57. 20. Fujita I, Tanaka M, Kanoh J. Identification of the functional domains of the
telomere protein Rap1 in Schizosaccharomyces pombe. PLoS One. 2012;7, e49151. Additional files J Cell Biol. 2009;187:413–27. 8. Chikashige Y, Yamane M, Okamasa K, Tsutsumi C, Kojidani T, Sato M, et al. Membrane proteins Bqt3 and −4 anchor telomeres to the nuclear envelope
to ensure chromosomal bouquet formation. J Cell Biol. 2009;187:413–27. 9. Chikashige Y, Hiraoka Y. Telomere binding of the Rap1 protein is required
for meiosis in fission yeast. Curr Biol. 2001;11:1618–23. 9. Chikashige Y, Hiraoka Y. Telomere binding of the Rap1 prot
for meiosis in fission yeast. Curr Biol. 2001;11:1618–23. 10. Cooper JP, Watanabe Y, Nurse P. Fission yeast Taz1 protein is required for
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inactivation of Pat1 kinase proceeds with aberrant nuclear positioning of
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activation after recruitment in fission yeast. Curr Biol. 2014;24:2006–11. Submit your next manuscript to BioMed Central
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The Contribution of Liver Sinusoidal Endothelial Cells to Clearance of Therapeutic Antibody
|
Frontiers in physiology
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cc-by
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The Contribution of Liver Sinusoidal Endothelial
Cells to Clearance of Therapeutic Antibody
James, Bethany H.; Papakyriacou, Pantelitsa; Gardener, Matthew J.; Gliddon, Louise;
Weston, Christopher J.; Lalor, Patricia F.
DOI: Citation for published version (Harvard):
James, BH, Papakyriacou, P, Gardener, MJ, Gliddon, L, Weston, CJ & Lalor, PF 2022, 'The Contribution of Liver
Sinusoidal Endothelial Cells to Clearance of Therapeutic Antibody', Frontiers in Physiology, vol. 12, 753833. https://doi.org/10.3389/fphys.2021.753833 Link to publication on Research at Birmingham portal General rights
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•User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and P
•Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of th When citing, please reference the published version. The Contribution of Liver Sinusoidal
Endothelial Cells to Clearance of
Therapeutic Antibody Bethany H. James1, Pantelitsa Papakyriacou1, Matthew J. Gardener2, Louise Gliddon2,
Christopher J. Weston1 and Patricia F. Lalor1* Bethany H. James1, Pantelitsa Papakyriacou1, Matthew J. Gardener2, Louise Gliddon2,
Christopher J. Weston1 and Patricia F. Lalor1* 1 Centre for Liver and Gastroenterology Research and National Institute for Health Research (NIHR) Birmingham Biomedical
Research Centre, Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, United Kingdom,
2 Antibody Pharmacology, Biopharm Discovery, Glaxo Smith Kline Research and Development, Stevenage, United Kingdom Many chronic inflammatory diseases are treated by administration of “biological”
therapies in terms of fully human and humanized monoclonal antibodies or Fc
fusion proteins. These tools have widespread efficacy and are favored because they
generally exhibit high specificity for target with a low toxicity. However, the design of
clinically applicable humanized antibodies is complicated by the need to circumvent
normal antibody clearance mechanisms to maintain therapeutic dosing, whilst avoiding
development of off target antibody dependent cellular toxicity. Classically, professional
phagocytic immune cells are responsible for scavenging and clearance of antibody via
interactions with the Fc portion. Immune cells such as macrophages, monocytes, and
neutrophils express Fc receptor subsets, such as the FcγR that can then clear immune
complexes. Another, the neonatal Fc receptor (FcRn) is key to clearance of IgG in vivo
and serum half-life of antibody is explicitly linked to function of this receptor. The liver
is a site of significant expression of FcRn and indeed several hepatic cell populations
including Kupffer cells and liver sinusoidal endothelial cells (LSEC), play key roles in
antibody clearance. This combined with the fact that the liver is a highly perfused organ
with a relatively permissive microcirculation means that hepatic binding of antibody has
a significant effect on pharmacokinetics of clearance. Liver disease can alter systemic
distribution or pharmacokinetics of antibody-based therapies and impact on clinical
effectiveness, however, few studies document the changes in key membrane receptors
involved in antibody clearance across the spectrum of liver disease. Similarly, the
individual contribution of LSEC scavenger receptors to antibody clearance in a healthy or
chronically diseased organ is not well characterized. This is an important omission since
pharmacokinetic studies of antibody distribution are often based on studies in healthy
individuals and thus may not reflect the picture in an aging or chronically diseased
population. The Contribution of Liver Sinusoidal
Endothelial Cells to Clearance of
Therapeutic Antibody Therefore, in this review we consider the expression and function of key
antibody-binding receptors on LSEC, and the features of therapeutic antibodies which
may accentuate clearance by the liver. We then discuss the implications of this for the
design and utility of monoclonal antibody-based therapies. Take down policy down policy
the University of Birmingham exercises care and attention in making items available there are rare occasions when an it
ded in error or has been deemed to be commercially or otherwise sensitive. this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access
ely and investigate. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we
the work immediately and investigate. Download date: 24. Oct. 2024 REVIEW REVIEW
published: 14 January 2022
doi: 10.3389/fphys.2021.753833 Keywords: liver, endothelium, antibody, therapy, disease Edited by:
Natalia A. Osna,
University of Nebraska Medical
Center, United States Reviewed by:
Srivatsan Kidambi,
University of Nebraska-Lincoln,
United States
Manuel Romero-Gómez,
Seville University, Spain *Correspondence:
Patricia F. Lalor
p.f.lalor@bham.ac.uk Specialty section:
This article was submitted to
Gastrointestinal Sciences,
a section of the journal
Frontiers in Physiology a section of the journal
Frontiers in Physiology
Received: 05 August 2021
Accepted: 25 October 2021
Published: 14 January 2022
Citation:
James BH, Papakyriacou P,
Gardener MJ, Gliddon L, Weston CJ
and Lalor PF (2022) The Contribution
of Liver Sinusoidal Endothelial Cells
to Clearance of Therapeutic Antibody. Front. Physiol. 12:753833. doi: 10.3389/fphys.2021.753833 Received: 05 August 2021
Accepted: 25 October 2021
Published: 14 January 2022 James BH, Papakyriacou P,
Gardener MJ, Gliddon L, Weston CJ
and Lalor PF (2022) The Contribution
of Liver Sinusoidal Endothelial Cells
to Clearance of Therapeutic Antibody.
Front. Physiol. 12:753833.
doi: 10.3389/fphys.2021.753833 Generation of Antibodies for Therapeutic Therapeutic
mAbs
have
similar
structure
to
endogenous
immunoglobulin, i.e., four polypeptide chains, two light and two
heavy, each with both a Fab fragment and an Fc region. These
form a complex Y-shaped structure (see Figure 1). The Fab
fragment is composed of one constant region and one variable
domain which make up the antigen binding site. The Fc region
at the tail end of the antibody binds to elements of the immune
system such as complement components and surface receptors
known as Fc receptors (FcRs). Historically, man-made antibodies
were generated using the hybridoma technique (Kohler et al.,
1976) to generate murine monoclonal reagents as exemplified by
OKT3 (Kung et al., 1979). This murine antibody targeting human
CD3 antigen on T cells was widely used in immunotherapeutic
contexts including management of allograft rejection. However,
it has since been withdrawn due to side effects and generation
of host anti-murine antibodies which reduced efficacy (Sgro,
1995). Subsequently, the disadvantages of murine mAbs were
partially overcome by generation of chimeric antibodies. Here
recombinant DNA technology was used to generate hybridized
reagents consisting of the variable region from a mouse antibody
fused to a human antibody constant region. This reduced the
potential for the generation of anti-murine antibodies. The first
chimeric mAb approved by the FDA, abciximab (Lu et al., 2020) is
a Fab fragment antagonist to glycoprotein IIb/IIIa receptor used
to inhibit platelet aggregation. This was soon followed by, the
first full length IgG chimeric antibody “rituximab,” an anti-CD20
antibody widely used as an immune modifier (Maloney et al.,
1997). To further reduce the risk of immunogenicity, the residual
proportion of mouse antibody has been further diminished by the
advent of complementarity determining region (CDR) grafting
approaches (Riechmann et al., 1988; Tsurushita et al., 2005). Despite the increased proportion of human sequence within such
antibodies, adverse reactions still occurred (Nechansky, 2010). y
However, adverse effects post-treatment are not uncommon,
and often relate to the pathway being targeted or the mode
of action of the drug itself. Importantly problems and adverse
events are not always predicted by preclinical screening strategies. Toxicity or adverse events may relate to biological function
of the target molecule [e.g., minor bleeds in patients treated
with anti-platelet agents such as abciximab (Tamhane and
Gurm, 2008)] or interaction with off-target tissues. Less specific
toxicity can also be explained by hypersensitivity responses to
immunogenic “non” human elements of therapeutics. INTRODUCTION demographics for likely clinical use are best represented. In this
article we will consider the underestimated role of the liver, and
specifically the sinusoidal endothelial cells in antibody clearance. We also consider strategies that could be utilized to minimize
hepatic clearance, and the impact of age or chronic disease on
endothelial: antibody interactions. We begin with a review of
therapeutic antibody generation and structure before considering
implications for hepatic targeting and explanations for reported
adverse events in clinical use. Citation: James BH, Papakyriacou P,
Gardener MJ, Gliddon L, Weston CJ
and Lalor PF (2022) The Contribution
of Liver Sinusoidal Endothelial Cells
to Clearance of Therapeutic Antibody. Front. Physiol. 12:753833. doi: 10.3389/fphys.2021.753833 January 2022 | Volume 12 | Article 753833 1 Frontiers in Physiology | www.frontiersin.org Sinusoidal Endothelial Antibody Interactions James et al. The Growing Importance of Therapeutic
Antibodies Monoclonal antibody-based therapies for a variety of conditions
have been available since the late 1980s. Therapeutic antibodies
are biopharmaceuticals that recognize and bind to a specific
antigen leading to either activation or inhibition of downstream
biological
pathways. Monoclonal
antibodies
(mAbs)
are
the most common clinical tool and represent the leading
treatment modality for diseases ranging from inflammatory and
autoimmune disease to cancer. Upon recognition of cognate
antigen they either trigger an antibody mediated cellular
cytotoxic (ADCC) and/or a complement-dependent cytotoxic
(CDC) effector response, or act to neutralize the intended
target antigen. Antibodies are large molecules, which generally
don’t
interact
with
transport
molecules
or
detoxification
enzymes, exhibit ion channel-related complications or cause
immunogenicity. Thus antibody-based therapeutics tend to be
potent and well tolerated (Catapano and Papadopoulos, 2013). Only three antibodies were approved by the FDA in 2013 and
four in 2014, whereas as of December 2019 a total of 79 mAbs
have met approval standards with over 500 currently undergoing
clinical trials around the world (Kaplon et al., 2020). Hence the
global therapeutic antibody market is predicted to generate over
$300 billion by 2025 (Lu et al., 2020). Frontiers in Physiology | www.frontiersin.org Generation of Antibodies for Therapeutic Monoclonal antibodies (left structure) are composed of four polypeptide chains, two light (L)
and two heavy (H), each both a Fab fragment and an Fc region (blue) joined by a hinge section to create a Y-shaped structure. The Fab fragment which recognizes
antigen is composed of constant (C) and variable (V) domains which make up the antigen binding site. Specific fragments are also shown. Fab fragments can be
bivalent or monovalent, and engineered bispecific antibodies can contain or lack an Fc portion. A good example of this approach is blinatumomab used in
treatment of lymphoblastic leukemia (Kantarjian et al., 2017). This antibody combines two antigen receptor epitopes to
recognize CD3+ effector T cells and CD19+ B cells to stimulate
recognition and elimination of B cell blasts. Although effective at
improving survival, this approach is not without adverse events
including elevation in liver enzymes (Kantarjian et al., 2017). The other formulation is the full-length IgG-like asymmetric
BsAb (Fc-based BsAbs, or BsMabs) which retain an Fc portion. Mosunetuzumab used in treatment of leukemia exemplifies this
approach again targeting both a B cell epitope (CD20) and
CD3 (Schuster, 2021), and also bears a Fc domain engineered
to minimize FcγR and complement binding. However, if a
strong immune response is required, intact Fc regions facilitate
interactions with FcR and C1q. The small size and dual antigen
specificity of such bispecific reagents places a target cell in
close proximity to the effector cells resulting in a more effective
response than more traditional mAbs. Hence such forms of
BsAb have low therapeutic concentrations and short half-life,
(Wang et al., 2019) which can meant that frequent infusions
are required possibly increasing potential for offtarget effects. More recently there have been attempts to improve specificity of
targeting by using gene therapy approaches to drive cell specific
expression of bispecific antibodies at the site of need. This is
particularly attractive if hepatospecific targeting is required, given
the high phagocytic activities and ready absorbance of liposomes gene repertoire is replaced with human counterparts leading to
development of transgenic lines (Lonberg et al., 1994; Mendez
et al., 1997) such as the Xeno-mouse. The huge potential of
this technology is exemplified by panitumumab, the first Xeno-
mouse reagent to gain FDA approval. This fully human IgG2
EGFR antibody is used in therapy for metastatic colorectal
cancer (Jakobovits et al., 2007). Currently 19 approved mAbs
have been developed using such transgenic mice. Generation of Antibodies for Therapeutic When
designing a new antibody-based therapy there is also a need
to minimize interactions with non-target molecules and tissues
other than the therapeutic target. These issues can be resolved
by careful engineering of antibody to reduce immunogenicity,
maximize efficacy, and minimize clearance. Similarly, choice
of administration route has an impact on its efficacy and
clearance. Intravenous administration rapidly delivers 100% of
antibody into the systemic circulation and generates high plasma
concentrations, but increases the potential for offtarget exposure,
hypersensitivity reactions and the cost of in-house treatment. In contrast, sub-cutaneous and intra-muscular administration
deliver antibody via the lymphatic system. Here formulation,
injection volume and physical factors such as age and weight
of the patient (Richter et al., 2012; Richter and Jacobsen,
2014) can impact on bioavailability. Antibodies destined for
use in chronic conditions need to have the longest possible
half-life and minimal clearance rates to support a favorable
administration strategy and ensure dosing frequency is not
prohibitive. Importantly preclinical pharmacokinetic testing of
new reagents in a disease specific model is vital to ensure patient This led to a drive to produce fully humanized reagents
through application of technologies such as phage or yeast display
of antibody peptide libraries (Smith, 1985; McCafferty et al.,
1990). This method is rapid and robust with libraries containing
1 × 1010 antibody fragments available and is now considered the
gold standard for recombinant antibody production. The anti-
TNF antibody Adalimumab was generated using this approach
and is currently one of the best-selling therapeutics in the
world, generating $20 billion in 2018 (Kempeni, 1999; Lu et al.,
2020). Similarly, immunization of transgenic rodents to generate
fully humanized antibodies is significant. Here the mouse IgG January 2022 | Volume 12 | Article 753833 Frontiers in Physiology | www.frontiersin.org 2 James et al. Sinusoidal Endothelial Antibody Interactions FIGURE 1 | Typical structure of monoclonal and bispecific antibodies. Monoclonal antibodies (left structure) are composed of four polypeptide chains, two light (L)
and two heavy (H), each both a Fab fragment and an Fc region (blue) joined by a hinge section to create a Y-shaped structure. The Fab fragment which recognizes
antigen is composed of constant (C) and variable (V) domains which make up the antigen binding site. Specific fragments are also shown. Fab fragments can be
bivalent or monovalent, and engineered bispecific antibodies can contain or lack an Fc portion. FIGURE 1 | Typical structure of monoclonal and bispecific antibodies. Frontiers in Physiology | www.frontiersin.org A Focus on Fc Receptors and
Mechanisms of Antibody Uptake and
Clearance y
The process for uptake and recycling of antibody is described
in Figure 2. Once bound to FcγR a monoclonal antibody is
internalized into an endosome. Here they encounter membrane
bound FcRn (Roopenian and Akilesh, 2007) which is responsible
for the protection of IgG catabolism, recycling the antibody to
the surface leading to an increased half-life. This binding is pH
dependent and will only occur in acidic endosomes, with a pH
at around 6–6.5. FcRn containing vesicles become exposed to
an increasing pH gradient until they reach the cell surface and
physiological pH. This causes the mAb and FcRn to dissociate
and the antibody is then released from the cell and recycled
back into circulation. mAbs that fail to be recycled by FcRn are
either cleared via the activation of C1q, and undergo clearance via
the classical complement pathway or are degraded by proteases
present within lysosomes (Leipold and Prabhu, 2019). Therefore
the FcRn is important to spare the mAb from degradation and
prolong the half-life (Haraya et al., 2019) potentially reducing
therapeutic dosing and frequency. Some studies have suggested
that it is FcRn that primarily impacts on pharmacokinetics
and that FcγRIIb has little impact on circulating antibody
distribution (Abuqayyas et al., 2013). However, it is important
to note that some studies with knockout animals deficient in
FcγRIIb tested antibodies at concentrations far below therapeutic
concentrations. Even in these circumstances there was an increase
in liver distribution (albeit variable) even at low dose suggesting
that within the liver FcγRIIb may be involved in clearance and
degradation of antibody (Abuqayyas et al., 2013). This seems to
be particularly important for antibody: antigen complexes which
are cleared into liver whilst antigen alone is not (Ljunghusen et al.,
1990). Thus, in the next section we describe the function of the
hepatic sinusoidal endothelial cells to highlight their potential
roles in antibody bioavailability. Highly charged cationic molecules like antibodies with poor
pharmacokinetic profiles are cleared reasonably quickly (Haraya
et al., 2019) and evidence suggests that this clearance takes place
in highly vascularized organs like the liver and spleen (Li et al.,
2014). The liver in particular is a major site for internalization
and catabolic clearance of therapeutic antibodies as they are
typically too large for renal elimination. This is facilitated in
part by an impressive scavenging system. Generation of Antibodies for Therapeutic This method
is advantageous as there is often no requirement for an affinity
maturation step for targets with high affinity, and full-length
IgG antibodies are made. However, if the antigen being used
to immunize is particularly toxic then phage display is the
preferred technique. To date human and humanized mAbs are
the dominant format of therapeutic antibodies accounting for,
respectively, 51 and 35% of all mAbs currently in clinical use
(Lu et al., 2020). Whilst traditional monoclonal antibodies bind to a single
antigen, bispecific tools have been engineered to improve
targeting [increase the efficacy of immune: target cell or
receptor:ligand interactions (Kang and Lee, 2021)] and exhibit
favorable tissue penetration. Different formats exist and each has
its own advantages and challenges. Fragment based bispecific
antibodies (BsAb) lack a Fc region but still contain two
independent antigen binding domains. As there is no Fc
region present, these BsAb are considerably smaller than
traditional mAbs allowing them to penetrate tissues easily. January 2022 | Volume 12 | Article 753833 3 Sinusoidal Endothelial Antibody Interactions James et al. and nanosomes within the liver. This approach is elegantly
exemplified by the work of Kruse et al. (2017) who generated
hepatitis B Ag : CD3 specific bispecific antibodies with antiviral
efficacy in vivo (Kruse et al., 2017). endothelium (Vaccaro et al., 2005), tissue macrophages and
Kupffer cells, enterocytes and some epithelial cells (Latvala
et al., 2017). It is atypical in that along with binding IgG it
also recognizes albumin and plays key roles in transcytosis
and recycling of both to maintain circulating concentrations
(Pyzik et al., 2019). A Focus on Fc Receptors and
Mechanisms of Antibody Uptake and
Clearance Cells of the hepatic
reticuloendothelial system express many receptors that can bind
and internalize antibodies either by target mediated clearance or
via non-specific uptake. As noted above, Fc receptors on a cell
surface generally recognize the Fc portion of antibody and as a
consequence activate and modulate immune responses or clear
immune complexes. This could take the form of destruction of
an opsonized target cell or the activation/regulation of cellular
effector responses. However, exaggerated antibody-dependent
autoimmune and hypersensitivity responses and circulating
therapeutic antibody pharmacokinetics are also impacted by the
action of these receptors (Hogarth and Pietersz, 2012). In the
context of antibody-based therapies, interaction with FcR is
important for specific targeting of an immune response. The Fc
gamma receptor (FcγR) family of proteins consists of six FcγRs in
humans which include FcγR1 (CD64), FcγRIIa,b and c (CD32a-
c) and FcγRIIIa and b (CD16a and b) (Brooks et al., 1989). Each
has a slightly different cellular distribution and affinity for IgG
(Hogarth and Pietersz, 2012). Human IgG1 and 3 bind more
effectively to FcγRs than IgG2 and 4 (Schwab et al., 2015) but
IgG1 antibodies are still the most commonly used for therapies
(Lucas et al., 2018). Clustering of antibody and target antigen may
be enhanced by binding to FcγRIIb (Stopforth et al., 2016). In
contrast, internalization, and catabolism of antibodies via FcγR
may be particularly important for antibodies with circulating
soluble antigens or which form large immune complexes with
target as these tend to bind well to FcRs (Lucas et al., 2018). Frontiers in Physiology | www.frontiersin.org Liver Sinusoidal Endothelial Cell
Structure and Function Although historically the role of
FcRn LSEC has not been well documented (Skogh et al., 1985),
hepatocyte intracellular FcRn (Pyzik et al., 2019) has been
linked to clearance and catabolism of antibody and albumin
transport. Interestingly we also see intracellular localization in
human hepatocytes (Figure 5) with increased peri-membranous
distribution in advanced disease (Blue arrows Figure 5). This may
reflect a response to hypergammaglobulinemia in cirrhosis and
liver disease (Alonso et al., 2012; Cacciola et al., 2018). FcRn also
plays roles in the pathology of toxic liver injury. Drugs including
paracetamol are transported bound to circulating albumin, and
blockade of the interaction between albumin and FcRn reduces
hepatotoxicity after paracetamol administration (Pyzik et al.,
2017). Interestingly LSEC also express a scavenger receptor
lectin, dendritic cell specific ICAM-3 grabbing non-integrin (DC-
SIGN) (Lai et al., 2006; Schwab and Nimmerjahn, 2013) which
has been demonstrated to be a coreceptor for some viruses
(Gramberg et al., 2007). This receptor also bind intravenously
administered therapeutic Immunoglobulin (IVIg) (Hogarth and
Pietersz, 2012; Schwab and Nimmerjahn, 2013), upregulates
expression of FcγRIIb and protects against immune-complex
mediated disease (Anthony et al., 2011). vessel wall such as presence of fenestrated endothelium and
basal lamina thickness. Junctional structure is also important
with the presence of endothelial cells containing tight junctions
limiting access, as is seen in the brain (Tabrizi et al., 2010). Thus,
the liver sinusoidal bed presents a particular challenge. Liver
sinusoidal endothelial cells (LSEC, Figure 3) which are exposed
to both systemic and portal blood are designed to maximize the
exchange of useful material from the blood into the liver and
vice versa (Shetty et al., 2018). They form part of the hepatic
reticuloendothelial system with roles in both the clearance of
detrimental pathogens and waste products and the transport
of important metabolic products to and from the proximal
hepatocytes. These activities are facilitated by the presence of
numerous macroscopic pores or “fenestrations,” organized into
sieve plates which transverse the full thickness of the endothelial
layer allowing transport of lipids and proteins (Hunt et al., 2019)
and also medicinal drugs such as lidocaine and paracetamol
(Mitchell et al., 2011). Importantly unlike the kidney (Satchell and
Braet, 2009) and other organs, the hepatic sinusoidal endothelial
fenestrations lack a diaphragm and basal lamina. This, plus
the ability of cells to rapidly regulate fenestration diameter
and number (O’Reilly et al., 2010; Cogger et al., 2016) further
regulates transport. Liver Sinusoidal Endothelial Cell
Structure and Function g
Liver sinusoidal endothelial cells also express an unusual
complement of scavenger receptors which recognize, bind, and
rapidly internalize an enormous diversity of extracellular ligands
(Shetty et al., 2018). These are characterized into classes A to J
depending on their ligand recognition and structural properties
(Patten et al., 2021) and LSEC express receptors in classes SR-
B, E, F, G, and H to support clearance of fatty acids, lipids ECM
proteins, glycosaminoglycan molecules and apoptotic cells. This
significant endocytic capability supports the immune regulation
(Knolle and Limmer, 2001), metabolic capacity (Li et al., 2011)
and “waste management” (Smedsrod, 2004) functions of the
liver. In the context of this article, it is important to note
that LSEC also express high levels of FcR under homeostatic
conditions. The FcR on LSEC can bind opsonized pathogens
and macromolecules to facilitate clearance, with blood-borne
immune complexes rapidly cleared from the circulation by
both Kupffer cells (KC) and LSEC (Smedsrod, 2004). Although
KC may be more efficient at clearing immune complexes, the
increased number of LSEC compared to KC within a liver
means that their total capacity may be similar (Johansson et al.,
2000). Circulating immune complex clearance can cause tissue
damage and inflammation in some conditions (Johansson et al.,
2000) and thus sinusoidal endothelial cells contribute to the
process of clearance via the FcR interaction (Johansson et al.,
2000). This may be particularly important when the load of
circulating IgG is high (Johansson et al., 2000). LSEC have
been suggested to express all three of the major Fcγ receptors
(Smedsrod, 2004) and it is estimated that up to 75% of all
the FcγRIIb within the body is expressed on LSEC (Ganesan
et al., 2012). Thus, this abundant receptor expression plays a
key role in removal of small immune complexes from blood. We have documented expression in human livers (Figure 4) and
confirm that expression is abundant and localized to LSEC in
the healthy liver. Expression is maintained in chronic disease All the evidence above suggests that in a healthy liver, the
LSEC are armed with key receptors and endocytic machinery
to bind and transport antibody and immune complexes. There is functional evidence to support this. For example,
studies of clearance of Bispecific antibodies in cynomolgus
monkeys suggest a role for both macrophages and LSEC in
clearance (Datta-Mannan et al., 2016). Liver Sinusoidal Endothelial Cell
Structure and Function One factor which remains challenging in the development
of antibody therapies relates to their pharmacokinetics and
clearance in tissue. This alters exposure to target antigen and
ultimately efficacy. Distribution within a tissue is impacted
upon by movement across the vessel wall and interaction
with endothelial cells and macrophages which express the
receptors described above. Tissues like the liver which have
fenestrated non-continuous endothelial cells, are highly perfused
and abundantly vascularized, will have greater exposure to
antibody (Datta-Mannan, 2019). The isoelectric point of an
antibody appears to particularly influence hepatic clearance, such
that engineering of antibody variants with high pI leads to
preferential sequestration and clearance by the liver (Ganesan
et al., 2012). Transport of antibody from blood into tissue is
dependent on local perfusion gradient and key features of the Engagement of receptor on immune cells generally induces
a cellular response via activation of immunoreceptor tyrosine-
based activation motif (ITAM) and SRC family kinase activation. In most cases this causes a pro-inflammatory response, but
FcγRIIb has inhibitory effects via activation of immunoreceptor
tyrosine-based activation motif (ITIM) (Hogarth and Pietersz,
2012), despite binding IgG with a relatively low affinity. In B
cells this can downregulate signals from the other FcR and
cause apoptosis. There are also descriptions of two variants
of FcγRIIb (b1 and b2) which have slight differences in the
ability to internalize antibody due to variance in structure of the
cytoplasmic domain of the receptor (Stopforth et al., 2016). The
neonatal Fc receptor (FcRn) seems to be more involved in antigen
presentation and IgG recycling within cells. It is expressed by January 2022 | Volume 12 | Article 753833 4 Sinusoidal Endothelial Antibody Interactions James et al. (Figure 4) but the distribution is altered in cirrhosis and
intensity of staining is reduced, which may suggest an impact
on function. FcRn has a more widespread hepatic distribution,
described to be present on epithelial cells, endothelium, and
immune cell populations (Pyzik et al., 2019) in animal studies. In agreement, our investigation of human liver (Figure 5)
confirms intense sinusoidal expression localized to Kupffer cells. Periportal immune cells are also positive with a degree of
intracellular staining in hepatocytes. Faint intracellular LSEC
staining is confirmed by confocal studies (Figure 5 final panel)
on cultured human LSEC. Frontiers in Physiology | www.frontiersin.org Liver Sinusoidal Endothelial Cell
Structure and Function LSEC have specialized pores in their cell surface (the fenestrations, blue arrows) which organize into sieve plates to facilitate direct exchange of materials between
the hepatic parenchyma and bloodstream. The LSEC also express unique profiles of cell surface scavenger receptors and Fc receptors (Black arrowheads) which
can interact with macromolecules within the slow flowing sinusoidal blood. Kupffer cells (KC) are specialized macrophages which patrol along the sinusoids to fulfil
their immune regulatory functions. In chronic disease or aged livers, the nature of the LSEC changes. They lose most of their fenestrations and alter abundance of
scavenger and Fc receptors. They also produce a more complex basement membrane. This restricts movement of materials into and out of the parenchyma. FIGURE 3 | The organization of the hepatic sinusoid. The hepatic sinusoids represent the capillary bed of the liver and are lined by specialized liver sinusoidal
endothelial cells (LSEC). These sit above the hepatocyte layer separated only by the Space of Disse which contains minimal basement membrane in a healthy liver. LSEC have specialized pores in their cell surface (the fenestrations, blue arrows) which organize into sieve plates to facilitate direct exchange of materials between
the hepatic parenchyma and bloodstream. The LSEC also express unique profiles of cell surface scavenger receptors and Fc receptors (Black arrowheads) which
can interact with macromolecules within the slow flowing sinusoidal blood. Kupffer cells (KC) are specialized macrophages which patrol along the sinusoids to fulfil
their immune regulatory functions. In chronic disease or aged livers, the nature of the LSEC changes. They lose most of their fenestrations and alter abundance of
scavenger and Fc receptors. They also produce a more complex basement membrane. This restricts movement of materials into and out of the parenchyma. Does Liver Sinusoidal Endothelial Cells
Biology Influence the Outcome of
Therapeutic Antibody Administration,
and Is This Important When Designing
Antibodies? Liver Sinusoidal Endothelial Cell
Structure and Function Here use of clodronate
to deplete macrophages did not have a great effect on antibody
clearance, suggesting that the contribution of macrophages was
marginal. This was confirmed by costaining of therapeutic
antibody with markers of LSEC to confirm co-localization
(Datta-Mannan et al., 2016) with little staining for the
bispecific antibodies observed in macrophages. Studies of
humanized mice which express human FcγR and are given a
humanized antiplatelet antibody confirm these findings with
no major effect after macrophage deletion (Schwab et al.,
2015). Clearance of opsonized pathogen too is linked to intact
FcγRIIb function on LSEC, with deficient mice exhibiting
slower pathogen clearance (Ganesan et al., 2012). A more
interesting question, however, is what impact LSEC have on
the pharmacokinetics and pharmacodynamics of therapeutic
antibodies? Also, whether newer antibody formulations can be
optimized to exhibit the most favorable dosing profiles and
minimize side effects by consideration of LSEC function in health
and disease? January 2022 | Volume 12 | Article 753833 Frontiers in Physiology | www.frontiersin.org 5 Sinusoidal Endothelial Antibody Interactions James et al. FIGURE 2 | Receptor mediated antibody uptake. The Fc portion of free antibody or antibody bound to soluble antigen to form an immune complex bind to FcγR at
the cell surface. Once bound antibody is internalized into an acidified endosome via fluid phase pinocytosis. The endosomes contain FcRn which binds via the heavy
chains in the Fc region in a pH sensitive manner. The FcRn can then recycle bound antibody back to the cell membrane where physiological pH of blood allows
uncoupling and release back into the circulation. Alternately mAbs that fail to be recycled by FcRn are either cleared via the activation of C1q, and the classical
complement pathway or are degraded by proteases present within lysosomes within the cell. FIGURE 3 | The organization of the hepatic sinusoid. The hepatic sinusoids represent the capillary bed of the liver and are lined by specialized liver sinusoidal
endothelial cells (LSEC). These sit above the hepatocyte layer separated only by the Space of Disse which contains minimal basement membrane in a healthy liver. LSEC have specialized pores in their cell surface (the fenestrations, blue arrows) which organize into sieve plates to facilitate direct exchange of materials between
the hepatic parenchyma and bloodstream. Liver Sinusoidal Endothelial Cell
Structure and Function The LSEC also express unique profiles of cell surface scavenger receptors and Fc receptors (Black arrowheads) which
can interact with macromolecules within the slow flowing sinusoidal blood. Kupffer cells (KC) are specialized macrophages which patrol along the sinusoids to fulfil
their immune regulatory functions. In chronic disease or aged livers, the nature of the LSEC changes. They lose most of their fenestrations and alter abundance of
scavenger and Fc receptors. They also produce a more complex basement membrane. This restricts movement of materials into and out of the parenchyma. FIGURE 2 | Receptor mediated antibody uptake. The Fc portion of free antibody or antibody bound to soluble antigen to form an immune complex bind to FcγR at
the cell surface. Once bound antibody is internalized into an acidified endosome via fluid phase pinocytosis. The endosomes contain FcRn which binds via the heavy
chains in the Fc region in a pH sensitive manner. The FcRn can then recycle bound antibody back to the cell membrane where physiological pH of blood allows
uncoupling and release back into the circulation. Alternately mAbs that fail to be recycled by FcRn are either cleared via the activation of C1q, and the classical
complement pathway or are degraded by proteases present within lysosomes within the cell. FIGURE 2 | Receptor mediated antibody uptake. The Fc portion of free antibody or antibody bound to soluble antigen to form an immune complex bind to FcγR at
the cell surface. Once bound antibody is internalized into an acidified endosome via fluid phase pinocytosis. The endosomes contain FcRn which binds via the heavy
chains in the Fc region in a pH sensitive manner. The FcRn can then recycle bound antibody back to the cell membrane where physiological pH of blood allows
uncoupling and release back into the circulation. Alternately mAbs that fail to be recycled by FcRn are either cleared via the activation of C1q, and the classical
complement pathway or are degraded by proteases present within lysosomes within the cell. FIGURE 3 | The organization of the hepatic sinusoid. The hepatic sinusoids represent the capillary bed of the liver and are lined by specialized liver sinusoidal
endothelial cells (LSEC). These sit above the hepatocyte layer separated only by the Space of Disse which contains minimal basement membrane in a healthy liver. Does Liver Sinusoidal Endothelial Cells
Biology Influence the Outcome of
Therapeutic Antibody Administration,
and Is This Important When Designing
Antibodies? For
example, studies of Humanized DR-5 antibodies (an apoptosis
inducing TNFR) with an engineered Fc fragment to enhance
FcγRIIb binding in mice engineered to express human FcγRIIb,
resulted in increased ALT/AST and mortality (Li and Ravetch,
2012) at supraphysiological doses. Here the FcR was important
for the hepatotoxicity. In other studies, humanized antibody
designed to target tumor cells by binding to a TNFR stimulatory
receptor (CD137) on immune cells to promote anti-tumor
immunity responses (Qi et al., 2019) such as Urlumab (Segal
et al., 2017) was also associated with liver toxicity, inflammation
and liver related adverse events. Mechanistic studies on such
antibodies suggest that LSEC expression of FcγRIIb increases
crosslinking and activatory effects of strong agonistic antibodies
to enhance liver toxicity (Qi et al., 2019). However, engineering
of Fab fragments that retain strong agonism minimizes this
effect. It is also important to consider potential target-related
toxicities alongside FcR-related hepatotoxicity in some cases. As
an example, antibodies against TNF were tested as potential
anti-inflammatory therapies in human alcoholic hepatitis but
some studies were terminated due to adverse outcomes (Blendis
and Dotan, 2004) or showed no mortality benefit over standard
therapies. There are reports of drug induced toxicity associated
with many formulations of anti-TNF antibodies (Lopetuso et al.,
2018), particularly in patients with autoimmune liver disease
(Tobon et al., 2007) and thus vasculotoxicity associated with antibody clearance could explain an underlying mechanism of
damage. However, it is also important to note that TNFα plays a
key role in hepatocyte regeneration (Fausto, 2000) and promotes
hepatic infiltration by immune cells which drive repair (Chauhan
et al., 2020) or fight sepsis which is a significant risk in alcoholic
hepatitis (Sharma et al., 2009). Thus biological inhibition of
hepatic repair mechanisms may also explain some of the adverse
outcomes associated with this approach. pp
GSK305002 is a humanized IgG antibody that neutralizes
the soluble chemokine CCL20 and was in development as a
potential therapy for inflammatory disease (Laffan et al., 2020). Although no safety signatures appeared in a phase 1 study
in humans, subsequent longer term escalating dose toxicity
studies in cynomolgus monkeys highlighted a significant vascular
inflammation in most subjects which is unexpected for an
antibody targeting soluble antigen. In the liver this presented as
moderate inflammation with immune deposits localized within
the sinusoids. Does Liver Sinusoidal Endothelial Cells
Biology Influence the Outcome of
Therapeutic Antibody Administration,
and Is This Important When Designing
Antibodies? antibodies to enhance interactions with FcRn and improve
pharmacokinetics. Fc receptor mediated clearance of immune
complex is often a desirable therapeutic strategy. Here cell
surface Fc receptors bind to the Fc portion of IgG antibodies
in immune complexes with their target, and these are cleared
from the circulation through uptake into macrophages and
endothelial cells in the liver (Lovdal et al., 2000; Ganesan et al.,
2012). However, in some situations internalization of therapeutic
antibodies via actions of FcγRIIb can reduce clinical efficacy, as
has been reported for the use of rituximab in some leukemias Evidence cited above from knockout animals which have
modified hepatic FcR expression confirm the contribution of the
liver to clearance. Therapeutic antibody development approaches
may include engineering of the Fc portion of humanized January 2022 | Volume 12 | Article 753833 Frontiers in Physiology | www.frontiersin.org 6 Sinusoidal Endothelial Antibody Interactions James et al. FIGURE 4 | Hepatic sinusoidal endothelial expression of FcγR2b alters in disease. Representative immunochemical (left panels, 10× original magnification Bar is
200 um) and immunofluorescent stains (right panels, 100× original magnification, Bar is 20 um) for FcγR2b on representative examples of healthy (top row) and
diseased liver [bottom row, cirrhotic explanted liver from patient with PSC (left) or ALD (right)]. FcγR is localized to the LSEC in both cases, but expression is more
intense and consistent across the sinusoid in a healthy context. In explanted cirrhotic human livers some areas of sinusoids lack expression completely. FIGURE 4 | Hepatic sinusoidal endothelial expression of FcγR2b alters in disease. Representative immunochemical (left panels, 10× original magnification Bar is
200 um) and immunofluorescent stains (right panels, 100× original magnification, Bar is 20 um) for FcγR2b on representative examples of healthy (top row) and
diseased liver [bottom row, cirrhotic explanted liver from patient with PSC (left) or ALD (right)]. FcγR is localized to the LSEC in both cases, but expression is more
intense and consistent across the sinusoid in a healthy context. In explanted cirrhotic human livers some areas of sinusoids lack expression completely. (Lim et al., 2011) and cancer models (Clynes et al., 2000). It
is also noteworthy that circulating immune complex clearance
can cause tissue damage and inflammation in some conditions
(Johansson et al., 2000). This may be particularly important when
the load of circulating IgG is high (Johansson et al., 2000). Frontiers in Physiology | www.frontiersin.org Does Liver Sinusoidal Endothelial Cells
Biology Influence the Outcome of
Therapeutic Antibody Administration,
and Is This Important When Designing
Antibodies? Target antigen did not appear to be contained
in these deposits and importantly anti-human antibodies were
not detected or were present at a level too low to explain the
findings (Laffan et al., 2020). This would suggest that localization
of FcR [or CCL20 (Shields et al., 1999)] on the LSEC may
have provided a focus for immune complex deposition and
complement mediated toxicity toward the LSEC. Vasculotoxicity
has also been seen with other antibody drugs and can present
as Sinusoidal Obstruction Syndrome (Jain and Litzow, 2018). This is damage to the sinusoidal endothelium, particularly in
central areas of the lobule which exposes the subendothelial cells
to blood constituents driving a necrotic response and vascular
occlusion. This may relate to drug conjugates bound to antibodies January 2022 | Volume 12 | Article 753833 Frontiers in Physiology | www.frontiersin.org 7 James et al. Sinusoidal Endothelial Antibody Interactions James et al. Sinusoidal Endothelial Antibody Interactions
FIGURE 5 | Hepatic expression of FcRn alters in disease. Representative immunochemical (top panels), and immunofluorescent stains (bottom left panel) for FcRn
on representative examples of healthy (top row) and diseased liver (middle row) or primary cultures of human LSEC. Both hepatocytes and sinusoidal cells express
FcRn but the intensity increases in disease (ALD, middle row). Hepatocellular membrane expression increases as disease progresses (blue arrowheads). Original
immunochemical stain images captured at 10× and 50× magnification (left and right panels, respectively). Cultured LSEC express FcRn (red stain) in an intracellular
vesicular pattern (white arrows). FIGURE 5 | Hepatic expression of FcRn alters in disease. Representative immunochemical (top panels), and immunofluorescent stains (bottom left panel) for FcRn
on representative examples of healthy (top row) and diseased liver (middle row) or primary cultures of human LSEC. Both hepatocytes and sinusoidal cells express
FcRn but the intensity increases in disease (ALD, middle row). Hepatocellular membrane expression increases as disease progresses (blue arrowheads). Original
immunochemical stain images captured at 10× and 50× magnification (left and right panels, respectively). Cultured LSEC express FcRn (red stain) in an intracellular
vesicular pattern (white arrows). particularly that expressed in B cells (Dudek et al., 2019), but
presence of endothelial cells is necessary to recreate the immune
activatory responses in in vitro assays (Dhir et al., 2012). to facilitate target cell toxicity (e.g., calicheamicin for inotuzumab
and gemtuzumab). Careful screening for
pre-existing disease in
patient populations.
Age-dependent
pharmacokinetic
assessment at Phase 1
testing
Use of FcRn blockers
to enhance IgG
degradation Does Liver Sinusoidal Endothelial Cells
Biology Influence the Outcome of
Therapeutic Antibody Administration,
and Is This Important When Designing
Antibodies? Perhaps the best example of a serious adverse
reaction to antibody therapy, the first human trials of the CD28
specific TGN1412 (Suntharalingam et al., 2006) also highlights
how important FcR binding is and how hard responses are to
predict. TGN1412 is a potent agonistic antibody developed for
use in treatment of some cancers and rheumatoid arthritis. Its
agonistic events are potentiated by interactions with FcγRIIb, Immune or toxic responses to biotherapeutics are complex
and can be target related or influenced by the structure and
clearance of the antibody itself. For this reason, all new
therapeutics are tested extensively in preclinical models and
healthy volunteers before proof of efficacy in a patient. However, January 2022 | Volume 12 | Article 753833 Frontiers in Physiology | www.frontiersin.org 8 Sinusoidal Endothelial Antibody Interactions James et al. there are still instances where preclinical models have failed to
accurately predict human responses or those in a specific patient
cohort or requirements for alternate dosing regimens in chronic
disease. Hepatic impairment and impact on antibody kinetics
may alter exposure, tolerability and effectiveness if metabolism
or excretion is altered (Sun et al., 2020). This may relate to
lower albumin production by a damaged liver impacting on
antibody exposure of factors which alter expression or function
of FcRn and FcγRs could also alter systemic exposure. However,
regulatory bodies in some cases suggest that validation of MAb
therapy in populations with renal or hepatic impairment is
not vital for licensing (Lucas et al., 2018). Moreover, there are
clear examples where prior liver injury or older age increase
the risk of adverse events of antibody-based treatments (Jain
and Litzow, 2018). This has meant that for some antibody-
based therapies where hepatotoxic side effects have been noted,
pre-existing clinical liver disease is considered an exclusion
for use. For example - tocilizumab (humanized IL-6 receptor antibody) and anakinra (IL-1R antagonist antibody) used as
anti-inflammatories in rheumatoid arthritis have potential, well
described hepatotoxic consequences in some patients (Mahamid
et al., 2011) particularly if other immunosuppressive drugs such
as methotrexate have been administered. The challenge remains being able to predict and explain such
toxicities, and then to be able to engineer a solution to them. Does Liver Sinusoidal Endothelial Cells
Biology Influence the Outcome of
Therapeutic Antibody Administration,
and Is This Important When Designing
Antibodies? It is important to note that the FcγRs are slightly different in
mice (Schwab et al., 2015) than humans and thus variations
in human receptors not represented in mice can mean that
rodent models are not perfect for predicting humanized antibody
activity and clearance. Similarly, IgG4 mAbs don’t interact
with monkey FcR’s and thus wouldn’t be picked up in species
specific screens (Hansel et al., 2010). Even in a human context,
individuals have polymorphisms in Fc: FcR interactions which
underpin interindividual variation in antibody clearance and
efficacy (Hansel et al., 2010). Levels of FcR expression change with
age and disease state. We note above that FcRn expression within
the liver is altered in cirrhosis and suggested this could relate
to circulating antibody concentration fluctuations in disease
(Holdstock et al., 1982) which is clearly associated with poor
prognosis (Cacciola et al., 2018). However, it may also be a
consequence of age or disease related sinusoidal capillarization
(Figure 3). Importantly not all scavenger receptors on LSEC
decrease with aging or capillarization. Thus whilst receptors
such as CD36 are increased on LSEC with age or development
of fatty liver disease (Sheedfar et al., 2014), expression of
mannose receptor decreases (Dini et al., 1990) and studies in
rats suggest Stabilin-1 and -2 are broadly similar in young
and old animals (Simon-Santamaria et al., 2010). Nevertheless,
decline in fenestration with age can reduce clearance of drugs
such as paracetamol (Mitchell et al., 2011). Similarly, clearance
of gut derived LPS is impaired in cirrhosis due to reduced
sinusoidal permeability leading to hyperactivation of plasma cells
and increased immunoglobulin production (Liu et al., 2015). Capillarization of LSEC also restrict access to hepatocyte FcRn
which normally transports antibody across epithelial barriers
and maintains circulating antibody concentration (Yeung et al.,
2009). Mice that are deficient in FcRn have reduced half-life
of administered antibodies (Israel et al., 1996). Coupled with
reduced expression of scavenger receptors such as DC-SIGN and
FcγR on diseased LSEC this could profoundly alter antibody
clearance kinetics. Similarly, occupancy of DC-SIGN by ligands
such as viral and bacterial antigens (Gupta and Gupta, 2012)
during infection could alter availability for binding antibody-
based therapies. In situations of hepatic autoimmunity or disease,
clearance of autoantibodies could be managed using FcRn
blockers to enhance IgG degradation to manage autoantibodies
or control clearance of therapeutic immunoglobulins (Vaccaro
et al., 2005). Engineering of Fc
portion to minimize
interaction or delay
internalization of
receptor
Inclusion of human cell
based or tissue array
screens in pre-trail
development stages Lack of clinical efficacy
upon testing in human
subjects Enhanced deposition
and clearance by LSEC
leading to
vasculotoxicity LSEC capillarization or
autoantibody
occupancy of FcRs
impacting on PK
Accelerated clearance
by hepatic FcγRIIb Modify Fc portion to
enhance interaction
with FcRn and improve
half life Modify Fc
portion to minimize
interaction with FcγRIIb
Analysis of Fc portion
and specific testing of
clearance by human
FcR to minimize
crosslinking and
activation in sinusoid
Careful screening for
binding to Fc receptors
on LSEC Immune complex
binding to LSEC and
cell apoptosis leading
to exposure of basal
lamina
LSEC capillarization,
reduction in hepatic
albumin production REFERENCES Datta-Mannan, A., Croy, J. E., Schirtzinger, L., Torgerson, S., Breyer, M., and
Wroblewski, V. J. (2016). Aberrant bispecific antibody pharmacokinetics linked
to liver sinusoidal endothelium clearance mechanism in cynomolgus monkeys. MAbs 8, 969–982. doi: 10.1080/19420862.2016.1178435 Abuqayyas,
L.,
Zhang,
X.,
and
Balthasar,
J. P. (2013). Application
of
knockout
mouse
models
to
investigate
the
influence
of
FcgammaR
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the
pharmacokinetics
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anti-platelet
effects
of
MWReg30,
a
monoclonal
anti-GPIIb
antibody. Int. J. Pharm. 444,
185–192. doi: 10.1016/j.ijpharm.2013.01.001 Datta-Mannan, A., Witcher, D. R., Tang, Y., Watkins, J., and Wroblewski, V. J. (2007). Monoclonal antibody clearance. Impact of modulating the interaction
of IgG with the neonatal Fc receptor. J. Biol. Chem. 282, 1709–1717. doi:
10.1074/jbc.M607161200 Alonso, M., Gomez-Rial, J., Gude, F., Vidal, C., and Gonzalez-Quintela, A. (2012). Influence of experimental alcohol administration on serum immunoglobulin
levels: contrasting effects on IgE and other immunoglobulin classes. Int. J. Immunopathol. Pharmacol. 25, 645–655. doi: 10.1177/039463201202500311 Dhir, V., Fort, M., Mahmood, A., Higbee, R., Warren, W., Narayanan, P., et al. (2012). A predictive biomimetic model of cytokine release induced by TGN1412
and other therapeutic monoclonal antibodies. J. Immunotoxicol. 9, 34–42. doi:
10.3109/1547691X.2011.613419 Anthony, R. M., Kobayashi, T., Wermeling, F., and Ravetch, J. V. (2011). Intravenous gammaglobulin suppresses inflammation through a novel T(H)2
pathway. Nature 475, 110–113. doi: 10.1038/nature10134 Dini, L., Lentini, A., and Devirgiliis, L. C. (1990). Binding and uptake of ligands
for mannose-specific receptors in liver cells: an electron microscopic study
during development and aging in rat. Mech. Ageing Dev. 56, 117–128. doi:
10.1016/0047-6374(90)90003-X Blendis, L., and Dotan, I. (2004). Anti-TNF therapy for severe acute alcoholic
hepatitis: what went wrong? Gastroenterology 127, 1637–1639. doi: 10.1053/
j.gastro.2004.09.089 Dudek, S., Weissmuller, S., Anzaghe, M., Miller, L., Sterr, S., Hoffmann, K.,
et al. (2019). Human Fcgamma receptors compete for TGN1412 binding that
determines the antibody’s effector function. Eur. J. Immunol. 49, 1117–1126. doi: 10.1002/eji.201847924 Brooks, D. G., Qiu, W. Q., Luster, A. D., and Ravetch, J. V. (1989). Structure and
expression of human IgG FcRII(CD32). Functional heterogeneity is encoded
by the alternatively spliced products of multiple genes. J. Exp. Med. 170,
1369–1385. doi: 10.1084/jem.170.4.1369 Fausto, N. (2000). Liver regeneration. J. Hepatol. 32, 19–31. doi: 10.1016/S0168-
8278(00)80412-2 Cacciola, I., Filomia, R., Alibrandi, A., Franze, M. S., Caccamo, G., Maimone,
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3649 Ganesan, L. FUNDING This manuscript presents independent research supported in
part by the National Institute for Health Research, NIHR
Birmingham Biomedical Research Centre at the University
Hospitals Birmingham, NHS Foundation Trust, and University
of Birmingham (Grant Reference Number BRC-1215-20009). BJ was funded by a BBSRC Industrial Case Ph.D. Studentship
with GSK (Reference BB/T508317/1). PP was funded from
the European Union’s Horizon 2020 Research and Innovation
Program under the Marie Skłodowska-Curie Grant Agreement
No. 766181, project “DeLIVER”. Does Liver Sinusoidal Endothelial Cells
Biology Influence the Outcome of
Therapeutic Antibody Administration,
and Is This Important When Designing
Antibodies? Alternately specific engineering of monoclonal or
bispecific antibodies to modify interactions with FcRn could also
be used to improve pharmacokinetics (Schutten et al., 1993;
Datta-Mannan et al., 2007; Lucas et al., 2018; Datta-Mannan,
2019). This may be particularly important in the context of
treating chronic disease if an antibody-based therapy needs to
be maintained at therapeutic levels for a long time. Indeed, anti-
FcγRIIb antibodies have been suggested as a strategy to reduce TABLE 1 | Clinical challenges associated with hepatic clearance of biological
therapies and strategies to mitigate risk during drug development. Clinical challenge
Explanation
Mitigating strategy
Impact of LSEC Fc
receptors on antibody
PK
Accelerated or delayed
clearance of circulating
antibody
Modify Fc portion to
enhance interaction
with FcRn and improve
half life Modify Fc
portion to minimize
interaction with FcγRIIb
Localized
hepatotoxicity or DILI in
reponse to antibody
therapy in humans
Enhanced deposition
and clearance by LSEC
leading to
vasculotoxicity
Analysis of Fc portion
and specific testing of
clearance by human
FcR to minimize
crosslinking and
activation in sinusoid
Complement mediated
toxicity/Sinusoidal
obstruction syndrome
associated with
antibody therapy
Immune complex
binding to LSEC and
cell apoptosis leading
to exposure of basal
lamina
Careful screening for
binding to Fc receptors
on LSEC
Altered antibody PK in
older patients or
patients with underlying
liver disease
LSEC capillarization,
reduction in hepatic
albumin production
Careful screening for
pre-existing disease in
patient populations. Age-dependent
pharmacokinetic
assessment at Phase 1
testing
Complications due to
autoantibody
production in hepatic
autoimmunity
LSEC capillarization or
autoantibody
occupancy of FcRs
impacting on PK
Use of FcRn blockers
to enhance IgG
degradation
Desire to improve half
life of therapeutic
antibody
Accelerated clearance
by hepatic FcγRIIb
Engineering of Fc
portion to minimize
interaction or delay
internalization of
receptor
Lack of clinical efficacy
upon testing in human
subjects
Reduced abilities of
rodent or primate
models to recreate
human hepatic
antibody clearance
Inclusion of human cell
based or tissue array
screens in pre-trail
development stages TABLE 1 | Clinical challenges associated with hepatic clearance of biological
therapies and strategies to mitigate risk during drug development. Immune complex
binding to LSEC and
cell apoptosis leading
to exposure of basal
lamina
LSEC capillarization,
reduction in hepatic
albumin production January 2022 | Volume 12 | Article 753833 Frontiers in Physiology | www.frontiersin.org 9 Sinusoidal Endothelial Antibody Interactions James et al. AUTHOR CONTRIBUTIONS clearance of therapeutic antibodies for prolonged administration. However, these were rapidly cleared from the circulation since
FcγRIIb is rapidly internalized once antibody binds (Williams
et al., 2013). Nevertheless, it is clear that new approaches
to antibody design are increasing our abilities to control
the pharmacokinetics and targeting of therapeutic antibodies
to maximize efficacy whilst minimizing offtarget effects. In
conclusion we have highlighted the often-underestimated role
of the liver sinusoidal endothelial cell to antibody clearance. We have also suggested how understanding the changing
nature of LSEC in health and disease may explain variations
in pharmacokinetics and toxicity in different populations and
preclinical models. Challenges to antibody discovery programs
are summarized in Table 1. Thus, it seems vital to ensure that
future drug development pathways incorporate testing in models
with truly representative features and cellular constituents to
address issues of poor kinetics, unexpected toxicity and poor
predictive ability. clearance of therapeutic antibodies for prolonged administration. However, these were rapidly cleared from the circulation since
FcγRIIb is rapidly internalized once antibody binds (Williams
et al., 2013). Nevertheless, it is clear that new approaches
to antibody design are increasing our abilities to control
the pharmacokinetics and targeting of therapeutic antibodies
to maximize efficacy whilst minimizing offtarget effects. In
conclusion we have highlighted the often-underestimated role
of the liver sinusoidal endothelial cell to antibody clearance. We have also suggested how understanding the changing
nature of LSEC in health and disease may explain variations
in pharmacokinetics and toxicity in different populations and
preclinical models. Challenges to antibody discovery programs
are summarized in Table 1. Thus, it seems vital to ensure that
future drug development pathways incorporate testing in models
with truly representative features and cellular constituents to
address issues of poor kinetics, unexpected toxicity and poor
predictive ability. All authors listed have made a substantial, direct, and intellectual
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Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner(s) are credited
and that the original publication in this journal is cited, in accordance with accepted
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Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere during the cold winter 2015/2016
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Redistribution of total reactive nitrogen in the lowermost
Arctic stratosphere during the cold winter 2015/2016 Helmut Ziereis1, Peter Hoor2, Jens-Uwe Grooß3, Andreas Zahn4, Greta Stratmann1,a, Paul Stock1,
Michael Lichtenstern1, Jens Krause2,b, Vera Bense2, Armin Afchine3, Christian Rolf3,
Wolfgang Woiwode4, Marleen Braun4, Jörn Ungermann3, Andreas Marsing1,2, Christiane Voigt1,2,
Andreas Engel5, Björn-Martin Sinnhuber4, and Hermann Oelhaf4 Michael Lichtenstern1, Jens Krause2,b, Vera Bense2, Armin Afchine3, Christian Rolf3,
Wolfgang Woiwode4, Marleen Braun4, Jörn Ungermann3, Andreas Marsing1,2, Christiane Voigt1,2,
Andreas Engel5, Björn-Martin Sinnhuber4, and Hermann Oelhaf4
1Institut für Physik der Atmosphäre, Deutsches Zentrum für Luft- und Raumfahrt (DLR),
Oberpfaffenhofen, Germany
2Institut für Physik der Atmosphäre, Johannes-Gutenberg-Universität Mainz, Mainz, Germany
3Institut für Energie- und Klimaforschung – Stratosphäre (IEK-7), Forschungszentrum Jülich, Jülich, Germany
4Institut für Meteorologie und Klimaforschung, Karlsruher Institut für Technologie, Karlsruhe, Germany
5Institut für Atmosphäre und Umwelt, Goethe Universität Frankfurt, Frankfurt, Germany
anow at: Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany
bnow at: Excelitas Technologies GmbH & Co. KG, Wiesbaden, Germany 2Institut für Physik der Atmosphäre, Johannes-Gutenberg-Universität Mainz, Mainz, Germany
3Institut für Energie- und Klimaforschung – Stratosphäre (IEK-7), Forschungszentrum Jülich, Jülich, Germany
4Institut für Meteorologie und Klimaforschung, Karlsruher Institut für Technologie, Karlsruhe, Germany
5Institut für Atmosphäre und Umwelt, Goethe Universität Frankfurt, Frankfurt, Germany
anow at: Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany
bnow at: Excelitas Technologies GmbH & Co. KG, Wiesbaden, Germany Correspondence: Helmut Ziereis (helmut.ziereis@dlr.de) Correspondence: Helmut Ziereis (helmut.ziereis@dlr.de) Received: 20 August 2021 – Discussion started: 22 September 2021
Revised: 5 January 2022 – Accepted: 26 January 2022 – Published: 17 March 2022 Abstract. During winter 2015/2016, the Arctic stratosphere was characterized by extraordinarily low temper-
atures in connection with a very strong polar vortex and with the occurrence of extensive polar stratospheric
clouds. From mid-December 2015 until mid-March 2016, the German research aircraft HALO (High Altitude
and Long-Range Research Aircraft) was deployed to probe the lowermost stratosphere in the Arctic region
within the POLSTRACC (Polar Stratosphere in a Changing Climate) mission. More than 20 flights have been
conducted out of Kiruna, Sweden, and Oberpfaffenhofen, Germany, covering the whole winter period. Besides
total reactive nitrogen (NOy), observations of nitrous oxide, nitric acid, ozone, and water were used for this
study. Total reactive nitrogen and its partitioning between the gas and particle phases are key parameters for
understanding processes controlling the ozone budget in the polar winter stratosphere. The vertical redistribu-
tion of total reactive nitrogen was evaluated by using tracer–tracer correlations (NOy–N2O and NOy–O3). Published by Copernicus Publications on behalf of the European Geosciences Union. Research article Atmos. Chem. Phys., 22, 3631–3654, 2022
https://doi.org/10.5194/acp-22-3631-2022
© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License. 1
Introduction POLSTRACC aimed to study the lower
Arctic stratosphere over a full winter–spring period (Oelhaf
et al., 2019). This winter was characterized by unusual low
temperatures of the Arctic polar vortex. The analysis of dif-
ferent datasets showed that the early winter of this season
was the coldest in the Arctic stratosphere in the last 68 years
(Matthias et al., 2016). The temperatures in the lower strato-
sphere were close to or at record low values between late De-
cember and early February (Manney and Lawrence, 2016). In extended areas of the Arctic stratosphere, temperatures
dropped below the existence temperature of ice (Voigt et
al., 2018). With the spaceborne lidar CALIOP on board the
CALIPSO satellite, extended regions of polar stratospheric
clouds have been observed between 15 and 26 km from De-
cember until end of January (Pitts et al., 2018; Voigt et al.,
2018). Extensive PSC abundance in the Arctic leads to the
activation and repartitioning of chlorine species (Johansson
et al., 2018, 2019; Marsing et al., 2019). Trace gas measure-
ments performed with the Aura Microwave Limb Sounder
(MLS) showed remarkable denitrification in the polar vortex
(Manney and Lawrence, 2016). A finding that was also illus-
trated by model simulations. With the EMAC atmospheric
chemistry–climate model, a strong denitrification of 4 to
8 ppb was simulated (Khosrawi et al., 2017). At the begin-
ning of March, a major final warming began, and the full
breakdown of the vortex occurred at the beginning of April
(Manney and Lawrence, 2016). In this sense, the conversion of gas-phase nitric acid into
the particle phase is of decisive importance. It not only sup-
plies the surface for heterogeneous reactions but also re-
moves nitric acid as reaction partner for processes deactivat-
ing chlorine compounds. Heterogeneous reactions also en-
able the de-noxification of the stratosphere by the conversion
of NOx to nitric acid. This process also contributes to the in-
hibition of chlorine deactivation (e.g. Solomon, 1990; Waibel
et al., 1999). Particle formation is followed by sedimentation,
leading to irreversible removal of nitric acid. The removal of
nitrogen compounds from the stratosphere allows for con-
tinuing ozone destruction that increases with increasing il-
lumination of the polar vortex at the end of the polar win-
ter. Although, temperatures in the Arctic winter stratosphere
are usually higher than in the Antarctic, and polar strato-
spheric clouds (PSCs) regularly occur as well (e.g. 1
Introduction tion of sedimenting particles (e.g. Fischer et al., 1997; Hintsa
et al., 1998; Waibel et al., 1999). During the winter, the den-
itrified air masses in the polar vortex sink down. Therefore,
heterogeneous processes at higher altitudes can lead to nitri-
fication and with a time lag to a denitrification of the upper
troposphere and lower stratosphere (UTLS). Reactive nitro-
gen species are key parameters in reaction cycles controlling
ozone concentration (e.g. Hegglin et al., 2006; Stratmann et
al., 2016). A redistribution of nitrogen oxides therefore af-
fects the chemistry at the UTLS, where even small changes
may have a significant impact on the radiative properties of
the atmosphere (Riese et al., 2012). Since the mid-1980s, observations in the Antarctic and later
in the Arctic region have revealed unprecedented ozone loss
in the polar stratosphere with the beginning of the spring sea-
son (e.g. Farman et al., 1985; Müller et al., 1996; Waibel et
al., 1999; Sinnhuber et al., 2000). The discovery of the so-
called ozone hole was the starting point of extensive mea-
surement campaigns with research aircraft, balloons, satel-
lites, and ground-based instruments to study the processes
that lead to this ozone decrease. It turned out that gas-phase
chemistry alone is not sufficient to explain these observa-
tions. Heterogeneous reactions on polar stratospheric clouds
were identified as key processes for the reactions involved
(e.g. Crutzen and Arnold, 1986; Solomon, 1999; Lowe and
MacKenzie, 2008). Particle surfaces serve as a platform to
convert inactive halogen compounds into halogen species
that are suitable to destroy ozone in catalytic cycles. Depend-
ing on temperature, composition, and physical state, different
types of polar stratospheric clouds can be distinguished: liq-
uid supercooled droplets, binary or ternary solutions (SBSs,
STSs), nitric acid hydrates (NAD, NAT), and water ice parti-
cles (e.g. Fahey et al., 2001; Hoyle et al., 2013; Khosrawi et
al., 2017; Tritscher et al., 2021). In recent decades, the Arctic was the target of several in-
tensive missions like SOLVE/THESEO in 2000 (Newman et
al., 2002) or RECONCILE in 2009/2010 (von Hobe et al.,
2013) just to give a few examples. During several winter
seasons, substantial ozone loss was observed (e.g. Sinnhu-
ber et al., 2000; Rex et al., 2006; Sinnhuber et al., 2011). These measurement campaigns were followed by the POL-
STRACC (Polar stratosphere in a Changing Climate) mission
in winter 2015/2016. measurements. The simulations confirm that the ensemble of all observations is representative of the vortex-wide
vertical NOy redistribution. measurements. The simulations confirm that the ensemble of all observations is representative of the vortex-wide
vertical NOy redistribution. Redistribution of total reactive nitrogen in the lowermost
Arctic stratosphere during the cold winter 2015/2016 The
trace gases are well correlated as long as the NOy distribution is controlled by its gas-phase production from
N2O. Deviations of the observed NOy from this correlation indicate the influence of heterogeneous processes. In early winter no such deviations have been observed. In January, however, air masses with extensive nitrifi-
cation were encountered at altitudes between 12 and 15 km. The excess NOy amounted to about 6 ppb. During
several flights, along with gas-phase nitrification, indications for extensive occurrence of nitric acid containing
particles at flight altitude were found. These observations support the assumption of sedimentation and subse-
quent evaporation of nitric acid-containing particles, leading to redistribution of total reactive nitrogen at lower
altitudes. Remnants of nitrified air masses have been observed until mid-March. Between the end of February
and mid-March, denitrified air masses have also been observed in connection with high potential temperatures. This indicates the downward transport of air masses that have been denitrified during the earlier winter phase. Using tracer–tracer correlations, missing total reactive nitrogen was estimated to amount to 6 ppb. Further, indi-
cations of transport and mixing of these processed air masses outside the vortex have been found, contributing
to the chemical budget of the winter lowermost stratosphere. Observations within POLSTRACC, at the bottom
of the vortex, reflect heterogeneous processes from the overlying Arctic winter stratosphere. The comparison
of the observations with CLaMS model simulations confirm and complete the picture arising from the present H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere
measurements. The simulations confirm that the ensemble of all observations is representative of the vortex-wide
vertical NOy redistribution. 3632 2.1.1
Total reactive nitrogen (gas and particle phases) Total reactive nitrogen (NOy) is the sum of all reactive ni-
trogen species in the atmosphere, namely NO, NO2, HNO3,
PAN, HNO2, HNO4, N2O5, ClONO2, and others. During
POLSTRACC, total reactive nitrogen was measured using
the AENEAS (AtmosphEric Nitrogen oxides mEAsuring
System) instrument. Since 2012, this measuring system has
been regularly operated on HALO during several missions
(Jurkat et al., 2016; Wendisch et al., 2016; Voigt et al., 2017;
Lelieveld et al., 2018). In addition, the observed reactive nitrogen was compared
with CLaMS model simulations. It was investigated how the
model–measurement comparison performs in the different
phases of the winter and how well the model describes the
different influences of heterogeneous processes on the nitro-
gen oxide distribution at the lowermost stratosphere. The detection of total reactive nitrogen is based on a well-
established technique, comprising catalytic conversion and
chemiluminescence. NOy species are reduced catalytically
on the surface of a heated gold tube to NO (e.g. Bollinger et
al., 1983; Fahey et al., 1985). As reducing agent, hydrogen
is added. Subsequently, NO is detected by a chemilumines-
cence detector (e.g. Ridley et al., 1974; Kley, 1980; Drum-
mond et al., 1985). At DLR this detector type has also been
used for observations from other aircraft like the DLR Fal-
con (Ziereis et al., 2000b) and the DLR Dornier 228 (Reiner
et al., 1999). A modified instrument was used on the Rus-
sian research aircraft Geophysica (Voigt et al., 2005, 2006;
Molleker et al., 2014). For more than 15 years, a NO / NOy
detector identical in construction has been operated on a
commercial airliner in the framework of IAGOS-CARIBIC
(In-service Aircraft for a Global Observing System: https: 2.1
Measurement techniques The research aircraft HALO (https://www.dlr.de/content/en/
missions/halo.html, last access: 21 July 2021) is based on
a Gulfstream G550 large business aircraft with a maximum
ceiling of about 15 km and range of more than 8000 km. The
payload of nearly 3 t comprised a set of remote sensing and
in situ instruments complementing each other. A detailed de-
scription can be found elsewhere (Oelhaf et al., 2019). For
the present analysis, experimental data from several instru-
ments have been used. The observation period provided a unique opportunity to
study the lowermost stratosphere over an entire winter pe-
riod. So, the following questions could be addressed. How
does the distribution of reactive nitrogen evolve from late
December to mid-March? What influence do heterogeneous
processes have on the distribution of reactive nitrogen com-
pounds at the bottom of the polar vortex? This extremely cold
winter favoured the formation of PSC particles and their sed-
imentation. So far, PSC particles containing reactive nitro-
gen compounds have been found almost exclusively at alti-
tudes above 15 km. Therefore, another goal of these measure-
ments was the search for PSC particles at the flight altitude
of HALO. Table 1. POLSTRACC mission phases. During the three mission phases, 21 science flights (some
with intermediate landings) with more than 150 flight hours
were performed. Most of the flights have been conducted out
of Kiruna in northern Sweden. More than 70 % of the data
have been obtained north of about 60◦N. Typical flight alti-
tudes ranged between about 12.5 and 14.5 km in the lower
stratosphere. A detailed description of the flights and the
whole mission is given in Oelhaf et al. (2019). titude and Long-Range Research Aircraft) as platform for
in situ and remote sensing instruments. POLSTRACC was
part of a combined mission called PGS (POLSTRACC-GW-
LCYCLE-SALSA) that also includes objectives with respect
to gravity waves and stratosphere–troposphere exchange. One major goal was to study the interrelationship between
climate and the polar stratosphere (Oelhaf et al., 2019). The investigation of the reactive nitrogen distribution at
the bottom of the polar vortex and the search for nitrate-
containing particles was a key issue of this mission, which
was pursued with in situ and remote sensing instruments
(Braun et al., 2019). With the AENEAS (Atmospheric Ni-
trogen Oxides Measuring System) in situ instrument, the to-
tal reactive nitrogen distribution was observed in the lower-
most stratosphere. Observations of N2O and O3 have been
used to interpret the nitrogen oxides measurements. Tracer–
tracer correlations are an important tool for studying pro-
cesses apart from transport and mere gas-phase chemistry. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere Table 1. POLSTRACC mission phases. Phase I
Phase II
Phase III
Early winter
Mid-winter
Late winter
8–21 Dec
12 Jan–2 Feb
26 Feb–18 Mar Table 1. POLSTRACC mission phases. hofen to Kiruna and ended with a flight out of Kiruna and
back on 2 February. The observations were suspended until
end of February. On 26 February, flights were resumed. The
late-winter mission phase ended with a flight on 18 March
from the HALO home base in Oberpfaffenhofen to Kiruna
and back. 1
Introduction Pitts et al.,
2018). During several aircraft campaigns, nitrate-containing
PSC particles have been observed at altitudes between 15 and
21 km (Northway et al., 2002; Voigt et al., 2005). Denitrified regions resulting from particle sedimentation
were found predominantly at elevations above 15 to 16 km
(e.g. Waibel et al., 1999; Fahey et al., 2001; Popp et al., 2001;
Jin et al., 2006; Woiwode et al., 2014). Observations at lower
altitudes are rare. Denitrified regions are associated with re-
gions of elevated nitrogen concentrations caused by evapora- POLSTRACC has been an extensive measurement cam-
paign using the German research aircraft HALO (High Al- https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3633 https://doi.org/10.5194/acp-22-3631-2022 2.1.3
Ozone Ozone was measured by the Fast and Accurate In Situ Ozone
Instrument (FAIRO). It combines a UV photometer and a
fast and precise chemiluminescence detector. The total un-
certainty is 1.5 %, and the typical precision is 0.5 % at 10 Hz
(Zahn et al., 2012; Oelhaf et al., 2019). The forward-facing inlet oversamples particles. The over-
sampling is caused by the sub-isokinetic sampling of parti-
cles. Due to the high ratio between true air speed of the air-
craft and the flow velocity inside the inlet line particles are
sampled with enhanced efficiency relative to the gas phase. This approach has been already used during earlier observa-
tions, e.g. from NASA ER-2 (Fahey et al., 2001; Northway
et al., 2002), NASA DC-8 (Weinheimer et al., 1998), or the
DLR Falcon (Feigl et al., 1999; Ziereis et al., 2004). It was
used to investigate the nitrate content of PSC particles as well
as that of cirrus ice particles. The enhancement factor de-
pends on the flow ratio as well as on ambient pressure, tem-
perature, and particle size. The corresponding relation was
derived by Belyaev and Levin (1974) and was adapted for
aircraft observations (Fahey et al., 1989; Feigl et al., 1999). The enhancement factor strongly increases with increasing
particle diameter and yields a maximum value for diameters
larger than about 10 µm. For larger particles, the enhance-
ment factor increases only slightly with diameter. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphe 3634 //www.iagos.org/iagos-caribic/, last access: 21 July 2021)
(Stratmann et al., 2016). //www.iagos.org/iagos-caribic/, last access: 21 July 2021)
(Stratmann et al., 2016). 2.1.4
Water vapour Water vapour was measured by the Fast In Situ Stratospheric
Hygrometer (FISH) based on a Lyman Alpha Photometer flu-
orescence technique and achieved a precision and accuracy
of 1 % and 5.6 %, respectively, during POLSTRACC (Zöger
et al., 1999; Oelhaf et al., 2019). The FISH instrument was
connected to a forward-facing inlet and thus can detect ice
particles (evaporated at the inlet walls) in addition to the gas-
phase water vapour (Afchine et al., 2018). 2.1.5
Nitric acid A separate measurement of gas-phase nitric acid (HNO3)
was performed by the Airborne chemical Ionization Mass
Spectrometer (AIMS) using SF−
5 as a reagent ion via another
backward-facing inlet. During POLSTRACC, the accuracy
was 16 % with a precision of 10 %–15 % at a time resolution
of 1.7 s (Jurkat et al., 2017; Marsing et al., 2019; Oelhaf et
al., 2019). Whenever the expression “NOy” is used in this study,
gas-phase NOy detected with the backward-facing inlet is
meant. Total reactive nitrogen observed with the forward-
facing inlet comprises gas-phase NOy and enhanced particu-
late NOy (NOy_P) as gas-phase equivalent. It is denoted as
NOy_tot. NOy_tot is not corrected for oversampling. Apart
from episodes when particles containing nitrogen oxides
were sampled with the forward-facing inlet, the NOy signal
detected with the two channels agreed within about 7 %. 2.1.2
Nitrous oxide The measurement of nitrous oxide (N2O) is based on a quan-
tum cascade laser infrared absorption spectrometer. The TRI-
HOP (TRacer In-situ quantum cascade laser absorption spec-
trometer/ Hydrogenperoxide and Organic Peroxide monitor)
instrument was operated during POLSTRACC with a preci-
sion of 1.84 ppb and total uncertainty of 2.7 ppb for the mea-
surement of N2O (Krause et al., 2018; Oelhaf et al., 2019). During POLSTRACC, AENEAS was operated with two
separate detector channels. Both channels of the instrument
were equipped with gold converters, allowing for the detec-
tion of NOy. The two separate NOy channels were connected
to forward- and aft-facing inlets. With the backward-facing
inlet, mainly gas-phase total reactive nitrogen is measured. The sampling of particles larger than about 1 µm is discrimi-
nated (Feigl et al., 1999). The overall uncertainty of the total
reactive nitrogen measurement depends on the actual ambi-
ent concentration. It is about 8 % for volume mixing ratios
of 0.5 ppb and about 6.5 % for about 1 ppb (Stratmann et al.,
2016; Oelhaf et al., 2019). 2
Instruments and methods The observation period with HALO extended from end of
December 2015 to mid-March 2016. The deployment of
HALO was divided into three phases: early, mid, and late
winter (Table 1). During the first phase in December, two
flights were conducted out of Oberpfaffenhofen to probe the
UTLS at mid and high latitudes. The mid-winter observation
phase started on 12 January with a flight from Oberpfaffen- https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 634
H Ziere Atmos. Chem. Phys., 22, 3631–3654, 2022 2.1.6
GLORIA Vertical distributions of nitric acid were retrieved from limb-
imaging observations by the GLORIA instrument (Gim-
balled Limb Observer for Radiance Imaging of the Atmo-
sphere; Friedl-Vallon et al., 2014; Riese et al., 2014). Char-
acteristics of the GLORIA 2D chemistry mode and dynam-
ics mode data presented here are discussed by Johansson et
al. (2018) and Krasauskas et al. (2021). Note that GLORIA
samples air volumes to the right-hand side of the aircraft
and not along the vertical projection of the flight path. Fur-
thermore, due to the limb-viewing geometry, which enables
detection of minor atmospheric constituents with low abun-
dances, the individual GLORIA profiles constitute horizon-
tally smoothed representations of the atmospheric scenery
perpendicular to the flight path. Thus, the GLORIA data at NOy_tot = EF · NOy_P + NOy,
(1) (1) where EF represents the enhancement factor. NOy_net is
defined as the difference between total NOy and gas-phase
NOy. Particulate nitrate, NOy_P, as gas-phase equivalent can
be derived from the difference between the signal obtained
with the forward- (NOy_tot) and aft-facing inlets (NOy) and
the enhancement factor, respectively. NOy_P = NOy_net/EF
(2) (2) NOy_P = NOy_net/EF Atmos. Chem. Phys., 22, 3631–3654, 2022 https://doi.org/10.5194/acp-22-3631-2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3635 ing POLSTRACC, the mean age of the probed air masses
ranged between about 1 and 5 years (Krause et al., 2018). flight altitude do not exactly match the geolocations and sam-
pling characteristics of the simultaneous in situ observations
on board HALO. Therefore, in the presence of small-scale
structures and horizontal trace gas gradients, differences be-
tween these different types of observations at flight altitudes
are possible and are not necessarily indicative of instrument
errors. In the present study, the redistribution of total reactive ni-
trogen in the lowermost stratosphere during POLSTRACC
was analysed using tracer–tracer correlations, namely the
correlation between total reactive nitrogen and nitrous ox-
ide. Photolysis and the reaction of nitrous oxide with O(1D)
are the main sources of total reactive nitrogen in the strato-
sphere (Keim et al., 1997; Greenblatt and Ravishankara,
1990). These processes lead to the formation of nitric ox-
ide. Subsequent reactions produce NO2 and finally nitric acid
(HNO3) as well as other reactive nitrogen species. These pro-
cesses are most effective in regions with high UV-radiation
as in the tropical stratosphere (Murphy et al., 1993). 3
Observations Strahan (1999) formulated this relationship as follows. 2.2
CLaMS For the interpretation of the observations and the underly-
ing processes, we employ simulations of the Chemical La-
grangian Model of the Stratosphere (CLaMS). The chemical
transport model CLaMS is based on the Lagrangian trans-
port concept and is described elsewhere (Grooß et al., 2014
and references therein). The Lagrangian concept is used in
two ways. First the chemical composition of the air is sim-
ulated for so-called air parcels that follow the wind and are
distributed irregularly in space. Second, the particles are also
simulated by the Lagrangian principle. The simulations fol-
low multiple representative NAT particle parcels, which are
transported by the wind and in addition are exposed to grav-
itational settling. As temperatures increase above TNAT, the
sedimented particle parcels evaporate and cause nitrification
of the surrounding air parcels. Similarly, the vertical redistri-
bution of water vapour by ice particles is included (Tritscher
et al., 2019). This simulation setup has shown to reproduce
the observed denitrification and nitrification (Grooß et al.,
2014). The CLaMS simulation is initialized on 1 November 2015
based on MLS satellite data, multiannual CLaMS simula-
tions (Pommrich et al., 2014) and tracer–tracer correlations
using a similar procedure as described by Grooß et al. (2014),
and it runs until March 2016. The simulation encompasses
the Northern Hemisphere from the surface to 900 K poten-
tial temperature with a vertical resolution of 100 m. Results
of this simulation have also been shown elsewhere (Grooß et
al., 2018; Braun et al., 2019; Johansson et al., 2019). An analysis for a large number of ER-2 flights showed that
the NOy–N2O correlation is linear down to about 170 ppb
(Strahan, 1999). Based on these observations, a semiempiri-
cal quantity called NO∗
y can be derived, estimating the con-
centration of expected NOy arising from observed N2O in
the stratosphere. 2.1.6
GLORIA The cor-
relation between total reactive nitrogen and nitrous oxide is
conserved during the transport of air masses from the tropics
to the polar regions as long as no sinks or sources for NOy are
effective (Murphy et al., 1993). The observed negative slope
of the relation between NOy and N2O can be understood as a
kind of conversion efficiency. It is the portion of N2O that is
converted into total reactive nitrogen by photolysis and sub-
sequent reactions and is of the order of 6 %–8 % (e.g. Fahey
et al., 1990b; Loewenstein et al., 1993; Weinheimer et al.,
1993; Fischer et al., 1997; Strahan, 1999). This correlation
is linear over a wide range of N2O concentrations. Above
about 25 to 30 km altitude, the loss reaction of NO with N
becomes increasingly important, leading to a deviation from
the linear relationship for N2O values below about 100 ppb
(Loewenstein et al., 1993). Observations within the lower-
most stratosphere of the Northern Hemisphere suggest some
seasonality of this slope (Hegglin et al., 2006). A stronger
seasonality was observed in the tracer–tracer correlation be-
tween O3 and N2O (Hegglin and Shepherd, 2007; Bönisch et
al., 2011). H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3636 Figure 1. PGS flight 5 on 21 December 2015. (a) Altitude and
N2O; (b) NOy (observed), NO∗y(calculated), and HNO3 (AIMS
instrument). The uncertainty range arising from the calculation of
NO∗y is shaded in grey. (c) NOy / O3 ratio and O3. Similar NOy concentrations, with values between about 1
and 2 ppb, have also been found during earlier aircraft mis-
sions in the winter Arctic lower stratosphere (Hübler et al.,
1990; Weinheimer et al., 1993; Arnold et al., 1998). During
the POLSTAR I mission in January 1997, NOy values up to
4 ppb were observed (Ziereis et al., 2000a). In the winter po-
lar stratosphere, NOy is mainly comprised (≥90 %) of nitric
acid as was found by aircraft and balloon-borne observations
(Wetzel et al., 2002; Schneider et al., 1999). For comparison,
nitric acid observed with the AIMS instrument (Jurkat et al.,
2016, 2017; Marsing et al., 2019) is also included in Fig. 1. During most parts of the flight, nitric acid and total reactive
nitrogen agree with each other within the uncertainty range
of both instruments, which confirms that stratospheric NOy
is mainly dominated by HNO3, while other NOy components
may gain importance near the tropopause. Figure 6a shows total reactive nitrogen plotted versus N2O
for the flight on 21 December. Also included in this figure
is the regression line resulting from a linear least squares
fit (R2 = 0.87). The range of its uncertainty is indicated by
dashed lines. As expected for undisturbed conditions, NOy
and N2O are anticorrelated. To exclude tropospheric values
that would affect the correlation, only values obtained in the
stratosphere have been used for this analysis. In 2016 the
tropospheric N2O concentration amounted to about 329 ppb
(Combined Nitrous Oxide data from the NOAA Global Mon-
itoring Laboratory, 2021). Therefore, the analysis was per-
formed only for N2O values smaller than 320 ppb. Figure 1. PGS flight 5 on 21 December 2015. (a) Altitude and
N2O; (b) NOy (observed), NO∗y(calculated), and HNO3 (AIMS
instrument). The uncertainty range arising from the calculation of
NO∗y is shaded in grey. (c) NOy / O3 ratio and O3. dNOy is defined as the difference between calculated NO∗
y
and observed gas-phase NOy. dNOy is defined as the difference between calculated NO∗
y
and observed gas-phase NOy. The slope of the regression line, corresponding to the fac-
tor f given in Eq. (3), is about 0.064. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere This value agrees
reasonably well with earlier observations performed with
these instruments. In late summer 2012, the HALO mission
TACTS (Transport and composition in the UT/LMS) (Müller
et al., 2016) was performed at northern midlatitudes. Nitrifi-
cation and denitrification could be excluded for this time of
the year and region. A linear least squares fit between NOy
and N2O for stratospheric values (N2O < 320 ppb) obtained
during the TACTS mission gave a slope of about 0.067. The
derived slope is also comparable to findings during earlier
observations in the winter Arctic region that were not af-
fected by nitrification or denitrification. During the AASE
missions in winter 1989 and 1991/1992, slopes between
0.064 and 0.078, respectively, have been observed (Fahey et
al., 1990a, b; Weinheimer et al., 1993). dNOy = NOy −NO∗
y
(4) (4) In the lower winter polar stratosphere, deviations of dNOy
from zero can be indicative for processes like nitrification
and denitrification, resulting from the formation of polar
stratospheric cloud particles, their sedimentation and subse-
quent evaporation. This relationship was derived from obser-
vations in stratospheric air masses; it is not expected to hold
in tropospheric air masses. In the lower winter polar stratosphere, deviations of dNOy
from zero can be indicative for processes like nitrification
and denitrification, resulting from the formation of polar
stratospheric cloud particles, their sedimentation and subse-
quent evaporation. This relationship was derived from obser-
vations in stratospheric air masses; it is not expected to hold
in tropospheric air masses. 3.1
Tracer–tracer correlations NO∗
y = (N2O(ts) −N2O(obs)) · f + NOy(ts). (3) (3) Tracer–tracer correlations of long-lived species are an es-
tablished method to study transformation processes of trace
gases in the lower stratosphere. The relation between chem-
ical species in the stratosphere is linear and compact as
long as their chemical lifetime is long compared to trans-
port timescales (Plumb and Ko, 1992). The relation between
total reactive nitrogen and nitrous oxide can be used in this
sense, because their lifetime is long compared to transport
timescales (Keim et al., 1997). The lifetime of nitrous oxide
is more than 100 years (Prather et al., 2015). The mean age
of an air parcel can be understood as average time since the
last contact with the troposphere (Ploeger et al., 2015). Dur- N2O(obs) is the observed N2O concentration, N2O(ts) is the
tropospheric concentration of N2O entering the stratosphere,
and f is the conversion efficiency. An additional term was
added accounting for the contribution of tropospheric NOy
to the observed concentrations in the stratosphere. As long as there are no additional processes, sources or
sinks, in the lower stratosphere affecting the NOy concentra-
tion, observed NOy should be very close to NO∗
y (within the
uncertainty range of observations). https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3.2.2
Mid-winter phase According to Eq. (3), the tropospheric concentration of
N2O is included in the calculation of NO∗
y. The tropospheric
N2O value is steadily increasing over the years. Therefore,
different air mass ages imply different tropospheric N2O
concentrations. During POLSTRACC, the age of the probed
air masses ranged between about 1 and 5 years (Krause et
al., 2018). Within 5 years, the tropospheric N2O concentra-
tion increased by about 1.5 % (Combined Nitrous Oxide data
from the NOAA Global Monitoring Laboratory, 2021). This
corresponds to a difference in tropospheric N2O of about
5 ppb and a difference in NO∗
y (assuming a conversion effi-
ciency of 0.067) of about 0.3 ppb. Although this contribution
to NO∗
y is comparatively small, the tropospheric N2O con-
centration in Eq. (3) was chosen according to its air mass
age. The second phase of the POLSTRACC mission started with
the transfer flight of HALO from Oberpfaffenhofen to Kiruna
in northern Sweden on 12 January 2016. Seven flights from
Kiruna were completed by 2 February 2016. More than 90 %
of these flight routes were lying north of 60◦N, with more
than 85 % of the total flight time in the lower stratosphere
with potential vorticity values of more than 2 PVU. The
height of the dynamical tropopause is commonly attributed
to the level where the potential vorticity equals this value. p
y q
During the mid-winter phase, the observed relation be-
tween NOy and N2O differs significantly from the situation
in the early-winter phase or from the observations at mid-
latitudes as during TACTS in 2012. This deviation was al-
ready observed during the first local flight out of Kiruna on
18 January. The flight went along the east side of Sweden and
then along the coast of Norway back to Kiruna. The main
scope of this mission flight was the probing of filamented
stratospheric air (Oelhaf et al., 2019). At altitudes above
12 km, significantly higher NOy concentrations were mea-
sured, with values up to about 10 ppb, than during the De-
cember flight, with maximum values up to 3.4 ppb (Fig. 2). In parallel, the N2O concentration was as low as 280 ppb. At the beginning and end of the flight, NOy and NO∗
y were
nearly identical. NOy and NO∗
y also agreed between about
41 600 and 44 000 s UTC when HALO flew close to the
tropopause. 3.2.1
Early-winter phase The equation describing the regression can be rewritten to
take the form of Eq. (3). In this formulation, the following
calculations of NO∗
y were performed. The slope obtained dur-
ing the midlatitude mission TACTS was chosen as conver-
sion efficiency f . The deviation from the value determined
during PGS flight 5 can serve as a measure for the uncer-
tainty in determining this slope; it is about 4 %. A further
uncertainty in the calculation of NO∗
y arises from the contri-
bution of tropospheric NOy. For the POLSTRACC observa-
tions, NOy(ts) was estimated to be about 0.78 ppb (derived
from the regression curve for tropospheric N2O values). This During the early-winter phase of the POLSTRACC mission,
two flights have been conducted out of Oberpfaffenhofen
on 17 and 21 December 2015. The first flight headed from
Oberpfaffenhofen to the north-west of Scotland. The flight
on 21 December led to Spitzbergen with the main objec-
tive being to perform a polar vortex survey in early winter. During this flight (at 12 to 14.4 km altitude), observed NOy
concentrations ranged between about 1 and 3.4 ppb (Fig. 1). The highest concentrations have been found at the northern
turnaround point of the flight at about 81◦N. https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 tion of total reactive nitrogen in the lowermost Arctic stratosphere H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3637 craft observations within the STREAM campaign in the Arc-
tic found values between 0.003 and 0.006 for this ratio in
undisturbed stratospheric air (Fischer et al., 2000). value lies well within the range spanned by previous obser-
vations. From the observations during the TACTS mission,
a tropospheric value of 0.65 ppb was derived. Strahan (1999)
derived tropospheric NOy(ts) of 0.44±0.22 ppb by averaging
measurements at the tropical tropopause. During STREAM-
97, the observed mean NOy mixing ratios have been about
0.7 ppb in the upper troposphere (Fischer et al., 2000). Ob-
servations with IAGOS-CARIBIC show a high variability
of reactive nitrogen in the upper troposphere (Stratmann et
al., 2016). Values depend on region and season and where
and when the observations were performed, and they range
between about 0.4 and 1.4 ppb. 3.2.1
Early-winter phase The uncertainty in the esti-
mation of NO∗
y resulting from the uncertainty of the tropo-
spheric NOy contribution is highest directly at the tropopause
where the relative contribution of tropospheric NOy to NO∗
y
is largest. With decreasing N2O concentration and increasing
stratospheric character of the air mass, NOy arising from the
photooxidation of N2O increases. At N2O values of 300 ppb
and less this uncertainty amounts to about 10 % or less. p
In December 2015, the polar vortex had already reached
temperatures that allowed for the formation of PSC particles
(Oelhaf et al., 2019). With CALIOP, large areas covered with
PSCs were observed between approximately 15 and 25 km
altitude (Pitts et al., 2018). Based on the present study of
the NOy–N2O correlation and NOy / O3 ratio, no indications
have been found that the lowermost Arctic stratosphere was
already affected by redistribution of reactive nitrogen species
at the beginning of winter. However, considering that only
one flight has been performed to the Arctic covering only
a small part of the sub-vortex region, redistribution in the
lowermost stratosphere already in December cannot be ruled
out by these observations. 3.2.2
Mid-winter phase Outside these periods, observed NOy was sig-
nificantly higher than calculated NO∗
y. In Fig. 6b, NOy is
shown versus N2O along with NO∗
y. For stratospheric N2O
concentrations, observed NOy levels were much higher than
NO∗
y by up to about 6 ppb. This excess NOy cannot be ex-
plained by any unknown additional tropospheric source as
the deviation from NO∗
y increases with decreasing N2O. The
excess NOy amounts to about 50 % of the whole gas-phase
total reactive nitrogen and is also reflected in the highly
variable NOy / O3 ratio during this flight. High ratios have
been found along with high values for dNOy. Values of the In Fig. 1b, measured NOy values are shown along with
calculated NO∗
y values. Also shown is the uncertainty range
of NO∗
y. During most of the time, both curves agree well
within the uncertainty range. A larger deviation at around
54 000 s UTC was found at high N2O values close to the
tropopause where this relation is not expected to be valid. Another diagnostic tool for characterizing the lowermost
stratosphere with respect to reactive nitrogen species is
the NOy / O3 ratio (Murphy et al., 1993). NOy and O3
are mainly produced in the tropical stratosphere and have
comparable lifetimes in the lower stratosphere. Their ratio
is more constant than the concentration of the individual
species itself. This can also be seen in Fig. 1. NOy and O3 ex-
hibit a substantial variability along the flight track while the
NOy / O3 ratio does not change a lot. Typical values for this
ratio in the lower stratosphere at high northern latitudes are
around 0.003–0.004 for undisturbed conditions (e.g. Mur-
phy et al., 1993; Fahey et al., 1996). Values of the same
magnitude were observed during the POLSTRACC flight in
December (Fig. 1). Here a median NOy / O3 ratio of about
0.0038 was measured. For comparison, in winter 1997 air- https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3638 Figure 2. PGS flight 7 on 18 January 2016. (a) Altitude and N2O;
(b) NOy (observed), NO∗y(calculated), and HNO3 (GLORIA instru-
ment); (c) NOy / O3 ratio and O3; and (d) HNO3 observed with
GLORIA. Figure 3. PGS flight 12 on 31 January 2016. NOy (observed) and
NO∗y (calculated). Figure 3. 3.2.2
Mid-winter phase PGS flight 12 on 31 January 2016. NOy (observed) and
NO∗y (calculated). flights of the mid-winter phase. A further example for a
flight with enhanced NOy is given in Fig. 3. The flight on
31 January led towards the north of Scotland and Ireland and
further north to about 76◦N. West of Ireland and Scotland,
HALO dived into the troposphere and encountered clean tro-
pospheric air masses with NOy and O3 values down to about
0.2 and 30 ppb, respectively. For this phase of the flight,
no NO∗
y was determined, because the above-given relation
is only valid for stratospheric conditions. Between roughly
44 000 and 52 000 s UTC, N2O values between about 300 and
260 ppb was observed. Concurrently, ozone and total reactive
nitrogen increased (not shown). During this period, observed
NOy values were up to more than twice as high than esti-
mated NO∗
y. dNOy ranged between about two and more than
6 ppb. Again, higher NOy concentrations are also reflected in
a higher NOy / O3 ratio. These findings suggest a substantial
redistribution of total reactive nitrogen in the lower Arctic
stratosphere. Particles containing total reactive nitrogen can
sediment down to HALO flight altitudes within a few days. The fall speed for particles with diameter of 10 µm and above
is more than 1 km d−1 (Fahey et al., 2001). Here, evaporation
of the particles leads to increased NOy volume mixing ratios. The interpretation of this observation as particle-based redis-
tribution is supported by lidar observations from space and
from HALO. CALIOP detected PSCs in the Arctic strato-
sphere between December and late January at altitudes be-
tween 15 and 26 km (Pitts et al., 2018). Figure 2. PGS flight 7 on 18 January 2016. (a) Altitude and N2O;
(b) NOy (observed), NO∗y(calculated), and HNO3 (GLORIA instru-
ment); (c) NOy / O3 ratio and O3; and (d) HNO3 observed with
GLORIA. NOy / O3 ratio changed from around 0.004 to values up to
about 0.01. Observations with the GLORIA instrument on
board HALO complement the in situ observations down to
upper troposphere and also show nitrification of the lower-
most stratosphere (Braun et al., 2019). Figure 2d shows a sec-
tion through the atmosphere below HALO’s flight altitude for
the flight on 18 January. For this flight, typical vertical res-
olution of ∼500–1000 m was diagnosed for the chemistry
mode data. 3.2.3
Late-winter phase The third mission phase covered the period between
26 February and 18 March 2016. In total 13 flights were per-
formed in late winter. Again, most of the flights led from
Kiruna to regions north of 60◦N. Late winter was charac-
terized by descending air masses with lower N2O concentra-
tions and higher potential temperature at HALO flight alti-
tudes. At the beginning of the third mission phase, the NOy
distribution in the lower stratosphere was characterized by
the transition of the influence of sedimentation and evapo-
ration of particles to the influence by downward-transported
air masses that have been denitrified before. The flight on
26 February (Figs. 4 and 6d) may serve as an example. One
objective of this flight was observation in a stratospheric
cold pool west of Greenland. On its way to Baffin Island
total reactive nitrogen was generally higher than calculated
NO∗
y. dNOy amounted to about 4 ppb. Over Baffin Island,
N2O dropped down to values of about 205 ppb while con-
currently potential temperature reached values of more than
about 395 K (not shown). In these air masses descended from
the stratosphere above, observed NOy was about 4 ppb below
calculated NO∗
y. On the way back to Kiruna, HALO again
encountered air masses with observed NOy levels exceed-
ing NO∗
y. Thus, during this flight, signatures of both nitrifi-
cation and denitrification were observed, demonstrating the
filamentous structure of the lower stratosphere. g
Figure 7a summarizes all concurrent NOy–N2O observa-
tions during POLSTRACC. The data are grouped in sub-
datasets according to the three mission phases. The NOy–
N2O data field seems to be split into two major branches di-
vided by the line representing NO∗
y. During the early-winter
phase (points in black), the NOy–N2O data pairs are essen-
tially grouped around the NO∗
y curve. The mid-winter pe-
riod (points in red) is mostly characterized by values lying
above NO∗
y, for N2O concentrations between about 250 and
300 ppb. The late winter (points in blue) shows values above
NO∗
y as well as values below. Values below NO∗
y are asso-
ciated with N2O values below about 250 ppb. As pointed out
earlier, this cannot be attributed to the deviation of NOy from
the linear correlation at low N2O concentrations which is
only expected for N2O concentrations below about 100 ppb
(Loewenstein et al., 1993). In Fig. 3.2.2
Mid-winter phase A typical total uncertainty (1σ) of the order of
10 %–20 % is estimated for this particular flight. Both verti-
cal resolution and total errors thereby depend on altitude and
observed scenery. NOy / O3 ratio changed from around 0.004 to values up to
about 0.01. Observations with the GLORIA instrument on
board HALO complement the in situ observations down to
upper troposphere and also show nitrification of the lower-
most stratosphere (Braun et al., 2019). Figure 2d shows a sec-
tion through the atmosphere below HALO’s flight altitude for
the flight on 18 January. For this flight, typical vertical res-
olution of ∼500–1000 m was diagnosed for the chemistry
mode data. A typical total uncertainty (1σ) of the order of
10 %–20 % is estimated for this particular flight. Both verti-
cal resolution and total errors thereby depend on altitude and
observed scenery. Lidar observations on board HALO also show extensive
regions of polar stratospheric clouds. On 22 January, a large
ice PSC was detected with a horizontal expanse of about
1400 km and thickness of up to 6 km between 18 and 24 km
altitude (Voigt et al., 2018). Observations of nitrification of
the lower stratosphere up to more than 10 ppb by evaporating
particles have also been made during previous airborne mis-
sions to the Arctic and Antarctic (Hübler et al., 1990; Fischer
et al., 1997; Arnold et al., 1998; Dibb et al., 2006; Molleker
et al., 2014). In Fig. 2b nitric acid obtained in the high spectral resolu-
tion “chemistry mode” of GLORIA for this flight is shown
along with in situ observed total reactive nitrogen. Although
air masses probed with GLORIA and AENEAS are not iden-
tical, a high agreement between both measurements was
found. The interpretation of the elevated level of NOy as remnants
of evaporated particles during POLSTRACC is also sup-
ported by the observation of particulate nitrate at flight alti- Substantial deviations of observed NOy from expected
NO∗
y have been observed during nearly all POLSTRACC https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3639 western part of Greenland. Calculated NO∗
y exceeds observed
NOy by up to 5 ppb. During ascent and descend of HALO, air
masses close to the tropopause or below were encountered,
where no correlation between NOy and N2O is expected. 3.2.2
Mid-winter phase Fig-
ure 6e shows NOy versus N2O for this flight. Down to about
260 ppb N2O, observed NOy and calculated NO∗
y agreed
within a reasonable uncertainty range. On average, the dif-
ference is about 0.08 ppb with a standard deviation of about
0.48 ppb. At lower N2O concentrations, observed NOy val-
ues were significantly lower than calculated NO∗
y. At around
180 ppb N2O, about 50 % of the calculated NO∗
y was miss-
ing. During the continuation of the flight from Kangerlussuaq
to Oberpfaffenhofen in Germany (not shown), the potential
temperature did not exceed 380 K, and N2O values remained
above 260 ppb. During this flight no deviation of NOy from
NO∗
y was found. As mentioned earlier, besides the flight
from Kangerlussuaq to Oberpfaffenhofen, most of the flights
stayed north of about 60◦N. One exemption was the flight
on 16 March to the Canary Islands. Within the accuracy of
the measurement, no significant deviation of observed NOy
from NO∗
y was found (not shown). In situ observations from
HALO revealing denitrified air masses fit well in the overall
picture of this winter. They complete the picture arising from
satellite observations and model simulations for this winter. Denitrified air masses have been observed with the Aura mi-
crowave limb sounder (MLS) (Manney and Lawrence, 2016). Simulations with the EMAC model showed both denitrified
zones in the middle stratosphere and regions with enhanced
total reactive nitrogen below for December to February. By
mid-March, the denitrified zone stretches down to pressures
higher than 100 hPa (Khosrawi et al., 2017). Figure 4. PGS flight 14 on 26 February 2016. NOy (observed) and
NO∗y (calculated). Figure 4. PGS flight 14 on 26 February 2016. NOy (observed) and
NO∗y (calculated). tude. During several flights, indications for the occurrence of
particles containing nitrate have been found (see Sect. 3.3). https://doi.org/10.5194/acp-22-3631-2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere Early winter: PGS flight 5 – 21 December 2015,
mid-winter: PGS flight 7 – 18 January 2016 and PGS flight 12 –
31 January 2016, late winter: PGS flight 14 – 26 February 2016
and PGS flight 19a – 13 March 2016. The uncertainty range aris-
ing from the linear least squares fit for PGS flight 5 is indicated by
dashed lines. Figure 5. PGS flight 19 on 13 March 2016. (a) Altitude and
N2O; (b) NOy (observed) and NO∗y(calculated); (c) NOy / O3 ra-
tio and O3. predominantly found between 340 and 370 K during the
flights in January. The largest denitrification was observed
in air masses with potential temperatures between 390 and
410 K in late winter. The highest values of nitrification or
denitrification, respectively were each found at the high-
est flight altitude of approximately 14 km. During previous
observations, evidence for denitrification has mainly been
found at higher altitudes in the stratosphere. For example, in
February 1995, measurements with the MIPAS-B balloon in-
strument found a 50 % reduction in NOy at altitudes between
16 and 22 km (Waibel et al., 1999). Denitrification was also
detected by satellite observations in winter 2009/2010 in the
Arctic between about 475 and 525 K (Khosrawi et al., 2011). With the CLaMS model, denitrification at about 500 K and
nitrification at about 400 K were simulated for this winter
(Grooß et al., 2014). Figure 6. NOy and NO∗y versus N2O for selected flights in three ob-
servational phases. Early winter: PGS flight 5 – 21 December 2015,
mid-winter: PGS flight 7 – 18 January 2016 and PGS flight 12 –
31 January 2016, late winter: PGS flight 14 – 26 February 2016
and PGS flight 19a – 13 March 2016. The uncertainty range aris-
ing from the linear least squares fit for PGS flight 5 is indicated by
dashed lines. The POLSTRACC mission covered the whole winter sea-
son from December to mid-March, providing the unique op-
portunity to probe the lowermost stratosphere under differ-
ent conditions. The distribution of reactive nitrogen changed
from undisturbed condition in early winter to a condition
with elevated concentrations (nitrification) and finally to a
condition with lowered concentrations (denitrification) in
late winter. This transformation of the reactive nitrogen dis-
tribution in the lower Arctic stratosphere is shown in Fig. 6. In this figure the NOy–N2O relation for five selected flights
is depicted. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3640 Figure 5. PGS flight 19 on 13 March 2016. (a) Altitude and
N2O; (b) NOy (observed) and NO∗y(calculated); (c) NOy / O3 ra-
tio and O3. predominantly found between 340 and 370 K during the
flights in January. The largest denitrification was observed
in air masses with potential temperatures between 390 and
410 K in late winter. The highest values of nitrification or
denitrification, respectively were each found at the high-
est flight altitude of approximately 14 km. During previous
observations, evidence for denitrification has mainly been
found at higher altitudes in the stratosphere. For example, in
February 1995, measurements with the MIPAS-B balloon in-
strument found a 50 % reduction in NOy at altitudes between
16 and 22 km (Waibel et al., 1999). Denitrification was also
detected by satellite observations in winter 2009/2010 in the
Arctic between about 475 and 525 K (Khosrawi et al., 2011). With the CLaMS model, denitrification at about 500 K and
nitrification at about 400 K were simulated for this winter
(Grooß et al., 2014). The POLSTRACC mission covered the whole winter sea-
son from December to mid-March, providing the unique op-
Figure 6. NOy and NO∗y versus N2O for selected flights in three ob-
servational phases. Early winter: PGS flight 5 – 21 December 2015,
mid-winter: PGS flight 7 – 18 January 2016 and PGS flight 12 –
31 January 2016, late winter: PGS flight 14 – 26 February 2016
and PGS flight 19a – 13 March 2016. The uncertainty range aris-
ing from the linear least squares fit for PGS flight 5 is indicated by
dashed lines. Figure 5. PGS flight 19 on 13 March 2016. (a) Altitude and
N2O; (b) NOy (observed) and NO∗y(calculated); (c) NOy / O3 ra-
tio and O3. Figure 5. PGS flight 19 on 13 March 2016. (a) Altitude and
N2O; (b) NOy (observed) and NO∗y(calculated); (c) NOy / O3 ra-
tio and O3. Figure 5. PGS flight 19 on 13 March 2016. (a) Altitude and
N2O; (b) NOy (observed) and NO∗y(calculated); (c) NOy / O3 ra- Figure 6. NOy and NO∗y versus N2O for selected flights in three ob-
servational phases. 3.2.3
Late-winter phase 7b, dNOy versus N2O is
presented for the three mission phases. The values range be-
tween about +6 and −6 ppb. In both figures, high values of
NOy and dNOy for N2O values close to 320 ppb indicate tro-
pospheric air masses where the relation between NOy and
N2O is not valid. During the further course of the POLSTRACC mission,
the influence of the sedimentation and evaporation of parti-
cles on the composition of the lowermost stratosphere fur-
ther decreased. On 13 March, the flight led from Kiruna to
Kangerlussuaq in Greenland (Fig. 5). Observed NOy was
nearly identical with calculated NO∗
y during large sections of
the flight. During the last quarter of the mission at altitudes
above 14 km, air masses with potential temperatures between
380 and 410 K have been encountered. Concurrently, N2O
dropped down to minimum values of about 180 ppb over the The influence of downward transport on nitrification or
denitrification is also reflected in Fig. 8. Here dNOy is shown
versus potential temperature. Positive values of dNOy are https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere The sequence of these figures illustrates the tem-
poral evolution of NOy at the bottom of the vortex. In early
winter (Fig. 6a) NOy and N2O are well correlated, reflecting the fact that the distribution is controlled by the gas-phase
production of NOy from N2O. In mid-winter (Fig. 6b and
c) the observed NOy exceeds the calculated NO∗
y by several
parts per billion (ppb), indicating the influence of the evapo-
ration of sedimenting particles containing nitric acid. In late
winter the distribution of NOy is controlled by the down-
ward transport of air masses that have undergone removal of
nitric acid by heterogeneous processes (Fig. 6e). Figure 6d
shows a flight typical for the transition between the mid- and
late-winter distribution of NOy. The observations at HALO
flight altitude reflect the processes in the stratosphere above. https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere ereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3641 Figure 7. (a) NOy and NO∗y versus N2O for all POLSTRACC flights. The three phases of the POLSTRACC mission are colour coded. Values are additionally given as means (averaged over 5 ppb N2O intervals) along with standard deviation. Calculated NO∗y is shown as a
solid line; the uncertainty range is indicated by dashed lines. (b) Same as (a) but with dNOy versus N2O. Figure 7. (a) NOy and NO∗y versus N2O for all POLSTRACC flights. The three phases of the POLSTRACC mission are colour coded. Values are additionally given as means (averaged over 5 ppb N2O intervals) along with standard deviation. Calculated NO∗y is shown as a
solid line; the uncertainty range is indicated by dashed lines. (b) Same as (a) but with dNOy versus N2O. Figure 8. dNOy colour-coded for the three POLSTRACC phases
(early, mid, and late winter) versus potential temperature. Denitrification in the middle stratosphere by heterogeneous
processes and removal by sedimenting particles happened at
nearly the same time as nitrification by evaporating was ob-
served at the lowermost stratosphere. However, descending
denitrified air masses at flight altitudes were first observed
with a time lag of several weeks in late winter. In a certain
way, the vertical distribution of NOy in the winter strato-
sphere was mapped to a temporal variation at HALO flight
altitude. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3642 Figure 9. Distribution of dNOy in coordinates of equivalent latitude and potential temperature (theta) during phase I (13 December–21 De-
cember), phase II (12 January–2 February), and phase III (26 February–18 March). The black contours show potential vorticity in PVU. Figure 9. Distribution of dNOy in coordinates of equivalent latitude and potential temperature (theta) during phase I (13 December–21 De-
cember), phase II (12 January–2 February), and phase III (26 February–18 March). The black contours show potential vorticity in PVU. were missing during this flight period. This is at least roughly
in accordance with a much more detailed simulation using
the CLaMS model. For this POLSTRACC flight, a chemi-
cal ozone depletion of more than 1 ppm or about 50 % was
estimated (Oelhaf et al., 2019). above, predominantly at potential temperature over 380 K
and equivalent latitudes over 50◦N. However, weak denitri-
fication with losses up to 1 ppb is also observed throughout
the whole latitude range above 360 K, even outside the vor-
tex. Similarly, at lower isentropes slightly positive values of
dNOy at lower equivalent latitudes are consistent with export
of former vortex air to lower latitudes (Hoor et al., 2004;
Krause et al., 2018). These findings indicate transport and
mixing of vortex processed air masses to the mid-latitude
lowermost stratosphere in late winter and early spring. The temporal evolution of the sub-vortex region is also vis-
ible in an ozone–nitrous oxide coordinate system. Figure 10
shows the correlation between these two trace gases for the
same flights as in Fig. 6. In addition, the regression line re-
sulting from a linear least squares fit analysis of the Decem-
ber flight is plotted. Similar criteria apply to this tracer re-
lationship as for the NOy–N2O correlation (Hegglin et al.,
2006; Hegglin and Shepherd, 2007; Bönisch, 2011). A slope
of −18.5 was derived from the December 2015 flight. This
compares quite well with the mid-latitude slope derived from
the September data during the TACTS flights (see above) of
−19.2. From late January through to the end of the mission,
the observed ozone concentrations deviate more and more
from the regression line to lower values. The difference in-
creases with decreasing nitrous oxide concentrations, parallel
to the evolution of the NOy–N2O correlation in Fig. 6. Thus,
the denitrification observed in the descending air masses is
also reflected in an ozone decrease. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere As was pointed out earlier, the NOy / O3 ratio is a further
diagnostic tool for studying processes in the lower strato-
sphere. Evaporating particles lead to a nitrification of the
lowermost stratosphere and this process finds its echo in the
increase of the NOy / O3 ratio as can be seen in Fig. 2. The
influence of the denitrification in the late-winter phase does
not form such an obvious signature in the NOy / O3 ratio. For
example, Fig. 5c shows the NOy / O3 ratio versus time for
the flight on 13 March. Before about 37 000 s UTC, NOy and
NO∗
y agree reasonably well. There are no indications for den-
itrification during this part of the flight. In the further course
of the flight the difference between NOy and NO∗
y increased
along with decreasing N2O concentrations, a clear signature
of denitrification. The NOy / O3 ratio, however, does not de-
crease during this period as one might expect but it increased
from about 3.9 × 10−3 to about 4.8 × 10−3. The obvious ex-
planation for these observations is that not only NOy was
removed from the air masses but also ozone. In the sense of
the NOy / O3 ratio the decrease in NOy by denitrification is
counterbalanced by the decrease in ozone. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere Air masses processed in the polar vortex can also be
transported to midlatitudes. Figure 9 shows dNOy in coor-
dinates of equivalent latitude and theta (potential tempera-
ture). Equivalent latitude takes advantage of the adiabatically
quasi-conserved nature of potential vorticity. It therefore re-
moves the variability in trace gas distributions that originates
from reversible deviations from the climatological mean due
to Rossby and smaller-scale waves (see Hegglin et al., 2006). The early-winter period shows a relatively undisturbed dis-
tribution of reactive nitrogen, the dNOy values are close to
zero. The mid-winter period is mostly characterized by posi-
tive dNOy values, particularly above 340 K and polewards
of 50◦N equivalent latitude. The late-winter period shows
a nitrified region at the same location but with weaker ni-
trification than in phase II. A denitrified region is located Figure 8. dNOy colour-coded for the three POLSTRACC phases
(early, mid, and late winter) versus potential temperature. https://doi.org/10.5194/acp-22-3631-2022 https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3643 Figure 10. O3 versus N2O for selected flights (same as in Fig. 6)
in three observational phases. Early winter: PGS flight 5 – 21 De-
cember 2015, mid-winter: PGS flight 7 – 18 January 2016 and PGS
flight 12 – 31 January 2016, late winter: PGS flight 14 – 26 Febru-
ary 2016 and PGS flight 19a – 13 March 2016. Additionally, the
regression line resulting from the December flight is given. The un-
certainty range of the regression is indicated by dashed lines. Figure 10. O3 versus N2O for selected flights (same as in Fig. 6)
in three observational phases. Early winter: PGS flight 5 – 21 De-
cember 2015, mid-winter: PGS flight 7 – 18 January 2016 and PGS
flight 12 – 31 January 2016, late winter: PGS flight 14 – 26 Febru-
ary 2016 and PGS flight 19a – 13 March 2016. Additionally, the
regression line resulting from the December flight is given. The un-
certainty range of the regression is indicated by dashed lines. Figure 11. POLSTRACC flight on 20 January 2016. During this
flight, particulate nitrate was observed. (a) Gas-phase NOy, (b) total
nitrate (including particulate nitrate) and gas-phase nitrate, (c) same
as (b) but with a smaller time interval, and (d) ratio of particulate
nitrate (corrected for enhancement) to gas-phase nitrate. obtained with backward-facing inlet. In Fig. 11b total NOy
d
i h h f
d f
i
i l
i
h
l
i h Figure 11. POLSTRACC flight on 20 January 2016. During this
flight, particulate nitrate was observed. (a) Gas-phase NOy, (b) total
nitrate (including particulate nitrate) and gas-phase nitrate, (c) same
as (b) but with a smaller time interval, and (d) ratio of particulate
nitrate (corrected for enhancement) to gas-phase nitrate. Figure 11. POLSTRACC flight on 20 January 2016. During this
flight, particulate nitrate was observed. (a) Gas-phase NOy, (b) total
nitrate (including particulate nitrate) and gas-phase nitrate, (c) same
as (b) but with a smaller time interval, and (d) ratio of particulate
nitrate (corrected for enhancement) to gas-phase nitrate. Figure 10. O3 versus N2O for selected flights (same as in Fig. 6)
in three observational phases. 3.3
Observations of particulate nitrate The POLSTRACC payload mainly comprised in situ gas-
phase and remote sensing instruments. It did not include in-
struments for specific measurements of particle parameters. However, the NOy detector offers an indirect method to ob-
serve particles containing reactive nitrogen compounds. For
these measurements, the oversampling characteristic of the
forward-facing inlet was used. With the backward-facing in-
let, only gas-phase total reactive nitrogen is measured (see
Sect. 2.1.1). Particles evaporating within the inlet system re-
lease nitrate molecules that are detected by the NOy instru-
ment. During one episode during the flight on 18 January, the As a plausibility check, with a rough back-of-the-envelope
calculation, the missing ozone can be estimated for this
flight. Between about 37 000 and 42 200 s UTC, the deni-
trification amounted to about 5 ppb, and the NOy / O3 ra-
tio increased to about 0.0048. Based on these observations
and assuming an “undisturbed” NOy / O3 ratio of 0.004, on
average, concentrations of the order of about 1 ppm ozone https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere The vapour pressure of nitric acid over NAT can be de-
rived from measured NOy, water vapour, ambient pressure,
and temperature using the expression given by Hanson and
Mauersberger (1988). For estimating the saturation ratio, one
can assume that observed NOy mainly consists of nitric acid
in the stratosphere as confirmed by simultaneous measure-
ments of nitric acid (Fig. 1). In Fig. 13 the saturation ratio of
nitric acid is plotted along with the gas-phase equivalent of
particulate nitrate. In general, a good agreement between the
occurrence of particulate nitrate and high NAT saturation ra-
tios was found. During the flight on 18 January, the saturation
ratio was up to about 0.25 indicating ongoing evaporation of
the particles. During the particle observations of the other
flights, the air masses have been supersaturated with respect
to NAT. As discussed in Sect. 2.1.1, the enhancement factor
reaches a maximum for particles larger than about 10 µm and
is then independent of diameter (Belyaev and Levin, 1974). It also increases strongly with increasing ambient pressure. The maximum enhancement factor ranged between about 50
and 85 for the instrumental setting during this mission at the
altitudes where particles were detected. For comparison, an
enhancement factor between about 13 and 22 was applied
for measurements on board of the ER-2 at lower ambient
pressures during AAOE (Airborne Antarctic Ozone Exper-
iment) in the Antarctic (Fahey et al., 1989). For observations
during POLSTAR at altitudes up to 13 km, a maximum en-
hancement factor of 140 was derived for particles larger than
10–20 µm (Feigl et al., 1999). To convert total NOy to an equivalent gas-phase NOy con-
centration, the particle size dependent enhancement factor
has to be known. As mentioned earlier, the HALO payload
did not include instruments for the independent measurement
of the particle size. The enhancement factor therefore can
only be estimated with some uncertainty from the signal ob-
tained with the forward-facing inlet. As pointed out earlier,
nitrate containing particles evaporate completely in the in-
let and converter and the released nitrate molecules are de-
tected. From this signal, the diameter of the particles can be
derived. This method was described in detail by Northway
et al. (2002). Due to wall effects particles do not evaporate
within the sampling frequency of the instrument (1 s). The
complete evaporation takes up to 20 s. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere The measured concentration enters the formula
with the units mol mol−1: D[m] = 4.28 × 10−3 NOy
1/3
(5) (5) (
,
;
g
,
)
The measurement approach, using the oversampling char-
acteristic of inlets, has also been used during earlier aircraft
missions in the Arctic. High NOy net values indicating par-
ticulate nitrate have been observed during the SOLVE mis-
sion in winter 1999/2000 from ER-2. Large fields of PSC
particles were found between about 15 and 21 km (North-
way et al., 2002). Also balloon-borne measurements con-
firmed the presence of NAT (Voigt et al., 2000) in that win-
ter while liquid ternary solution particles (Schreiner et al.,
1999) were observed in the THESEO winter before. During
the VINTERSOL-EUPLEX mission in February 2003, sin-
gle NAT particles have been observed at altitudes between
about 18 and 20 km on board Geophysica (Voigt et al., 2005). Compared to previous observations, during POLSTRACC
particulate nitrate was found at lower altitudes between about
10 and 14.5 km. This corresponds roughly to potential tem-
peratures between about 310 and 370 K (Fig. 12). A similar approach was also chosen for analysing particles
from observations from Geophysica (Voigt et al., 2005). As
was shown by Northway et al. (2002), this method can only
be applied as long as the evaporation periods of the individ-
ual particles do not overlap. During POLSTRACC, the fre-
quency of evaporating particles was that high that for most
time this approach could not be used to estimate the parti-
cle diameter. At the edge of some particle episodes, how-
ever, individual evaporation events could be identified. Using
the above given formula, particle diameters between about 9
and 18 µm have been derived. The particle diameters derived
from the POLSTRACC observations are roughly in the same
order of magnitude as those measured during previous air-
craft missions to the Arctic. Particle diameters, between 5
and 20 µm, have been derived from measurements with the
ER-2 during the SOLVE mission at altitudes between 15 and
21 km (Northway et al., 2002; Fahey et al., 2001). During
RECONCILE in January 2010, particle diameters between
10 and 24 µm have been concluded from observations on
board of the research aircraft Geophysica (Molleker et al.,
2014). Particles with diameter smaller than 6 µm have been
observed from Geophysica in February 2003 (Voigt et al.,
2005). H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere Early winter: PGS flight 5 – 21 De-
cember 2015, mid-winter: PGS flight 7 – 18 January 2016 and PGS
flight 12 – 31 January 2016, late winter: PGS flight 14 – 26 Febru-
ary 2016 and PGS flight 19a – 13 March 2016. Additionally, the
regression line resulting from the December flight is given. The un-
certainty range of the regression is indicated by dashed lines. obtained with backward-facing inlet. In Fig. 11b total NOy
measured with the forward-facing inlet is shown along with
gas-phase NOy. NOy_tot is not corrected for enhancement
and shows values up to 100 ppb. In total, the areas in which
particulate NOy was observed during this flight extended
over about 2000 km. Outside these episodes NOy obtained
with the forward- and aft-facing inlet, respectively, agreed
within about 7 % percent. resulting chemiluminescence signal was even so high that it
could not be processed by the detection electronics. During the mid-winter phase of POLSTRACC, particu-
late nitrate has been observed during four flights using this
measurement approach. The flight on 20 January went from
Kiruna north towards Spitzbergen, then westwards towards
Iceland and back to Kiruna. One mission objective was to
probe forecasted nitrification of the lowermost stratosphere. Gas-phase NOy ranged between about 1 and 8 ppb at alti-
tudes between 12.5 and 14.3 km (see Fig. 11). During four
periods, the signal obtained with the forward-facing inlet
was up to a factor of 50 higher than the gas-phase signal Particulate nitrate was observed in situ on HALO only dur-
ing the mid-winter phase. In addition to the 20 January flight,
particulate nitrate was also observed during the flights on 18,
25 and 31 January. The large-scale particulate events were
observed over distances of approximately 800 to 1400 km. Widespread polar stratospheric clouds of different composi-
tions have also been observed with the WALES lidar instru- https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3644 ment (WAter vapour Lidar Experiment in Space – airborne
demonstrator) on board HALO above flight altitude (Voigt et
al., 2018). Between December and end of January, PSC par-
ticles have been detected with spaceborne lidar between 15
and 26 km (Pitts et al., 2018; Voigt et al., 2018). tor of the formula combines density and specific instrument
parameters. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3645 Figure 12. NOy_net versus altitude (a) and NOy_net versus potential temperature (b) for all flights with the occurrence of particulate nitrate
(18, 20, 25, 31 January 2016). Figure 12. NOy_net versus altitude (a) and NOy_net versus potential temperature (b) for all flights with the occurrence of particulate nitrate
(18, 20, 25, 31 January 2016). cess dNOy and NOy was about 20 %. The highest values
for particulate nitrate amounted to about 1.5 ppb. The high-
est peak ratio between particulate nitrate and gas-phase ni-
trate was up to more than 0.7. Averaged over a longer pe-
riod (50 800 to 53 500 s UTC), particulate nitrate amounted
to around 0.2 ppb. This corresponds to a ratio between par-
ticulate and gas-phase nitrate of about 0.14. High dNOy val-
ues were not necessarily observed at the same time as high
particulate nitrate, indicating that the particles have been al-
ready completely evaporated. With the exception of the peak
values, more reactive nitrogen was found in the gas-phase
than in the particulate phase. smaller for the smaller NAT particles compared to ice. This
might affect the enhancement factor used for the estimation
of the gas-phase equivalent of particulate nitrate and there-
fore might affect the quantitative determination of the ratio
between particulate and gas-phase nitrate. However, this un-
certainty does neither affect the fact that nitrate particles have
been observed nor does it affect the estimate of the particle
diameter. It also has no influence on the gas-phase measure-
ments of nitrified and denitrified air masses. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere Therefore, for estimat-
ing the diameter of the particle, the signal has to be integrated
over the complete time of evaporation. Assuming NAT den-
sity and for the specific parameters of the AENEAS NOy
detector the particle diameter can be estimated. The prefac- Following the above-sketched assumptions, the gas-phase
equivalent of the observed particulate nitrate can be esti-
mated as is presented for the observations during the flight
on 20 January (Fig. 14). This allows us to establish a kind
of NOy partitioning. The observed total reactive nitrogen in
the lower stratosphere comprises three contributions: (a) gas-
phase NOy arising from the photooxidation of N2O – “undis-
turbed NO∗
y”, (b) NOy from already evaporated nitrate par-
ticles, and (c) particulate nitrate. For the flight on 20 Jan-
uary, observed NOy exceeds calculated NO∗
y by up to 6 ppb. During the last part of this flight, up to about 60 % of ob-
served NOy can be attributed to evaporated particles. Aver-
aging over the whole flight, the median ratio between ex- https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 https://doi.org/10.5194/acp-22-3631-2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphe It corresponds to simulated NOy without considering
heterogeneous reactions, and subsequent redistribution. both in the observations during the campaign but also in the
simulation. As time proceeds, these NOy-rich structures are
diluted and mixed with ambient air. Due to the limited knowl-
edge of NAT formation nuclei, small-scale temperature dis-
tributions and also due to the resolution of the model, an ex-
act and detailed simulation of the nitrification filaments is not
possible. In the simulation, all of the HNO3 released from
NAT particle evaporation is collected in the air parcels that
have about 100 km distance. In some cases, the interpolation
of the resulting simulated NOy to a specific location as the
flight path may result in unrealistically high values. In general, again model and measurement capture the
same large-scale features. Both observations and simulations
indicate an enhancement of NOy during the same times or lo-
cations of the flight path. Typical NOy enhancements are of
the order of 2–3 ppb, but also the nitrification peak of 10 ppb
NOy was reproduced by the model. Deviations can be found
at a smaller scale where the nitrification patterns are not con-
gruent at all times. During episodes that are not affected by
nitrification, deviations between simulation and observation
are below 1 ppb. First, we compare in situ NOy measurements with model
values simulated along the flight paths. In Fig. 15 measured
and simulated values are presented for selected flights for the
three winter phases. During the early-winter phase, the lower
stratosphere does not show any signature for nitrification or
denitrification as was shown in Sect. 3.2.1. As an example,
the flight from Oberpfaffenhofen to the Arctic on 21 Decem-
ber was chosen (Fig. 15a). Up to that time, no vertical NOy
redistribution was simulated by CLaMS; therefore, NOy and
NO∗
y are identical. The larger structures of the observed in
situ NOy variations along the flight path have been largely
reproduced by the simulations. There are a few smaller-scale
variations that are not met by the model; for example, a larger As example for the late-winter period, the 13 March flight
was chosen. The air masses probed in the March flight
show indications of denitrification over longer periods. Both
observations and model simulations reveal the patchy fila-
mented structure of the lower stratosphere. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphe H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3646 Figure 13. NOy_net, ambient temperature, saturation ratio, and
TNAT for all flights where particulate nitrate was observed. During
the second half of the flight, PGS flight 8 the NAT saturation tem-
perature, TNAT, could not be calculated because of missing data. Figure 13. NOy_net, ambient temperature, saturation ratio, and
TNAT for all flights where particulate nitrate was observed. During
the second half of the flight, PGS flight 8 the NAT saturation tem-
perature, TNAT, could not be calculated because of missing data. Figure 14. NOy partitioning for PGS flight 8. Gas-phase NOy
dNOy = NOy −NO∗y (calculated) and enhancement corrected par
ticulate nitrate are shown. (a) Absolute values; (b) dNOy / NO
ratio, NOy_P / NOy ratio, with 1 s values and as running averag
(30 s). deviation of about 1 ppb NOy in absolute numbers is found
between about 50 000 and 55 000 s UTC. For the mid-winter phase, the flight on 18 January has been
chosen for comparison (Fig. 15b). During this flight, indi
cations for particulate nitrate have been found by the AE Figure 14. NOy partitioning for PGS flight 8. Gas-phase NOy,
dNOy = NOy −NO∗y (calculated) and enhancement corrected par-
ticulate nitrate are shown. (a) Absolute values; (b) dNOy / NOy
ratio, NOy_P / NOy ratio, with 1 s values and as running average
(30 s). Figure 14. NOy partitioning for PGS flight 8. Gas-phase NOy,
dNOy = NOy −NO∗y (calculated) and enhancement corrected par-
ticulate nitrate are shown. (a) Absolute values; (b) dNOy / NOy
ratio, NOy_P / NOy ratio, with 1 s values and as running average
(30 s). deviation of about 1 ppb NOy in absolute numbers is found
between about 50 000 and 55 000 s UTC. Figure 13. NOy_net, ambient temperature, saturation ratio, and
TNAT for all flights where particulate nitrate was observed. During
the second half of the flight, PGS flight 8 the NAT saturation tem-
perature, TNAT, could not be calculated because of missing data. For the mid-winter phase, the flight on 18 January has been
chosen for comparison (Fig. 15b). During this flight, indi-
cations for particulate nitrate have been found by the AE-
NEAS observations. Based on tracer–tracer correlation, the
air masses encountered have been affected by nitrification
(Sect. 3.2.2). Besides simulated NOy also NO∗
y CLaMS is
shown. 4
Model simulations Besides the above-sketched dependences, the estimation
of the enhancement factor might be hampered by the fact that
the aircraft itself has an influence on the sampling character-
istic of the inlets on the top of the fuselage. This was shown
in a publication studying the influence of the inlet position on
the quantitative determination of the ice water content (Af-
chine et al., 2018). Depending on size of the ice particles,
overestimation as well as underestimation of the ice water
content was found for the specific inlet position of the FISH
instrument. Due to the different positions of the water and
reactive nitrogen inlet, as well as the different particle size
distributions of the NAT PSC and cirrus ice crystals, these
findings cannot easily be transferred to the present measure-
ments. However, an influence of the aircraft on the particle
sampling cannot be excluded although this effect might be The distribution of reactive nitrogen in the lowermost win-
ter Arctic stratosphere was also simulated with the Chemi-
cal Lagrangian Model of the Stratosphere (CLaMS). CLaMS
was developed at the Forschungszentrum Jülich as a modular
chemistry transport model. With CLaMS, denitrification in
the Arctic stratosphere in winter 2002/2003 and 2009/2010
was simulated and compared to in situ measurements from
Geophysica (Grooß et al., 2005, 2014). The simulation of the impact of vertical settling of NAT
particles is challenging as it depends critically on tempera-
ture and also the heterogeneous nucleation (Tritscher et al.,
2021). For example, nitrification occurs when the sedimen-
tation of NAT particles falls into altitudes with temperatures
above TNAT. This results in a horizontally and vertically fil-
amentary small-scale structure in the NOy distribution, seen https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3647 Figure 15. Comparison between observed NOy and simulated NOy values with the CLaMS model. Additionally, NO∗y calculated with
CLaMS is given. Shown are examples for three POLSTRACC flights from the three observation phases. Early winter: PGS flight 5 –
21 December 2015, mid-winter: PGS flight 7 – 18 January 2016, late winter: PGS flight 19a – 13 March 2016. Figure 15. Comparison between observed NOy and simulated NOy values with the CLaMS model. Additionally, NO∗y calculated with
CLaMS is given. Shown are examples for three POLSTRACC flights from the three observation phases. Early winter: PGS flight 5 –
21 December 2015, mid-winter: PGS flight 7 – 18 January 2016, late winter: PGS flight 19a – 13 March 2016. a strong nitrification peak of about 14 ppb NOy that is not
present in the observations. A detailed investigation revealed
that this nitrification filament in the simulation belongs to
one short episode on this flight west of the coast from Green-
land (71.7◦N, 56.6◦W; θ = 404.1 K) where the flight path is
very close to one specific air model air parcel (distance of
7 km). In this air parcel, all evaporating HNO3 is collected
over the range given by the horizontal model resolution of
about 100 km. Small uncertainties in the temperature of the
reanalyses could cause a small displacement of such a fila-
ment. It may be that there is a similar nitrification filament
close to the flight path, but this cannot be clarified here. these two evaluations, vortex average and average at the flight
path, overlap well, we conclude that the ensemble of all ob-
servations are representative of the vortex-wide vertical NOy
redistribution. Finally, Fig. 17 combines observed and simulated (along
the flight paths) NOy and dNOy correlation with N2O. In
general, observations and simulations agree well with devia-
tions below about 1 ppb. Only few measurement points were
obtained below N2O values of 200 ppb, which could explain
the larger discrepancy between simulated and observed val-
ues. The comparison between observations and model simu-
lations is a multistep process. First, we were able to show,
through the comparisons along the flight paths, that the pro-
cesses underlying the model simulations are so well under-
stood that the observations can be reproduced. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere In a second
step, we showed that these simulations along the flight path
are representative of the vortex-wide NOy distribution. This,
in turn, suggests that the observations, although limited in
time and space to individual flights, provide a good descrip-
tion of the distribution of nitrogen oxides in the subpolar re-
gion during this winter. g
p
Besides these comparisons along individual flight paths,
we used the CLaMS simulation to investigate if the verti-
cal NOy redistribution on a vortex-wide scale can be under-
stood. This is done by examining the correlation on NOy
and dNOy with the inert tracer N2O. The simulations were
used to put the observations in a broader context. Especially
they can contribute to what extent the observations represent
the global development within the polar vortex. To do that,
the simulations were evaluated as vortex average and also
interpolated to the HALO flight paths. For comparing the
time development of denitrification and nitrification in the
three campaign phases, we evaluated the model averages at
central dates for each of the campaign phases (17 Decem-
ber, 22 January, and 8 March). Similar to the observations
combined in Fig. 7, Fig. 16 shows the correlation of NOy
and dNOy with N2O as interpolated to the flight path and
time obtained with CLaMS. For the comparison, two short
10 min periods out of the over 250 flight hours were ex-
cluded (within 5 min of the times 31 January 2016 14:17 and
13 March 2016 11:20 UTC), and they were close to a single
model air parcel with likely an overestimation of NOy as de-
scribed above. The right panels of Fig. 16 show two kinds
of averages for N2O bins: triangles show the average NOy
as interpolated to the flight path position, and thick coloured
lines show the average vortex NOy mixing ratios. The error
bars and the thin coloured lines indicate the ± 1σ range. As H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphe The simulation
indicates that denitrification reveals a NOy reduction by 2–
4 ppb throughout the second half of the flight. Also, in this
case, the NOy deviations between the simulation and ob-
servations are generally around 1 ppb with a few excep-
tions. Around 11:20 UTC (40 800 s), the simulation indicates https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere 3648 Figure 16. Correlations of NOy (a, b) and dNOy (c, d) with N2O from CLaMS simulations. The left panels show all dates in different
colours for the three campaign phases. Data within 5 min of the times 31 January 2016 14:17 and 13 March 2016 11:20 UTC are omitted
(see text). The right panels show the averages for N2O bins ± 1σ standard deviations of these values as filled triangles with error bar signs. Also shown are the vortex average correlation for central dates of the campaign phases as thick coloured lines, ± 1σ standard deviation is
indicated as thin coloured lines. Figure 16. Correlations of NOy (a, b) and dNOy (c, d) with N2O from CLaMS simulations. The left panels show all dates in different
colours for the three campaign phases. Data within 5 min of the times 31 January 2016 14:17 and 13 March 2016 11:20 UTC are omitted
(see text). The right panels show the averages for N2O bins ± 1σ standard deviations of these values as filled triangles with error bar signs. Also shown are the vortex average correlation for central dates of the campaign phases as thick coloured lines, ± 1σ standard deviation is
indicated as thin coloured lines. Figure 17. (a) NOy and (b) dNOy averaged over 5 ppb N2O intervals along with standard deviations for the three mission phases. Observed
values (circles) are shown along with values from the CLaMS model simulation (triangles). Figure 17. (a) NOy and (b) dNOy averaged over 5 ppb N2O intervals along with standard deviations for the three mission phases. Observed
values (circles) are shown along with values from the CLaMS model simulation (triangles). gen did not show any indications for deviations from undis-
turbed conditions controlled by gas-phase chemistry and
transport. This changed during the second mission phase in
January and beginning of February. During several flights,
enhanced NOy values have been observed. Using NOy–N2O
tracer correlations, nitrification could be clearly identified. Observed NOy values have been up to 6 ppb higher than ex-
pected without redistribution of nitrogen species. This is also
reflected in the NOy / O3 ratio that was up to more than a fac- tor of 2 higher in January than in December. This could be
interpreted in terms that the sub-vortex region was affected
by heterogeneous processes taking place in the stratosphere
above. 5
Summary During the course of the extremely cold winter 2015/2016,
aircraft-based measurements with the German research air-
craft HALO have been performed in the lowermost strato-
sphere of the Arctic region within the POLSTRACC mission. The observation period covered the whole winter–spring sea-
son from December to March. This extended observational
period offered the unique opportunity to study the changing
distribution of total reactive nitrogen in the lowermost strato-
sphere with time. Tracer–tracer correlations, the relation between NOy and
N2O and O3, respectively, have been used as a tool to study
and interpret the observed temporal evolution of the UTLS
composition. In December, the distribution of reactive nitro- https://doi.org/10.5194/acp-22-3631-2022 Atmos. Chem. Phys., 22, 3631–3654, 2022 H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere At least one of the (co-)authors is a mem-
ber of the editorial board of Atmospheric Chemistry and Physics. The peer-review process was guided by an independent editor, and
the authors also have no other competing interests to declare. Nitrification of the lowermost stratosphere in mid-winter
and denitrification in late winter are linked together by het-
erogeneous processes in the above-lying stratosphere. While
nitrification caused by sedimenting particles was already ob-
served in mid-winter at flight altitude, the result of the deni-
trification at higher altitudes was not observed at the bottom
of the vortex before the end of February. Concurrently with
denitrification, lower ozone concentrations were observed in
the sinking air masses, indicating ozone destruction at higher
altitudes. Disclaimer. Publisher’s note: Copernicus Publications remains
neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Special issue statement. This article is part of the special is-
sue “The Polar Stratosphere in a Changing Climate (POLSTRACC)
(ACP/AMT inter-journal SI)”. It is not associated with a conference. In situ observed total reactive nitrogen has been compared
with the results of simulations with the Chemical Lagrangian
Model of the Stratosphere (CLaMS). In general, CLaMS
simulations reproduced the observed overall NOy structures
and concentrations. This is true for undisturbed conditions in
December as well as for nitrified conditions in January and
denitrified conditions in February and March. The compari-
son with the model simulations suggests that the observations
during POLSTRACC have been representative of the vortex-
wide vertical NOy redistribution. Acknowledgements. We would like to thank all colleagues of
the DLR Flight Department for their excellent support, which
made this successful flight campaign possible. We thank the entire
POLSTRACC-GW-LCYCLE-SALSA team for the very productive
and pleasant collaboration. We thank all colleagues who partici-
pated in the meteorological flight planning. The authors would like
to thank the Earth System Modelling (ESM) project for funding the
CLaMS simulations by providing computation time on the ESM
partition of the JUWELS supercomputer. We thank Martin Dameris
(internal review) for many helpful comments. y
Thus, the present measurements provide a comprehensive
picture of the temporal evolution of the reactive nitrogen dis-
tribution during a whole winter period. They allowed us to
observe the transition of the lowermost sub-vortex region
from the undisturbed to the nitrified and finally denitrified
state. Financial support. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere halo-db.pa.op.dlr.de (last access: 21 July 2021; POLSTRACC
mission, 2021). Global CLaMS model results are available
from https://doi.org/10.26165/JUELICH-DATA/FBZRVO (Grooß,
2022). ERA-Interim reanalysis data are available at ECMWF https:
//www.ecmwf.int (last access: 21 July 2021; ECMWF, 2021). Along with enhanced gas-phase values, particulate nitrate
was observed during mid-winter at flight altitudes between
about 10 and 14 km. The occurrence of PSC particles at such
altitudes is rare. Particulate nitrate was observed over wide
regions during four flights out of Kiruna. The diameter of
these PSC particles ranged between about 9 and 18 µm. The
occurrence of particulate nitrate at flight altitude and the ni-
trification of the lowermost stratosphere fit into the picture of
other observations made in this winter. Extended PSC cover-
age has been already observed during December by CALIOP
(Pitts et al., 2018). Particulate nitrate formed in the middle
stratosphere was also observed by lidar on board HALO and
simulated by models (Voigt et al., 2018; Khosrawi et al.,
2017). However, the uncertainty of the sampling character-
istic of the inlet makes a quantitative determination of the
ratio between particulate and gas-phase nitrate difficult. Author contributions. HZ, GS, PS, and ML were responsible for
the NOy measurements. HZ was responsible for the further data
analysis and the writing of the article. PH and JK performed the
N2O measurements. VB performed part of the N2O analysis. JUG
performed the CLaMS model simulations. AZ was responsible for
the ozone measurements. AA and CR carried out the water observa-
tions. Air mass age was provided by AE, and HNO3 measurements
with the AIMS instrument were performed by AM and CV. HNO3
observations by GLORIA were provided by WW, MB, JU, and the
GLORIA team. HO and BMS were responsible for the scientific
flight planning and GLORIA observations. All co-authors were in-
volved in review and editing of the paper. Nitrified regions at the lowermost stratosphere have also
been found in the late-winter phase. Along with nitrified re-
gions, subsidence of air masses from the polar vortex con-
trolled more and more the distribution of reactive nitrogen at
flight altitudes. Using tracer–tracer correlations, substantial
denitrification could be derived in subsiding air masses with
minimum values of down to about −5 ppb. This means that
up to about 50 % of the undisturbed NOy was missing. Competing interests. H. Ziereis et al.: Redistribution of total reactive nitrogen in the lowermost Arctic stratosphere Particles falling down from the PSC regions evapo-
rate and released gas-phase NOy, leading to a nitrification of
the lowermost stratosphere. This means that during some pe-
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Assessing green manure impact on wheat productivity through Bayesian analysis of yield monitor data
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Frontiers in plant science
| 2,024
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cc-by
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TYPE Original Research
PUBLISHED 27 March 2024
DOI 10.3389/fpls.2024.1323124 TYPE Original Research
PUBLISHED 27 March 2024
DOI 10.3389/fpls.2024.1323124 OPEN ACCESS REVIEWED BY
Shahbaz Khan,
Colorado State University, United States
Pabitra Kumar Mani,
Bidhan Chandra Krishi Viswavidyalaya, India
Swaraj Kumar Dutta,
Bihar Agricultural University, India Niko Gamulin*, Miroslav Zoric´ , Ðura Karagic´ and Sreten Terzic´
Agro R&D, Login EKO d.o.o., Aradac, Serbia Agronomy research traditionally relies on small, controlled trial plots, which may
not accurately represent the complexities and variabilities found in larger, real-
world settings. To address this gap, we introduce a Bayesian methodology for the
analysis of yield monitor data, systematically collected across extensive
agricultural landscapes during the 2020/21 and 2021/22 growing seasons. Utilizing advanced yield monitoring equipment, our method provides a detailed
examination of the effects of green manure on wheat yields in a real-world
context. The results from this comprehensive analysis reveal significant insights
into the impact of green manure application on wheat production,
demonstrating enhanced yield outcomes across varied landscapes. This
evidence suggests that the Bayesian approach to analyzing yield monitor data
can offer more precise and contextually relevant information than traditional
experimental designs. This research underscores the value of integrating large-
scale data analysis techniques in agronomy, moving beyond small-scale trials to
offer a broader, more accurate perspective on agricultural practices. The
adoption of such methodologies promises to refine farming strategies and
policies, ultimately leading to more effective and sustainable agricultural
outcomes. The inclusion of a Python script in the appendix illustrates our
analytical process, providing a tangible resource for replicating and extending
this research within the agronomic community. CITATION
Gamulin N, Zoric´ M, Karagic´ Ð and Terzic´ S
(2024) Assessing green manure impact on
wheat productivity through Bayesian analysis
of yield monitor data. Front. Plant Sci. 15:1323124. doi: 10.3389/fpls.2024.1323124 COPYRIGHT
© 2024 Gamulin, Zoric´ , Karagic´ and Terzic´ . This is an open-access article distributed under
the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or reproduction
is permitted which does not comply with
these terms. KEYWORDS green manure, soil fertility, Bayesian analysis, telemetry data, MCMC Assessing green manure
impact on wheat productivity
through Bayesian analysis
of yield monitor data
Niko Gamulin*, Miroslav Zoric´ , Ðura Karagic´ and Sreten Terzic´
Agro R&D, Login EKO d.o.o., Aradac, Serbia OPEN ACCESS
EDITED BY
Tarik Mitran,
Indian Space Research Organisation, India
REVIEWED BY
Shahbaz Khan,
Colorado State University, United States
Pabitra Kumar Mani,
Bidhan Chandra Krishi Viswavidyalaya, India
Swaraj Kumar Dutta,
Bihar Agricultural University, India
*CORRESPONDENCE
Niko Gamulin
niko.gamulin@logineko.com
RECEIVED 17 October 2023
ACCEPTED 04 March 2024
PUBLISHED 27 March 2024
CITATION
Gamulin N, Zoric´ M, Karagic´ Ð and Terzic´ S
(2024) Assessing green manure impact on
wheat productivity through Bayesian analysis
of yield monitor data. Front. Plant Sci. 15:1323124. doi: 10.3389/fpls.2024.1323124
COPYRIGHT
© 2024 Gamulin, Zoric´ , Karagic´ and Terzic´ . This is an open-access article distributed under
the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the OPEN ACCESS
EDITED BY
Tarik Mitran,
Indian Space Research Organisation, India
REVIEWED BY
Shahbaz Khan,
Colorado State University, United States
Pabitra Kumar Mani,
Bidhan Chandra Krishi Viswavidyalaya, India
Swaraj Kumar Dutta,
Bihar Agricultural University, India
*CORRESPONDENCE
Niko Gamulin
niko.gamulin@logineko.com
RECEIVED 17 October 2023
ACCEPTED 04 March 2024
PUBLISHED 27 March 2024
CITATION
Gamulin N, Zoric´ M, Karagic´ Ð and Terzic´ S
(2024) Assessing green manure impact on
wheat productivity through Bayesian analysis
of yield monitor data. Front. Plant Sci. 15:1323124. doi: 10.3389/fpls.2024.1323124
COPYRIGHT
© 2024 Gamulin, Zoric´ , Karagic´ and Terzic´ . This is an open-access article distributed under
the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or reproduction
is permitted which does not comply with COPYRIGHT
© 2024 Gamulin, Zoric´ , Karagic´ and Terzic´ .
This is an open-access article distributed under
the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or reproduction
is permitted which does not comply with
these terms. green manure, soil fertility, Bayesian analysis, telemetry data, MCMC 1 Introduction This variability can be attributed to diverse
environmental conditions and agronomic practices. Bayesian statistics is a “yet another” and powerful framework
for making informed decisions from the data Kruschke (2015). It
allows researchers to incorporate existing knowledge, beliefs, or
previous data into the model. For example, existing a priori
knowledge about physiological parameters of the crop, soil
properties, as well as weather variables can be used for the
improvement of the prediction accuracy. Furthermore, Bayesian
statistics provides a framework for explicit modeling uncertainty of
various sources such as weather conditions or occurrence of pests
and plant diseases Bi and Chen (2011). Agricultural researchers traditionally conducted small-scale
field trials using appropriate experimental designs in order to
evaluate or compare the performance of different treatments, crop
varieties, or management practices. Classical experimental designs
are based on Fisher’s principles: randomization, replication and
local control Fisher et al. (1960). Statistical analysis employed by
appropriate linear model approaches is based on the key
assumption that the model errors are independent, identically
distributed and with constant variance Piepho et al. (2013). Frequently, all these assumptions are violated due to omnipresent
within-field spatial variability. It implying the presence of the small
scale variability or among plot correlation. Gilmour et al. (1997)
divided the experimental design variation into three meaningful
types of variation i.e., (i) local trend which reflect small variations in
soil fertility and moisture; (ii) large scale variation reflects the global
trend of variation typically along the row or column directions and
(iii) extraneous variation caused from agricultural management
practices that may have a recurrent pattern (for example,
direction of planting or harvesting). Availability of the linear
mixed models lead the development of the powerful
methodologies and approaches for analysis of data from well-
designed randomized experiments Besag and Kempton (1986);
Grondona et al. (1996) which gained in precision and power of
the conclusions Marchant et al. (2019). Bayesian statistics played a crucial role in our proposed model
for estimation of the green manure effect on the commercial crop. Our approach, which involves updating the probability for a
hypothesis as more evidence or information becomes available,
allowed us to incorporate both the inherent variability in data and
the uncertainty in our prior beliefs into Bayesian model. This
provided a distinct and robust understanding of the effectiveness
of green manure, and allowed us to make more informed
recommendations for its use on production fields. 1 Introduction Green manure is the practice of incorporating plants into the soil as a nutrient source,
has gained increasing attention in recent years due to its potential to enhance soil fertility,
improve crop productivity, and promote sustainable agricultural practices Ye et al. (2014); Cai et al. (2019); Ma et al. (2021). Additionally, the escalating expenses linked
with chemical fertilizers have underscored the significance of exploring and Frontiers in Plant Science 01 frontiersin.org Gamulin et al. 10.3389/fpls.2024.1323124 implementing green manure practices Tanveer et al. (2019); Li
et al. (2020); Lei et al. (2022). Green manure offers ecological
services by harnessing the power of natural processes to protection
against soil erosion, reduction of nutrient losses, improvement of
soil and water quality, and to some extent, the reduction of
occurrence of pests and weeds Dabney et al. (2001); Hartwig
and Ammon (2002); Ryder and Fares (2008); Dorn et al. (2015). Crops such as leguminous species have been used widely as green
manures to increase the available nitrogen in the soil and organic
matter in general Vyn et al. (2000); Thorup-Kristensen
et al. (2003). the possibilities for conducting of the on-farm experiments to
compare the different agronomic practices or to test the
conclusions from a small-scale field experiments with advanced
possibilities in data collection with a spatial resolution such as the
yield monitor data throughout the standard machineries Paccioretti
et al. (2021); Hegedus et al. (2023). Previously mentioned techniques for analysis small-scale
experiments cannot be readily applied for data analysis of the on-
farm experiments due to their complexity, heterogeneity and spatial
scale variability. There are large number of methodological
approaches for the analysis of on-farm experiments ranging from
geospatial regression models to Bayesian statistical methods
Kyveryga (2019); Cho et al. (2021); Paccioretti et al. (2021);
Hegedus et al. (2023). The positive impact of field peas as green manure crops on
wheat yield and quality is well-established. Wheat stands as a
pivotal crop in human civilization, being the most extensively
cultivated cereal globally, with over 220 million hectares planted
annually across various climatic zones on all continents. Therefore,
even a modest enhancement in wheat production sustainability,
such as through greenhouse gas emission reduction, can exert a
significant impact on the global environment. However, the effects
of legume crops on subsequent cereals are highly variable
Kirkegaard et al. (2008); Preissel et al. (2015) and pose challenges
in predictability. Abbreviations: MCMC, Markov Chain Monte Carlo, NUTS, No-U-Turn
Sampler; TF, Test Fields; CF, Control Fields. 1 Introduction Green manure is an important natural and sustainable tool for
maintaining the production of the healthier food while reducing the
negative environmental impact of agriculture on living
environment and making agriculture more resilient on climate
change. To our best knowledge this is first large-scale study to
compare the effectiveness of the green manure on yield of
commercial crop using Bayesian estimation model and spatial
resolution of yield monitor data. The primary objective of this
study is to demonstrate the proposed model functionality using
three real field datasets from two growing seasons. Secondly, the
theoretical behind of our proposed Bayesian model will be outlined. The Python script for the easy implementation of the proposed
model will be described. In contrast to small-scale field trials, the large-scale field or on-
farm trials are more variable and less precise but more
representative when compared with a standard agronomic
practice in a given production region Piepho et al. (2011). Intensive developments in the field of precision agriculture open frontiersin.org 2.1 Site description The sites (Figure 1) climatic zone is characterized by a long-
term annual average temperature of 11.1°C and a frost-free period
extending over 180 days. The region typically receives an annual
precipitation sum of 580 mm. However, the year 2021 experienced Frontiers in Plant Science 02 frontiersin.org 10.3389/fpls.2024.1323124 Gamulin et al. FIGURE 1
Aerial view of agricultural fields in Mužlja and Ðurđevo Vojvodina, Serbia, where data was analyzed for the year 2022. The legend differentiates
between fields treated with green manure (Test) and untreated fields (Control), providing a visual representation of the experimental design for the
assessment of green manure’s impact on crop yield. FIGURE 1
Aerial view of agricultural fields in Mužlja and Ðurđevo Vojvodina, Serbia, where data was analyzed for the year 2022. The legend differentiates
between fields treated with green manure (Test) and untreated fields (Control), providing a visual representation of the experimental design for the
assessment of green manure’s impact on crop yield. May through the first decade of August for DJ. The annual
temperature also increased by 2.1°C, affecting both soil moisture
and air humidity (Figure 2). precipitation levels exceeding this multi-year average by 88 mm. Despite this overall increase, a critical drought period lasting from
late May through June significantly affected the critical growth
stages of wheat, namely flowering and grain filling, due to reduced
nutrient availability and constrained yields. This drought was
exacerbated by an increase in the annual air temperature by 1.4°C
above the long-term average, further stressing the crops. In contrast,
2022 saw a reduction in precipitation, with the Mužlja (MU) and
Ðurdevo¯ (DJ) locations receiving 153 mm and 175 mm less than
the multi-year average, respectively. Drought periods were notably
severe, especially from May to June and June-July for MU, and from The soil across the experimental sites is classified as Pellic
Vertisol (Aric, Mollic, Gleyic, Raptic) (VR-pe-ai.mo.gl.rp)
according to the WRB classification. This heavy clay soil, with a
combined silt and clay content ranging from 76-85%, exhibits
significant physical characteristics that influence its agricultural
potential. Detailed information on the soil texture is provided in
Table 1. The soil’s structurelessness when wet and its propensity to
crack deeply (up to 50 cm) when dry, alongside high bulk density, FIGURE 2
Comparison of seasonal weather data for MU (2021 and 2022) and DJ (2022), illustrating average monthly air temperatures, precipitation sums, and
freeze probabilities. 2.2 Data Our study meticulously curated yield monitoring data from
agricultural fields for a detailed analysis of crop yields influenced by
green manure application. The initial phase in 2021 concentrated on
fields within MU, where we established two sets of fields: test fields
(TF) which had green manure applied, and control fields (CF) which
did not, to set a benchmark for yield comparison within this locale. For the 2022 season, we extended our observation to include a
different set of fields in MU, alongside newly incorporated fields
from DJ, maintaining the division into TF and CF in both areas for
our comparative study. These fields were selected based on their
historical management sequences being consistent across both TF and
CF, ensuring comparability with the sole variable being the application
of green manure. This selection process, in conjunction with aerial
imagery presented in the article, allowed for a detailed bifurcated
analysis of two geographically proximate locations—separated by
roughly six kilometers—thereby enhancing the robustness of our
comparative framework. The dataset’s comprehensive details, such
as the number of measurements, field counts, and total area sizes for
each location, are systematically listed in Table 5. substantial total porosity, and very low water infiltration rates,
underscore a prevalence of micropores. These features contribute
to its high water holding capacity, as detailed in Table 2, yet hinder
drainage and soil aeration. During spring, the soil remains cool and
moist for an extended period, effectively shortening the
vegetation season. Acidic in nature, the soils at both locations have a very low
calcium carbonate (CaCO3) content and a moderate to high level of
organic matter, contributing to their fertility profile. Available
phosphorus levels are very low, while potassium levels are
adequate (Table 3). It is important to note that the last
application of synthetic mineral fertilizers occurred in the autumn
of 2018, as part of the transition to organic farming practices,
ensuring that residual effects on soil nutrient status during our
study period were minimized. To further refine our experimental design, as depicted in
Figure 3, the fields were segmented into precisely measured
polygons to serve as distinct observational units. These polygons
were dimensioned to align with the width of the harvesting
equipment and the length determined by the interval of
measurement combined with the harvester’s speed. This granular
method of field division was critical in capturing the subtle
variances within each field. 2.2 Data Through this structured approach, we
ensured that the fields within each pair—TF and CF—were not only
similar in terms of past management practices and soil types, with
slight variations between MU and DJ, but also that they were
geographically aligned to minimize environmental variability. Thus, our experimental design allowed for a nuanced analysis
that could accurately evaluate the impact of green manure on
crop yield variations, controlling for other potential
confounding factors. 2.1 Site description The x-axis categorizes months alongside freeze probabilities, while dual y-axes display temperatures and
precipitation, respectively. FIGURE 2
Comparison of seasonal weather data for MU (2021 and 2022) and DJ (2022), illustrating average monthly air temperatures, precipitation sums, and
freeze probabilities. The x-axis categorizes months alongside freeze probabilities, while dual y-axes display temperatures and
precipitation, respectively. FIGURE 2
Comparison of seasonal weather data for MU (2021 and 2022) and DJ (2022), illustrating average monthly air temperatures, precipitation sums, and
freeze probabilities. The x-axis categorizes months alongside freeze probabilities, while dual y-axes display temperatures and
precipitation, respectively. 03 Frontiers in Plant Science frontiersin.org Gamulin et al. 10.3389/fpls.2024.1323124 10.3389/fpls.2024.1323124 10.3389/fpls.2024.1323124 TABLE 1 Soil texture (%) up to 30 cm in depth. and semi-arid climates, making it a viable option for various
agricultural conditions, including organic farming systems. and semi-arid climates, making it a viable option for various
agricultural conditions, including organic farming systems. Location
Coarse
sand
(2-
0.2
mm)
Fine sand
(0.2-
0.02
mm)
Silt
(0.02-
0.002
mm)
Clay
(<0.002
mm)
MU
0.60
13.90
24.07
61.44
DJ
1.09
22.43
26.68
49.80 2.1.1 Plant material description
2.1.1.1 Green manure crop Field peas (Pisum sativum var. sativum), the local cultivar NS
Mraz, were utilized as a green manure crop. This cultivar is a winter
semidwarf field pea variety, characterized by the development of
lush vegetative biomass reaching an average height of 75-80 cm at
the flowering stage (BBCH-scale 65). It is an early-maturing variety,
with relatively thick and sturdy stems, exhibiting good standing
ability and favorable tolerance to major pea diseases. Additionally, it
demonstrates excellent tolerance to low temperatures, even in the
absence of snow cover. The achieved yields of green biomass, dry
matter, content of key macroelements, and ash are presented
in Table 4 TABLE 2 Physical attributes of soil up to 30 cm in depth. Frontiers in Plant Science 2.2.1.2 Sampling In our study, we employed Bayesian analysis, a statistical
approach that combines prior knowledge with observed data to
update the probability of a hypothesis Kruschke (2015); West
(2016). This method is particularly advantageous when dealing
with complex systems or limited data, as it facilitates a more
comprehensive interpretation of results Kruschke (2010). In MU22 and DJ22 datasets, we encountered a significant
disparity in area sizes between TF and CF, which necessitated the
implementation of a sampling process to equilibrate the
representation of each field within our analysis. For MU22, we
had 423 measurements for the TF and 188 for the CF, while for
DJ22, both the TF and CF contained 964 measurements each. We also utilized Markov Chain Monte Carlo (MCMC)
techniques, a class of algorithms designed to approximate
complex probability distributions often encountered in Bayesian
analysis Besag et al. (1995). MCMC methods generate random
samples from a target distribution by simulating a Markov chain, a
sequence of random variables where each variable depends solely on
its immediate predecessor Karras et al. (2022). Over time, the chain
converges to the desired distribution, enabling the estimation of
various quantities of interest Robert (1995). To address this, we employed a stratified random sampling
technique. Each field, whether a test or control, was treated as a
stratum from which we randomly selected a proportional number
of measurements. This approach ensured that each field contributed
equally to the final analysis, irrespective of its size, thus maintaining
the integrity of the comparison between the TF and CF groups. For
MU22, this meant random sampling from the TF and CF
measurements to balance the two groups. In contrast, for DJ22,
since the number of measurements was already balanced, we
ensured that the selection was random and representative. In the context of evaluating the effectiveness of green manure,
these statistical techniques were instrumental in analyzing the
extensive yield monitor data. Traditional agronomic research
often relies on comparing control and test groups comprising
microplots, which may not adequately account for field
heterogeneity or other factors influencing crop yields. This sampling strategy served three key purposes: 1. It guaranteed that each field, regardless of acreage, had an
equivalent influence on the overall results, thereby
preventing larger fields from unduly affecting the
analytical outcome. By harnessing the capabilities of Bayesian analysis and MCMC
techniques, our study surmounted these limitations. 2.2.1 Data preparation
2.2.1.1 Filtering The French winter soft wheat (Triticum aestivum L.) variety
Solenzara has been utilized across all environments. It is recognized
for its notable yield potential and good disease tolerance. It
demonstrates satisfactory performance even in heavy clay soils Our data filtering process aimed to bolster the reliability and
validity of the subsequent analysis. Initially, outliers were rigorously TABLE 2 Physical attributes of soil up to 30 cm in depth. Location
Physical Properties
Water retention (% weight)
Specific weight
(g cm-3)
Bulk density
(g cm-3)
Porosity total
(vol. %)
Filtration
(K-Darcy
(cm s-1)
33 kPa
625 kPa
1500 kPa
MU
2.38
1.55
30.16
5.43x10-4
47.24
40.34
35.61
DJ
2.52
1.38
35.13
9.16x10-4
45.55
41.67
28.82 04 Gamulin et al. 10.3389/fpls.2024.1323124 TABLE 3 Chemical attributes of soil up to 30 cm in depth. Location
pH in
1M KCl
ECe 25°C
dS/m
CaCO3
(%)
SOC
(%)
Total N
(%)
AL-P2O5
(mg/100g)
AL-K2O
(mg/100g)
MU
5.05
0.479
0.11
4.07
0.261
4.74
33.86
DJ
5.71
0.502
0.24
3.38
0.17
3.04
23.19 identified by applying a statistical threshold based on the 3-sigma
rule. This rule posits that in a normal distribution, nearly all values
(99.7%) lie within three standard deviations (sigma) of the mean. Measurements falling outside this range were deemed outliers and
excluded from the datasets. This criterion was chosen as it effectively
removes extreme deviations that could disproportionately influence
the analysis, ensuring a focus on data that accurately represents the
central tendency and variability of our sample populations. In summary, this meticulous filtering and sampling process was
designed to refine our datasets, ensuring that they accurately reflect
the true effects of green manure application on crop yield without
distortion from outliers or uneven field representation. Frontiers in Plant Science E 4 Winter field peas, cv. NS Mraz, aboveground biomass yield and chemical composition at flowering stage (BBCH-scale 65). 2.2.1.2 Sampling The extensive
yield monitor measurements offered a more detailed and accurate
representation of each field, facilitating a deeper understanding of
the relationships between green manure application and crop yields. This approach allowed us to account for field heterogeneity and
other confounding factors, yielding more robust and statistically
significant results. 2. It reduced the potential for bias that might favor the test
group if the TF areas were significantly larger, as larger
sample sizes can lead to overestimation of effects. 3. It increased the comparability between the TF and CF
groups, thereby enhancing the reliability and validity of
the study’s conclusions. Location
Fresh matter yield
(kg m−2)
Dry matter yield
(kg m−2)
Macronutrients content (%)
Ash content (%)
N
P
K
C
MU21
2.20
0.431
3.867
0.29
2.081
41.74
9.64
MU22
2.48
0.610
3.58
0.43
2.69
39.04
9.79
DJ22
2.83
0.481
3.977
0.334
2.014
42.01
9.27 05 Gamulin et al. 10.3389/fpls.2024.1323124 TABLE 5 Summary of dataset characteristics per location. TABLE 5 Summary of dataset characteristics per location. Season
Location
Number of fields
Abbrev. Total area
(ha)
Total number of meas. Sample size
2021
MU
21
MU21
190
15847
Test: 1104, Ctrl.: 644
2022
MU
13
MU22
110
80137
Test: 964, Ctrl.: 964
2022
DJ
4
DJ22
110
40959
Test: 423, Ctrl.: 188
In this table, “Abbrev.” stands for abbreviation, “Meas.” stands for Measurements, AND “Ctrl.” stands for Control. P(qjD) = P(Djq)P(q)
P(D)
(1) 2.3.1.1 Estimating priors (1) In this study, the posterior distribution is derived from the dry
yield measurements observed in both scenarios - with and without
the application of green manure. Each measurement is modeled as a
function of the mean (µ) and standard deviation (s). These
parameters, µ and s, are priors that are computed given the
posterior, under the assumption that the distribution follows a
Normal (Gaussian) pattern. The direct analytical calculation of
prior values µ and s can be complex. Therefore, it is standard
practice to estimate these values using MCMC methods, which
provide a powerful and efficient approach for approximating
these parameters. The components of Equation (1) can be delineated as: •
P(q|D) is the posterior distribution of the parameters (q)
given the data (D). This is what we want to compute. •
P(D|q) is the likelihood of the data given the parameters. •
P(q) is the prior distribution of the parameters. •
P(D) is the probability of the data, also known as
the evidence. •
P(q) is the prior distribution of the parameters. •
P(D) is the probability of the data, also known as
the evidence. MCMC methods, including NUTS, make it possible to sample
from the posterior distribution without needing to compute the
evidence P(D), which can be computationally expensive or
even intractable. MCMC methods are a class of algorithms for sampling from a
probability distribution Van Ravenzwaaij et al. (2018). They
construct a Markov chain that has the desired distribution as its
equilibrium distribution. The states of the chain, after a large
number of steps, are then used as samples from the
desired distribution. By employing MCMC on the dry yield measurements, we can
derive estimates for the most probable values of µ and s. In essence,
the absolute discrepancy between the reconstructed dry yield values
and the observed measurements should be minimized. This process
allows us to achieve an optimal estimation of the parameters,
thereby providing a robust model for the underlying dry
yield distribution. The MCMC method operates in the following manner: 1. Initialization: Start from any position. This could be a
random position or an educated guess. 2. Iteration: For each iteration of the algorithm, propose a new
position. The method to propose new positions is specific
to the MCMC algorithm being used. 2.3.1.2 Calculating contrast distribution Frontiers in Plant Science 2.3.1.3 Statistical considerations of the impact of the
unobserved factors It is important to note that the yield data we analyzed were not
direct observations of the actual yield, but rather of yield monitor
measurements. As such, there is a potential for error introduced by
the yield monitor system. However, the precision of the yield
monitor system is sufficiently high, minimizing the impact of this
potential error impact on our analysis. For the growing season of MU21, the application of green
manure resulted in a TF yield average of 3540 kg/ha, compared to
the CF average of 2550 kg/ha. The calculated contrast mean yield,
standing at 1034 kg/ha, represents the mean of the differences
between matched pairs of TF and CF, rather than the difference of
the group means. This nuanced approach, which is discernible
across 68% of the observed area, underpins the agronomic benefits
of green manure, as evidenced in Figure 4. In addition to yield monitor error, we also considered the
heterogeneity of the observed fields and other unobserved factors
such as variations in humidity and other environmental conditions. These factors can significantly influence crop yields and, if not
properly accounted for, could introduce bias into results. To mitigate the impact of these unobserved factors, we adopted
a comparative approach for each observation. Specifically, we
compared neighboring fields, ensuring that any unobserved
factors would have a similar impact on both the test and control
sets. This approach allowed us to isolate the effect of green manure Moving on to MU22, the mean yields for the TF and CF groups
were 5990 kg/ha and 3220 kg/ha, respectively. The contrast mean
was notably higher at 2620 kg/ha, with the effects being statistically
evident in an impressive 87% of the observed area. This season’s
results, depicted in Figure 5, further corroborate the efficacy of
green manure in enhancing crop productivity. Lastly, in DJ22, the mean yields for the TF and CF groups were
2480 kg/ha and 1170 kg/ha, respectively. The contrast mean here
was 1263 kg/ha, and the effects were statistically discernible in 80%
of the observed area, as illustrated in Figure 6. FIGURE 3
This image showcases the meticulous segmentation of agricultural
fields into discrete polygons, illustrated by a detailed example with a
yield of 4040 kg/ha over an 86.6 m2 area. Such precise partitioning
is vital for capturing the subtle variability across the field, allowing
for a nuanced analysis of crop yield determinants. 2.3.1.2 Calculating contrast distribution Upon determining the statistical properties of the yield
measurements distribution for both scenarios-with and without
the application of green manure - simulations were conducted to
reconstruct the statistics for both cases. The effectiveness of green
manure was evaluated by comparing the mean values of posterior
distributions, achieved by calculating the contrast distribution. 3. Acceptance or rejection: Based on the likelihood of the new
position (which is calculated from the desired distribution),
decide whether to move to the new position or stay at the
current position. This decision is made using the
acceptance rule, which, in the case of the Metropolis-
Hastings algorithm, for example, accepts movements that
increase the likelihood and also sometimes accepts
movements that decrease it. The process of calculating the contrast distribution of the mean
for dry yield data in test and control groups of wheat fields is
outlined in the provided Python code snippet (Listing 2). The
process commences with the extraction of the necessary dry yield
data, followed by the establishment of probabilistic models for both
the test and control groups. Subsequent to the generation of samples
from the posterior distributions of these groups, the computation of
the contrast distribution of the mean is concluded. This process is repeated many times. The positions form a
Markov chain, where each position is dependent only on the
previous one, and after a large number of iterations, the
distribution of positions will approximate the desired distribution. F4 illustrates the reconstructed dry yield distributions for wheat
fields MU21, comparing fields that utilized green manure (TF) to
those that did not (CF). The central image represents the contrast
distribution, alternatively referred to as the posterior distribution of
the difference. On the right, the posterior contrast is displayed,
signifying the likelihood of measurements in the TF surpassing In the context of Bayesian inference, MCMC is used to sample
from the posterior distribution of the parameters. In the provided
code, the No-U-Turn Sampler (NUTS) Hoffman et al. (2014), an
extension of the Hamiltonian Monte Carlo, is used for this purpose. The foundation of MCMC technique is anchored in Bayes’ theorem
Harney (2003), as presented in Equation (1). 06 frontiersin.org Gamulin et al. 10.3389/fpls.2024.1323124 those in the CF. Essentially, this illustrates the proportion of Test
group measurements that exceed those of the CF. application on crop yield from the effects of these
unobserved factors. 3 Results The results of our methodology are summarized in Table 6 and
Figures 4–6, including mean yield values for both the TF and CF
groups, as well as the contrast mean for MU21, MU22, and DJ22. In
each case, the mean values are accompanied by the percentage of
the total observed area where the effects are statistically evident,
leaving the remaining percentage not statistically discernible due to
other factors affecting yield. 2.3.1.2 Calculating contrast distribution The posterior distribution of the difference between the TF and
CF groups serves as a robust metric, accurately quantifying the
effectiveness of the treatment administered to the Test group in
contrast to the CF. It is not permissible to simply compare the
overlap in distributions. The contrast distribution indeed embodies
this difference. To further address field heterogeneity, we divided each field into
smaller polygons (Figure 3, with each polygon represented by a
single yield monitor measurement. This approach allowed us to
compare yields at the polygon level rather than at the field level,
effectively canceling out the impact of field heterogeneity on
our results. When evaluating the wheat fields MU22, the mean difference
value between fields with and without wheat stands at 2860, a value
significantly and reliably above zero. However, it is important to
note that the overlapping distributions of the TF and CF groups do
not denote a reliable difference. The contrast distribution essentially
comprises the distribution of the difference between the simulated
measurement pairs for the TF and CF groups. Furthermore, the
overlap of distributions should not be misconstrued as an indication
of identical distributions. By adopting these strategies, we were able to conduct a more
robust and accurate analysis of the impact of green manure on crop
yields, accounting for potential sources of error and bias. This
rigorous approach to data analysis underscores the validity of our
findings and their implications for sustainable agricultural practices. frontiersin.org 2.3.1.3 Statistical considerations of the impact of the
unobserved factors The central panel in each of Figures 4–6 represents the contrast
distribution - a Bayesian estimation of the difference between yields
in TF and CF, computed for each matched pair rather than derived
from the simple difference in group means. This estimation
accounts for the variability within each pair, providing a more
accurate depiction of the effect size. The figures printed on the
central panel indicate the HDI, which contains the range of most
credible values for the contrast mean. The right-most panels in these figures reflect the posterior
distribution of the contrast estimates, with the percentages
indicating the probability of the TF yields exceeding the CF
yields. It’s noteworthy that these distributions appear non-
normal, which may be attributed to the Bayesian estimation
process that accounts for prior information and the data’s FIGURE 3
This image showcases the meticulous segmentation of agricultural
fields into discrete polygons, illustrated by a detailed example with a
yield of 4040 kg/ha over an 86.6 m2 area. Such precise partitioning
is vital for capturing the subtle variability across the field, allowing
for a nuanced analysis of crop yield determinants. FIGURE 3
This image showcases the meticulous segmentation of agricultural
fields into discrete polygons, illustrated by a detailed example with a
yield of 4040 kg/ha over an 86.6 m2 area. Such precise partitioning
is vital for capturing the subtle variability across the field, allowing
for a nuanced analysis of crop yield determinants. 07 Frontiers in Plant Science frontiersin.org Gamulin et al. 10.3389/fpls.2024.1323124 TABLE 6
Experimental results for the effectiveness of green manure on crop yield. TABLE 6
Experimental results for the effectiveness of green manure on crop yield. Observation
Yield (kg/ha)
Gain (%)
µTest
µControl
µContrast mean
MU21
3540
2550
1034
39
MU22
5990
3220
2620
86
DJ22
2480
1170
1263
112 inherent variability, rather than assuming a normal distribution for
the yield data. inherent variability, rather than assuming a normal distribution for
the yield data. encompass a wide range of experimental results, vary from -0.2 to
+3.1 Mg/ha extra yield (-11 to +156% of the reference yield) for
temperate sites and from -2.1 to +3.0 Mg/ha extra yield (-44 to
+265% of the reference yield) for Mediterranean sites Preissel et al. (2015). Peoples and Herridge (1990) cite studies by various authors
that highlight the beneficial effects of warm-season legumes used as
preceding crops on wheat grain yield. 2.3.1.3 Statistical considerations of the impact of the
unobserved factors The observed increase in
wheat grain yield, compared to a cereal-cereal cropping sequence,
varied from 0.27 to 1.6 t/ha, with a relative increase ranging between
10% and 98%. In our research, as soil conditions degrade, the
efficacy of green manure application becomes increasingly
pronounced. Particularly in scenarios marked by poor soil quality,
wherein the reference grain yield of wheat notably falls below the
national average of 4,900 kg/ha Vucicevic (2023), the utilization of
green manure demonstrates remarkable effectiveness. The
amplification in wheat grain yield associated with the winter pea
green manure application spanned from 1034 to 2620 kg/ha, and
positive effects varied between 39% and 112%. Blanco-Canqui et al. (2012) reported that summer cover crops increased crop yields,
particularly at low rates of N application. Without additional
application of N mineral fertilizer, wheat yield was increased by
1.60 times. The incorporation of green manure crops into the soil at
an optimal depth range of 20-25 cm likely indicates improved soil
management practices, which in turn lead to enhanced soil
structure, reduced compaction, and improved soil aeration
Rinnofner et al. (2008); Tanveer et al. (2019). These favorable
conditions foster the activity of microorganisms responsible for
organic matter mineralization, ultimately resulting in increased
mineral nitrogen provision Lyu et al. (2023). However, the novelty
of our work lies not merely in reinforcing the advantageous These comprehensive results, through rigorous statistical
analysis, consistently reinforce the positive impact of green
manure on crop yields across different locales and seasons. The
visual comparison provided in Figure 7 through box plots of the
yield values for the TF and CF in MU21, MU22, and DJ22, conveys
the substantial and consistent yield improvement associated with
green manure application, underscoring its potential as a
sustainable agronomic practice. 4 Discussion In this research, we endeavored to elucidate the effects of green
manure application on wheat yields over two consecutive growing
seasons, integrating novel methods that move beyond traditional
trial plots to encompass larger field scales. Our results affirm the
benefits of green manure, echoing the findings of N’Dayegamiye
and Tran (2001), who reported similar enhancements in wheat
yields and nitrogen uptake. Mineral nitrogen provision is a key factor in the response of
cereals following legumes compared with cereals following non-
legumes Peoples and Herridge (1990); Chalk (1998); Evans et al. (2001); Peoples et al. (2009). However, the response in wheat grain
yield may not be entirely due to plant available N. Improvements in
soil structure, phosphorus mobilization, the breaking of pest and
disease cycles which afflict cereal monoculture, and phytotoxic and
allelopathic effects of different crop residues have all been implicated
in the yield response Ma et al. (2021). The yield benefits, which FIGURE 4
Mean yield values for MU21, showing the TF mean (3540 kg/ha), CF mean (2550 kg/ha), and contrast mean (1034 kg/ha). The effects are statistically
evident in 68% of the total observed area. FIGURE 4
Mean yield values for MU21, showing the TF mean (3540 kg/ha), CF mean (2550 kg/ha), and contrast mean (1034 kg/ha). The effects are statistically
evident in 68% of the total observed area. 08 Frontiers in Plant Science frontiersin.org Gamulin et al. 10.3389/fpls.2024.1323124 FIGURE 5
Mean yield values for MU22, illustrating the TF mean (5990 kg/ha), CF mean (3220 kg/ha), and contrast mean (2620 kg/ha). The effects are
statistically evident in 87% of the total observed area. Mean yield values for MU22, illustrating the TF mean (5990 kg/ha), CF mean (3220 kg/ha), and contrast mean (2620 kg/ha). The effects are
statistically evident in 87% of the total observed area. monitor data were assumed to be minor and comparable across
TF and CF, thus not substantially influencing the comparative
analysis. Nevertheless, this assumption warrants further scrutiny,
as any systematic discrepancies could affect the robustness of our
conclusions. Our findings contribute to a growing body of evidence
that supports the use of green manure as a sustainable agricultural
practice, capable of boosting crop yields across diverse growing
conditions. However, this study’s scope, confined to Mužlja and
Ðurđevo, suggests the need for broader research across varying soil
types, climates, and management practices to generalize these
results. 4 Discussion Future investigations should also delve into the long-term
impacts of green manure on soil health and nutrient dynamics,
which could offer insights into the sustainability of these practices. Moreover, advanced statistical models, such as dependency-
extended two-part models Rodrigues-Motta and Forkman (2022),
could refine our understanding of the intricate relationships within
agricultural data. In conclusion, while acknowledging the limitations
and assumptions inherent in our methodology, our research
presents a compelling case for the adoption of yield monitor data
in large-scale agricultural settings. By extending the application of
rigorous, large-scale field analyses and advanced statistical outcomes of green manure but in the adoption of an innovative
approach that evaluates these effects across expansive agricultural
areas. The granularity of our field measurements, facilitated by
dividing the fields into smaller polygons, allowed for a more
detailed and accurate representation of each field, thereby
overcoming the challenge of field heterogeneity—a factor often
neglected in conventional small-scale trials. This methodological
advancement has enabled us to detect yield variations with greater
precision, as each polygon could be considered an independent
experimental unit, providing a high-resolution dataset that captures
the intricate interplays within a crop’s environment. Our use of
Bayesian analysis and MCMC techniques represents a significant
methodological leap in agricultural research. These techniques, as
applied in the works of Besag and Higdon (1999) and Rodrigues-
Motta and Forkman (2022), allow for a sophisticated interpretation
of data that classical statistical methods may not fully capture. By
integrating prior knowledge and considering the probability
distributions of our data, we have unearthed a deeper
understanding of the yield responses to green manure. It is crucial
to note that yield monitor data, while invaluable, are not infallible
proxies for actual yields. The potential errors inherent in yield FIGURE 6
Mean yield values for DJ22, with the TF mean (2480 kg/ha), CF mean (1170 kg/ha), and contrast mean (1263 kg/ha). The effects are statistically
evident in 80% of the total observed area. FIGURE 6 FIGURE 6
Mean yield values for DJ22, with the TF mean (2480 kg/ha), CF mean (1170 kg/ha), and contrast mean (1263 kg/ha). The effects are statistically
evident in 80% of the total observed area. 09 Frontiers in Plant Science frontiersin.org Gamulin et al. 10.3389/fpls.2024.1323124 FIGURE 7
Box plots of the yield values for the test and control groups in MU21, MU22, and DJ22. Besag, J., and Higdon, D. (1999). Bayesian analysis of agricultural field experiments.
J. R. Stat. Soc. Ser. B: Stat. Method. 61, 691–746. doi: 10.1111/1467-9868.00201 Data availability statement The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation. Author contributions techniques, future research can continue to unravel the complexities
of sustainable farming practices, thereby enhancing the global
agricultural landscape. NG: Data curation, Formal Analysis, Investigation,
Methodology, Software, Visualization, Writing – original draft. ĐK: Methodology, Resources, Supervision, Validation, Writing –
review & editing. MZ: Conceptualization, Investigation,
Methodology, Validation, Writing – original draft, Writing –
review & editing. ST: Writing – review & editing, Supervision. Publisher’s note All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. Conflict of interest Authors NG, ĐK, MZ, and ST were employed by company
Login EKO d.o.o. Funding The author(s) declare that no financial support was received for
the research, authorship, and/or publication of this article. The
study was conducted as part of the activities in LoginEKO Research
and Development Center, located in Aradac, Serbia. 4 Discussion the figure illustrates the differences in yield associated with
the application of green manure (2021 MU GM, 2022 MU GM, and 2022 DJ GM) and control groups (2021 MU Control, 2022 MU Control, and 2022
DJ Control). FIGURE 7
Box plots of the yield values for the test and control groups in MU21, MU22, and DJ22. the figure illustrates the differences in yield associated with
the application of green manure (2021 MU GM, 2022 MU GM, and 2022 DJ GM) and control groups (2021 MU Control, 2022 MU Control, and 2022
DJ Control). 5 Conclusion This study signifies a pivotal shift in agricultural research
methodologies, integrating sophisticated statistical techniques
such as Bayesian analysis and MCMC to evaluate green manure’s
impact on large-scale fields over two growing seasons. These
methodological advancements enhance the reliability of our
findings, demonstrating the agronomic benefits of green manure
through rigorous assessment of yield monitor data and field
heterogeneity. Our results not only confirm the effectiveness of
green manure in boosting crop yields but also highlight its role in
promoting sustainable farming practices. The adoption of these
advanced statistical methods provides a more solid foundation for
decisions in sustainable agriculture, advocating for green manure’s
integration into farming systems. Looking ahead, it’s crucial to
explore green manure’s broader applications and its potential to
improve soil health and agricultural sustainability. Our study
presents a compelling case for the use of green manure as a
scientifically backed strategy to enhance both yield and
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observed= dry_yield_measurements) dry_yield
pm. Normal ( dry_yield , mu mu, sd sigma,
observed= dry_yield_measurements) observed= dry_yield_measurements) d i s t r i b u t i o n
samples_test = np. concatenate ( # Perform MCMC sampling with model : [np. random. normal (m, s, 100) for m, s in zip (t r a c e _ d f _ t t r a c e = pm. sample (1000, tune =1000) Appendix mean (dry_yield_measurements) n t r o l])
# Generating samples for the control group using the p o s t e r i o
r d i s t r i b u t i o n
samples_control = np. concatenate (
[np. random. normal (m, s, 100) for m, s in zip (t r a c e _ d f _ c
o n t r o l [‘mu ‘], t r a c e _ d f _ c o n t r o l [‘ sigma ‘])])
samples_control = np. random. choice (samples_control, size
=1000, replace =True)
# Generating samples for the t e s t group using the p o s t e r i o r
d i s t r i b u t i o n
samples_test = np. concatenate (
[np. random. normal (m, s, 100) for m, s in zip (t r a c e _ d f _ t
e s t [‘mu’], t r a c e _ d f _ t e s t [‘ sigma ‘])])
samples_test = np. random. choice (samples_test, size =1000,
replace =True)
# Calculating the contrast d i s t r i b u t i o n of the mean
contrast_mean = t r a c e _ d f _ t e s t [‘mu’] −t r a c e _ d f _ c o n t r
o l [‘mu’]
contrast_samples = samples_test −samples_control s t d _ p r i o r = np. std (dry_yield_measurements)
# Create a PyMC3 model s t d _ p r i o r = np. std (dry_yield_measurements) # Generating samples for the control group using the p o s t e r i o
r d i s t r i b u t i o n # Create a PyMC3 model with pm. Model () as model : samples_control = np. concatenate ( # Prior for mean and standard deviation mu = pm. Normal (‘mu’, mu=mu_prior, sd= s t d _ p r i o r)
sigma = pm. Exponential (‘ sigma ‘, 1/mu_prior) mu = pm. Normal (‘mu’, mu=mu_prior, sd= s t d _
sigma = pm. Appendix mu = pm. Normal (‘mu’, mu=prior_mean, sd= p r i o r _ s t d)
sigma = pm. Exponential (‘ sigma ‘, 1/prior_mean) mu = pm. Normal (‘mu’, mu=prior_mean, sd= p r i o r _ s t d)
sigma = pm Exponential (‘ sigma ‘ 1/prior mean) dry_yield = pm. Normal (‘ dry_yield ‘, mu=mu, sd=sigma,
observed= dry_yield_control) dry_yield = pm. Normal (‘ dry_yield ‘, mu=mu, sd=sigma,
observed= dry_yield_control) Listing 1. Estimating µ and s
import pymc3 as pm
import numpy as np Listing 1. Estimating µ and s import pymc3 as pm import pymc3 as pm t r a c e _ c o n t r o l = pm. sample (1000, tune =1000) t r a c e _ c o n t r o l = pm. sample (1000, tune =1000) import numpy as np import numpy as np t r a c e _ d f _ c o n t r o l = pm. trace_to_dataframe (t r a c e _ c
o n t r o l) t r a c e _ d f _ c o n t r o l = pm. trace_to_dataframe (t r a c e _ c
o n t r o l) # Assume that dry y i e l d measurements are stored in t h i s
numpy array dry_yield_measurements = np. array ([…]) # replace
with actual data t r a c e _ d f _ c o n t r o l [‘ c l a s s ‘] = s e l
f. name_control_group t r a c e _ d f _ c o n t r o l [‘ c l a s s ‘] = s e l
f. name_control_group # Calculate prior mean and standard deviation from the
measurements # Concatenating the trace data frames for the t e s t and control
groups trace_df = pd. concat ([t r a c e _ d f _ t e s t, t r a c e _ d f _ c o
n t r o l]) mu_prior = np. mean (dry_yield_measurements) mu_prior = np. t r a c e = pm. sample (1000, tune =1000) t r a c e = pm. sample (1000, tune =1000) e s t [‘mu’], t r a c e _ d f _ t e s t [‘ sigma ‘])]) # Convert the trace to a DataFrame for easier analysis
and manipulation samples_test = np. random. choice (samples_test, size =1000,
replace =True) trace_df = pm. trace_to_dataframe (t r a c e) # Calculating the contrast d i s t r i b u t i o n of the mean
contrast_mean = t r a c e _ d f _ t e s t [‘mu’] −t r a c e _ d f _ c o n t r
o l [‘mu’]
l
l
l
l Listing 2. Contrast Distribution Calculation # Extracting the dry y i e l d data from the t e s t and
control groups d r y _ y i e l d _ t e s t = s e l f. g d f _ y i e l d _ t e s t [‘ DryYield
‘]. to_numpy () dry_yield_control = s e l f. gdf_yield_control [‘ DryYield ‘]. to_numpy () dry_yield_control = s e l f. gdf_yield_control [‘ DryYield ‘]. to_numpy () # Calculating the prior mean and standard deviation from the
grouped dry y i e l d data # Calculating the prior mean and standard deviation from the
grouped dry y i e l d data prior_mean = s e l f. gdf_dry_yield_grouped [‘ DryYield ‘]. mean () prior_mean = s e l f. gdf_dry_yield_grouped [‘ DryYield ‘]. mean () p r i o r _ s t d = s e l f. gdf_dry_yield_grouped [‘ DryYield ‘]. std () # Defining the marginal l i k e l i h o o d s for the t e s t and
control groups m a r g i n a l _ l i k e l i h o o d _ t e s t = 1
marginal_likelihood_control = 1
# Constructing the p r o b a b i l i s t i c model for the t e s t group
with pm. Model () as test_group_model :
mu = pm. Normal (‘mu’, mu=prior_mean, sd= p r i o r _ s t d)
sigma = pm. t r a c e = pm. sample (1000, tune =1000) Exponential (‘ sigma ‘, 1/prior_mean) m a r g i n a l _ l i k e l i h o o d _ t e s t = 1
marginal_likelihood_control = 1 # Constructing the p r o b a b i l i s t i c model for the t e s t group
with pm. Model () as test_group_model : dry_yield = pm. Normal (‘ dry_yield ‘, mu=mu, sd=sigma,
observed= d r y _ y i e l d _ t e s t) dry_yield = pm. Normal (‘ dry_yield ‘, mu=mu, sd=sigma,
observed= d r y _ y i e l d _ t e s t) t r a c e _ t e s t = pm. sample (1000, tune =1000) t r a c e _ d f _ t e s t = pm. trace_to_dataframe (t r a c e _ t e s t) t r a c e _ d f _ t e s t [‘ c l a s s ‘] = s e l f. name_test_group # Constructing the p r o b a b i l i s t i c model for the
control group with pm. Model () as control_group_model : frontiersin.org Frontiers in Plant Science 12
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https://openalex.org/W4289389254
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https://zenodo.org/records/1464855/files/EOM-5-2018-article_4.pdf
|
Russian
| null |
Study of Exchange Processes in Ion Exchangers in Electric Fields via Computer Resistometrics. Part 2. Methods and Technology
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,018
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cc-by
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__________________________________________________________________
Кошель Н.Д., Смирнова Е.В., Кошель С.А., Электронная обработка материалов, 2018, 54(5), 56–67. 56 56 Исследование обменных процессов в ионитах
в электрических полях методом компьютерной
резистометрии. Часть 2. Методы и техника
*Н. Д. Кошель, Е. В. Смирнова, С. А. Кошель
ГВУЗ «Украинский государственный химико-технологический университет»,
г. Днепр (Днепропетровск), 49005, Украина, *e-mail: kkknd@ua.fm Рассмотрена техника экспериментов для изучения кинетики процессов ионного обмена в
слабых электрических полях на ионообменных смолах методом компьютерной резистометрии. В процессе ионного обмена в системе ионит-модельный раствор NaCL+NaOH зарегистри-
рована динамика изменения химического состава раствора путем измерения сопротивления
раствора резистометром с проточным сенсором и последующей компьютерной обработкой
сигнала. Обсуждается схема взаимодействия электрического и диффузионного полей ионов в
твердой фазе ионита. Показано, что при наложении слабого направленного электрического
поля на сферическую частицу ионообменника нарушается сферическая симметрия скорости
перемещения ионов и изменяется баланс ионных потоков. Следствием этого является ускоре-
ние обменного процесса в направлении вектора электрического поля. Рассмотрена экспери-
ментальная установка для изучения динамики ионного обмена, включающая конвективный
реактор, встроенную колонку с порцией ионита, систему термостатирования, проточный
сенсор электрического сопротивления раствора, электронный резистометр, потенциостат-
гальваностат. Описаны три метода эксперимента – нестационарный с суспензией ионита в
замкнутом объеме, нестационарный с фильтрацией через ионит в колонке, метод открытой
фильтрации через колонку. Показано, что в суспензионном режиме обмена на основном интер-
вале времени процесса скорость ионного потока в твердой фазе постоянна и не зависит от
степени насыщения ионита. Эффект обусловлен действием закона электронейтральности на
два потока ионов с зарядами одного знака и с противоположными градиентами концентрации. Ключевые слова: ионит, ионный обмен, обменная емкость, электрическое поле, диффузия,
сопротивление, сенсор, резистометр. УДК 541.183.12:541.135
DOI: 10.5281/zenodo.1464855
ВВЕДЕНИЕ УДК 541.183.12:541.135
DOI: 10.5281/zenodo.1464855
ВВЕДЕНИЕ из-за параллельного протекания тока по иониту и
раствору часть тока протекает в растворе. Доля
тока, протекающего через ионит, приблизи-
тельно пропорциональна доле его электропро-
водности в суммарной величине, поэтому эффек-
тивность полезного действия тока меньше
приведенных оценок, особенно в растворах
высоких концентраций, например при регене-
рации ионитов (С 1 М). В этих условиях
эффект ускорения обмена тоже существует 1 и
проявляется при более высоких токах. Можно грубо оценить условия существования
эффекта ускорения процесса. Скорость движе-
ния иона под действием напряженности электри-
ческого поля определяется 2, 3 как Описанные
особенности
воздействия
электрического поля на процесс ионного обмена,
а также многообразие возможных форм электри-
ческих полей, типов ионитов и характера обмен-
ных
процессов
требуют
более
детального
анализа результатов взаимодействия электри-
ческого и диффузионного полей в ионитах. Для
изучения явлений, возникающих при наложении
электрического поля на специфический двойной
диффузионный процесс в ионитах, необходимы
специальные нетрадиционные методы, соответ-
ствующее оборудование и техника экспери-
ментов. Рассматриваемая далее техника исследо-
ваний ионообменных процессов позволяет суще-
ственно ускорить определение свойств ионитов,
которое выполняется обычно 4, 5 традицион-
ными химико-аналитическими методами. λ
,
z
dE
v
F
dx
(1) (1) где множитель перед скобками – физическая
подвижность данного типа ионов v0 (скорость
движения в поле напряженностью 1 В/см),
определяемая из уравнения Нернста-Эйнштейна;
z – заряд иона; F = 96500 Кл/моль – число Фара-
дея; – эквивалентная электропроводность иона. Для ионов (кроме ионов Н+ и ОН–) предельное
значение в очень разбавленных водных раство-
рах составляет около 40–60 Ом–1см2·моль–1, а в
твердой фазе ионита – на 1–2 порядка меньше. Отсюда физическую подвижность однозарядного
иона в ионите можно оценить величиной
v0 = /F 0,00005 см2·(Вс)–1. Если принять
коэффициент
диффузии
в
твердой
фазе
D 10–6 см2/с, то есть на порядок меньшим, чем
в растворе, скорость диффузионного потока
ионов vдиф можно приблизительно оценить для
частиц радиусом r = 0,05 см как vдиф D/r =
2·10–5 cм/c. Оценка по соотношению (1) показы-
вает, что электромиграционная составляющая
скорости ионов vмигр, соизмеримая с диффу-
зионной скоростью, возникает при напряжен-
ности электрического поля 1–2 В/см и плот-
ностях тока менее 1 мА/см2. Однако реально В настоящем сообщении изложен достаточно
широкий круг вопросов техники исследований с
тем, чтобы не описывать их подробно в сообще-
ниях о конкретных результатах. УДК 541.183.12:541.135
DOI: 10.5281/zenodo.1464855
ВВЕДЕНИЕ соида. Эта геометрическая фигура сдвинута в
направлении градиента потенциала электри-
ческого
поля
dE/dx
относительно
центра
частицы. Этот сдвиг тем больше, чем больше
градиент потенциала поля. Асимметрия профиля
скоростей относительно поверхности частицы
нарушает баланс ионных потоков через границу
раздела фаз и вызывает ускорение потока ионов. Эффект соответствует разности двух участков
площади
сечения
эллипсоида,
разделенных
точками его пересечения с контуром частицы и
расположенных на фронтальной и тыльной
сторонах частицы. Наибольший эффект ускоре-
ния будет заметен в направлении вектора
градиента электрического поля, наименьший – в
перпендикулярном направлении (рис. 1). Ионообменные технологии на предприятиях
различных отраслей промышленности и энерге-
тики широко используются для целей сорбции,
водоподготовки
и
водоочистки. Особенно
эффективны ионообменные системы для глубо-
кой очистки воды в фармацевтической промыш-
ленности и ядерно-химических технологиях. Известно, что кинетика процессов ионного
обмена контролируется скоростью диффузии
ионов в твердой фазе и сильно различается у
многочисленных типов ионитов. В [1] на
примере регенерации катионита КУ-2-8 было
показано, что ускорить диффузионный процесс в
твердой фазе возможно при наложении слабых
электрических полей. Описанная
простейшая
картина
взаимо-
действия полей в действительности намного
сложнее. В колонке частицы контактируют
между собой, их электрические поля перекрыва-
ются и деформируются. Кроме того, поток
жидкой фазы между частицами вносит дополни-
тельный фактор диффузионного торможения в
жидкости. Еще один отрицательный дейст-
вующий фактор – возникновение электрического Некоторые простейшие закономерности этого
явления можно представить качественно. На
рис. 1 можно увидеть механизм эффекта ускоре-
ния обменного процесса. При наложении одно-
родного электрического поля на отдельную
частицу ионита трехмерная поверхность, образо-
ванная векторами результирующей скорости
подвижных ионов, приобретает форму эллип- (а)
(б)
Рис. 1. Схема взаимодействия диффузионного и электрического полей в сферической частице ионита в однородном
электрическом поле. (а) – ориентация диффузионного (1), электрического (2) полей и результирующего поля скоростей
подвижных ионов (3) в диаметральном сечении сферической частицы ионита. (б) – сечение эллипсоидного поля скоростей
подвижных ионов (F) относительно сечения частицы (S). 57 57 (б) (а) (а) (б) Рис. 1. Схема взаимодействия диффузионного и электрического полей в сферической частице ионита в однородном
электрическом поле. (а) – ориентация диффузионного (1), электрического (2) полей и результирующего поля скоростей
подвижных ионов (3) в диаметральном сечении сферической частицы ионита. (б) – сечение эллипсоидного поля скоростей
подвижных ионов (F) относительно сечения частицы (S). поля в электропроводной жидкой фазе. Ток в
такой двухфазной дискретной системе протекает
параллельно в обеих фазах. При этом часть тока
в фазе ионита, которая как раз и образует эффект
ускорения
ионного
обмена,
уменьшается
пропорционально возрастанию электропровод-
ности жидкой фазы. ЭКСПЕРИМЕНТАЛЬНАЯ УСТАНОВКА Схема установки для исследования динамики
ионного обмена показана на рис. 2. Установка
находится в цилиндрическом реакторе (1) из
нержавеющей стали с внутренним диаметром 58 100 мм и высотой 103 мм. На боковую поверх-
ность реактора, изолированную стеклотканью,
для обогрева намотана тонкая нихромовая
проволока (2), закрытая снаружи теплоизолиру-
ющим асбестовым слоем. Регулировка и стаби-
лизация температуры обеспечиваются блоком из
контактного термометра (3) и электронного
усилителя УКТ-4У2 (4), функционирующего в
режиме реле. Система терморегулирования рабо-
тает от трансформатора (5) с выходным напря-
жением до 50 В и имеет регулируемую мощность
до 50 Вт. В реакторе предусмотрена циркуляция
раствора пропеллерной мешалкой (6) с электро-
двигателем,
работающим
от
регулируемого
источника постоянного напряжения (7). Цирку-
ляция раствора предназначена для обеспечения
однородности температурного и концентрацион-
ного полей в реакторе. 100 мм и высотой 103 мм. На боковую поверх-
ность реактора, изолированную стеклотканью,
для обогрева намотана тонкая нихромовая
проволока (2), закрытая снаружи теплоизолиру-
ющим асбестовым слоем. Регулировка и стаби-
лизация температуры обеспечиваются блоком из
контактного термометра (3) и электронного
усилителя УКТ-4У2 (4), функционирующего в
режиме реле. Система терморегулирования рабо-
тает от трансформатора (5) с выходным напря-
жением до 50 В и имеет регулируемую мощность
до 50 Вт. В реакторе предусмотрена циркуляция
раствора пропеллерной мешалкой (6) с электро-
двигателем,
работающим
от
регулируемого
источника постоянного напряжения (7). Цирку-
ляция раствора предназначена для обеспечения
однородности температурного и концентрацион-
ного полей в реакторе. деленный потенциал, так как у гидроксида
серебра меньшее произведение растворимости,
чем у хлорида. Для
непрерывного
мониторинга
состава
растворов использовали предложенный нами
ранее
метод
компьютерной
резистометрии,
теория которого подробно рассмотрена в 6. Метод основан на измерениях удельного сопро-
тивления смеси растворов двух электролитов,
NaCl + NaOH, с последующей компьютерной
обработкой сигнала. Удельные электропровод-
ности солей и щелочей или солей и кислот
существенно отличаются 7, 8 вследствие
аномального механизма переноса ионов Н+ и
ОН– в водных растворах. Это различие и позво-
ляет использовать их для точного определения
состава смешанного бинарного раствора. Оно
сохраняется при любых температурах, однако
характер температурной зависимости электро-
проводности
значительно
различается
для
растворов NaCl и NaOH 7. Рис. 2. Функциональная схема установки с мини-колонкой
УМК-3 и проточным низкочастотным резистометрическим
сенсором РСН. Программа компьютерной обработки сигнала
резистометрического сенсора позволяет опреде-
лить
концентрации
отдельных
компонентов
раствора путем сравнения измеренной удельной
электропроводности раствора с ее теорети-
ческим значением для заданной температуры. Теоретическое значение удельной электропро-
водности вычисляется путем аппроксимации
известных
справочных
данных
7,
8. ЭКСПЕРИМЕНТАЛЬНАЯ УСТАНОВКА В алгоритме аппроксимации использованы две
модели
эквивалентной
электропроводности
раствора – молекулярной, как суммы эквива-
лентных электропроводностей NaCl и NaOH, и
ионной, как суммы электропроводностей ионов
Na+, Cl– и OH–. Конечным результатом расчет-
ного алгоритма является нелинейная зависи-
мость доли NaCl в смешанном растворе,
S = CS/CSA, от его удельной электропроводности
κSA. Рис. 2. Функциональная схема установки с мини-колонкой
УМК-3 и проточным низкочастотным резистометрическим
сенсором РСН. Для предотвращения контакта химической
системы реактора с внешней атмосферой в
плотно закрытую крышкой внутреннюю камеру
реактора подавали чистый водород от мембран-
ного электролизера (14) через емкость с водой
(15), поглощающей пары электролита. Такая
система
препятствовала
попаданию
воздуха
внутрь реактора и предотвращала возможную
карбонизацию раствора, содержащего ионы ОН–,
и, как следствие – искажение результатов экспе-
риментов. Для регистрации динамики быстрых обмен-
ных процессов непосредственно в реакторе без
отбора проб был разработан специальный низко-
частотный сенсорный резистометр РСН. Прибор
работает
на
частоте
импульсного
знако-
переменного тока частотой 4 кГц. Из-за отсут-
ствия
электрохимических
реакций
стало
возможным
использование
двухэлектродного
сенсора. В работе были использованы проточные
сенсоры, изготовленные в стеклянных трубках,
которые дают надежные показания в растворах
малых (РСН-4 до 0,05 М) или больших (РСН-16,
РСН-17 – до 1 М) концентраций модельных
растворов. Наиболее надежные результаты полу-
чаются в разбавленных растворах (до 0,02 М),
для которых имеются общепринятые справочные
данные, полученные в прецизионных измере- КОНТРОЛЬ СОСТОЯНИЯ
СИСТЕМЫ ИОНИТ-РАСТВОР Он пред-
ставляет собой произведение показания сенсора
на его константу kS = L/Q (отношение эффектив-
ной длины измерительной камеры к ее сечению),
определяемую при калибровке. Однако ввиду
некоторой нелинейности зависимости 1/R = f(C)
«константа» сенсора непостоянна и зависит от
концентрации анализируемого раствора. В связи
с этим использование какой-либо постоянной
«константы» сенсора может давать значитель-
ную ошибку при обработке данных, полученных
в растворах разных концентраций. Ошибку легко
исключить для модельной бинарной смеси
компонентов с постоянной суммарной концен-
трацией CSA, используя трехточечный метод. Он
основан на трех измерениях – измерения в неиз-
вестной точке S = CS/CSA и в двух крайних
точках CS = CSA и CА = CSA. Фактически это соот-
ветствует двум дополнительным калибровочным
опытам в каждой измеряемой точке зависимости
S = f(C). На практике в больших сериях
измерений в динамическом процессе измерения
в двух крайних точках выполняются только один
раз и практически не усложняют технику экспе-
римента. Очевидно, что трехточечный метод
пригоден для любых концентраций. ниях 4, 5. Поэтому в отдельных измерениях на
концентрированных растворах есть возможность
при необходимости уточнить результат путем
измерения сопротивления пробы, разбавленной
до нужного уровня (CS < 0,01 M). В электронной схеме резистометра процессор
(ATmega328 на плате ARDUINO UNO R3)
формирует
стабилизированное
напряжение
U0 = 5 0,005 В. Это напряжение поступает на
два отдельных входа процессора, который
программно
формирует
одинаковые
пары
прямоугольных импульсов напряжением 5 В с
противоположными полярностями и с нулевым
суммарным током. Импульсный ток проходит
через делитель напряжения, который состоит из
двух последовательных сопротивлений – сопро-
тивления раствора RХ в камере сенсора и
резистора R2. В положительном импульсе тока
измеряется падение напряжения U2 на резисторе
R2, по которому установленной в процессор
программой вычисляется значение сопротив-
ления раствора RХ: 0
2
2
2
. X
U
R
R
R
U
(2) (2) (2) На рис. 3 показаны результаты калибровки в
растворе NaCl высокоомного сенсора, рассчи-
танного для работы с разбавленными раство-
рами. Как видно на рисунке, в области концен-
траций даже до 0,04 М экспериментальные
данные и результаты расчета по обеим моделям
раствора совпадают. При более высоких концен-
трациях расчет по аппроксимационным моделям
уже неприменим. Однако прямая пропорцио-
нальность между обратным сопротивлением 1/R,
пропорциональным
удельной
электропровод-
ности раствора, и концентрацией сохраняется в
области довольно высоких концентраций до 1 М
(рис. 4). Поэтому расчетный алгоритм для таких На рис. 5 показаны калибровочные графики
сенсора в смешанном растворе NaCl+NaОН. КОНТРОЛЬ СОСТОЯНИЯ
СИСТЕМЫ ИОНИТ-РАСТВОР Изучение динамики обменных процессов
требует непрерывного контроля состава обраба-
тываемого раствора. Химический анализ доста-
точно трудоемкий и неудобный при анализе
быстрых процессов. Неприменимым оказался
метод рН-метрии для определения концентрации
ионов ОН–, так как в щелочных растворах в
присутствии ионов Cl– потенциал стеклянного
электрода зависит от их концентрации. Анало-
гично
неприменим
и
потенциометрический
метод для определения концентрации ионов Cl–. Электроды сравнения, обратимые относительно
ионов Cl–, в щелочном растворе имеют неопре- Рис. 3. Калибровочные графики высокоомного сенсора
РСН-16 (kS = 7 см–1) в растворах NaCl. 1 – график линей-
ного тренда по экспериментальным данным в области
концентраций до 0,1 М; 2 и 3 – результаты обработки
экспериментальных данных по моделям раствора 2 и 3-х
Рис. 4. Корреляция между обратным сопротивлением
сенсора РСН-17 (kS = 18,5 см–1) и концентрацией NaCl. Температура 24,2оС. 59 59 Рис. 4. Корреляция между обратным сопротивлением
сенсора РСН-17 (kS = 18,5 см–1) и концентрацией NaCl. Температура 24,2оС. 9 Рис. 3. Калибровочные графики высокоомного сенсора
РСН-16 (kS = 7 см–1) в растворах NaCl. 1 – график линей-
ного тренда по экспериментальным данным в области
концентраций до 0,1 М; 2 и 3 – результаты обработки
экспериментальных данных по моделям раствора 2 и 3-х
частиц. Температура 24,2оС. Рис. 4. Корреляция между обратным сопротивлением
сенсора РСН-17 (kS = 18,5 см–1) и концентрацией NaCl. Температура 24,2оС. условий может быть более простой. Он пред-
ставляет собой произведение показания сенсора
на его константу kS = L/Q (отношение эффектив-
ной длины измерительной камеры к ее сечению),
определяемую при калибровке. Однако ввиду
некоторой нелинейности зависимости 1/R = f(C)
«константа» сенсора непостоянна и зависит от
концентрации анализируемого раствора. В связи
с этим использование какой-либо постоянной
«константы» сенсора может давать значитель-
ную ошибку при обработке данных, полученных
в растворах разных концентраций. Ошибку легко
исключить для модельной бинарной смеси
компонентов с постоянной суммарной концен-
трацией CSA, используя трехточечный метод. Он
основан на трех измерениях – измерения в неиз-
вестной точке S = CS/CSA и в двух крайних
точках CS = CSA и CА = CSA. Фактически это соот-
ветствует двум дополнительным калибровочным
опытам в каждой измеряемой точке зависимости
S = f(C). На практике в больших сериях
измерений в динамическом процессе измерения
в двух крайних точках выполняются только один
раз и практически не усложняют технику экспе-
римента. Очевидно, что трехточечный метод
пригоден для любых концентраций. условий может быть более простой. КОНТРОЛЬ СОСТОЯНИЯ
СИСТЕМЫ ИОНИТ-РАСТВОР Заметим, что алгоритм определения концен-
траций по трехточечной схеме может быть
применен для растворов даже неопределенного
состава, например природных вод 9, при
условии,
что
в
изучаемом
динамическом
процессе,
например
выпаривании
или
мембранном разделении, соотношение раство-
ренных компонентов не изменяется. В этом
варианте, в отличие от аппроксимационных
моделей, не возникает необходимости в повы-
шении точности определения электропровод-
ности 10 путем уточнения моделей раствора. Описанная
выше
экспериментальная
установка и ее элементы разработаны таким
образом, чтобы сделать возможным организацию
различных режимов процесса ионного обмена. Далее представлены три наиболее важных
режима, которые различаются условиями проте-
кания процесса и особенностями получаемой
информации. Заметим, что алгоритм определения концен-
траций по трехточечной схеме может быть
применен для растворов даже неопределенного
состава, например природных вод 9, при
условии,
что
в
изучаемом
динамическом
процессе,
например
выпаривании
или
мембранном разделении, соотношение раство-
ренных компонентов не изменяется. В этом
варианте, в отличие от аппроксимационных
моделей, не возникает необходимости в повы-
шении точности определения электропровод-
ности 10 путем уточнения моделей раствора. Характерная
особенность
суспензионного
режима состоит в том, что в нем невозможно
создать внутри частиц ионита одинаково ориен-
тированное электрическое поле из-за хаоти-
ческого движения частиц в свободном конвек-
тивном потоке. Если же наложить постоянное
электрическое поле на вращающуюся суспензию,
его влияние, по-видимому, можно рассматривать
как действие вращающегося электрического поля
на неподвижные частицы, расположенные в
различных
геометрических
положениях. Вероятно, эффект воздействия такого поля
можно обнаружить, хотя из-за неоднородности
системы его анализ существенно усложняется. Есть сведения о том, что воздействие нестацио-
нарного и неориентированного электрического
поля на процессы в ионитах дает положительный
эффект. Описанная
выше
экспериментальная
установка и ее элементы разработаны таким
образом, чтобы сделать возможным организацию
различных режимов процесса ионного обмена. Далее представлены три наиболее важных
режима, которые различаются условиями проте-
кания процесса и особенностями получаемой
информации. Суспензионный
режим
ионного
обмена,
позволяющий выделить только кинетические
свойства твердой фазы, особенно полезен для
определения наиболее важных характеристик
обменных процессов – обменной емкости разных
форм, изотерм распределения ионов, констант
равновесия и т.д. При моделировании обменных
процессов результаты экспериментов в электри- КОНТРОЛЬ СОСТОЯНИЯ
СИСТЕМЫ ИОНИТ-РАСТВОР Из графиков можно видеть, что при постоянном
значении суммарной концентрации линейность
зависимости = f(S) соблюдается даже при
довольно высоких концентрациях. Заметное
расхождение данных, полученных обработкой по
трем разным алгоритмам, наблюдается только
при высоких долях щелочи в смешанном
растворе. Характерно, что отклонение от линей-
ности видно только при использовании в 60 Суспензионный режим обеспечивает равно-
мерное и интенсивное омывание каждой частицы
по всей поверхности, резко усиливает конвекцию
жидкости вблизи поверхности частиц и сводит к
минимуму концентрационные ограничения в
растворе, то есть дает информацию о кинетике
обменных процессов только в твердой фазе. Это
важное преимущество, характерное только для
результатов,
получаемых
в
суспензионном
режиме. Суспензионный режим имеет некото-
рую качественную аналогию с условиями работы
вращающегося дискового электрода в электро-
химии,
позволяющего
разделять
эффекты
диффузионной и активационной кинетики. расчетах аппроксимационных моделей раствора
2 и 3-х частиц. Это закономерно, поскольку
аппроксимационные модели применимы только
в разбавленных растворах, а отклонения от
теоретических моделей более сильно выражены
именно по щелочи. При использовании трехто-
чечной схемы расчета зависимость = f(S)
значительно ближе к линейной, и результат
обработки имеет одинаковую точность во всей
области значений S. Рис. 5. Калибровочный график сенсора РСН-16 в смешан-
ном растворе NaCl+NaОН. 1 – расчет данных измерений
трехточечным методом; 2 и 3 – расчет по математическим
моделям раствора 2 и 3-х частиц. CSA = 0,11 M, температура
17,5оС. Так
как
процесс
обмена
протекает
в
замкнутом объеме, он является нестационарным
и в общем виде описывается уравнением первого
порядка: (3) (3) α / τ
(
,α ). S
SA
S
d
d
f C
(3) Конкретный вид решения этого уравнения: Конкретный вид решения этого уравнения: α
(
,τ)
S
SA
f C
(4) (4) Рис. 5. Калибровочный график сенсора РСН-16 в смешан-
ном растворе NaCl+NaОН. 1 – расчет данных измерений
трехточечным методом; 2 и 3 – расчет по математическим
моделям раствора 2 и 3-х частиц. CSA = 0,11 M, температура
17,5оС. определяется условиями кинетики процесса –
зависимостью скорости обмена от концентрации
бинарного раствора CSA, от соотношения концен-
траций компонентов S, от требований гидроди-
намики. Установление вида зависимости (4) и
есть задача эксперимента. определяется условиями кинетики процесса –
зависимостью скорости обмена от концентрации
бинарного раствора CSA, от соотношения концен-
траций компонентов S, от требований гидроди-
намики. Установление вида зависимости (4) и
есть задача эксперимента. ОБМЕНА В ЗАКРЫТОМ РЕАКТОРЕ Наличие системы регулируемой циркуляции
раствора по замкнутой схеме в конвективном
реакторе дает возможность исследовать обмен-
ные процессы в суспензионном режиме, когда
порция ионита свободно вращается в растворе. 61 практически линейную форму. Это необычная
форма
для
нестационарных
диффузионных
процессов, и она показывает, что решением
дифференциального
уравнения
(3)
является
константа, имеющая смысл скорости обменного
процесса. Кажущаяся необычность состоит в
том, что, согласно уравнению Фика, плотность
потока диффузии, то есть скорость процесса
обмена, должна во времени изменяться, так как
изменяются градиенты концентрации обоих
компонентов по мере насыщения или регене-
рации ионита: ческих полях интерпретируются не однозначно
из-за неоднородности поля. На рис. 6 приведены первичные результаты
экспериментов, выполненных в суспензионном
режиме при регенерации полностью истощен-
ного ионита АН-2-ФН растворами NaOH разных
концентраций. Перед опытом ионит переводили
в хлоридную форму выдержкой 1 час в реакторе
в перемешиваемом растворе 1 М NaCl, затем
промывали в дистиллированной воде. Регист-
рацию данных каждого эксперимента начинали с
момента погружения порции ионита в предвари-
тельно нагретый до постоянной температуры
перемешиваемый раствор одного компонента, в
данном примере – NaOH. В процессе обмена
периодически записывали показания резисто-
метра и момента времени замера. . dC
j
D dx
(5) (5) Линейность графиков иллюстрирует интерес-
ную особенность двойной диффузии в зерне
ионита. В твердой фазе под действием соответ-
ствующих градиентов концентрации dC/dx в
модельной системе NaCl+NaOH движутся два
потока ионов одинаковых зарядов (Cl– и ОН–)
параллельно, но в противоположных направле-
ниях. Для того чтобы соблюдалось требование
закона
электронейтральности,
оба
потока
должны двигаться с одинаковой скоростью. Это
требование удовлетворяется за счет того, что
градиенты концентрации обоих ионов изме-
няются синхронно, но в разных направлениях –
один растет в процессе регенерации (для иона
ОН–), а второй (ион Cl–) уменьшается. При этом
среднее значение диффузионного потока, общее
для обоих ионов, остается одинаковым и посто-
янным, независимо от изменяющегося состава
ионной среды в твердой фазе. Одинаковыми
будут эффективный коэффициент диффузии и
градиенты концентрации обоих ионов, отлича-
ющиеся только знаком. А это означает постоян-
ную скорость диффузии в соответствии с урав-
нением (5). Таким образом, линейность основ-
ного участка графика динамики является одно-
значным доказательством того, что в процессах
переноса в твердой фазе лимитирует гелевая (в
твердой фазе) диффузия. Рис. 6. Динамика регенерации анионита АН-2-ФН в
ОН-форму растворами NaOH в области концентраций
CSA 0,1 М (числа на графиках). Суспензионный режим
процесса. Рис. 6. Динамика регенерации анионита АН-2-ФН в
ОН-форму растворами NaOH в области концентраций
CSA 0,1 М (числа на графиках). Суспензионный режим
процесса. ОБМЕНА В ЗАКРЫТОМ РЕАКТОРЕ Форма всех графиков динамики ионного
обмена немонотонна, что указывает на различ-
ные кинетические механизмы, действующие на
разных этапах. На начальных участках графиков
наблюдается нелинейная динамика в форме,
близкой к логарифмической. Можно показать,
что в первые минуты скорость обмена контроли-
руется диффузией ионов в растворе. Конечные
участки всех графиков представляют собой гори-
зонтальные линии, соответствующие установле-
нию на границе фаз равновесного состояния,
когда процесс сорбции прекращается. Пара-
метры
равновесного
состояния
являются
наиболее важной частью информации, на осно-
вании которой далее можно рассчитать все
характерные параметры ионита и обменного
процесса в целом. Детали таких оценок требуют
отдельного обсуждения и анализа и здесь не
рассматриваются. Описанный механизм двойного ионного
потока в ионите очень похож на известный
механизм возникновения двойного электри-
ческого или диффузионного слоя на границе
между растворами простого электролита с
разными концентрациями 3. Ионные потоки в
плоском диффузионном слое в одномерном
случае в простом электролите описываются
уравнениями, включающими диффузионную и
электромиграционную составляющие: (
)
;
dC
z F
dE
j
D
D C
dx
RT
dx
(6)
(
)
;
dC
z F
dE
j
D
D C
dx
RT
dx
(7) (6) Следует, однако, обратить внимание на сред-
ние участки графиков – они имеют характерную (7) 62 где
D+,
D–
–
коэффициенты
диффузии;
С+, С– – концентрации; z+, z– – заряды ионов
(с учетом знака). Интегрированием этой системы
уравнений находят величину диффузионного
потенциала и значение эффективного коэффи-
циента диффузии DEFF. Диффузионный потен-
циал ED = dE/dx замедляет диффузионный поток
более быстрых ионов и ускоряет поток более
медленных. В результате в пограничном слое
устанавливается
некоторый
эффективный
коэффициент диффузии, одинаковый для обоих
ионов (знак z– здесь берется положительным): специальных вспомогательных операций. Мини-
колонка дает возможность проводить процесс
при неизменном расположении частиц, то есть
моделировать работу в условиях, близких к
промышленным. Работу мини-колонки можно видеть на схеме
рис. 2. Через слой ионита предусмотрено
пропускание
тока
произвольной
формы
и
направления между внутренним (9) и внешним
(10) платиновыми электродами. Ток генерирует-
ся электронным потенциостатом (11). Раствор в мини-колонку подается из рабочей
камеры реактора через специальный проточный
сенсор (12) перистальтическим насосом (13)
Heidolph B-V 01-03 с регулируемой производи-
тельностью до 16,7 мл/мин (0,088 см/с на полное
сечение) и выходит из колонки в ту же камеру
реактора. В экспериментах с целью ограничения
концентрационной поляризации использовали
режим
максимальной
производительности
насоса. ОБМЕНА В ЗАКРЫТОМ РЕАКТОРЕ Таким образом, в реакторе с замкнутой
схемой циркуляции процесс протекает в неста-
ционарном режиме, в котором состав раствора
непрерывно изменяется во времени. Благодаря
фиксированному расположению частиц ионита в
мини-колонке можно создавать электрическое
поле
заданной
напряженности,
ориентации,
формы и динамики. (
)
. EFF
z
z
D D
D
z D
z D
(8) (8) Оба иона электролита, имеющие противопо-
ложные знаки, диффундируют против градиента
концентрации в одном направлении и с равными
скоростями, не нарушая закона электроней-
тральности. Отличие этого механизма от меха-
низма ионных потоков в ионите состоит в том,
что в диффузионном слое в одном направлении
движутся ионы противоположных знаков, а в
ионите – движутся ионы одного знака, но в
противоположных направлениях. Равенство ион-
ных потоков и коэффициентов диффузии в обоих
объектах регулируется возникающим внутрен-
ним электрическим полем. В диффузионном слое
скачок потенциала легко измеряется на границе,
разделяющей два раствора. В ионите действие
внутреннего электрического поля проявляется
косвенно – в форме равенства диффузионных
потоков двух ионов. Таким образом, в реакторе с мини-колонкой
создаются такие же условия, как и в реакторе с
суспензионным режимом, поэтому динамические
характеристики
процесса
должны
иметь
качественно аналогичную форму. Здесь исполь-
зуется и аналогичная техника подготовки и
проведения
опытов,
регистрации
данных. Однако
в
мини-колонке
промежуточные
операции с ионитом (промывки, регенерация)
технически более простые. РЕЖИМ ИОННОГО ОБМЕНА
В МИНИ-КОЛОНКЕ С ПРОТОКОМ
ПО ОТКРЫТОЙ СХЕМЕ Третий вариант режима работы ионообмен-
ника – традиционный проточный режим погло-
щения примеси из раствора или регенерации
ионита в мини-колонке. В схеме с открытым
протоком состояние системы также нестацио-
нарное, поэтому на выходе мини-колонки сопро-
тивление раствора тоже должно регистриро-
ваться непрерывно. При этом в колонках боль-
шой высоты основным информативным пара-
метром является «время до проскока», фиксиру-
ющее момент, когда в растворе на выходе появ-
ляются непоглощенные ионы. До этого момента
на
выходе
фиксируется
квазистационарное
состояние с постоянной нулевой концентрацией
поглощаемого иона. Время между началом пере-
ходного процесса (начальный момент фиксации
проскока) и его окончанием (достигнуто состо-
яние равновесия между фазами раствора и
ионита и прекращение процесса обмена) в
колонках большой высоты обычно незначи-
тельно. Рис. 7. Динамика абсорбции ионов Cl– анионитами
Purolyte A-400 (1) и АН-2-ФН (2) из раствора 0,02 М NaCl в
колонке. Температура 24,2С. Рис. 8. Динамика накопления ионов ОН– (абсорбции ионов
Cl–) из раствора 0,2 М NaCl анионитом Purolyte A-400 в
электрическом поле в мини-колонке. Числа на графиках –
плотность тока, мА/см2. Температура 24,2оС. Рис. 7. Динамика абсорбции ионов Cl– анионитами
Purolyte A-400 (1) и АН-2-ФН (2) из раствора 0,02 М NaCl в
колонке. Температура 24,2С. Рис. 7. Динамика абсорбции ионов Cl– анионитами
Purolyte A-400 (1) и АН-2-ФН (2) из раствора 0,02 М NaCl в
колонке. Температура 24,2С. Рис. 8. Динамика накопления ионов ОН– (абсорбции ионов
Cl–) из раствора 0,2 М NaCl анионитом Purolyte A-400 в
электрическом поле в мини-колонке. Числа на графиках –
плотность тока, мА/см2. Температура 24,2оС. В отличие от высоких колонок, в описанной
выше мини-колонке ситуация иная. Слой ионита
в первом приближении работает как условно
«тонкий» и однородный по всей длине, он посте-
пенно меняет свое состояние в процессе. При
этом на выходе мини-колонки сразу же возни-
кает постоянный «переходный процесс» – состав
раствора изменяется непрерывно, от начала
процесса до достижения состояния равновесия. Информационным параметром является скорость
обмена, определяемая как производная сигнала
по времени. Рис. 8. Динамика накопления ионов ОН– (абсорбции ионов
Cl–) из раствора 0,2 М NaCl анионитом Purolyte A-400 в
электрическом поле в мини-колонке. Числа на графиках –
плотность тока, мА/см2. Температура 24,2оС. Метод с открытым протоком также позволяет
накладывать на ионит ориентированное электри-
ческое поле, определять влияние разных факто-
ров на кинетику процесса и равновесные пара-
метры. При наложении электрического поля на
процесс в мини-колонке форма динамических
кривых обменного процесса остается такой же,
как и без действия поля. На рис. 8 показана
динамика обменного процесса для анионита
Purolyte
A-400. РЕЖИМ ИОННОГО ОБМЕНА В ЗАКРЫТОМ
РЕАКТОРЕ С МИНИ-КОЛОНКОЙ Важной частью установки является съемная
мини-колонка (8, рис. 2), имеющая вид стеклян-
ного цилиндра диаметром 20 мм, в нижней части
которого расположен слой ионита высотой
3–4 см между двумя плотно вставленными
сетками. При скорости потока раствора 0,1 см/с
время прохождения порции раствора через
мини-колонку составляет не более 10–20 секунд,
поэтому ионит по всей толщине находится в
одинаковых условиях и практически однороден
по степени насыщения или регенерации. Мини-
колонка специально и сконструирована так,
чтобы за счет однородности упростить и сделать
более точным и достоверным анализ экспери-
ментальных данных. р
На рис. 7 показана динамика степени насы-
щения двух анионитов относительно их теорети-
ческой обменной емкости (ТОЭ – по техни-
ческому паспорту продукта), полученная в реак-
торе в мини-колонке. Как видно из рисунка,
форма
динамической
кривой
в
АН-2-ФН
несколько иная, чем в суспензионном режиме –
монотонная, а средний линейный участок не
выражен. Длительность процесса незначительно
увеличилась, что указывает на наиболее вероят-
ную причину – более высокое сопротивление
переносу в жидкой фазе из-за плотности распо-
ложения частиц и небольшой концентрации
абсорбируемых
ионов. По-видимому,
моно-
тонная форма динамической кривой отражает
особенности межфазного обмена в условиях
колонки – неравнодоступность поверхности
частиц в плотном слое, вследствие чего диффу-
зионное сопротивление в растворе проявляется Небольшая толщина слоя ионита и его незна-
чительное гидравлическое сопротивление слоя
позволяют практически полностью избавиться от
застойных явлений, а для сильно набухающих
ионитов легко их преодолевать без применения 63 на всем протяжении процесса. На ионитах,
характеризующихся большой скоростью обмена
(Purolyte A-400), процесс абсорбции идет так
быстро, что нелинейность рабочего участка
можно заметить только в растянутом масштабе
по оси времени. изводимости, снижение обменной емкости при
наложении электрического поля. изводимости, снижение обменной емкости при
наложении электрического поля. РЕЖИМ ИОННОГО ОБМЕНА
В МИНИ-КОЛОНКЕ С ПРОТОКОМ
ПО ОТКРЫТОЙ СХЕМЕ В
данном
случае
эффект
ускорения, оцененный по моменту выхода на
горизонтальный участок, выражен слабо. При
большой скорости процесса существенное влия-
ние на форму динамических кривых оказывали
неконтролируемые
длительности
начальных
операций – погружение колонки в реактор,
закрепление, соединение с насосом и т.д. На
фоне малой длительности процесса, 5–6 минут,
вклад подготовительных операций маскирует
эффект. Можно отметить также некоторое
незначительное, практически в пределах воспро- В открытой схеме раствор с выхода из мини-
колонки направляется в проточный сенсор. На рис. 9 показана динамика абсорбции ионов
Cl– в открытой схеме на более емкой лабора-
торной колонке (сухая масса анионита Purolyte
A-400 50 г, высота слоя 8 см, сечение 9 см2). Эта
конструкция колонки имела принципиальные
недостатки – неоднородность распределения
скорости жидкости по сечению, электрофорети-
ческая деформация формы слоя ионита, неодно-
родность электрического поля по сечению,
капиллярные эффекты при вытекании раствора и
др. Тем не менее полученные в ней результаты 64 ческий эффект даже при большой скорости
процесса. иллюстрируют особенности формы графиков
динамики процесса и характер получаемой
информации в открытой схеме протока. Эта
аналогия сохраняется качественно и в мини-
колонке, где указанные негативные явления
отсутствуют. Характер динамических кривых колонки СА()
на рис. 9 показывает, что на рабочем участке
(на правом склоне) они могут быть аппрок-
симированы экспоненциальной функцией: Рис. 9. Динамика десорбции ионов ОН– (абсорбции ионов
Cl–) в проточной колонке из раствора 0,02 М NaCl [6]. Анионит Purolyte A-400. Температура 27оС. Числа на
кривых – плотность тока, мА/см2. exp( τ / τ*),
А
m
С
C
(9) (9) где Сm – параметр, близкий к концентрации в
точке максимума; * – характерный масштаб
времени переходного процесса. Для кривых
2, 1, 0 на рис. 9 значения * равны соответ-
ственно 11, 33, 51 минуте. Отметим, что эти
числа зависят от конкретной массы ионита в
колонке
и
скорости
протекания
раствора. Поэтому они имеют значение только для сравне-
ния влияния различных условий процесса как
упрощенная числовая характеристика формы
графиков динамики процесса в конкретной
колонке. где Сm – параметр, близкий к концентрации в
точке максимума; * – характерный масштаб
времени переходного процесса. Для кривых
2, 1, 0 на рис. 9 значения * равны соответ-
ственно 11, 33, 51 минуте. Отметим, что эти
числа зависят от конкретной массы ионита в
колонке
и
скорости
протекания
раствора. Поэтому они имеют значение только для сравне-
ния влияния различных условий процесса как
упрощенная числовая характеристика формы
графиков динамики процесса в конкретной
колонке. РЕЖИМ ИОННОГО ОБМЕНА
В МИНИ-КОЛОНКЕ С ПРОТОКОМ
ПО ОТКРЫТОЙ СХЕМЕ Как видно из сравнения графиков, при нало-
жении электрического поля обменная емкость
анионита
Purolyt
A-400,
соответствующая
интегралам функций СА(), заметно умень-
шается. Рис. 9. Динамика десорбции ионов ОН– (абсорбции ионов
Cl–) в проточной колонке из раствора 0,02 М NaCl [6]. Анионит Purolyte A-400. Температура 27оС. Числа на
кривых – плотность тока, мА/см2. Рис. 10. Динамика абсорбции ионов Cl– в проточной
колонке из раствора 0,02 М NaCl. Анионит АН-2ФН [11]. Температура 27оС: 1 – без тока; 2 – плотность тока
2,2 мА/см2. Общая форма динамических кривых С()
имеет пик на начальном участке. При установ-
лении режима из колонки вначале выходит вода,
первоначально
заполнявшая
поровое
прост-
ранство. Далее на выходе колонки концентрация
СА продукта реакции обмена ОН– быстро увели-
чивается. Максимум концентрации отмечается,
когда достигает выхода фронт потока с началь-
ной концентрацией NaCl CS = CSA. В данном
случае в максимуме степень преобразования
раствора,
равная
отношению
концентрации
продукта (ОН–) СА к суммарной концентрации
CSA
=
CS+CA,
неполная
и
составляет
0,01/0,02 = 0,5. Сочетание общей небольшой
высоты слоя 8 см и относительно большой
скорости протока 1,3 см/мин недостаточно для
полного
поглощения
хлорид-ионов. После
достижения максимума на основном участке
динамической
кривой
скорость
обменной
реакции и концентрация продукта постепенно
снижаются. Можно заметить, что максимум на
графиках рис. 9 расположен примерно на 12–15
минуте и соответствует времени насыщения
смолы в мини-колонке на графиках рис. 7 и 8. Таким образом, длительность восходящих участ-
ков на графиках рис. 9 соответствует времени
насыщения частицы ионита. Можно также заме-
тить, что наклон начального участка, пропорци-
ональный скорости обменного процесса, при
действии электрического поля увеличивается, то
есть обнаруживается положительный кинети- Рис. 10. Динамика абсорбции ионов Cl– в проточной
колонке из раствора 0,02 М NaCl. Анионит АН-2ФН [11]. Температура 27оС: 1 – без тока; 2 – плотность тока
2,2 мА/см2. На этой же открытой колонке можно видеть
(рис. 10), что динамика процесса (здесь – погло-
щение хлорид-ионов слабоосновным анионитом
АН-2ФН) имеет форму, отличающуюся от
формы графиков на рис. 6–8 [11]. Здесь вначале
на выходе колонки регистрируется участок паде-
ния концентрации абсорбируемого иона. Он
соответствует
начальному
нестационарному
процессу вытеснения из колонки воды после
промывки рабочим раствором с начальной
концентрацией CS = CSA. Далее формируется
основной участок кривой с приблизительно
постоянной степенью извлечения хлорид-ионов
(1–S) = (1–CS/CSA). В конце этого участка
наблюдается скачок до значения S 0,8. 65 извлекался до уровня, меньшего предела обна-
ружения
химическим
анализом. РЕЖИМ ИОННОГО ОБМЕНА
В МИНИ-КОЛОНКЕ С ПРОТОКОМ
ПО ОТКРЫТОЙ СХЕМЕ Широкого
промышленного использования метод EDI пока
не получил главным образом из-за постепенного
отравления мембран 17, 18 и большого расхода
энергии. Мы не будем вникать в детали этой
важной и интересной технической проблемы, а
обратим внимание только на то, что в устройстве
EDI ионообменная смола находится в зоне
действия весьма сильного электрического поля,
около 800 В/м 17. Поэтому транспортные
свойства ионита должны очень сильно отли-
чаться от тех, которые у него можно определять
в обычном состоянии вне действия поля. А определить влияние электрического поля по
параметрам режима EDI трудно из-за сложности
самого устройства, в котором последовательно
включено
несколько
разных
сопротивлений
ионным потокам. Поэтому оценки параметров
переноса в ионитах, полученные с использо-
ванием математических моделей процесса EDI с
большим количеством произвольных априорных
предположений,
весьма
условны. В
этом
смысле описанные здесь методы компьютерной
резистометрии выгодно отличаются тем, что в
них конструктивно и технологически исключа-
ются побочные явления, искажающие инфор-
мацию о процессах переноса внутри частиц
ионита. Это важное достоинство, так как опреде-
ление обменных свойств ионитов представляет
собой
трудоемкий
процесс,
традиционно
связанный с выполнением длительных хими-
ческих анализов. Использование безреагентного
метода компьютерной резистометрии позволяет
намного сократить время выполнения этой
традиционной задачи, а также получать досто-
верную информацию о характере воздействия
электрических полей на процесс ионного обмена. Момент возникновения скачка на колонке можно
ориентировочно определить как «время до
проскока» в колонках большой высоты. Сравни-
вая две приведенные на рис. 10 динамические
кривые процесса, можно видеть, что на основном
участке средняя глубина извлечения хлорид-
ионов
(1–S)
при
наложении
постоянного
электрического поля увеличилась почти в два
раза. Примерно так же увеличилось и время до
момента скачка на кривых динамики, то есть
условное «время до проскока». Оба эффекта
показывают значительное возрастание обменной
емкости ионита при наложении постоянного
электрического поля. Из анализа данных, полученных в мини-
колонке в конвективном реакторе и в проточной
колонке и показанных на рис. 7–10, можно
сделать предварительное заключение о том, что
эффекты воздействия слабых электрических
полей на процессы ионного обмена в большей
мере проявляются на слабоосновных ионитах с
заторможенным обменным процессом. Таким
образом,
используя
комбинацию
разных экспериментальных режимов проведения
обменного процесса, можно наиболее полно и
достоверно оценивать свойства ионообменных
материалов. ЛИТЕРАТУРА 1. Кошель Н.Д., Магдыч Е.А., Акимов А.М. Вопросы
химии и химической технологии. 2010, 5, 137–139. 2. Организация процесса в мини-колонке
(слой 3–4 см, сечение 3,14 см2), встроенной в
конвективный реактор. В этом варианте легко
формировать ориентированное электрическое
поле и фиксировать геометрическую структуру
плотного слоя ионита для оценки промыш-
ленных условий. Форма получаемой инфор-
мации качественно аналогична предыдущему
варианту. 2. Newman John S. Electrochemical system. New Jersey,
USA: Premtice-Hall Inc. Englewood Cliffs, 1973. 3. Cynthia G. Zoski. Handbook of Electrochemistry. Las
Cruces, New Mexico, USA: Elsevier, 2007. 935 p. 4. Demirbas Ayhan, Pehlivan Erol, Gode Fethiye, Altun
Turkan et al. J Colloid Interface Sci. 2005, 282(1),
20–25. 5. Pehlivan Erol, Altun Turkan. J Hazard Mater. 2006,
134(1–3), 149–156. 3. Открытая схема протока раствора через
мини-колонку с регистрацией сопротивления
непосредственно на выходе. 6. Кошель Н.Д., Костыря М.В. ЭОМ. 2017, 53(2),
103–109. р
Легко организовать еще один интересный
режим, который потребует некоторых конструк-
тивных изменений системы – режим взвешен-
ного или кипящего слоя или режим псевдоожи-
жения. Он давно используется в промышленно-
сти для интенсификации процессов массообмена
20. В электрохимии он оказался эффективным
при извлечении металлов из очень разбавленных
растворов 21, 22. Для организации режима
взвешенного слоя поток раствора через колонку
следует направить снизу вверх с помощью более
мощного насоса с регулируемой производитель-
ностью. Поток раствора нужно подбирать таким,
чтобы частицы ионита занимали весь объем
рабочей камеры, почти свободно перемещаясь во
взвешенном состоянии и сталкиваясь между
собой. В этих условиях на каждую частицу
действуют две примерно равные силы – сила
тяжести, направленная вниз, и сила давления
потока раствора, направленная вверх. Перемещая
внутри колонки ограничительную сетку, легко
создать необходимое оптимальное соотношение
между объемом рабочей камеры колонки и
объемом ионита, то есть порозность взвешенного
слоя (50–70%). По-видимому, результат должен
быть близким к тому, который получается в
суспензионном режиме. Режим взвешенного
слоя может дать полезную информацию при
моделировании некоторых процессов в промыш-
ленных ионообменных колоннах, например, при
технологических
операциях
взрыхления
и Легко организовать еще один интересный
режим, который потребует некоторых конструк-
тивных изменений системы – режим взвешен-
ного или кипящего слоя или режим псевдоожи-
жения. Он давно используется в промышленно-
сти для интенсификации процессов массообмена
20. В электрохимии он оказался эффективным
при извлечении металлов из очень разбавленных
растворов 21, 22. Для организации режима
взвешенного слоя поток раствора через колонку
следует направить снизу вверх с помощью более
мощного насоса с регулируемой производитель-
ностью. ОБСУЖДЕНИЕ И ВЫВОДЫ Рассмотренные в этом сообщении техника и
технология эксперимента по определению
обменных свойств ионитов разрабатывались в
первую очередь для того, чтобы получать
надежные данные о влиянии электрических
полей на обменные процессы в ионитах. В классических промышленных технологиях
ионного
обмена
воздействие
электрических
полей не используется, хотя может быть в
определенных условиях выгодным 1. Поэтому
в научной и технической литературе проблема
воздействия электрических полей на процессы
ионного обмена практически игнорируется. Тем
не менее само явление существует и оказывает,
бесспорно, очень сильное влияние на работу уже
давно известных гибридных устройств 12,
совмещающих электродиализ с ионообменными
мембранами
и
ионитом,
размещаемым
в
пространстве между мембранами. В литературе
эти процессы известны под названием «электро-
деионизация» (EDI), или непрерывная деиони-
зация (CDI) 13. Интерес к таким системам
сохраняется и сейчас 13–19. Имеются примеры
детального
анализа
применения
гибридных
систем в промышленной гальванотехнике для
извлечения ионов никеля из промывных вод
17, 18, а в 19 ранее сообщалось, что никель р
р
Сущность метода компьютерной резисто-
метрии заключается в непрерывной регистрации
сопротивления раствора встроенным в конвек-
тивный
реактор
сенсором
и
последующей
компьютерной обработке сигнала. Обработка
сигналов по специальным программам позволяет
определять на первом этапе динамику изменения
состава раствора в обменном процессе, а на
втором этапе – все обменные характеристики
ионита. Для получения этой информации разра-
ботаны программируемый микропроцессорный
резистометр РСН и проточные сенсоры сопро-
тивления. Прибор на частоте 4 кГц генерирует
пары одинаковых прямоугольных импульсов
противоположной полярности напряжением 5 В
с нулевым фарадеевским током. Импульсы
поступают
на
два
электрода
сенсора,
а
программа микропроцессора преобразует ответ-
ный сигнал в цифровую форму сопротивления. 66 промывки смолы между сменой направления
цикла работы. Для надежности и достоверности результатов
анализа процессов ионного обмена удобно
использовать комплекс методов, отличающихся
способом организации процесса и характером
получаемой информации. Наиболее информа-
тивны три метода. Получаемая информация после первичной
обработки экспериментальных данных представ-
лена для методов 1–3 в качестве иллюстрации
формы первичного сигнала. Она позволяет
оценить возможности методов и оптимально
планировать
эксперименты
для
оценивания
характеристик материалов и процессов ионного
обмена на конкретных материалах. Получаемая информация после первичной
обработки экспериментальных данных представ-
лена для методов 1–3 в качестве иллюстрации
формы первичного сигнала. Она позволяет
оценить возможности методов и оптимально
планировать
эксперименты
для
оценивания
характеристик материалов и процессов ионного
обмена на конкретных материалах. 1. Организация в конвективном реакторе
суспензионного режима с малым количеством
ионита. Метод позволяет устранить торможение
переноса в растворе и получать информацию
только о свойствах твердой фазы. Процесс в
системе нестационарный, регистрируется дина-
мика изменения состава раствора во времени ЛИТЕРАТУРА Поток раствора нужно подбирать таким,
чтобы частицы ионита занимали весь объем
рабочей камеры, почти свободно перемещаясь во
взвешенном состоянии и сталкиваясь между
собой. В этих условиях на каждую частицу
действуют две примерно равные силы – сила
тяжести, направленная вниз, и сила давления
потока раствора, направленная вверх. Перемещая
внутри колонки ограничительную сетку, легко
создать необходимое оптимальное соотношение
между объемом рабочей камеры колонки и
объемом ионита, то есть порозность взвешенного
слоя (50–70%). По-видимому, результат должен
быть близким к тому, который получается в
суспензионном режиме. Режим взвешенного
слоя может дать полезную информацию при
моделировании некоторых процессов в промыш-
ленных ионообменных колоннах, например, при
технологических
операциях
взрыхления
и 7. Справочник химика. Химическое равновесие и
кинетика
свойства
растворов. Электродные
процессы. Том 3. Издание 2. М.-Л.: Химия, 1965. 1005 с. 8. Washburn E.W. International Critical Tables of
Numerical Data, Physics, Chemistry and Technology. N-Y: National research council of the USA, 2003. (6),
511 p. 9. McCleskey Blaine R. J Chem Eng Data. 2011, 56(2),
317–327. 10. Hunt Robert C. How to Increase the Accuracy of
Solution
Conductivity
Measurements. Sensor
Development, Inc. 1995. 17 p. www.aitsouthwest.com 11. Koshel N.D., Smyrnova O.V., Kostyrya M.V. The
study of ion exchange kinetics on the anionite in weak
electric
fields. Abstracts
of
8th
international
conference “Materials Science & Condensed Matter
Physics”. Chisinau, 12–16 September, 2016. Р. 340. 12. Шапошник В.А., Решетникова А.К., Золо-
тарева Р.И., Исаев Н.И. Журнал прикладной химии. 1973, 46(12), 2659–2663. 13. Özgür Arar, Ümran Yüksel, Nalan Kabay, Mithat
Yüksel. Desalination. 2014, 342, 16–22. 14. Abdelaziz
Smara, Rachid
Delimi, Eric
Chai-
net, Jacqueline Sandeaux. Sep Purif Technol. 2007,
57(1), 103–110. 15. Akrama Mahmoud, Andrew F.A. Hoadley. Water
Research. 2012, 46(10), 3364–3376. 16. Lucía Alvarado, Aicheng Chen. Electrochim Acta. 2014, 132, 583–597. 17. Dzyazko Yu.S., Belyakov V.N. Desalination. 2004,
162, 179–189. 67 18. Dzyazko Yu.S., Ponomaryova L.N., Rozhdest-
venskaya L.M., Vasilyuk S.L. et al. Desalination. 2014, 342, 43–51. tion of the electric field vector. The test unit to study the
ion exchange dynamics, which includes a convective
reactor, a built-in column with the portion of the
exchanger, a temperature control system, a flowing sensor
of the electrical resistance of the solution, an electronic
resistometer, a potentiostat – galvanostat, has been
described. ЛИТЕРАТУРА Three experimental methods have been
described: 1 – with the circulating suspension of
exchanger in the closed volume of thr convective reactor,
which makes it possible to limit the influence of diffusion
resistance in the solution; 2 – with the filtration of the
solution through the exchanger in the built-in column in
the closed volume of the convective reactor; and 3 – the
open filtration of the solution through the column. In all
methods the exchange process proceeds in a nonstationary
mode. The methods differ by the form of the information
obtained about the dynamics of the process, which is
illustrated by examples. It is shown that the speed of the
ion flux in the solid phase is constant and does not depend
on the saturation value of the exchanger in the suspension
regime of exchange in the base interval time of the
process. The effect is caused by the action of the principle
of electroneutrality on two ion fluxes with the charges of
the same sign but with opposite concentration gradients. Computer resistometrics can be effective as a nonchemi-
cal method of an accelerated test of exchange properties
of ion-exchange materials. 19. Spoor P.B., Grabovska L., Koene L., Janssen L.J. Chem Eng J. 2002, 89(1–3), 193–202. 20. Гельперин Н.И., Айнштейн В.Г., Кваша В.Б. Основы техники псевдоожижения. М.: Химия,
1967. 664 с. 21. Shvab N.A., Stefanjak N.V., Kazdobin K.A.,
Wragg A.A. J Appl Electrochem. 2000, 30(11),
285–1292. 22. Shvab N.A., Stefanjak N.V., Kazdobin K.A.,
Wragg A.A. J Appl Electrochem. 2000, 30(11),
1293–1298. Поступила 12.06.18 Summary The experimental technique and equipment to study
the kinetics of the ion exchange processes in weak electric
fields on the ion-exchange resins by computer resisto-
metrics method have been described. The interaction
scheme of electrical and diffusion ion fields in the solid
phase of the ion exchanger has been discussed. It is
shown that during the superposition of a weak electric
field to the ion exchanger globule, the vectors of the total
speed of displacement of ions generate the ellipsoidal
surface, displaced to the side of the vector of the electric
field strength relative to the center of the particle. The
asymmetry of the velocity field illustrates a change in the
balance of ionic flows and their acceleration in the direc- Keywords: exchanger, ion exchange, exchange
capacity, electric field, diffusion, conductivity, sensor,
resistometer.
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https://openalex.org/W2044568872
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https://zenodo.org/records/2160057/files/article.pdf
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German
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Über den Mechanismus der Wirkung von Salpetersäure auf Grenzkohlenwasserstoffe
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Berichte der Deutschen Chemischen Gesellschaft
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public-domain
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?) Vergl. M. Koiiowaloff, lot^. cit.; nucli Joiirn. (I. Iluss. Phys.-chzni.
G1.s. YG, 232 [1904]. 1372 1372 'j Diesc Bericlite 28, 1561 [lS95]. - Journ. d. Ihs. PIiys.-chcni. G c ~
26, 93 [1894]. 1373 1373 ainyls durch konzentrierte Salpetersiiure I), und es ist rnir gelungen ZII
zeigen, daB bei Wirkung von konzentrierter Salpetersaure auf Hexa-
rnethylen S t i c k s t o f f o x y d u l und zwnr in einer der Theorie entspre-
chenden Menge ehtweicht. Diese letzte Tatsache scheint ririr eine voll-
standige Betatigung tler oben erwiihnten Deutung unserer Reaktion zii
bieten. Es verlauft natnlich in allen Fallen, in welchen die Salpeter-
saure die Gruppen CH2 (oder CI13) angreift, die Reaktion durch fol-
gende Stden : Snlpetersiure
Nitroverbindung. Gretizkolilen \\ asserstoff
illdehyd (Keton). /I
+
Isonitroverbindung
I V
Carbonsaure. Snlpetersiure Gretizkolilen \\ asserstoff Carbonsaure. Die Untersuchung wurde init Hexamethylen (nach S a b a t i e r )
begonuen, doch wurde sie nachher auch au€ andere Verbindungen ge-
sjittigten Chxrakters crweitert. Die Ergehnisse dieser Untersnchungen
11 errle in liurzer Zeit ~nitzuteilen. 1 ) Journ. d. Kuss. Phys.-chtm. Ges. 38, 134 [1906]; C. B. 1906, 11, 314. 210. S. Nametkin: tfber den Mechanismus der Wirkung von
SalpetersiLure auf C3renakohlenwasserstoffe. [Ais dem J.aboratorium fhr Organ. Cheniic au tier Univer>it:tt zii Moskau.)
{Eingegnngen ani 9. Mirz 1909.) M. lionowalofF, der die nitrierende N'irkung \ o n S:cdpeteraii\ire
a i i f i::renzkohien\vn~serstof~~ riittleckte, war tler Afeinung, dalJ tler
rrste Yorgang bei der Wirkiiug von Snlpetersiiure ;itif Grenzkohleu-
wasserstoffe eine Nitrierung sei, iiud dafj die Erscheiuungen der Ox!-
chtion erst nls sekundiire Renktion gedncht wertlen niiisoeii '). Betrachten wir diese Ileaktion von I ~ u n o w a l o f
fs Stnndprinkte
ails, so ist es zu erwnrteu, tlaO einerseits mit der \.ermirideriing (lei-
relativen Mnsse von Salpetersiiire die Ausbeiiteii nn Nitrovsrbintliingei~
\vachsen werden, ardererseits, daO bei der Osydatioti VOII Nitrover-
bindringen dieselben S h r e n ') erhalten werden , (lie stet5 als Nehrir-
I'rodukte bei der Nitrieriing tler I<ohlenwasaerstoffe aukreten. Die unten nngegebeuen Versuclisdnteu bestiitigen vollkoniiiien, die
erste yon diesen Behnuptungen. IXe zweitr hat sich auch bestiitigt,
(loch nur z t m Teil. Rein1 liochen von reinem Nitro-hexanietliyle II
mit Salpetersiiure wurde tatsiichlich A (1 i p i II s ii II re , jedocli in eineiii
hochst iinbedeutenden Quantum erhalten. Kin ganz antleres Resultat
ergab die 0xyd:ition des Isonitro-liex:~~netliylens. liier verlief
tlie Reakt.ion recht leicht sowohl in s;iiirern nls :ruch in alltnlischern Me-
dium. 1)aIjei erschien als Hauptprodukt der Osydation .4 dipinsiiiire. 1:s entstand nun also die Prage: biltlet sich nicht etwa wiihrend der
ersten Phase des Prmxesses bei Wirkuug yon SdpetersBure nnf Hexn-
inethylell eine I s o n i t roverbindung? \'on
diesern Stantlpunkte aus
lviirden zu allererst die beiden oben angefulirten Uehnuptiingen er-
klRrlich eracheinen. Doch gestattet dieser Standpunkt auch eine wei-
tere esperimentelle Priifung. Wenn narnlich wirklich :iuf der ersten
Stufe unserer Reaktion eine Isonitroverbindung entsteht, so mu0 sie
sich nntiirlich unter den Bedingungen tler Reaktion sogleich teils in
tlie stabile Form einer Nitroverbindiiug isomerisieren ~ teils eine Zer-
setzung nnch folgendeni Schema erleiden (N e f): g
2R.C~II:NO.OlI = 2R.CHO + NsO + HzO. 2RzCNO.OII = 21tzC:O + N20 + HzO. Tatskchlich beojachtete X. l i o n cm w alo f f die Hilclung eines Al-
drhgdes der l.(i.6-Trimethyl-heptylsZiire bei der Nitrierirng des Di-iso- Tatskchlich beojachtete X. l i o n cm w alo f f die Hilclung eines Al-
drhgdes der l.(i.6-Trimethyl-heptylsZiire bei der Nitrierirng des Di-iso- 'j Diesc Bericlite 28, 1561 [lS95]. - Journ. d. Ihs. PIiys.-chcni. G c ~
26, 93 [1894]. li: s 1' e r i ni e n t e 11 e s. 1. 15infltild cler hlasse bei d e r W i r k u n g v o n S a l p e t e r s a u - e
it u f G r e n z k o h l e n w a s s e r s t o f f e. 1Ou ccni (76: g) H e x a n t e t h y l e n wurden bei jedem Versuche gleich-
wlliig in Glasrohren verteilt, in welchen sich je 30 ccrn Salpetersaurr
(spez. Gew. 1.2) befanden. Die %ah1 der Rohren bei dem ersten Ver-
siiche war 5, beirn zweiten 20. Die Rohren wurden irn ICanonen-
ofen 9 Stunden lang bis 103-1Q5° in dem Versuch I, bis 107-110''
in den1 Versuch I1 erwiirlnt. Alle 3 Stunden wurden die RGhren vor-
sichtigerweise geoffnet, wobei der Druck sich als betrachtliah erwies. ~~
-
.___
.- . ~
_ . ~. ~
. .. ~
. ,
,
Vcrsuch I
1
T'crsuch 11
Genommen Hexamethyleu . . . . . . . ~
100 ccni
1
100 c a n
))
Salpetturshro (spez. Gew. 1.2) . . ~
150 ccni
~
600 ccnl
42.5 g
rohe Nitroproduktc
. . . . . . . i
17g
I
33.5g
d. h. 35.9 0;"
2 1 Adipinsgurr, Schnip. 150-15P
. . . ~
6.7g
'
17.5g
\ andere Siuren . . . , . . . . . ,
6.0g
1 2 3 g
zurickerhnlten : RohlenwasserstoEI . . . 55.5 g
35.5 g
'
! reagiert : Kohlenwaserstoff . . . . . . . '
22.5 g
j d. 11. 49.3 o/o
2 \ Mono-nitro~erbinduii~ . . . . . . I
I5 g
19 g
' d. 11. 43.5 " / o
' d. h. 29.1 '1.0 2
2 .inn. tl. (,'hem. 302, l i [lSSS]. 1374 Die Ausbeuten wurden nur nach deni ;in der lteaktion beteiligten
Kohlenwasserstoff berechnet. Wir sehen also, daB die Verrnehruug der Masse YOU Salpeter-
siltire ein Sinken der Ausbeuten an Nitroprodukten hervorrnft. Zu-
gleich entspricht aber, \vie es auch zii erwnrten stand, dieser Vermin-
clerung der Aiisbeuten an Sitroprodukten, ein Steigen der Energie
tler ~)sydationsprozesse. Der Gesanitbetrag an organischen Saiiren,
die m:in i n dem Versucli I1 erbnlten bat, ist riiehr als doppelt so hoch
irii Tergleich zii dem Betrage an Siiuren in den1 Versuch I. Das reioe N i t r o - h e x a m e t h y l e n : das man in &n beiden letzten
1,'ersiichen erhiilt, ist eine farblose Fliissigkeit, \on eigenartigem Ge-
riich. Seiii Siedepunkt ist 109.5O bei 40 rnni Driick: bei 742 mm
siedete (Ins Nitroprodukt bei 200"" niit rnerklicher Zersetzunq. Spez. Gea. d! = 1.0P53: dig = 1.0681): I:t?flnkt.-Kol.ffiz. n,, -- 1.4612:
tidier Mo1.-Refr. 33.19; nach (lei Thcoiii. fiir CeIill NO? 33.257 %ur B(.stimrnung dcs spczifischtm Gew'chtca:
Snbstanx bci 0" 1.2983 g ; lwi 19') 1.27SO g. Wasser
x
O0 1.1964 "; x
4fl 1.1966 x %ur B(.stimrnung dcs spczifischtm Gew'chtca: %ur B(.stimrnung dcs spczifischtm Gew'chtca: Snbstanx bci 0" 1.2983 g ; lwi 19') 1.27SO g. Wasser
x
O0 1.1964 "; x
4fl 1.1966 x Die hnalyse dcs Sitrohnxanicthylens erg& fc~lgmde ltesultate : Die hnalyse dcs Sitrohnxanicthylens erg& fc~lgmde ltesultate :
0.1706 g Sbst.: 0.4594 g COz, 0.2260 p H?O. - 0.2052 g Sbht.: 20.3 ~I'III
S (220, 748 mm). Ber. C 55.81, 11 8.52, X 10.85. Gef. n 55.44, )) 8.39, )) 11.03. Markownikoff ') gibt fiir Nitronaphthen xns der Frakt,ioii 80-32*
tle:: kaukasischen Erdiils ein vie1 zii niedriges spezilisches Gewicbt
(do = 1.0759) und (dzO = I.Ot;O.i),
das zweifellos aiif das Vorhanden-
sein einer gewissen Menge ron Nivoparaffinen in seineni Praprat
hin\veist. Das reine Nitrohexamethylen liist sich beim Erairrnen ruck-
htanddos in konzentrierter Alknliliisung. Aus dieser Liisung scheiden
schwache Saiiren (CO, 11. a.) mieder die Nitroverbindling nus, stark? tlngepen rufen eine kompliziertere Reaktion hervor (vgl. S. 1372). Doch gelingt es bei guteni Abkiihlen , die ckarakteristische Reaktion
:tiif Isonitroverbindungen rnit Eisenchlorid zu erzielen. '2. U b e r die W i r k u n g d e r O x y d a t i o n aiif N i t r o - irntl
Is o n it r o v e r b i n d u n g e 11. Die Keaktion voii Salpetersiure mit Nitrohelamethylen verlanft
iiiillerst langsarn. Bei eineni Versuche wurden nach neunstundiger Er-
wirrnung :iuf deni Sandbade ails 13 g CsHI1hT()? I
I
I
~
50 ccrn Sal- petersailre (spez. Gew. 1 3 ) 11 g rohen Nitroproduktes erhalten, aus
denen sich 10 g bei 40 mni bis 11Sc abdestillieren lieBen. Nach dern
Eindarnpfen der Shureschicht nuf dern Wasserbade erhielt man 2 g
Shure, die nach dem Umkrystallisieren aus Nasser ihren Schmelz-
punkt bei 150-152°
hatte. Die Analyse des Silbersalzes bestatigte,
d d die Szure, z u m wesentlichen Teile wenigstens, aiis B d i p i n s Z a r e
hestand. 0.2587 g Shst.: 0.1;540 g Ag. 0.2587 g Shst.: 0.1;540 g Ag. C ~ H 8 0 s A g p . Ber. <Ig 59.97. Gef. Ag 59.53. Bei weitem energischer wirkt die Oxydation a d I so n i t r o hexa-
nietbylen im Augenblick seines Entstehens und ebenso auf seine Me-
tnllderivnte. '/*-?/. Liisung yon Kaliumperrnangnnat entfarht sich fast
angenblicklicli, weun sie zur alkalischen Liisung einer Nitroverbindung
zugetropft wird. Das lleaktionsprodukt erwies sich hier XIS Adipin -
s a u re. Dieselbe Slure bildet. sich in beinahe theoretischen Meugen
(33 O/O der l'heorie), wenn man tropfenweise die alkalische Losuug
des Nitroproduktrs in siedende Salpetersaure (spez. Gew. 1.3) bringt. (Schmelzpunkt und Andyse des Silbersslzes). Es fragt sich nan, kann nicht etwn. unter diesen 13edingnugen die
Nitroverbindung als Zwischenprodnkt hei der Oxydntion von Hexa-
methyleu znr Adipinskure betrachtet werden ? Diese Frage sol1 der
nachstehende Versiich beantworten. 100 ccrn Hexamethylen wurden
ti Stunden mit 100 ccni Salpetersaure (spez. Gew. 1.4) gekocht. Nach
dem Eindampfen der Shureschicht erhielt m a n 1 g rohe, krystallinische
S u r e , d. h. doppelt so viel, aIs in derii entsprechenden Versuche nrit
I3 g Nitrohexamethylen bei netinstiindigem Kochen (vergl. oben). Es bildet sich also die AdipinsZure aus Nitrohesarnethylen viel lang-
aarner, als sie sich bei indirekter Wirkung von Salpetersaure auf
Hexamethylen bilden kann. Daher kann das Nitrohexamethylen kein
Zwischenprodukt in unserer Reaktion sein. 3. l j b e r die bei d e r W i r k u n g v o n S a l p e t e r s i i u r e auf H e x a -
met h y 1 en en t we i c h e n d e n G as e. 3. l j b e r die bei d e r W i r k u n g v o n S a l p e t e r s i i u r e auf H e x a -
met h y 1 en en t we i c h e n d e n G as e. n e r Apparat., welchen ich bei der Untersnchung von gasformigen
Produkten, die bei dieser Reaktion erhalten werden, benutzte, bestand
aus einern dickwandigen, riinden Kolben (300 ccrn), an den eine gas-
leitende Riihre mit einem Hahne, ein Tropftrichter und ein Kuhler
angeschrnolzen waren. Das obere Ende des Kuhlers ging in eine
nach untenvgebogene Rijhre iiber. an deren freiern Ende ich die erste
Waschflasche
rnit konzentrierter Schwefelsaure
(fiir NO*) ange-
schrnolzen babe; die letzte enthielt Paraffiniil, das die rnitgerissenen
Darnpfe des Koblenwvasserstoffs zaruckhalten .solhe. Sie wurde mit 1376 eineni gewcihnlichen Stickstoffsaniniler in Verbindnng gesetzt. Die
T;jft
wurde aus dem ganzen Apparate zuvorderst durch trockne
Kohlensiiure (nus NaHCOa) verdrangt. Sodnnn wurde durch den
‘I’ropft.richter Salpetersaure rind dann Kohlenwasserstoff in den Kolheri
eiogefuhrt. Der gasleitende Halin \v:irde gescblossen und das E:r-
warinen hi2gonnen. Ziiletzt warttrn tlie Gase (lurch Kohlens5ure ver-
dlrsngt iind zii Z9iten i n eine gro0e Hiirette yon H u n t e mit konzen-
t r I er t e im A tz I< n I i ii t)ergef u h r t . g
Die \.orprohe zeigte, dalJ man tatsiichlich I)eiin ICochen des Hes:i-
iiiethyleus niit Salpetersaure (spez. c:ew. 1.4) uuter anderen Renktions-
1)roduliteii aucli ein farbloses Gas erhllt, das aeder von konzentriertrii
TAsunKeil von llisenvitrinl, iioch von rtlkalischen 1,osungen des Pyrci-
g:rllols aiifgenoinineii nird. hlit Iinhleiiwasserstoff~i~mpfen hildetr
das Gas ein explosives Gemisch; ein gliuiniender Holzspau flaniintr
in ihoi ;tuf. liiirz, es hlieb keiti Zweifel, d;iB d:is Gas tatsachlich
S t i c k s t o f f n s j- du 1 w:ir. (duarititative l-ksultate: E s tvrirden in eineni Versuche 254 CCIII
( ; a s (bei 00 iind 7fiO nini Druck) iind 1.75 g krptallinische Siiiire er-
tialten. Es zeigte sicli, dais dem Gase rtivaa Iiolilenw~isjerstoffdiiluple bei-
gemecgt xareii, ail Wertlem deutete (lie lteaktion niit, 1-prwentigrr
1.osung voii Palladiiiinclllorid nuf tlie Anwesenheit, ron Iiolilenoxyd I I I
dem G:ise. In deiii (.;nap
ivurtie der Gehalt a n Stickstoff ermittelt
(inittels lbrehschinid tb 1’IatincapiIl;ire) I). Da nun iiiiser Gas Iiein Stickstoffoxyd rntliielt (Kdition niit
Lisenvitriollijsung),
niuWte das Voluni des erhaltenen St.ickstoEFs drin
\.‘alum des ari der H.eaktion heteiligten Stickstoffoxydiils gleich seiii. Tlie G:isaii:ilysr ivurde iiher Quecksilber im Apparate von H e ni pe I
aiisgefiihrt. Es zeigte sich, da13 das Casgemiscli 93.3Ol0 El0 nnd ti.7’/0
e.:ner Alischung voii l~ohlen\rasserstoffdaiiipfen iind Kohlenoxyd en t-
Iiielt. I) Iinorye wid ;\i.iidt, diese Bericht.: 21, 2136 [1899]. 3. l j b e r die bei d e r W i r k u n g v o n S a l p e t e r s i i u r e auf H e x a -
met h y 1 en en t we i c h e n d e n G as e. Fiihren wir
niif (iriind dieser Daten eine Iiorrektion in die
oben angegebene (::tsnienge ein, SO wird sicli erweisen, daO iiian auf
1.7.5 g roher Siiure c?:%
ccrn N?O erhalt. Bei einein aiideren nnalogeri
1-ersuche .wiirden
1.65 g rohe Saure und 221 ccm NZO erhalteri. Hechnen wir ein ivlolekiil Stickstoffoxydul auf ein XIolekiil Adipinsaurr,
sn betrageii die husheuten aq StickstofEoxydul
8 8 O i 0 , d. h. sie sinti
beinahe doppelt so groll als diejenigen, (lie, nacli Nefs Gleichuiig
erwartet werden konnten. Man ist also genijtigt anz~nehiiien, daW eine Oxydation
zweiten CH?-C;ruppe zur Carboxylgruppe stattfindet, und
noch eiiier
w a r nach I) Iinorye wid ;\i.iidt, diese Bericht.: 21, 2136 [1899]. 1377 1377 derselben Art, wie eti niit der ersten C h p p e geschah. In die ange-
fiihrten Berechnungen der Ausbeuten von Stickstoffoxydul mu8 aber
ncich eine sachliche Korrektion eingefiihrt werden. Es zeigte aicb,
da13 auUer dem Hauptprodukte. der Oxydation - der Adipinsaure,
sich dabei auch ca. 7010 B e r n s t e i n s i u r e (Schrnp. 1 8 3 O ; Anhydrid,
aach Auschiitz I) hergestellt, scbrnolz bei 119-120°)
und 12.6OiO
4:lutarsau r e (Schmp. 91-94O;
Analyse des Silbersalzes, Titration)
tinter den Osydationsprodiikttii vorfanden. Nach der Analogie rnussen
wir annehrnen, da13 jede Methplengruppe bei Wirkuog von Salpeter-
siiare dieselben Urnwandlungen erleidet, die sie bei der Uildung von
Adipinsaure erleiden niu13. Unter Beriicksichtigung dieses Umstandes
werden x i r finden, daB die .4iisbeuten voii Stickstoffoxydul 74.5O10 der
theoretischen ausrnachen. Die Arbeit wird in tlem 1,aboratoriuni fiir
f )rpnische Chernir
an der Universitiit hiosknu fortgesetzt. Hr. Prof. N. Z e l i n s k y kani
mir stets freundlichst rnit R:it
i i n d 'Tat entgegen. Ilafiir sei dern hoch-
geachteteti Iirn. Professor nuch RU dieser Stelle mein innigster Dank
aosgesprochen. 211. E. Noelting, E. Grandmougin und 11. Freimann:
Zur Kenntnis der Reduktionsprodukte der $-Naphthochinon-
hydrazone. ,
2, Diese Berichte 24, 1592 [1891]: Bull. 50,:. intl. hlulh. 1892, 127. j ) Auu. d. Cheni. 226, 8 [I884]. (Eingegangen am 1. April 1909) (Eingegangen am 1. April 1909) Vor nahezu 30 Jahren waren wir (Noelting und G r a n d m o u g i n )
tier Frage der K o n s t it u ti o n d e r (3- Nap h t hociiinonh y d raz o n e
&her getreten und danials nu€ Grund verschiedener experimentel1r.r
Vntersuchungeu ?) Zuni Resultat zelangt, da13 diese Yerbiodungen al>
u--kzoderivnte d e s re-Naphthols auizufassen sind, eine Auffassung,
die wohl jetzt ziernlich allgernein angenomnien wird. Wir liatteu damals :tuch unter anderen Beweisen ernihnt, da13 die
k t h e r dieser Hydrazonc, die Hir in reinem Zustande und aohlkr>-
stallisiert eihaltrn hatten, SauerstoffStIier sind, denn bei der reduzie-
renden Spaltung mit Zinnchlortir und Salzsaure erlialt man neben den
Amidonaphtholathern stets Anilin, und nicbt ein alkgliertes Anilin. Auch diese Spaltungsprodukte wurden analpsenrein erhalteo und
eingeheoder charakterisiert.
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https://openalex.org/W3114152213
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https://journal.uinjkt.ac.id/index.php/arabiyat/article/download/17444/pdf
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English
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ARABIC LEARNING AT MADRASAH ALIYAH BASED ON THE 2013 CURRICULUM
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Arabiyat : Jurnal Kebahasaaraban dan Pendidikan Bahasa Arab/Arabiyat: Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban
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cc-by-sa
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1 Mushoffan Prasetianto, “CLIL: Suggested English Materials for Curriculum 2013”, Language
Circle, Journal of Language and Literature, Vol. 8, No. 2, 2014.
2 M. Fadlilah, Implementasi Kurikulum 2013 dalam Pembelajaran SD/MI, SMP/MTs dan SMA/MA,
(Yogyakarta: Ar-Ruzz Media, 2014), 29-30. ARABIC LEARNING AT MADRASAH ALIYAH
BASED ON THE 2013 CURRICULUM
Iis Susiawati, Moch. Hasyim Fanirin
Indonesian Islamic Institute of Az-Zaytun (AL-AZIS) Indramayu, Indonesia
Mekarjaya, Alzaytun, Kabupaten Indramayu, Jawa Barat, 45264, Indonesia
Corresponding E-mail: umifaiz@gmail.com Iis Susiawati, Moch. Hasyim Fanirin
Indonesian Islamic Institute of Az-Zaytun (AL-AZIS) Indramayu, Indonesia
Mekarjaya, Alzaytun, Kabupaten Indramayu, Jawa Barat, 45264, Indonesia
Corresponding E-mail: umifaiz@gmail.com Arabiyat : Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban
Vol. 7 No. 2, December 2020, 251-263
P-ISSN: 2356-153X; E-ISSN: 2442-9473
doi: http://dx.doi.org/10.15408/a.v7i2.17444 Arabiyat : Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban
Vol. 7 No. 2, December 2020, 251-263
P-ISSN: 2356-153X; E-ISSN: 2442-9473
doi: http://dx.doi.org/10.15408/a.v7i2.17444 ARABIC LEARNING AT MADRASAH ALIYAH
BASED ON THE 2013 CURRICULUM
Iis Susiawati, Moch. Hasyim Fanirin
Indonesian Islamic Institute of Az-Zaytun (AL-AZIS) Indramayu, Indonesia
Mekarjaya, Alzaytun, Kabupaten Indramayu, Jawa Barat, 45264, Indonesia
Corresponding E-mail: umifaiz@gmail.com Abstract
Th The purpose of this study was to obtain information and data about the
implementation of the 2013 curriculum from the aspects of planning, implementation
and evaluation as well as the factors that influence the process of implementing the
2013 curriculum at Madrasah Aliyah Nurul Hikmah Haurgeulis Indramayu. The
study was conducted in Madrasah Aliyah Nurul Hikmah Haurgeulis as a
representation of madrasas that have implemented the 2013 Curriculum in learning
Arabic. This research was a field research with a qualitative approach. Data obtained
from three sources, namely observation, interviews and documentation. The subjects
of this study were the principal, the curriculum section and the tenth grade students
of Madrasah Aliyah Nurul Hikmah Haurgeulis Indramayu. The results of this study
indicate that the implementation of the 2013 Curriculum at Madrasah Aliyah Nurul
Hikmah Haurgeulis Indramayu, especially in learning Arabic, was still not running
optimally according to the parameter criteria. There were several factors that
influence the implementation process of the 2013 Curriculum, namely teachers,
students and infrastructure. the 2013 curriculum, Arabic learning, Madrasah Aliyah Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 on noble values, academic values, the needs of students and society. The curriculum is
oriented towards competency development. The second, juridical aspects that is a
foundation used as a legal umbrella in the preparation and development of the
curriculum. One of the juridical foundations is Permendikbud Number 81A of 2013
concerning Implementation of the 2013 Curriculum. The third, conceptual aspect, a
foundation based on ideas or ideas that are abstracted from concrete events. on noble values, academic values, the needs of students and society. The curriculum is
oriented towards competency development. The second, juridical aspects that is a
foundation used as a legal umbrella in the preparation and development of the
curriculum. One of the juridical foundations is Permendikbud Number 81A of 2013
concerning Implementation of the 2013 Curriculum. The third, conceptual aspect, a
foundation based on ideas or ideas that are abstracted from concrete events. Curriculum and learning are two important aspects of educational activities. Both discussed what education is and how it should be implemented. 3 Therefore the
important role of the curriculum in the educational process should be anticipatory and
adaptive to changes and developments and advances in science and technology. Both
require understanding and study in their application and implementation in schools. Therefore, the curriculum is an important factor in the learning process. Because the
curriculum is a set of various subjects that must be studied by students. 4 The
curriculum must keep abreast of developments in science and technology, so that it
can be better applied in the learning process. The 2013 Curriculum is basically a refinement of the previous curriculum. The
target of curriculum change is the teacher as the direct implementers in the classroom. While curriculum is the planned program, teachers are the actors that implement the
program through teaching and learning process. This is the relationship between
curriculum, teachers, and learning.5 The target of the 2013 Curriculum is to increase and balance between
attitudes, skills, and knowledge competencies. In accordance with Law no. 20 of 2013
as stated in the explanation of Article 35: graduate competence is a qualification of
graduate abilities that includes attitudes, knowledge and skills according to agreed
national standards. This is also in line with the development of the Competency-Based
Curriculum (CBC) which was initiated in 2004 which includes competency in attitudes,
knowledge and skills in an integrated manner. 3 Azkia Muharom Albantani, “Implementasi Kurikulum 2013 pada Pembelajaran Bahasa Arab di
Madrasah Ibtidaiyah”, Arabiyat : Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, Vol. 2 No. 2, 2015.
4 Ahmad Janan Asifudin, Mengukir Pilar-Pilar Pendidikan Islam, (Tinjauan Filosofis), (Yogyakarta:
Suka Press, 2010), 111.
5 Maulidia Rachmawati Nur and Ahmad Madkur, “Teachers Voices on the 2013 Curriculum for
English Instructional Activities”, IJEE, Vol. 1, No. 2, 2014.
6 Wina Sanjaya, Kurikulum dan Pembelajaran, (Jakarta: Kencana Prenada Media Group, 2009), 197. Introduction The 2013 curriculum is formulated using methods that suit the needs of
students, with real learning and students playing an active role. It is expected to
produce a thought process that continues to develop well. The 2013 curriculum shows
that a certain subject can be integrateed to other subject.1 The 2013 curriculum in its preparation is based on the following aspects 2. The
first is philosophical aspect. The 2013 Curriculum in this context is education based 7 Wahyu Bagja Sulfemi, Manajemen Kurikulum di Sekolah: Modul Pembelajaran Program Studi
Administrasi Pendidikan STKIP Muhammadiyah Bogor, (Bogor: Visi Nusantara Maju, 2018), 32. 8 Andi Prastowo, “Transformasi Kurikulum Pendidikan Dasar dan Menengah di Indonesia
(Kurikulum Tingkat Satuan Pendidikan Menuju Kurikulum 2013 hingga Kurikulum Ganda)”, JIP: Jurnal
Ilmiah PGMI, Vol. 4, No. 2, 2018. Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 of the curriculum, namely the support of the principal, support of peer teachers and
internal support in the classroom.7 of the curriculum, namely the support of the principal, support of peer teachers and
internal support in the classroom.7 The 2013 curriculum is a new curriculum which began its implementation in
2013/2014. While the implementation of the 2013 Curriculum in madrasas is in
accordance with the Circular of the Director General of Education of the Ministry of
Religion of the Republic of Indonesia No. SE / Dj.I / PP.00 / 50/2013 (Dirjen
Pendis Kemenag RI, 2013) which was signed by the Director General of Islamic
Education on July 8, 2013 by considering personnel preparation and budget readiness
at the Ministry of Religion only started in 2014/2015 lesson year specifically for class
I, IV, VII and X only.8 This curriculum is a development of the existing curriculum,
namely the Competency-Based Curriculum (KBK) which was initiated in 2004 and the
Education Unit Level Curriculum (KTSP) in 2006. It's just that the emphasis in the
2013 curriculum is the improvement and balance of soft and hard skills. skills which
include aspects of competence attitudes, knowledge and skills. As stated by Putra and Gunawan (in Gunawan), the 2013 Curriculum
emphasizes greatly in building student‟s characters, developing relevant skills based on
student interests and needs and developing a thematic approach that benefits student‟s
cognitive abilities.9 The scientific approach as a modern pedagogic dimension in learning is also
emphasized in this 2013 Curriculum. The scientific approach includes: observing,
asking, reasoning, trying, and forming networks. The learning process in the 2013
curriculum touches three domains, namely attitudes, skills and knowledge. As for the
assessment process using authentic assessment, which is a significant measurement of
the learning outcomes of students in the three assessed domains. Saddhono in Dewi
stated that this scientific approach is a learning process that support creativity. 10 The
2013 curriculum entry emphasizes the activities based learning. Therefore, the
assessment will have more emphasis on the process in the aspect of attitudes,
knowledge, and skill.11 7 Wahyu Bagja Sulfemi, Manajemen Kurikulum di Sekolah: Modul Pembelajaran Program Studi
Administrasi Pendidikan STKIP Muhammadiyah Bogor, (Bogor: Visi Nusantara Maju, 2018), 32. j
8 Andi Prastowo, “Transformasi Kurikulum Pendidikan Dasar dan Menengah di Indonesia
(Kurikulum Tingkat Satuan Pendidikan Menuju Kurikulum 2013 hingga Kurikulum Ganda)”, JIP: Jurnal
Ilmiah PGMI, Vol. 4, No. 2, 2018. 9 Imam Gunawan, “Indonesian Curriculum 2013: Instructional Management, Obstacles Faced
by Teachers in Implementation and the Way Forward”, ICET, Atlantis Press, Advences in Social Science,
Education and Humanities Research, Vol. 128 10 Dewi Kusumaningsih, “Indonesian Text Role as Draft Science in Curriculum 2013:
Assessment Introduction Text Structure Strategies in an Indonesian Book”, Asian Journal of Social Sciences
& Humanities, Vol. 2, No. 4, 2013. 11 Fauzan and Anis Fuadah Zuhri, “Analysing The Essence of Fiqh Subjects in Curriculum
2013”, Ahkam, Vol. 17, No. 1, 2017. 7 Wahyu Bagja Sulfemi, Manajemen Kurikulum di Sekolah: Modul Pembelajaran Program Studi
Administrasi Pendidikan STKIP Muhammadiyah Bogor, (Bogor: Visi Nusantara Maju, 2018), 32.
8 Andi Prastowo, “Transformasi Kurikulum Pendidikan Dasar dan Menengah di Indonesia
(Kurikulum Tingkat Satuan Pendidikan Menuju Kurikulum 2013 hingga Kurikulum Ganda)”, JIP: Jurnal
Ilmiah PGMI, Vol. 4, No. 2, 2018.
9 Imam Gunawan, “Indonesian Curriculum 2013: Instructional Management, Obstacles Faced
by Teachers in Implementation and the Way Forward”, ICET, Atlantis Press, Advences in Social Science,
Education and Humanities Research, Vol. 128
10 Dewi Kusumaningsih, “Indonesian Text Role as Draft Science in Curriculum 2013:
Assessment Introduction Text Structure Strategies in an Indonesian Book”, Asian Journal of Social Sciences
& Humanities, Vol. 2, No. 4, 2013.
11 Fauzan and Anis Fuadah Zuhri, “Analysing The Essence of Fiqh Subjects in Curriculum
2013”, Ahkam, Vol. 17, No. 1, 2017. Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 Where as in language learning, especially
Arabic has its own approach which is an implementation of linguistic theory and
learning psychology, especially for novice learners. The 2013 Curriculum policy is aimed at improving the previous curriculum. Entering the new academic year 2014/2015 until now, the implementation of the 2013
Curriculum still faces major obstacles. Basically, the application of the curriculum in
learning is not only seen from the curriculum design, but more importantly, in reality. In addition, there are also several factors that can affect the process of learning
activities including teacher, student, facilities, tools and available media. 6 Meanwhile,
according to Mars in Sulfemi, there are three influential factors in the implementation 252 ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 The purposes of the 2013 Curriculum12 are to improve the quality of education
by balancing soft skills and hard skills through the ability of attitudes, skills and
knowledge in order to face growing global challenges; to form and increase
productive, creative and innovative human resources as capital for the development of
the nation and state of Indonesia; to relieve educators in delivering material and
preparing teaching administration, where the government has prepared all curriculum
components along with textbooks used in learning; to increase the participation of
central and local governments as well as community members in a balanced manner in
determining and controlling quality in the implementation of the curriculum at the
educational unit level; and to increase healthy competition between education units
regarding the quality of education to be achieved. Schools are given the flexibility to
develop the 2013 Curriculum according to the conditions of the education unit, the
needs of students and regional potential. Implementation in realizing curriculum purposes needs organizing learning,
choosing the right learning approach, determining learning procedures, establishing
competencies effectively, establishing success criteria, and educating who is required
to be able to design effective and fun (meaningful) learning. Problems in learning Arabic can be seen from two aspects: 1) linguistic
problems which include speech, qawa'id nahwiyyah sharfiyyah, ta'addud al-ma'na,
mutaradifat, and others. According to Fachrurrozi and Mahyudin (in Hasyim), this
problem of learning Arabic must be fixed because a person's understanding of the
components of language determines how he teaches a language. 2) non-linguistic
problems include approaches, methods, media, teaching materials, motivation in
learning, time allocated for learning and learning objectives.13 Arabic is the language used by Arabs and their peninsula as a means of
communication between them in expressing certain goals so that they are able to
interact based on geographic location, culture and civilization.14 According to A. Fuad
Effendy, Arabic is the Koran, the language of communication between Muslims,
whose role is not only as a means of communication between humans and others, but
also as a means of communication for humans who believe in Allah, which is
manifested in the form of ritual and worship.15 According to al-Ghulayain, Arabic are
sentences used by Arabs to express their goals (thoughts and feelings). 12 M. Fadlilah, Implementasi Kurikulum 2013 dalam Pembelajaran SD/MI, SMP/MTs dan SMA/MA,
24.
13 M. Hasyim, “Andragogi dalam Pembelajaran Bahasa Arab”, Arabiyat : Jurnal Pendidikan Bahasa
Arab dan Kebahasaaraban, Vol. 2, No. 1, 2015.
14 Zulhanan, Paradigma Baru Pembelajaran Bahasa Arab (Kajian Teoritis dan Praktis), (Bandar
Lampung: An-Nur Press, 2004), 1.
15 A. Fuad Effendy, Metodologi dan Strategi Alternatif (Pembelajaran Bahasa Arab), (Malang: Misykat,
2005), 17-18. Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 9 Imam Gunawan, “Indonesian Curriculum 2013: Instructional Management, Obstacles Faced
by Teachers in Implementation and the Way Forward”, ICET, Atlantis Press, Advences in Social Science,
Education and Humanities Research, Vol. 128 10 Dewi Kusumaningsih, “Indonesian Text Role as Draft Science in Curriculum 2013:
Assessment Introduction Text Structure Strategies in an Indonesian Book”, Asian Journal of Social Sciences
& Humanities, Vol. 2, No. 4, 2013. 11 Fauzan and Anis Fuadah Zuhri, “Analysing The Essence of Fiqh Subjects in Curriculum
2013”, Ahkam, Vol. 17, No. 1, 2017. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 253 12 M. Fadlilah, Implementasi Kurikulum 2013 dalam Pembelajaran SD/MI, SMP/MTs dan SMA/M 12 M. Fadlilah, Implementasi Kurikulum 2013 dalam Pembelajaran SD/MI, SMP/MTs dan SMA/MA,
24 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 narrated by experts.16 Meanwhile, according to Taufiq, Arabic is the language used by
communities in the Arabian Island.17 narrated by experts.16 Meanwhile, according to Taufiq, Arabic is the language used by
communities in the Arabian Island.17 The purpose of learning Arabic is to master the knowledge of language and
Arabic language proficiency, such as muthala'ah, muhadatsah, insya', nahwu and sharf. Learning Arabic is an ideal discipline, which includes ideology (religion), science, and
social life. Madrasah Aliyah (MA) Nurul Hikmah Haurgeulis Indramayu is one of the
private madrassas that has implemented the 2013 Curriculum. According to the results
of interviews with the Head of Madrasah, so far no one has researched the application
of the 2013 curriculum in madrasas to Arabic language subjects, while there have been
other subjects, namely biology. Therefore the authors are interested in raising the
problem of implementing the 2013 Curriculum in learning Arabic for class X
Madrasah Aliyah Nurul Hikmah Haurgeulis Indramayu and want to find out whether
the implementation of the 2013 Curriculum in madrasah has gone well in planning,
implementation and evaluation as well as the factors that influence the process. 22 Angga Virgiana, Strategi Pengembangan Kompetensi Widyaiswara dalam Pengelolaan Pembelajaran di
Lembaga Pusat Pengembangan dan Pemberdayaan Pendidik dan Tenaga Kependidikan Ilmu Pengetahuan Alam,
(Bandung: Universitas Pendidikan Indonesia, 2013). Suharsimi Arikunto, Prosedur Penelitian Suatu Pendekatan Praktek, (Jakarta: Rineka Cipta, 2002) Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 And these
goals have reached us how to translate it, and al-Quran al-Karim and al-Hadith As-
Sharif protect it for us, and something in the form of prose and poetry that has been 24. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 254 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 16 Musthafa al-Ghulayain, Jami’ ad-Durus al-‘Arabiyah, (Al-Iskandariyah: Dar Al-„Aqidah, 2014), 5.
17 WildanTaufiq, Metode Penelitian Bahasa Arab, (Bandung: PT Refika Aditama, 2018), 11-12.
18 Lexy J. Moleong. Metodologi Penelitian Kualitatif. Bandung: Remaja Rosdakarya, 2002, h. 3
19 Dudung Abdurrahman, Pengantar Metode Penelitian, (Yogyakarta; Kurnia Kalam Semesta, 2003),
7.
20 Sanapiah Faisol, Format-Format Penelitian Sosial, (Jakarta: Raja Grafindo Persada, 2007), 18.
21 Suharsimi Arikunto, Prosedur Penelitian Suatu Pendekatan Praktek, (Jakarta: Rineka Cipta, 2002),
120.
22 Angga Virgiana, Strategi Pengembangan Kompetensi Widyaiswara dalam Pengelolaan Pembelajaran di
Lembaga Pusat Pengembangan dan Pemberdayaan Pendidik dan Tenaga Kependidikan Ilmu Pengetahuan Alam,
(Bandung: Universitas Pendidikan Indonesia, 2013). napiah Faisol, Format-Format Penelitian Sosial, (Jakarta: Raja Grafindo Persada, 2007), 18. 16 Musthafa al-Ghulayain, Jami’ ad-Durus al-‘Arabiyah, (Al-Iskandariyah: Dar Al-„Aqidah, 2014), 5.
17 WildanTaufiq, Metode Penelitian Bahasa Arab, (Bandung: PT Refika Aditama, 2018), 11-12.
18 Lexy J. Moleong. Metodologi Penelitian Kualitatif. Bandung: Remaja Rosdakarya, 2002, h. 3
19 Dudung Abdurrahman, Pengantar Metode Penelitian, (Yogyakarta; Kurnia Kalam Semesta, 2003),
20 S
i h F i
l F
t F
t P
liti
S i l (J k t
R j G fi d
P
d
2007) 18 21 Suharsimi Arikunto, Prosedur Penelitian Suatu Pendekatan Praktek, (Jakarta: Rineka Cipta, Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 about the state of educational institutions that are the object of research and other
supporting notes in the 2013 Curriculum. about the state of educational institutions that are the object of research and other
supporting notes in the 2013 Curriculum. To obtain accurate data and in accordance with the subject matter under study,
the researchers used several data collection methods which complement each other,
including: observation, interviews and documentation. The analysis in this study was
an inductive approach, a method that departs from specific facts, from concrete
events and then from these facts or events general generalizations are drawn.23 a) Improvement of Content Standards From the results of documentation and observation, it was found that the
material for Arabic lessons in class X of Madrasah Aliyah Nurul Hikmah Haurgeulis
Indramayu was in accordance with the 2013 Curriculum Content Standards, because
the madrasah had used textbooks that had been provided by the Ministry of Religion. The material taught is in accordance with the established syllabus and curriculum. although the material taught is tailored to the abilities of students who have
heterogeneous educational backgrounds. And judging from the existing RPP is in
accordance with the 2013 Curriculum. As stated Nur Ahid et. al. that many factors and
elements of the success of an education in educational institutions, one the factors and
elements is the curriculum.25 Implementation of the 2013 Curriculum at Madrasah Aliyah Nurul Hikmah Haurgeulis Implementation of the 2013 Curriculum at Madrasah Aliyah Nurul Hikmah Haurgeulis Based on the results of observations, interviews and documentation at
Madrasah Aliyah (MA) Nurul Hikmah Haurgeulis Indramayu for two months, from
November 2019 to January 2020, the following data were obtained: Taking into account the existing conditions, potentials and problems, and in
line with the vision, mission and long-term goals of the madrasah, Madrasah Aliyah
Nurul Hikmah haurgeulis establishes strategic goals to be achieved according to the
eight National Education Standards,24 which are increasing Content Standards,
increasing Process Standards, increasing the Outcomes Competency Standards,
increasing the Standards for Educators and Education Personnel, increasing the
Standards for Facilities and Infrastructure, increasing Management Standards,
increasing Financing Standards and improving Education Assessment Standards. The
details are: a) Improvement of Content Standards y
J
f
y
25 Nur Ahid, et al, “Evaluation of Curriculum 2013 with Context Input Process Product Model
in Schools of Kediri, Indonesia”, International Journal of Psychosocial Rehabilitation, Vol. 24, No. 7, 2020. 23 Winarno Surahmad, Pengantar Penelitian Dasar dan Metode Teknik, (Bandung: Tarsito, 1982). 24 Said Hamid Hasan, “History Education in Curriculum 2013: A New Approach to Teaching
History”, Historia International Journal of History in Education, Vol. 14, No. 2, 2013, 164. Method This research was qualitative in nature, which is a research approach that
produces descriptive data in the form of written or oral data from people and
observable behavior.18 According to the type, this research is field research, namely
research that aims to conduct an in-depth study of a social unit in such a way as to
produce a well-organized and complete picture of the social unit. 19 The form of this
research is descriptive research (descriptive research), which is research that describes
an object with regard to the problem under study without questioning the relationship
between research variables.20 In this research, the authors used case research which aims to study intensively
about the background of a person, group or institution, in detail and in depth about a
particular organization, institution or phenomenon.21 Virgiana states that the subject
of research is an object, thing or person attached to data about the object of
research.22 Sources of data in this study are the subjects from which data can be
obtained and collected, namely literary data sources, data sources obtained from books
written by experts in accordance with the problem under study, including scientific
papers, papers and publications related to the curriculum. 2013. Includes documents ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 255 23 Winarno Surahmad, Pengantar Penelitian Dasar dan Metode Teknik, (Bandung: Tarsito, 1982).
24 Said Hamid Hasan, “History Education in Curriculum 2013: A New Approach to Teaching
History”, Historia International Journal of History in Education, Vol. 14, No. 2, 2013, 164.
25 Nur Ahid, et al, “Evaluation of Curriculum 2013 with Context Input Process Product Model
in Schools of Kediri, Indonesia”, International Journal of Psychosocial Rehabilitation, Vol. 24, No. 7, 2020. Winarno Surahmad, Pengantar Penelitian Dasar dan Metode Teknik, (Bandung: Tarsito, 1982).
24 Said Hamid Hasan, “History Education in Curriculum 2013: A New Approach to Teaching
History”, Historia International Journal of History in Education, Vol. 14, No. 2, 2013, 164.
25 Nur Ahid, et al, “Evaluation of Curriculum 2013 with Context Input Process Product Model Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 knowledge and skills. Learning is a process, way, act of making people or living things
learn.26 According to Suyono and Hariyanto, as quoted by Fadillah, the term learning
comes from the basic word learning, which is an activity or a process to acquire
knowledge, improve skills, improve behavior, attitudes and strengthen personality. 27
Finally it can be concluded that learning is a process of interaction between educators
and students as well as between students that can be done with various media and
learning resources that support the learning success of students. knowledge and skills. Learning is a process, way, act of making people or living things
learn.26 According to Suyono and Hariyanto, as quoted by Fadillah, the term learning
comes from the basic word learning, which is an activity or a process to acquire
knowledge, improve skills, improve behavior, attitudes and strengthen personality. 27
Finally it can be concluded that learning is a process of interaction between educators
and students as well as between students that can be done with various media and
learning resources that support the learning success of students. Three things must be considered in the standard process, namely planning,
implementation and evaluation. Planning carried out by class X Arabic teacher MA
Nurul Hikmah haurgeulis, Mr. Mansyur Fadhli, in between has prepared a syllabus and
lesson plans that are relevant to the standard 2013 Curriculum process, the syllabus
and lesson plans are designed to achieve effective learning and according to the needs
of students. But with regard to learning resources, as the results of interviews with the
principal and a review of the learning process in class the school has not provided
textbooks according to the needs of students. Text books are only held by the teacher. Students note the material that is delivered and what the teacher writes on the
blackboard. The learning implementation did not run optimally as planned in the RPP; it
still looked like the Education Unit Level Curriculum. Actually, the teacher has tried to
use interactive, inspirational, fun, creative, challenging and motivating methods of
students, but the students don't seem enthusiastic. This illustrates that Arabic learning
at Madrasah Aliyah Nurul Hikmah Haurgeulis there are inhibiting factors in the
learning process, including those who are not fluent in reading Arabic texts and the
unavailability of Arabic text books for each student. 26 Pusat Pembinaan dan Pengembangan, Kamus Besar Bahasa Indonesia, (Jakarta: Balai Pustaka,
1995), 15.
27 M. Fadlilah, Implementasi Kurikulum 2013 dalam Pembelajaran SD/MI, SMP/MTs dan SMA/MA,
15
28 Heri Retnawati, Samsul Hadi, and Ariadie Chandra Nugraha, “Vocational Hight School
Teachers Difficulties in Implementing Assessment in Curriculum 2013 in Yogyakarta Province of
Indonesia”, International Journal of Instruction, Vol. 9, No. 1, 2016. 28 Heri Retnawati, Samsul Hadi, and Ariadie Chandra Nugraha, “Vocational Hight School
Teachers Difficulties in Implementing Assessment in Curriculum 2013 in Yogyakarta Province of
Indonesia”, International Journal of Instruction, Vol. 9, No. 1, 2016. 26 Pusat Pembinaan dan Pengembangan, Kamus Besar Bahasa Indonesia, (Jakarta: Balai Pustaka,
1995), 15.
27 M. Fadlilah, Implementasi Kurikulum 2013 dalam Pembelajaran SD/MI, SMP/MTs dan SMA/MA,
15
28 Heri Retnawati, Samsul Hadi, and Ariadie Chandra Nugraha, “Vocational Hight School b) Improvement of Process Standards b) Improvement of Process Standards Learning activities are educational processes that provide opportunities for
students to develop their potential into increasing abilities in terms of attitudes, Learning activities are educational processes that provide opportunities for
students to develop their potential into increasing abilities in terms of attitudes, ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 256 29 Afiful Ikhwan, “Management of Learning Assessment Using Curriculum 2013 (Case Study in
Islamic Primary School (MI)Muhammadiyah 5 Wonosari Ponorogo - East Java - Indonesia)”, Muaddib:
Studi Kependidikan dan Keislaman, Vol. 8, No. 2, 2018.
30 Loeloek Indah Poerwanti dan Sofyan Amri, Panduan Memahami Kurikulum 2013, (Jakarta: PT.
Prestasi Pustakarya, 2013), 202-213. Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 The evaluation of the learning process was carried out periodically and
continuously following the learning outcomes. Among other things, by asking some
students to come to the front of the class to reread and explain the material that has
been delivered, questions and answers about the material that has been delivered at
the previous meeting and about the subject matter that was just delivered before the
lesson ends or closes and checks the student notebooks. Because, the keyword in 2013
Curriculum that has been implemented is authentic asessment. Authentic asessment is
a process of assessing global awareness and the process requires that the students
should demonstrate a deeper understanding of the thinking, the motivation, and the
actions of various culture in order succesfully respond to the communities and the
workplaces that extend well beyond their current comfort level. 28 ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 257 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 Pedagogic competence includes the ability of teachers to conduct process
assessment and learning of student and utilize the results of assessment and evaluation
of student learning for the interests of student learning.29 The purpose of curriculum evaluation is to examine the overall curriculum
performance in terms of various criteria. The performance indicators that are
evaluated are not only limited to effectiveness, but also relevance and efficiency. One
of the curriculum components that is important to evaluate is related to the learning
process and learning outcomes. Sukmadinata in Poerwanti and Amri suggested three
approaches in curriculum evaluation, namely: research (comparative analysis),
objective approaches and multivariate mixed approaches. 30 There are three main
components in the scope of assessment in the 2013 Curriculum, namely the
assessment of attitudes, knowledge and skills. c) Improvement of Graduate Competency Standards c) Improvement of Graduate Competency Standards After undergoing an integral learning process, graduates of Madrasah Aliyah
Nurul Hikmah Haurgeulis Indramayu are expected to have graduate abilities which
include attitudes, knowledge and skills. The madrasah and teachers hope that students
can achieve the expected academic targets and be able to develop their potential as
members of society with the knowledge gained at madrasah. In particular the
competence of graduates related to Arabic learning outcomes while studying at
Madrasah Aliyah, it is hoped that students will be able to read Arabic script properly
and correctly, be able to practice Arabic knowledge in everyday life according to what
they have obtained in school and be able to develop it according to situations and
conditions. in society and have noble character in social life as the guidance and
direction of the teacher and the school when he was a student at Madrasah Aliyah
Nurul Hikmah. d) Increasing the Standards for Educators and educational staff d) Increasing the Standards for Educators and educational staff Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 Because the learning process is not only related to transferring knowledge, but also to
the methods and approaches used during the learning process in the classroom. Because the learning process is not only related to transferring knowledge, but also to
the methods and approaches used during the learning process in the classroom. e) Improvement of Standard for Facilities and Infrastructure From the results of observations and interviews with both the principal and
the Arabic teacher, the standard of facilities and infrastructure at Madrasah Aliyah
Nurul Hikmah Haurgeulis Indramayu was still inadequate, including the lack of
facilities and books in the library. The books available were old, obsolete and dusty. It
seems that the library was not maintained and rarely visited. In addition, and its
position in the office of the teacher and principal's room, seems narrow like a
warehouse limited only by bookshelves. The unavailability of textbooks for each
student to be brought home as study material, the absence of a language laboratory
and the lack of learning media used during learning, especially in Arabic subjects. However, the madrasa is in good condition and is well maintained and the madrasa
continues to provide improvements. f) Improvement of Management Standards f) Improvement of Management Standards The performance of madrasah management at MA Nurul Hikmah Haurgeulis
Indramayu was based on strong teamwork and partnerships with a clear vision and
mission under the auspices of the Nurul Hikmah foundation and is known by all
parties. Madrasah Aliyah Nurul Hikmah Haurgeulis Indramayu already has guidelines
that regulate the curriculum for each unit and syllabus, educational calendar,
organizational structure, division of tasks among educators, academic regulations,
rules of conduct, code of ethics for relationships between fellow citizens in the
madrasa environment and educational operational costs. The Nurul Hikmah
Foundation houses four educational institutions in Haurgeulis and Gantar, including
Madrasah Ibtidaiyah Nurul Hikmah Haurgeulis, Madrasah Tsanawiyah Nurul Hikmah
Haurgeulis, Madrasah Tsanawiyah Nurul Hikmah Gantar and Madrasah Aliyah Nurul
Hikmah Haurgeulis. The four educational institutions are managed and integrated
respectively. in the Nurul Hikmah foundation which is headed by a chairman of the
foundation. At any time, a meeting can be held to evaluate the performance of these
institutions. d) Increasing the Standards for Educators and educational staff The standards of educators and educational staff at MA Nurul Hikmah
Haurgeulis Indramayu have met the standards, namely the fulfillment of the number
of educators and educational staff, the qualifications of educators and educational staff
as well as the competence of educators and educational staff that are adequate and
meet both feasibility and mental. But there is a little note, that the class X Arabic
teacher at Madrasah Aliyah Nurul Hikmah Haurgeulis Indramayu is not an Arabic
teacher educator background, but he is a graduate of the LIPIA Syari'a Faculty,
although from a scientific aspect there is no doubt his competence. Although it would
be nice if Arabic lessons were taught by educators with Arabic language educational
backgrounds in accordance with applicable regulations in the field of education. 258 ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 with the competencies that will be achieved, but also associated to the learning model
that has been applied.31 with the competencies that will be achieved, but also associated to the learning model
that has been applied.31 In terms of educational assessment standards, Madrasah Aliyah Nurul Hikmah
Haurgeulis Indramayu has created an assessment system for students both in the
academic and non-academic fields, the assessment has an impact on the teaching and
learning process and the involvement of parents in their child's learning process. Academic assessments include daily assessments when students learn and have
activities in school related to the material being taught, assessment of the
completeness of notebooks, assessment of assignments given to students as a form of
repetition of students for the material taught to them, assessment of midterm and end-
semester tests. The non-academic assessment include assessment of discipline, morals
and personality, accuracy in carrying out tasks, activeness in arts, sports and
organizational activities. Ramayulis stated that there are six curriculum components, namely objectives,
curriculum content, media (facilities and infrastructure), strategy, learning process and
evaluation.32 The rate of economic growth and increase in national productivity is
influenced by the establishment of competency standards and national education
quality standards. If competency quality standards and education quality standards
have been developed in accordance with the vision, mission and goals of national
education, then this is set forth in the curriculum and supported by professional
teachers and school principals, education is expected to produce quality human
resources (HR) capable of bringing Indonesia out of the crisis. and can keep up with
the acceleration of information in the era of globalization.33 31 Amat Jaedun, V. Lilik Hariyanto, and Nuryadin, E.R. “An Evaluation of The Implementation
of Curriculum 2013 at The Building Construction Department of Vocational High Schools in
Yogyakarta ”, Journal of Education, Vol. 7, No. 1, 2014.
32 Ramayulis, Ilmu Pendidikan Islam, (Jakarta: Kalam Mulia, 2010), 234-236.
33 E. Mulyasa, Pengembangan dan Implementasi Kurikulum 2013, Cet. Ke 6, (Bandung: PT Remaja
Rosdakarya, 2015), 23. g) Improvement of Financing Standards g) Improvement of Financing Standards Madrasah has financial planning according to standards and seek additional
financial support. Madrasah Aliyah Nurul Hikmah Haurgeulis is one of the madrasahs
that receive BOS funds from the government in the management of their educational
institutions. Madrasah Aliyah Nurul Hikmah Haurgeulis also manages teacher and
student cooperatives to meet daily needs and learning at school. h) Improvement of Education Assessment Standards An Authentic assessment has to be implemented on an ongoing process and
integrated in learning. Therefore, an authentic assessment is not only in accordance ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 259 31 Amat Jaedun, V. Lilik Hariyanto, and Nuryadin, E.R. “An Evaluation of The Implementation
of Curriculum 2013 at The Building Construction Department of Vocational High Schools in
Yogyakarta ”, Journal of Education, Vol. 7, No. 1, 2014. y
(J
)
33 E. Mulyasa, Pengembangan dan Implementasi Kurikulum 2013, Cet. Ke 6, (Bandung: PT Remaja
Rosdakarya, 2015), 23. gy
, J
f
,
,
,
32 Ramayulis, Ilmu Pendidikan Islam, (Jakarta: Kalam Mulia, 2010), 234-236. gy
, J
f
,
,
,
32 Ramayulis, Ilmu Pendidikan Islam, (Jakarta: Kalam Mulia, 2010), 234-236.
33 E. Mulyasa, Pengembangan dan Implementasi Kurikulum 2013, Cet. Ke 6, (Bandung: PT Remaja
Rosdakarya, 2015), 23. Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 and understand the Arabic material taught by the teacher. Coupled with the lack of
textbooks that should be able to take home and study at home both before the
material is taught and after learning as repetition. So it can be understood if in the
learning process it appears that students are not enthusiastic about participating in
learning. There are even students who have not been able to read Arabic writing well. How will you be able to follow the learning material that is taught properly if you just
have difficulty reading it. But the madrasa continues to strive for guidance for students
who have not been able to read Arabic writing with the Qur‟an Read and Write
program. The third, facility and infrastructure factors. To support the learning process,
the facilities and infrastructure at MA Nurul Hikmah Haurgeulis are inadequate. Such
as the absence of a language laboratory, the lack of facilities and books in the library,
the unavailability of text books for each student, the lack of learning media used such
as LCD, computers and the internet. The classrooms used for the learning process
were all in good condition. In each class, there are windows for air and light
ventilation so that they can freely enter and make classroom conditions comfortable to
use for learning. Factors Affecting the Implementation of 2013 Curriculum in Learning Arabic Based on the results of observations, interviews, and documentation, there
were several factors that influence the process. The first, teacher factor. Class X
Arabic teacher MA Nurul Hikmah Haurgeulis Indramayu did not meet the standards
of Arabic language teachers, because he did not have a higher education background
for Arabic teachers according to the subject matter he taught. His undergraduate
certificate from the faculty of syari'ah at LIPIA. The second, student factors. For some students of class X MA Nurul Hikmah
Haurgeulis Indramayu, Arabic is a material that they have just encountered and they
learned at Madrasah Aliyah. Because their educational background is from public
schools where there are no Arabic subjects. This makes it difficult for them to follow ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 260 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 7(2), 2020 that by correcting these deficiencies, better results will be achieved in learning Arabic
based on the 2013 Curriculum at Madrasah Aliyah Nurul Hikmah Haurgeulis.[] that by correcting these deficiencies, better results will be achieved in learning Arabic
based on the 2013 Curriculum at Madrasah Aliyah Nurul Hikmah Haurgeulis.[] that by correcting these deficiencies, better results will be achieved in learning Arabic
based on the 2013 Curriculum at Madrasah Aliyah Nurul Hikmah Haurgeulis.[] Conclusion After discussing and analyzing the data, this research concluded the following
research results. In general, the implementation of the 2013 Curriculum in Arabic class
X learning at Madrasah Aliyah Nurul Hikmah Haurgeulis Indramayu is in accordance
with the provisions and rules of the 2013 Curriculum, namely using a scientific
approach consisting of observing, asking, experimenting, association and
communication and has reached three domains in the learning process, namely
attitudes, knowledge and skills. Whereas in the assessment, the teacher has used
authentic assessment, which is a significant measurement of students in the three areas
being assessed, namely attitude assessment, knowledge assessment and skills
assessment. Although there are several things that cause the implementation of the
2013 Curriculum at Madrasah Nurul Hikmah Haurgeulis to be not optimal, especially
in learning Arabic, because there are several factors that affect the implementation
process of implementing the 2013 Curriculum. Factors that influence the application
of the 2013 curriculum in Arabic learning at the madrasah among them the teacher
factors, the student factors and the facilities and infrastructure factors as previously
explained. For Madrasah Aliyah Nurul Hikmah Haurgeulis, it is hoped that in the future
it will continue to improve everything that is still lacking, including the linearity of
education for Arabic teachers who teach Arabic, which should be adjusted to the
subjects they are teaching. Likewise with the supporting and supporting facilities and
infrastructure in the implementation of the 2013 Curriculum, at least a handbook for
each student so that they can be brought home and studied at home before and after
the material is delivered in class, especially for beginner students who have never
previously studied Arabic. and of course learning becomes more efficient. It is hoped ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 261 REFERENCES Abdurrahman, Dudung. Pengantar Metode Penelitian. Yogyakarta: Kurnia Kalam
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Barriers to and facilitators of implementing complex workplace dietary interventions: process evaluation results of a cluster controlled trial
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Fitzgerald et al. BMC Health Services Research (2016) 16:139
DOI 10.1186/s12913-016-1413-7 Fitzgerald et al. BMC Health Services Research (2016) 16:139
DOI 10.1186/s12913-016-1413-7 Open Access © 2016 Fitzgerald et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Barriers to and facilitators of implementing
complex workplace dietary interventions:
process evaluation results of a cluster
controlled trial Sarah Fitzgerald*, Fiona Geaney, Clare Kelly, Sheena McHugh and Ivan J. Perry * Correspondence: sarahfitzgerald@ucc.ie
Department of Epidemiology and Public Health, University College Cork, 4th
Floor, Western Gateway Building, Western Road, Cork, Ireland Background restricting options and provision of real incentives (i.e. price discounts) [14, 23]. These complex high-intensity
interventions are informed by empirical evidence and
theories and have a multi-level approach where they are
specifically developed to target all stakeholders within an
organisation (e.g. employers, caterers, employees) [7]. g
The increasing prevalence of diet-related diseases is a
major global public health problem. The growing burden
on population health and unsustainable cost escalation
is crippling healthcare systems worldwide [1–4]. The
causal factors of diet-related diseases are inherently
complex and require complex solutions [5]. Behavioural
interventions aim to improve dietary behaviours and
reduce the associated burden of diet-related diseases at a
population-level [6–8]. The Medical Research Council
(MRC) advocate the importance of combining the evalu-
ation of outcomes and processes when evaluating com-
plex interventions [7]. Process evaluations monitor and
evaluate the fidelity of interventions and can provide an in-
depth understanding of factors that lead to the success or
failure of implementing complex interventions [7, 9–11]. g
( g
p y
p y
) [ ]
The available evidence on process evaluation of low-
intensity workplace interventions has focused mainly on
the effectiveness of interventions rather than on why in-
terventions succeed or fail [24, 25]. The limited available
evidence indicates that contextual factors, particularly
structural and organisational changes can greatly influ-
ence the implementation of workplace interventions
[26–29]. Evidence further suggests that in order to suc-
cessfully implement workplace healthy eating interven-
tions, it is vital to secure engagement by the catering
team. Securing this engagement requires the research
team to provide substantial support and understanding
to the catering team [23]. The complexities of the mod-
ern working environment including on-going structural
changes and competing work projects have also been in-
dicated as factors that can impede intervention imple-
mentation. In contrast, active involvement of managers
in implementation, negotiation skills, consideration of
workplace culture and assessing readiness for change
can serve as facilitators of implementation [27]. It has
also been suggested that ensuring there is transparency
in the implementation plan regarding roles and respon-
sibilities of each team member can help facilitate inter-
vention implementation [11, 27]. Similarly, contextual
factors were also identified as influential in the imple-
mentation of a health promotion intervention in four
Danish industrial canteens and structural changes which
resulted in downsizing, high employee turnover and job
insecurity impeded successful implementation [29]. Background g
The workplace has been identified as an important
health promotion setting as individuals spend long pe-
riods of time in their work environments and it also al-
lows
targeted
health
promotion
programmes
reach
specific population groups [2, 8, 12, 13]. The workplace
provides access to a stable population in a controlled
setting, making it conducive to the implementation of
complex interventions [14]. However, uncertainty exists
regarding the effectiveness of complex workplace dietary
interventions. Previous interventions have demonstrated
limited efficacy with small effect sizes [15–17]. Al-
though, some studies have reported that workplace in-
terventions can have moderate positive effects on dietary
behaviour in terms of healthier food choices and increas-
ing fruit and vegetable consumption [8, 16–20], significant
uncertainty remains regarding the long-term effects on
dietary behaviour, health status outcomes and cost-
effectiveness [8, 17, 21]. These interventions failed to
include detailed process evaluations but recommended
that future workplace interventions should integrate rigor-
ous qualitative and quantitative evaluation methods to ex-
plore reasons for ambiguous findings [15–18, 22]. y
p
p
There are a number of change theories and frame-
works which describe the implementation of interven-
tions within organisations. These theories suggest that
fully understanding processes of change within organisa-
tions is critical for the successful development and im-
plementation of workplace health promotion initiatives
[30, 31]. Lewin’s model of organisational change is one
such theory and involves, unfreezing of current attitudes
to change, implementing the new intervention and re-
freezing new attitudes and behaviour by supporting and
reinforcing change [32, 33]. This theory suggests that
assessing organisational readiness for change and mini-
mising the restraining factors of tacit organisational
cultures are central for successful implementation of in-
terventions and for achieving sustained change [30–33]. Schein’s theory on organisational change further sug-
gests that in order to embed change, the intervention
needs to become part of the culture of the organisation
[31]. The principles of these theories are reinforced in Very few comprehensive process evaluations of work-
place dietary interventions have been conducted. Fur-
thermore, few studies explore the opinions of those
directly involved in workplace dietary interventions ei-
ther as a decision maker or a participant. The evidence
base consists mainly of process evaluations that evaluate
low-intensity workplace health promotion interventions
or
workplace
stress
interventions. Abstract Background: Ambiguity exists regarding the effectiveness of workplace dietary interventions. Rigorous process
evaluation is vital to understand this uncertainty. This study was conducted as part of the Food Choice at Work trial
which assessed the comparative effectiveness of a workplace environmental dietary modification intervention and
an educational intervention both alone and in combination versus a control workplace. Effectiveness was assessed
in terms of employees’ dietary intakes, nutrition knowledge and health status in four large manufacturing
workplaces. The study aimed to examine barriers to and facilitators of implementing complex workplace
interventions, from the perspectives of key workplace stakeholders and researchers involved in implementation. Methods: A detailed process evaluation monitored and evaluated intervention implementation. Interviews were
conducted at baseline (27 interviews) and at 7–9 month follow-up (27 interviews) with a purposive sample of
workplace stakeholders (managers and participating employees). Topic guides explored factors which facilitated or
impeded implementation. Researchers involved in recruitment and data collection participated in focus groups at
baseline and at 7–9 month follow-up to explore their perceptions of intervention implementation. Data were
imported into NVivo software and analysed using a thematic framework approach. Results: Four major themes emerged; perceived benefits of participation, negotiation and flexibility of the
implementation team, viability and intensity of interventions and workplace structures and cultures. The latter three
themes either positively or negatively affected implementation, depending on context. The implementation team
included managers involved in coordinating and delivering the interventions and the researchers who collected
data and delivered intervention elements. Stakeholders’ perceptions of the benefits of participating, which
facilitated implementation, included managers’ desire to improve company image and employees seeking health
improvements. Other facilitators included stakeholder buy-in, organisational support and stakeholder cohesiveness
with regards to the level of support provided to the intervention. Anticipation of employee resistance towards
menu changes, workplace restructuring and target-driven workplace cultures impeded intervention
implementation. Conclusions: Contextual factors such as workplace structures and cultures need to be considered in the
implementation of future workplace dietary interventions. Negotiation and flexibility of key workplace stakeholders
plays an integral role in overcoming the barriers of workplace cultures, structures and resistance to change. Trial registration: Current Controlled Trials: ISRCTN35108237. Date of registration: 02/07/2013 Keywords: Process evaluation, Implementation, Facilitators, Barriers, Workplace dietary intervention Page 2 of 13 Page 2 of 13 Fitzgerald et al. BMC Health Services Research (2016) 16:139 Background By
design,
low-
intensity workplace health promotion interventions tend
to focus solely on information provision and fail to in-
vestigate the effects of environmental approaches, such
as food modification [14]. In contrast, high-intensity in-
terventions are complex in nature and typically consist
of a number of different interacting components. These
components can include both information provision and
environmental approaches such as, food modification, Fitzgerald et al. BMC Health Services Research (2016) 16:139 Page 3 of 13 Page 3 of 13 intervention which comprised of three elements; group pre-
sentations,
individual
nutrition
consultations
and
the
provision of detailed nutrition information (traffic light
menu-labelling, posters, leaflets and emails). The third
workplace received an environmental dietary modification
intervention which consisted of five elements 1) menu
modification (restriction of fat, saturated fat, sugar and salt),
2) increase in fibre, fruit and vegetables, c) price discounts
for fresh fruit, d) strategic positioning of healthier alterna-
tives and e) portion size control [21]. Table 1 outlines the
allocation of the interventions. The intervention design was
developed by the research team who had specific expertise
in public health nutrition and dietetics and was advised by
catering stakeholders (Catering Managers Association of
Ireland (CMAI)). The research team collaborated with the
workplace stakeholders (human resources (HR) and cater-
ing managers) to implement the FCW interventions within
each individual workplace. Each workplace was assigned a
research workplace leader who was based on-site and col-
laborated with the workplace stakeholders to co-ordinate
data collection for rotating shift schedules and monitor ad-
herence to the interventions. Implementation was moni-
tored and evaluated in all four workplaces using a detailed
process evaluation throughout the intervention period, ana-
lysing perspectives of management stakeholders, participat-
ing
employees
and
research
assistants. Steckler
and
Linnan’s conceptual framework guided the process evalu-
ation and was based on the components of context, reach,
dose delivered, dose received, fidelity and recruitment [9]. implementation frameworks which outline the enablers
and barriers to successful implementation within organi-
sations [34]. Stakeholder buy-in, organisational support,
supportive organisational culture, monitoring and evalu-
ation are defined as enablers of implementation. The ex-
ternal environment, resistance to change and vested
interests are outlined as barriers to implementation
within organisations [34]. Methods
Conte t The current study was carried out as part of the Food
Choice at Work (FCW) study, a cluster controlled trial
conducted in four large manufacturing workplaces in
Cork, Ireland. Details of the FCW study have been pub-
lished elsewhere [21]. Briefly, the FCW study assessed
the comparative effectiveness of a workplace environ-
mental dietary modification intervention and an educa-
tional intervention both alone and in combination
versus a control workplace on employees dietary behav-
iours, nutrition knowledge and health status. Changes in
employees’ dietary intakes and health status (BMI, waist
circumference and blood pressure) outcomes were mea-
sured at baseline, follow-up at 3–4 months and 7–9
months. As the focus of the FCW study was to imple-
ment a complex dietary intervention in an environment
that could tolerate different interacting intervention com-
ponents, workplaces were purposively selected and allo-
cated interventions. Workplaces were deemed eligible if
they were manufacturing workplaces who employed more
than 250 employees, had a daily workplace canteen,
located in Cork, represented on the Industrial Develop-
ment Authority of Ireland (IDA) website and were able to
commit to all components of the complex intervention
for the duration of the study [21]. In order to ensure that
the participating workplaces and employees were repre-
sentative of the general Irish workforce, demographic vari-
ables of non-participating employees were examined. Background To improve the implementation of complex, high-
intensity workplace dietary interventions and achieve
sustainable organisational change, it is imperative that
factors which facilitate and impede the implementation
process are identified by exploring the opinions of those
directly involved [9]. The aim of this study was to define
and explore the facilitators of and barriers to the imple-
mentation of complex, high-intensity workplace dietary
interventions from the perspectives of key workplace
stakeholders, participating employees and research assis-
tants delivering the intervention. Participants For the process evaluation, purposive sampling was used
to recruit management stakeholders who were involved
in the intervention either through initial consultation,
decision-making or on-going collaboration with the re-
searchers who collected data. Employees who partici-
pated in the intervention were selected using random
number generation software. At baseline 27 face-to-face
semi-structured
interviews
(13
managers
and
14
employees) were conducted and 27 interviews (12 man-
agers and 15 employees) were conducted post interven-
tion implementation. Where feasible the same people
were interviewed at follow-up stage, however this was
dependent on availability of participants. Research assis-
tants who conducted the interviews were involved in re-
cruitment and data collection but were not known to
the participants they interviewed. Table 2 outlines the
characteristics of managers and employees who took
part. Purposive sampling was used to recruit research as-
sistants for the focus groups. All research assistants
involved in the FCW study were invited to participate at
baseline and at follow-up stage. Nine out of eleven
research assistants took part at baseline and four out of
six research assistants took part at follow-up. The reason In the control workplace, data was collected at base-
line and at each stage of follow-up. Participants in this
workplace were informed that they were involved in a
university-led study to observe employees dietary behav-
iours. The second workplace received a nutrition education Page 4 of 13 Fitzgerald et al. BMC Health Services Research (2016) 16:139 Table 1 Allocation of FCW interventions across the workplaces and description of interventions
Workplace
Intervention implemented
Description of interventions
Control (Food &
beverage industry)
Control site
Monitored employees eating behaviours. Education (Health
industry)
Nutrition education
intervention
Nutrition education consisted of three elements: 1) monthly group presentations, 2)
individual nutrition consultations and 3) detailed nutrition information (shopping cards,
posters, leaflets and emails), including the application of a healthy eating traffic light cod
system to daily menus and vending machines. This displayed the number of calories an
nutritional breakdown of the meal or food item. Environmental
(Automotive industry)
Environmental dietary
modification intervention
Environmental dietary modification consisted of five elements: 1) restriction of fat, satura
fat, sugar and salt, 2) increase fibre, fruit and vegetables, 3) price discounts on whole fre
fruit, 4) strategic positioning of healthier alternatives and 5) portion size control. Combined (IT industry)
Combined intervention
All the elements of the nutrition education intervention and the environmental dietary
modification intervention were implemented. Data collection Semi-structured face-to-face interviews were conducted
at baseline between February and April 2013 and at
follow-up stage between April and July 2014. Interviews
were conducted in the workplaces and lasted between
40 and 60 min. The baseline focus group was conducted
in May 2013 and the follow-up focus group was con-
ducted post intervention implementation in August
2014. These were hosted in University College Cork by
an independent moderator and lasted for 1 h. An assist-
ant moderator took observational notes. In the inter-
views and focus groups probes were used to initiate
discussion when there was a pause and also to further
explore points of interest. Topic guides A co-investigator involved in the FCW study developed
semi-structured topic guides for the interviews and focus
groups (Additional files 1, 2, 3, 4, 5 and 6). As previously
outlined, Steckler and Linnan’s conceptual framework
was used to guide the process evaluation plan. Thus, the
topic guides were based on the six components of the
framework; context, reach, dose delivered, dose received,
fidelity and recruitment [9]. These topic guides were
reviewed and refined by research assistants on the study. Pilot interviews that were conducted at baseline and at
follow-up stage, overall study objectives, preliminary
analysis of baseline data and researchers’ experience of
intervention implementation further informed the topic
guides. For the interviews, the topic guides were used to
explore facilitators of and barriers to the implementation Participants Nutrition education consisted of three elements: 1) monthly group presentations, 2)
individual nutrition consultations and 3) detailed nutrition information (shopping cards,
posters, leaflets and emails), including the application of a healthy eating traffic light coding
system to daily menus and vending machines. This displayed the number of calories and
nutritional breakdown of the meal or food item. Environmental dietary modification consisted of five elements: 1) restriction of fat, saturated
fat, sugar and salt, 2) increase fibre, fruit and vegetables, 3) price discounts on whole fresh
fruit, 4) strategic positioning of healthier alternatives and 5) portion size control. All the elements of the nutrition education intervention and the environmental dietary
modification intervention were implemented. of the interventions from the perspective of management
stakeholders and employees. For the focus groups, the
topic guides were used to explore the experiences of the
research assistants delivering a complex intervention in
the workplace. for non-participation in the focus groups was the part-
time availability of research assistants and there were
fewer researchers employed at follow-up stage. For the interviews, individuals were contacted by email
and follow-up telephone call when necessary. The focus
group moderator emailed research assistants and invited
them to participate. All participants provided written in-
formed consent. Data were digitally recorded and tran-
scribed verbatim. To preserve confidentiality, data were
anonymised. Analytical tools Both managers and employees highlighted the benefits
of participating in the study. Managers had a desire to
improve company image and foster employee loyalty
while employees had a desire to improve their health. The perception of a long-term benefit rather than the
benefit itself facilitated implementation in the short-term
as it encouraged engagement and fostered buy-in. Verba-
tim examples of this theme are included in Table 3. 1) Familiarisation: Three researchers (SF, FG and CK)
conducted the interviews. Researchers became
familiar with the data by re-reading transcripts,
audio tapes, field notes and observational notes. Recurring themes and initial ideas were noted in an
analytical memo. y
The perception of a long-term benefit rather than the
benefit itself facilitated implementation in the short-term
as it encouraged engagement and fostered buy-in. Verba-
tim examples of this theme are included in Table 3. 1. Concern with company image: Managers had a
vested interest in ensuring successful
implementation of the interventions as they had a
strong desire to portray a positive company image to
both industry and employees. Managers believed
that participation in the study would be a means of
achieving this objective. Managers wanted to depict
an image of a progressive company both nationally
and internationally in the manufacturing industry. This desire facilitated implementation as managers
were supportive of the interventions and they
facilitated access to employees by releasing them
from work activities to attend study
appointments. Managers felt involvement in a
university-led study would be regarded as presti-
gious by other companies. They expressed pride
in being ‘chosen’ to participate and believed that
it created a sense of elitism in the manufacturing
industry. According to some of the researchers
who collected data, a concern with company
image motivated workplace stakeholders to pro-
vide recruitment and implementation support. 2) Identification of a thematic framework: Four
researchers (SF, SMH, FG and CK) undertook initial
coding of a selection of transcripts (one
management stakeholder and one employee
participant). These were subject to inter-coder reli-
ability as one of the researchers (SMH) was not in-
volved in data collection. Open coding allowed for
an inductive approach. The preliminary coding
framework was developed by discussing the conver-
gence and divergence of codes. The researchers
redefined this framework for subsequent stages of
coding. 3) Indexing: This stage involved the indexing of specific
parts of the data to correspond to the emerging
themes. Analytical tools The framework approach was used for analysis of data
[9, 35]. This was considered appropriate as the process
evaluation had pre-specified objectives while it also per-
mitted the emergence of unexpected themes. Framework
analysis is dynamic, allowing for change throughout the Table 2 Characteristics of baseline and follow-up interviews conducted with managers and employees
Managers
Employees
Workplace
Baseline
Follow-up at 7–9 months
Baseline
Follow-up at 7–9
months
Control
2 (Occupational health and administrative
managers)
3 (Occupational health and HR managers)
4 (2 male and 2
female)
4 (2 male and 2
female)
Education
3 (Occupational health, HR and catering
managers)
3 (Occupational health, HR and catering
managers)
3 (2 female and 1
male)
4 (3 male and 1
female)
Environmental 4 (Managing director, HR and catering
managers)
3 (Managing director, HR and catering
managers)
4 (2 female and 2
male)
4 (2 male and 2
female)
Combined
4 (Occupational health and catering
managers)
3 (Occupational health and catering
managers)
3 (1 female and 2
male)
3 (1 male and 2
female) Table 2 Characteristics of baseline and follow-up interviews conducted with managers and employees
Managers
Employees
Workplace
Baseline
Follow-up at 7–9 months
Baseline
Follow-up at 7–9
months
Control
2 (Occupational health and administrative
managers)
3 (Occupational health and HR managers)
4 (2 male and 2
female)
4 (2 male and 2
female)
Education
3 (Occupational health, HR and catering
managers)
3 (Occupational health, HR and catering
managers)
3 (2 female and 1
male)
4 (3 male and 1
female)
Environmental 4 (Managing director, HR and catering
managers)
3 (Managing director, HR and catering
managers)
4 (2 female and 2
male)
4 (2 male and 2
female)
Combined
4 (Occupational health and catering
managers)
3 (Occupational health and catering
managers)
3 (1 female and 2
male)
3 (1 male and 2
female) cs of baseline and follow-up interviews conducted with managers and employees Fitzgerald et al. BMC Health Services Research (2016) 16:139 Page 5 of 13 Page 5 of 13 Page 5 of 13 analytical process while its systematic nature provides
transparency. This was beneficial as multiple researchers
were involved in data collection, analysis and interpret-
ation. The following steps were completed [9]: Perceived benefits of participation Both managers and employees highlighted the benefits
of participating in the study. Managers had a desire to
improve company image and foster employee loyalty
while employees had a desire to improve their health. Analytical tools Data was imported into NVivo software
(QSR International Pty Ltd) for coding. The refined
coding framework was systematically applied to the
data and the main thematic categories and sub-
categories were formed. appointments. Managers felt involvement in a
university-led study would be regarded as presti-
gious by other companies. They expressed pride
in being ‘chosen’ to participate and believed that
it created a sense of elitism in the manufacturing
industry. According to some of the researchers
who collected data, a concern with company
image motivated workplace stakeholders to pro-
vide recruitment and implementation support. 4) Charting: The coded data was further abstracted and
synthesised during the charting process by two of
the researchers. This involved arranging themes into
illustrative charts based on headings included in the
thematic framework. 2. 2. Managers’ personal interest: In some workplaces key
workplace stakeholders expressed a personal interest
in maintaining a healthy lifestyle. Occupational
health stakeholders in the control and combined
workplaces had a professional background in
nursing and had great interest in supporting
initiatives that would enhance health consciousness
in the workplace. Similarly in the education
workplace, a HR stakeholder had professional
training and interest in nutritional sciences. This
interest was a driver for workplace participation and
ensured that implementation of the interventions
received organisational support. 2. Managers’ personal interest: In some workplaces key
workplace stakeholders expressed a personal interest
in maintaining a healthy lifestyle. Occupational
health stakeholders in the control and combined
workplaces had a professional background in
nursing and had great interest in supporting
initiatives that would enhance health consciousness
in the workplace. Similarly in the education
workplace, a HR stakeholder had professional
training and interest in nutritional sciences. This
interest was a driver for workplace participation and
ensured that implementation of the interventions
received organisational support. 5) Mapping and interpretation: The charts provided a
schematic diagram of the process evaluation which
guided data interpretation. Interpretations were
checked and discussed by two researchers. The
interpretation of the themes was guided by the
specific objectives of the study and also by the
unexpected themes that emerged during analysis. 5) Mapping and interpretation: The charts provided a
schematic diagram of the process evaluation which
guided data interpretation. Interpretations were
checked and discussed by two researchers. The
interpretation of the themes was guided by the
specific objectives of the study and also by the
unexpected themes that emerged during analysis. Major themes 3.Fostering employee loyalty: “If you’re trying to convince employees that you’re interested and trying to engage with them, show
them that you care about their health and well-being so that’s a good engagement tool” (Occupational health, nutrition
education site—baseline stage). “If we can keep our employees healthy, they’ll be happier, they’ll produce better work, they’ll hit their efficiencies a lot better and
they’re more likely to be in here” (HR, nutrition education site—follow-up stage). 4. Health concerns among employees: “We don’t have the luxury in this modern day and age of getting to 54, in days of old you’d
get to this age and you pull back a little, there’s young and progressive people coming up underneath you and they take the
pressure and that, that doesn’t happen today. They are going to work people until they’re 65” (Employee, nutrition education
site—follow-up stage). 1. Flexibility: The flexibility and adaptability of the
researchers manifested itself in a number of ways. To facilitate timely data collection, it was critical for
the researchers to adapt to the structure and
practices of each worksite. Researchers were
required to schedule appointments that
complemented rotating shift patterns. Similarly,
monthly group nutrition presentations were
delivered multiple times each day to also
complement rotating shifts. Data collection often
occurred during busy times on site such as ‘end of
quarter’. On these occasions, employees frequently
rescheduled appointments and researchers had to
facilitate these late changes. At the outset, managers
were concerned that the target-driven culture of
manufacturing workplaces would not be suitable for
implementing a study that requires employee inter-
action and significant logistical planning. However,
researchers’ adaptability to changes facilitated
implementation. would foster employee loyalty and boost morale
within the workplace which could result in financial
benefits for the company by reducing absenteeism
and increasing productivity. It was anticipated that
this could be achieved by managers promoting
participation in elements such as the healthy-eating
group presentations. g
p p
4. Health concerns among employees: The main
reasons for employees participating included age
concerns, individual health concerns (weight,
cholesterol level, blood pressure, and digestive
disorders) and lifestyle concerns. Older participating
employees felt pressure to keep up with younger
employees in their fast-paced working environments. Employees were seeking health improvements in an
effort to curtail any negative effects of ageing and
the need to ‘slow down’ their working pace. Major themes 3. g
3. Fostering employee loyalty: A desire to improve
relations between employers and employees was a
motivating factor for participation. Managers
identified the study as an opportunity to improve
relations with employees. In order to demonstrate
their support for the study to employees, they
released staff from work activities for appointments
and provided resources for the study. They believed
that driving health consciousness among employees 3. Fostering employee loyalty: A desire to improve
relations between employers and employees was a
motivating factor for participation. Managers
identified the study as an opportunity to improve
relations with employees. In order to demonstrate
their support for the study to employees, they
released staff from work activities for appointments
and provided resources for the study. They believed
that driving health consciousness among employees Four major themes emerged; 1) perceived benefits of par-
ticipation, 2) negotiation and flexibility of the implementa-
tion team, 3) viability and intensity of intervention and 4)
individual workplace structures and cultures. Depending
on context, the latter three themes were found to have
both a positive and negative impact on implementation
and are discussed as either facilitators or barriers. Findings
are presented from the perspective of management stake-
holders, employees and research assistants. Page 6 of 13 Fitzgerald et al. BMC Health Services Research (2016) 16:139 Table 3 Theme of ‘perceived benefits of participation’ and verbatim examples
Theme
Verbatim Examples
Perceived benefits of
participation
1. Concern with company image: “We were one of the ones to be chosen, that’s a huge cannon feather in our cap you know we’re
thrilled about that and you know again to promote the fact that it’s not everybody that was selected….we were chosen as a
company for a particular reason and we’re honoured to be included” (HR manager, Environmental site—follow-up stage). 2. Managers’ personal interest: “I would have been the person who pushed it to say ‘let’s go and do this, it’s an opportunity, yeah’…
having dieticians on site, having access to all this expertise you know, and it is a great pile of health promotion going on in the
background” (Occupational health, Control site—follow up stage). 3.Fostering employee loyalty: “If you’re trying to convince employees that you’re interested and trying to engage with them, show
them that you care about their health and well-being so that’s a good engagement tool” (Occupational health, nutrition
education site—baseline stage). Major themes Employees appreciated the investment their
employers made in the study as it provided them
with a unique opportunity to have a nutritional
consultation and a free health check-up during their
working hours. It reassured employees that their
employer concerns went beyond generating profit
hence they felt obliged to participate. p
2. Negotiation: The researchers also perceived
negotiation as central to successful implementation. It was necessary for the researchers to negotiate a
level of change that was agreeable to mangers,
caterers and the researchers themselves. In some
instances this resulted in changes to the planned
intervention components or the scale of change. Effective communication with managers was
necessary to reach a compromise with regards to
what intervention elements were implemented and
to what degree they were implemented, particularly
for the environmental modification intervention. For
example, the proposed portion size restrictions were
heavily negotiated between the researchers and
catering staff with compromises being made by all
parties. Willingness to change among catering staff 2. Major themes “If we can keep our employees healthy, they’ll be happier, they’ll produce better work, they’ll hit their efficiencies a lot better and
they’re more likely to be in here” (HR, nutrition education site—follow-up stage). 4. Health concerns among employees: “We don’t have the luxury in this modern day and age of getting to 54, in days of old you’d
get to this age and you pull back a little, there’s young and progressive people coming up underneath you and they take the
pressure and that, that doesn’t happen today. They are going to work people until they’re 65” (Employee, nutrition education
site—follow-up stage). Theme
Verbatim Examples
Perceived benefits of
participation
1. Concern with company image: “We were one of the ones to be chosen, that’s a huge cannon feather in our cap you know we’re
thrilled about that and you know again to promote the fact that it’s not everybody that was selected….we were chosen as a
company for a particular reason and we’re honoured to be included” (HR manager, Environmental site—follow-up stage). 2. Managers’ personal interest: “I would have been the person who pushed it to say ‘let’s go and do this, it’s an opportunity, yeah’…
having dieticians on site, having access to all this expertise you know, and it is a great pile of health promotion going on in the
background” (Occupational health, Control site—follow up stage). 3.Fostering employee loyalty: “If you’re trying to convince employees that you’re interested and trying to engage with them, show
them that you care about their health and well-being so that’s a good engagement tool” (Occupational health, nutrition
education site—baseline stage). “If we can keep our employees healthy, they’ll be happier, they’ll produce better work, they’ll hit their efficiencies a lot better and
they’re more likely to be in here” (HR, nutrition education site—follow-up stage). 4 Health concerns among employees: “We don’t have the luxury in this modern day and age of getting to 54 in days of old you’d . Managers’ personal interest: “I would have been the person who pushed it to say ‘let’s go and do this, it’s an opportunity, yeah’
aving dieticians on site, having access to all this expertise you know, and it is a great pile of health promotion going on in the
ackground” (Occupational health, Control site—follow up stage). Flexibility and negotiation Enthusiasm of caterers towards
the intervention further facilitated the progress of
implementation. Support of the catering company in
their workplace stemmed from caterers realising that
involvement in the study could be a valuable learn-
ing opportunity and serve as a foundation on which
to enhance the knowledge of the catering staff. 1. Stakeholder buy-in: Employees recognised the
importance of receiving ‘buy-in’ from catering and
management stakeholders in order for the interven-
tion to be successfully implemented. This was also
highlighted by the researchers who acknowledged
their flexibility and willingness to change as a crucial
facilitating factor. Enthusiasm of caterers towards
the intervention further facilitated the progress of
implementation. Support of the catering company in
their workplace stemmed from caterers realising that
involvement in the study could be a valuable learn-
ing opportunity and serve as a foundation on which
to enhance the knowledge of the catering staff. 3. 3. High-level workplace management support: Due to
the target-driven culture in the manufacturing in-
dustry, supervisors were reluctant to release produc-
tion staff to attend appointments. A disruption on
the production line could lead to knock-on effects
for overall site-level efficiencies. However, supervi-
sors were instructed by managers to adapt to the
demands of the intervention for the duration of the
study period. To ensure that catering staff adhered
to the intervention elements, management needed to
reinforce the commitment that the workplace had
made to the study. This was particularly evident in
the environmental and combined workplaces, where
environmental modification elements were imple-
mented and more negotiation was needed in these
workplaces. Stakeholder cohesiveness with regards
to organisational support was central to achieving
successful implementation. 3. High-level workplace management support: Due to
the target-driven culture in the manufacturing in-
dustry, supervisors were reluctant to release produc-
tion staff to attend appointments. A disruption on
the production line could lead to knock-on effects
for overall site-level efficiencies. However, supervi-
sors were instructed by managers to adapt to the
demands of the intervention for the duration of the
study period. To ensure that catering staff adhered
to the intervention elements, management needed to
reinforce the commitment that the workplace had
made to the study. This was particularly evident in
the environmental and combined workplaces, where
environmental modification elements were imple-
mented and more negotiation was needed in these
workplaces. Stakeholder cohesiveness with regards
to organisational support was central to achieving
successful implementation. Flexibility and negotiation Negotiation: “Changing down to nearly half, we just couldn’t, there would be uproar…we did a taste test, we put three plates
out one with what we serve now, one with what UCC wanted us to serve and something somewhere in the middle that we felt
we could serve and get away with, that’s the way we made our choice” (Occupational health, combined intervention site
baseline stage). “The breakfast option alright was something that you couldn’t change too much. I suppose from their side they were just afraid
that there would be a lot of backlash from the employees and there at the front line then dealing with it” (Researcher 2 - follow
up stage) 3. High-level workplace management support: “I found it very, very hard to get product builders released for their sessions. That was a huge struggle for me, it’s the team leaders and they’re all about their metrics, they want to have, net efficiencies,
be on target” (Occupational Health—nutrition education site—follow-up stage). and researcher negotiation skills facilitated
compromises being reached. management were actively involved in the study, it en-
couraged employee participation and secured more buy-
in from production supervisors and team leaders. In the
environmental workplace, the support of HR managers
went beyond providing basic logistical support and HR
contacts became involved in providing recruitment sup-
port. Organisational restructuring and a ‘traditional’ work-
place culture had a negative effect on implementation. Verbatim examples of this theme are included in Table 5. The researchers described how certain meals
appeared to be non-negotiable in the environmental
and combined workplaces. The cooked breakfast was
part of the workplace culture and researchers found
reaching an agreement on modifying this option
challenging. A compromise was eventually reached
on reducing the portion size of the cooked breakfast
and cooking method was changed from frying to
baking when possible. In this instance, workplace
culture was identified as a barrier to full-scale
implementation. Catering stakeholders anticipated
employee resistance to change in response to changes
being made to the breakfast options. This expectation
persisted and impeded the implementation of some of
the environmental modification elements. 1. Stakeholder buy-in: Employees recognised the
importance of receiving ‘buy-in’ from catering and
management stakeholders in order for the interven-
tion to be successfully implemented. This was also
highlighted by the researchers who acknowledged
their flexibility and willingness to change as a crucial
facilitating factor. Flexibility and negotiation Catering stakeholders anticipated that their involve-
ment would impress the head office of their catering
company as staff will have the opportunity to apply
the knowledge and skills they gained on how to pro-
duce healthy menus after the study period and also
in future interventions. This long term potential
benefit garnered buy-in from catering stakeholders
and facilitated intervention implementation as they
were more invested in making the intervention a
success in their workplace. 2. Production work: Both managers and employees
perceived shift work to be a barrier to
implementation. This was due to the logistical
problems of arranging appointments for shift
workers outside standard office hours. However, it
emerged that it was the nature of production work 2. Production work: Both managers and employees
perceived shift work to be a barrier to
implementation. This was due to the logistical
problems of arranging appointments for shift
workers outside standard office hours. However, it
emerged that it was the nature of production work Flexibility and negotiation The researchers who collected data and were involved in
coordination and delivery of intervention elements were
adaptable to dynamic workplace environments which
facilitated implementation. This flexibility enabled the
researchers to successfully negotiate with workplace
managers on degrees of change that were agreeable to
all parties and ensured the study received organisational
support. Verbatim examples of this theme are included
in Table 4. Fitzgerald et al. BMC Health Services Research (2016) 16:139 Page 7 of 13 Table 4 Theme of ‘negotiation and flexibility’ and verbatim examples
Theme
Verbatim Examples
Negotiation and
flexibility
1. Flexibility: “You need to adapt and be understanding because schedules do change so you go in with your full schedule and you
mightn’t get all of them or people last minute can’t make it and you’re getting annoyed when you’re there on site waiting but out
on site things are changing constantly so you really have to adapt”. (Researcher 2 - follow-up stage)
2. Negotiation: “Changing down to nearly half, we just couldn’t, there would be uproar…we did a taste test, we put three plates
out one with what we serve now, one with what UCC wanted us to serve and something somewhere in the middle that we felt
we could serve and get away with, that’s the way we made our choice” (Occupational health, combined intervention site
baseline stage). “The breakfast option alright was something that you couldn’t change too much. I suppose from their side they were just afraid
that there would be a lot of backlash from the employees and there at the front line then dealing with it” (Researcher 2 - follow-
up stage)
3. High-level workplace management support: “I found it very, very hard to get product builders released for their sessions. That was a huge struggle for me, it’s the team leaders and they’re all about their metrics, they want to have, net efficiencies,
be on target” (Occupational Health—nutrition education site—follow-up stage). 1. Flexibility: “You need to adapt and be understanding because schedules do change so you go in with your full schedule and y
mightn’t get all of them or people last minute can’t make it and you’re getting annoyed when you’re there on site waiting but o
on site things are changing constantly so you really have to adapt”. (Researcher 2 - follow-up stage) 2. Workplace structures and cultures Production work: “There’s a big, discrepancy between the support staff and the people who work on the line, in that the
support staff have that freedom to, to go to these things” (Occupational health, nutrition education site—follow-up stage). 3.Organisational restructuring: “Those who are in charge they’d have the overall influence because they’re the ones bringing
in the stock and stuff, so they have to be behind it 100 %. Like if there was opposition from the management that could
hinder it” (Employee, nutrition education site—baseline stage). “Many employees they left the company and were moved to other departments, so it was hard to get them back for the last 2. Production work: “There’s a big, discrepancy between the support staff and the people who work on the line, in that the
support staff have that freedom to, to go to these things” (Occupational health, nutrition education site—follow-up stage). 3.Organisational restructuring: “Those who are in charge they’d have the overall influence because they’re the ones bringing
in the stock and stuff, so they have to be behind it 100 %. Like if there was opposition from the management that could
hinder it” (Employee, nutrition education site—baseline stage). “Many employees they left the company and were moved to other departments, so it was hard to get them back for the last
stage of the study but we got agreement from the managers in order to allow us to complete the last stage” (Researcher 3 -
follow-up stage). 4.Workplace culture: “Well it’s another concern, its more rural here, people are a bit more conservative about their food, I
mean we’ve been asked over the years for stuff like Panini’s, honestly, I’d give them a week and they just don’t go” (Catering
Manager, environmental site baseline stage). rather than the shift cycles that impeded
implementation. agree to all modifications. Catering stakeholders
were cautious when agreeing changes which resulted
in the cooked breakfast menu option not being fully
modified in the workplace. However, as previously
mentioned researchers overcame this by reaching
compromises on method of cooking, portion size
and reducing the number of days that chips were
available in the workplaces. p
3. Organisational restructuring: Conversely, a number
of workplace factors were identified as aspects that
impeded implementation. Two of the largest
workplaces (education and combined) underwent
major restructuring during the study. Workplace structures and cultures This involved
the relocation of a large number of employees from
both workplaces, which resulted in them being
ineligible to participate in the study as they were no
longer exposed to the intervention. As a direct result
of the restructuring, a large proportion of the
remaining employees changed shift patterns. In
order to deal with these effects researchers had to
liaise with management on how to best minimise
loss to follow-up and had to adapt elements of the
study to these changes. This involved researchers
creating an appointment schedule to facilitate
changes in shift work patterns to encourage em-
ployees to complete all stages of data collection. The
time it took to liaise with management regarding
restructuring changes had a direct impact on the time-
line of the study. Adjusting to the restructuring changes
and the delays in recruitment meant that data collection
timelines had to re-evaluated, however getting approval
from the management stakeholders for these readjust-
ments proved to be very time consuming. Workplace structures and cultures Individual workplace structures and cultures had an im-
pact on implementation. In workplaces where senior Fitzgerald et al. BMC Health Services Research (2016) 16:139 Page 8 of 13 Table 5 Theme of ‘workplace structures and cultures’ and verbatim examples
Theme
Verbatim Examples
Workplace structures and
cultures
1. Stakeholder buy-in: “We had really good contacts with HR, they helped with recruitment, they helped schedule some
participants…..that was probably the easiest site in terms of scheduling and recruiting…. if someone didn’t turn up all
I had to do was go downstairs and tell one of the HR people and they would actually go and get the employee”
(Researcher 1 - follow-up stage). 2. Production work: “There’s a big, discrepancy between the support staff and the people who work on the line, in that the
support staff have that freedom to, to go to these things” (Occupational health, nutrition education site—follow-up stage). 3.Organisational restructuring: “Those who are in charge they’d have the overall influence because they’re the ones bringing
in the stock and stuff, so they have to be behind it 100 %. Like if there was opposition from the management that could
hinder it” (Employee, nutrition education site—baseline stage). “Many employees they left the company and were moved to other departments, so it was hard to get them back for the last
stage of the study but we got agreement from the managers in order to allow us to complete the last stage” (Researcher 3 -
follow-up stage). 4.Workplace culture: “Well it’s another concern, its more rural here, people are a bit more conservative about their food, I
mean we’ve been asked over the years for stuff like Panini’s, honestly, I’d give them a week and they just don’t go” (Catering
Manager, environmental site baseline stage). Table 5 Theme of ‘workplace structures and cultures’ and verbatim examples
Theme
Verbatim Examples Table 5 Theme of ‘workplace structures and cultures’ and verbatim examples
Theme
Verbatim Examples
Workplace structures and
cultures
1. Stakeholder buy-in: “We had really good contacts with HR, they helped with recruitment, they helped schedule some
participants…..that was probably the easiest site in terms of scheduling and recruiting…. if someone didn’t turn up all
I had to do was go downstairs and tell one of the HR people and they would actually go and get the employee”
(Researcher 1 - follow-up stage). 2. Viability and intensity of interventions The design of the interventions also impacted how they
were implemented. The sustainability of the interventions
and the ability of workplaces to tailor the interventions to
meet the needs of their workplace facilitated implemen-
tation. The anticipated employee resistance to change in
response to the environmental modification impeded
implementation of the interventions. The intensity of the
interventions also affected implementation. The high-
intensity intervention (combined intervention) was well
received by employees. However, the low-intensity inter-
ventions (education and environmental) did not meet
employee expectations which impeded implementation. Verbatim examples of this theme are included in Table 6. 1. Sustainability of interventions: Intervention design
had impact on implementation. At the outset,
catering staff were apprehensive about implementing
environmental modification elements as they
anticipated it would cause a significant increase in
workload. However, it transpired that any extra
workload initially created dissipated once the
intervention was in place and as a result the study
was easier to maintain. Environmental modification
elements became part of the normal catering routine
within workplaces even after the study, with
workplaces sustaining elements. Similarly, the
environmental modification site maintained the 1. Sustainability of interventions: Intervention design
had impact on implementation. At the outset,
catering staff were apprehensive about implementing
environmental modification elements as they
anticipated it would cause a significant increase in
workload. However, it transpired that any extra
workload initially created dissipated once the
intervention was in place and as a result the study
was easier to maintain. Environmental modification
elements became part of the normal catering routine
within workplaces even after the study, with
workplaces sustaining elements. Similarly, the
environmental modification site maintained the 4. Workplace culture: According to the researchers
involved in data collection, the workplace culture
provided challenges during implementation. This
manifested itself particularly in the environmental
modification site, with the majority of employees
described as having ‘traditional’ eating habits. The
cooked breakfast menu options and side portion of
chips were described as part of the tradition of the
workplace. The expectation of poor uptake of the
interventions made catering stakeholders reticent to Fitzgerald et al. BMC Health Services Research (2016) 16:139 Page 9 of 13 Table 6 Theme of ‘viability and intensity of intervention design’ and verbatim examples
Theme
Verbatim Examples
Viability and intensity of
intervention design
1. Viability and intensity of interventions Information at a glance: “People are in a hurry so it was a perfect situation where you were rushing in and out you
could still see at a glance what your options were in terms of healthy choices” (Occupational health, nutrition education
site—follow-up stage). 4. Employee resistance to change: “The glazed loin of bacon, we took it off for 2 weeks and we had something like 300
common cards or something you know it’s like, ‘where is bacon’ because it would always be on a Monday or Tuesday”
(Catering manager, combined intervention site—follow—up stage). 4. Employee resistance to change: “The glazed loin of bacon, we took it off for 2 weeks and we had something like 300
common cards or something you know it’s like, ‘where is bacon’ because it would always be on a Monday or Tuesday”
(Catering manager, combined intervention site—follow—up stage). “I suppose from their side they were just afraid that there would be a lot of backlash from the employees and there at
the front line then dealing with it but to be fair when we spoke again with them there wasn’t too much backlash”
(Researcher 2 - follow-up stage). “I suppose from their side they were just afraid that there would be a lot of backlash from the employees and there at
the front line then dealing with it but to be fair when we spoke again with them there wasn’t too much backlash”
(Researcher 2 - follow-up stage). “I suppose from their side they were just afraid that there would be a lot of backlash from the employees and there at
the front line then dealing with it but to be fair when we spoke again with them there wasn’t too much backlash”
(Researcher 2 - follow-up stage). 5. Intervention intensity: “It’s not very regular, should I say and it’s not very intrusive, you know what I mean… it’s the
idea of, you know, getting weighed in once a week and kind of like the competition type thing” (Employee, environmenta
site—follow-up stage). 5. Intervention intensity: “It’s not very regular, should I say and it’s not very intrusive, you know what I mean… it’s the
idea of, you know, getting weighed in once a week and kind of like the competition type thing” (Employee, environmental
site—follow-up stage). Viability and intensity of interventions dietary behaviour, regardless of participation in the
study with all employees being exposed to the
intervention in the canteen. healthy default menu options, increased the number
of ‘chip free’ days per week in the workplace and
removed free-flowing sugar and salt from the can-
teen. The catering staff in the combined intervention
decided to keep elements that modified the nutri-
tional quality of food in terms of fat, saturated fat,
sugar and salt. 3. Information at a glance: Employees outlined how the
traffic light system enabled them to make informed
decisions with regards to healthy or unhealthy menu
options. It provided information at a glance in a fast-
paced environment which was particularly helpful to
production workers as their lunch times were very
restrictive. This visibility of the intervention was de-
scribed as a talking point among employees and they
discussed their clinical measurements, progress and
feedback with each other. Displays of nutritional in-
formation in the canteen and the daily email of
healthy options were considered effective. The traffic
lights created a social desirability response as em-
ployees were reluctant to choose a menu option that
was coded as red when they were eating in a group. It also emerged that since the study finished in the
workplaces, employees and catering stakeholders
found the absence of intervention very noticeable,
mainly the traffic light coding system and the nutri-
tional information that was displayed in the canteen. The design of the intervention in terms of its inclu-
sive and visible nature was perceived to be a key fa-
cilitator for successful implementation. 3. However, there was a perception among the
researchers that catering stakeholders in the
combined workplace found the initial
implementation of the intervention burdensome in
terms of extra workload. Researchers suggested that
this caused a delay in implementation at the outset
which was overcome through negotiation of
elements that were more feasible for the catering
staff to implement. p
2. Tailoring of interventions: The advantage of being
able to tailor the intervention to address certain
needs was also alluded to by the employees. An
employee being able to ‘pick and choose’ to engage
with certain elements was not an intended feature of
the study design. Viability and intensity of interventions Sustainability of interventions: “It was much easier than I thought it was going to be…I was a little bit scared at
the start of all the changes that would have to be made, but actually it was fine, it was fine, it was all quite
manageable” (Catering manager, environmental site—follow-up stage). 2. Tailoring of interventions: “Even though the study is over it still continued, there was no dramatic okay that’s done
go back to the old ways, pretty much there’s a lot of things that we kept on board” (Catering manager, combined
intervention site—follow-up stage). 3. Information at a glance: “People are in a hurry so it was a perfect situation where you were rushing in and out you
could still see at a glance what your options were in terms of healthy choices” (Occupational health, nutrition education
site—follow-up stage). 4. Employee resistance to change: “The glazed loin of bacon, we took it off for 2 weeks and we had something like 300
common cards or something you know it’s like, ‘where is bacon’ because it would always be on a Monday or Tuesday”
(Catering manager, combined intervention site—follow—up stage). “I suppose from their side they were just afraid that there would be a lot of backlash from the employees and there at
the front line then dealing with it but to be fair when we spoke again with them there wasn’t too much backlash”
(Researcher 2 - follow-up stage). 5. Intervention intensity: “It’s not very regular, should I say and it’s not very intrusive, you know what I mean… it’s the
idea of, you know, getting weighed in once a week and kind of like the competition type thing” (Employee, environmen
site—follow-up stage). 2. Tailoring of interventions: “Even though the study is over it still continued, there was no dramatic okay that’s done
go back to the old ways, pretty much there’s a lot of things that we kept on board” (Catering manager, combined
intervention site—follow-up stage). 3. Information at a glance: “People are in a hurry so it was a perfect situation where you were rushing in and out you
could still see at a glance what your options were in terms of healthy choices” (Occupational health, nutrition education
site—follow-up stage). 3. Viability and intensity of interventions BMC Health Services Research (2016) 16:139 Page 10 of 13 Page 10 of 13 Page 10 of 13 combined intervention workplace reported that
employees’ resistance to change was largely in
response to the removal of some of the unhealthy
options on the menu. This impeded the
implementation of the intervention slightly as
caterers were reluctant to introduce a further chip
free day that had been suggested during the
negotiation with the researchers. However, catering
stakeholders were determined to implement the
agreed intervention elements to an extent they
thought was feasible. The expectation of resistance
to change was one of the main reasons cited for
negotiating the degrees of change in the workplace. There was a perception among researchers that the
‘backlash’ was not as great as expected. Researchers
suggested that any resistance that occurred was due
to a small minority in the workplaces and the
catering company were capable of dealing with it. production work rather than restrictive shift cycles im-
peded implementation of a complex workplace dietary
intervention. Organisational restructuring caused delays to
the study timeline, attrition and disruptions to schedules. These barriers persisted throughout the study but were
eased by the flexibility and negotiation skills of the re-
searchers. The adaptability of the implementation team was
a vital facilitator for implementation and helped accommo-
date the impact of extensive organisational restructuring. Despite consensus in the literature that workplace
dietary process evaluations should be conducted concur-
rently with evaluations of outcomes, the current evi-
dence base is extremely limited [25]. However, findings
from this study are consistent with process evaluations
of other types of organisational interventions. The struc-
tural environment can act as a major barrier to imple-
mentation if it cannot tolerate the intervention that is
being implemented [34]. Previous research indicates that
contextual factors have significant influence on the im-
plementation of workplace interventions. Complexities
of the modern working environment including structural
changes, competing projects, employee turnover and
downsizing have all been outlined as potential barriers
to implementation [27, 29]. Workplaces are dynamic en-
vironments and their contexts cannot be controlled. The
flexibility and adaptability of the researchers were im-
portant factors that helped the study overcome context-
ual barriers [23]. 5. . Intervention intensity: Catering stakeholders and
employees in the education and environmental
workplaces felt that the study lost momentum
towards the end of the study period. Viability and intensity of interventions The
interventions implemented in the education and
environmental workplaces were low intensity by
design compared to the high intensity intervention
that was implemented in the combined workplace. Employees and catering stakeholders in the
education and environmental workplaces felt that
the interventions would have benefited from more
regular stages of data collection and suggested that
more emphasis should be placed on physical
measurements and weight loss to increase
intervention intensity. The low intensity
interventions delivered in these workplaces did not
meet employee expectations. Employees felt that
delays in data collection and long stages of follow-up
resulted in a loss of interest and focus in the study. The findings are consistent with research that suggests
stakeholder buy-in and supportive organisational cultures
facilitate implementation [23, 27, 29]. Managers perceived
benefits and personal interest in the study fostered their
buy-in and support which facilitated implementation. Stakeholder consultation and buy-in is critical for success-
ful implementation [34]. The implementation team openly
consulted with each other throughout recruitment, inter-
vention allocation and intervention implementation. This
consultation process was beneficial for the researchers
collecting data and coordinating and delivering the inter-
vention as they were able to assess the capacity and suit-
ability of each workplace for particular intervention
elements. The process also assisted in workplaces provid-
ing organisational support to the study. Supportive organ-
isational structures and systems are a key enabler of
successful implementation [34]. This study reported the
presence of strong organisational support from one of the
workplaces whereby the HR manager assisted in recruiting
and scheduling of employees for their appointments
which facilitated timely implementation. Viability and intensity of interventions This occurred naturally throughout
the study as employees reported that different
elements of the intervention worked for them, for
example, some employees found the health eating
chat table more beneficial to them compared to the
monthly group nutrition presentations. Employees
also appreciated that participation in the study was
open to all employees in the workplace, regardless of
job position. This inclusive study design which
allowed employees to adapt elements to meet their
own requirements was perceived as a key facilitating
factor for implementation by employees and
management stakeholders. The intervention created
scope to positively impact all employees in terms of 2. Tailoring of interventions: The advantage of being
able to tailor the intervention to address certain
needs was also alluded to by the employees. An
employee being able to ‘pick and choose’ to engage
with certain elements was not an intended feature of
the study design. This occurred naturally throughout
the study as employees reported that different
elements of the intervention worked for them, for
example, some employees found the health eating
chat table more beneficial to them compared to the
monthly group nutrition presentations. Employees
also appreciated that participation in the study was
open to all employees in the workplace, regardless of
job position. This inclusive study design which
allowed employees to adapt elements to meet their
own requirements was perceived as a key facilitating
factor for implementation by employees and
management stakeholders. The intervention created
scope to positively impact all employees in terms of The design of the intervention in terms of its inclu-
sive and visible nature was perceived to be a key fa-
cilitator for successful implementation. 4. Employee resistance to change: The potential for
employee ‘backlash’ in response to choice restriction
impeded implementation. Caterers anticipated that
the implementation of choice restriction may create
a sense of perceived powerlessness amongst
employees. They also anticipated employee ‘backlash’
in reaction to the introduction of chip free days and
reduced portion size. Some of these concerns were
both anticipated and realised concerns. The 4. Employee resistance to change: The potential for
employee ‘backlash’ in response to choice restriction
impeded implementation. Caterers anticipated that
the implementation of choice restriction may create
a sense of perceived powerlessness amongst
employees. They also anticipated employee ‘backlash’
in reaction to the introduction of chip free days and
reduced portion size. Some of these concerns were
both anticipated and realised concerns. The Fitzgerald et al. Discussion However, in practice, the
authors agree that employees should be made fully aware
of what the intervention entails at the outset. This study has several strengths and limitations. To
ensure rigour, Guba’s framework for ensuring trust-
worthiness in qualitative research was adhered to [36]. This framework proposes four criteria for assessing
trustworthiness; credibility, transferability, dependability
and confirmability. Credibility is concerned with asses-
sing the internal validity of the findings, ensuring they
are congruent with reality [36]. In an attempt to ensure
credibility, well established research methods were used. These methods included the use of random sampling
when appropriate, holding regular debriefing discussions
during data collection and triangulating findings from
different stakeholders. Transferability refers to the extent
to which findings can be generalised or applied to other
contexts [36]. These findings may be generalisable nation-
ally and transferable internationally as the workplaces in-
cluded are multi-national manufacturing companies with
similar worldwide structures and operations. Dependabil-
ity addresses the reliability of the study and whether or
not the same results would be achieved if the study were
repeated [36]. In this study dependability is concerned
with the repeatability of the methods [36, 37]. Both an in-
depth methodological description which reported exten-
sively on processes used and a comprehensive description
on how changing contexts affected the implementation of
interventions were provided. Based on the results of this study, it is vital that future
intervention teams consider individual workplace cul-
tures and structural changes during the development
and implementation of interventions. The effects of
structural changes need to be monitored regularly
throughout the study. Workplaces need to be able to
tailor the intervention to meet their own specific needs
with minimal effort [11]. Consultation with key stake-
holders should be an integral aspect of complex work-
place interventions prior to implementation and can
assist in considering the challenges of manufacturing
work and in assessing an organisations readiness for
change. Stakeholders need to be aware of the demands
of the study and researchers need to determine if the
workplace structure can tolerate all aspects of the inter-
vention. Understanding the feasibility of implementing
the FCW interventions will help researchers and work-
place stakeholders anticipate future barriers of imple-
menting multisite workplace dietary interventions. The fourth construct of confirmability is concerned
with the objectivity of the research [36]. Discussion This study aimed to establish what factors facilitated or
impeded implementation of complex workplace dietary
interventions. Four principal themes emerged; perceived
benefits of participation, negotiation and flexibility of the
implementation team, viability and intensity of interven-
tion design and workplace structures and cultures. Contextual factors were found to heavily influence im-
plementation. Tacit workplace cultures including ‘trad-
itional’ menu preferences and anticipated and realised
resistance to change prevented full-scale implementation
of the environmental intervention. The target-driven
culture of manufacturing workplaces impeded imple-
mentation as the researchers involved in data collection
experienced challenges in arranging appointments with
employees. Our
results
suggest
that
manufacturing Our findings are in line with Lewin’s theory of organisa-
tional change which suggests that sustained organisational
change is achieved by workplaces achieving an appropriate
balance between minimising restraining factors and pro-
moting facilitating factors [31–33]. Tacit organisational Fitzgerald et al. BMC Health Services Research (2016) 16:139 Page 11 of 13 Page 11 of 13 cultures such as resistance to change and fragmented
relationships between workplace stakeholders need to be
managed. Resistance to change is a key barrier to achieving
sustainable organisational change [34]. This stage can also
be referred to as the ‘Unfreezing’ stage of the Lewin’s theory
whereby organisations need to recognise the need to
change the current situation [31]. This was achieved
through initial consultations between the research team
and workplace stakeholders. The second stage is referred to
as the ‘Transition’ stage and involves the actual implemen-
tation of the intervention which should promote new be-
haviours,
values or attitudes. This
was achieved by
implementing the FCW interventions and in order to over-
come resistance, negotiation on degrees of change occurred
during the implementation process. Restrictive factors can
be overcome by key workplace stakeholders reinforcing the
benefits of participation and by negotiation and comprom-
ise to minimise negative internal politics. This step can also
be referred to as the ‘Re-freezing’ stage where the change
becomes fixed in the workplace culture of the organisation. Schein’s theory of organisational change is also reflected in
our results as such positive reinforcement and minimising
of restrictive facts can help the change to become embed-
ded in the workplace culture [31]. blinded to their interventions during the FCW trial, the re-
searchers were unable to clarify the employees’ expectations
of the different interventions. Discussion In this study, re-
searcher bias cannot be ruled out as some of the authors
were involved in the overall FCW study and were familiar
with participants. Efforts were made to remain as object-
ive as possible with researchers conducting interviews in
workplaces that they did not visit for data collection. Furthermore, there were a number of members of the
multidisciplinary FCW research team involved in the ana-
lysis and interpretation of findings. However, the inclusion
of respondent validation may have been useful as respon-
dents’ interpretation of emerging results can help refine
findings and strengthen conclusions. Consideration also needs to be given to employee ex-
pectations. Employees’ expectations of an intervention
can impact how it is implemented and received. The
control, education and environmental workplaces re-
ceived low intensity interventions and employees in
these workplaces felt that the momentum of the study
was lost over time. Employees had anticipated an inter-
active intervention that would be of high intensity with
more frequent physical assessments. This perceived loss
of momentum impeded implementation as employees’
interest in the study declined. As the employees were Acknowledgements The authors would like to sincerely thank the workplaces, management and
catering stakeholders and employees who were involved in the FCW study
and the process evaluation. Authors’ contributions All authors worked on the design of this study. SF, FG and CK were
responsible for data collection. SF and SMH were primarily responsible for
data analysis. SMH acted as the inter-rater during analysis and moderator of
the focus groups. All authors provided feedback on interpretation of analysis. SF drafted the paper and all authors provided feedback and approved the
final manuscript. References Ethical approval was granted by the Clinical Research
Ethics Committee of the Cork Teaching Hospitals in
Ireland, March 2013. 1. Capacci S, Mazzocchi M, Shankar B, et al. Policies to promote healthy eating
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CMAI: Catering Managers Association of Ireland; FCW: food choice at work
study; HR: human resources; IDA: Industrial Development Authority of
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guidance/ph6/chapter/3-Recommendations. Accessed 24th Feb 2015. Competing interests The authors declare that there are no conflicts of interest. Conclusion Cohesiveness between different
stakeholders within the workplace and between the imple-
mentation team (stakeholders involved in co-ordination
and delivery of interventions and researchers involved in
data collection and delivery of intervention elements) is
essential for successful implementation. Intervention im-
plementation within organisations is largely influenced by
contextual factors. To achieve organisational change, these
factors need to be carefully considered prior to implemen-
tation along with an assessment of readiness for change. This study provides an in-depth understanding of the im-
plementation context to further illuminate the findings of
the FCW study. Our results may also inform the imple-
mentation of future workplace dietary interventions for
the development of sustainable diet-related disease pre-
vention and provide an opportunity for scaling of similar
interventions for use in practice) for use in practice. Additional file 5: Topic Guide for Follow-up Focus Group. (DOC 33 kb)
Additional file 6: Topic Guide for Managers (Post implementation
stage). (DOCX 27 kb) Additional file 5: Topic Guide for Follow-up Focus Group. (DOC 33 kb)
Additional file 6: Topic Guide for Managers (Post implementation
stage). (DOCX 27 kb) Funding This work is supported by the HRB Centre for Health & Diet Research grant
(HRC2007/13) which is funded by the Irish Health Research Board and by the
Department of Agriculture, Fisheries and Food. This work was also
conducted as part of the HRB Scholar Programme in Health Services
Research under Grant No. PHD/2007/16. Research bursaries were awarded by
the Irish Heart Foundation and Nutrition and Health Foundation. Dr Sheena
Mc Hugh is funded by the Health Research Board Research Leader Award in
Diabetes (RL/2013/7). Received: 23 June 2015 Accepted: 19 April 2016 Received: 23 June 2015 Accepted: 19 April 2016 Conclusion The findings of this study can be used to support the
argument that process evaluations should be carried
out concurrently with effectiveness studies for work-
place interventions [25]. This study demonstrates how
process evaluations can be used to explore factors
that
may
influence
implementation
in
controlled
intervention studies and highlights the complexities
associated
with
implementing
complex
workplace Fitzgerald et al. BMC Health Services Research (2016) 16:139 Page 12 of 13 dietary interventions. Perceived benefits of participa-
tion, stakeholder buy-in and organisational support
are intrinsic facilitators of implementing workplace
dietary interventions. Flexibility and negotiation play a
pivotal role in overcoming the barriers of individual
workplace
cultures,
structures
and
resistance
to
change. Interventions also need to be adaptable as
the manufacturing companies need to tailor interven-
tions to meet specific structural and cultural require-
ments of their workplaces. Workplace stakeholders
play a central role in achieving organisational change
by
reinforcing
benefits
and
providing
fundamental
organisational support. Cohesiveness between different
stakeholders within the workplace and between the imple-
mentation team (stakeholders involved in co-ordination
and delivery of interventions and researchers involved in
data collection and delivery of intervention elements) is
essential for successful implementation. Intervention im-
plementation within organisations is largely influenced by
contextual factors. To achieve organisational change, these
factors need to be carefully considered prior to implemen-
tation along with an assessment of readiness for change. This study provides an in-depth understanding of the im-
plementation context to further illuminate the findings of
the FCW study. Our results may also inform the imple-
mentation of future workplace dietary interventions for
the development of sustainable diet-related disease pre-
vention and provide an opportunity for scaling of similar
interventions for use in practice) for use in practice. dietary interventions. Perceived benefits of participa-
tion, stakeholder buy-in and organisational support
are intrinsic facilitators of implementing workplace
dietary interventions. Flexibility and negotiation play a
pivotal role in overcoming the barriers of individual
workplace
cultures,
structures
and
resistance
to
change. Interventions also need to be adaptable as
the manufacturing companies need to tailor interven-
tions to meet specific structural and cultural require-
ments of their workplaces. Workplace stakeholders
play a central role in achieving organisational change
by
reinforcing
benefits
and
providing
fundamental
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interventions: Beyond traditional methods. Work Stress. 2007;21(4):348–62. 28. Cox T, Karanika M, Griffiths A, et al. Evaluating organizational-level work stress
interventions: Beyond traditional methods. Work Stress. 2007;21(4):348–62. 29. Nielsen K, Fredslund H, Christensen KB, et al. Success or failure? Interpreting
and understanding the impact of interventions in four similar worksites. Work Stress. 2006;20(3):272–87. 30. Heward S, Hutchins C, Keleher H. Organizational change—key to capacity
building and effective health promotion. Health Promot Int. 2007;22(2):170–8. 31. Batras D, Duff C, Smith BJ. Organizational change theory: implications for
health promotion practice. Health Promot Int. 2016;31(1):231-41 32. Siler-Wells G. An implementation model for health system reform. Soc Sci
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and we will help you at every step: Submit your next manuscript to BioMed Central
and we will help you at every step: 33. Weick KE, Quinn RE. Organizational change and development. Annu Rev
Psychol. 1999;50:361–86. • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
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Submit your manuscript at
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and we will help you at every step: 34. Burke K, Morris K, McGarrigle L. An Introductory Guide to Implementation: Terms,
Concepts and Frameworks. Dublin: Centre for Effective Services (CES); 2012. 35. Gale NK, Health G, Cameron E, et al. Using the framework method for the
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Res Methodol. 2013;13:117. 36. Guba E. Criteria for assessing the trustworthiness of naturalistic inquiries. ECTJ. 1981;29(2):75–91. 36. Guba E. Criteria for assessing the trustworthiness of naturalistic inquiries. ECTJ. 1981;29(2):75–91. 37. Shenton AK. Strategies for ensuring trustworthiness in qualitative research
projects. Educ Inf. 2004;22:63–75. 37. Shenton AK. Strategies for ensuring trustworthiness in qualitative research
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Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
http://dx.doi.org/10.1590/2175-623675528
1515 O Lugar de onde falamos, o Território que habitamos e
a nossa Problematização Neste texto, desejamos compartilhar a experiência de atuarmos
como pesquisadores1 em Psicologia Institucional, em uma escola muni-
cipal de Educação de Jovens e Adultos na cidade de Vitória (ES). Nessa
atuação, utilizando-nos de experimentações corporais-artísticas ins-
piradas nas obras de Lygia Clark e Hélio Oiticica, pudemos tecer mo-
mentos de produção de sentido, de modo a enfrentarmos o problema da
presença do niilismo nos corpos. Nesta pesquisa, atuamos segundo a perspectiva da pesquisa-in-
tervenção, para a qual toda investigação tem caráter político, na me-
dida em que deva se inserir em espaços onde haja embates de força e
enfrentamento das questões sociais atuais. Segundo Paulon e Romag-
noli (2010), na pesquisa-intervenção se assume o compromisso social
do pesquisador, que investiga implicado com os efeitos não neutros de
sua atuação, prescindindo da dicotomia sujeito x objeto. Romagnoli
(2014) ainda diz que a postura crítica, o combate ao reducionismo e a
busca pela desnaturalização são características das ações do pesquisa-
dor-interventor. Na pesquisa-intervenção, portanto, o pesquisador não
vai a seu campo de pesquisa apenas para coletar dados, adotando uma
postura laboratorial, eximindo-se de interferir ou ser contaminado pelo
que se passa na realidade: o pesquisador-interventor atua implicando-
-se com as questões que lhe tocam, buscando problematizá-las e procu-
rando construir soluções que as transformem. Partindo desta perspectiva, é que adotamos a cartografia como
metodologia de trabalho específica de pesquisa-intervenção. A carto-
grafia compreende a pesquisa como ação num campo de imanência,
onde o cruzamento de distintas linhas de força (culturais, midiáticas,
biológicas, afetivas, tecnológicas etc.) compõe uma realidade singular. Deleuze (1997), diz que cartografar é criar uma listagem ou mapa de
afetos experimentados em um deslocamento, o qual revela os impasses,
aberturas e devires de uma realidade. Para o autor, o trajeto percorri-
do na cartografia “[...] se confunde não só com a subjetividade dos que
percorrem um meio, mas com a subjetividade do próprio meio” (Deleu-
ze, 1997, p. 73). Destarte, a cartografia também ultrapassa a dicotomia
objetividade x subjetividade. Por isso, compreendemos como Barros e
Kastrup (2010), que cartografar implica acompanhar processos já em
curso, de modo que o cartógrafo se lance em um território disposto a
sempre (re)começar pelo meio, entre pulsações. Assim, Barros e Bar-
ros (2013) lembram que a cartografia porta uma dimensão clínica, pois
ela implica ações capazes de transformar o campo de forças em que é
realizada. Educação Antiniilista: corpo e arte
produzindo sentido na escola Fernando YonezawaI
Márcia CuevasI IUniversidade Federal do Espírito Santo (UFES), Vitória/ES – Brasil IUniversidade Federal do Espírito Santo (UFES), Vitória/ES – Brasil RESUMO – Educação Antiniilista: corpo e arte produzindo sentido na
escola. Neste texto objetivamos compartilhar a experiência de atuarmos
como pesquisadores-interventores junto a uma escola municipal de Educa-
ção de Jovens e Adultos da cidade de Vitória (ES), onde estivemos realizan-
do oficinas corporais-artísticas inspiradas nas obras de Lygia Clark e Hélio
Oiticica. Utilizando-nos do método cartográfico de pesquisa em Psicolo-
gia Institucional, também baseados nos conceitos filosóficos de Deleuze,
Guattari e Nietzsche, problematizamos o niilismo presente na educação e
nos corpos dos estudantes, assim como buscamos afirmar a possibilidade
de produzir sentido no processo educacional a partir destas intervenções
corporais-artísticas. p
Palavras-chave: Educação. Arte. Corpo. Produção de Sentido. ABSTRACT – Antinihilist Education: body and art producing sense in
school. In this text we aim to share the experience of acting as researchers-
-interveners at a municipal school of Youth and Adult Education in the city
of Vitória (ES), where we were performing body-artistic workshops inspired
by Lygia Clark’s and Hélio Oiticica’s works. Using the cartographic method
of research in Institutional Psychology, also based on the philosophical
concepts of Deleuze, Guattari and Nietzsche, we problematize the nihilism
presented in the education and in the students’ bodies, as well we try to
affirm the possibility of producing sense in the educational process from
these corporal-artistic interventions. p
Keywords: Education. Art. Body. Sense Production. Educação Antiniilista O Lugar de onde falamos, o Território que habitamos e
a nossa Problematização Logo, a cartografia não retrata a realidade, mas a produz, en-
quanto nela age. Desta maneira, nossa pesquisa nesta escola tem se realizado a
partir da participação ativa em reuniões de gestão, encontros de forma-
ção e planejamento docente, aulas e seminários especiais. É preciso di-
zer que, nesta escola, onde se adota os princípios da educação popular Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1516 Yonezawa; Cuevas de Paulo Freire como norteadores, os educadores trabalham em dupla,
procurando transversalizar seus conteúdos de áreas de conhecimento
distintas. Com o intento de produzir sujeitos críticos, a escola sempre
aborda temas políticos em sala de aula, como os problemas ambientais
e a violência contra a mulher. Ademais, tendo como um de seus gran-
des princípios ético-políticos promover a inclusão de todos aqueles que p
p
p
p
q
q
– em suas palavras – tiveram o direito à educação negado, essa escola
se configurou de modo a ir atender localmente os bairros onde fossem
encontradas demandas de educação para jovens e adultos. A institui-
ção atende nos três turnos do dia e se espalhou por 14 diferentes locais
da cidade, colocando-se a trabalhar em espaços e contextos distintos
uns dos outros. Têm-se, por exemplo, turmas dentro da associação de
catadores de material reciclável, ou num centro de convivência da ter-
ceira idade, ou ainda dentro do Centro de Referência à População de
Rua da cidade. Por isso, seu público é bastante diverso, de modo a haver
salas em que jovens de 19 anos estudam com idosos de 80 anos, bem
como se acolhem também adolescentes vinculados a medidas socioe-
ducativas, jovens portadores de necessidades especiais, moradores de
rua e, como diz a escola, todos os que foram afastados da educação pelo
fato do sistema pedagógico tradicional não ter dado conta de suas espe-
cificidades. As aulas têm 3 horas de duração e acontecem de segunda
à quinta-feira, pois as sextas-feiras são inteiramente dedicadas à for-
mação de seus educadores, que estudam temas como sexualidade, cul-
tura afro-brasileira e produção de saúde na sociedade contemporânea. Buscando práticas capazes de trabalhar na transversalidade de tantas
especificidades e necessidades, a escola também tem como princípios
o dialogismo e a compreensão de que os tempos de aprendizagem são
sempre diversos. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1517 O Lugar de onde falamos, o Território que habitamos e
a nossa Problematização Com efeito, tínhamos a
impressão de que, para os estudantes, não compreender algo denuncia-
ria sua suposta incompetência ou ignorância, condições absolutamente
condenáveis dentro da lógica da escola clássica: o não saber é o crime
maior que marca todos os corpos escolarizados. Por isso, concordamos
com a hipótese dos educadores da escola, mas consideramos ainda que
também constitui o arranjo de fechamento afetivo dos estudantes toda
a produção de modos de vida do capitalismo contemporâneo, caracteri-
zada por uma sofisticada espécie de niilismo. Conforme Guattari (1990,
p. 8), é a relação da subjetividade com “[...] sua exterioridade – social,
animal, vegetal, cósmica – que se encontra assim comprometida numa
espécie de movimento geral de implosão e infantilização regressiva”. Nas situações que descrevemos, sentíamo-nos num terrível im-
passe de diversas ordens: afetiva, pedagógica, política, ética etc. Víamos
que as boas intenções da educação popular, baseada em dialogismo,
democracia e inclusão do universo existencial dos estudantes não ga-
rantiam a sensibilização dos corpos e a produção de pensares engaja-
dos. Evidentemente, não se tratava de julgar a posição dos estudantes
e impor-lhes outro modo de pensar supostamente melhor; tampouco
parecia plausível sermos espontaneístas, crendo que seria suficiente a
retirada do autoritarismo do educador para que os estudantes se mos-
trassem interessados. “O que nos incomodava era que, renunciando ao
juízo, tínhamos a impressão de nos privarmos de qualquer meio para
estabelecer diferenças entre existentes, entre modos de existência,
como se a partir daí tudo se equivalesse” (Deleuze, 1997, p. 153). Logo, seria ingênuo, ainda, confiar que não termos temas plane-
jados tornaria a aula mais democrática e pior seria ignorar que existem
temas urgentes a serem levados para a sala de aula, como a problemati-
zação do racismo e da violência de gênero. Ora, Deleuze (2006) diz que
aprender algo implica em enfrentar um campo problemático, forçar o
pensamento a pensar. Algo só é aprendido sob diversas tonalidades afe-
tivas, envolvendo a sensibilidade e a experiência do encontro do corpo
com os afetos da ideia em questão. “Mas, em sua primeira característi-
ca, e sob qualquer tonalidade, ele só pode ser sentido” (Deleuze, 2006, p. 203), destaca o filósofo sobre aquilo que nos força a pensar. Quer dizer,
pensar é sempre pensar de um novo modo, mas a partir de um tensio-
namento desconfortante da sensibilidade. O Lugar de onde falamos, o Território que habitamos e
a nossa Problematização Neste sentido é que aposta em não punir ou excluir os
estudantes, de forma a insistir no diálogo como meio de transformação
dos efeitos que a violência e a exclusão social e pedagógica deixam nos
corpos. Neste território singular é que, semanalmente acompanhando a
dupla de Geografia e Artes em uma das turmas, víamos os estudantes
com idades entre 16 a 19 anos, sempre se mostrando extremamente de-
sinteressados pelos temas trabalhados, ainda que os educadores se fi-
zessem afetuosos, preocupados em dialogar, convidando-os a compor
a aula com elementos de suas vivências. Neste trajeto de participação
em aulas, viemos notando grande dificuldade de se trabalhar, especial-
mente com os jovens, temas que pediam um deslocamento dos conteú-
dos disciplinares comuns. Todas as vezes em que propúnhamos discutir
temas artísticos ou políticos, sentíamos como se aparecesse um muro
de indiferença ou rechaço entre nós e os estudantes. Sentíamos que, nos
corpos dos jovens, havia um fechamento para toda diferença. Diante
do que estranhavam, adotavam a postura de ignorar ou simplesmente
afastar: começavam a mexer em seus telefones celulares, ou deitavam
a cabeça sobre os braços cruzados em cima da mesa, recusando-se a
dialogar conosco e com os professores. Não se sentiam impelidos a per-
guntar, ou a nos questionar e demonstravam negação e desgosto; afetos Educação Antiniilista que, sem expressar rebeldia, apenas paralisavam as relações. Quando
não compreendiam algo, ou eram intensamente afetados, repeliam o
que os afetava. No limite, refugiavam-se sob o teto da sagrada opinião
pessoal, falaciosa instituição pseudodemocrática do capitalismo. A re-
jeição amarga se direcionava, pois, a tudo o que era novo, tinha caráter
artístico, ou então vinha questionar suas certezas. que, sem expressar rebeldia, apenas paralisavam as relações. Quando
não compreendiam algo, ou eram intensamente afetados, repeliam o
que os afetava. No limite, refugiavam-se sob o teto da sagrada opinião
pessoal, falaciosa instituição pseudodemocrática do capitalismo. A re-
jeição amarga se direcionava, pois, a tudo o que era novo, tinha caráter
artístico, ou então vinha questionar suas certezas. No tocante a estas questões, circula na escola a ideia de que os es-
tudantes jovens viriam muito marcados pelos ranços da escola tradicio-
nal conteudista e por isso reagiriam desse modo. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1518 O Lugar de onde falamos, o Território que habitamos e
a nossa Problematização Ainda nas palavras críticas
de Deleuze, a imagem dogmática do pensamento faz crer que haja uma
boa vontade inerente ao pensador e, portanto, bastaria que se ativasse
essa boa vontade para que o pensamento fosse capaz de pensar. Mas, Yonezawa; Cuevas dentro desta imagem do pensamento, pensar é apenas reconhecer, isto
é, pensar é fazer recognição, refletir a imagem de uma ideia pronta e
essencial tomada como verdadeira e primordial. dentro desta imagem do pensamento, pensar é apenas reconhecer, isto
é, pensar é fazer recognição, refletir a imagem de uma ideia pronta e
essencial tomada como verdadeira e primordial. Ora, de certa maneira, também não seria essa a crença pedagó-
gica da escola? Preocupada em não culpabilizar os estudantes, não es-
tariam, por outro lado, caindo numa espécie de mito do bom selvagem
do pensamento? Lembremos, pois, que uma verdade é sempre histórica
e social, uma verdade só é verdade quando já assentada sobre modos
de relação e pensamento dominantes. Então, fazer do pensamento uma
atividade de recognição derivada de uma boa vontade pressuposta é
torná-lo dócil, moça recatada, capaz apenas de reiterar verdades ins-
tituídas, repetir jargões, conteúdos ou fórmulas esvaziadas de sentido,
da força de criação e da potência de pensar diferentemente. Faz-se aí
um pensamento respondente, que só pensa respostas esperadas sem
enfrentar problemas, sem ser tensionado. “É um preconceito infantil,
segundo o qual o mestre apresenta um problema, sendo nossa tarefa
resolvê-lo e sendo o resultado desta tarefa qualificado de verdadeiro ou
de falso por uma autoridade poderosa” (Deleuze, 2006, p. 228). Quer dizer, a imagem clássica do pensamento perfaz uma con-
cepção de sujeito infantilizado e passivo. Não à toa, quando se falava
de conteúdos disciplinares tradicionais em sala de aula, víamos os es-
tudantes engajarem-se em realizar as atividades preparadas pelos edu-
cadores. O rechaço ou amargor só se expressava quando se deparavam
com imagens, tarefas, experiências desconhecidas, deslocantes. Trata-
-se, pois, de vermos que a imagem dogmática do pensamento impregna
a sensibilidade dos estudantes, bem como é mais ou menos reproduzi-
da pela escola, na intenção de reverter a culpabilização produzida pela
educação tradicional de onde, na maioria das vezes, vieram transferi-
dos estes estudantes. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1519 Niilismo nos Corpos e na Educação Tão logo, é preciso esclarecer o que quisemos dizer quando in-
dicávamos a presença do niilismo na postura afetiva dos estudantes. Então, sigamos a Nietzsche para compreendermos a concepção de nii-
lismo. Nas palavras do filósofo, o niilismo é uma forma de produção de
tipos de vida, que está ligada diretamente a valores morais-ascéticos e
a uma modulação das forças de uma cultura e dos corpos. O niilismo é
o valor dado à vida a partir do que Nietzsche (2009) chama de vontade
de nada, vontade de não mais lutar, ou criar, vontade de não mais de-
sejar, não mais apropriar-se de realidade, vontade de que o fim venha
logo, ou de que tudo no mundo esteja terminado, sem mais movimento
que implique tensionamento de força. Quanto ao conceito de força, é
Deleuze (2001) quem melhor o esclarece, a partir de sua leitura da obra
nietzschiana: força é tudo o que age de modo a apropriar-se de uma
quantidade de realidade. Destarte, no niilismo, encontra-se minguada
a vontade de ampliar a realidade ou de potencializar a vida, estado de
forças que se justifica sobre princípios que valoram o amansamento e o
acanhamento das forças. Trata-se, diz Nietzsche (2009), de valores que
só podem advir de homens cansados, cujo estado de forças corporais é
doentio e enfraquecido e, por isso, não suportam ver corpos nos quais
as forças estejam vigorosas, desejosas por mais força, mais realidade. Numa cultura niilista, os corpos se encontram sob a modulação carac-
terística de uma vida que definha e chega ao fim, uma constituição de
forças na qual a vontade se volta contra a vida (Nietzsche, 2009), ou seja,
contra si própria. Portanto, o niilismo é um estado de negação da vida. “A vida mesma é, para mim, instinto de crescimento, de duração,
de acumulação de forças, de poder: onde falta a vontade de poder, há
declínio” (Nietzsche, 2007, p. 13). Para o filósofo, o niilismo quer con-
servar o que já está em tempo de perecer, quer defender o enfraqueci-
mento, em detrimento das forças expansão e potencialização da vida. Desse modo, se faz do nada um valor, se deseja que a vida seja medida
pelo nada, isto é, por uma abstração, uma noção imaginária; ou então
se quer que a vida se iguale a valores supremos, inscritos nas leis de um
mundo do além, um mundo metafísico. O Lugar de onde falamos, o Território que habitamos e
a nossa Problematização É preciso considerar, por isso, que o desgosto des-
tes estudantes é efeito da forma clássica de educação, na qual se viola
também as potências do corpo, a partir de intensa individualização,
infantilização e produção da sensação de incapacidade. Partimos, por-
tanto, da posição de que termos esta compreensão, embora explique,
não legitima o estado de anestesia e fechamento dos corpos, pois se tra-
ta de uma modulação afetiva extremamente despotencializante, tanto
para os próprios estudantes, quanto para nós que, junto dos professo-
res, estávamos no lugar de educadores. Assim, foi tomando o desafio de
produzir algum estremecimento nas sensibilidades presentes em sala
de aula, que nossa pesquisa-intervenção passou a estar focada na re-
lação entre arte, corpo e produção de sentido na educação, apostando
em realizar experimentações corporais-artísticas nos espaços de aula. Apresentado o território que habitamos e o problema que nos mo-
biliza, traremos, a seguir, os conceitos filosóficos em que embasamos
nossas reflexões nesta pesquisa e, mais adiante, compartilharemos
uma destas experimentações realizadas junto à escola, nesta turma,
onde acompanhamos os educadores de Geografia e Artes. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1519 Educação Antiniilista Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1520 Niilismo nos Corpos e na Educação “Mas não se diz ‘nada’: se diz
‘além’; ou ‘Deus’, ou ‘a verdadeira vida’...” (Nietzsche, 2007, p. 14). Dito de
outra forma, o niilismo deforma a vida, prefigura o absurdo de identifi-
cá-la a valores que a tornariam mais verdadeira, mais digna, mais vida
do que ela mesma em seu movimento próprio de criação de si, como
se esse movimento mesmo fosse sujo, vil e, por isso, precisasse ser pu-
rificado, corrigido e tornado inofensivo. “O niilismo começa com um
deslocamento do centro de gravidade da vida em direção a uma outra
esfera que não ela mesma” (Pelbart, 2013, p. 94). O problema, contudo
é que, denuncia Nietzsche (2007, p. 16), “[...] a vontade de fim, a vontade
niilista quer alcançar o poder” e, para tanto, inventa verdades e virtudes
últimas, eternas, ou mesmo primordiais, imemoriais, às quais os ho-
mens deveriam se submeter. Notemos, aqui, que esta busca à verdade Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1520 Yonezawa; Cuevas presente no niilismo é encontrada como fundamento da imagem dog-
mática do pensamento, de que falamos anteriormente. presente no niilismo é encontrada como fundamento da imagem dog-
mática do pensamento, de que falamos anteriormente. A partir do momento em que cria valores a serem impostos, o nii-
lismo, de modo sub-reptício crê na ausência de valor da vida (Nietzsche,
2008), na impossibilidade de haver valores que possam afirmá-la. En-
tão, o niilismo se mostra sempre ambíguo e adulterante: por um lado,
ele descredita qualquer valor; principalmente descrê que haja sentido
na própria existência da vida; por outro, ele impõe valores e sentidos
forjados a partir desta primeira noção. Contudo, para Nietzsche (2008),
existem dois tipos de niilismo: primeiramente, há um niilismo passivo,
esse propriamente resultante do sentido de enfraquecimento e deca-
dência das forças e da vontade; mas, a partir de outra modulação vital,
haveria ainda um niilismo ativo, o qual duvida de todos os valores até
agora criados, pois vê muito bem que foram criados para e por um tipo
de homem, tendo-o como centro e como medida. Neste caso, o niilismo
viria justamente denunciar que a existência dos sentidos vistos como
presentes na essência das coisas mundanas não seria mais do que pro-
jeção de nossa própria vontade, a qual colocaria nas coisas o valor que
bem lhe convém para a conservação do tipo de vida por nós cultivada. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1521 Niilismo nos Corpos e na Educação Então, a diferença entre os dois niilismos está tanto no sentido, quanto
no sentimento envolvido. No niilismo passivo, o sentido de que nada
vale vem antes de tudo e, com isso, não se encontra sentido na vida a
não ser nos valores morais, diga-se, no dogmatismo. Advindo dos sen-
timentos de fraqueza de corpo, da falta de gosto, do amargor e recha-
ço perante a vida, o niilismo passivo resolveria a condição moribunda
criando mundos sem força, tensões e problemas, “[...] de modo que tudo
se refresca, cura, apazigua, entorpece [...]” (Nietzsche, 2008, p. 37). Já no
niilismo ativo, o sentido de ausência de valor vem como efeito da desco-
berta de que todos os valores são criados para afirmar um tipo de vida
desejado; sendo assim, todos os valores se tornam passíveis de serem
derrubados, para que sempre um novo valor possa emergir e vigorar,
para afirmar a vida que germina, quer nascer e fortalecer-se: os senti-
mentos que o constituem são de alegria, potência e vigor. Ora, a descrença e desconfiança nos valores é tão niilismo quan-
to a crença fanática num valor final e universal (Nietzsche, 2012). Em
outras palavras, o niilismo se revela tanto na impetuosa exigência de
certeza da ciência, quanto na exigência de apoio, de suporte, típica do
“instinto de fraqueza” (Nietzsche, 2012, p. 214), o qual se agarra aflito a
misticismos dogmáticos, porque se sente incapaz de criar valores. Tan-
to em um, como em outro, “[...] se encontra o nada por trás de todos os
ideais do ser humano” (Nietzsche, 2006, p. 81); e, no entanto, simulta-
neamente, encontrar este nada é justamente o primeiro passo de um
niilismo ativo, o qual se sente capaz de, por consequência desse (des)
encontro, levar a cabo a força do artifício e, desta maneira, criar novos
valores. No niilismo ativo, um inicial pessimismo ante os valores dados
se transforma em confiança na força inventiva da vida. “Por um lado, o
niilismo é um sintoma de crescente fraqueza, por outro, de força ascen-
dente” (Pelbart, 2013, p. 101). Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1521 Educação Antiniilista Por isso, a saída ao niilismo não estaria nem na forma da crendice
típica de religiosos e maus cientistas, nem no pessimismo dos depres-
sivos e adoentados. Niilismo nos Corpos e na Educação Vemos, pois, que o niilismo passivo falseia a vida,
inverte a condição de forças que permite criar mais vida, colocando a
impotência acima do sentido de força. A depreciação niilista da vida e
sua “[...] necessidade de negação da ‘vontade’” (Nietzsche, 2006, p. 75)
constituem uma vida dócil e peçonhenta, que se contradiz a si própria,
valorando e elevando o sentido de desprezo, enquanto rebaixa e recha-
ça as forças de criação. Ora, são estas linhas componentes de um niilismo passivo que
sentimos passar pelos corpos dos estudantes. Ressaltemos, porém,
que entendemos que estávamos diante de uma composição de forças
específica, histórica, cultural, que constituía os corpos daquele modo,
incluindo-se, aí, os nossos próprios; ou seja, não se trata de essencia-
lizar o niilismo nos corpos dos estudantes. Estes, ao se negarem a co-
nectar-se com as aulas focadas em temas artísticos ou políticos, além
de expressarem desprezo e amargor em seus rostos e corpos deitados
sobre as mesas, em nenhum momento mostravam revolta ou crítica. Os
educadores, por sua vez, ao compreenderem apenas reflexivamente de
onde advinham essas posturas, não ultrapassavam muito o sentido de
benevolência para com os desvalidos que Nietzsche denuncia ser sinto-
ma típico presente no niilismo. Os educadores de Artes e Geografia que
acompanhávamos, mesmo assim, diziam se sentir muito impotentes e
sem saber o que fazer diante desta composição de forças na sala de aula. Em ambos, assim como por nosso corpo, parecia passar apenas a sensa-
ção passiva de que é preciso continuar apesar de tudo, afinal, esse é nosso
fardo de educadores brasileiros. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1522 Embate: corpos passando, corpos (trans)passados Então, em diálogo com o educador de Artes, dissemos estar muito
incomodados com o amargor dos estudantes, o qual fazia com que as
aulas, às segundas-feiras pela manhã, parecessem eternas e entedian-
tes, como se o tempo se arrastasse com muito peso, estranhamente de
modo idêntico ao que todos nós já sentimos em nossos corpos ao fre-
quentarmos as aulas disciplinares das escolas tradicionais; porém, so-
bretudo nos incomodava que estivéssemos passivos. Diante disso tudo,
decidimos criar uma experimentação corporal-artística inspirada nas
obras de Lygia Clark e Hélio Oiticica, que tinha o intento de fazer estre-
mecer a opacidade afetiva dos estudantes e instigar as suas sensibilida-
des. Assim, neste trecho a seguir, apresentamos partes de nosso diário
de campo, para compartilhar a experiência da proposição desta oficina. Tivemos a ideia de construir um túnel de sensações e, para tanto,
convidamos o educador de Artes a compô-lo junto conosco. Ele topou
sorridente e, ante a necessidade de prepararmos todo o aparato estético
do túnel para uma aula de segunda-feira, passamos uma tarde de sába-
do toda juntos, em uma das salas do espaço em que escola mantinha
aquela turma, empilhando carteiras, colando tecidos sobre elas para Yonezawa; Cuevas formar o túnel, pendurando garrafas, espalhando materiais no chão,
colando cartazes. O túnel que montamos com tecidos marrons e vermelhos possuía
cinco fases, separadas também por tecidos. Cada fase tinha pendurada,
em suas paredes, questionamentos provocativos para serem lidos. Ha-
via também um áudio repetitivo com uma doce voz feminina recitando
tais frases. Na primeira parte do túnel, o chão foi coberto com muitos
balões coloridos; na segunda, bolinhas de gel roxas e alaranjadas, do ta-
manho de bolinhas de gude, forravam o piso; na terceira cabine, pendu-
ramos no teto diversas garrafas plásticas transparentes com água colo-
rida de diferentes cores (roxo, amarelo, rosa, verde); na quarta saleta do
túnel, dispusemos três grandes mapas, um do Estado do Espírito Santo,
outro do Brasil e outro do mundo. No mapa do Espírito Santo colamos
fotos de locais turísticos (praias, cachoeiras e prédios históricos) que
se localizam bem próximos a Vitória, passíveis de serem visitados com
uma passagem de ônibus urbano. Já na quinta e última parte, cobrimos
o chão com gel azul e grãos de feijão. As frases que dispusemos foram
as seguintes: - Qual o seu sonho na vida? - O que você mais deseja? Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1523 Embate: corpos passando, corpos (trans)passados - Quais lugares você tem vontade de conhecer? - O que você quer dar para as pessoas e para o mundo? - Você tem paixão pelo que? - Para que serve tanto desânimo e desgosto? - O que você quer da escola? - O que você quer dos professores? - Esta escola é igual a todas? - Aqui você é tratado do mesmo jeito que em outras escolas que
você esteve? - Aqui você é tratado do mesmo jeito que em outras escolas que
você esteve? Essas frases têm relação com sensações que tínhamos junta-
mente aos estudantes. Sentíamos, por exemplo, que esses jovens, ao se
manterem em posturas arredias, não percebiam muitas vezes o quan-
to, nesta escola, eram tratados de modo muito mais atencioso e afetivo,
sem a típica massificação e indiferença da maioria das escolas. Tam-
bém percebíamos que, devido ao não acesso a certas informações e à
restrição de circulação que invisivelmente são impostas às pessoas de
classes sociais baixas – das quais fazem parte estes estudantes –, muitos
deles sequer tinham saído dos bairros onde residiam e estudavam, não
podendo, então, passear e conhecer lugares capazes de potencializar o
corpo ou trazer novos conhecimentos. Por isso é que dispusemos, sobre
o mapa do Espírito Santo, paisagens de belos locais próximos a Vitória. Notando estas condições concretas de (im)possibilidade é que compre-
endemos que o niilismo não é produzido psicologicamente apenas, mas
também como composição e agenciamento de corpos, de espaços, de
circulação e de fluxos. O niilismo como cultura do enfraquecimento
dos corpos é, sobretudo, constituído como agenciamento de realida- Educação Antiniilista de. Alguns dos meninos desta escola mal podiam sair de seus bairros
porque, ao circularem por outras regiões, estavam ameaçados de mor-
te pelos atores do tráfico de drogas. Mas, mais que isso, uma pesada
atmosfera de ausência de possibilidades para a vida parecia cair sobre
seus corpos e modos de viver. Percebíamos que tais clausuras subme-
tiam os estudantes a passarem seus dias apenas sendo impregnados pe-
los discursos e imagens idiotizantes da TV, ou pelos novos movimentos
cristãos-evangélicos, os quais, embora se tornem comumente a opção
possível de encontros sociais e lazer, inundam as comunidades perifé-
ricas de um discurso conservador, escravizante e moral. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1524 Yonezawa; Cuevas Yonezawa; Cuevas Ao chegarmos perto da porta, Madalena sorria muito, vibrante e
querendo entrar logo. Dulce ficou pelo meio do corredor: Você não vem,
não?! Perguntamos em tom provocativo. Eu não! ela respondeu sorrin-
do e encolhendo o corpo. Já sentíamos nela uma mistura de medo com
uma crescente vontade de saber do que se tratava. Perguntamos para
Madalena se estava com medo. Agora estava mais agitada, tinha um
pouco de medo também, mas sorria muito. Contraiu o corpo e os bra-
ços em direção ao peito e disse Ai!. Seguramos sua mão, abrimos a porta
até a metade. Dissemos Entra!. Entrou e logo começou a vibrar num es-
tado emocional muito diferente. Seu rosto sorria: estava compenetra-
da, olhava para baixo, para os balões, um sorriso muito discreto. Era
possível sentir como estava emocionada. Ficou um bom tempo com os
balões, chutando-os e brincando com seus pés. Também começávamos
a nos emocionar. Parecia que a coisa estava funcionando, que estava
afetando. Deixamos que ela curtisse os balões por alguns minutos. Se-
guramos sua mão e a levamos para a cabine das bolinhas. Ela novamen-
te fixou o olhar, parada. Dissemos que podia pisar. Pisou com as dificul-
dades de equilíbrio que seus limites impunham, mas pisou. Pegamos
algumas para ela ter nas mãos. Tudo se passava em muito silêncio, mas
era possível sentir a vibração, os afetos passando por ela, por nós. Passamos para as garrafas, ela apenas as olhou. Parecia gostar das
cores, parecia tocada. Quando chegamos aos mapas, fomos explicando
quais lugares eram aqueles das imagens. Perguntamos se ela conhecia
algum lugar e ela respondeu que não. Logo passamos para a cabine do
gel. Dissemos que ela podia pisar também. Ela pisou, encolheu-se um
pouco, mas ficou saboreando a sensação. Ajudamos a limpar os pés na
toalha que havíamos deixado no fim do túnel e, na saída, perguntamos
o que ela sentiu. Mostrou com as mãos, fazendo gestos circulares, de
baixo para cima e para frente sobre seu peito. Dos seus fonemas qua-
se ininteligíveis, só compreendemos mo no meio da primeira frase. Ela
estava com os olhos cheios de lágrimas. Na segunda frase, disse clara-
mente, amor. Pedimos que repetisse o que disse primeiro, novamente
não compreendemos e perguntamos Emocionada? Disse sim com a ca-
beça. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1525 Embate: corpos passando, corpos (trans)passados Pois bem, na manhã em que iriamos experimentar nosso túnel,
estávamos apreensivos, ansiosos: sabíamos que aquilo tudo era uma
grande provocação, uma tentativa de escoriar impiedosamente uma
constituição afetiva e corporal. Tudo podia ser hostilizado, ser tomado
como coisa de playboy, inclusive pela própria escola, caso quisesse dar
lugar a um discurso rigidamente pautado na valorização da identidade
de classe social. Nossa intenção, contudo, não era reafirmar uma dita
identidade de classe ou origem popular, mas trazer ampliações de rea-
lidade, potencializações produzidas por encontros com o insólito, com
outra espécie de não sentido, com o desconfortar da sensibilidade insti-
tuída, com afetos novos e estrangeiros. Naquela manhã, após o intervalo do lanche, saímos com os es-
tudantes da sala de aula e, quando chegamos ao corredor que levaria à
sala de nossa instalação, Dulce2, uma das jovens estudantes, foi paran-
do pelo caminho, enquanto Madalena, uma estudante de 54 anos com
deficiência intelectual e fonoaudiológica, avançou. Dulce disse que es-
tava curiosa, mas muito nervosa, com medo. Mostrou suas mãos, sor-
rindo. Olha minhas mãos como é que estão! Estavam geladas! Madalena
sorria, estava vibrando, agitada e também curiosa, com ímpeto de con-
tinuar. Antes dos estudantes entrarem no túnel, explicamos que aquela
experiência fora inspirada em dois artistas brasileiros, Lygia Clark e Hé-
lio Oiticica. Dissemos que ambos os artistas acreditavam que não deve-
ríamos apenas observar as obras de arte, mas também deveríamos po-
der interagir com as obras, tocá-las, manuseá-las, entrar nelas. Falamos
rapidamente dos penetráveis de Oiticica e das esculturas manipuláveis
de Lygia Clark. Dissemos aos estudantes que era uma experimentação:
pedimos que entrassem descalços e apenas tentassem sentir a vivência
e responder às perguntas com o coração. João, como sempre, não mostrava muito interesse. Homero não
chegava; escrevemos uma mensagem pelo telefone celular dizendo que
tínhamos preparado algo legal com arte e que seria massa se ele viesse;
mas, respondeu que não iria, pois estava ocupado naquele dia. Insis-
timos que desse uma passada rápida; ele explicou que não iria porque
precisava fazer um corre, ir atrás de R$ 200,00 que estava devendo. Fi-
camos preocupadíssimos: sabíamos que tinha a ver com o tráfico, com
seu passado. Essa notícia deixou nosso coração opaco por um bom tem-
po. Homero era um estudante vindo de medida socioeducativa, que di-
zia gostar muito de arte; por isso nos importava muito que pudesse estar
presente naquele dia. Yonezawa; Cuevas Procuramos não forçar interpretações e nem colocar palavras em
sua boca para substituir aquilo que não entendíamos do que ela dizia. Saiu sorridente, muito tocada. Também estávamos emocionados. Em seguida, chamamos Dulce. Ela seguia nervosa. Dissemos que
iriamos juntos e pegamos em sua mão gelada. Quando entramos, tam-
bém ficou silenciosa, mas sorria com um pouco de ironia. Perguntou o
que queríamos dizer com aquilo. A ironia nos machucava. Responde-
mos que ela é que precisava dizer, deixando-se sentir, saboreando deva-
gar, ouvindo as perguntas. Logo passamos para as bolinhas e, quando
estávamos mudando de cabine, ela ouviu Para que serve tanto desâni-
mo e desgosto? e de cara disse, apontando o dedo em direção à fonte do
som: Essa pergunta é para mim!. Nas bolinhas de gel, não quis pisar, Eu
não vou pisar nisso! Dissemos para experimentar, insistimos um pouco. Ela pisou sem muita convicção. Também demos algumas bolinhas para
que ela tocasse com as mãos. Dissemos que ovos de peixe pareciam com Educação Antiniilista aquilo. Na cabine das garrafas coloridas, ela apenas as olhou e pareceu
achar bonito. Vimos que ia lendo às perguntas. Quando chegamos nos
mapas, novamente um momento importante. Perguntamos se ela co-
nhecia algum daqueles lugares; respondeu que nunca foi para nenhum,
que nunca sai de casa. Explicamos onde era cada lugar, mostramos no
mapa, contamos que era fácil ir com uma passagem de ônibus comum,
que dava para ir e voltar no mesmo dia. No gel, ela disse que não pisa-
ria de jeito nenhum. Fincou os pés fora da área gelatinosa e paralisou. Brincamos e demos uma empurradinha. Ela riu e saltou por cima da
poça de gel. João entrou e quis logo ir passando. Dissemos para ter calma, para
saborear. Desacelerou um pouco, mas passou um tanto rapidamente
pelos balões. Na fase seguinte, porém, deteve-se por muito tempo me-
xendo nas bolinhas de gel com os pés. Perguntamos o que sentia ou
pensava e ele disse que via ali nas bolinhas muitas pessoas aglomera-
das, juntas, enquanto outras saíam e, sozinhas, seguiam um caminho
diferente. Quando olhou as garrafas, parecia também achar bonito. Era
surpreendente que eles todos se detinham e pareciam ficar impressio-
nados com a cabine dos mapas. João foi reconhecendo os mapas, Esse
aqui é do Brasil, esse é do mundo, esse é do Espírito Santo. Eu já fui aqui. É Santa Tereza? Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1526 Yonezawa; Cuevas Yonezawa; Cuevas Conta que já experimentou ir a festas, mas não gosta de bagunça. Fa-
lamos, então, que ela não experimentou, por exemplo, conhecer aque-
les lugares bonitos que mostramos nos mapas. Ela confirma, diz se ver
muito fechada mesmo, que sua mãe reclama disso também; fala ainda
que é simpática e afetuosa com todo mundo, mas guarda coisas que não
abre para ninguém. Interceptamos: Você está se abrindo nesse momento. Wiliam sorri e reafirma. Ela concorda discretamente; diz saber que é
muito desanimada, mas não entende por que se sente assim. A conversa anda, a professora Elen fala de meditação. Dulce se
anima de fazermos, de experimentarmos. João diz que não tem paciên-
cia. Afirmamos que podemos fazer e Elen fala de trazermos colchone-
tes. Todos se animam. Lembramos que na segunda-feira seguinte não
estaríamos e Dulce diz: Então, não nesta segunda, mas na outra! Senti-
mo-nos vibrantes com a possibilidade de experimentarmos, de sairmos
da carteira, de seguirmos nos afetando por outras práticas corporais. Terminamos a manhã de aula sentindo que conseguíramos produzir
alguma diferença para aqueles meninos, para o trabalho da escola. g
ç p
q
p
Pois bem, notamos que os corpos dos estudantes eram (trans)
passados por novos sentidos, à medida que iam passando pelo túnel es-
tético. Entendemos que, ali, as forças dos corpos entravam em embate
silencioso com as forças atuantes em cada cabine do túnel. Vemos, por
isso, que o não sentido operado pela arte se difere muito do não sentido
da pasmaceira niilista. Na arte, o não sentido é propriamente destrui-
ção dos sentidos dados, desacomodação da sensibilidade e transborda-
mento de força; ao passo que, no niilismo, o não sentido é tão somente
ausência de força, nadificação da vida. A arte cuida, portanto, de dar
expressão a um niilismo ativo, proliferador de sentidos a partir do não
sentido. Como dizem Deleuze e Guattari (1992), a função da arte é jus-
tamente destituir os clichês da opinião, isto é, desmontar a máxima de
que, dentro dos sentidos dados, todos podem ter bom senso a partir de
uma opinião pessoal. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1527 Yonezawa; Cuevas Foi das poucas coisas que disse. Respondemos que sim
e perguntamos sobre os outros lugares. Ele não sabia. Apontamos para
Guarapari e ele disse que já tinha ido. E aqui? Perguntou, apontando. Respondemos: É Barra do Jucu. Disse que nunca foi. Falamos que era
perto, que dava para ir de ônibus. O momento mais interessante foi
quando passamos para a poça de gel. Ele hesitou por um tempo. Sorria
e contraía o corpo, com certa mistura de nojo e medo. Colocava só a
ponta do pé, como quando se testa a água fria de uma piscina. Várias
vezes fez isso, rindo. Nós o apertávamos. Vai! Coragem! Muito devagar
foi pisando. No fim colocou os pés todos, ficou muito pouco tempo e
saiu. Olhou para nós, fazendo um sim com a cabeça e disse: Os negros
né, a lama. Respondemos que sim. Quando saímos nos disse que o gel
podia ser preto, para parecer mais com a lama que os negros pisavam. Depois da experimentação, voltamos à sala de aula, que tinha as
carteiras organizadas em roda: o professor Wiliam pergunta E aí? O
que acharam? Por alguns instantes tudo parecia ter voltado ao que era
de costume: Dulce deitada sobre a carteira, João mexendo no telefone;
mas, diante da pergunta, Dulce se mostrou extremamente tocada. Le-
vantou o tronco e disse que ficou pensando no que mais deseja da vida. Disse que a pergunta estava rodando sem parar em sua cabeça, que não
sabia responder. Respondemos, então, que algumas perguntas a gente
passa a vida respondendo, que precisamos continuar procurando e nos
perguntando. Ela se impressiona: É?!. O professor Wiliam provoca João,
E você, João, o que achou? Ele, por um instante, sai do foco do telefone,
arregala os olhos, levanta o tronco e diz: Achei interessante! Lembrei dos
negros e da lama! O educador se impressiona. Daí, João volta a se fechar
sobre o telefone. Mas a conversa segue com Dulce: ela diz que sua vida é
muito ir de casa para a escola, da escola para casa e de casa para a igreja. Yonezawa; Cuevas Para os autores, ainda, a arte trava um combate
com o caos, ao mesmo tempo em que o desposa, de modo a emoldu-
rar porções dele, criando seres de sensação (Deleuze; Guattari, 1992);
e o que desejávamos com esta intervenção junto dos estudantes era de
fato introduzir um pouco de caos desconfortante no lago lodoso e des-
vitalizado que haviam se tornado as aulas naquele espaço. Com a arte,
instalam-se objetos e estes, sendo copos de caos, fazem emergir o que
Lygia Clark (1960) chama de vazio-pleno, a percepção de que toda forma
implica um preenchimento intrínseco de sentido. Além disso, tal como
Hélio Oiticica (1962, p. 19), acreditamos nas formas de arte em que “[...]
já não quer o sujeito (espectador) resolver a sua contradição em relação
ao objeto pela pura contemplação”. Ao elaborarmos um túnel em que os
corpos fossem levados a ser passados por sensações deslocantes, pre-
tendíamos, numa só tacada, combater a ausência de sentido niilista e
oferecer campos de sensibilidade a serem experimentados; pretendía-
mos trazer um momento no qual se pudesse “[...] transformar o que há
de imediato na vivência cotidiana em não mediato” (Oiticica, 1962, p. 11), colocando em xeque a dominância do sentido de baixa potência e ucação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1527 Educação Antiniilista amargura, que atrelava todos os corpos presentes na aula a um derro-
tismo. Do mesmo modo que Lygia Clark (1965b) acreditamos que poder
colocar o homem comum na posição de artista é meio de torná-lo um
ser mais pleno e, tão logo, pôr em cheque [sic] o vazio espiritual sem signi-
ficado aparente que nos cerca. Não se tratou, portanto, de democratizar
a arte, mas de fazê-la habitar lugares e funções improváveis para o meio
escolar, tirando-a do lugar de ser mais uma disciplina a ser lecionada,
para torná-la operadora e conversora de linhas de força. Diante disso, cabe agora compreender com um pouco mais de mi-
núcia de que maneira esta experiência contribuiu para escoriar o nii-
lismo e germinar novos sentidos nos corpos. Para tanto, traremos mais
elementos do pensamento de Deleuze e Guattari, de modo que possam
ser postos em movimento juntamente às linhas produzidas nesta expe-
riência. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1528 Passagem Educativa: arte e produção de sentido O sentido daquela parafernália não podia emergir se-
não de sua agitação junto aos corpos, na imanência desse encontro e,
ainda assim, este sentido aí produzido não era um nexo, ou reconhe-
cimento de uma ideia a ser captada como significado oculto, como um
isso quer dizer. Deleuze (2003) bem destaca que é o bom senso quem
encobre o sentido de significados, mas ele não é capaz de doar sentido. “Qual é então o papel do artista? Dar ao participante o objeto que não
tem importância em si mesmo e que só terá na medida em que o par-
ticipante atuar. É como um ovo que só revela sua substância quando
abrimos” (Clark, 1965a, p. 1). tidades fixas. Ora, não é esta a violência que vimos emergir quando do
encontro dos corpos dos estudantes com os materiais do túnel? Boli-
nhas coloridas e gelatinosas, gel, garrafas com líquidos coloridos trans-
lúcidos, perguntas provocativas, nada disso tinha só um nexo racional
assentado sobre um liame sensato e existente anteriormente à passa-
gem dos corpos. O sentido daquela parafernália não podia emergir se-
não de sua agitação junto aos corpos, na imanência desse encontro e,
ainda assim, este sentido aí produzido não era um nexo, ou reconhe-
cimento de uma ideia a ser captada como significado oculto, como um
isso quer dizer. Deleuze (2003) bem destaca que é o bom senso quem
encobre o sentido de significados, mas ele não é capaz de doar sentido. “Qual é então o papel do artista? Dar ao participante o objeto que não
tem importância em si mesmo e que só terá na medida em que o par-
ticipante atuar. É como um ovo que só revela sua substância quando
abrimos” (Clark, 1965a, p. 1). Porém, a ausência de sentido na produção de sentido se dá, não
apenas porque a cada estudante coube atuar na experiência (relativis-
mo), mas principalmente por causa do chamado “[...] paradoxo da proli-
feração indefinida” (Deleuze, 2003, p. 32), pelo qual o sentido sempre se
expressa nos corpos, mas, ao fazê-lo, se impulsiona a um novo sentido,
expresso, por sua vez, em outra composição de corpos: um sentido só
faz sentido diferenciando-se infinitamente. Então, o paradoxo se carac-
teriza por esticar o sentido em duas direções de desdobramento ao mes-
mo tempo (Deleuze, 2003). Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1529 Passagem Educativa: arte e produção de sentido Com efeito, dizemos que esta experimentação feita junto a esta
escola foi intensiva, não só porque presenciamos a movimentação de
emoções fortes, como no caso de Madalena. Víamos, em seu silêncio,
a mais alta potência de ser afetada sendo mobilizada. A estudante que,
pelo fato de ter problemas cognitivos e fonoaudiológicos, supostamente
seria menos apta para compreender uma forma de arte tão abstrata ou
enigmática, fora a que mais afetos empreendeu na passagem pelo túnel,
de modo a experimentar um transbordamento em seu corpo e, precisa-
mente por isso, ter se emocionado. Certamente, experiências intensivas fazem emergir uma emocio-
nalidade; contudo, o intenso não pode ser identificado à presença de
sentimentos e, muito menos, se pode cair na interpretação fácil de que
emoções melífluas e deleitosas sejam o maior indicativo da força inten-
siva de uma experiência. Quando Deleuze diz que uma aprendizagem
só pode emergir sendo sentida, é claro que ele também quis nos fazer
entender que apenas a partir de uma movimentação dos afetos é que
se pode gerar conhecimento. Portanto, acreditamos, sim, que Madale-
na apreendera novos sentidos em seu corpo, os quais ultrapassam as
emoções emergidas. Contudo, Deleuze não restringe o sentido aos sen-
timentos envolvidos. Para o filósofo os sentidos são efeitos incorporais,
os quais nascem das misturas de corpos, mas não se identificam a essas
misturas ou estados de coisa (Deleuze, 2003). As misturas dos corpos
são expressões dos sentidos, mas este, o expresso, não se confunde com
sua expressão. Ora, Deleuze lembra que o sentido é sempre paradoxal, isto é, ele
extrapola os significados dados e os dados significados, pois estes, por
sua vez, se assentam sobre a forma histórica ou socialmente hegemô-
nica do pensamento. Em outras palavras, o sentido emerge como não
sentido, como tensão ruidosa e ruinosa ao pensamento. O paradoxo do
sentido, diz Deleuze (2003), destrói o bom senso como sentido único e,
em seguida, destrói também o senso comum como designação de iden- Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 20
1528 Yonezawa; Cuevas tidades fixas. Ora, não é esta a violência que vimos emergir quando do
encontro dos corpos dos estudantes com os materiais do túnel? Boli-
nhas coloridas e gelatinosas, gel, garrafas com líquidos coloridos trans-
lúcidos, perguntas provocativas, nada disso tinha só um nexo racional
assentado sobre um liame sensato e existente anteriormente à passa-
gem dos corpos. Passagem Educativa: arte e produção de sentido Se, por exemplo, uma flor desabrocha, esse
desabrochar – verbo puro, devir que acontece à flor – é o sentido que faz
com que, num mesmo desenrolar, ela esteja mais expandida que antes e
menos aberta do que está se tornando. Desta forma, nosso túnel de sensações estica os corpos em dire-
ção a esvaziar sua modulação afetiva dominante, enquanto simulta-
neamente empurra-os a preencherem-se de sentidos estrangeiros, que
lhes incluem novas variedades afetivas. O trabalho da arte é justamente
acrescentar sempre novas variedades de sensação ao mundo (Deleuze;
Guattari, 1992). Portanto, quando afirmamos a força intensiva desta ex-
perimentação, estamos querendo destacar o fato de que ela propiciou
desvios, germinações, desacomodações, tensionamentos, problemati-
zações, fissuras e inquietações. A experimentação com o túnel foi in-
tensiva porque, a partir dela, algo aconteceu, a modulação lânguida e
anêmica dos corpos foi machucada e descontinuada, ao mesmo tempo
em que foi preenchida pelo vigor de sentidos insuspeitos. Ora, vimos
João ser afetado de tal modo com finura que, mesmo sendo um dos es-
tudantes que vinham às aulas com mais desinteresse e flacidez, pôde
conectar aquilo que aprendera sobre a escravidão negra – certamente
estudada nas aulas de História – com as sensações que afetavam seu
corpo no contato com os materiais da cabine. Ademais, a lama em que,
segundo sua fala, os negros pisavam, seguramente era um pequeno
elemento delirante que ele acrescentara à proliferação sensitiva que a
cabine trouxera. Igualmente, poderíamos dizer que a colônia de boli-
nhas coloridas fez germinar em seu corpo um sentido de multidão, bem Educação Antiniilista como de solidão e afirmação de caminhos distintos. Ora, não cremos
que essas imagens ou sensações trazidas pelo estudante esgotem o sen-
tido, elas são apenas as significações que expressam parcamente à mul-
tiplicidade esquizofrênica mais ampla e constituinte do sentido que por
ali passava. Elas não são o sentido em si, mas são signos da produtivi-
dade multiplicitária do sentido que se produzia ali. Como diz Deleuze
(2006), o extenso não dá nunca conta do intensivo como tal (talvez por
isso, as coisas ditas pelos estudantes sejam também poucas). Por outro
lado, também só podemos conhecer o intensivo desenvolvido numa ex-
tensão que o explica através de qualidades (Deleuze, 2006). Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1530 Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1531 Passagem Educativa: arte e produção de sentido Desta forma, tampouco cremos que o não senso contido na ex-
perimentação artística deva se confundir com as qualidades dos ma-
teriais (gelatinosos, coloridos, leves) ou com a fácil combinação aleató-
ria destes. Em primeiro lugar, parte das combinações foi relativamente
pensada: as perguntas sobre o que se deseja e as fotos nos mapas, por
exemplo, vieram para questionar posicionamentos específicos dos es-
tudantes. Mas, mais além, compreendemos que as qualidades dos ma-
teriais impliquem uma profusão de linhas mais finas e aleatórias de
afetação, as quais, quando tomadas como corpos de forças misturadas
com as forças dos corpos, se tornam maquinismo de produção de sen-
tido. De fato, como mostra Lygia Clark (2008), as mãos, a boca, todas as
partes corporais são inseparáveis de experiências intensivas, cujo nas-
cedouro se dá no encontro com objetos e porções concretas de mundo
extensivo. Dessa forma, nossa instalação fazia com que cada objeto e
cada corpo vigorassem numa composição, “[...] que o afasta de seu uso,
instituindo-o na sua poética” (Clark, 2008, p. 117). A partir disso, no trajeto da experimentação, também vimos Dul-
ce ser mobilizada pelas cores e texturas dos materiais, bem como pelas
perguntas ditas em voz doce. Seus próprios depoimentos posteriores
à experimentação mostram como sua sensibilidade fora tensionada, a
ponto de sair com o coração instigado com algumas das questões feitas. Destaquemos especialmente o fato de ter se afetado pelas perguntas
que diziam respeito ao que mais se deseja na vida e ao desgosto. Re-
alizar certos questionamentos para este público desta escola pode ser
uma intervenção extremamente transformadora, como foi para Dul-
ce. Com efeito, são indagações políticas, ao mesmo tempo em que são
educativas, pois, além de retorquirem a aceitação pré-concebida de que
naquele contexto social os sujeitos não têm ou não terão opções signifi-
cativas para suas vidas, elas também abrem possiblidades de afetação,
cognição, pensamento e, sobretudo, de movimento existencial. Per-
guntar sobre o que se deseja no caminho da existência deve ser tomado
como ação eminentemente educativa, a qual mobiliza meios de bus-
car conhecimento e, ao mesmo tempo, cuida de questões afetivas que
atravessam a modulação corporal com a qual se entra na sala de aula. Numa tal proposta, a aprendizagem promovida não é apenas de conteú-
dos curriculares, mas de elementos essenciais para organizar um modo
potente de vida. Yonezawa; Cuevas Yonezawa; Cuevas É preciso, portanto, que tenhamos uma ampliação radical da
educação em direção ao trabalho de promover o aprendizado de mais
e novos afetos, saindo da concepção conteudista, cogitocêntrica e disci-
plinar da educação. Educar, assim, seria ampliar a realidade pela qual
os corpos são afetados, isto é, educar seria mais do que somente ensinar
ideias que retratam uma realidade já dada. Portanto, se dizemos que nosso túnel ofereceu uma experiência
intensiva é mais porque vimos os estudantes ultrapassarem um estado
dado de sensibilidade, para realizarem o que Deleuze (2006, p. 333) cha-
ma de exercício transcendental do pensamento, ou “[...] pedagogia dos
sentidos”, pela qual, a sensibilidade acessa a força originalmente insen-
sível e inefável do sentido e os corpos tornam-se matéria de um devir. Ora, o que é característico do intensivo é que ele esteja sempre a afir-
mar a diferença, colocando-a como realidade, como caldo de cultura do
real, onde se compreende o desigual em si (Deleuze, 2006). Então, algo é
tanto mais intenso, quanto mais seja capaz de guardar simultaneidades
de sentido. Embora os relatos dos estudantes tenham sido significati-
vos, nos permitimos ver intensividade na experimentação feita pelo fato
desses depoimentos terem sido parcos, porém, poéticos: houve um ex-
cedente de afetação em detrimento dos significados atribuídos. Havia,
pois, um campo invisível de sentidos que coexistia (insistiam) às falas
expressas: Dulce, por exemplo, permanecia mobilizada com a experi-
ência. Educação mais que Escolar: educação afetiva e (micro)
política Apesar da discussão profundamente filosófica realizada aqui, é
preciso lembrar que a problematização do niilismo é imediatamente
política. Ora, a força de diminuição da vida não é senão expressão de
um (micro)fascismo, isto é, expressão do ódio à diferença e da tentati-
va dos modos adoentados e enfraquecidos de vida fazerem prevalecer a
submissão e passividade, ao se passarem por potentes, caricaturando a
imagem da força e do poder. Assim, consideramos que, com experimen-
tações como esta apresentada aqui, fomentamos uma inclusão estética
e afetiva, a qual, ao invés de ser nova forma de adaptação ao mundo do-
minante e pacto com uma pré-concepção misericordiosa sobre os estu-
dantes supostamente desvalidos e injustiçados, é impiedosa ampliação
de sensibilidade, de possibilidade, e pacto com a força de poder mais, de
tomar aos estudantes como capazes sempre de mais afeto, mais potên-
cia e mais realidade. Estamos cientes de que, de fato, estes estudantes
têm muito pouco acesso e contato com inúmeros bens culturais, tan-
to que, em aula de campo realizada com eles para conhecer os prédios
históricos do centro de Vitória, muitos disseram nunca ter entrado num
museu ou teatro. Porém, mesmo considerando o fato de que há barrei-
ras sociais invisíveis para o acesso de determinadas classes sociais a
bens culturais, não podemos incorrer na postura de alimentar senti-
mentos caridosos para com este público. O que se coloca em jogo, então, Educação Antiniilista com intervenções artísticas como as que empreendemos, é a possibili-
dade de, ao se fazer penetrar os corpos em porções de caos, se fomentar
também instantes de liberdade, de ampliação do pensamento e da sen-
sibilidade. “Não se trata aqui da participação pela participação, nem da
agressão pela agressão, mas de que o participante dê um significado ao
seu gesto e de que seu ato seja alimentado por um pensamento, nesse
caso a enfatização de sua liberdade de ação” (Clark, 1965a, p. 2). Como dizíamos, trata-se, nisso tudo, de realizar um trabalho polí-
tico, de se enfrentar ao um (micro)fascismo. Ora, Guattari (1985) lembra
que, na medida em que o agigantamento da produção capitalista des-
territorializou o corpo individual, liberou-se também uma revolucioná-
ria energia molecular, da qual não conhecemos ainda a capacidade de
operar uma revolução maquínico-semiótica. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018.
1532 Educação mais que Escolar: educação afetiva e (micro)
política 34). é engendrada por operadores de qualquer natureza ou tamanho, está
manufaturada de modo a premunir a existência contra toda intrusão de
acontecimentos suscetíveis de atrapalhar e perturbar a opinião” (Guat-
tari, 1990, p. 34). Com efeito, o enfraquecimento da vitalidade que vimos em nossos
estudantes, a ausência de valor ou sentido dado a questionar, aprender,
perguntar e ser afetado já nos parece ser parte deste insidioso esquema
de espiritualização da servidão que o autor denuncia. É algo que interes-
sa muito à continuidade da produção de modos de vida do capitalismo
a secreção de corpos-zumbis, amedrontados, ou arredios à diferença,
anestesiados por imagens midiáticas incessantes, ou empedrados por
concepções religiosas dogmáticas. Não se trataria, pois, de ideologia,
mas de modelização dos campos de sensibilidade pelos quais os corpos
se afetam, o que promove uma forma de assujeitamento ainda mais rí-
gida e sedutora, embora mais inclusiva e aparentemente menos violen-
ta. O capitalismo é o sistema social propriamente niilista, na medida em
que leva os corpos a esgotarem suas forças vitais dentro de um duplo
maquinismo de produção-consumo incessante, frenético e exasperado. Por isso é que, em nosso trabalho junto à escola, o enfrentamento
do niilismo passa a ser questão educacional seminal. Nossa militância é
por uma prática pedagógica que se afirme na fabricação de novas esté-
ticas, cuja potência faça frente às formas de captura das sensibilidades e
dos corpos, formas essas baseadas em amortecimento da sensibilidade,
acompanhada do oferecimento de imagens e signos caricatos, estereo-
tipados e docilizantes. Portanto, afirmamos uma política educacional
que envolva uma educação afetiva, isto é, uma prática que, ultrapas-
sando a escolarização conteudista e disciplinar, se incumba de violar
os clichês semióticos nos quais os corpos são envolvidos, para secretar
afetos insuspeitos, devirulentos, potentes o suficiente para promover
uma aprendizagem de sentidos novos, instigantes, avivadores das for-
ças. Tratar-se-ia, pois, de uma educação de natureza artística; afetiva
não porque seja carinhosa e doce, mas por implicar o exercício de ex-
pansão da sensibilidade em direção a uma multiplicidade de sentidos e
capacidades de ser afetado. Com efeito, trata-se de batalha que não se
finda em algumas intervenções e tampouco encontra garantia num es-
tado final de formação subjetiva. O trabalho de ampliar as sensibilida-
des é cambaleante prática educacional, realizada como aposta na pro-
dução de diferença. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1533 Educação mais que Escolar: educação afetiva e (micro)
política Para o autor, o crescimento
estrondoso das máquinas produtivas também tornou as formas de sub-
metimento muito mais poderosas, na medida em que se sutilizaram e
se miniaturizaram. Neste contexto, não é mais o homem, como indi-
víduo, que trabalha, mas é um agenciamento de órgãos, partes, peças,
funções que se articulam com outras tantas funções e peças técnicas. Isso, em contrapartida, possibilita que as verdadeiras revoluções sejam
operadas em termos de transformação de sentido, de mutação das me-
nores relações constituintes dos agenciamentos de submissão. “A análise molecular não pode ser senão a expressão de um agen-
ciamento de potências moleculares, associando teoria e prática” (Guat-
tari, 1985 p. 182). Para Guattari (1985), tais análises da molecularidade
implicam em compreender, sobretudo, como o fascismo se moleculari-
zou também, dando continuidade a um tipo de funcionamento social
totalitário, desta vez, sob outras formas, estéticas e dimensões. O autor
alerta para o quanto, cada vez mais, são as “[...] técnicas de impregnação
audiovisuais” (Guattari, 1985, p. 53), que fazem o trabalho de submeter
os corpos com suavidade, introduzindo-os muito cedo numa política
semiótica dominante; processo transversal, cuja força não privilegia re-
cair somente sobre uma ou outra classe social. Há, para o autor, uma
homogeneização das competências semióticas operada no capitalismo,
com vistas à perpetuação do seu sistema produtivo e modo de vida. Quer dizer, a servidão no capitalismo é constituída através de catalisa-
dores de sentido, não apenas por sistemas mecânicos; “[...] não se tra-
ta somente de operações concretas de aprendizagem, mas também da
aquisição de esquemas abstratos, de esquemas relacionais, de toda uma
iniciação à axiomática do capital” (Guattari, 1985, p. 54). Como afirma Guattari (1985), o capitalismo atual já não depen-
de apenas de meios de coerção centralizados em formações estatais: os
meios atuais de sujeição operam através de condensadores semióticos
flutuantes e informacionais, os quais fazem com que os próprios sujei-
tos assumam os meios de controle, repressão e modelização da ordem
dominante. Entendemos que esta reprodução semiótica dos modos de
vida hegemônicos do capitalismo seja uma modulação do niilismo, pois
se expressam nos corpos através exatamente da tentativa de contenção
da produção de novos sentidos. “A subjetividade capitalísitica, tal como Yonezawa; Cuevas é engendrada por operadores de qualquer natureza ou tamanho, está
manufaturada de modo a premunir a existência contra toda intrusão de
acontecimentos suscetíveis de atrapalhar e perturbar a opinião” (Guat-
tari, 1990, p. Educação mais que Escolar: educação afetiva e (micro)
política Através destas ações experimentais, acreditamos
operar uma forma de inclusão destes estudantes no universo de riqueza
estética e afetiva das linguagens artísticas, as quais costumeiramente
são levadas somente até galerias de arte elitizadas; além disso, estamos
promovendo também uma delicada raspagem das impregnações semi-
óticas amortecedoras da sensibilidade dos corpos. Sendo assim, ressal-
tamos que intervenções de caráter estético-político como esta devam
estar presentes em todos os níveis e modalidades educacionais, não po-
dendo ser pensadas como específicas à Educação de Jovens e Adultos,
ou ao ensino regular, por exemplo. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1533 Educação Antiniilista Afinal, nesta educação antiniilista, trata-se de liberar as sensibi-
lidades e de avivar as forças dos corpos em direção a uma concepção de
mundo mais artística, rica em sentidos e menos ortopédica. Com prá-
ticas de multiplicação estética e produção de sentidos fazemos frente a
um sistema de controle dos corpos baseado numa política de homoge-
neização dos signos. Recebido em 07 de agosto de 2017
Aprovado em 17 de novembro de 2017 Recebido em 07 de agosto de 2017
Aprovado em 17 de novembro de 2017 Notas 1 Pesquisa amparada pela CAPES e pela Fundação de Amparo à Pesquisa e Ino-
vação do Espírito Santo (FAPES). 2 Todos os nomes aqui são fictícios para que as identidades dos estudantes e
educadores sejam preservadas. Referências BARROS, Letícia; BARROS, Maria Elizabeth. O Problema da Análise em Pesqui-
sa Cartográfica. Fractal Revista de Psicologia, Rio de Janeiro, v. 25, n. 2, p. 373-
390, maio/ago. 2013. BARROS, Letícia; BARROS, Maria Elizabeth. O Problema da Análise em Pesqui-
sa Cartográfica. Fractal Revista de Psicologia, Rio de Janeiro, v. 25, n. 2, p. 373-
390, maio/ago. 2013. BARROS, Laura Pozzana; KASTRUP, Virgínia. Cartografar é Acompanhar Pro-
cessos. In: PASSOS, Eduardo; KASTRUP, Virgínia; ESCOSSIA, Liliana. Pistas do
Método da Cartografia: pesquisa-intervenção e produção de subjetividade. Porto Alegre: Sulina, 2010. P. 52-75. CLARK, Lygia. O Vazio Pleno. Três Rios: Associação Cultural O Mundo de Ly-
gia Clark, 1960. Disponível em: <http://www.lygiaclark.org.br/arquivo_detPT. asp?idarquivo=16>. Acesso em: 28 fev. 2017. CLARK, Lygia. A Propósito da Magia do Objeto. Três Rios: Associação Cultural
O Mundo de Lygia Clark, 1965a. Disponível em: <http://www.lygiaclark.org.br/
arquivo_detPT.asp?idarquivo=20>. Acesso em: 28 fev. 2017. CLARK, Lygia. Um Mito Moderno: o instante como nostalgia do cosmos. Três
Rios: Associação Cultural O Mundo de Lygia Clark, 1965b. Disponível em:
<http://www.lygiaclark.org.br/arquivo_detPT.asp?idarquivo=22>. Acesso em:
01 mar. 2017. CLARK, Lygia. Breviário sobre o Corpo. Arte & Ensaios, Rio de Janeiro, n. 16, p. 114-123, jul. 2008. DELEUZE, Gilles. Crítica e Clínica. São Paulo: Editora 34, 1997. DELEUZE, Gilles. Nietzsche e a Filosofia. Porto: Rés-Editora, 2001. DELEUZE, Gilles. Lógica do Sentido. São Paulo: Perspectiva, 2003. DELEUZE, Gilles. Diferença e Repetição. Rio de Janeiro: Graal, 2006. DELEUZE, Gilles; GUATTARI, Félix. O que é a Filosofia? Rio de Janeiro: Editora 34, 1992. GUATTARI, Félix. Revolução Molecular: pulsações políticas do desejo. São Pau-
lo: Editora Brasiliense, 1985. GUATTARI, Félix. As Três Ecologias. Campinas: Papirus, 1990. NIETZSCHE, Friedrich. Crepúsculo dos Ídolos. São Paulo: Companhia das Le-
tras, 2006. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1534 Yonezawa; Cuevas NIETZSCHE, Friedrich. O Anticristo e Ditirambos de Dionísio. São Paulo: Com-
panhia das Letras, 2007. NIETZSCHE, Friedrich. A Vontade de Poder. Rio de Janeiro: Contraponto, 2008. NIETZSCHE, Friedrich. Genealogia da Moral. São Paulo: Companhia das Le-
tras, 2009. NIETZSCHE, Friedrich. A Gaia Ciência. São Paulo: Companhia das Letras, 2012. OITICICA, Hélio. Penetráveis. Rio de Janeiro: Centro de Arte Hélio Oiticica,
1962. PAULON, Simone; ROMAGNOLI, Roberta. Pesquisa-Intervenção e Cartografia:
melindres e meandros metodológicos. Estudos e Pesquisas em Psicologia, Rio
de Janeiro, n. 1, p. 85-102, jan./abr. 2010. PELBART, Peter Pal. O Avesso do Niilismo: cartografias do esgotamento. São
Paulo: n-1 Edições, 2013. ROMAGNOLI, Roberta. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1515-1535, out./dez. 2018. 1535 Referências O Conceito de Implicação e a Pesquisa-Intervenção Ins-
titucionalista. Psicologia e Sociedade, Belo Horizonte, v. 26, n. 1, p. 44-52, jan./
abr. 2014. Fernando Yonezawa é psicólogo, Mestre em Educação pela Universidade Fernando Yonezawa é psicólogo, Mestre em Educação pela Universidade
Federal do Rio Grande do Sul, Doutor em Psicologia pela Universidade de
São Paulo e pós-doutorando do Programa de Pós-Graduação em Psicologia
Institucional da Universidade Federal do Espírito Santo. E-mail: fefoyo@yahoo.com.br E-mail: fefoyo@yahoo.com.br Márcia Cuevas é psicóloga, Doutora em Educação pela Universidade Fede-
ral do Espírito Santo e Professora do Programa de Pós-Graduação em Psico-
logia Institucional da Universidade Federal do Espírito Santo. g
p
E-mail: marcia.roxana@hotmail.com Este é um artigo de acesso aberto distribuído sob os termos de uma Li-
cença Creative Commons Atribuição 4.0 Internacional. Disponível em:
<http://creativecommons.org/licenses/by/4.0>.
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https://openalex.org/W3097258484
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https://www.frontiersin.org/articles/10.3389/fonc.2020.553536/pdf
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English
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Identification of Spindle and Kinetochore-Associated Family Genes as Therapeutic Targets and Prognostic Biomarkers in Pancreas Ductal Adenocarcinoma Microenvironment
|
Frontiers in oncology
| 2,020
|
cc-by
| 14,161
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ORIGINAL RESEARCH
published: 02 November 2020
doi: 10.3389/fonc.2020.553536 Yi Liu1*, Zong-rui Jin2, Xing Huang3, Ye-cheng Che4 and Qin Liu5* Yi Liu1*, Zong-rui Jin2, Xing Huang3, Ye-cheng Che4 and Qin Liu5* 1 Department of Gastrointestinal Surgery, Guangxi Medical University Cancer Hospital, Guangxi Clinical Research Center
for Colorectal Cancer, Nanning, China, 2 Department of Hepatobiliary Surgery, The First Affiliated Hospital of Guangxi Medical
University, Nanning, China, 3 Department of Radiotherapy, The Second Affiliated Hospital of Guangxi Medical University,
Nanning, China, 4 Department of Emergency Medicine, First People’s Hospital of Fuzhou, Fuzhou, China, 5 Department
of Medical Ultrasonics, Second People’s Hospital of Guilin, Guilin, China Aim: The role of spindle and kinetochore-associated (SKA) genes in tumorigenesis
and cancer progression has been widely studied. However, so far, the oncogenic
involvement of SKA family genes in pancreatic cancer and their prognostic potential
remain unknown. Edited by:
Lucia Conti,
National Institute of Health (ISS), Italy Edited by:
Lucia Conti,
National Institute of Health (ISS), Italy Edited by:
Lucia Conti,
National Institute of Health (ISS), Italy
Reviewed by:
Kawaljit Kaur,
University of California, Los Angeles,
United States
Niccola Funel,
University of Pisa, Italy
*Correspondence:
Qin Liu
liuqinchaosheng@163.com
Yi Liu
liuyi@stu.gxmu.edu.cn Reviewed by:
Kawaljit Kaur,
University of California, Los Angeles,
United States
Niccola Funel,
University of Pisa, Italy Methods: Here, we carried out a meta-analysis of the differential expression of SKA
genes in normal and tumor tissue. Univariate and multivariate survival analyses were
done to evaluate the correlation between SKA family gene expression and pancreas
ductal adenocarcinoma (PDAC) prognosis. Joint-effect and stratified survival analysis as
well as nomogram analysis were used to estimate the prognostic value of genes. The
underlying regulatory and biological mechanisms were identified by Gene set enrichment
analysis. Interaction between SKA prognosis-related genes and immune cell infiltration
was assessed using the Tumor Immune Estimation Resource tool. *Correspondence:
Qin Liu
liuqinchaosheng@163.com
Yi Liu
liuyi@stu.gxmu.edu.cn *Correspondence:
Qin Liu
liuqinchaosheng@163.com
Yi Liu
liuyi@stu.gxmu.edu.cn Specialty section:
This article was submitted to
Cancer Immunity and Immunotherapy,
a section of the journal
Frontiers in Oncology Results: We find that SKA1–3 are highly expressed in PDAC tissues relative to non-
cancer tissues. Survival analysis revealed that high expression of SKA1 and SKA3
independently indicate poor prognosis but they are not associated with relapse-
free survival. The prognostic value of SKA1 and SKA3 was further confirmed by
the nomogram, joint-effect, and stratified survival analysis. Analysis of underlying
mechanisms reveals that these genes influence cancer-related signaling pathways,
kinases, miRNA, and E2F family genes. Notably, prognosis-related genes are inversely
correlated with several immune cells infiltrating levels. Received: 19 April 2020
Accepted: 08 September 2020
Published: 02 November 2020 Keywords: biomarker, spindle and kinetochore associated, prognosis, pancreatic cancer, bioinformatics analysis,
immune infiltration Survival Analysis Kaplan–Meier (KM) survival analysis and multivariate Cox
regression analysis were used to evaluate the SKA gene prognostic
value. Samples in each dataset were divided into high and low
expression groups based on each gene’s median expression value. Kaplan–Meier survival analysis was used to screen out clinical
factors related to PDAC prognosis, and the clinical parameters
with a P < 0.05 were selected into the Cox regression analysis. Next, multivariate survival analysis adjusted by prognostic-
related clinical features was done. As the clinical information of
GSE62452 and GSE28735 datasets was inadequate, these datasets
were used to validate the prognostic value of each gene by
KM survival analysis only. Afterward, to evaluate combined
predictive potential on patient overall survival (OS), joint-effect
survival analysis for the combination of mRNA transcriptional
level and prognosis-related clinical factors was performed. Citation: Liu Y, Jin Z, Huang X, Che Y and
Liu Q (2020) Identification of Spindle
and Kinetochore-Associated Family
Genes as Therapeutic Targets
and Prognostic Biomarkers
in Pancreas Ductal Adenocarcinoma
Microenvironment. Conclusion: We find that SKA1 and SKA3 expression correlates with prognosis
and immune cell infiltration in PDAC, highlighting their potential as pancreatic cancer
prognostic biomarkers. November 2020 | Volume 10 | Article 553536 1 Frontiers in Oncology | www.frontiersin.org New Pancreatic Cancer Prognostic Biomarkers Liu et al. INTRODUCTION Platform (UCSC Xena).3 RNA-seq raw data were normalized by
DESeq package (24). Pancreas ductal adenocarcinoma SKA gene
expression profile microarrays, normalized using limma package
(25), were retrieved from Gene Expression Omnibus (GEO).4
All the expression profile data were log2-transformed before
further analysis. Pancreatic cancer is one of the most aggressive malignancies in
the world and is associated with a high rate of metastasis and
mortality. Its 5-year survival rate is estimated to be 5% globally,
and 11.7% in China (1–3). It is estimated that in 2018, there
were 458,918 new pancreatic cancer cases and 432,242 patient
deaths worldwide (4). Pancreatic cancer management is mainly
by surgical resection, which is thought to improve the 5-year
survival to 20–30% (5). Non-etheless, surgical resections are
recommended for <20% of pancreatic cancer cases since the
disease is often diagnosed at the advanced stage (5–7). Therefore,
strategies for early detection and treatment of pancreatic cancer
are urgently needed. Bioinformatic Analysis Using SKA Family
Genes The online tools of Gene Multiple Association Network
Integration Algorithm (GeneMANIA)5 and the Search Tool
for the Retrieval of Interacting Genes/Proteins (STRING)6
were used to structure interaction networks in gene–gene and
protein–protein, respectively (36–39). A co-expression matrix
of SKA mRNA was constructed using the TCGA, GTEx, and
GSE62452 datasets. Given the limited treatment options for pancreas ductal
adenocarcinoma (PDAC), novel biomarkers are needed to
enhance treatment and prognosis. Previous reports show that
SKA family genes may influence cancer treatment response
and prognosis. We therefore speculated that SKA family genes
might be prognostic in PDAC. To test this possibility, we used
bioinformatics to investigate their expression in PDAC and how
it correlates with prognosis. Meta-Analysis The following search terms were used to retrieve relevant datasets
from GEO: “pancreatic cancer,” “mRNA,” and “pancreas.” The
inclusion criteria for PDAC microarrays were as follows: (1) it
must contain both normal and tumor samples, (2) expression
profile data are available, (3) samples are all human, and (4) ≥10
samples available. A schematic of the search process is shown in
Supplementary Figure 1. Details of the included microarrays are
shown in Supplementary Table 1. Eleven previously published
datasets were retrieved (GSE71729, GSE28735, GSE15471,
GSE62165,
GSE62452,
GSE16515,
GSE32676,
GSE1542,
GSE74629, GSE91035, and GSE101462) (26–35). The clinical
information of GSE62452 and GSE28735 was also downloaded to
explore the potential prognostic value of SKA genes. After adding
the TCGA PDAC cohort, a total of 715 PDAC samples and 297
non-cancer pancreatic samples from 12 datasets were combined
and meta-analysis was used to compare gene expression between
PDAC and normal tissue. g
y
It is well-known that mitosis is a common biological process
in eukaryotic cells. During mitosis, the spindle ensures that sister
chromatids are correctly distributed between daughter cells (8). SKA1–3 family of genes are essential for the accurate timing of
late mitosis. Spindle and kinetochore-associated (SKA) complex
produced by the SKA genes maintain stability of metaphase plate
or spindle checkpoint silencing (9, 10). Upregulation of SKA1
triggers nucleation of interphase microtubules, while depletion
of the SKA1 complex results in abnormal mitosis (11, 12). The association between SKA1 and cancer has been widely
investigated. It has been reported that SKA1 overexpression may
lead to the development of pancreatic cancer in mouse models
(13). SKA1 upregulation has also been observed in multiple
malignancies, including gastric, oral, bladder, non-small cell lung,
hepatocellular, and prostate cancer. Elevated levels of SKA1 have
been shown to promote cancer cell proliferation and influence
(14–20). 1https://www.gtexportal.org/home/
2http://cancergenome.nih.gov Acquisition of Public RNA-Seq and Gene
Microarray Data A schematic representation of our study outline is shown
in Figure 1. Violin plots and RNA-seq data of SKA gene
expression in normal tissues were obtained from Genotype-
Tissue Expression (GTEx)1 (21, 22). RNA-seq raw data for
149 samples (145 PDAC tissue and 4 non-tumor pancreatic
tissue) were obtained from The Cancer Genome Atlas (TCGA)2
(23). Matched clinical information of the PDAC patients
was obtained from University of California Santa Cruz Xena 3http://xena.ucsc.edu/
4https://www.ncbi.nlm.nih.gov/geo/
5http://genemania.org/
6https://string-db.org/cgi/input.pl 3http://xena.ucsc.edu/
4https://www.ncbi.nlm.nih.gov/geo/
5http://genemania.org/
6https://string-db.org/cgi/input.pl 1https://www.gtexportal.org/home/
2http://cancergenome.nih.gov 2http://cancergenome.nih.gov November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 2 Liu et al. New Pancreatic Cancer Prognostic Biomarkers FIGURE 1 | Flowchart presenting the general work flow of this study. FIGURE 1 | Flowchart presenting the general work flow of this study. expression of prognosis-related genes and clinicopathology
features
of
PDAC
using
the
datasets
with
adequate
clinical information. Stratified survival analysis with multiple clinicopathological
features was also used to evaluate the predictive value of the genes
in PDAC patients. Nomogram All
PDAC
patient
data
from
TCGA
and
its
matched
clinicopathological features were combined with prognosis-
related genes to construct a nomogram. Each prognosis-related
gene was classified into the high or low expression groups based
on median gene expression. The nomogram can calculate the
total score of each patient based on existing information and
predict survival probability. In addition, the nomogram can
evaluate how each parameter affects the probability of survival. Samples
from
TCGA
were
divided
into
two
categories
according to median expression values of prognosis-related
SKA genes. Gene set enrichment analysis (GSEA)7 was then
done to explore potential biological mechanisms (40, 41). Gene sets of Kyoto Encyclopedia of Genes and Genomes
(KEGG) (c2.all.v6.2.symbols.gmt) and gene ontology (GO)
(c5.all.v6.2.symbols.gmt) were used in this study (42, 43). The
criteria for statistically significance were a nominal P value < 0.05
and FDR < 0.25. (44). Next, kinase–target, transcription factor–
target, and miRNA network enrichment analyses were done
on the LinkedOmics database8 online tool (45). The top five
outcomes from each gene set are shown. 7http://www.broadinstitute.org/gsea/index.jsp
8http://www.linkedomics.org/login.php Prognostic Value Evaluation and Clinical
Relevance of SKA1 and SKA3 Area under the curve (AUC) of the receiver operating
characteristic (ROC) curve was done to assess the accuracy
of prognostic-related genes in predicting patient survival using
survivalROC package. We compared the correlation between 7http://www.broadinstitute.org/gsea/index.jsp
8http://www.linkedomics.org/login.php November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 3 New Pancreatic Cancer Prognostic Biomarkers Liu et al. 9https://www.cbioportal.org/
10https://bioinformatics.mdanderson.org/public-software/estimate/
11https://cistrome.shinyapps.io/timer/ Survival Analysis We first analyzed TCGA dataset and removed a case lacking
the survival time so that 145 eligible PDAC patients were
enrolled at last. After performing KM survival analysis with the
clinicopathological indicators of PDAC, the P value less than 0.05
clinical features including radical resection, radiation therapy,
and targeted molecular therapy were selected out, which were
further involved in the Cox regression analysis for adjustment
(Table 1). Then, the KM method was also used for SKA1–3 gene
survival analysis, indicating that all the genes were significantly
associated with prognosis in PDAC patients except for SKA2, and
high expression suggest a shorter survival time (Figures 4A–C). The median survival time (MST) for low and high expression
groups of SKA genes were SKA1 (695 vs 485 days), SKA2 (568 vs
518 days), and SKA3 (698 vs 485 days) (Table 2). Similarly, the
Cox regression analysis outcomes showed that SKA1 (adjusted
P = 0.04; adjusted HR = 1.656, 95% CI: 1.024–2.677) was an
independent prognostic factor for PDAC patients as well as in
SKA3 (adjusted P = 0.034; adjusted HR = 1.688, 95% CI: 1.040–
2.742), while it was not with SKA2 (adjusted P = 0.837; adjusted
HR = 0.952, 95% CI: 0.592–1.529). Survival analysis from GEO
datasets of GSE62452 and GSE28735 showed that high expression
of SKA1 and SKA3 was significantly correlated with a poor
OS, except for SKA1 in GSE28735 due to the limited sample
(Figures 4D–I). To estimate the role of SKA genes in recurrence
of PDAC, relapse-free survival (RFS) time was used to structure
the KM curve, which suggests that the high expression group of
SKA1 or SKA3 was not associated with the recurrence rate of
PDAC compared with the low expression group (Figures 4J–L). Analysis of Genomic Alterations and
Methylation Level of Prognosis-Related
Genes for meta-analysis. This analysis showed that SKA1–3 expression
is higher in PDAC tissue relative to non-tumor pancreatic
tissue [SKA1: SMD = 0.35 (0.20–0.50), I2 = 0.0%, Figure 2A;
SKA2: SMD = 0.64 (0.32–0.96), I2 = 74.1%, Figure 2C; SKA3:
SMD = 0.70 (0.31-1.09), I2 = 82.6%, Figure 2E]. The AUCs
of the sROC of SKA1, SKA2, and SKA3 as calculated by
diagnostic meta-analysis were 0.69, 0.77, and 0.78, respectively
(Figures 2B,D,F). Heterogeneity for meta-analysis of SKA2
and SKA3 was significantly derived from GSE74629 and
GSE15471, respectively, whereas no heterogeneity existed in the
comprehensive analysis of SKA1. To elucidate the potential regulatory mechanism of gene
expression, gene mutations, and copy number variations (CNVs),
data were accessed on cBioportal.9 The calculation method
of CNVs was Genomic Identification of Significant Targets in
Cancer 2.0 (GISTIC2) (46). Next, DNA methylation data for the
genes with matched RNA-seq expression profile (log2(count + 1))
were accessed from UCSC Xena and analyzed by Infinium
Human Methylation 450 BeadChip. The methylation assessment
for the genes was expressed as β-values. Correlation analysis and
KM survival analysis were used to explore potential methylation
sites regulating gene expression. Bioinformatics Analysis Gene
and
protein
interaction
analysis
SKA1–3
using
GeneMANIA and STRING, respectively, revealed a significant
degree interaction, as well as protein homology (Figures 3A,B). Furthermore, there existed strong co-expression of SKA genes
not only in the normal dataset of GTEx but also in tumor
datasets of TCGA and GSE62452 (Figures 3C–E), especially the
correlation between SKA1 and SKA3 (r = 0.62, 0.6, and 0.69). Statistical Analysis Stata (version 12.0) was used to plot forest plots and summary
ROC (sROC) curve of the meta-analysis. The standard mean
difference (SMD) and 95% confidence interval (CI) were used to
identify differentially expressed SKA genes in cancer and non-
cancer tissues. Summary ROC was used to evaluate the capacity
of genes to discriminate between cancer and non-cancer tissues. All statistical analysis was carried out on SPSS (version 22.0). Survival differences were identified by hazard ratio (HR) and
95% CI. Two or multiple groups of continuous variables were
compared by Student t test and one-way ANOVA, respectively. All the correlation analysis method used Spearman’s correlation. P value < 0.05 was considered statistically significant. Exploration of Tumor Immune Infiltration RNA-seq data were analyzed using ESTIMATE (estimation of
stromal and immune cells in malignant tumor tissues using
expression data)10 algorithm to calculate cellular component in
tumor tissues and estimate tumor microenvironment (TME)
scores for stromal and immune cells (47). The immune cells
analyzed to assess tumor immunity are B cells, CD4+ T
cells, CD8+ T cells, dendritic cells (DCs), neutrophils, and
macrophages. Tumor Immune Estimation Resource (TIMER)11
was then used to evaluate infiltration by the immune cells
(48). Next, the correlation between immune cell infiltration and
prognosis-related gene expression in PDAC was evaluated. 9https://www.cbioportal.org/ Expression Level Distribution and
Meta-Analysis of SKA1–3 Relative
to
other
human
normal
organ
tissues,
SKA1–
3
expression
was
lower
in
pancreatic
tissues
in
GTEx
(Supplementary Figures 2A–C). To obtain stability results
of differential expression analysis of SKA genes between PDAC
tissues and non-tumor tissues, we integrated multiple datasets We further explored the effect of the gene combination on
the prognosis of patients and divided samples into three groups. KM survival curves showed that group 3 (MST: 394 days)
with high expression of SKA1 and SKA3 genes had the worst
prognosis, while group 1 (MST: 695 days) with low expression
of the two genes had the best prognosis, suggesting that their
high expression represents a high risk of death (Table 2 and November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 4 New Pancreatic Cancer Prognostic Biomarkers Liu et al. et al. New Pancreatic Cancer Prognostic Biomarke
IGURE 2 | Meta-analysis of 12 datasets of pancreatic cancer. (A) Forest plot showing SKA1 expression difference. (B) sROC curve of SKA1. (C) Forest plot
howing SKA2 expression difference. (D) sROC curve of SKA2. (E) Forest plot showing SKA3 expression difference. (F) sROC curve of SKA3. gure 5A). The combination of SKA1 or SKA3 with prognosis-
ated clinical characteristics can better show the difference in
radiation therapy, and residual resection. High expression o
SKA1 suggested a poor survival time in patients with an ag FIGURE 2 | M t
l
i
f 12 d t
t
f
ti
(A) F
t
l t h
i
SKA1
i
diff
(B) ROC
f SKA1 (C) F
t
l t FIGURE 2 | Meta-analysis of 12 datasets of pancreatic cancer. (A) Forest plot showing SKA1 expression difference. (B) sROC curve of SKA1. (C) Forest plot
showing SKA2 expression difference. (D) sROC curve of SKA2. (E) Forest plot showing SKA3 expression difference. (F) sROC curve of SKA3. Figure 5A). The combination of SKA1 or SKA3 with prognosis-
related clinical characteristics can better show the difference in
PDAC prognosis, which improved the predictive performance for
prognosis (Figures 5B–I and Tables 3, 4). radiation therapy, and residual resection. Nomogram Patients were grouped into three categories as mentioned
previously, and patient mRNA expression data with matched
clinical features were used to build a nomogram. Each variable
had a score that was denoted by a line length. The higher the
score, the greater the effect of the gene on prognosis. This
analysis reveals that that mRNA expression of SKA1 and SKA3
can significantly affect patient survival (Figure 6C). g
Subsequently, we evaluated correlation between prognosis-
related genes and clinicopathological parameters. Analysis of the
GSE62452 dataset revealed SKA1 upregulation in stage II patients
relative to stage I patients (Figures 7F,G). In terms of histologic
grade, analysis of the GSE62452 dataset showed that SKA1
and SKA3 expression were significantly higher in G3/G4 PDAC
relative to G1/G2 disease stage and SKA1 and SKA3 only showed
an upregulated trend in G3 samples in TCGA (Figures 7H,I). Moreover, relative to tumor-free survival patients, tumor-bearing
patients significantly expressed high levels of SKA1 and SKA3 Expression Level Distribution and
Meta-Analysis of SKA1–3 Next, SKA1 and SKA3 expression
scatter plots, survival status scatter diagrams, and expression heat
maps were used to visualize the genes’ prognostic value. This
analysis revealed that increased SKA expression levels correlated
with reduced survival time (Figure 7E). Expression Level Distribution and
Meta-Analysis of SKA1–3 High expression of
SKA1 suggested a poor survival time in patients with an age
of <60 [HR (95% CI): 21.674 (2.002–14.599)] and non-radical
resection [HR (95% CI): 3.213 (1.284–8.037)], and similarly,
poor prognosis was significantly correlated with high expression
of SKA3 in patients with G3 [HR (95% CI): 2.810 (1.132–6.975)], g
g
Stratified
analysis
with
various
clinical
pathological
parameters
was
adjusted
by
targeted
molecular
therapy, November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 5 Liu et al. New Pancreatic Cancer Prognostic Biomarkers FIGURE 3 | Interaction networks and co-expression matrix of spindle and kinetochore associated genes. (A) Gene–gene interaction network created using the Gene
Multiple Association Network Integration Algorithm (GeneMANIA). (B) Protein–protein interaction network created using the Search Tool for the Retrieval of
Interacting Genes/Proteins (STRING). (C–E) Co-expression matrix of SKA genes in TCGA, GTEx, and GSE62452 datasets. FIGURE 3 | Interaction networks and co-expression matrix of spindle and kinetochore associated genes. (A) Gene–gene interaction network created using the Gene
Multiple Association Network Integration Algorithm (GeneMANIA). (B) Protein–protein interaction network created using the Search Tool for the Retrieval of
Interacting Genes/Proteins (STRING). (C–E) Co-expression matrix of SKA genes in TCGA, GTEx, and GSE62452 datasets. 3 years: 0.637, 0.603, and 0.564) at 1-, 2-, and 3-year survival were
medium as shown in Figures 7A,B. The time-dependent ROC
curve produced from the GSE62452 dataset showed that SKA1
(1, 2, and 3 years: 0.523, 0.675, and 0.805) and SKA3 (1, 2, and
3 years: 0.614, 0.795, and 0.844) were effective predictors of 3-year
PDAC survival (Figures 7C,D). Next, SKA1 and SKA3 expression
scatter plots, survival status scatter diagrams, and expression heat
maps were used to visualize the genes’ prognostic value. This
analysis revealed that increased SKA expression levels correlated
with reduced survival time (Figure 7E). no radiation therapy [HR (95% CI): 1.978 (1.150–3.402)], alcohol
history [HR (95% CI): 3.924 (1.940–7.937)], age of < 60 [HR
(95% CI): 5.050 (1.308–19.490)], non-radical resection [HR
(95% CI): 3.275(1.384–7.958)], and male [HR (95% CI): 2.161
(1.083–4.321)] (Figures 6A,B). 3 years: 0.637, 0.603, and 0.564) at 1-, 2-, and 3-year survival were
medium as shown in Figures 7A,B. The time-dependent ROC
curve produced from the GSE62452 dataset showed that SKA1
(1, 2, and 3 years: 0.523, 0.675, and 0.805) and SKA3 (1, 2, and
3 years: 0.614, 0.795, and 0.844) were effective predictors of 3-year
PDAC survival (Figures 7C,D). Gene Enrichment Analysis Gene set enrichment analysis analysis of the potential mechanism
by which prognosis-related genes influence PDAC revealed that
in the high SKA1 expression group, there was significant
enrichment for cell cycle-related biological processes (GO:
cell division, cell cycle checkpoint, and cell cycle phase
transition,
Figures
8A–C)
and
tumor-related
signaling
pathways (KEGG: cell cycle, P53 signaling pathway, and
DNA replication, Figures 8D–I). GO term analysis of the high
SKA3 expression group revealed enrichment for processes
involved in negative regulation of T cell proliferation and
positive regulation of the WNT pathway (Figures 8J,K). KEGG annotation showed the high SKA3 expression group
participated in tumor-related signaling pathways (pancreatic
cancer, pathways in cancer, toll-like receptor pathway, and Prognostic Value and Clinical Relevance
of Prognosis-Related Genes In TCGA cohort, the time-dependent ROC curve of SKA1 (1,
2, and 3 years: 0.645, 0.558, and 0.544) and SKA3 (1, 2, and November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 6 New Pancreatic Cancer Prognostic Biomarkers Liu et al. TABLE 1 | Clinical characteristics of pancreatic ductal adenocarcinoma patients. Variables
Events/total (n = 145)
MST (days)
HR (95% CI)
Log-rank P value
Age (years)
0.406
<60
23/42
592
1
≥60
60/103
568
1.405 (0.887,2.226)
Missing
0
Gender
0.412
Female
43/69
570
1
Male
40/76
660
0.784 (0.55,1.118)
Missing
0
TNM stage
0.905
Stage I
6/12
498
1
Stage II
74/125
592
1.608 (0.813,3.182)
Stage III + IV
3/7
545
2.333 (1.069,5.090)
Missing
1
Histologic grade
0.164
G1/G2
56/104
607
1
G3
27/41
485
1.366 (0.966,1.932)
Missing
0
Radiation therapy
0.023
Yes
58/94
481
1
No
17/37
702
2.322 (1.418,3.802)
Missing
14
Targeted molecular therapy
< 0.001
Yes
27/35
239
1
No
52/99
684
1.489 (1.055,2.1)
Missing
11
Alcohol history
0.923
No
29/50
532
1
Yes
46/83
598
1.263 (0.794,2.007)
Missing
12
Radical resection
0.009
Yes
46/83
614
1
No
33/53
394
1.691 (1.098,2.604)
Missing
9
MST, median survival time; HR, hazard ratio; CI, confidence interval. (Figure 7J) and both of them did not corelate with advanced
pathological T (T3/4) (Figure 7K). (Figure 7J) and both of them did not corelate with advanced
pathological T (T3/4) (Figure 7K). adherens junction) (Figures 8L–O). The enrichment results
of network analyses on the LinkedOmics database indicated
that SKA1 and SKA3 are mainly regulated by the same
transcription factors (V$E2F1_Q6, V$E2F_Q4, V$E2F_Q6,
V$E2F_Q4_01),
networks,
and
kinases,
including
cyclin-
dependent kinase 1 (CDK1), polo-like kinase 1 (PLK1),
cyclin-dependent kinase 2 (CDK2), and aurora B kinase
(AURKB). The miRNA-target network for SKA1 was related
to MIR-185, MIR-512-3p, MIR-507, MIR-218, and MIR-96,
while SKA3 was associated with MIR-507, MIR-119A, MIR-513,
MIR-338, and MIR-137 (Table 5). Frontiers in Oncology | www.frontiersin.org Genomic Alterations and DNA
Methylation Level of Prognosis-Related
Genes Analysis of SKA gene mutation in the 153 PDAC patients
showed that 12 (7%) of them were mutation carriers (Figure 9A). Furthermore, box plot analysis of CNV data from the PDAC November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 7 New Pancreatic Cancer Prognostic Biomarkers Liu et al. FIGURE 4 | Kaplan-Meier survival curves showing the association of spindle and kinetochore related genes with the overall survival and relapse-free survival of
pancreatic ductal adenocarcinoma patients from TCGA and GEO datasets. Overall survival in TCGA: (A) SKA1; (B) SKA2; (C) SKA3; Overall survival in GEO62452:
(D) SKA1; (E) SKA2; (F) SKA3; Overall survival in GEO28735: (G) SKA1; (H) SKA2; (I) SKA3; Relapse-free survival in TCGA stratified by (J) SKA1; (K) SKA2;
(L) SKA3. FIGURE 4 | Kaplan-Meier survival curves showing the association of spindle and kinetochore related genes with the overall survival and relapse-free survival of
pancreatic ductal adenocarcinoma patients from TCGA and GEO datasets. Overall survival in TCGA: (A) SKA1; (B) SKA2; (C) SKA3; Overall survival in GEO62452:
(D) SKA1; (E) SKA2; (F) SKA3; Overall survival in GEO28735: (G) SKA1; (H) SKA2; (I) SKA3; Relapse-free survival in TCGA stratified by (J) SKA1; (K) SKA2;
(L) SKA3. patients revealed that increased copy number of the genes
poorly correlates with higher expression of prognosis-related
genes (Figures 9B,C). First, We found poor correlation between
DNA methylation and gene expression in pure PDAC (data
not show). Next, we explored whether DNA methylation
status influenced gene expression in 185 pancreatic cancer
samples by analyzing 11 and 15 CpG sites in the SKA1 and
SKA3 DNA locus, respectively. Five of the 11 sites in the
SKA1 DNA locus had high methylation with β values > 0.6,
while two sites (cg18558188: β ± SD = 0.84 ± 0.04, | r|
= 0.254; cg18742986: β ± SD = 0.89 ± 0.06, | r|
= 0.19)
exhibited significant negative correlation with gene expression,
indicating that DNA methylation may regulate SKA1 expression
(Figure 9D). When samples were divided into two groups based on median methylation level, loci cg18558188 and
cg18742986 methylation were not associated with PDAC
OS, while higher total CpG methylation levels of SKA1
correlated with improved patient survival (Supplementary
Figures 3A–C). All SKA3 CpG sites exhibited very low
methylation
(β-value
<
0.4)
and
were
not
explored
further (Figure 9E). Prognosis-Related Genes Correlated
With Tumor Immune Microenvironment
and Key Gene Mutations GSEA revealed that SKA3 modulates T cell activity. Next,
we explored relationship between prognosis-related genes and November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 8 New Pancreatic Cancer Prognostic Biomarkers Liu et al. TABLE 2 | Prognostic value of single and combined of spindle and kinetochore-associated genes expression in pancreatic ductal adenocarcinoma patient OS from
TCGA. Gene
Events/total
(n = 145)
MST/MRS
(days)
Crude HR (95% CI)
Crude
P value
Adjusted HR (95% CI)a
Adjusted
P valuea
SKA1
Low
37/72
695
1
1
High
46/73
485
1.711 (1.095,2.671)
0.018
1.656 (1.024,2.677)
0.04
SKA2
Low
39/72
568
1
1
High
44/73
518
1.149 (0.745,1.771)
0.530
0.952 (0.592,1.529)
0.837
SKA3
Low
40/72
598
1
1
High
52/73
485
1.495 (0.968,2.308)
0.069
1.688 (1.040,2.742)
0.034
Group 1
SKA1low + SKA3low
26/53
695
1
1
Group 2
SKA1low + SKA3high or SKA1high + SKA3low
16/38
598
1.200 (0.641,2.246)
0.568
1.010 (0.482,2.115)
0.980
Group 3
SKA1high + SKA3high
41/54
394
1.797 (1.098,2.942)
0.02
1.587 (0.932,2.702)
0.089
CI, confidence interval; SKA, spindle and kinetochore associated; HR, hazard ratio; MST, median survival time; OS, overall survival. aAdjusted for radical resection, radiation therapy, and targeted molecular therapy. TABLE 2 | Prognostic value of single and combined of spindle and kinetochore-associated genes expression in pancreatic ductal adenocarcinoma patient OS from
TCGA. Gene
Events/total
(n = 145)
MST/MRS
(days)
Crude HR (95% CI)
Crude
P value
Adjusted HR (95% CI)a
Adjusted
P valuea
SKA1
Low
37/72
695
1
1
High
46/73
485
1.711 (1.095,2.671)
0.018
1.656 (1.024,2.677)
0.04
SKA2
Low
39/72
568
1
1
High
44/73
518
1.149 (0.745,1.771)
0.530
0.952 (0.592,1.529)
0.837
SKA3
Low
40/72
598
1
1
High
52/73
485
1.495 (0.968,2.308)
0.069
1.688 (1.040,2.742)
0.034
Group 1
SKA1low + SKA3low
26/53
695
1
1
Group 2
SKA1low + SKA3high or SKA1high + SKA3low
16/38
598
1.200 (0.641,2.246)
0.568
1.010 (0.482,2.115)
0.980
Group 3
SKA1high + SKA3high
41/54
394
1.797 (1.098,2.942)
0.02
1.587 (0.932,2.702)
0.089
CI, confidence interval; SKA, spindle and kinetochore associated; HR, hazard ratio; MST, median survival time; OS, overall survival. aAdjusted for radical resection, radiation therapy, and targeted molecular therapy. nd combined of spindle and kinetochore-associated genes expression in pancreatic ductal adenocarcinoma patient OS from TABLE 2 | Prognostic value of single and combined of spindle and kinetochore-associated genes expression in pancreatic ductal adenocarcinoma patient OS from
TCGA. Prognosis-Related Genes Correlated
With Tumor Immune Microenvironment
and Key Gene Mutations TABLE 2 | Prognostic value of single and combined of spindle and kinetochore-associated genes expression in pancreatic ductal adenocarcinoma patient OS from
TCGA FIGURE 5 | Joint-effect survival analysis for overall survival of patients with pancreatic ductal adenocarcinoma. (A) Combination of SKA1 and SKA3. Combination of
SKA1 and prognosis-related clinical factors: (B) histologic grade; (C) radiation therapy; (D) targeted molecular therapy; (E) radical resection. Combination of SKA3
and prognosis-related clinical factors: (F) histologic grade; (G) radiation therapy; (H) targeted molecular therapy; (I) radical resection. FIGURE 5 | Joint-effect survival analysis for overall survival of patients with pancreatic ductal adenocarcinoma. (A) Combination of SKA1 and SKA3. Combination of
SKA1 and prognosis-related clinical factors: (B) histologic grade; (C) radiation therapy; (D) targeted molecular therapy; (E) radical resection. Combination of SKA3
and prognosis-related clinical factors: (F) histologic grade; (G) radiation therapy; (H) targeted molecular therapy; (I) radical resection. November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 9 New Pancreatic Cancer Prognostic Biomarkers Liu et al. TABLE 3 | Joint-effect survival analysis of SKA1 expression and clinical variables in pancreatic ductal adenocarcinoma patient overall survival. Group
SKA1
Variables
Events/total
(n = 145)
MST
(days)
Crude HR (95% CI)
Crude P
value
Adjusted HR (95% CI)a
Adjusted
P valuea
Histologic grade
a
Low
G1 + G2
20/50
913
1
1
b
Low
G3
12/22
592
1.413 (0.686,2.911)
0.349
2.030 (0.924,4.459)
0.078
c
High
G1 + G2
36/53
532
1.717 (0.991,2.975)
0.054
1.505 (0.828,2.734)
0.180
d
High
G3
16/20
470
2.459 (1.272,4.753)
0.007
1.759 (0.856,3.618)
0.125
Radiation therapyb
i
Low
No
23/48
684
1
1
ii
Low
Yes
7/18
702
0.718 (0.307,1.678)
0.444
1.002 (0.417,2.405)
0.997
iii
High
No
36/47
381
2.015 (1.192,3.407)
0.009
1.472 (0.848,2.554)
0.170
iv
High
Yes
10/18
691
0.839 (0.398,1.766)
0.644
0.817 (0.354,1.884)
0.636
Targeted molecular therapyc
A
Low
No
7/11
467
1
1
B
Low
Yes
25/57
695
0.239 (0.101,0.570)
0.001
0.287 (0.116,0.710)
0.007
C
High
No
20/24
219
1.512 (0.634,3.604)
0.351
1.659 (0.691,3.979)
0.257
D
High
Yes
28/42
603
0.290 (0.122,0.691)
0.005
0.329 (0.130,0.831)
0.019
Radical resectiond
I
Low
Yes
21/44
695
1
1
II
Low
No
10/23
607
1.104 (0.518,2.354)
0.798
0.877 (0.384,2.006)
0.756
III
High
Yes
25/39
596
1.156 (0.641,2.084)
0.630
0.791 (0.425,1.469)
0.457
IV
High
No
24/31
366
2.949 (1.619,5.371)
< 0.001
2.440 (1.273,4.679)
0.007
SKA1, spindle and kinetochore-associated complex subunit 1; MST, median survival time; HR, hazard ratio; CI, confidence interval. Prognosis-Related Genes Correlated
With Tumor Immune Microenvironment
and Key Gene Mutations Tumor cells have
the capacity to evade clearance by macrophages through the
upregulation of antiphagocytic surface proteins including cluster
of differentiation 47 (CD47), programed death-ligand 1 (PD-L1), Prognosis-Related Genes Correlated
With Tumor Immune Microenvironment
and Key Gene Mutations aAdjusted for radical resection, radiation therapy, targeted molecular therapy. bInformation of radiation therapy was unavailable in 14 patients. cInformation of targeted molecular therapy was unavailable in 11 patients. dInformation of radical resection was unavailable in 8 patients. KA1 expression and clinical variables in pancreatic ductal adenocarcinoma patient overall survival. programmed cell death protein 1 (PD1), cytotoxic T-lymphocyte-
associated protein 4 (CTLA4), and beta-2-microglobulin (B2M)
(49–52). We explored whether SKA1 and SKA3 had correlation
with these antiphagocytic molecules. SKA1 and SKA3 were
positively correlated with the expression of CD47, PD-L1,
and B2M in TCGA dataset, respectively. Similar results were
displayed in the GSE625452 dataset (Supplementary Table 2). These results may explain why high expression of SKA1 and
SKA3 could result in reduced infiltration levels of immune cells
in PDAC. Mutation analysis revealed that high SKA1 expression
highly correlates with the KRAS mutation group but not with
TP53 mutations. High SKA3 expression in the KRAS and TP53
mutant groups was significantly associated with the mutant type
(Figures 10H,I). tumor immunity, including immune cell infiltration and immune
scores. Results from TIMER analysis show that CD8+ T
cells, CD4+ T cells, and macrophages exhibit a significant
negative correlation with SKA1 mRNA expression and its
CNVs significantly impact immune cell infiltration levels
(Figures 10A,B). In particular, SKA3 expression significantly
exhibits a strongly negative correlation with all immune cell
infiltration levels except for B cell. Infiltration of natural killer
(NK) cells is not related to SKA gene expression (Supplementary
Figures 4A–D). Additionally, infiltration levels for B cell and
CD4+ T cell can be affected by SKA3 CNVs (Figures 10C,D). Then, scatter plot analysis was used to visualize the distribution
of CD4+ T and CD8+ T cell between the groups expressing
high and low levels of prognosis-related genes. This analysis
revealed that CD8+ T cells were significantly fewer in the high
expression group, but no difference in CD4+ T cell distribution
was observed between the groups (Figures 10E,F). So, we
speculated that high expression levels of SKA1 and SKA3, to some
extent, may mediate tumor escape and inhibit the infiltration
levels of immune cells. To test this possibility, we calculated the
tumor immune scores using the RNA-seq data from the TCGA
cohort. Consistently, immune score negatively correlated with
SKA3 expression but did not correlate with SKA1 expression,
indicating that SKA3 has a great influence on immune cell
infiltration in PDAC tissues (Figure 10G). Frontiers in Oncology | www.frontiersin.org DISCUSSION In this study, we combined PDAC cohorts’ data from GEO
and TCGA and used bioinformatics to evaluate the potential
prognostic value of SKA genes in PDAC. Our meta-analysis
found that all SKA genes are significantly upregulated in
PDAC and exhibited medium diagnostic value for PDAC. Survival analysis showed that overexpression of SKA1 and SKA3
is associated with poor prognosis. The prognostic value of
the two genes was further studied by ROC, joint-effect, and
stratified analysis among other strategies. Our data highlight
SKA1 and SKA3 as potential therapeutic targets against PDAC. In this study, we combined PDAC cohorts’ data from GEO
and TCGA and used bioinformatics to evaluate the potential
prognostic value of SKA genes in PDAC. Our meta-analysis
found that all SKA genes are significantly upregulated in
PDAC and exhibited medium diagnostic value for PDAC. Survival analysis showed that overexpression of SKA1 and SKA3
is associated with poor prognosis. The prognostic value of
the two genes was further studied by ROC, joint-effect, and
stratified analysis among other strategies. Our data highlight
SKA1 and SKA3 as potential therapeutic targets against PDAC. November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 10 New Pancreatic Cancer Prognostic Biomarkers Liu et al. TABLE 4 | Joint-effect survival analysis of SKA3 expression and clinical variables in pancreatic ductal adenocarcinoma patient overall survival. DISCUSSION Group
SKA3
Variables
Events/total
(n = 145)
MST
(days)
Crude HR (95% CI)
Crude P
value
Adjusted HR (95% CI)a
Adjusted
P valuea
Histologic gradea
e
Low
G1 + G2
21/48
607
1
1
f
Low
G3
14/22
598
1.320 (0.667,2.611)
0.425
1.407 (0.666,2.974)
0.371
g
High
G1 + G2
35/55
532
1.614 (0.937,2.782)
0.085
1.731 (0.944,3.173)
0.076
h
High
G3
14/20
460
2.791 (1.412,5.517)
0.003
3.393 (1.605,7.172)
0.001
Radiation therapyb
v
Low
No
24/48
607
1
1
vi
Low
Yes
7/16
702
0.799 (0.343,1.861)
0.604
1.138 (0.448,2.891)
0.786
vii
High
No
35/47
375
2.268 (1.344,3.827)
0.002
2.094 (1.223,3.585)
0.007
viii
High
Yes
10/20
691
0.819 (0.391,1.715)
0.597
1.011 (0.451,2.270)
0.978
Targeted molecular therapyc
E
Low
No
10/15
467
1
1
F
Low
Yes
23/49
702
0.826 (0.412,1.654)
0.589
0.254 (0.112,0.574)
0.001
G
High
No
17/20
160
4.317 (2.069,9.008)
< 0.001
2.090 (0.942,4.638)
0.070
H
High
Yes
30/50
627
1.016 (0.517,1.995)
0.964
0.409 (0.182,0.917)
0.030
Radical resectiond
V
Low
Yes
21/43
695
1
1
VI
Low
No
12/23
592
1.139 (0.578,2.242)
0.707
1.170 (0.527,2.598)
0.700
VII
High
Yes
25/40
517
1.347 (0.777,2.336)
0.289
0.718 (0.718,2.442)
0.369
VIII
High
No
22/31
378
3.427 (1.903,6.172)
< 0.001
1.702 (1.702,6.433)
<0.001
SKA3, spindle and kinetochore-associated complex subunit 3; MST, median survival time; HR, hazard ratio; CI, confidence interval. aAdjusted for radical resection, radiation therapy, and targeted molecular therapy. bInformation of radiation therapy was unavailable in 14 patients. cInformation of targeted molecular therapy was unavailable in 11 patients. dInformation of radical resection was unavailable in 8 patients. New Pancreatic Cancer Prognostic Biomarkers New Pancreatic Cancer Prognostic Biomarkers. KA3 expression and clinical variables in pancreatic ductal adenocarcinoma patient overall survival. and DNA replication, processes that significantly correlate with
cancer progression (64–67). Our data also associated high SKA1
expression with advanced cancer phenotypes such as stage II,
G3 + G4, and patients survived with tumor. SKA2, a novel cell
cycle gene, has been proposed as a biomarker and therapeutic
target against cancer (68). It has been reported that suppressed
SKA2 expression triggers kinetochore fiber instability, leading
to mitotic failure (9). A study by Ren et al. found that SKA2
overexpression induces epithelial–mesenchymal transition in
breast cancer, promoting cell invasion and metastasis (69). SKA2
also influences proliferation, migration, and invasive capacity
of gastric and lung cancer cells (70, 71). While SKA2 was also
highly expressed in our study, no correlation was found with
PDAC prognosis. Frontiers in Oncology | www.frontiersin.org DISCUSSION A probable reason for this may be inter-
tumor heterogeneity, which requires additional investigation. SKA3 mediates appropriate mitotic exit by interacting with the
NDC80 complex, which regulates meiotic spindle migration and
anaphase spindle stability (55, 72, 73). SKA3 upregulation in lung
adenocarcinoma cells correlates with increased metastases and
tumor growth (74). Similarly, it has been reported that high SKA3
expression promotes lung cancer cell proliferation and predicts
patient outcomes. Through bioinformatics analysis, Tang et al. found that SKA3 is associated with elevated susceptibility to
breast cancer brain metastasis and negatively correlates with
breast cancer survival (75). Our data further validate the
correlation between high SKA3 expression and advanced clinical
features of G3/G4. Our findings show that SKA3 may be involved Additionally, we explored the potential mechanisms regulating
SKA1 and SKA3 expression and found that DNA methylation
may influence SKA1 expression and patients’ OS. Possible
mechanisms include cancer- and immune-related pathways, and
potential regulators include cancer-related kinases, miRNA, and
the E2F family. Finally, we explored the association between
prognosis-related genes and tumor immune microenvironment. Biochemically, SKA1 is known to directly bind microtubules
through its C-terminal domain to stimulate oligomerization (11,
53). Spindle and kinetochore-associated complex deficiency is
associated with chromosome congression failure and cell death
(54–56). Multiple studies have reported that SKA1 promotes
cancer progression in a variety of tumors (13, 19, 57–61). An
in vitro study found that SKA1 accelerates cell proliferation
and cancer progression in glioma via tumor-associated signaling
pathways, including cell cycle and Wnt/β-catenin signaling (62). An immunohistochemical study of 126 hepatocellular carcinoma
patients revealed that SKA1 expression is significantly elevated in
tumor tissues, and it can regulate the hepatocellular carcinoma
cell cycle and contributes to poor prognosis (63). Qin et al. used targeted small interfering RNA to knockdown SKA1 and
observed hepatocellular carcinoma cell cycle arrest in the G0/G1
phase (17). Besides, SKA1 has been reported to contribute to
chemotherapy resistance in lung carcinoma through prevention
of cisplatin-induced apoptosis (19). Here, we find that high
SKA1 expression predicts poor PDAC prognosis and participates
in cell cycle, cell cycle checkpoint, P53 signaling pathway, November 2020 | Volume 10 | Article 553536 11 New Pancreatic Cancer Prognostic Biomarkers Liu et al. g
GURE 6 | The relationship of spindle and kinetochore-associated genes with the clinical information. (A,B) Stratified survival analysis of SKA1 and SKA3 in vari
nicopathological parameters. Frontiers in Oncology | www.frontiersin.org DISCUSSION (C) A prognostic nomogram based on SKA1 and SKA3 for predicting the 1-, 2-, and 3-year overall survival rate of patients with
ancreatic ductal adenocarcinoma. HR, hazard ratio; CI, confidence interval; L/L: SKA1low + SKA3low; L/H: SKA1low + SKA3high; H/L: SKA1high + SKA3low; H/H:
KA1high + SKA3high. Wnt signaling, pancreatic cancer, pathways in cancer, toll-like
eptor signaling pathway, and adherens junction, which are
own to play a crucial role in tumor progression (76–78). Kinases regulate various processes such as genomic stability
cycle, cell differentiation, and somatic reprogramming
Previous investigators have shown that dysregulation of C
leads to G2 phase arrest and promotes tumor progres
making it an ideal biomarker and therapeutic target FIGURE 6 | The relationship of spindle and kinetochore-associated genes with the clinical information. (A,B) Stratified survival analysis of SKA1 and SKA3 in various
clinicopathological parameters. (C) A prognostic nomogram based on SKA1 and SKA3 for predicting the 1-, 2-, and 3-year overall survival rate of patients with
pancreatic ductal adenocarcinoma. HR, hazard ratio; CI, confidence interval; L/L: SKA1low + SKA3low; L/H: SKA1low + SKA3high; H/L: SKA1high + SKA3low; H/H:
SKA1high + SKA3high. cycle, cell differentiation, and somatic reprogramming (79). Previous investigators have shown that dysregulation of CDK1
leads to G2 phase arrest and promotes tumor progression,
making it an ideal biomarker and therapeutic target (80,
81). CDK1 binds Ndc80, thereby phosphorylating SKA3 and
recruiting SKA to kinetochores to facilitate mitotic progression in Wnt signaling, pancreatic cancer, pathways in cancer, toll-like
receptor signaling pathway, and adherens junction, which are
known to play a crucial role in tumor progression (76–78). Kinases regulate various processes such as genomic stability,
mitosis, and the cell cycle. CDK1, a member of the cyclin-
dependent kinase protein family, participates in mitosis, cell November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 12 New Pancreatic Cancer Prognostic Biomarkers Liu et al. Liu et al. g
FIGURE 7 | Analysis of the prognostic value and clinical relevance of SKA1 and SKA3 in pancreatic ductal adenocarcinoma patients. (A–D) Time-dependent receiver
operating characteristic (ROC) curves of SKA1and SKA3 showing the 1-, 2-, and 3-year overall survival rate of patients with PDAC from TCGA and GSE62452
datasets. (E) From top to bottom are the expression values of SKA1, patients’ survival status distribution, and the expression heat map of SKA1 in the low- and
high-expression groups. Frontiers in Oncology | www.frontiersin.org DISCUSSION The expression distribution of SKA1 and SKA2 genes in different (F,G) AJCC stages and (J,K) grades in TCGA and GSE62452 datasets. H,I) The expression distribution of SKA1 and SKA2 genes in different pathological T grade and cancer status in TCGA cohort. *p < 0.05, **p < 0.01, ***p < 0.001. FIGURE 7 | Analysis of the prognostic value and clinical relevance of SKA1 and SKA3 in pancreatic ductal adenocarcinoma patients. (A–D) Time-dependent receiver
operating characteristic (ROC) curves of SKA1and SKA3 showing the 1-, 2-, and 3-year overall survival rate of patients with PDAC from TCGA and GSE62452
datasets. (E) From top to bottom are the expression values of SKA1, patients’ survival status distribution, and the expression heat map of SKA1 in the low- and
high-expression groups. The expression distribution of SKA1 and SKA2 genes in different (F,G) AJCC stages and (J,K) grades in TCGA and GSE62452 datasets. (H,I) The expression distribution of SKA1 and SKA2 genes in different pathological T grade and cancer status in TCGA cohort. *p < 0.05, **p < 0.01, ***p < 0.001. (82). Another study
by Hou
et
al. showed
that SKA3
interacted with CDK2 and inhibited P53 phosphorylation,
thereby regulating proliferation of liver cancer cell (83). PLK1,
a tumorigenic factor, is a mitotic cyclin-independent serine
threonine kinase that has been proposed as a potential
therapeutic target for pancreatic cancer (84, 85). Another
kinase, AURKA, participates in pancreatic carcinogenesis via
the MAPK1/ERK2 signaling pathway (86). The same regulatory network of kinases (CDK1, CDK2, PLK1, and AURKA) was
identified in our study as potential regulators of SKA1 and
SKA3 in PDAC. The E2F family is part of the transcription
factors that regulate gene expression. It participates in the cell
cycle process, and activated E2F initiates oncogenic signaling
in several cancer types (87, 88). Several approaches have been
developed to directly or indirectly target E2F1 with the aim of
modifying malignant phenotypes of PDAC (89–92). Herein, E2F1 Frontiers in Oncology | www.frontiersin.org November 2020 | Volume 10 | Article 553536 13 New Pancreatic Cancer Prognostic Biomarkers Liu et al. FIGURE 8 | Gene set enrichment analysis of SKA1 and SKA3 in TCGA dataset. (A–C,J,K) GSEA results of C5 gene sets for high SKA1 and SKA3 expression
groups; (D–I,L–O) GSEA results of C2 gene sets for high SKA1 and SKA3 expression groups; NES, normalized enrichment score; FDR, false discovery rate. DISCUSSION FIGURE 8 | Gene set enrichment analysis of SKA1 and SKA3 in TCGA dataset. (A–C,J,K) GSEA results of C5 gene sets for hig
groups; (D–I,L–O) GSEA results of C2 gene sets for high SKA1 and SKA3 expression groups; NES, normalized enrichment scor hment analysis of SKA1 and SKA3 in TCGA dataset. (A–C,J,K) GSEA results of C5 gene sets for high SKA1 and SKA3 expression
sults of C2 gene sets for high SKA1 and SKA3 expression groups; NES, normalized enrichment score; FDR, false discovery rate. FIGURE 8 | Gene set enrichment analysis of SKA1 and SKA3 in TCGA dataset. (A–C,J,K) GSEA results of C5 gene sets for high SKA1 and SKA3 expression
groups; (D–I,L–O) GSEA results of C2 gene sets for high SKA1 and SKA3 expression groups; NES, normalized enrichment score; FDR, false discovery rate. and alter genetic content, causing different phenotypes. In
this study, CNVs of SKA1 and SKA3 were found to be
weakly correlated with gene expression. DNA methylation
is a crucial epigenetic mechanism, which maintains genome
stability, chromatin structure, and pluripotency in human cells. Changes in DNA methylation levels often accompany neoplasm
development (105, 106). The role of DNA methylation in
SKA genes has not been investigated. In this preliminary
study, we found that two sites in SKA1 were significantly
involved with gene expression. High total DNA methylation
level of SKA1 predicts favorable prognosis in PDAC. We
thus hypothesize that DNA methylation may be an important
regulatory mechanism for SKA1 that influences the OS of
patients with PDAC. However, no DNA methylation sites were was found to work with SKA1 and SKA3 to jointly regulate
cell cycle and aggravate PDAC. MicroRNA, an endogenous
small RNA with a length of about 20–24 nucleotides, has been
implicated in human carcinogenesis (93). The miRNAs identified
in our study have been associated with deteriorated neoplastic
malignant phenotype such as proliferation, cell cycle, invasion,
drug resistance, and angiogenesis (94–98). In fact, miR-185, miR-
96, miR-218, miR-137, and miR-338 have been proven to exhibit
therapeutic and prognostic value in PDAC, indicating that the
SKA gene may be one of the target genes for these miRNAs
(99–103). A research team found that genomic alteration is a
common phenomenon in human tumors (104). Copy number
variations, induced by genomic rearrangement, disrupt genes November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 14 New Pancreatic Cancer Prognostic Biomarkers Liu et al. DISCUSSION TABLE 5 | The kinase and transcription factor-target networks of SKA1 and SKA3 in TCGA dataset (LinkedOmics). Gene
Enriched Category
Gene Set
Leading-EdgeNum
FDR
SKA1
Kinase Target
Kinase_CDK1
69
0
Kinase_PLK1
30
0
Kinase_CDK2
58
0
Kinase_AURKB
25
0
Kinase_CHEK1
23
0
Transcription Factor Target
V$E2F1_Q6
58
0
V$E2F_Q4
47
0
V$E2F_Q6
47
0
V$E2F_Q4_01
45
0
V$E2F_02
50
0
miRNA Target
TCTCTCC,MIR-185
54
0
CAGCACT,MIR-512-3P
66
0
GTGCAAA,MIR-507
42
0
AAGCACA,MIR-218
136
0
GTGCCAA,MIR-96
99
0
SKA3
Kinase Target
Kinase_CDK1
66
0
Kinase_AURKB
31
0
Kinase_PLK1
30
0
Kinase_CDK2
67
0
Kinase_ATR
15
0
Transcription Factor Target
V$E2F_Q3_01
44
0
V$E2F1_Q4_01
43
0
V$E2F_Q4_01
44
0
V$E2F1_Q6
58
0
V$E2F_Q6
45
0
miRNA Target
GTGCAAA,MIR-507
52
0
CTACTGT,MIR-199A
42
0.007
CCTGTGA,MIR-513
37
0.009
ATGCTGG,MIR-338
28
0.013
AAGCAAT,MIR-137
59
0.013
LeadingEdgeNum, the number of leading edge genes; FDR, false discovery rate from Benjamini and Hochberg from gene set enrichment analysis (GSEA). V$, the
annotation found in Molecular Signatures Database (MSigDB) for transcription factors (TF). TABLE 5 | The kinase and transcription factor-target networks of SKA1 and SKA3 in TCGA dataset (LinkedOmics). LeadingEdgeNum, the number of leading edge genes; FDR, false discovery rate from Benjamini and Hochberg from gene set enrichment analysis (GSEA). V$, the
annotation found in Molecular Signatures Database (MSigDB) for transcription factors (TF). LeadingEdgeNum, the number of leading edge genes; FDR, false discovery rate from Benjamini and Hochberg from gene set enrichment analysis (GSEA). V$, the
annotation found in Molecular Signatures Database (MSigDB) for transcription factors (TF). found to regulate SKA3 gene expression, which may need more
work to test it. regulated T cells. Thus, we assessed the association between
immune cell infiltration and SKA prognosis-related genes in
PDAC. Results showed that SKA1 was negatively correlated with
CD8+ T cell and macrophages, and the infiltration level of CD8+
T cells was significantly lower in the high SKA1 expression group,
indicating an immunosuppressive state. Similarly, the abundance
of CD4+ T cells, CD8+ T cells, and macrophages was decreased in
the SKA3 high expression group. There is a widespread belief that
CD4+ T cells compromise the majority of T cells in pancreatic
cancer and are positively associated with tumor metastasis and
negatively associated with OS (113). Some subsets of CD4+ T
cells may also be needed for antitumor immunity. Frontiers in Oncology | www.frontiersin.org DISCUSSION CD4+ helper
T cells may promote and maintain CTL memory, amplify T and
B cells, and help CTL resist negative regulation (114). CD4+ T
lymphocytes may inhibit tumor cell growth by cytolysis or by
regulating the TME (115). More detailed studies are necessary to
illustrate the specific role of each CD4+ T lymphocyte subset in
pancreatic cancer. Macrophages, participating in the production,
mobilization, activation, and regulation of immune effector
cells, have at least three major functions: antigen presentation, In recent years, immunotherapy has shown great promise
for cancer patients, especially those with refractory cancer. The
complexity of immune cells in tumor tissue influences the host
biological behavior and the outcome of immunotherapy (107). The TME, composed of non-cancerous cells in and around the
tumor, also performed an important role in the genomic analysis
of various tumors (108). CD8+ cytotoxic T lymphocytes (CTLs)
can specifically recognize major histocompatibility complex
(MHC) antigens, which are widely used in targeted therapy (109). A pioneer study revealed that infiltration of CD4+/CD8+ T
cells correlated with good prognosis in PDAC (110). Likewise,
a study by Masugi et al. (111) measured densities of CD8+
T cells in different locations of tumor. They found that high
CD8+ T cell density was significantly associated with prolonged
survival of 214 patients with pancreatic cancer (111). Wu
et al. also reported that the low infiltration level of CD4+ T
lymphocytes was associated with poor prognosis of pancreatic
cancer patients (112). GSEA suggested that SKA3 negatively November 2020 | Volume 10 | Article 553536 15 Liu et al. Liu et al. New Pancreatic Cancer Prognostic Biomarkers GURE 9 | Genomic alterations and DNA methylation level of spindle and kinetochore-associated genes. (A) OncoPrint of SKA1–3 alterations in TCGA cohort. ferent types of genetic alterations shown in different colors. (B,C) SKA1 and SKA3 expression in different CNV groups. Data were obtained from the cBioportal
tps://www.cbioportal.org/). (D,E) Two heatmaps showing the methylation profile of 11 CpG sites in SKA1 DNA locus and 15 CpG sites in SKA3 DNA locus. Data
ere obtained from UCSC Xena (http://xena.ucsc.edu/) using Infinium HumanMethylation450 BeadChip. FIGURE 9 | Genomic alterations and DNA methylation level of spindle and kinetochore-associated genes. (A) OncoPrint of SKA1–3 alterations in TCGA cohort. Different types of genetic alterations shown in different colors. (B,C) SKA1 and SKA3 expression in different CNV groups. Data were obtained from the cBioportal
(https://www.cbioportal.org/). Frontiers in Oncology | www.frontiersin.org DISCUSSION (D,E) Two heatmaps showing the methylation profile of 11 CpG sites in SKA1 DNA locus and 15 CpG sites in SKA3 DNA locus. Data
were obtained from UCSC Xena (http://xena.ucsc.edu/) using Infinium HumanMethylation450 BeadChip. phagocytosis, and immunomodulation (116). Cytokines such as
IL-1, IL-6, TNF-α, interferon (IFN)-α/β, IL-10, IL-12, and IL-18
released from macrophages could participate in the regulation of
immune/inflammatory responses. IL-12 stimulates proliferation
of activated T and NK cells, enhances NK and lytic activity of
CTLs, and induces IFN-γ production by T and NK cells. In
addition, they produce chemokines that stimulate lymphocyte
movement and regulation of migration lymphocytes from the
blood to tissues (117). Similarly to macrophages, DCs have
long been established as indispensable antigen-presenting cells (APCs), which act as systemic sentinels capable of responding
to endogenous and exogenous “danger” signals to initiate and
propagate immune responses to inciting antigens or induce
immune tolerance (118, 119). On sensing of appropriate cues,
DCs mature and express chemokine receptors and costimulatory
molecules under normal conditions. DCs promote immunity or
tolerance by sampling and presenting antigens to T cells and by
providing immunomodulatory signals through cell–cell contacts
and cytokines (120, 121). DCs are often associated with superior
cross-presentation of antigens, which results in stronger CD8+ November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 16 New Pancreatic Cancer Prognostic Biomarkers Liu et al. Liu et al. IGURE 10 | The impact of SKA1 and SKA3 gene expression and mutation on tumor immunity. (A) SKA1 expression showed significant negative correlation with
filtration levels of CD8+ T cells and macrophages. (B) SKA1 CNV influenced the infiltration levels of all the immune cells. (C) SKA3 expression showed significant
egative correlation with infiltration levels of CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. (D) Changes in SKA3 CNV altered infiltration
evels of B cells and CD4+ T cells. (E–G) Infiltration level of CD4+ T and CD8+ T cell and distribution of immune scores in high expression and low expression groups
f SKA1 and SK A3 in TCGA datasets. Immune scores were calculated using the ESTIMATE algorithm. (H,I) The expression distribution of SKA1 and SKA3 genes in
ifferent mutation status of TP53 and KRAS in TCGA dataset. *p < 0.05, **p < 0.01, ***p < 0.001. FIGURE 10 | The impact of SKA1 and SKA3 gene expression and mutation on tumor immunity. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fonc. 2020.553536/full#supplementary-material Supplementary Figure 1 | A flow diagram showing the selection process
of GEO datasets. Collectively, high expression of SKA1 and SKA3 predicts poor
prognosis of PDAC and may therefore be potential biomarkers
for this disease. These genes could regulate cancer-related
signaling pathways and inhibit immune infiltration within the
tumor in PDAC. Further prospective studies are required to verify
these molecular mechanisms. Supplementary Figure 2 | The distribution of (A) SKA1, (B) SKA2, (C) SKA3
genes in human normal organ tissues. Supplementary Figure 2 | The distribution of (A) SKA1, (B) SKA2, (C) SKA3
genes in human normal organ tissues. Supplementary Figure 3 | Kaplan-Meier survival curves showing the survival
significance of SKA1 DNA methylation in pancreatic cancer patients. (A) Total
methylation level of SKA1; (B) cg18558188; (C) cg18742986. Supplementary Figure 4 | The impact of SKA1 and SKA3 gene expression and
mutation on tumour immunity. (A,B) SKA1 expression showed no correlation with
infiltration levels of natural killer (NK) cells. (C,D) SKA3 expression is not
associated with infiltration levels of natural killer (NK) cells. ACKNOWLEDGMENTS This is a short text to acknowledge the contributions of specific
colleagues, institutions, or agencies that aided the efforts of the
authors. We would like to thank Sheng-xin self-study network for
providing technical support. ETHICS STATEMENT Ethical review and approval was not required for the study
on human participants in accordance with the local legislation
and institutional requirements. Written informed consent for
participation was not required for this study in accordance with
the national legislation and the institutional requirements. AUTHOR CONTRIBUTIONS XH, YC, and YL performed the data analysis work and aided
in writing the manuscript. QL and ZJ designed the study and
assisted in writing the manuscript. YL and QL edited the
manuscript. All authors read and approved the final manuscript. yp
Finally, some limitations exist in this study. First, this study
was performed using retrospectively collected data, which may
contain selection bias and recall bias. Secondly, our results
are based on bioinformatics analysis and thus underlying
biological mechanisms remain undefined. Thirdly, the protein
expression levels of SKA1 and SKA3 and their involvement
in the pathogenesis and progression of PDAC deserve further
studies. Despite these limitations, this is the first study to reveal
the potential correlation between SKA genes and PDAC tumor
immune escape and comprehensively explore the prognostic
value of SKA genes in patients with pancreatic cancer. Our results
may be informative for future research and clinical management
of PDAC patients. Despite these limitations, this is the first
study to reveal the association of SKA genes with immune
function regulation of PDAC patients. There have been no
reports demonstrating that SKA genes could regulate the immune
infiltration of PDAC. DATA AVAILABILITY STATEMENT Supplementary Table 1 | Details of the GEO datasets included in this study. Supplementary Table 1 | Details of the GEO datasets included in this study. The
datasets
presented
in
this
study
can
be
found
in
online repositories. The names of the repository/repositories Supplementary Table 2 | Correlations between prognosis-related gene and
Gene Markers of cancer escape in TCGA and GSE62452. Supplementary Table 2 | Correlations between prognosis-related gene and
Gene Markers of cancer escape in TCGA and GSE62452. DISCUSSION (A) SKA1 expression showed significant negative correlation with
nfiltration levels of CD8+ T cells and macrophages. (B) SKA1 CNV influenced the infiltration levels of all the immune cells. (C) SKA3 expression showed significant
negative correlation with infiltration levels of CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. (D) Changes in SKA3 CNV altered infiltratio
evels of B cells and CD4+ T cells. (E–G) Infiltration level of CD4+ T and CD8+ T cell and distribution of immune scores in high expression and low expression grou
of SKA1 and SK A3 in TCGA datasets. Immune scores were calculated using the ESTIMATE algorithm. (H,I) The expression distribution of SKA1 and SKA3 genes
different mutation status of TP53 and KRAS in TCGA dataset *p < 0 05 **p < 0 01 ***p < 0 001 FIGURE 10 | The impact of SKA1 and SKA3 gene expression and mutation on tumor immunity. (A) SKA1 expression showed significant negative correlation with
infiltration levels of CD8+ T cells and macrophages. (B) SKA1 CNV influenced the infiltration levels of all the immune cells. (C) SKA3 expression showed significant
negative correlation with infiltration levels of CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. (D) Changes in SKA3 CNV altered infiltration
levels of B cells and CD4+ T cells. (E–G) Infiltration level of CD4+ T and CD8+ T cell and distribution of immune scores in high expression and low expression groups
of SKA1 and SK A3 in TCGA datasets. Immune scores were calculated using the ESTIMATE algorithm. (H,I) The expression distribution of SKA1 and SKA3 genes in
different mutation status of TP53 and KRAS in TCGA dataset. *p < 0.05, **p < 0.01, ***p < 0.001. T cell immunity, and DCs can additionally support T helper 1
cell polarization of CD4+ T cells (122, 123). In tumor patients,
DCs acquire, process, and present tumor-associated antigens on
MHC molecules and provide costimulation and soluble factors to
shape T cell responses. However, a number of active mechanisms in the TME perturb DC functions, resulting in insufficient T cell
activation and, potentially, the induction of T cell tolerance to
tumor-associated antigens (119). Lymphocytes and macrophages
decreased significantly in patients with high expression of SKA1
and/or SKA3 in this study, leading to reduction of activation November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 17 Liu et al. DISCUSSION New Pancreatic Cancer Prognostic Biomarkers of immune cells, including CD8+ T and CD4 + T cells and
NK and lytic activity of CTLs, suggesting that they might have
an immune-excluded phenotype where CD4+ T/CD8+ T cells
were maintained in the stroma, restricting cancer immunity. Interestingly, the infiltration of cytotoxic CD8+ T cells might
be modified by immunotherapy. The above mechanism may
be potentially responsible for short-term survival in PDAC
patients correlates with increased SKA1 and/or SKA3 expresses. Analysis of immune scores in TME yielded similar outcomes
as those of immune infiltration. In addition, SKA1 and SKA3
were positively correlated with the expression of antiphagocytic-
related genes (CD47, PD-L1, and B2M). In general, these
findings indicate that SKA1 and SKA3 play a significant role in
the recruitment and regulation of immune-infiltrating cells in
PDAC, which may eventually influence patients’ survival time. Thus, we hypothesized that patients with high expression of
SKA1 or SKA3 might benefit from immunotherapy than those
with low expression. Further research is required to address
this hypothesis. and
accession
number(s)
can
be
found
in
the
article/
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absence of any commercial or financial relationships that could be construed as a
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presenting cells critical for T cell immunity. Cancer Cell. (2014) 26:638–52. doi: 10.1016/j.ccell.2014.09.007 Copyright © 2020 Liu, Jin, Huang, Che and Liu. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these terms. 123. Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H, Kohyama
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science.1164206 November 2020 | Volume 10 | Article 553536 Frontiers in Oncology | www.frontiersin.org 22
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EHD1 wt Allele
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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios EHD1 wt Allele National Cancer Institute National Cancer Institute Qeios ID: JDV791 · https://doi.org/10.32388/JDV791 Source National Cancer Institute. EHD1 wt Allele. NCI Thesaurus. Code C126474. Human EHD1 wild-type allele is located in the vicinity of 11q13 and is approximately 37 kb
in length. This allele, which encodes EH domain-containing protein 1, plays a role in
endocytic recycling. Qeios ID: JDV791 · https://doi.org/10.32388/JDV791 1/1
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Microalbuminuria and left ventricular hypertrophy among newly diagnosed black African hypertensive patients: a cross sectional study from a tertiary hospital in Uganda
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* Correspondence: jgnabbaale@gmail.com
1Uganda Heart Institute, Mulago Hospital, P.O. Box 7051, Kampala, Uganda
Full list of author information is available at the end of the article Nabbaale et al. BMC Research Notes (2015) 8:198
DOI 10.1186/s13104-015-1156-2 Nabbaale et al. BMC Research Notes (2015) 8:198
DOI 10.1186/s13104-015-1156-2 © 2015 Nabbaale et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. RESEARCH ARTICLE Open Access Microalbuminuria and left ventricular hypertrophy
among newly diagnosed black African
hypertensive patients: a cross sectional study
from a tertiary hospital in Uganda Microalbuminuria and left ventricular hypertrophy
among newly diagnosed black African
hypertensive patients: a cross sectional study
from a tertiary hospital in Uganda Juliet Nabbaale1*, Davis Kibirige2, Emmanuel Ssekasanvu3, Elias S Sebatta1, James Kayima1, Peter Lwabi1
and Robert Kalyesubula3,4 Study population and sampling technique The reasons for this growing burden are multiple,
ranging from socio-economic changes and genetic in-
fluence. At a genetic level, there is growing evidence
showing an association between elevated diastolic BP
and CaMK4 affecting endothelial functions like controlling
vascular resistance hence increasing the risk of HT [4]. The study population comprised of 256 newly diagnosed
eligible adult hypertensive patients attending the outpatient
hypertension clinic or admitted on the cardiology ward at
Mulago national referral and teaching hospital and the
Uganda Heart Institute in Kampala, Uganda. The inclusion
criteria into the study were: newly diagnosed adult hyper-
tensive patients (aged ≥18 years of age) and those who gave
written informed consent to participate in the study. Recently, a burgeoning burden of HT has been de-
scribed both in rural and urban areas of Uganda. One
earlier community based study conducted in the former
Teso district in 1941 reported a very low frequency of
HT of 2.9 % among adults aged 21-50 years [5]. Another
similar population based study was conducted in the
early 1960’s in Kasangati, a rural community outside of
Kampala city revealed prevalence of 13.7 % [6]. Recent
similar studies have documented age standardized HT
prevalence to range between 14.6 % to 30.5 % [7–10]. In
one rural study, a pre HT, an antecedent to clinically de-
tected HT was reported to occur in 33.9 % of the study
participants [9]. Patients
with
documented
chronic
kidney
disease,
catheterization, fever (temperature above 37.5 °C), con-
gestive cardiac failure, female patients who were menstru-
ating, active urinary tract infection, patients with diabetes
mellitus, those taking ACEIs, obese patients and those
who had been under vigorous exercise in the past 24 h
were excluded from the study (Fig. 1). Vigorous exercise
was defined as that which could cause sweating, heavy
breathing or fast heart beat like brisk walking in our Afri-
can context. They were consecutively recruited over a period of
5 months (June to October 2012) until the calculated
sample size was attained. Microalbuminuria is an early indicator of renal damage
and has been demonstrated as one of the principal pre-
dictive factors of cardiovascular (CV) complications, all
cause and cardiovascular mortality independent of the
traditional risk factors like dyslipidemia, hypertension
[11, 12]. Study design and setting Recent evidence demonstrates that cardiovascular diseases
(CVD) are the commonest cause of deaths globally. Low- and middle-income countries are disproportion-
ally affected, with over 80 % of CVD deaths occurring
in these countries [1, 2]. Hypertension (HT) is a grow-
ing public health problem and it is now being widely
reported in many rural and urban parts of Sub Saharan
Africa (SSA) as one of the commonest cause of morbidity
and mortality [3]. This cross sectional study was carried out in the out-
patient hypertension clinic, the cardiology ward of Mulago
national referral and teaching hospital, Kampala Uganda
and the Uganda Heart Institute, Kampala Uganda. Mulago
hospital serves as the national referral hospital and univer-
sity teaching hospital for the Makerere University College
of Health Sciences. It has a 1500 bed capacity. Study population and sampling technique Left ventricular hypertrophy (LVH) determined
either by standard 12-lead electrocardiography (ECG) or
echocardiography is also a marker of subclinical organ
damage related to hypertension and an independent pre-
dictor of cardiovascular morbidity/mortality [13]. Abstract Background: Microalbuminuria is an early manifestation of kidney damage and independently predicts cardiovascular
disease (CVD). Left ventricular hypertrophy (LVH) is also an early marker of cardiac manifestation of target organ
damage among hypertensive patients. The prognostic significance of microalbuminuria and its correlation with left
ventricular hypertrophy has not been extensively studied in African adult hypertensive populations. This study aimed at
determining the prevalence of microalbuminuria, LVH in patients with microalbuminuria and the correlation between
microalbuminuria and LVH among newly diagnosed black adult hypertensive patients attending a large outpatient
hypertension clinic or admitted on the cardiology ward at Mulago national referral and teaching hospital and Uganda
Heart Institute in Kampala, Uganda. Methods: In this cross-sectional study, 256 newly diagnosed eligible black adult hypertensive patients attending the
outpatient hypertension clinic or admitted on the cardiology ward at Mulago national referral and teaching hospital
and the Uganda Heart Institute, Kampala Uganda were consecutively recruited over a period of 5 months. Data on
socio-demographics, clinical and laboratory findings of the study participants was collected using a pre tested
questionnaire. Two spot urine samples were collected to assess for microalbuminuria. Echocardiography (ECHO)
was done to assess for the left ventricular mass index using the formula of Teicholz as evidence for early hypertensive
heart disease. Results: The mean age/standard deviation of the study participants was 54.3 ± 6.2 years with a female predominance
(162, 63.3 %). The prevalence of microalbuminuria among newly diagnosed hypertensive patients was 39.5 %. The
prevalence of LVH among patients with microalbuminuria was found to be 17 %. There was a positive correlation
between microalbuminuria and left ventricular hypertrophy among the newly diagnosed adult hypertensive patients
at Mulago Hospital (r = 0.185, p = 0.003). Conclusions: This study demonstrates that microalbuminuria is highly prevalent among newly diagnosed black
hypertensive patients and in the presence of LVH. There is also a positive correlation between microalbuminuria and
LVH among newly diagnosed hypertensive patients. Since it is a less costly and readily available test, it can be used to
predict presence of LVH especially in resource limited settings where ECHO services are not readily available. Keywords: Microalbuminuria, left ventricular hypertrophy, Left ventricular mass index, Newly diagnosed hypertensive
patients, Cross sectional study, Africa, Uganda Page 2 of 7 Page 2 of 7 Nabbaale et al. BMC Research Notes (2015) 8:198 Sample size calculation Using prevalence of 21.3 % of microalbuminuria among
newly diagnosed hypertensive patients reported by Rayner
B et al in South Africa in 2006 [16], with an 80 % power
and a two sided α < 0.05, a sample size of 256 was esti-
mated using the Kish Leslie (1965) formula. Regression of microalbuminuria and LVH with standard
anti hypertensive therapy (mainly with angiotensin con-
verting enzyme inhibitors) is associated with a significant
reduction in the associated CV morbidity and mortality
[14]. Beta blockers especially the cardio selective ones also
possess a role in LVH regression, albeit a minor role [15]. Data collection
A
d A pre tested questionnaire was used to collect the
socio-demographic and clinical data of the study partic-
ipants. Blood pressure was measured using standard
procedures using a standard Manual Dekamet Accoson
MK3® mercury sphygmomanometer machine. Using stand-
ard methods, anthropometric measurements of weight in
kilograms (kg) and the height in meters (m) were also per-
formed and body mass index (BMI) calculated using the
formula, BMI = weight in kg/height in m2. There is paucity of studies on the burden of microalbu-
minuria and its correlation with LVH among black African
newly diagnosed adult HT patients in Uganda. This study
therefore aimed at examining the burden of microalbumi-
nuria and its correlation with LVH in newly diagnosed
adult HT patients attending the outpatient hypertension
clinic or admitted on the cardiology ward at Mulago na-
tional referral and teaching hospital and the Uganda Heart
Institute, Kampala Uganda. Ethical consideration
h
d This study was approved by the department of medicine,
College of Health Sciences Makerere University and the
research and ethics committee of the School of Medi-
cine, Makerere University Kampala Uganda. All the
study participants provided written informed consent to
participate in this study. Assessment of microalbuminuria The study participants were first instructed about the
process of collecting a midstream urine sample. They Page 3 of 7 Nabbaale et al. BMC Research Notes (2015) 8:198 History, exam and questionnaire
filled. Enrolled 350 newly diagnosed
hypertensive patients at MOPD,
UHI, 3BEM, 4C Cardiology
12 Active UTI
5 Febrile T>37.5°C
20 Documented kidney
disease
9 CCF
21 DM
7 Menstruating females
20 Obese
Urinalysis for microscopy and
microalbuminuria and then cardiac
ECHO performed for LVH. 256 participants fulfilled the
inclusion criteria. Fig. 1 Patient flow chart on recruitment Enrolled 350 newly diagnosed
hypertensive patients at MOPD,
UHI, 3BEM, 4C Cardiology 256 participants fulfilled the
inclusion criteria. History, exam and questionnaire
filled. Urinalysis for microscopy and
microalbuminuria and then cardiac
ECHO performed for LVH. Fig. 1 Patient flow chart on recruitment Fig. 1 Patient flow chart on recruitment were provided with two urine containers and requested
to provide two samples of about 10 mls of mid-stream
urine. One sample was collected in a sterile bottle for
urinalysis including microscopy of the urine sediment. The second one was collected in a urine container with
a preservative called 0.2 % sodium-azide for measuring
of urinary albumin. The urine samples were sent to the
Uganda Heart Institute laboratory for analysis which was
done by the Immuno-turbidimmetric Method (RAN-
DOX). Microalbuminuria was defined as a random urine
albumin level between 30 and 299 mg/dl. determine strength of association for categorical risk
factors. A p-value of less than 0.05 was considered
significant. Association between microalbuminuria, LVH and the
study variables were compared to test the strength of
the association between them and the variables that in-
cluded age, sex, education level, marital status and occu-
pation. The
prevalence
of
microalbuminuria
was
determined using proportions. The correlation between
microalbuminuria
and
LVH
was
determined
using
Spearman’s rank correlation analysis technique. Echocardiography (ECHO) performance for assessing LVH
A trans thoracic ECHO was done by an experienced car-
diologist with experience in echocardiography and clin-
ical cardiology using a GE Vivid 7 echocardiography
machine LV mass index was calculated using Teicholz
formula where increased LV Mass index was values >
125 g/m2. Data analysis Data was coded and double entered in EpiData, cleaned
and exported to STATA10 for analysis. Descriptive ana-
lysis of the study population was conducted. Pearson
chi-square X2 and odds ratio analysis was applied to Fig. 1 Patient flow chart on recruitment Prevalence of LVH among study participants with
microalbuminuria and according to age categories Seventy two percent (72 %) of the study participants
with controlled hypertension had microalbuminuria,
while 8 % of those who had stage 1 hypertension had
microalbuminuria. Only 2 % of tho study participants
who had stage 2 hypertension had microalbuminuria. Sixty eight (68 %) of the study participants who had con-
trolled hypertension had LVH while 12 % of the partici-
pants who had stage 1 hypertension had LVH and 85 %
of the participants in stage 2 hypertension had LVH. The prevalence of LVH among the study participants
with microalbuminuria was 17 %, higher than the overall
study prevalence of LVH of 14.1 % (shown in Table 3). LVH was more prevalent in the older age groups of 55–74
(15, 41.7 %) and 75–94 years (6, 16.7 %) compared to the
younger age groups (Table 4). Table 1 Socio-demographic characteristics of study participants
Characteristics
Frequency
Percentage (%)
Gender
Female
162
63.3
Male
94
36.7
Age
18 – 34
34
13.3
35 – 54
110
43
55 – 74
85
33.2
75 – 94
27
10.5
Level of education
No Formal education
42
16.4
Primary education
88
34.4
Secondary education
66
25.8
Tertiary education
60
23.4
Marital Status
Single
30
11.7
Married
165
64.5
Divorced
8
3.1
Cohabiting
1
0.4
Separated
13
5.1
Widowed
39
15.2 Table 1 Socio-demographic characteristics of study participants
Characteristics
Frequency
Percentage (%) Socio demographic characteristics of study participants The mean age/standard deviation of the study partici-
pants was 54.3 ± 6.2 years with a female predominance
(162, 63.3 %). The age range of the study participants Page 4 of 7 Nabbaale et al. BMC Research Notes (2015) 8:198 Table 2 Age-adjusted prevalence of microalbuminuria in age
groups
Age
Frequency (N)
Non–age adjusted (%)
Age-adjusted
prevalence (%)
18–34
34
12
1.6
35–54
110
42
18
55–74
85
32
10.6
75–94
27
14
1.48
Total
256
100
31.7 % was from 17 to 94 years with majority aged between 35–54
years (43.0 %) and 55–74 years (33.2 %). Table 1 and Fig. 1
summarize the socio demographic characteristics and the
flow chart of the study participants respectively. Prevalence of microalbuminuria and LVH The overall prevalence of microalbuminuria and LVH
among the study participants was 39.5 % and 14.1 % re-
spectively. The age adjusted prevalence of microalbumi-
nuria (31.7 %) was lower compared to the unadjusted
prevalence as shown in Table 2. Prevalence microalbuminuria and LVH according to BMI Prevalence microalbuminuria and LVH according to BMI
Microalbuminuria was detected in 72.3 %, 18.2 % and
9.1 % of the study participants with a normal BMI,
under weight and overweight respectively. LVH occurred
in 66.7 %, 19.4 % and 13.9 % of study participants with
normal BMI, underweight and overweight respectively. According to the different categories of HT, 72 %, 8 %
and 2 % of the study participants with controlled HT,
stage 1 and stage 2 HT had microalbuminuria respect-
ively. LVH was highly prevalent among the patients with
stage 2 hypertension (85 %) and controlled HT (12 %). Only 12 % of the participants who had stage 1 hyperten-
sion had LVH. Prevalence of hypertensive heart failure clinically and by
echocardiography 52.3 % of the study participants had diastolic heart failure
and 18.7 % of the study participants had systolic heart
failure on echocardiography. However, 46.7 % of the
same study participants had Class 1 NYHA symptoms,
24.1 % were in class 11, 9.2 % were in class 111 and
only 2 % of the study participants were in class 1 V. Discussion To our knowledge, this is the first study to assess the bur-
den of microalbuminuria and its correlation with LVH
among newly diagnosed adult HT patients in Uganda, a
country with a growing burden of CVD. It unequivocally
illustrates a high overall prevalence of microalbuminuria
and LVH of 39.5 % and 14.1 % respectively, both features
of target organ damage among the study participants in
Uganda. These proportions represent a significant group
of newly diagnosed adult hypertensive individuals who are
at an increased risk of CV morbidity and mortality. The
study also noted a positive correlation between LVH and
microalbuminuria. In another similar study done in Nigeria, the preva-
lence of microalbuminuria in 96 newly diagnosed non
diabetic hypertensive patients was 32.3 % compared to
6.3 % in age and sex matched controls [19]). In a multi
racial South African study among 1901 ambulatory
hypertensive patients, the overall prevalence of microal-
buminuria was 21.3 % [16]). The independent predictors
of microalbuminuria were severe hypertension (OR 2.9,
p < 0.0001), type 2 diabetes (OR 2.5, p < 0.002), and
Asian ethnic group (OR 2.0, p = 0.02). The reported prevalence of microalbuminuria in our
study is comparable to what has been reported in one
large international multi-center study [17] and in other
African hypertensive populations [16, 18, 19]. In the inter-
national, observational, practice-based study i-SEARCH
(Survey for Evaluating Microalbuminuria Routinely by
Cardiologists in patients with Hypertension) that was
designed to assess the frequency with which microalbumi-
nuria occurred in a large outpatient population of 21,050
patients who were currently treated or newly diagnosed
with hypertension and were under professional care in 26
countries, a high overall prevalence of microalbuminuria
of 58.4 % noted [17]. LVH in our study was documented in 14.1 % of the
study participants, a finding comparable to that in the
South African study (18.9 %) [16]. In our study, LVH was
also more frequent in patients with microalbuminuria
compared to those without (17 % vs. 12.2 %). A higher fre-
quency of LVH in patients with microalbuminuria has also
been described in other similar studies [19–21]. Correlation between microalbuminuria and LVH among
study participants As shown in Table 5, there was a positive correlation be-
tween microalbuminuria and LVH (r = 0.185, p = 0.003). This close association is further illustrated by the bivariate
scatter plot (Fig. 2). Page 5 of 7 Nabbaale et al. BMC Research Notes (2015) 8:198 Di
i
d
ib d
b
(i SEARCH
d )
h
l
f
Table 4 Prevalence of LVH among different age categories
LVH
15–34
35–54
55–74
75–94
Total
Present
Frequency
2 (5.6 %)
13 (36.1 %)
15 (41.7 %)
6 (16.7 %)
36 (14.1 %)
Absent
Frequency
32 (14.5 %
97 (44.1 %)
70 (31.8 %)
21 (9.6 %)
220 (85.9 %)
Total
34
110
85
27
256 described above (i-SEARCH study), the prevalence of
microalbuminuria among the 476 patients recruited
from 40 cardiology centers was 67.8 % [18]. This high
prevalence in the Moroccan study compared to the one
documented in the global study (58.4 %) could probably
be explained by the high proportion of patients with
suboptimal blood pressure control (81.4 %) and presence
of multiple co-existing CV risk factors like diabetes
mellitus, dyslipidemia and obesity which influence
microalbuminuria (≥65 % of the patients had ≥3 CV
risk factors). Discussion Microalbuminuria was significantly related to the pres-
ence of specific predictors, including male gender, abnor-
mally high waist circumference, increased blood pressure
levels (systolic ≥120 mmHg, diastolic ≥100 mmHg), cre-
atinine clearance ≥50 ml/min, or clinical conditions such
as diabetes mellitus, congestive heart failure, history of
cerebral pathology, and peripheral arterial disease. The correlation between microalbuminuria and LVH
in our study was found to be positive with a correlation
coefficient of 0.185 and p value of 0.003. Similarly,
microalbuminuria has been shown to positively correlate
with LVH in other large studies [22, 23] and other car-
diac abnormalities like left ventricular dysfunction and
ischemic heart disease [24]. Among African hypertensive populations, in a study
performed in Morocco that was part of the global study Microalbuminuria as a marker of early renal damage
reflects widespread vascular damage (microangiopathy),
a pro inflammatory state (with increased levels of IL-6,
TNF-α, CRP and fibrinogen) and ensuing endothelial
dysfunction. In addition to hypertension, microalbumi-
nuria can also result from other unfavorable metabolic
profile like hyperinsulinaemia and dyslipidemia [11, 12]. This hence augments development and progression of
atherosclerotic disease. Table 5 Correlation between microalbuminuria and LVH
Microalbuminuria LVH
Spearman's
rank
correlation
Microalbuminuria Correlation
Coefficient
1.000
0.185
p-value
0.003
N
256
256
LVH
Correlation
Coefficient
0.185
1.000
p-value
0.003
N
256
256 Table 5 Correlation between microalbuminuria and LVH
Microalbuminuria LVH With advancing stages of HT, it has been illustrated
that both microalbuminuria and LV mass increase with
worsening BP levels. LVH as a marker of target organ Page 6 of 7 Nabbaale et al. BMC Research Notes (2015) 8:198 Fig. 2 Scatter plot showing the correlation between microalbuminuria and LVH microalbuminuria can be more simply achieved by
using an efficient and accurate method of taking a spot
urine sample or an early morning urine specimen to
minimize changes that occur during the day, this
method does not require any special preparation prior
to collecting the urine sample. However, 24-h and over-
night urine collections are time consuming and often
inaccurate particularly because incomplete collections
are frequent. Vigorous exercise in 24 h preceding the
albuminuria test could have increased the prevalence in
our study patients however there was an attempt to
assess this cause, albeit subjectively. Discussion damage and a well known predictor of CV events has an
independent association with a pro inflammatory state,
and in particular elevated fibrinogen levels, which is as-
sociated with development of atherosclerosis [25]. Other than persistently increased volume or pressure
overload on the left ventricle as occurs in HT, LVH can
develop in aortic stenosis, hypertrophic cardiomyopathy
and physiological LVH as seen in athletes. Abbreviations
d Abbreviations
BMI: Body mass index (BMI) which is calculated using the formula
BMI = weight in kg/height in m2.; CVD: Cardiovascular disease.;
CV: Cardiovascular.; ECHO: Echocardiography.; ECG: Electrocardiography.;
HT: Hypertension.; IVS: Inter ventricular septal thickness.; LVH: Left
ventricular hypertrophy.; LVPW: Left ventricular posterior wall thickness.;
SSA: Sub Saharan Africa.. Screening for urine albumin excretion should be ex-
tensively adopted in clinical care as a relatively simple,
readily available and cheap process to facilitate early
vascular disease detection as a strategy of averting HT
related complications like LVH and optimal management
in Uganda and SSA. Conclusions This study demonstrates a high prevalence of microalbu-
minuria and LVH, predictors of CV morbidity and mortal-
ity in this patient subgroup in Uganda and a corresponding
positive correlation between both conditions. Competing interests Competing interests
The authors declare that they have no competing interests. Authors’ contributions
f Microalbuminuria was assessed on a single occasion
using a spot urine sample although guidelines recom-
mend triple testing (2 out of 3 tests need to be posi-
tive). However, it has been suggested that diagnosis of JN, ES, SE, JK made significant contributions to the conception and design
of the study. PL and ES made substantial contribution to data analysis. JN
drafted the initial manuscript. All authors reviewed the manuscript and
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1 1Uganda Heart Institute, Mulago Hospital, P.O. Box 7051, Kampala, Uganda. 2Department of Medicine, Uganda Martyrs Hospital Lubaga, Kampala,
Uganda. 3Department of Medicine, Makerere University College of Health
Sciences, Kampala, Uganda. 4Department of Physiology, Makerere University
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Uganda. 3Department of Medicine, Makerere University College of Health
Sciences Kampala Uganda 4Department of Physiology Makerere University 1Uganda Heart Institute, Mulago Hospital, P.O. Box 7051, Kampala, Uganda. 2Department of Medicine, Uganda Martyrs Hospital Lubaga, Kampala,
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and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
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and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
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and take full advantage of: 18. Habbal R, Sekhri A, Volpe M. Prevalence of microalbuminuria in
hypertensive patients and its associated cardiovascular risk in clinical
cardiology: Moroccan results of the global i-SEAR CH survey - a sub-analysis
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of a survey with 21 050 patients in 26 countries worldwide. Cardiovasc J Afr. 2010;21:200–5. • Convenient online submission 19. Busari O, Opadijo G, Olarewaju T, Omotoso A, Jimoh A. Electrocardiographic
correlates of microalbuminuria in adult Nigerians with essential
hypertension. Cardiol J. 2010;17(3):281–7. 20. Forlemu A, Menanga A, Ashuntantang G, Kingue S. Urinary Protein
Excretion Is Associated with Left Ventricular Hypertrophy in Treatment-
Naïve Hypertensive Patients in an African Hospital Setting. Cardiorenal
Med. 2013;3:57–62. 20. Forlemu A, Menanga A, Ashuntantang G, Kingue S. Urinary Protein
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Med. 2013;3:57–62.
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Mangotoxin production of Pseudomonas syringae pv. syringae is regulated by MgoA
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© 2014 Carrión et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. * Correspondence: cazorla@uma.es
1Departamento de Microbiología, Facultad de Ciencias, Instituto de
Hortofruticultura Subtropical y Mediterránea “La Mayora”-Universidad de
Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC),
Universidad de Málaga, Campus de Teatinos, 29071 Málaga, Spain
Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Open Access Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Mangotoxin production of Pseudomonas syringae
pv. syringae is regulated by MgoA Víctor J Carrión1,3,4, Menno van der Voort3, Eva Arrebola2, José A Gutiérrez-Barranquero1,5, Antonio de Vicente1,
Jos M Raaijmakers3,4 and Francisco M Cazorla1* Keywords: Antimetabolite toxin, mgo operon, GacS/GacA, Plant-microbe interaction Keywords: Antimetabolite toxin, mgo operon, GacS/GacA, Plant-microbe interaction Abstract Background: The antimetabolite mangotoxin is a key factor in virulence of Pseudomonas syringae pv. syringae
strains which cause apical necrosis of mango trees. Previous studies showed that mangotoxin biosynthesis is
governed by the mbo operon. Random mutagenesis led to the identification of two other gene clusters that affect
mangotoxin biosynthesis. These are the gacS/gacA genes and mgo operon which harbors the four genes mgoBCAD. Results: The current study shows that disruption of the nonribosomal peptide synthetase (NRPS) gene mgoA
resulted in loss of mangotoxin production and reduced virulence on tomato leaves. Transcriptional analyses by
qPCR and promoter reporter fusions revealed that mbo expression is regulated by both gacS/gacA and mgo genes. Also, expression of the mgo operon was shown to be regulated by gacS/gacA. Heterologous expression under the
native promoter of the mbo operon resulted in mangotoxin production in non-producing P. syringae strains, but
not in other Pseudomonas species. Also introduction of the mbo and mgo operons in nonproducing P. protegens
Pf-5 did not confer mangotoxin production but did enhance transcription of the mbo promoter. Conclusions: From the data obtained in this study, we conclude that both mbo and mgo operons are under the
control of the gacS/gacA two-component system and that the MgoA product acts as a positive regulator of
mangotoxin biosynthesis. Background syringae
UMAF0158 and derivatives mutants in mboA, mgoA and
gacA genes were used to obtain initial cultures in 10 ml of
LB broth. The bacterial strains were grown during 24 h at
28°C to prepare an optimal bacterial inoculum with an op-
tical density of 0.8 at 600 nm (approximately 109 cfu ml-1). One ml from these bacterial inocula was used to inoculate
100 ml of PMS broth. The cultures were incubated at 22°C
under orbital shaking at 150 rpm until the stationary phase. Samples were collected every 6 or 12 h to monitor the bac-
terial growth. Bacterial cfu per sample were determined by
10-fold serial dilutions on KMB plates. At the same time,
the mangotoxin production assessment was performed by a
cell-free filtrate dilution sequence at 50%. The mangotoxin
production is measured using arbitrary units, which can be
defined as the relative toxic volume of cell free filtrates of li-
quid cultures, which produces an inhibition halo of 18 mm
in diameter under standard assay conditions [2]. The meth-
odology presented a detection threshold of 0.5 toxic units,
due to the diameter of the wells where the cell-free filtrate
were deposited (9 mm). type III secretion and effector genes [24,25]. However, in
P. syringae pv. syringae B728a, GacA appears not to be
required for hrp gene expression [25]. The mgo operon is composed of four genes, mgoBCAD
[4,7]. Mutants in each gene belonging to the mgo operon
showed an alteration (mgoB mutant) or lack of mango-
toxin production (mgoC, mgoA and mgoD mutants). These genes encode for different hypothetical proteins
with predicted domains for a haem oxygenase (MgoB), a
p-aminobenzoate N-oxygenase (MgoC), a nonribosomal
peptide synthetase (MgoA), and a polyketide cyclase/
dehydrase or lipid transporter (MgoD) [4,7]. The pre-
dicted amino acid sequence of MgoA suggests only one
amino acid activation module and 14 conserved do-
mains, including aminoacyl adenylation, condensation,
thiolation, and additional reduction domains [4]. Genes
homologous to the mgo operon have been found in the
genomes of most Pseudomonas spp., with the exception
of P. protegens Pf-5 and CHAO [26,27]. Recent studies
on the pvf gene cluster in P. entomophila, a homologue
of the mgo operon, suggested that it affects virulence
[28]. Almost all the fluorescent Pseudomonas spp. lack
the mbo operon [29,30], but the mgo operon is con-
served
in
all
of
them
(except
P. protegens
Pf-5)
[4,7,26-28]. Background operon) is composed of six genes, mboABCDEF. Disruption
of each of these genes resulted in mangotoxin deficient mu-
tants and constitutive expression of the mbo operon in
non-mangotoxin producing P. syringae strains conferred
mangotoxin production [6]. Screening of the random mu-
tant library also led to the identification of several other
genes that may be involved in the regulation of mangotoxin
biosynthesis [4]. These included the gacS/gacA genes and
the so-called mangotoxin generating operon mgo [6,7]. Pseudomonas syringae is one of the most ubiquitous plant
pathogens, causing various economically important diseases
[1]. The present study focuses on P. syringae pv. syringae
UMAF0158 (CECT 7752) which causes apical necrosis of
mango [2,3]. The antimetabolite mangotoxin is a key viru-
lence factor of strain UMAF0158 [4,5]. This toxin is pro-
duced in the early exponential growth phase and inhibits
ornithine N-acetyl transferase, a key enzyme belonging to
the ornithine/arginine biosynthetic pathway [2]. The GacS/GacA two-component regulatory system is
highly conserved in Gram-negative bacteria and is in-
volved in a variety of functions, including pathogenicity
[8], quorum sensing [9,10], secondary metabolite pro-
duction
[11-14]
and
biofilm
formation
[15-17]. In
Pseudomonas syringae, the GacS/GacA two-component
system regulates the production of the phytotoxins syr-
ingomycin and syringopeptin [18-20], tabtoxin [21,22]
and phaseolotoxin [23]. In P. syringae pv. tomato
DC3000, GacS/GacA regulate the hrpR, hrpS, and hrpL
genes, which are required for the activation of the Hrp Random mini-Tn5 mutagenesis followed by cloning, se-
quencing and heterologous expression recently led to the
identification of the gene cluster that governs mangotoxin
biosynthesis [6]. The mbo operon (mangotoxin biosynthetic * Correspondence: cazorla@uma.es
1Departamento de Microbiología, Facultad de Ciencias, Instituto de
Hortofruticultura Subtropical y Mediterránea “La Mayora”-Universidad de
Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC),
Universidad de Málaga, Campus de Teatinos, 29071 Málaga, Spain
Full list of author information is available at the end of the article Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Page 2 of 13 initially incubated at 22°C for 24 h, and then at 37°C for an
additional 24 h [2]. To evaluate mangotoxin activity, the
same plate bioassay was carried out with the addition of
100 μl of a 6 mM solution of N-acetyl-ornithine or
L-ornithine to the double layer of E. coli [2]. To deter-
mine growth characteristics of representative strains, the
wild type mangotoxin-producing P. syringae pv. Bacterial strains and culture conditions The wild type strain P. syringae pv. syringae UMAF0158
(CECT 7752) and the collection of selected derivative
mutants used in this study (Table 1) were grown on
Pseudomonas agar F (Difco) plates, in liquid King’s
medium B (KMB) [31] or in Pseudomonas minimal
medium (PMS) [32] at 28°C. Escherichia coli strain
DH5α was used as a host for plasmid complementation
experiments. It was routinely grown on Luria-Bertani
(LB) plates or in LB broth at 37°C. Antibiotics for selec-
tion of P. syringae pv. syringae UMAF0158 and E. coli
derivatives were ampicillin (100 mg L-1), kanamycin
(50 mg L-1), gentamycin (30 mg L-1) or tetracycline
(25 mg L-1). Complementation experiments
A f
f
l Complementation experiments
DNA fragments of approximately 7 kb containing the mgo
and mbo operons, including the promoter and terminator
regions, were obtained by PCR using specific primers
(Additional file 1: Table S1) and high fidelity polymerase
(Phusion DNA polymerase, Finnzymes). The PCR amplifi-
cation products were cloned in pGEM-T Easy (Promega),
and the plasmids obtained were digested with XbaI for the
mgo operon and with EcoRI and PstI for the mbo operon. After the digestion, both operons fragment were obtained
from gel with the NucleoSpin kit (GE Healthcare) and
cloned into the correspondent shuttle vectors, pBBR1MCS-
5 [36] for the mgo operon and pMP220 [37] for the mbo
operon, which were digested, dephosphorylated (shrimp al-
kaline phosphatase; Promega), and purified with the
NucleoSpin kit according to the manufacturer’s instruc-
tions. E. coli DH5α was transformed with the plasmids ob-
tained, by heat shock transformation [38], and transformed
colonies were selected on LB agar plates supplemented with
gentamicin (30 mg L-1) in the case of pBBR1MCS-5 and
tetracycline (25 mg L-1) for pMP220. Plasmids with the
mgo and mbo operon cloned were obtained (Table 1). Cor-
rect integration and orientation of the fragments was veri-
fied by PCR and restriction analysis of isolated plasmids
(data not shown). The pLac-mgoBCAD construct was sub-
sequently electroporated into the mboA, mgoA and gacA Methods Bacterial strains and culture conditions Background To date, however, the functions of mgo op-
eron are yet unknown. The overall objective of this study was to get insight
into the role of the mgo operon in regulation of mango-
toxin production in P. syringae pv. syringae UMAF0158
and unravel the interplay between mgo, mbo and the
gacS/gacA two-component regulatory system. Mangotoxin production assay protegens
Pf-5
Non mangotoxin producer, mbo and mgo operon absent
[35] Plasmids
pBBR1MCS-5
4.7 kb broad-host-range cloning vector, Gmr
[36]
pGEM-T
3.0 kb cloning vector, Apr
Invitrogen
pGEM-TBCAD
mgoBCAD cloned in pGEM-T, Apr
This study
pLac-
mgoBCAD
mgoBCAD cloned in pBBR1MCS-5 downstream the lacZ promoter in the vector, mgo operon expression under its own
and PLAC promoter, Gmr
This study
pLac-
mboABCDEF
mboABCDEF cloned in pBBR1MCS-5 downstream the lacZ promoter in the vector, mbo operon expression under its own
and PLAC promoter, Gmr
[6]
pLac-
mboFEDCBA
mboABCDEF cloned in pBBR1MCS-5 in the opposite direction than the lacZ promoter in the vector, mbo operon expres-
sion under its own promoter, Gmr
[6]
pMP220
Promoter-probe vector containing a promoterless LacZ gene, Tetr
[37]
pMP-
mboABCDEF
mboABCDEF cloned in promoter-probe vector containing a promoterless LacZ gene, mbo operon expression under its
own promoter, Tetr
This study
pMP::PmboI
pMP220 vector containing the mbo operon promoter, Tetr
[6]
aCECT: Spanish Type Culture Collection, Spain. mboABCDEF cloned in promoter-probe vector containing a promoterless LacZ gene, mbo operon expression under its
own promoter, Tetr aCECT: Spanish Type Culture Collection, Spain. Mill.) cv. Hellfrucht Frühstamm maintained in vitro
using
Murashige
and Skoog
medium
(MS,
Sigma-
Aldrich) [4,5]. Bacterial suspensions from exponentially
growing cultures were adjusted to 108 cfu ml-1. The leaf-
lets were inoculated by placing six 10 μl drops of the
bacterial suspension on six different points on the same
leaflet. Inoculations were then carried out by piercing
through the droplets with a sterile entomological pin. The leaflets were maintained in MS media at 22°C and a
16:8-h light: dark photoperiod. Six tomato leaflets were
used to evaluate each strain. Detached leaflets only inoc-
ulated with sterile distilled water were included in all
experiments as a control. These experiments were
repeated three times. The development of necrotic
symptoms at the inoculation points (n = 108) was mutants, and the wild-type strains P. syringae pv. syringae
UMAF0158 and P. protegens Pf-5. The pMP-mboABCDEF
construct was transformed in P. protegens Pf-5 which previ-
ously contain the pLac-mgoBCAD, therefore this bacteria fi-
nally harbored both operons, the mgo and mbo operon. Transformed cells were selected on KMB agar supple-
mented with correspondent antibiotics. The presence of the
different plasmids was confirmed by PCR analysis with spe-
cific primers for pBBR1MCS-5 and pMP220 and plasmid
profiling. Mangotoxin production assay protegens
Pf-5
Non mangotoxin producer, mbo and mgo operon absent
[35]
Plasmids
pBBR1MCS-5
4.7 kb broad-host-range cloning vector, Gmr
[36]
pGEM-T
3.0 kb cloning vector, Apr
Invitrogen
pGEM-TBCAD
mgoBCAD cloned in pGEM-T, Apr
This study
pLac-
mgoBCAD
mgoBCAD cloned in pBBR1MCS-5 downstream the lacZ promoter in the vector, mgo operon expression under its own
and PLAC promoter, Gmr
This study
pLac-
mboABCDEF
mboABCDEF cloned in pBBR1MCS-5 downstream the lacZ promoter in the vector, mbo operon expression under its own
and PLAC promoter, Gmr
[6]
pLac-
mboFEDCBA
mboABCDEF cloned in pBBR1MCS-5 in the opposite direction than the lacZ promoter in the vector, mbo operon expres-
sion under its own promoter, Gmr
[6]
pMP220
Promoter-probe vector containing a promoterless LacZ gene, Tetr
[37]
pMP-
mboABCDEF
mboABCDEF cloned in promoter-probe vector containing a promoterless LacZ gene, mbo operon expression under its
own promoter, Tetr
This study
pMP::PmboI
pMP220 vector containing the mbo operon promoter, Tetr
[6]
aCECT S
i h T
C lt
C ll
ti
S
i Table 1 Bacterial strains and plasmids used in this study
Strain or
plasmid
Relevant characteristics
Reference/
source
Strains
E. coli
DH5α
E. coli [F’ Φ80lacZ ΔM15 Δ(lacZYA-argF)U169 deoR recA endA1 hsdR17 (rK-mK+)phoA supE44 lambda- thi-1]
[33]
CECT831
Indicator strain for mangotoxin production
CECTa
P. syringae pv. syringae
B728a
Complete genome, non-mangotoxin producer
[34]
UMAF0158
Wild type, isolated from mango, mangotoxin producer, Nfr
[2]
mboA-
Derivative mutant of UMAF0158 by insertion in mboA, Kmr, Nfr (named mboA-)
[6]
ΔmgoA
Derivative mutant of UMAF0158 by deletion of mgoA, Nfr (named ΔmgoA)
[7]
2βB7
miniTn5 mutant of UMAF0158 in gacA defective in mangotoxin, Kmr, Nfr (named gacA-)
[4]
3αE10
miniTn5 mutant of UMAF0158 in gacS defective in mangotoxin, Kmr, Nfr(named gacS-)
[2]
3γH1
miniTn5 mutant of UMAF0158, defective in mangotoxin production, Kmr, Nfr
[2]
4βA2
miniTn5 mutant of UMAF0158, defective in mangotoxin production, Kmr, Nfr
[2]
5αC5
miniTn5 mutant of UMAF0158, defective in mangotoxin production, Kmr, Nfr
[2]
6γF6
miniTn5 mutant of UMAF0158, defective in mangotoxin production, Kmr, Nfr
[2]
P. Mangotoxin production assay Antimetabolite toxin production was assayed by the indica-
tor technique previously described [32]. Briefly, a double
layer of the indicator microorganism E. coli CECT 831 was
prepared; after solidification, the P. syringae pv. syringae
strains to be tested were stab-inoculated. The plates were Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Page 3 of 13 Page 3 of 13 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Table 1 Bacterial strains and plasmids used in this study
Strain or
plasmid
Relevant characteristics
Reference/
source
Strains
E. coli
DH5α
E. coli [F’ Φ80lacZ ΔM15 Δ(lacZYA-argF)U169 deoR recA endA1 hsdR17 (rK-mK+)phoA supE44 lambda- thi-1]
[33]
CECT831
Indicator strain for mangotoxin production
CECTa
P. syringae pv. syringae
B728a
Complete genome, non-mangotoxin producer
[34]
UMAF0158
Wild type, isolated from mango, mangotoxin producer, Nfr
[2]
mboA-
Derivative mutant of UMAF0158 by insertion in mboA, Kmr, Nfr (named mboA-)
[6]
ΔmgoA
Derivative mutant of UMAF0158 by deletion of mgoA, Nfr (named ΔmgoA)
[7]
2βB7
miniTn5 mutant of UMAF0158 in gacA defective in mangotoxin, Kmr, Nfr (named gacA-)
[4]
3αE10
miniTn5 mutant of UMAF0158 in gacS defective in mangotoxin, Kmr, Nfr(named gacS-)
[2]
3γH1
miniTn5 mutant of UMAF0158, defective in mangotoxin production, Kmr, Nfr
[2]
4βA2
miniTn5 mutant of UMAF0158, defective in mangotoxin production, Kmr, Nfr
[2]
5αC5
miniTn5 mutant of UMAF0158, defective in mangotoxin production, Kmr, Nfr
[2]
6γF6
miniTn5 mutant of UMAF0158, defective in mangotoxin production, Kmr, Nfr
[2]
P. β-galactosidase assays determined after 10-day. The severity symptoms were
evaluated by the analysis of the total necrotic area per
leaflet induced by the inoculated strains after 10 days of
incubation. For severity measurement, the necrotic areas
of the inoculation points were digitally analyzed on the
six leaflets, using the computer image software VISILOG
5.0 (Noesis Vision Inc.). At the same time, two inocu-
lated leaflets were used to estimate the daily develop-
ment of the total bacterial population. For that purpose,
whole tomato leaflets were homogenized in sterile water
and bacterial counts were determined plating by 10-fold
serial dilutions on KMB plates. Bacterial growth inside
the plant tissue was recorded after H2O2 leaf surface dis-
infection. Colony counts growth based on the typical
morphology of P. syringae pv. syringae UMAF0158 were
recorded after incubation at 28°C for 48 h. determined after 10-day. The severity symptoms were
evaluated by the analysis of the total necrotic area per
leaflet induced by the inoculated strains after 10 days of
incubation. For severity measurement, the necrotic areas
of the inoculation points were digitally analyzed on the
six leaflets, using the computer image software VISILOG
5.0 (Noesis Vision Inc.). At the same time, two inocu-
lated leaflets were used to estimate the daily develop-
ment of the total bacterial population. For that purpose,
whole tomato leaflets were homogenized in sterile water
and bacterial counts were determined plating by 10-fold
serial dilutions on KMB plates. Bacterial growth inside
the plant tissue was recorded after H2O2 leaf surface dis-
infection. Colony counts growth based on the typical
morphology of P. syringae pv. syringae UMAF0158 were
recorded after incubation at 28°C for 48 h. β g
y
To study the mbo operon expression in different genetic
backgrounds, the mbo operon promoter (PmboI) cloned
into pMP220 [19] as previously described [6] was used. The derivative mutants in mgoA, gacA and gacS genes
were transformed with plasmid pMP::PmboI which con-
tains the PmboI. The plasmid pLac-mgoBCAD (harboring
the mgo operon) was also used to complement the
mgoA, gacA and gacS mutants and finally the β-
galactosidase activity of PmboI was measured. In order to
evaluate the effect of the mgo operon on the activity of
PmboI, P. protegens Pf-5 was used due to the absence of
the two operons in its genome. First, P. β-galactosidase assays protegens Pf-5
was transformed with the pMP::PmboI and the promoter
activity was measured, and secondly to measure the ef-
fect on the mbo operon transcription, this strain con-
taining the plasmid pMP::PmboI, was also transformed
with the plasmid pLac-mgoBCAD (mgo operon under
pLac
regulation). As
a
negative
control
the
β-
galactosidase activity was measured for the wild type
strain P. syringae pv. syringae UMAF0158 and each
strain used in this assay, transformed with empty vector
pMP220. β-galactosidase activities were quantified by
the Miller method [41]. Briefly, an overnight culture ob-
tained as previously described in growth curve and
toxins assay section were prepared. The samples were
collected at 18 h, and the cells were harvested and sus-
pended in assay buffer to eliminate any error in the de-
tection of β-galactosidase activity due to the effects of
different carbon sources present in the growth medium. The results presented are from three separate experi-
ments, each conducted in triplicate. Virulence evaluation The virulence of different mangotoxin producing or
non-producing P. syringae pv. syringae strains were ana-
lyzed in detached tomato leaflets (Solanum lycopersicum Page 4 of 13 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Transcriptional analysis From PMS cultures described above, cells from 2 ml
cultures were collected and spun down at 12,000 rpm
(1 min) from the wild type strain and the derivative
mutants in gacA and mgoA. The cells were frozen in
liquid N2 and stored at -80°C. For the RNA isolations
and cDNA synthesis, three biological replicates were
used for each time point. For the transcriptional ana-
lyses, RNA was isolated from the frozen bacterial cells
with Trizol reagent (Invitrogen), followed by DNase I
(GE Healthcare) treatment. One μg of RNA was used
for cDNA synthesis with Superscript III (Invitrogen)
according to the manufacturer’s protocol. For the
real-time quantitative PCR (Q-PCR), conducted with
the 7300SDS system from Applied Biosystems, the
SYBR Green Core kit (Eurogentec) with a final con-
centration of 3.5 mM MgCl2 was used according to
the manufacturer’s protocol. The concentration of the
primers was optimized (400 nM final concentration
for all of them), and a dissociation curve was per-
formed to check the specificity of the primers. The
primers used for the Q-PCR are listed in Additional
file 1: Table S1. To correct for small differences in
template concentration, rpoD was used as the refer-
ence housekeeping gene. The
cycle in which the
SYBR green fluorescence crossed a manually set cycle
threshold
(CT)
was
used
to
determine
transcript
levels. For each gene, the threshold was fixed based
on the exponential segment of the PCR curve. The
CT value of mboA was corrected for the housekee-
ping gene rpoD as follows: ΔCT = CT (mboA) - CT
(rpoD); the same formula was used for the other
genes studied. The relative quantification (RQ) values
were
calculated
by
the
following
formula:
RQ =
2−[ΔCT(mutant) −ΔCT(wild type)] [39,40]. Q-PCR analysis was
performed in duplicate (technical replicates) on three
independent RNA isolations (biological replicates). Phylogeny of the mgoA gene Phylogeny of the mgoA gene
In order to identify the presence of the mgoA gene in the
different genomes of Pseudomonas strains, the mgoA gene
from P. syringae pv. syringae UMAF0158 was used in
BLASTP [42] comparisons with whole genome sequences
of Pseudomonas spp. available in the databases. Once the
amino acid sequences of all the orthologous mgoA genes
were obtained, the putative adenylation domains were iden-
tified using the PKS/NRPS Analysis Web-site (http://nrps. igs.umaryland.edu/nrps) [43]. Other adenylation domains
of which the activated amino acid is already known were
obtained from the database and from De Bruijn met al. [44]. Two phylogenetic analyses were done, the first was
using the adenylation domain of all the NRPSs (328 resi-
dues) and the second was using the almost entire sequence
of MgoA (1015 residues). Amino acid sequences were
aligned with Muscle (MEGA5 software) and determination
of the optimal amino acid substitution model and phylo-
genetic tree construction were done using MEGA5 soft-
ware [45]. Neighbor-joining, maximum parsimony and
maximum-likelihood phylogenetic trees of the individual Page 5 of 13 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 t-test was carried out (p = 0.05), using the IBM.SSPS 19
software (IBM® Company). gene sequences were generated in MEGA5 by using the op-
timal model parameters and the option of complete dele-
tion to eliminate positions containing gaps. Confidence
levels for the branching points were determined using
1,000 bootstrap replicates. Results Involvement of mbo genes in mangotoxin production and
virulence in P. syringae pv. syringae UMAF0158 Involvement of mbo genes in mangotoxin production and
virulence in P. syringae pv. syringae UMAF0158
Six mangotoxin deficient mutants of P. syringae pv. syrin-
gae UMAF0158, were previously obtained and character-
ized for mangotoxin production (Table 1 and Figure 1). Mangotoxin characterization showed that although these
mutants did not show mangotoxin production, a slight pro-
duction of a yet unknown antimicrobial compound was ob-
served for mutants 4βA2 (mboB) and 5αC5 (mboD)
(Figure 1). For two mutants (3γH1 and 6γF6), the Tn5 in-
sertion was located in mgoC and mgoA respectively. Two
other non-mangotoxin producing mutants were disrupted
in the genes encoding the GacS/GacA two-component
regulatory system (3αE10 and 2βB7 respectively). Growth
of the mgoA mutant was shown to be similar to that of the Bioinformatics and statistical analysis y
Searches for sequence similarity in the NCBI databases
were carried out using BLAST algorithms [42]. Genome
and nucleotide sequences were visualized and manipu-
lated using the Artemis genome browser [46] and com-
pared using ACT [47] in combination with WebACT
[48]. The statistical analysis of incidence was performed
by SAS9.2 software (SAS Institute Inc.) by Enterprise
Guide 4.2 using generalized linear model analysis. The
β-galactosidase and the necrotic area data were statisti-
cally analyzed using an analysis of variance, followed by
Fisher’s least significant difference test (p = 0.05), and for
β-galactosidase activity on P. protegens Pf5, a Student’s Figure 1 Mangotoxin production by random miniTn5 insertional mutants. Three pairs of mutants in different genes of the mbo and mgo
operon, and in the gacS/gacA two-component regulatory system, obtained in previous works and tested for mangotoxin production. The
corresponding disrupted gene is detailed in brackets. The P. syringae pv. syringae strains UMAF0158 (mangoxin-producing wilt-type strain) and
B728a (nonproducing) were used as references. Mangotoxin production was evaluated using PMS minimal medium supplemented or not with
ornithine. The results are indicated as follows: - absence of inhibition halo, + presence of inhibition halo, -* slight toxicity which was not reverted
by addition of ornithine. Toxic activity reverted in presence of ornithine denotes the production of mangotoxin. Figure 1 Mangotoxin production by random miniTn5 insertional mutants. Three pairs of mutants in different genes of the mbo and mgo
operon, and in the gacS/gacA two-component regulatory system, obtained in previous works and tested for mangotoxin production. The
corresponding disrupted gene is detailed in brackets. The P. syringae pv. syringae strains UMAF0158 (mangoxin-producing wilt-type strain) and
B728a (nonproducing) were used as references. Mangotoxin production was evaluated using PMS minimal medium supplemented or not with
ornithine. The results are indicated as follows: - absence of inhibition halo, + presence of inhibition halo, -* slight toxicity which was not reverted
by addition of ornithine. Toxic activity reverted in presence of ornithine denotes the production of mangotoxin. Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Carrión et al. BMC Microbiology 2014, 14:46 Page 6 of 13 Figure 2 Transcriptional analysis and mbo operon promoter activity. mboA, mboC and mboE (A), belonging to the mbo operon and mgoB
and mgoA (B), belonging to the mgo operon transcript levels in the wild type strain P. syringae pv. syringae UMAF0158 and mgoA and gacA
mutants. Bioinformatics and statistical analysis (C) Comparison of the described consensus motif (5′-CANGGANG-3′) for P. fluorescens [49-51]: The search was done in front of each start
codon of the mgo and mbo genes. (D) β-galactosidase activity of the mbo operon promoter in the wild-type strain UMAF0158 and mgoA, gacS
and gacA mutants. These strains were transformed with the mbo operon promoter named pMP::PmboI and the empty promoter-probe vector
pMP220 was used as a control. The different mutants were also transformed with the vector pLac-mgoBCAD. Log2RQ represents the expression
levels of the studied genes by relative quantification scores. Values below 0 indicates lower expression than the housekeeping gene used for
normalization of data. The results are average of three independent experiments performed in triplicate. Error bars indicate standard deviation. Data were analysed for significance using an arcsine square root transformation with analysis of variance followed by Fisher’s least significant
difference test (P = 0.05). Values of bars with different letter designations represent a statistically significant difference. Figure 2 Transcriptional analysis and mbo operon promoter activity. mboA, mboC and mboE (A), belonging to the mbo operon and mgoB
and mgoA (B), belonging to the mgo operon transcript levels in the wild type strain P. syringae pv. syringae UMAF0158 and mgoA and gacA
mutants. (C) Comparison of the described consensus motif (5′-CANGGANG-3′) for P. fluorescens [49-51]: The search was done in front of each start
codon of the mgo and mbo genes. (D) β-galactosidase activity of the mbo operon promoter in the wild-type strain UMAF0158 and mgoA, gacS
and gacA mutants. These strains were transformed with the mbo operon promoter named pMP::PmboI and the empty promoter-probe vector
pMP220 was used as a control. The different mutants were also transformed with the vector pLac-mgoBCAD. Log2RQ represents the expression
levels of the studied genes by relative quantification scores. Values below 0 indicates lower expression than the housekeeping gene used for
normalization of data. The results are average of three independent experiments performed in triplicate. Error bars indicate standard deviation. Data were analysed for significance using an arcsine square root transformation with analysis of variance followed by Fisher’s least significant
difference test (P = 0.05). Values of bars with different letter designations represent a statistically significant difference. Figure 2 Transcriptional analysis and mbo operon promoter activity. Bioinformatics and statistical analysis mboA, mboC and mboE (A), belonging to the mbo operon and mgoB
and mgoA (B), belonging to the mgo operon transcript levels in the wild type strain P. syringae pv. syringae UMAF0158 and mgoA and gacA
mutants. (C) Comparison of the described consensus motif (5′-CANGGANG-3′) for P. fluorescens [49-51]: The search was done in front of each start
codon of the mgo and mbo genes. (D) β-galactosidase activity of the mbo operon promoter in the wild-type strain UMAF0158 and mgoA, gacS
and gacA mutants. These strains were transformed with the mbo operon promoter named pMP::PmboI and the empty promoter-probe vector
pMP220 was used as a control. The different mutants were also transformed with the vector pLac-mgoBCAD. Log2RQ represents the expression
levels of the studied genes by relative quantification scores. Values below 0 indicates lower expression than the housekeeping gene used for
normalization of data. The results are average of three independent experiments performed in triplicate. Error bars indicate standard deviation. Data were analysed for significance using an arcsine square root transformation with analysis of variance followed by Fisher’s least significant
difference test (P = 0.05). Values of bars with different letter designations represent a statistically significant difference. Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Page 7 of 13 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 virulence to wild type level (Additional file 3: Figure
S2B and S2C). wild type strain, with cell densities of up to 1011 cfu ml-1 in
liquid medium after 108 h of growth at 22ºC (Additional
file 2: Figure S1A). In contrast, the gacA mutant presented
an altered growth, with cell densities in the stationary phase
reaching only 109 cfu ml-1 (Additional file 2: Figure S1A). The dynamics of the mangotoxin production in relation to
bacterial growth was followed during four days of incuba-
tion. Mangotoxin production was detectable after 24 h of
growth, increased up to 1.4 toxic units (T.U.), then reduced
slightly upon entry of the stationary phase and then stabi-
lized (Additional file 2: Figure S1B). Mangotoxin production and transcriptional regulation in
the gacA and mgoA mutant To study the role of mgoA and gacA in mangotoxin biosyn-
thesis, transcription of the mboACE and mgoBA genes was
analyzed for the wild type strain, and for the mgoA and
gacA derivative mutants. Time course experiments showed
that the mbo genes in the wild type are expressed at the
highest level after 12 to 24 h (Additional file 4: Figure S3). Therefore all comparisons between wild type and mutants
were performed at 18 h of growth. Transcript levels of the
mboACE genes after 18 h of growth were significantly lower
in the gacA and the mgoA mutants than in the wild type
(Figure 2A). Also the transcript levels of mgoB and mgoA
were significantly lower in the gacA mutant (Figure 2B). The mgoA mutation did not affect transcription of gacS/
gacA (data not shown). Also mboA, mboC, or mboE muta-
tions did not significantly affect transcription of gacS/gacA
or mgoA (data not shown). These results indicate that the
GacS/GacA two-component regulatory system affects tran-
scription of both the mbo and mgo genes and that the prod-
uct of the mgo operon influences transcription of the mbo
genes. To further study if the GacS/GacA two-component
regulatory system could regulate the mgo and mbo genes
via RNA repressor binding proteins [49-51], the upstream In order to know if the virulence of the derivative mu-
tants mboA- and mgoA was reduced in comparison with
the wild type strain, detached tomato leaflets were arti-
ficially inoculated. Artificial inoculation experiments
using detached tomato leaflets [4] showed that bacterial
growth inside the tomato leaflets of the mboA- and
ΔmgoA mutants as well as their complemented derivatives
followed similar dynamics (Additional file 3: Figure S2A). When inoculations were performed, development of
necrotic lesions was observed on the leaf. Disease sever-
ity, represented by the necrotic area, showed that both
mangotoxin defective mutants were less virulent than
the wild type UMAF0158 (Additional file 3: Figure S2B
and S2C). When derivative strains were complemented
with the mboA and mgoA genes disease severity in-
creased but complementation did not fully restore Table 2 Toxic activity of P. syringae pv syringae UMAF0158 mutants and mgo operon complemented strains
Strains
E. Mangotoxin production and transcriptional regulation in
the gacA and mgoA mutant coli inhibition assay
Mangotoxin
production
PMS
PMS + ornithine
Wild type strain and derivative mutants
UMAF0158
+
-
Yes
mboA-
-*
-*
No
ΔmgoA
-
-
No
gacA-
-
-
No
gacS-
-
-
No
Transformed with empty vector
UMAF0158
+
-
Yes
mboA-
-*
-*
No
ΔmgoA
-
-
No
gacA-
-
-
No
gacS-
-
-
No
Transformed with pLac-mgoBCAD
UMAF0158
++
-
Yes
mboA-
-*
-*
No
ΔmgoA
++
-
Yes
gacA-
-
-
No
gacS-
-
-
No
The results are indicated as follows: - absence of inhibition halo, + inhibition halo between 5-10 mm, ++ inhibition halo bigger 10 mm, -* slight toxicity which did
not revert in presence of ornithine. Toxic activity, which reverts in the presence of ornithine, denotes the production of mangotoxin. c activity of P. syringae pv syringae UMAF0158 mutants and mgo operon complemented strains Table 2 Toxic activity of P. syringae pv syringae UMAF0158 mutants and mgo operon complemen
St
i
E
li i hibiti Transformed with pLac-mgoBCAD The results are indicated as follows: - absence of inhibition halo, + inhibition halo between 5-10 mm, ++ inhibition halo bigger 10 mm, -* slight toxicity which did
not revert in presence of ornithine. Toxic activity, which reverts in the presence of ornithine, denotes the production of mangotoxin. The results are indicated as follows: - absence of inhibition halo, + inhibition halo between 5-10 mm, ++ inhibition halo bigger 10 mm
not revert in presence of ornithine. Toxic activity, which reverts in the presence of ornithine, denotes the production of mangotoxin. Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Page 8 of 13 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Page 8 of 13 and mgo operons (Figure 2C). It must be taken into account
that the described consensus sequence is from P. protegens
[49], and nothing is known yet about the recognition site of
RNA binding proteins in P. syringae. regions of the mgo and mbo genes were inspected for the
presence of the described consensus motif (5′-CANG-
GANG-3′) previously described in P. protegens CHAO
[49]. This motif allows the binding of the repressor to the
RNA, and these repressor proteins can be removed by Gac/
Rsm. The complete consensus sequence was not detected
upstream of any of the mbo/mgo genes (Figure 2C). How-
ever, consensus GGA motifs for binding of the RNA bind-
ing proteins [49-51] were detected upstream of the mbo As the transcription of the mgo operon was substan-
tially lower in the gacA mutant (Figure 2B), we subse-
quently tested whether introduction of extra copies of
the mgo operon in the gacS or gacA mutant could re-
store mangotoxin production. When the mgo operon Figure 3 Phylogeny of the MgoA adenylation domain. Neighbor-joining tree, constructed with MEGA5 using the adenylation domains
extracted from nonribosomal peptide synthetases involved in syringomycin, syringopeptin, massetolide A, arthrofactin synthesis and mangotoxin
biosynthesis (MgoA). The presence (+) or absence (-) of the mbo operon is shown in the phylogenetic tree. The boxes indicate the different
groups of Pseudomonas species which are able to produce mangotoxin when were transformed with pLac-mboABCDEF (mbo operon under its
own and PLAC promoter expression) or pLac-mboFEDCBA (mbo operon under its own promoter expression). Also is indicated the signature
sequence of the adenylation domains in each strain. The evolutionary history was inferred using the Neighbor-Joining method [52]. MgoA phylogeny and mangotoxin production in other
strains The amino acid sequence of a typical non-ribosomal pep-
tide synthetase (NRPS) displays an adenylation (A) domain
responsible for recognition and subsequent activation of an
amino acid substrate. It also contains the typical thiolation
(T) and condensation (C) domains. Finally, the thioesterase
(TE) domain releases the final molecule from the NRPS as-
sembly line. Based on the specific signature sequences de-
scribed previously for A domains, analysis of MgoA did not
allow prediction of the amino acid to be activated. There-
fore, a phylogenetic analysis was performed with multiple
A domains from NRPSs of which activated amino acids are
known and with MgoA from other Pseudomonas species
(Figure 3 and Additional file 5: Figure S4). The results
showed that the A domains from the different MgoA
orthologues grouped in the same cluster, separate from
other A domains for which the activated amino acid residue
is known (Figure 3). Figure 4 Heterologous expression and production of Figure 4 Heterologous expression and production of
mangotoxin. (A) The mbo operon promoter activity in P. protegens
Pf-5 transformed with the mbo operon promoter (pMP::PmboI) and
with the empty promoter-probe vector pMP220 was used as a
control. To check the positive regulation of the mgo operon, the
strain Pf-5 was transformed with the vector pLac-mgoBCAD. The
result is the average of three independent experiments performed
in triplicate. Error bars indicate standard deviation. (B) Mangotoxin
production of P. protegens Pf-5 transformed with pLac-mboABCDEF
(mbo operon under its own and PLAC promoter expression), pLac-
mboFEDCBA (mbo operon under its own promoter expression) and
pLac-mgoBCAD (mgo operon under its own and PLAC promoter
expression) and pMP220-mboABCDEF (mbo operon under its own
promoter expression). Data were analysed for significance using a
Student’s t-test (P = 0.05). Value of bar with an asterisk designation
represent a statistically significant difference to the other bar value. To determine if mgoA present in other Pseudomonas
species can regulate the mbo operon, reporter constructs
pLac-mboABCDEF (mbo operon under its own and under
pLac promoter expression) and pLac-mboFEDCBA (mbo
operon only under its own promoter expression) were
used. Firstly, only specific P. syringae pathovars harbor the
mbo operon, and almost all strains from these pathovars
produce mangotoxin [29], with or without the introduc-
tion of the mbo operon containing plasmids (Figure 3). Our results showed that other P. The mgo operon is a positive regulator of mbo operon
transcription To further investigate if the mgo operon is able to
regulate the expression of the mbo operon, we intro-
duced the mbo operon promoter reporter construct
(pMP::PmboI) and the mgo genes in P. protegens Pf-5,
which lacks both the mgo and the mbo operons in its
genome. Compared to the promoter activity in the wild-
type Pf-5 background, a two-fold increase in ectopic
mbo promoter activity was observed when Pf-5 was
complemented with the mgo operon (Figure 4A). When
P. protegens Pf-5 was transformed with pLac-mboABC-
DEF (mbo operon under pLac regulation), it produces
mangotoxin. However, when P. protegens Pf-5 was trans-
formed with pMP-mboFEDCBA (mbo operon under only To further elucidate the role of the mgo operon in the regu-
lation of mangotoxin biosynthesis, expression assays were
carried out using a plasmid reporter construction consisting
of the mbo operon promoter fused to a promoterless lacZ
gene. When the plasmid reporter was transferred into the
wild type strain, high levels of β-galactosidase activity were
found, whereas for the mgoA, gacA and gacS mutants this
activity was substantially lower (Figure 2D). For the mgoA
mutant, complementation with the mgo operon restored β-
galactosidase activity to similar levels as in the wild type
strain (Figure 2D). In contrast, no restoration of the β-
galactosidase activity was found when the mgo operon was
introduced in the gacS/gacA, confirming results described
above (Table 2). Figure 4 Heterologous expression and production of
mangotoxin. (A) The mbo operon promoter activity in P. protegens
Pf-5 transformed with the mbo operon promoter (pMP::PmboI) and
with the empty promoter-probe vector pMP220 was used as a
control. To check the positive regulation of the mgo operon, the
strain Pf-5 was transformed with the vector pLac-mgoBCAD. The
result is the average of three independent experiments performed
in triplicate. Error bars indicate standard deviation. (B) Mangotoxin
production of P. protegens Pf-5 transformed with pLac-mboABCDEF
(mbo operon under its own and PLAC promoter expression), pLac-
mboFEDCBA (mbo operon under its own promoter expression) and
pLac-mgoBCAD (mgo operon under its own and PLAC promoter
expression) and pMP220-mboABCDEF (mbo operon under its own
promoter expression). Data were analysed for significance using a
Student’s t-test (P = 0.05). Value of bar with an asterisk designation
represent a statistically significant difference to the other bar value. Transformed with pLac-mgoBCAD The
evolutionary distances were computed using the JTT matrix-based method [53] and are in the units of the number of amino acid substitutions per site. The variation rate among sites was modelled with a gamma distribution. The analysis involved 126 amino acid sequences. There were a total of 328
positions in the final dataset. Evolutionary analyses were conducted in MEGA5 [45]. Bootstrap values (1,000 repetitions) are shown on branches. Figure 3 Phylogeny of the MgoA adenylation domain. Neighbor-joining tree, constructed with MEGA5 using the adenylation domains
extracted from nonribosomal peptide synthetases involved in syringomycin, syringopeptin, massetolide A, arthrofactin synthesis and mangotoxin
biosynthesis (MgoA). The presence (+) or absence (-) of the mbo operon is shown in the phylogenetic tree. The boxes indicate the different
groups of Pseudomonas species which are able to produce mangotoxin when were transformed with pLac-mboABCDEF (mbo operon under its
own and PLAC promoter expression) or pLac-mboFEDCBA (mbo operon under its own promoter expression). Also is indicated the signature
sequence of the adenylation domains in each strain. The evolutionary history was inferred using the Neighbor-Joining method [52]. The
evolutionary distances were computed using the JTT matrix-based method [53] and are in the units of the number of amino acid substitutions per site. The variation rate among sites was modelled with a gamma distribution. The analysis involved 126 amino acid sequences. There were a total of 328
positions in the final dataset. Evolutionary analyses were conducted in MEGA5 [45]. Bootstrap values (1,000 repetitions) are shown on branches. Page 9 of 13 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 either vector, they only produced mangotoxin when the
mbo operon was expressed constitutively but not when
they were transformed with the mbo operon with its na-
tive promoter (Figure 3). was introduced in the mgoA mutant mangotoxin pro-
duction was restored, which was not the case for the
mboA, gacA and gacS mutants (Table 2). MgoA phylogeny and mangotoxin production in other
strains syringae pathovars, that
do not contain the mbo operon, are all able to produce
mangotoxin when they were transformed with pLac-
mboABCDEF and pLac-mboFEDCBA (Figure 3). When
different P. fluorescens strains were transformed with Page 10 of 13 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Page 10 of 13 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 system receives a yet unknown signal that activates a set of
small RNAs [8,50,54]. The expression of genes regulated by
the GacS/GacA might be mediated through the Rsm path-
way [55,56]. In fact, components of this pathway such as
the three small RNAs RsmX, RsmY and RsmZ and two
RNA-binding proteins (RsmA and RsmE) were found in
the genome of P. syringae pv. syringae UMAF0158 (Unpub-
lished data). Transcriptional analysis of the mgo, mbo
and gac genes showed that the mbo genes were mark-
edly down-regulated in both the gacA and mgoA mu-
tants. On the other hand, the transcriptional levels of
mgoB and mgoA, also showed down-regulation in the
gacA mutant, indicating that the mgo operon is also
under regulation by the GacS/GacA two-component
regulatory system. These data suggest that GacS/GacA
is regulating the mbo operon expression via the mgo
operon, however direct regulation of the mbo operon
by the two-component regulatory system gacS/gacA
cannot be excluded (Figure 5). its own promoter expression) it was not able to produce
detectable amounts of mangotoxin, neither in absence
nor in presence of the mgo operon of P. syringae pv. syr-
ingae UMAF0158 (Figure 4B). Therefore, the presence
of the mbo and mgo operons in P. protegens Pf-5 would
be not
sufficient for the production of detectable
amounts of mangotoxin. Discussion
h
l The results of our study show that the regulation of
mangotoxin biosynthesis in the plant pathogenic P. syr-
ingae pv. syringae strain UMAF0158 is governed by a
complex
interplay
between
the
GacS/GacA
two-
component regulatory system, the nonribosomal peptide
synthetase mgoA and the mangotoxin biosynthesis op-
eron mbo. We showed that disruption of the mbo bio-
synthesis genes leads to reduced virulence. Introduction
of the mbo operon in these biosynthesis mutants re-
stored mangotoxin production but did not lead to full
restoration of virulence on tomato leaflets. Multiple cop-
ies of the plasmid with the mbo operon could lead to
overproduction of mangotoxin which may affect the
regulation or production of other virulence factors such
as syringomycin and syringopeptin. Transcriptional analysis with a lacZ fusion on the pro-
moter of the mbo operon (PmboI), revealed that the product
of the mgo operon could acts as positive regulator of mbo
transcription. Interestingly, the pvfC gene (homologue of
mgoA) is considered a regulator of virulence in P. enthomo-
phila, but appears not to be part of the GacS/GacA regula-
tory cascade [28]. In strain UMAF0158, introduction of the
mgo operon in a gac mutant could not restore mangotoxin
production or mbo-promoter activity, suggesting that next Taken together the obtained results of this work and the
previously described data [4,6,7], a simplified model for the
interplay among these genes can be constructed (Figure 5). In this model, the GacS/GacA two-component regulatory Figure 5 Proposed model for regulation of mangotoxin biosynthesis in P. syringae pv. syringae. In this model, GacS/GacA
two-component regulatory system activates directly or indirectly the transcription of the mgo operon. And the mgo operon could synthetize a
positive regulator of the mbo operon transcription. The mbo operon produces mangotoxin which acts as virulence factor. Figure 5 Proposed model for regulation of mangotoxin biosynthesis in P. syringae pv. syringae. In this model, GacS/GacA
two-component regulatory system activates directly or indirectly the transcription of the mgo operon. And the mgo operon could synthetize a
positive regulator of the mbo operon transcription. The mbo operon produces mangotoxin which acts as virulence factor. Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Page 11 of 13 Carrión et al. BMC Microbiology 2014, 14:46
http://www.biomedcentral.com/1471-2180/14/46 Additional file 3: Figure S2. Additional file 3: Figure S2. Virulence analysis of the wild type strain
P. syringae pv. syringae UMAF0158 and corresponding derivatives using a
detached tomato leaf assay. (A) In planta growth inside the tomato
leaflets after H2O2 surface disinfection of the wild type strain UMAF0158,
mgoA and mboA mutants, and their respective complemented
derivatives. (B) Severity of necrotic symptoms (necrotic area) on tomato
leaflets inoculated with wild type strain UMAF0158, the mutants in mboA
and mgoA with their respective complemented derivatives. The total
necrotic area (mm2) from 30 inoculated points on tomato leaflets was
measured 10 days after inoculation and used to compare the severity of
necrotic symptoms produced by the different strains. (C) Representative
pictures of the necrotic lesions produced by the wild type strain and the
different mutants at 10 dpi. Different letters denote statistically significant
differences at p = 0.05, according to analysis of variance followed by
Fisher’s least significant difference test. Phylogenetic analysis of MgoA and the adenylation do-
mains suggested an evolutionary specialization of this
protein into the Pseudomonas genus. In this context, it
is worth noting that the transformation of the mbo op-
eron under the expression of its own promoter only con-
fers mangotoxin production in the P. syringae group and
not in the P. fluorescens group. Therefore, it seems that
the NRPS MgoA is involved in different signal transduc-
tion pathways depending of the Pseudomonas species. In
the case of P. syringae, MgoA appears to activate man-
gotoxin production. It remains to be studied if MgoA is
also involved in the regulation and production of other
antimetabolites in the P. syringae group, such as tabtoxin
and phaseolotoxin. The positive regulation of the mbo
operon promoter activity in the presence of the mgo op-
eron in Pf-5, combined with the lack of detectable
amounts of mangotoxin suggests that additional factors
for mangotoxin biosynthesis or its export are not present
in the P. fluorescens group. Additional file 4: Figure S3. mboACE transcript levels in the wild type
strain UMAF0158. Relative expression of the genes involved in the
mangotoxin biosynthesis at the different time points during the growth
curve. For each time point, mean values of four biological replicates are
given; the error bars represent the standard errors of the mean. Additional file 4: Figure S3. mboACE transcript levels in the wild type
strain UMAF0158. Acknowledgements This work was supported by grants from the Regional Government of
Andalucía (Spain), grants from CICE - Junta de Andalucía, Ayudas Grupo
PAIDI AGR-169, and Proyecto de Excelencia (P07-AGR-02471) and Plan Nacional
de I + D + I del Ministerio de Ciencia e Innovacion (AGL2011-30354-C02-01)
cofinanced by FEDER (EU). Plan Propio of the University of Málaga funded a
stay of VJC in the Wageningen University, The Netherlands. This is publication
No. 5584 of the Netherlands Institute of Ecology (NIOO-KNAW). VJC was Authors’ contributions VJC, MV, EA, AV, JMR and FMC conceived the study. VJC and EA did all the
cloning and genetics of this study. VJC and MV did the Q-PCR experiments
and analysis. VJC and JAG did complementation and reporter construct
experiments. JMR and AV supported the research. VJC, MV, JMR and FMC
wrote the manuscript. VJC, EA, MV, AV, JMR and FMC coordinated and critically
revised the manuscript. All authors read and approved the manuscript. Competing interests
W
h
h
h
b We the authors hereby declare that there is no conflict of interests
concerning this manuscript. We the authors hereby declare that there is no conflict of interests
concerning this manuscript. Additional file 5: Figure S4. P Additional file 5: Figure S4. Phylogenetic analysis of the MgoA of
different Pseudomonas spp. Neighbor-joining tree was constructed with
MEGA5 using a partial sequence of MgoA. The boxes indicate the
different groups of Pseudomonas and the presence (mbo +) or absence
(mbo -) of the mbo operon. The evolutionary history was inferred using
the Neighbor-Joining method [52]. The evolutionary distances were
computed using the JTT matrix-based method [53] and are in the units
of the number of amino acid substitutions per site. The rate variation
among sites was modelled with a gamma distribution. The analysis
involved 126 amino acid sequences. There were a total of 1015 positions
in the final dataset. Evolutionary analyses were conducted in MEGA5 [45]. Burkholderia cenocepacia J2315 was used as the outgroup. Bootstrap
values (1,000 repetitions) are shown on the branches. Conclusions
f In summary, for P. syringae pv. syringae UMAF0158, the
GacS/GacA two-component system regulates transcription
of the mgo and mbo operons and thereby mangotoxin bio-
synthesis. At the same time, the mgo operon product seems
to act as a positive regulator of the mbo operon. The pro-
posed model for mangotoxin biosynthesis is a simplified
and initial overview of the interaction between the gac, mgo
and mbo gene products based on the results obtained in
the current study. This is the first evidence of the interplay
between MgoA and the GacS/GacA two-component regu-
latory
system
in
the
regulation
of
the
mangotoxin
biosynthesis. Additional files to the mgo operon, additional factors are regulated by the
gac system that influence mangotoxin production. It is
worth noting that P. entomophila and P. syringae pv. syrin-
gae harbor two different genetic backgrounds, adapted to
different environments. The first is found in diverse envi-
ronments such as soil, aquatic ecosystems, rhizosphere, and
in pathogenic interactions with Drosophila melanogaster
[57]. The second is adapted for plant infection and epi-
phytic survival [3]. Therefore, the regulatory roles of these
orthologues can substantially differ between these two
Pseudomonas species. On the other hand, the fact that both
PvfC and MgoA are involved in the regulation of virulence
could indicate that in other Pseudomonas spp. these factors
would be involved in the regulation of virulence and/or sec-
ondary metabolite production. Additional file 1: Table S1. Primers used in this study. Additional file 1: Table S1. Primers used in this study. Additional file 2: Figure S1. Growth characteristics of P. syringae pv. syringae strain UMAF0158 and the derivatives mgoA and gacA mutants. (A) Growth of the wild type strain UMAF0158 and the mgoA (ΔmgoA)
and gacA (2βB7) mutants at 22ºC in PMS. At each time point, the
bacterial density was estimated by serial dilutions and colony counts on
plates of selective medium and expressed as log cfu ml-1 of culture. (B)
UMAF0158 mangotoxin production at 22ºC in PMS. At each time point,
the mangotoxin production was estimated using cell-free filtrate and
represented as the previously defined toxic units (T.U.). The dashed line
represents the detection limit of the technique. Mean values for three
replicates are given; the error bars represent the standard errors of the
mean. Additional file 2: Figure S1. Growth characteristics of P. syringae pv. syringae strain UMAF0158 and the derivatives mgoA and gacA mutants. (A) Growth of the wild type strain UMAF0158 and the mgoA (ΔmgoA)
and gacA (2βB7) mutants at 22ºC in PMS. At each time point, the
bacterial density was estimated by serial dilutions and colony counts on
plates of selective medium and expressed as log cfu ml-1 of culture. (B)
UMAF0158 mangotoxin production at 22ºC in PMS. At each time point,
the mangotoxin production was estimated using cell-free filtrate and
represented as the previously defined toxic units (T.U.). The dashed line
represents the detection limit of the technique. Mean values for three
replicates are given; the error bars represent the standard errors of the
mean. Additional file 3: Figure S2. Relative expression of the genes involved in the
mangotoxin biosynthesis at the different time points during the growth
curve. For each time point, mean values of four biological replicates are
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de Hortofruticultura Subtropical y Mediterránea “La Mayora”-Universidad de
Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC),
Estación Experimental La Mayora, Algarrobo-Costa, 29750 Málaga, Spain. 3Laboratory of Phytopathology, Wageningen University, 6708 PB
Wageningen, The Netherlands. 4Department of Microbial Ecology, The
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and take full advantage of: 53. Jones DT, Taylor WR, Thornton JM: The rapid generation of mutation
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Katharine Susannah Prichard’s Coonardoo and Rachel de Queiroz’s The Year Fifteen: a settler colonial reading
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DOI: http://dx.doi.org/10.5007/2175-8026.2019v72n1p87 DOI: http://dx.doi.org/10.5007/2175-8026.2019v72n1p87 Key words: Settler colonialism; Coonardoo; Katharine Susannah Prichard;
he Year Fiteen; Rachel de Queiroz. KATHARINE SUSANNAH PRICHARD’S COONARDOO AND
RACHEL DE QUEIROZ’S THE YEAR FIFTEEN: A SETTLER
COLONIAL READING1 Déborah Scheidt1*
1Universidade Estadual de Ponta Grossa, Ponta Grossa, Paraná, Brasil Esta obra tem licença Creative Commons Two writers from the margins Brazilian and Australian literatures are far from “universal”, to use a
reductionist, Eurocentric expression that, nonetheless, still has power. Not being
universal means that the “literary classics” from those countries are virtually
unknown outside Brazil and Australia. And as Renata Wasserman (12) observes
in her assessment of Brazilian women writers, Brazilian literature can hardly be
seen to belong to the canon of “world literature”, in which, to be up-to-date in the
cultural circles one should necessarily have read Brazilian (and, for that matter,
Australian) iction, poetry or drama. Although in times of “English as a Global
Language” Australian literature might have a much wider reach and fare better in
the latter aspect compared to a literature written in Portuguese, very much like
their Brazilian counterparts, Australian women writers, would be, as Wasserman
(12) puts it, “at the margin of the margin”. he double challenge of being “culturally subaltern” in relation to Europe
and, more recently, the USA, and “gender subaltern” in their own patriarchal
societies, might be a good point of departure to approximate the writers
Katharine Susannah Prichard (1883-1969) and Rachel the Queiroz (1910-
2003). Several parallels can be drawn between them in that respect: at a time
when, more in Brazil than in Australia, as we shall see, men dominated the
realm of letters, not only did Queiroz and Prichard manage to make a living out
of writing iction, journalistic texts and criticism, among other genres, but they
also became prominent igures in the cultural milieus of their countries, living
long, active and, at times, polemic lives and having their works translated into
several languages. heir achievements are impressive. Prichard’s legacy includes thirteen novels,
an autobiography, four collections of short stories, fourteen plays, two volumes
of poetry, besides dozens of articles, critical texts, essays, pamphlets, interviews
and political statements. Queiroz published, among other works, eight novels,
four children’s books, ive plays, more than a hundred crônicas (sketches), an
autobiography (in partnership with her sister Maria Luiza de Queiroz Salek),
several works published in partnership, articles, interviews and reviews. She was
also the translator of dozens of novels, plays, biographies and other works into
Portuguese. Both women were awarded literary prizes and grants. Although Prichard was well into her twenties when Queiroz was born, they
published their best-known novels within a one-year period: Coonardoo was
published in 1929 and O Quinze in 1930. Abstract Settler Colonial Studies is a theoretical approach being developed in
Australia by Lorenzo Veracini (2010, 2015, 2016), inspired by Patrick
Wolfe’s (1999, 2016) precursor theories. It proposes a diferentiation
between “colonialism” and “settler colonialism” based on the premise
that the latter involves land dispossession and the literal or metaphorical
disappearance of Indigenous Others, while the former is mainly concerned
with the exploitation of Indigenous labour and resources. he fact that
settlers “come to stay” is a crucial element in positing settler colonialism
as “a structure”, whereas colonialism would be “an event” in the lives
of the colonised Others. his paper adopts settler colonial theories to
propose a comparative study of two modernist “social” novels by women
writers in Australia and Brazil: Katharine Susannah Prichard’s Coonardoo
(1929) and Rachel de Queiroz’s he Year Fiteen (1930). Both novels
deal with exploitation, discrimination, racism and the dispossession of
the Indigenous Other and their miscegenated descendants, from a non-
Indigenous, i.e. “settler”, perspective. Elements that are crucial for settler
colonialism, such as ambivalence, indigenisation and mechanisms of
disavowal and transfer in several of their guises, are examined, compared
and contrasted. Key words: Settler colonialism; Coonardoo; Katharine Susannah Prichard;
he Year Fiteen; Rachel de Queiroz. * Professora titular do Departamento de Estudos da Linguagem da Universidade Estadual de Ponta Grossa. Possui título de Doutora em Estudos Literários pela UFPR e de Mestre em Literaturas de Língua Inglesa também
pela UFPR. De julho a dezembro de 2017 realizou Estágio Pós-Doutoral na University of Sydney, Department
of English, com auxílio inanceiro da CAPES. Seu e-mail é deborahscheidt@yahoo.com.br. ORCID: 0000-0002-
8077-5257. 88 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... Two writers from the margins Unlike other of Queiroz’s novels, there
isn’t a complete translation of O Quinze into English. However, Darlene Sadlier
(1992) published extracts of the novel under the title he Year Fiteen, a title I
will adopt in this paper. Coonardoo and he Year Fiteen have made their authors
famous and have been continuously reprinted, especially the latter, which has had
more than 100 Brazilian editions in 87 years. Under a male pseudonym, Prichard’s Coonardoo (in a tie with M. Barnard
Eldershaw) won the Bulletin Magazine’s novel competition in 1928, a signiicant Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 89 feat considering that there were 542 contestants (hrossel 53), and, mainly, the
fact that the novel was the irst to consistently portray an Aboriginal female
protagonist, whereas the Bulletin proudly displayed the “Australia for the White
Man” motto on its irst page. Queiroz was only 18 when she became a celebrity
writer. he laconic style and the contents of her irst novel made it very diferent
from the sentimental (“sugary water”, as Brazilians say) plots expected from a
woman writer in Brazil at the time. hose factors led some of her irst critics
and admirers, such as the modernist icon Mario de Andrade, to think that he
Year Fiteen was the work of a man under a female pseudonym. Nearly 50 years
later, under great clamour, Queiroz would become the irst woman to be allowed
into the Brazilian Academy of Letters, in 1977, one year ater the regulation that
permitted only men into the Academy was revoked. Queiroz’s admittance into
the academy is remarkable, even to today’s standards. Out of 288 members since
it was founded in 1897, eight have been women so far, that is, 3%, in a country
where 51% of the population is female. Ideologically, the trajectories of both women resemble each other’s in even
more speciic ways. Prichard and Queiroz were attracted to communism, both
contributed to the inauguration of the Communist Party in their regions and both
felt betrayed by it. When Queiroz showed the manuscript for her second novel
João Miguel (1932) to her hierarchical superiors in the Party, they condemned,
among other things, the fact that it portrayed the incarceration of a labourer for
killing a workmate. hey recommended that Queiroz should edit the text, making
the wealthy boss the villain instead of the worker. he social/documental novel in Brazil and Australia Prichard and Queiroz’s ideological commitment and disillusion seem to be,
therefore, part of the Zeitgeist of the 1930s and the following decades. In Brazil,
the period was characterised by recession, high inlation and harsh military
intervention. Democracy was not consolidated, the old rural oligarchies that had
ruled the country since before the republic, in 1889, still fought each other for
inluence. Coups were expected events of national life. As an inlux of international
investment encouraged the industrialisation process, long-established monocrop
and pastoralist agricultural practices were challenged. However, the huge social
and economic gaps between social classes remained unaddressed. Literature
relected the period’s turbulences and inequalities and the 1930s came to be
associated with the so-called “social/regionalist” novel. For administrative purposes, Brazil is divided into ive macro regions
established oicially in the 1970s: North, Northeast, Centre-East, Southeast and
South. Although these regions are territorially vast and present a great deal of
internal diversity, Brazilians are frequently bound to stereotypically identify
them (especially if they are not their own region) with a few of their positive
or negative characteristics; thus the North Region could be associated with
forests and Indigenous peoples – as well as deforestation and land conlicts; the
South Region with cold weather, pampas vegetation, cattle farms and European
immigration – and with separatist movements, and so on. Difering from the Australian context, in which “regionalism” seems to have
mostly a political connotation, regionalismo in Brazil is very much related to
cultural life, the fact that, as Luis Augusto Fischer (7, my translation) explains,
a cultural product “has sprung from any of the [Brazilian] regions, one among
a number of others (although in general, the scenery depicted is predominantly
rural)”.2 In the Brazilian imagination, regionalist characters are instantly
associated with rural (unsophisticated and easily deridable) types: the caipira (the
country bumpkin from several parts of the Brazilian countryside), the sertanejo
(the north-eastern “bushman”), the gaúcho (the southern Brazilian version of the
cowboy), and so on. And as “region” is deined against “a (supposed) universalism
represented only in the art of the Court, the Centre, he Metropolis, always of
the City” (7, my translation),3 regionalist literature, as well as much of the 1930s
iction, have come to be viewed as minor art. Two writers from the margins In indignant response to this
interference with her freedom of expression, Queiroz let the Party. Censured by
her communist colleagues for being insuiciently communist, her work failed to
please the militarist federal government for the very opposite reason, and in 1937
the Vargas administration, accusing her of subversion, sent her to prison and
withdrew her irst three novels from the market. In Australia’s less truculent political context, Prichard went through
similar dilemmas, struggling to maintain her political views even when their
reception was unfavourable. In the 1930s she travelled extensively in Russia to
gather material for her pamphlet he Real Russia (published in 1934). When it
was suggested that she should submit the manuscript to the Communist Party
prior to its publication, Prichard refused to comply. According to her son and
biographer Ric hrossell “she could no more overstate the political content of a
novel to please Communist critics, than omit political views which she believed
to be a real part of her subject to meet the wishes of Australian and British critics”
(157). In the 1950s the Menzies administration tried to dissolve the Communist
Party in Australia, Australian armies joined the Korean War and the conservative
mentality started to see “the Red menace” everywhere. In a 1950 letter to her son,
Prichard wrote about her anxieties, joking about the “red witch” label that her
Perth neighbours had attached to her, and mentioning her fear of “interference
with [her] normal way of life”. Fortunately, that intervention never came to pass
(hrossell 156). Unlike Queiroz, who according to her own words (1999 27, 90 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... my translation) “became a sweet anarchist” ater Trotski was killed, Prichard
remained loyal to the Communist Party her whole life. he social/documental novel in Brazil and Australia Aiming at a less reductionist and derogatory view of the 1930s novel in
Brazil, Luís Bueno conducts an extensive examination of the ictional production
of that decade and inds, as a common ground for most of the novels, the fact
that they tend to, in a way or another, express the “problem of the observation
of the other”. his 1930s “other” in Brazil, he claims, consists mainly of the poor
(euphemistically called “proletarian”), but also of other marginalised people and 91 Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 groups, such as racial and ethnic minorities, children, adolescents, homosexuals,
people with mental health issues and women. Difering from previous decades,
the other who now appears in Brazilian literature is no longer characterized by
his/her folk interest, or by being a mere accessory role, but he/she becomes a
protagonist on his/her own right (Bueno, 16, my translations). he 1930 decade in Australia was characterised by the shit from the popular
poetry and short stories, that since the 1890s had been considered mainstream
literary forms, to the novel. Unlike the Brazilian scene, where women writers were
still a minority, the lourishing of the Australian novel in that period also meant
a better balance in terms of gender. Drusilla Modjeska (5) surveys the novels
produced from 1928 to 1939, concluding that the period was unprecedented in
the history of Australian literature, as nearly half of the novels were written by
women, who also stood out in terms of the quality of their work. Miles Franklin,
Marjorie Barnard, Flora Eldershaw, Christina Stead, Eleanor Dark, Henry
Handel Richardson and Prichard herself are some of the novelists who became
prominent in the decade. As David Carter (370-371) points out, from the 1930s to the early 1950s,
social disturbances, exacerbated by the Depression, fascism and the possibility
of war, were regarded by female and male Australian authors alike as imminent
threats to culture. As an opposition to traditional, “well-made” plots based on
heroism and romantic interest, a new literary mode emerged, shaped by an
impulse towards documentary writing. It was a time for the discussion of the
role of literature and intellectuals in society: “[s]tyle and subject-matter were
politically motivated as writers attempted to represent forcefully, in literature
discourse, facts and attitudes which they believed literature had conventionally
excluded and falsiied” (371). he social/documental novel in Brazil and Australia Rhetorically based on diferent premises from
those pertaining high modernism, the “documental novel” was shunned by some
modernists. What comes to mind is Patrick White’s deinition of these novels
as “the dreary, dun-coloured ofspring of journalistic realism” (16). Along the
same line as Bueno’s argument in defence of the 1930s novel in Brazil, Carter
(374) posits that the thematic and stylistic distinctiveness and diversity revealed
by 1930 and 1940s novels undermines that kind of criticism. Diversity was also
relevant to counteract the tendency to homogeneity pervasive in traditional
Australian literary histories. he diverse aspects of Australian life that Prichard sought to reveal in
Coonardoo were shocking occurrences in remote areas that urban coastal
Australians and traditional iction had preferred to overlook. he novel centres
on the relationship between Coonardoo, an Indigenous woman, and her white
boss, Hugh Watt. Coonardoo belongs to the Gnarler (alternative spelling for
Ngarla or Ngaala-wangga) People, whose ancestral lands at the time of the
narrative are within the territory of the ictional Wytaliba Station in the Pilbara
region, in North-Western Australia. According to he Australian Concise Oxford
Dictionary (1997), a station is a “tract of grazing land, usually having a discernible
centre of occupation”; “an extensive sheep or cattle raising establishment.” 92 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... Wytaliba is owned and run by Hugh’s strong-minded and independent mother,
Mrs Bessie. he novel mixes ictional events, events inspired in actual occurrences
and observations, in Prichard’s characteristic anthropological/poetic style, of
diferent aspects of station life and the life in the uloo, or native camp, where the
Gnarler Mob attempt to hold on to their traditional ways. Prichard used her own
travel experiences in the region as inspiration for the novel. Coonardoo spends her days as a house-servant and goes back to the uloo in
the evening to be with her people. Ater Mrs Bessie’s death, she continues to serve
Hugh’s wife, Mollie. Hugh and Coonardoo have been life-long friends and have
developed a strong afective connection, as well as a common love of the land. However, a single sexual encounter between them, occurred sometime before
Hugh’s marriage, leads to catastrophic consequences. Ater the marriage falls
apart and Mollie returns to Perth, Coonardoo and Winni, her child with Hugh,
leave the uloo and move permanently into the homestead. he social/documental novel in Brazil and Australia Although the common
view is that Coonardoo is Hugh’s “black velvet” (Aboriginal mistress), Hugh, for
years, represses his sexual drives towards her. During one of Hugh’s absences,
Sam Geary, the lustful owner of a neighbouring station, has sex with Coonardoo. he unresolved sexual and emotional tension between Hugh and Coonardoo
result in violence and Coonardoo’s ultimate moral and physical degradation in
exile. By the time Coonardoo returns to her country to die, Wytaliba, ravished by
drought and deprived of the people who genuinely love it (since Hugh has sold it
to Sam Geary and the Gnarler have let), is only a shadow of its initial grandeur. Space is also a central element of the so-called literatura da seca (drought
literature) in Brazil, of which he Year Fiteen is one of the best-remembered
titles. Like Coonardoo, he Year Fiteen has a historical backdrop: the drought of
1915 in Ceará State, one of the most devastating and long-lasting occurrences
of this kind in the sertão. he term sertão designates the semi-arid backlands of
the Northeast region. Whilst droughts are recurrent in the Northeast, they are
especially intense in Ceará, a State with 94% of its territory located in Brazil’s
semi-arid zone. he 1915 drought was witnessed by Queiroz herself when she
was a little girl on her family’s farm. he prolonged dry spell sets two plots in motion. he irst one initially points
to the romance formula between cousins and childhood sweethearts Vicente
and Conceição, whose families own cattle farms in the sertão. Conceição, a self-
educated and quite erudite young woman, spends most of the year in Fortaleza,
Ceará State’s capital city, where she works as a teacher. Vicente manages his
parents’ farm but refuses to do what most of his neighbours are doing at a time of
crisis: release, in order to cut costs, the few head of cattle that have survived. his
action also means dismissing the live-in farmhands and their families, making
them instantly unemployed and homeless. he second plot depicts the struggles of one of these impoverished, landless
families. Unable to aford train tickets, Chico Bento and his family set of on a
long walk from Quixadá, in the sertão, towards Fortaleza, on the coast, around
100 miles away. On this journey they cross paths with dozens of other forced 93 Ilha do Desterro v. 72, nº 1, p. he social/documental novel in Brazil and Australia 087-114, Florianópolis, jan/abr 2019 migrants, known in Brazil as retirantes (“retreaters”). hese travellers share
stories of poverty, famine, disease, death and injustice. he trip takes a heavy
toll on Chico Bento’s family: one of his sons, ravished by hunger, eats poisonous
roots and dies, another runs away towards an unknown, probably criminal future
and Chico’s sister-in-law stays behind, seemingly driven to prostitution. he two
plots come together in Fortaleza, where the family, like thousands of others, seeks
shelter in the so-called “concentration camp”. In 1930 the expression did not have
a Nazi connotation, being used to describe encampments of people guarded by
the police and built – or, rather, improvised – to stop drought refugees from
entering the town. Conceição, who volunteers in the camp, comes across them
and, ater adopting their youngest child, helps organise their migration to São
Paulo. Instead of pursuing a relationship with Vicente, Conceição chooses a path
that does not involve marriage. Although a love story between two people from similar social classes and
ethnic backgrounds could be more easily fulilled here than in Coonardoo,
Queiroz does not provide a traditional romantic solution to the plot. While
both Coonardoo and he Year Fiteen present heavy social comment on their
respective societies, they also challenge the romance formula as well as the
readers’ expectations. Coonardoo and settler colonialism In 1969, Catherine Duncan heard that Prichard was planning to destroy
some of her personal papers and manuscripts. In a letter that would prove quite
prophetic, she tried to dissuade her friend: “Is the writer capable of being his
own critic? […] Who is KSP ater all? Not a solid, one-sided slab in any case,
but with an ininite number of transparencies and obliquities which she reveals
briely in her writing and to friends […]. In ity years, dearest Kattie, the KSP
you are will have become someone else, she will have escaped you” (Duncan,
apud Bird, 2000, p. xi). Half a century ater her death, the multifaceted author that KSP has become
still incites controversy. he Austlit database displays more than 880 entries of
critical texts on Katharine Susannah Prichard.4 Examining some of those texts
pertaining to Coonardoo, it is possible to notice that various of the assertions
about the novel can be counteracted with diametrically opposed argumentation,
either by a single critic or by diferent ones: the novel – or parts of it – is censured
and praised; its degree of verisimilitude can be played up or down; it is viewed
alternately as subjective or as objective, realist or romantic, reticent or bold in its
assertions; it is considered strongly and consistently committed to an ideology
or loosely and mistakenly so; in the cultural studies realm it can be placed at
the cutting edge, supporting Indigenous and/or feminist causes, or it would
be reactionary, actually doing a disservice to the minorities it is supposed to
represent (for some of these readings see Bird, 2003; Corbould, 1999; Hodge,
1991; Kossew, 2004; Leane, 2014; Lever, 2000). 94 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... his dissonance might be rooted in the fact that Prichard is a non-
Indigenous woman trying to tell a story from the perspective of an Indigenous
one (her entitlement to do so is also a matter of contention). For the Wiradjuri
author and professor Jeanine Leane, inconsistencies in Coonardoo and other
Australian novels written by “whitefellas” who have attempted to look candidly
or sympathetically at Indigenous characters occur because along with their representations of Aboriginal people, these works are more
signiicantly journeys into the interior of the settler mind and consciousness
and its understanding of the phenomena of “the Aborigine” and are deeply
involved with questions of authority and power. Coonardoo and settler colonialism 087-114, Florianópolis, jan/abr 2019 apart from the metropolitan model and safeguarding their entitlement to the
Other’s land. In other words, “as it is coming from elsewhere and as it sees itself
as permanently situated, the settler collective is indigenous and exogenous at the
same time” (Veracini, 2010, 20). From that perspective, while in some theoretical approaches textual
occurrences such as “duality”, “contradiction”, “ambiguity”, “ambivalence”, “double
bind” – and other such variations – might be read as conceptual or structural
defects, settler colonial theory sees them as inherent aspects of the settler frame
of mind. Another relevant aspect is that settler colonial studies regard ictional
texts as legitimate sources for the observation and analysis of settler colonial
phenomena. In fact, ictional texts might even have the upper-hand in that
respect: “As non-historical approaches sometime display an analytical sharpness
that is seldom within the reach of traditional historical narratives, a ictional
reconstruction can perhaps better frame an encounter that is more imagined
than practiced (Veracini, 2010, 84). herefore, ictional plots with a factual background such as Coonardoo
provide a rich ield for settler colonial studies. he novel comprises a historically
identiiable period that has been thoroughly analysed by Marion Austin-Crowe. According to Austin-Crowe’s estimation, the events depicted by Prichard would
have happened roughly between 1885 to the mid-1920s and the Watts would
have bought Wytaliba from Saul Hardy, the irst European settler, around 1877. As the settlement of the Australian North-West had started in the 1860s, Wytaliba
station and the Gnarler uloo within it would constitute, thus, a “near-pioneering
community” (Austin Crowe, 23-26). In the following conversation, Sam Geary
and Mrs Bessie reminisce about the Watts’ droving years, prior to the purchase
of the station: “Ted’d drive the ration cart and she’d drive the bullocks with a couple of
boys — black imps — about ten and twelve,” he said. “And when some of
the chaps got on to Ted for letting Mrs Bessie ride ater the bullocks, she
said, ‘Here, what are you chippin’ about? When Ted’s with the ration cart I
know where he is, and when I’m with the bullocks he knows where I am’”
(Prichard, 47). his anecdote on the reversal of gender stereotypes foreshadows Mrs Bessie
being nicknamed Mumae by the Wytaliba workers (whereas the word sounds
like Hugh’s childhood name for his mother, it actually means “father” in the
Gnarler language). Coonardoo and settler colonialism “he Aborigine” is the irst
renaming and therefore representation of us (Leane 1). Because it focuses on the settler imagination and problematises the
representations of the settler and the Other created by it, the recent critical ield of
Settler Colonial Studies is a suitable analytical tool with which to study Coonardoo
and other complex novels. he main premise of Melbourne-based historian
and political scientist Lorenzo Veracini’s seminal book Settler Colonialism:
A heoretical Overview, is that settler colonialism should be understood and
analysed not as a branch of colonialism but as a separate category of European
domination that shares some characteristics with colonialism (Veracini, 2010,
9). Veracini’s theories further develop Australian anthropologist Patrick Wolfe’s
thesis proposed in 1999 that, while colonialism is based on the idea that colonisers
will exploit Indigenous labour and resources to, eventually, go back to where
they came from, settlers “come to stay” (Wolfe, 2016, 65; Veracini, 2015, 74). For Wolfe, colonialism would, thus, constitute an “event” in the history of the
colonised. Settler colonialism, on the other hand, would be “a structure”: he primary object of settler-colonization is the land itself rather than the
surplus value to be derived from mixing native labour with it. hough,
in practice, Indigenous labour was indispensable to Europeans, settler-
colonization is at base a winner-take-all project whose dominant feature
is not exploitation but replacement. he logic of this project, a sustained
institutional tendency to eliminate the Indigenous population, informs
a range of historical practices that might otherwise appear distinct –
invasion is a structure, not an event (Wolfe, 1999, 163). One of the complexities of settler societies lies in the fact that settlers “uneasily
occup[y] a place caught between two First Worlds, two origins of authority
and authenticity”(Johnston; Lawson qtd. in Veracini, 2010, p. 9). Also very
importantly, in Veracini’s theory (2015, 68), “settler colonialism is not inished”. European descendants in settler colonies have inherited the predicaments of
their ancestors: as they aspire to escape from imperial rule and strive to establish
and maintain their agency over indigenous lands, they are also ready, when
convenient, to evoke metropolitan authority in their dealings with Indigenous
Others, as well as to appropriate Indigenous traits when trying to set themselves 95 Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 Ilha do Desterro v. 72, nº 1, p. Coonardoo and settler colonialism In a style that evokes the nationalist “Lawson Tradition”5, the
cheerful, yarn-like account of the mateship between husband and wife disregards
any conlicts that might have existed or still existed between settlers and the
Indigenous custodians. here seems to be no qualms about the exploitation of
the labour of Indigenous children or their separation from their families so that
they can go droving with the settlers. In fact, the excuse for the exploitation of
those children is at the same time revealed and sotened by the adoption of the
ironic term of endearment “black imps”. 96 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... Hiding the sins of settler colonialism behind the guise of frontier myth,
romantic narratives of conquest, or nostalgia for the “good old times” are denial
mechanisms adopted by settlers. he encounter with Indigenous peoples,
becomes, thus, “premised on a foundational disavowal [that] can be better
described as a non-encounter” (Veracini, 2010, 84). One type of discourse that
contributes to the efacement of the injustices committed against Indigenous
peoples maintains that they would be dying races, doomed to disappear from
the outset; another poses Australia as the “Quiet Continent”, passively awaiting
the arrival of the Europeans (Veracini, 2010, 90-91). Prichard’s ictional world
draws from both types of disavowing discourses. Coonardoo’s tragic demise
and the disappearance of the uloo in Wytaliba Station are in line with Prichard’s
assertions, in her foreword to the novel, that “the poor, degenerate and degraded
creatures” that “the blacks” had become would be a “result of contact with
towns and the vices of white people.” She also resorts to the authority of Social
Darwinist theories to predict their disappearance: “In other words”, she claims in
her preface, “the Australian aboriginal stands somewhere near the bottom rung
of the great evolutional ladder we have ascended” (Prichard xxvi). As for the passivity of the Australian Continent, the protagonist herself
would stand for the quiet land, starting with her deeply symbolic name: She could see low brown huts down there beside the well, a deep narrow
well the Gnarler had dug long ago at a little distance from the creek. Coonardoo they called it, the dark well, or the well in the shadows. Coonardoo had been named ater the well near which she was born. he
huts were the huts of her people (Prichard 2). he “well in the shadow” is the main leitmotif along the narrative. Coonardoo and settler colonialism he combined
characteristics of depth and darkness suggest the mysterious ontological
bond with the land, something settlers are eager to possess. Forging a similar
connection between the European and the colonised land would validate, once
and for all, the settlers’ territorial rights, a self-bequeathed entitlement that is
always shadowed by the presence of the original custodians (Veracini, 2010, 43). Furthermore, in the harsh environment of the Western Australian outback
a shadowy well could allude to a miniature oasis, a source of life and relief in the
midst of extreme aridity. his connotation could be associated with the status of
Coonardoo as a house servant, an occupation that implies attention to life’s basic
needs in a context of near invisibility or, at least, of being taken for granted by
the employers/masters. Coonardoo is both a shadow to Mrs Bessie and Hugh,
always at their beck and call, and a source of care and comfort to the Watt family,
especially to Molly, Hugh’s wife. Molly is a frustrated city girl whose initial
dislike of station life turns gradually into hatred, and who eventually leaves the
management of the household and the care of her ive daughters to Coonardoo. Although Hugh and the omniscient narrator at times express disapproval
of Molly’s abuse of Coonardoo’s willingness to help, Coonardoo’s own (either 97 Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 inner or spoken) voice is not made to question the pertinence of her loyalty, an
exploitation that comes to the brink of slavery: hrough all the nervy restlessness and fury of Mollie’s discontent
Coonardoo was her slave. Silently, with slow grace and dignity, she waited
on and worked for Hugh’s wife, very oten not getting the rest at midday
with the other gins, it was so diicult for her to go without. An expression
of sufering and fortitude deepened on her face (Prichard 144) Another connotation of the word “well” is sexual. he word signiicantly
reappears in the subtle and understated description of Coonardoo and Hugh’s
sexual encounter, which is also the turning point to the narrative. he episode
occurs before Hugh’s marriage, when he is half-crazed, mourning the death of
his mother and “goes walkabout”, miming, in a clumsy way, the Aboriginal rite
of passage into manhood, when a boy embarks on a long, lonely walk in the
outback. Coonardoo and settler colonialism She was a stake, something to hang on to. More than that, the only stake he could hang on to. He had to remind
himself of her dark skin and race. Hugh had never been able to think
of Coonardoo as alien to himself. She was the old playmate; a force in
the background of his life, silent and absolute. Something primitive,
fundamental, nearer than he to the source of things: the well in the
shadows. (Prichard 140-141) Hugh’s attempt to (sexually) hold on to Coonardoo, something “nearer than
he to the source of things”, as well as his secret attachment to their child, is a
movement towards what Terry Goldie (13) calls “indigenization”. he need to
“become native” and “to belong” is one more of the complications posed by the
settler situation. Goldie (12), using White Canadians and the Canadian “Indian”
as paradigms, puts it in the following terms: “he White Canadian looks at the
Indian. he Indian is Other and therefore alien. But the Indian is indigenous
and therefore cannot be alien. So the Canadian must be alien. But how can
the Canadian be alien within Canada?”. White writers, Goldie goes on, have
tried to solve that impasse “through writing about the humans who are truly
indigenous” (13). Veracini also drawing from Goldie’s deinition, develops the
idea of “indigenisation” as the “crucial need to transform an historical tie (“we
came here”) into a natural one (“the land made us”) (2010, 21-22). Another attempt at indigenisation is discursive. In this respect, the novel
seems to be following the period’s ethnographic and anthropologic trends, as W.B. Spencer and F.J. Gillen’s book, originally published in 1899 as he Native Tribes of
Central Australia is re-launched as he Arunta in 1927, followed by he Vanished
Tribes, by James Devaney in 1929 and David Unaipon’s Myths and Legends of the
Australian Aboriginals in 1930. In Coonardoo, the Gnarler language appears in
songs, in expressions used by the Indigenous characters and in sparse words along
the text, for which Prichard provides translation and a glossary. Coonardoo and settler colonialism Coonardoo – who has promised Mrs Bessie to take her place and look
ater Hugh – quietly follows him from a distance and is eventually discovered: his was a childish adventure they were on. His gratitude shook him as
he thought of how she had followed and watched over him during the last
weeks. It yielded to yearning and tenderness. Deep inexplicable currents
of his being lowed towards her. g
“Coonardoo! Coonardoo!” he murmured. “Coonardoo! Coonardoo!” he murmured. Awakened, she came to kneel beside him, her eyes the fathomless shining
of a well in the shadows. Hugh took her in his arms, and gave himself to
the spirit which drew him, from a great distance it seemed, to the common
source which was his life and Coonardoo’s. hey slept beside the ire near the clump of dead mulga until it was
morning. Hugh started up to ind Coonardoo stirring embers of the ire. hey had walked back into the camp then. (Prichard 81-82) he sequence of the death of the mother, followed closely by a sexual encounter
with her surrogate (to be later followed by guilt, rejection and rage), has clear
psychoanalytical inferences. he sexual act and its outcome, the “half-caste” Winni,
(“the son of the whirlwind”), become, for Hugh, both a source of guilt (since he has
sworn he will never behave like Sam Geary in relation to Indigenous women) and
a conirmation of his own deeper connection with the land. he ambivalent feelings of guilt and desire to own the land are pointed out by
Veracini as characteristics of the settler mindset. herefore, when Hugh compares
his feelings towards Coonardoo and Winni to those towards Molly and their
eldest daughter Phyllis, the former seem to him more “rooted” into the earth: Mollie’s baby, fresh and pink-and-white, was a fairy creature. Hugh loved
her; but she was less real, much less his own than that son of a whirlwind. Always as he leant over, played with and held the baby, he thought of
Winni. His afection for the boy plagued him. Was it because he reproached
himself for the existence of the child? Perhaps. Hugh could not tell. Did he
reproach himself really? 98 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... Coonardoo had been the one sure thing in his life when his mother went
out of it. He had grasped her. Coonardoo and settler colonialism he novel opens
with Prichard’s transcription of an Indigenous song (about kangaroos coming over
to feed and making a devil dance with their feet), while, alone at dawn, the nine-
year-old Coonardoo laments the imminent departure of her playmate Youie (Hugh)
to school in the city, “[a]way and away, farther than Coonardoo could think, beyond
the blue backs of the hills, mulga scrub, and again away, and away, to the sea”: “Poodinyoober mulbeena, mulbeena, mulbeena!” she wailed, while yellow
moths beat the air before her, black markings of their wings lickering,
jiggling with little feet of the kangaroos, and white threads of the blossom
which were falling. Little feet, luttering wings, threads of falling blossom
wreathed a cobwebby sleepiness over her. Very drowsily, the faint reedy
voice twanged. Coonardoo’s head drooped, the ine silky jet of curled
lashes swept her cheek. Her singing ran out, and started again in a lurry
(Prichard 3). “Poodinyoober mulbeena, mulbeena, mulbeena!” she wailed, while yellow
moths beat the air before her, black markings of their wings lickering,
jiggling with little feet of the kangaroos, and white threads of the blossom
which were falling. Little feet, luttering wings, threads of falling blossom
wreathed a cobwebby sleepiness over her. Very drowsily, the faint reedy
voice twanged. Coonardoo’s head drooped, the ine silky jet of curled
lashes swept her cheek. Her singing ran out, and started again in a lurry
(Prichard 3). he efect of these lyrics, woven into the narrative voice’s own poetic prose, is
slightly surrealist, materialising Coonardoo’s sleepy state and providing a glimpse 99 Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 into her world. However imperfect and idealised, Prichard’s attempt at depicting
the Indigenous consciousness was a new development in Australian literature
in the 1930s. For Goldie (54-55) the innovation in Coonardoo was not thematic,
as many critics claim, but it would lie in the omniscient narration concentrating
on diferent characters, an Aboriginal woman among them. he song works as a
frame to the novel, reappearing in the inal chapter, about 40 years later, with a
new connotation. Lonely and dreamy once again, but also nostalgic and broken
by venereal disease and the hostile conditions of the life she’s led since Hugh
banished her from Wytaliba, Coonardoo goes back home and the kangaroo song
of her childhood becomes her requiem. Coonardoo and settler colonialism She was disgusted by practices she 100 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... considered immoral, until she began to understand a diference to her own
in the aboriginal consciousness of sex. She was surprised then, to ind in it
something impersonal, universal, of a religious mysticism (Prichard 26). She likes to see herself as the benign, open-minded, culture-sensitive settler
and she is aware of the damage caused by what Veracini (2010, 44) has called
“transfer by coerced lifestyle”, a type of transfer that does not necessarily involve
geographical movement, being a way of removing (transferring) the Indigenous
Others’ lifestyle and/or social and political organisation. In several instances, Mrs Bessie’s positioning seems to be uncharacteristically
tolerant for the time: “Mrs Bessie would not allow any Christianizing of the
aborigines on Wytaliba. She had never seen a native who was better for breaking
with his tribal laws and beliefs” (Prichard 16). he continuation of the paragraph,
however, counteracts that open-mindedness by revealing the impossibility of
maintaining those traditions in settler societies reliant on Indigenous labour, as
well as Mrs Bessie’s deep-seated value judgement regarding the primitivism of
Indigenous customs: “although all day Coonardoo was Mrs Bessie’s shadow, and
learned to wait on and do everything for her, bring her tools, make her baths and
her camp-ires, always at sunset she went of with her people and slept with the
dogs by her father’s camp-ire” (Prichard 16). Although, in thesis, settler societies do not rely primarily on Indigenous
labour or slavery, as Austin-Crowe’s study (65) concludes, settlers in North-
Western Australia were very historically dependent on Indigenous peoples for
the running and maintenance of both their stations and homesteads, adopting a
labour system that would be today seen as akin to slavery. hrough Mrs Bessie,
Prichard implies that even for the most well-meaning of station owners, respect
for the Other’s lifestyle seems to go only as far as it does not interfere with the
settlers’ own comfort and proit. By “exchanging” Indigenous labour for clothes,
rations, blankets, knick-knacks and occasionally a horse or other livestock, Mrs
Bessie helps promote the permanent “sedentarisation” of a society that is partly
nomadic. She also makes its members dependent on her own “benevolence”,
hence Indigenous characters’ half-starved, dirty and ragged state when Hugh
returns to the Station ater his long recovery period in the city (Prichard 101). Coonardoo and settler colonialism Prichard uses Coonardoo’s tragic fate as an allegory for the demise of the
Indigenous Other in Australia, a clear settler colonial strategy. Settler colonialism
is a project based mainly on land appropriation and the subconscious, ambivalent
or earnest desire for the disappearance of the Indigenous Others. his is a crucial
object for settlers, as the presence of Others brings forth a moral dilemma and
causes permanent anxiety. Indigenous Others “challenge with their very presence
the basic legitimacy of the settler entity” (Veracini, 2010, 33). To secure its
implementation and perpetuity, settler colonialism relies on varied strategies. Although the irst ideas that come to mind regarding Indigenous disappearance
are bloodshed and genocide, overtly violent means are not always the case. Veracini (2010, 33) uses the expression “transfer” to refer to several strategies
that settlers can adopt to make the Other vanish, in literal or metaphorical terms. One of Prichard’s objectives in making Coonardoo a protagonist seems to be
to try to counteract the efects of what Veracini (2010, 37) has called “perception
transfer”, or the attempt to ignore the (present or past) presence of Indigenous
peoples. By bringing to the surface at least two situations urban coastal Australians
were willing to ignore – the possibility of interethnic love and the sexual
exploitation of Aboriginal women – Prichard stirred Australians out of their
complacency and disturbed what the anthropologist W. E. H. Stanner (189) in
the 1960s called “he Great Australian Silence”, or white Australia’s unwillingness
to discuss the moral consequences of dispossession. Critical response and the
stacks of complaint letters that the Bulletin received ater the publication of the
story attest to the successful outcome of this aim. he ambivalent attitudes of the novel’s main landowners Mrs Bessie and Sam
Geary also make them “agents of transfer”. Mrs Bessie tries to come to terms
with her own ambiguous feelings towards some of the practices that would be
instantly considered taboo by Western standards – and thus, subject to transfer
– such as the early sexual initiation of girls, which Prichard describes in some
detail in chapter 3: Mrs Bessie had its of loathing the blacks. Although she had lived and
worked like a man, so long in the Nor’-West, without the least respect for
conventional ideas which hampered her in anything she wanted to do, her
white woman’s prejudices were still intact. Coonardoo and settler colonialism Giving the impression that Indigenous people cannot survive without the settlers’
kindness is another strategy to justify dispossession. At irst sight, Sam Geary would be on the opposite side of the virtue
spectrum when compared to Mrs Bessie, and yet his depiction by Prichard is
no less complex. On the one hand he is the abject sexual predator type, always
trying to negotiate with Indigenous men in order to acquire new women. He
ofers “old Joey Koonarra, Coonardoo’s father, a rile, blankets and tobacco for
the girl” (Prichard 32), but is stopped by Mrs Bessie. Invoking the Bible to justify
his polygamy with eleven Indigenous women, he also treats miscegenation
lightly, having fathered several half-caste children, who will not, obviously,
acquire any inheriting rights of his property, Nuniewarra. On the other hand, his
outspokenness about racial relations and the fact that he deies interethnic taboos 101 Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 (which are apparent in his dealings with Indigenous women, such as his current
lover, whom he calls “he Queen of Sheba”), contrast greatly with the hypocrisy
sustained by the supposedly benevolent settlers: Sheba had been with Geary two or three years now. She kept the keys of the
store-room. Before Sheba there had been Sarah and Tamar. Now Sheba and
Tamar both had corrugated-iron huts on Nuniewarra, although Sheba spent
most of her time at the homestead with Sam. She made tea for visitors, and
Geary took her with him when he went into Karrara, engaged rooms for her
at the hotel and gave her money to buy silk dresses. She went to the races
with him. But here in Wytaliba, Sheba had to eat at the kala miah [wood
heap] with the other gins [Aboriginal women] (Prichard 65) But Coonardoo refuses to become Sam Geary’s “black velvet”. And yet, single,
puzzling, half-consensual sexual intercourse with the abject settler results in
metaphorical and literal transfer, which takes the form of banishment, exile and
physical and moral decline. In Prichard’s telluric but tragic view of interracial relations, Coonardoo’s
approximation to the abject settler and distancing from the virtuous one result in
the deterioration of her own and Hugh’s connection with the land, and, ultimately,
in the decay of the land itself. he Year Fiteen: an attempt at a settler colonial reading Rachel de Queiroz’s reception, like Prichard’s, has been highly controversial. Heloisa Buarque de Hollanda (2016, my translation) ascribes that to the fact
that Queiroz “always excelled in driving against the traic of History”.6 Several
interviews given by Queiroz reinforcing again and again her ideas seem to
suggest that she did not mind and even felt a certain satisfaction in the polemic
her statements could incite. Hollanda (2016) and Wasserman (2007 57) believe
that her personal views have had too much inluence on the critical readings of
her work. In relation to the regularity of these critical readings, Hollanda (2016)
observes that from 1930 to 1960 there was a great wave of criticism on Queiroz,
an interest boosted by the novelty and quality of her work. With the proliferation
of graduate literature degrees, ater the 1960s (high times of military dictatorship
in Brazil), a new generation of critics, involved with and/or inluenced by the
establishment of an “academic canon” started to consider Queiroz an awkward
object of study. Hollanda (2016) attributes that rejection not to the response to
her iction itself, but to the apprehension created by her conlicting relationship
with feminism, her acquaintances in places of power, her free traic through the
backstage of Brazilian literature and politics, and her contentious, sometimes
inconsistent, political ideas and public statements. Scholars did not know how to
juggle Queiroz, the writer of iction, and Queiroz, the outspoken persona. Queiroz’s outspokenness was notorious. Albeit one of the most important
modernist writers in Brazil, she used to bale her interlocutors by claiming that 102 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... she “didn’t like writing”, that she “wrote only for the money”, that she thought
of herself more as a journalist than as a novelist (Hollanda, 2016), and so on. Not only did she try to disengage herself from any gendered views of her iction,
but she declared, more than once, that she “hated” feminism. She found it a
“badly-oriented” movement, so much so that, if “a feminist gave an interview
bad-mouthing a man; [she] found a way to claim that [she] liked him, just to keep
[her] position” (Queiroz, 1997, 26, my translation).7 And yet, her ictional work,
illed with what Queiroz herself classiied as “extraordinary women” (Queiroz,
1997, 26, my transltaion),8 is an invitation to feminist readings. he Year Fiteen: an attempt at a settler colonial reading In Hollanda’s
assessment (2016, my translation), her protagonists “are all strong, self-suicient
women, who obstinately follow the paths that lead to destinies marked by
independence and power.”9 Invariably, for Queiroz’s young protagonists, marriage
is not a formula for female happiness. And among her older female characters are
the matriarchs, rural women who are widowed or whose husbands are frequently
absent, and who “managed, with an iron grip, the large sugar and cattle farms in
the [Northeast] region” (Hollanda, 2016, my translation).10 Feminist readings of
Queiroz’s novels, however, are still infrequent. In relation to her political views, Queiroz’s positioning is even more
contentious. Having been persecuted, arrested and censured during the Vargas
presidential term for her communist ideas, and considering his successor, João
Goulart/Jango, a continuator of Vargas’s policies, she ended up, as she herself puts
it (long ater she had abandoned communism), “conspiring with the generals
for the overthrowing of Jango” (Queiroz, 1997, 29). 11 Her justiications for this
position are based on the fact that she had many friends and family among the
military and their sympathisers, who even gathered in her home to articulate their
moves. General Castelo Branco, Jango’s successor, was her friend and called her
“cousin” (Queiroz, 1997, 29). A recent example of the controversy that Queiroz’s
positions still incite happened on 17 November 2017. To mark Queiroz’s birthday
and targeting students of Brazilian literature preparing for university entrance
exams, the Facebook page Lítera re-posted a photo of Queiroz with the following
subtitles: “At 17 I wrote for newspapers, at 18 I published my irst novel, and I
was the irst woman to enter the Brazilian Academy of Letters. Nice to meet you. Rachel de Queiroz” (my translation). he post had been shared by more than 800
people. Among the comments, fans, some considering her a “Northeast Diva”,
and critics discussed the relevance (or not) of her collaboration with dictatorship
to the appreciation and evaluation of her iction.12 For Hollanda (2016, my translation), Queiroz’s “deep intimacy with power”
was inherited from the tradition of compadrio, or “godparenting”,13 a profoundly
ingrained trait in Brazilian personal and familial relations, especially in the
Northeast, a society historically dominated by archaic, oligarchical, landed
gentry. Compadrio derives from Catholic baptism rites, a compromise that the
godparent assumed to provide for a child in the case of the parents’ absence. he Year Fiteen: an attempt at a settler colonial reading In the
large landed estates, that practice was extended to an “almost feudal relationship
between godchildren and their godparents, who were the land-owners or the 103 Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 educated elite.” he objective was to “develop, within the family, strategies of power
distribution and ways of safeguarding the protection of household members and
godchildren” (Hollanda, 2016, my translation). he expressions compadre and its
feminine version comadre also have a more widespread, non-religious, meaning,
being used all over Brazil as a term of endearment to express esteem or friendship
(Houaiss). In interviews it is possible to perceive how compadrio afects the way Queiroz
sees herself, her family and her family’s social position and function in Ceará: I was born into a family of farmers, but our farms were always poor, cattle
farms, we never had the abundance of the farms in Bahia or Pernambuco
[neighbouring States to Ceará], where the [sugar cane] mill owner was
a dignitary. In spite of any ideologies, we were always friends with our
employees, we were “compadres” of our stockmen [godparents to the
stockmen’s children]. In our midst there were never any land problems,
because we always gave land for our tenants to cultivate. I may have a lot
of defects but I never charged for a grain of bean from a worker of mine. (Queiroz, 1997, 28)14 Landlessness and inequalities that were (and still are) happening in the
Northeast become, for Queiroz, not a political matter of land distribution, but
a personal question of lack of generosity on the part of the large landowners
who do not “give” (an obvious euphemism for “loan”) land for the tenants to
cultivate, who do not become godparents to their children or who charge for
all the “beneits” they concede to them. Queiroz also transposes this idealised
view of the relationship between the landed gentry and the landless in Ceará, a
manifestation of what Veracini calls “disavowal”, into her iction. In this paper,
these relationships will be approximated to the ones between the settler and
the Indigenous Other. his type of idealisation serves as a screen to profound
power and economic inequalities, evoking one of the disavowing settler strategies
described by Veracini (2010, 14). Analogously to Coonardoo, idealisation in he
Year Fiteen is emphasised by the contrast between the benevolent and the abject
landowners. he Year Fiteen: an attempt at a settler colonial reading he former are represented by Vicente and Conceição’s families, and
the latter by Mrs Maroca, the matriarch of the Aroeiras Farm and Chico Bento’s
boss, who is described as a “disgraceful” old rag and a miser (Queiroz, 2011, 90,
my translation).15 he wide social gaps in Brazil are – as much as their disavowal – deep-rooted,
as Queiroz’s statement above makes clear. Social class proiling goes hand-in-
hand with racism and oten overpowers it as a criterium for exclusion and
discrimination. According to sociologist Darcy Ribeiro, social inequality in Brazil
started to take shape in the sixteenth Century, when Portugal, a country with few
inhabitants, even for European standards, found itself in need to populate (and
therefore, protect from the grasp of competing powers, such as the Spanish, the
French and the Dutch), a huge mass of land overseas (in its current coniguration,
Brazil is 92 times larger than Portugal). he Crown’s solution was to grant huge 104 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... allotments of land (dozens of craggy leagues), the so-called donatarias, to a few
individuals considered loyal and wealthy enough to be able to settle on the land
with their own inancial means: he grantee was a high nobleman invested with feudal powers by the king
to govern his estate for thirty leagues in every direction; with the political
power to found villages, grant pieces of land, and license artisans and
merchants; with economic power to develop his lands directly or through
intermediaries, and even with the right to impose capital punishment. (Ribeiro 54) his method, put into practice as early as 1532, can be seen as the inception of the
land and income distribution problems that have plagued Brazil since then. he
donatarias system is also the root of the abusive political power that the landed
gentry have maintained along the history of Brazil, especially in the Northeast,
where some landowners would become known as coronéis. In spite of the
military term, coronelismo can be deined, according to the Houaiss Dictionary,
as a “social/political practice, characteristic of rural environments and small
interior towns, that lourished during the First Republic (1889-1930), a form of
bossism in which an elite, symbolically embodied in a landowner, controls the
means of production, detaining the local economic, political and social power”. he Year Fiteen: an attempt at a settler colonial reading Add slavery to this system of privileges and we have a society that, although still
resembling a settler colonial one in important ways, departs from the settler
colonial paradigm in others. Some of the characteristics of the Portuguese venture in Brazil are clearly
those of settler colonialism: the Portuguese came to stay, they made a point
of eliminating the Indigenous Others and introduced African and, later on,
Exogenous Others of diferent nationalities. However, Veracini (2010, 30) and
other scholars have acknowledged the diiculties of applying settler colonialism
theories to South American countries. According to Michael Goebel (139),
mestiçagem (miscegenation) and the colonisers’ primary intention of exploiting
labour for a proit, leaving the settlement itself as a secondary beneit, would make
it more diicult to it the Spanish and Portuguese-origin societies in America
into Veracini’s model. Mestiçagem is indeed a very complex matter in Brazil. When comparing
ethnic categorisations in Australia and Brazil, Wolfe (2016, 203), inds the
Brazilian system “extravagant” and “baroque in its excess”. While a division
between black and white (and, at most, “half-caste”) might generally suice
in Australia and many other hybridized societies, in Brazil there can be up to
500 diferent terms to describe racial types (although many of them are highly
specialised and localised in speciic micro-regions). And yet, Wolfe (2016, 204)
ponders, most of those terms refer to African + White variations and very few
to the admixture Amerindians + White. Caboclo is the main term to refer to
the latter (although mameluco, caiçara or curiboca are also sparsely used). his
disparity serves settler colonial purposes especially well. Settlers, as Veracini 105 Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 (2010, 26) points out, rely on the presence of Exogenous Others to feel entitled to
the land. Wolfe (2016, 204) explains the usefulness, in Brazil, of the one-drop rule
in creating and emphasising distinctions among African-Brazilians to foster the
social exclusion that comes from those distinctions. As a constant reminder of
the injustices of land appropriation, however, the presence of Indigenous Others
and the political conlicts that they might inspire
causes discomfort and (2010, 26) points out, rely on the presence of Exogenous Others to feel entitled to
the land. Wolfe (2016, 204) explains the usefulness, in Brazil, of the one-drop rule
in creating and emphasising distinctions among African-Brazilians to foster the
social exclusion that comes from those distinctions. he Year Fiteen: an attempt at a settler colonial reading As a constant reminder of
the injustices of land appropriation, however, the presence of Indigenous Others
– and the political conlicts that they might inspire – causes discomfort and
insecurity for settlers. heir elimination/assimilation into mainstream society
through miscegenation (and a much more limited range of terms to describe
their speciic ethnicity) becomes very much desirable. – and the political conlicts that they might inspire – causes discomfort and
insecurity for settlers. heir elimination/assimilation into mainstream society
through miscegenation (and a much more limited range of terms to describe
their speciic ethnicity) becomes very much desirable. Historically, miscegenation started as early as the arrival of the irst Portuguese
settlers. Ribeiro (49-50) observes that the establishment of the Portuguese colony
in South America was only possible because settlers took ruthless advantage of
the Indigenous practice known as cunhadismo (“in-lawism”). Many Amerindian
societies considered it a sign of esteem to “give” a male visitor a wife from their
clan. In taking the woman, the settler was no longer a foreigner and became
immediately an “in-law” to all her relatives on her parents’ side. As polygamy was
also a standard practice, a Portuguese man could easily form a complex network
of “relatives” to serve and work for him. More importantly, he could quickly
produce dozens of children. While settler violence and European diseases rapidly
decimated the Amerindian populations, it was this “widespread class of people of
mixed blood, who efectively occupied Brazil” (Ribeiro 50). In spite of the demographic signiicance that they soon achieved, these
caboclos or mamelucos were displaced individuals, becoming victims of two types
of rejection: First was that of their fathers, with whom they wanted to identify but who
looked down on them as impure sons of the land, taking good advantage of
their work while they were children and youths and later integrating them
into the bandeira expeditions [16th and 17th century colonial expeditions
into the interior of Brazil to search for precious metals and gather slaves
among the Indigenous Peoples] of which many made a career. he second
rejection was that of their maternal people. he Indians’ concept was that
a woman is simply the sack into which the male deposits his seed. he one
who is born is the child of the father and not of the mother, as the Indians
see it. he Year Fiteen: an attempt at a settler colonial reading Unable to identify himself with either of his ancestral lines, which
both rejected him, the mameluco fell into a no-man’s-land out of which he
shaped his identity as a Brazilian. So it was that by means of cunhadismo carried to extremes, a new human
breed was created, which was not recognized or seen as such by Indians,
by Europeans, or by blacks. (Ribeiro 70) In the oicial Brazilian historiography, until recently heavily inluenced by
Gilberto Freyre’s 1933 thesis that miscegenation had created in Brazil a less cruel
and racist society than the ones in North America (Freyre 1956), the fact that
the irst Brazilians ater European settlement were displaced half-caste children
enslaved by their own fathers and turned into enslavers of their Indigenous
countrymen (sometimes of their own mothers’ people), is easily “forgotten”. 106 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... Furthermore, a lot more emphasis is placed on African rather than on Indigenous
slavery. Self-perceptions of miscegenation say a great deal about Brazilians’
attitudes towards race and social class. Miscegenation has not meant the integration of the hybridised individuals
into a more privileged social stratum. As Deborah de Magalhães Lima (10)
elicits, the permanence, in Brazilian society, of the use of the term caboclo
as an expression of contempt in relation to the Other evinces that. here is a
contemporary generalised meaning to the term caboclo, current not only in the
regions with higher Indigenous demographics, but all over Brazil, that detaches
it from Indigenous associations, relating it to a person considered “more rural,
indigenous or rustic in relation to the speaker” (Lima, 1999, p. 7, my translation).16
Caboclo is, therefore, a regional (in the Brazilian sense) derogatory term that
is not usually self-attributed. In fact, when using it to describe someone, the
speaker is implicitly asserting his superior status as a “non-caboclo”. Signiicantly,
the caboclos, taken collectively, are not a social group found in real life, neither are
they a distinctive entity that could, like the Indígenas, or the African-Brazilians,
ight for political rights. Rather than a social group, caboclo is a social category,
“an abstraction, a unit in a classiication system intended to portray diferences
between people in a society” (Lima, 8, my translation).17 Miscegenation and variation in demographic estimation criteria make
it very diicult to determine the populational progression of the caboclos
in Brazil. he Year Fiteen: an attempt at a settler colonial reading he owner dismounted, with the same clumsy indolence […]. Vicente, sitting
on a hammock, the cigarette in between his hands, watched him come closer
and replied to the caboclo’s babbled greeting: – Good aternoon, compadre. Sit
down! he stockman sat on a wooden bench, close to the window. He’d come
to propose a deal… a few head of cattle he had in the Aroeiras and wanted to
sell… – So, is it true that you are going away? he caboclo wailed in a mournful
tone –Yes, sir… he owner gave orders for the cattle to be released…Today I
opened the gates… (Queiroz, 2011, 28, my translation). he horse stopped under the dried piece of white wood that served as shade. he owner dismounted, with the same clumsy indolence […]. Vicente, sitting
on a hammock, the cigarette in between his hands, watched him come closer
and replied to the caboclo’s babbled greeting: – Good aternoon, compadre. Sit
down! he stockman sat on a wooden bench, close to the window. He’d come
to propose a deal… a few head of cattle he had in the Aroeiras and wanted to
sell… – So, is it true that you are going away? he caboclo wailed in a mournful
tone –Yes, sir… he owner gave orders for the cattle to be released…Today I
opened the gates… (Queiroz, 2011, 28, my translation). he horse stopped under the dried piece of white wood that served as shade. he owner dismounted, with the same clumsy indolence […]. Vicente, sitting
on a hammock, the cigarette in between his hands, watched him come closer
and replied to the caboclo’s babbled greeting: – Good aternoon, compadre. Sit
down! he stockman sat on a wooden bench, close to the window. He’d come
to propose a deal… a few head of cattle he had in the Aroeiras and wanted to
sell… – So, is it true that you are going away? he caboclo wailed in a mournful
tone –Yes, sir… he owner gave orders for the cattle to be released…Today I
opened the gates… (Queiroz, 2011, 28, my translation). Vicente, pitying the cowboy, buys the cattle and the garment, although he
bargains for a lower price for the former. Not only in this passage but in his
interactions with the other characters whom he sees as igures of authority, Chico
is inarticulate and displays humbleness bordering on submission. he Year Fiteen: an attempt at a settler colonial reading Caboclo was a category in the oicial censuses of 1870 and 1890,
but was encompassed by the umbrella term pardo (a term that refers to an
indeinite brownish colour rather than to a racial category, used to designate any
combinations between White, Black and Indigenous), from 1940 on. However,
it is safe to say that the two regions with the largest proportion of Indigenous
peoples, respectively North and Northeast, are also the ones with the largest
number of caboclos. he complex character of Brazilian miscegenation makes it
hard to establish the triple structure of settler colonialism in racial/ethnic terms,
as Veracini’s theory claims. Yet, it is still possible to detect settler colonial traits in
the dynamics of the relations between the descendants of the white landowning
settlers and the hybridised descendants of Indigenous Others and caboclos, a
racial/ethnic phenomenon turned into a social-class matter in Brazil. While far
from claiming to come to a solution to the problem of settler colonialism and
the Portuguese settlement in South America, this paper will attempt to conduct
a settler colonial reading of he Year Fiteen based, as already asserted, on an
analogy between the settler/Indigenous Other relationship and that of the white
landowner/landless caboclo. In he Year Fiteen, Chico Bento as well as other people, men and women,
who work for the farmers or live on the farm premises, are referred to as
caboclos. he depiction of Chico’s knowledge of the land and skilful dealings
with the livestock (a familiarity with nature that he could have inherited from his
Indigenous ancestors, however distant they may be), and which secures him the
job of head stockman of the Aroeiras Farm, could be part of Queiroz’s project, Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 107 if we consider her as a 1930s regionalist writer, to “focus on the dry interior as
the traditional space par excellence that gave the Northeast its originality and
identity” (Albuquerque Jr. 78). Despite Chico’s knowledge of the land, he reveals
poor social skills in dealing with upper-class people. In the following episode,
Chico approaches Vicente in the hope of selling him his traditional sertanejo
leather garment and his remaining head of cattle, so that he and his family can set
of on their migration: he horse stopped under the dried piece of white wood that served as shade. he Year Fiteen: an attempt at a settler colonial reading In settler-
colonial terms, the stereotypical portrayal of the caboclo in his dependence on
the generosity of the benign landowner can be viewed as a disavowing technique
to justify landlessness and social inequality. Like Hugh Watt, the white landowner Vicente is characterised as the
benevolent boss who has developed a special connection with the land and who
not only looks ater his own employees, but also cares for other farmers’ neglected
workers (he is also godfather to Chico’s youngest son). He is pictured both as a
typical sertanejo and as a caboclo by Conceição: All day on horseback, cheerful and hard-working, Vicente had always
been like that, a friend of the bush, of the sertão, of everything that was
uncouth and rough. She had always known him wanting to be a cowboy,
like an unambitious caboclo, in spite of the displeasure that that caused to
his family (Queiroz, 2011, 21, my translation).18 Conceição, as will be later revealed, is ambivalent about Vicente’s lifestyle, while
Vicente’s family openly disapprove of it. he tensions between the city and the country, between modernity and
tradition, which in Coonardoo are personiied by Hugh’s girlfriend’s and
wife’s inadaptation to life at Wytaliba, are voiced here by Vicente’s family’s
disappointment in his choice of being “a nobody”,19 when compared to their pride
in his brother’s career as a small-town judge. his evokes the settler’s predicament. As a white landowner and, thus, inheritor of settler privileges, Vicente aspires to
indigeneity, in the form of the caboclo lifestyle; meanwhile his family consider
caboclos subhuman and unnoticeable. Both movements, contradictory as they 108 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... may seem, contribute to the same efect: the disavowal of Indigenous/landless
people’s rights and the justiication for the historical land dispossession practices
that have created the huge social gaps in Brazil. Vicente represents the North-
Easterner who likes to see himself as “made by the land” (Veracini 2010, 21-22)
and whose rights and privileges are not questioned, even when in the face of
the forced exile of the “true” caboclos. he combination of European and caboclo
characteristics – Vicente’s sex appeal revealed in his sunburnt skin, horse-riding
skills and typical sertanejo attire – excite Conceição’s interest in him. Conceição, in turn, is a compelling female character. he Year Fiteen: an attempt at a settler colonial reading A self-made twenty-
two-year-old intellectual with writing ambitions at the beginning of the twentieth
century, she is criticised by her grandmother for her lack of interest in marriage
and, mainly, for her readings, which include socialist and proto-feminist authors,
and which would generate “the worst of [her granddaughter’s] strange and
absurd” ideas (Queiroz, 2011, 14, my translation).20 Conceição makes a point in
holding a teaching job, even though there is no evidence that she depends on it
inancially. She also works for the well-being of the community, volunteering in
the concentration camp. In Chapter One Queiroz describes Conceição as a young
woman who “was used to thinking for herself [and] to living isolated, having
created her own prejudices and ideas, which were sometimes large, sometimes
daring” but also very much rooted in her own time and place, in spite of her avant-
gardism (Queiroz, 2011,14, my translation).21 his warning becomes meaningful
further along the novel, mainly in the episode where Conceição hears rumours
that Vicente might be involved with Josefa, the daughter of one of the farm-
hands. his triggers an assessment of Josefa as a “lowly, slutty cunhã [Indigenous
girl] with kinky hair and rotten teeth” (Queiroz, 2011, 64, my translation).22 She
also talks about it with her grandmother: – […]here has been a lot of talk about Vicente with Zé Bernardo’s Josefa… […]here has been a lot of talk about Vicente with Zé Bernardos Josefa…
he grandmother raised her eyes: he grandmother raised her eyes: he grandmother raised her eyes: – I had heard of it... A young man’s foolishness! he girl became irritated [...]: – It’s not foolishness! So, do you think it’s foolishness for a white man to
dirty himself with black women? Mrs Inácia smiled, in a conciliatory tone: Mrs Inácia smiled, in a conciliatory tone: – But, my dear, this happens to everyone... A white man, in the sertão
h
l
l k
h
d
h ’
bl
k
h ’
l h – But, my dear, this happens to everyone... A white man, in the sertão
– there are always stories like that…Besides, she’s not black; she’s a light-
shaded cabocla... – Well, I think it is shameless! And Vicente, with that saintly façade, is
worse than the others! he Year Fiteen: an attempt at a settler colonial reading (Queiroz, 2011, 66, my translation).23 Even ater discounting the fact that Conceição is clearly jealous, these
judgements that demean at once social class, race and gender may be disappointing
for contemporary readers, who would expect a diferent attitude from a heroine
who seems to be, otherwise, sensitive and open-minded. Later on, Conceição
will reconsider her reaction, and her inal decision not to reciprocate Vicente’s
love interest will be based on her desire to have a “room of her own”, in Virginia Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 109 Woolf’s terms, i.e., to have her own space to develop an intellectual life. It is clear
that in spite of Queiroz’s contempt for feminism, Conceição’s relections echo
some of Woolf’s ideas in her famous 1929 essay. he girl imagines herself married
to Vicente (who, she ponders, in terms of reading, is only interested in “cattle
account books”) and realises how incompatible their interests would be, and the
traditional role that would be expected from her as his wife. And yet, from a
settler-colonial perspective Conceição’s ambivalent behaviour in the excerpt
above makes sense. As a white upper-class woman who forms a connection with
the sertão and its people, her plight, as Bueno (128, my translation) puts it, is
feeling “squeezed between opposing requests: life in the country and the city,
intellectual achievement and maternity”.24 As a member of an older generation, however, her grandmother’s lack of
surprise and more unyielding attitude in measuring the gravity of Vicente’s
“normal” indiscretion according to skin colour variation is a clear remnant
of the historical sexual exploitation of female Indigenous and Exogenous
Others in colonial Brazil. It was in the countryside that the culture of slavery,
abolished only 27 years before the events of he Year Fiteen, survived more
openly. Slavery and its contemporary efects in terms of race and social class
are jokingly implied in the novel in the episode in which Vicente’s family’s
employees are sent to fetch the matriarch at the station and bring a litter (sedan
chair), to carry her to her farm: Mrs Inácia came to sit on the sedan chair and exclaimed: – Where are
the donkeys? Don’t you know that I only like to ride on a chair carried by
donkeys? he cowboy answered: – Dear godmother, don’t you have your
caboclos to carry you? he Year Fiteen: an attempt at a settler colonial reading Why would one use animals if we are here? Mrs
Inácia retorted: – But I don’t like it. It doesn’t do any good to my nerves. It
looks like they [the caboclos] are tired to death… he men laughed: – Of
course, my godmother’s weigh can kill eight men! he cowboy added: –
he pitiful donkeys that have survived [the drought] are no good…they
fall down for nothing…25 (Queiroz, 2011, 150, my translation) Mrs Inácia came to sit on the sedan chair and exclaimed: – Where are
the donkeys? Don’t you know that I only like to ride on a chair carried by
donkeys? he cowboy answered: – Dear godmother, don’t you have your
caboclos to carry you? Why would one use animals if we are here? Mrs
Inácia retorted: – But I don’t like it. It doesn’t do any good to my nerves. It
looks like they [the caboclos] are tired to death… he men laughed: – Of
course, my godmother’s weigh can kill eight men! he cowboy added: –
he pitiful donkeys that have survived [the drought] are no good…they
fall down for nothing…25 (Queiroz, 2011, 150, my translation) To disavow basic human rights and conceal the immorality of slavery, its
promoters turn to the reiication of Indigenous and Exogenous Others. Although,
taken iguratively, Mrs Inácia’s statement can be read in a mock humanitarian
sense, her words literally suggest that the reason informing her preference is the
caboclos’ alleged laziness when compared to donkeys. Accordingly, the general structure of the secondary plot implies the
reiication of the caboclo. While the natural phenomenon of the drought afects
all the characters in the novel, during the worst periods the landowning families
take refuge in urban areas, where many have real estate or can aford to rent
a second home. Unlike their impoverished workers, these landed gentry have
the prerogative of returning to their farms to recover their heritage and their
status quo ater the crisis is over. It is not a coincidence that Chico Bento’s family’s
perilous journey to Fortaleza is portrayed as analogous to that of the hungry 110 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... cattle, which lacking water and pastures, are released by the farmers to survive
or perish on their own. he members of the family who survive ind only the
concentration camp at the entrance to the city. he Year Fiteen: an attempt at a settler colonial reading Historically, the Alagadiço camp
was set up by the Ceará State government with the supposed intention of giving
assistance to the waves of migrants leeing the parched sertão. According to
Frederico de Castro Neves (105), however, the government fell very short of its
goal, and the camp became popularly known as a human “corral”, aimed at hiding
thousands of bedraggled people from the view of city dwellers by stopping them
from entering the newly gentriied city of Fortaleza (Ponte 27-70). With very little government assistance and at the mercy of the community’s
charity, not that much diferent from their future Nazi namesakes, the camps became
huge depositories of malnourished people in very precarious sanitary conditions. Along the history of the camps, thousands of people died of hunger, smallpox and
other contagious diseases. But managing to leave the concentration camps behind
did not guarantee a much better prospect. In Queiroz’s re-creation of the drought of
1915, Chico Bento’s family’s inal destination, like that of thousands of other landless
people in the irst half of the twentieth century – historically caboclos, mulatos and
other miscegenated individuals in their majority – are the large metropoles, such
as the city of São Paulo, where, it is implied, they will become one more family to
swell the wretched shantytowns today known as favelas. Suggestive of Coonardoo’s
trajectory, Chico’s forced migration results in permanent geographical transfer and
transfer by coerced lifestyle, as the family members lose their connections with the
land – along with their identities – along the way. Although in highly hybridized Brazil – unlike in the near-pioneering society
depicted in Coonardoo – it becomes diicult to pinpoint the Indigenous Other
so that he/she can neatly it the settler colonial scheme, oppositions and veiled
conlicts between the white landed gentry and miscegenated landless social
categories very much resemble those between the settler and the Indigenous Other. Conclusion Due to the problematic position of European settlers in between two worlds
and the eagerness with which they attempt to legitimise their claims to the land
and exploitation of Indigenous labour, societies created by settler colonialism
are sites of permanent insecurities, contradictions and paradoxes. As Veracini
has observed, those complexities appear not only relected, but ampliied in the
production of ictional writers. his paper examined the representations of the
settler and the Other produced by two authors writing from almost opposite
corners of the world. Despite their geographical distance, Katharine Susannah
Prichard and Rachel de Queiroz felt analogous needs to incorporate to their
ictional plots turbulent events in the rural history of their countries and the
conlicting transitions from pastoralism to urban modernity that modiied
the ties of people with the land. In the process of ictionally recreating these
occurrences, as white, middle-class women talking about the Indigenous or 111 Ilha do Desterro v. 72, nº 1, p. 087-114, Florianópolis, jan/abr 2019 111 miscegenated Other, they reveal, directly or indirectly, the marks of their settler
colonial heritages. Both the white landowners and the landless Others in Coonardoo and he Year
Fiteen are ambivalently portrayed. he characterisations of Mrs Bessie, Hugh,
Vicente, Conceição, and even Sam Geary move between benign and perverse,
fair and exploitative, tolerant and racist, modern and reactionary. Attempts at
indigenising the white settler by conferring him/her with Aboriginal and caboclo
traits are a recurrent manoeuvre to justify their entitlement to the Other’s land. And if, on the one hand, some transfer tactics described by Veracini as settlers’
subterfuges to make the Other disappear – such as perception transfer or transfer
by coerced lifestyle – are poignantly exposed and condemned by both authors,
on the other, the landless Indigenous peoples or caboclos display accommodating
natures and submissiveness that seem to conirm Prichard’s evolutionist beliefs
and Queiroz’s paternalistic views on social strife. he comparison of the novels
demonstrates that the settler imagination is a compelling apparatus, able to create,
manipulate and disseminate representations of the settler self and the Other that
at the same time justify and question settler hegemony. Notes 1. Many thanks to CAPES, the Brazilian Ministry of Education’s Coordination for the
Improvement of Higher Education Personnel, for having provided the inancial
means for my research on settler colonialism and the composition of this article,
during my postdoctoral stay at the University of Sydney, Australia, from June to
December 2017. I also thank Cassandra Madderom for her invaluable help in
language revision. I am solely responsible for any lapses. 2. “obra haver brotado de uma região qualquer, uma entre outras (se bem que em
geral se trata de cenário com dominância rural)”. 3. “(suposto) universalismo, que estaria representado apenas na arte da Corte, do
Centro, da Metrópole, sempre da Cidade.” 4. Austlit.edu.au. Web. 01 Dec. 2017. 5. Although the atmosphere here is very diferent from the gloominess that is Barbara
Baynton’s trademark, this passage evokes the irony of the gender role reversal in the
opening paragraph of “Squeaker’s Mate”. See SCHEIDT, Déborah. “Mateship and the
Female Body in Barbara Baynton’s ‘Squeaker’s Mate’”. Ilha do Desterro: A Journal of
English Language, Literatures in English and Cultural Studies, v. 68, 2015. pp. 67-74. 6. “sempre primou em andar na contramão da História” (Kindle edition). 7. “às vezes uma feminista dava uma entrevista falando mal de um homem; pois eu
achava um jeito de dizer que gostava do atacado só para marcar minha posição.” 7. “às vezes uma feminista dava uma entrevista falando mal de um homem; pois eu
achava um jeito de dizer que gostava do atacado só para marcar minha posição.”
8. “mulheres danadas” 9. “mulheres fortes, autossuicientes, que percorrem com obstinação, os caminhos
que levam aos destinos marcados pela independência e pelo poder.” 10. “dirigiam com mãos severas as grandes fazendas de gado e açúcar da região.” 11. “eu conspirei com os generais para a derrubada do Jango” 12. Web: https://www.facebook.com/LiteraBrasil1/photos/a.352148138280656.1073
741828.349691378526332/823920297770102/?type=3&theater . 10 Dec. 2017. 112 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... 13. “relação quase feudal entre ailhados e seus padrinhos que eram donos da terra ou
doutores” / “desenvolver, no interior do quadro familiar, estratégias de distribuição
de poder e formas de garantia de proteção aos agregados e ailhados”). 14. “Nasci em família de fazendeiros, mas as nossas fazendas sempre foram pobres,
fazendas de gado, nunca tivemos aquela fartura das fazendas baianas ou
pernambucanas, onde o senhor de engenho era uma personalidade. A despeito
das ideologias, sempre fomos amigos dos nossos empregados, éramos compadres
dos nossos vaqueiros. Notes – Mas, minha ilha, isso acontece com todos... Homem branco, no sertão - sempre
saem essas histórias... Além disso não é uma negra; é uma caboclinha clara... – Pois eu acho uma falta de vergonha! E o Vicente, todo santinho, é pior do que os
outros!” 24. “espremida entre diferentes solicitações: a vida no campo e na cidade, a realização
intelectual e a maternidade”. 25. “Dona Inácia veio se sentar na cadeirinha, admirou-se: – Que é dos jumentos? Vocês
não sabem que eu só gosto de andar de cadeirinha levada por jumento? O vaqueiro
acudiu: – Minha madrinha não tem os seus caboclos pra carregarem a senhora? Por
que se havia de botar animal, tendo nós? Dona Inácia teimou: – Mas eu não gosto. Faz-me mal aos nervos. Parece que vão morrendo de cansaço... Os cabras riram-se:
– Está-se vendo! o peso de minha madrinha mata oito homens! O vaqueiro ajuntou:
– Mesmo porque os jumentinhos que escaparam não dão pra nada... Ainda estão
caindo...” 25. “Dona Inácia veio se sentar na cadeirinha, admirou-se: – Que é dos jumentos? Vocês
não sabem que eu só gosto de andar de cadeirinha levada por jumento? O vaqueiro
acudiu: – Minha madrinha não tem os seus caboclos pra carregarem a senhora? Por
que se havia de botar animal, tendo nós? Dona Inácia teimou: – Mas eu não gosto. Faz-me mal aos nervos. Parece que vão morrendo de cansaço... Os cabras riram-se:
– Está-se vendo! o peso de minha madrinha mata oito homens! O vaqueiro ajuntou:
– Mesmo porque os jumentinhos que escaparam não dão pra nada... Ainda estão
caindo...” Albuquerque Jr., Durval Muniz de. he Invention of the Brazilian Northeast. Translated
by Jerry Dennis Metz. Duke University Press, 2014. Notes No nosso meio nunca houve esse problema de terra, porque
a gente sempre deu a terra para o morador plantar. Eu posso ter muita coisa, mas
nunca cobrei um caroço de feijão de um trabalhardor meu.” 15. In the original version: “– Aquela velha é uma desgraça! Tenho fé em Deus que o
dinheiro que ela poupa ainda há de lhe servir pra comer em cima duma cama...”. 16. “mais rural, indígena ou rústica em relação ao locutor ou à locutora”. 17. “uma abstração, uma unidade de um sistema de classiicação social projetado para
retratar as diferenças entre as pessoas na sociedade.” 18. “Todo o dia a cavalo, trabalhando, alegre e dedicado, Vicente sempre fora assim,
amigo do mato, do sertão, de tudo o que era inculto e rude. Sempre o conhecera
querendo ser vaqueiro como um caboclo desambicioso, apesar do desgosto que
com isso sentia a gente dele.” 18. “Todo o dia a cavalo, trabalhando, alegre e dedicado, Vicente sempre fora assim,
amigo do mato, do sertão, de tudo o que era inculto e rude. Sempre o conhecera
querendo ser vaqueiro como um caboclo desambicioso, apesar do desgosto que
com isso sentia a gente dele.” 19. In the original version: Vicente teimava em não querer ser gente, or “Vicente
insisted on not wanting to be a person” (Queiroz, 2011, 22). 20. “as piores das tais ideias, estranhas e absurdas” 20. “as piores das tais ideias, estranhas e absurdas” 21. “Acostumada a pensar por si, a viver isolada, criara para seu uso ideias e preconceitos
próprios, às vezes largos, às vezes ousados”. 22. “Uma cabra, uma cunhã à-toa, de cabelo pixaim e dente podre!...” 23. “ – Diz que estão falando muito do Vicente com a Josefa do Zé Bernardo... A avó levantou os olhos:
– Eu já tinha ouvido dizer... Tolice de rapaz! A moça exaltou-se [...]:
– Tolice, não senhora! Então Mãe Nácia acha uma tolice um moço branco andar se
sujando com negras? Dona Inácia sorriu, conciliadora:
– Mas, minha ilha, isso acontece com todos... Homem branco, no sertão - sempre
saem essas histórias... Além disso não é uma negra; é uma caboclinha clara... – Pois eu acho uma falta de vergonha! E o Vicente, todo santinho, é pior do que os
outros!” – Tolice, não senhora! Então Mãe Nácia acha uma tolice um moço branco andar se
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NAEA v. 2, n. 2, dezembro 1999. pp. 5-32. Mitchell, Adrian. Fiction. he Oxford History of Australian Literature. Edited by
Kramer, Leonie. Oxford University Press, 1981. 114 Déborah Scheidt, Katharine Susannah Prichard’s Coonardoo... Modjeska, Drusilla. Exiles at Home: Australian Women Writers, 1925-1945. Harper
Collins, 2001. Neves, Frederico de Castro. “Curral dos Bárbaros: os Campos de Concentração no
Ceará (1915-1932)”. Revista Brasileira de História. v. 15, n. 29. 1995. pp. 93-122. Ponte, Sebastião Rogério. Fortaleza Belle Epoque: reformas urbanas e controle social
1860-1930. Fundação Demócrito Rocha, 1993. Prichard, Katharine Susannah. Coonardoo. Angus and Robertson, 2013. Queiroz, Rachel de. “As Três Rachéis”. Cadernos de Literatura Brasileira. n. 4. São
Paulo: Instituto Moreira Sales, 1997. pp. 21-39. ______. From he Year Fiteen. Translated by Darlene Sadlier. One Hundred Years
Ater Tomorrow: Brazilian Women’s Fiction in the 20th Century. Edited by Darlene
Sadlier. Indiana University Press, 1992. pp. 36-49. ______. O Quinze. José Olympio, 2011. ___. O Quinze. José Olympio, 2011. Ribeiro, Darcy. he Brazilian People: he Formation and Meaning of Brazil. Trandlated
by Gregory Rabassa. University Press of Florida, 2000. Stanner, W. E. H. “he Great Australian Silence”. he Dreaming and Other Essays. Black Ink Agenda, 2011. pp. 182-192. hrossell, Ric. Wild Weeds and Windlowers: he Life and Letters of Katharine
Susannah Prichard. Angus and Robertson, 1982. Veracini, Lorenzo. Settler Colonialism: A heoretical Overview. Palgrave Macmillan,
2010. ____. “Settler Colonialism as a distinct mode of domination”. Edited by Cavanah, Edward, Veracini, Lorenzo. he Routledge Handbook of the History of Settler
Colonialism. Routledge, 2016. p. 1-8. ______. Settler Colonialism Today. Palgrave Macmillan, 2015. Wasserman, Renata R. Mautner. Central at the Margin: Five Brazilian Women Writers. Bucknell University Press, 2007. White, Patrick. “he Prodigal Son.” Patrick White Speaks. Jonathan Cape, 1990. Wolfe, Patrick. Settler Colonialism and the Transformation of Anthropology. Cassel,
1999. __. Traces of History: Elementary Structures of Race. Verso, 2016. Recebido em: 28/02/2018
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Fragment C of Tetanus Toxin: New Insights into Its Neuronal Signaling Pathway
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International journal of molecular sciences
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Int. J. Mol. Sci. 2012, 13, 6883-6901; doi:10.3390/ijms13066883 Int. J. Mol. Sci. 2012, 13, 6883-6901; doi:10.3390/ijms13066883 International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
OPEN ACCESS International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
OPEN ACCESS Fragment C of Tetanus Toxin: New Insights into Its Neuronal
Signaling Pathway Ana C. Calvo 1, Sara Oliván 1, Raquel Manzano 1, Pilar Zaragoza 1, José Aguilera 2 and
Rosario Osta 1,* 1. Introduction The Clostridial neurotoxin family comprises tetanus neurotoxin and seven distinct botulinum
neurotoxins, which cause the diseases tetanus and botulism [1,2]. Regarding neuroparalytic clostridia,
Clostridial botulinum and Clostridial tetani produce the most potent toxins, botulinum and tetanus,
due to their remarkable neurospecificity and their catalytic cleavage at low concentrations of neuronal
substrates, which can oscillate between 10−12 and 10−13 M. The main difference between these toxins is
in the intensity and duration of muscle paralysis. In fact, severe tetanus is characterized by violent and
persistent spasms of the head, trunk and limb muscles [2]. The infectious nature of tetanus toxin was well documented before 1906, and the necessity of
producing a tetanus antitoxin was decisive during the First World War [3]. The promising protective
effects of eosin, tested in vitro and in vivo [4,5], and the development of serums against the toxin,
mainly obtained in horses [6], undoubtedly opened a door to the unexplored field, at that time, of its
molecular mechanism of action. In 1905, Sherrington described the mechanism of action of the toxin
on spinal reflexes as a conversion of inhibition into excitation, thus destroying coordination [7]. Additionally, he investigated the effects of strychnine because this substance had a similar effect on
the central nervous system as the tetanus toxin. In 1942, Acheson and collaborators concluded for the
first time that the toxin was carried to the spinal cord through peripheral nerves when it was injected
intramuscularly in cats; that is, the toxin traveled selectively through the segments of the spinal cord
that supplied the innervation of the injected area [8]. Ana C. Calvo 1, Sara Oliván 1, Raquel Manzano 1, Pilar Zaragoza 1, José Aguilera 2 and
Rosario Osta 1,* 1 LAGENBIO (Laboratory of Genetics and Biochemistry), Faculty of Veterinary-I3A, Aragonese
Institute of Health Sciences (IACS), University of Zaragoza, Miguel Servet 177, 50013
Zaragoza, Spain; E-Mails: accalvo@unizar.es (A.C.C.); soligar@unizar.es (S.O.);
rmanzano@unizar.es (R.M.); pilarzar@unizar.es, osta@unizar.es (P.Z.) 1 LAGENBIO (Laboratory of Genetics and Biochemistry), Faculty of Veterinary-I3A, Aragonese
Institute of Health Sciences (IACS), University of Zaragoza, Miguel Servet 177, 50013
Zaragoza, Spain; E-Mails: accalvo@unizar.es (A.C.C.); soligar@unizar.es (S.O.);
rmanzano@unizar.es (R.M.); pilarzar@unizar.es, osta@unizar.es (P.Z.) 1 LAGENBIO (Laboratory of Genetics and Biochemistry), Faculty of Veterinary-I3A, Aragonese
Institute of Health Sciences (IACS), University of Zaragoza, Miguel Servet 177, 50013
Zaragoza, Spain; E-Mails: accalvo@unizar.es (A.C.C.); soligar@unizar.es (S.O.);
rmanzano@unizar.es (R.M.); pilarzar@unizar.es, osta@unizar.es (P.Z.) 2 Institute of Neurosciences, Department of Biochemistry and Molecular Biology, Universitat
Autònoma de Barcelona (UAB), Center of Biomedical Research Network in Neurodegenerative
Diseases (CIBERNET), 08193, Cerdanyola del Vallès, Spain; E-Mail: jose.aguilera@uab.cat * Author to whom correspondence should be addressed; E-Mail: osta@unizar.es;
Tel.: +34-976-761621; Fax: +34-976-762949. Received: 28 March 2012; in revised form: 8 May 2012 / Accepted: 23 May 2012 /
Published: 7 June 2012 Received: 28 March 2012; in revised form: 8 May 2012 / Accepted: 23 May 2012 /
Published: 7 June 2012 Abstract: When Clostridium tetani was discovered and identified as a Gram-positive
anaerobic bacterium of the genus Clostridium, the possibility of turning its toxin into a
valuable biological carrier to ameliorate neurodegenerative processes was inconceivable. However, the non-toxic carboxy-terminal fragment of the tetanus toxin heavy chain
(fragment C) can be retrogradely transported to the central nervous system; therefore,
fragment C has been used as a valuable biological carrier of neurotrophic factors to
ameliorate neurodegenerative processes. More recently, the neuroprotective properties
of fragment C have also been described in vitro and in vivo, involving the activation of
Akt kinase and extracellular signal-regulated kinase (ERK) signaling cascades through
neurotrophin tyrosine kinase (Trk) receptors. Although the precise mechanism of the
molecular internalization of fragment C in neuronal cells remains unknown, fragment C
could be internalized and translocated into the neuronal cytosol through a clathrin-mediated
pathway dependent on proteins, such as dynamin and AP-2. In this review, the origins,
molecular properties and possible signaling pathways of fragment C are reviewed to
understand the biochemical characteristics of its intracellular and synaptic transport. Int. J. Mol. Sci. 2012, 13 6884 Int. J. Mol. Sci. 2012, 13 Int. J. Mol. Sci. 2012, 13 1. Introduction Supporting this result, Brooks and collaborators,
15 years later, studied the spinal inhibitory mechanisms based on five types of inhibition [9]: rect inhibition of motoneurons by impulses in Group I a afferent fibers of antagonistic mus (a) direct inhibition of motoneurons by impulses in Group I a afferent fibers of antagon (a) direct inhibition of motoneurons by impulses in Group I a afferent fibers of antagonistic muscles;
(b) inhibition by impulses in the Group I b afferent fibers from muscles of the same limb; (b) inhibition by impulses in the Group I b afferent fibers from muscles of the same li n by impulses in the Group I b afferent fibers from muscles of the same limb; (c) inhibition of extensor motoneurons by impulses in Groups II and III muscle afferent fibers and
in cutaneous afferent fibers; and (c) inhibition of extensor motoneurons by impulses in Groups II and III muscle afferent fibers and
in cutaneous afferent fibers; and (d) inhibition of motoneurons following the activation of Renshaw cells by volleys in axon c These five forms of spinal inhibition were diminished and eventually abolished 8–10 h after
injecting the toxin either peripherally into a mixed nerve trunk or directly into the spinal cord, which
indicated that the toxin diffused slowly across the spinal cord, taking several hours to move 1 mm. From these results, it was also concluded that the toxin moved much faster longitudinally in nerve
trunks and within the spinal cord [9]. The nature of the action of tetanus toxin has been widely described in different animal models [10–15],
exploring its effect not only in the spinal cord but also in the cerebral cortex [16]. Different
pharmacological substances have been used to ameliorate its symptoms in addition to eosin, such as
acetylcholine and eserine [12]. These data were the starting point to attempt to characterize a protein
that was completely necessary for the toxin to exert its effect, the tetanus-toxin receptor. Wassermann
and Takaki, in 1898, observed that the toxin was fixed in the nervous tissue; in particular, brain tissue Int. J. Mol. Sci. 2012, 13 6885 was more effective than the spinal cord, and gray matter was more effective than white matter [17]. Eight years later, Landsteiner and Botteri believed that phrenosine was the receptor of the tetanus
toxin [18]. 1. Introduction However, in 1959 van Heyningen reinvestigated phrenosine as a possible receptor of
the toxin, and he realized that the phrenosine concentrations in gray and white matter were inversely
proportional to their capacity to fix the toxin. Based on his experiments in bovine brain samples, he
concluded that the receptor was a highly specific substance that was mainly present in the gray matter, its
interaction with the toxin was unaffected by temperature and it tended to associate with cerebrosides,
although it was distinct from them [19,20]. Two years later, he confirmed that the sialic acid residues of
the gangliosides were essential for toxin fixation and that the toxin did not appear to change the
ganglioside molecule [21]. Although further studies supported this fact [22,23], currently, the identity
of this receptor remains unknown despite knowledge of its agonist, tetanus toxin. 2. Molecular Structure and Properties of Fragment C: Toward the Discovery of Fragment C Tetanus toxin is a single peptide of approximately 150 kDa, which consists of 1315 amino-acid
residues. The toxin forms a two-chain activated molecule composed of a heavy chain (HC) and a light
chain (LC) linked by a disulfide bond. The catalytic domain of the toxin resides in the LC, while the
translocation and receptor-binding domains are present in HC [24–27] (Figure 1). Tetanus and
botulinum toxins are zinc metalloproteases that cleave SNARE (soluble NSF attachment receptor)
proteins, which interfere with the fusion of synaptic vesicles to the plasma membrane and ultimately
blocks neurotransmitter release in nerve cells [28]. Figure 1. Diagram of the tetanus toxin molecule. The targeting and the translocation
domains are located in the heavy-chain (HC), whereas the catalytic domain is located in the
light-chain (LC) of the molecule. Its proteolytic activity is Zn2+-dependent, and heavy-metal
chelators generate inactive apo-neurotoxins. The position of the cleavage of the tetanus-toxin
molecule by papain is indicated. The digestion yields two fragments; one of them, fragment
C, is approximately 50 kDa [29]. The mechanism of cell internalization and catalytic activity of tetanus toxin can be summarized in
few steps [24,25] (Figure 2): The mechanism of cell internalization and catalytic activity of tetanus toxin can be summarized in
few steps [24,25] (Figure 2): Int. J. Mol. Sci. 2012, 13 6886 (a) cell binding, mainly mediated by the ganglioside-recognition domain in the C-terminal region
of HC; (a) cell binding, mainly mediated by the ganglioside-recognition domain in the C-terminal region
of HC; (b) internalization into neuronal cells. Different internalization mechanisms have been described:
the mechanism that tetanus toxin possibly follows to internalize into neurons may be a
clathrin-mediated pathway, which is dependent on the proteins dynamin, AP-2 and AP180 [30]. Other potential receptor molecules will be discussed in the next section. (c) membrane translocation from mature endosomes into the neuronal cytoplasm. After incorporation
of the toxin within endosomes, a structural change of the toxin is induced by the acidification of
the endosomal environment; thus, a membrane-spanning pore forms. At this step, the
oligomerization of four toxin amphipathic alpha-helices is required for channel formation; and (d) target recognition and catalytic cleavage of neuronal substrates. Once the toxin reaches the
cytoplasm, it specifically cleaves neuronal proteins integral to vesicular trafficking and
neurotransmitter release. In particular, the synaptic vesicle protein synaptobrevin (VAMP) is the
target of tetanus toxin. 2. Molecular Structure and Properties of Fragment C: Toward the Discovery of Fragment C This protein belongs to a family of proteins that facilitate exocytosis
in neurons known as SNARE proteins. The other members of this family are syntaxin and
SNAP-25, which are the main molecular targets of botulinum toxin. SNARE proteins are
formed by coiled-coil interactions of the alpha-helices of its members, which is required for
membrane fusion [31–33]. Figure 2. Proposed internalization pathway of tetanus toxin. The ganglioside-recognition
domain in the C-terminal region of HC allows the toxin to be internalized into the neuron. The light chain of the toxin (LC) cleaves the soluble NSF attachment receptor (SNARE)
complex, inhibiting neurotransmitter release. Botulinum toxins are produced by the anaerobic bacterium Clostridium botulinum, which is
considered to be a potent blocker of synaptic transmission in peripheral cholinergic nervous system Botulinum toxins are produced by the anaerobic bacterium Clostridium botulinum, which is
considered to be a potent blocker of synaptic transmission in peripheral cholinergic nervous system Botulinum toxins are produced by the anaerobic bacterium Clostridium botulinum, which is
considered to be a potent blocker of synaptic transmission in peripheral cholinergic nervous system Int. J. Mol. Sci. 2012, 13 6887 synapses. There are seven serologically distinct botulinum isoforms (denoted A–G), which exhibit
strong amino-acid sequence similarity. The mature toxin is characterized by three main structural
domains, an N-terminal light chain Zn2+-metalloprotease and the heavy chain that contains the
N-terminal, of approximately 50-kDa, translocation domain, and the C-terminal, of approximately
50-kDa, receptor-binding domain. This receptor-binding domain comprises two subdomains, a β-sheet
jelly-roll fold and a β-tree foil-fold carboxy subdomain. The seven botulinum toxin serotypes cleave
specific residues on one of three SNARE proteins: botulinum serotypes B, D, F and G cleave VAMP,
botulinum serotypes A and E cleave SNAP25, and botulinum serotype C cleaves SNAP25 and
syntaxin 1a [26,28]. 2.1.1. Molecular Binding As previously mentioned, tetanus toxin can form channels in lipid membranes at low pH in the
endosomal compartment of nerve cells. These channels are actually pores, which are involved in the
transport of the toxin to the cytosol [34]. The ability to form pores in lipid vesicles resides in the
hydrophobic domain of LC and the N-terminal of HC, which allows for the translocation of the toxin
through the lipid membrane. However, in the absence of fragment C, the toxin retains little ability
to paralyze neuromuscular transmission [35]. The lipids which are most sensitive to the action of
the toxin are, in descending order: phosphatidylinositol > phosphatidylserine > phosphatidylcholine and
cholesterol [36]. Furthermore, tetanus toxin increases its binding and insertion into lipid bilayer at acidic
pH, suggesting that the toxin can penetrate into cells through a low pH intracellular compartment [37]. What is the molecular structure of fragment C that makes it essential for toxin behavior? The early step in tetanus-toxin internalization is cell binding. Lipid rafts are microdomains of the
plasma membrane enriched in sphingolipids (gangliosides are members of this group of lipids),
cholesterol and glycosylphosphatidylinositol (GPI)-anchored proteins. Lipid rafts behave as specialized
domains for tetanus-toxin binding and internalization into neurons. In particular, gangliosides constitute
the main part of mammalian plasma membranes. Their oligosaccharide residues and their ubiquity at the
outer leaflet of the membrane allow them to function as bioactive signal transducers. The affinity of
fragment C for gangliosides, which resides in the last 34 residues, has been widely tested in different
animal models and in different tissues. For example, greater interaction was observed with long-chain
gangliosides from rat brain than with similar gangliosides in rabbit kidney [38]. Furthermore, it is
suggested that tetanus toxin, mainly through fragment C, and gangliosides undergo significant
conformational changes upon their interaction, leading to the formation of macromolecular aggregates. In particular, cerebrosides, sulfatides, sphingomyelin and phosphatidylserine seem to increase the
percentage of α-helices in the toxin [39]. Interestingly, studies on the ganglioside-binding properties of fragment C have demonstrated that
polysialic acids within the gangliosides, such as GD1b (disialic acid residues attached to the internal
galactose residue) and GT1b (disialic acid residues attached to the terminal galactose residue), but not
GM1 (monosialic acid residue attached to the internal galactose residue), are necessary for the binding
process [40,41]. Specifically, the amino-acid residues tryptophan 1288, histidine 1270 and aspartate Int. J. Mol. Sci. 2.1.1. Molecular Binding 2012, 13 6888 1221 were found to be critical for the binding of fragment C to ganglioside GT1b [42]. GT1b is a
trisialo sphingolipid with a branched carbohydrate structure containing a single N-acetylneuraminic
acid (NeuAc) on one arm and a NeuAc dimer on the other (Figure 3). The strongest and most specific
ganglioside association with tetanus toxin is with GT1b because the targeting domain of the toxin contains
two binding sites that can accommodate NeuAc residues separated by a distance of approximately
25 Å [43]. Studies based on atomic-force microscopy/total internal-reflection fluorescence microscopy
(TMAFM/TIRFM) reinforce this result, suggesting that the membrane activity of fragment C is dependent
on both the ganglioside concentration in the membrane and the pH of the medium [44]. Figure 3. A schematic representation of the structures of gangliosides GT1b, GM1 and
GD1b. The strongest and most specific ganglioside association with fragment C of tetanus
toxin is with GT1b, since the targeting domain of the toxin contains two binding sites that
can accommodate NeuAc residues. Additionally, the receptor-binding domain of tetanus and botulinum A and B toxins is structurally
similar, containing two subdomains: an amino-terminal lectin-like jelly-roll subdomain and a
carboxyl-terminal beta-trefoil subdomain linked by a single chain (Figure 4). Each of these
subdomains is composed of beta-sheets joined by loops that protrude from the molecule. Regarding
tetanus toxin, and based on previous studies [19–21,45], four distinct carbohydrate-binding sites for
lactose, galactose, sialic acid and N-acetyl-galactosamine (NGA) were determined with X-ray
crystallography [40,41]. In particular, the beta-trefoil subdomain seems to have a more relevant role in
ganglioside binding than does the amino-terminal lectin-like subdomain, which was demonstrated by Int. J. Mol. Sci. 2012, 13 6889 analyzing the localization of these binding domains, showing that the binding function resided in the
beta-trefoil domain [40,46] (Figure 4), and mutant proteins of fragment C [41]. Figure 4. (a) Image from the RCSB PD of PDB ID 1A8D [47]; (b) Illustration of the
carbohydrate-binding sites for lactose (LAC), galactose (GAL), sialic acid (SIA) and
N-acetyl-galactosamine (NGA) in tetanus toxin. These binding sites are localized in the
beta-trefoil domain of fragment C (residues 865–1315). Adapted from [40]. 2.1.1. Molecular Binding (a)
(b)
More recent studies have suggested that gangliosides are functional dual receptors for tetanus toxin
and they are necessary for high-affinity binding to neuronal and non-neuronal cells. In particular, these
studies showed that HC of the toxin bound gangliosides via two carbohydrate-binding sites, the lactose
binding site called W-pocket and the sialic acid binding site called R-pocket. Both W- and R-pockets
are the binding sites for GM1a and GD3 gangliosides [53]. Al h
h
h
ffi i
f f
C f
li
id
h
b
id l
h
i
d
h (b) (a) (a) (a) (b) (b) (a) (b) More recent studies have suggested that gangliosides are functional dual receptors for tetanus toxin
and they are necessary for high-affinity binding to neuronal and non-neuronal cells. In particular, these
studies showed that HC of the toxin bound gangliosides via two carbohydrate-binding sites, the lactose
binding site called W-pocket and the sialic acid binding site called R-pocket. Both W- and R-pockets
are the binding sites for GM1a and GD3 gangliosides [53]. Although the affinity of fragment C for gangliosides has been widely characterized, another
hypothesis could suggest that a high-affinity protein receptor can be involved in tetanus toxin
internalization. Schiavo and co-workers showed that the N-glycosylated 15-kD receptor protein has
also been described as a surface glycoprotein that interacts with tetanus toxin in neuronal cell-lines and
motor neurons. The C-terminal subdomain of fragment C of tetanus toxin is sufficient and necessary
for cell binding and interaction with the 15-kD putative receptor, highlighting the importance of this
domain of fragment C for the neurospecific interaction of the toxin [54]. The same group also suggested
that GPI anchored protein Thy-1, a highly expressed glycoprotein that can interact with tetanus toxin to
mimic ganglioside binding [55]. In addition, HC of the toxin can be retrograde-trafficked within motor
neurons in Rab-7 positive structures that are shared with the neurotrophin receptors p75NRT and TrkB [56]. Swaminathan and co-workers showed that a tri-peptide Tyr-Glu-Trp bound to the binding-receptor domain
of the toxin with interactions to Arg1226, suggesting that a protein as well as a ganglioside can bind to
this domain [49]. Similarly to tetanus toxin, botulinum toxin Types A, B, C and F bind gangliosides GT1b, GD1b and
GD1a, whereas botulinum Type E binds GT1b and GT1a, botulinum Type D binds
phosphatidylethanolamine (PE) and botulinum Type G recognizes all the gangliosides with an
approximately similar affinity [26]. 2.1.1. Molecular Binding (a)
(b) (a) (a) (b) (b) (b) (a) Based on studies that included a combination of computational methods, these four
carbohydrate-binding sites are localized in two different sites on the surface of fragment C: Site-1, a
common site for all clostridial neurotoxins through which fragment C binds lactose and part of GT1b
(Gal-GalNAc); and the other well-characterized site Site-2, through which fragment C binds
sialyllactose, lactose, disialyllactose (DiSia), a tripeptide Tyr-Glu-Trp (YEW) and the Gal-NAc part of
GT1b. Site-2 has been shown to be vital for the toxicity of the toxin [48]. Furthermore, Tyr 1290 and Trp
1289 in Site-1 and Asp 1147 and Arg 1226 in Site-2 play a key role in ganglioside binding [49] (Figure 5). In particular, DiSia binding characterizes ganglioside binding to fragment C and could be the
binding site for the ganglioside sugar moiety GD3 [48,50]. If ligands that simultaneously bind to two
adjacent sites are identified, they could be used to develop bi-dentate reagents for tetanus toxin, as
demonstrated by Cosman and collaborators. Of the six small molecules they studied, three were found
to be the best ligands to use for preparing bi-dentate detection agents: doxorubicin or 3'-sialyllactose
(binding to Site-1), lavendustin A, YEW and MP-biocytin (binding to Site-2) and the peptide
Ser-Gln-Asn-Tyr-Pro-Ile-Val (SQNYPIV) (binding to a third independent site). Doxorubicin presents
more advantages than do the other molecules because it tends to bind fragment C over a wider range of
temperatures, solvent conditions and concentrations [50]. Based on studies that included a combination of computational methods, these four
carbohydrate-binding sites are localized in two different sites on the surface of fragment C: Site-1, a
common site for all clostridial neurotoxins through which fragment C binds lactose and part of GT1b
(Gal-GalNAc); and the other well-characterized site Site-2, through which fragment C binds
sialyllactose, lactose, disialyllactose (DiSia), a tripeptide Tyr-Glu-Trp (YEW) and the Gal-NAc part of
GT1b. Site-2 has been shown to be vital for the toxicity of the toxin [48]. Furthermore, Tyr 1290 and Trp
1289 in Site-1 and Asp 1147 and Arg 1226 in Site-2 play a key role in ganglioside binding [49] (Figure 5). Int. J. Mol. Sci. 2012, 13 6890 Figure 5. (a) Image from the RCSB PDB of PDB ID 1FV3 regarding
Site-1 of fragment C [51]; (b) Image from the RCSB PDB of PDB ID 1YYN regarding
Site-2 of fragment C [52]. 2.1.1. Molecular Binding Furthermore, the described surface-protein receptors, involved in
neuronal specificity of botulinum toxins, are related to protein components of the synaptic vesicle
membrane. Interestingly, the botulinum serotypes that exhibit highest sequence similarity share the
same protein receptor. This is the case of botulinum Types A, E and F, which bind SV2, a family of Int. J. Mol. Sci. 2012, 13 6891 synaptic-vesicle membrane proteins, whereas botulinum Types B, and G bind SytI and SytII, which is
the calcium sensor that triggers synaptic vesicle fusion. No protein receptor has been associated with
botulinum Types C and D. The general pathway of nerve entry begins with the preinsertion conformation
of the translocation domain, followed by the translocation of the light-chain protease across
endosomes. This translocation is highly dependent on the pH gradient, redox gradient and the
transmembrane potential. The completion of translocation ends when the C-terminus of the light chain
enters the cytosol, where SNARE cleveage takes place and this is the last portion translocated that exit the
channel. The disulfide bridge between light and heavy chains is crucial for botulinum toxicity and is
required for chaperone function, acting as a principal determinant for cargo translocation and release [26]. 2.1.2. Retrograde Axonal Transport Proposed pathway for the hybrid protein β-Gal-fragment C. Once the hybrid
protein is injected intramuscularly, it is found in large uncoated vesicles and then transported
retrogradely to the endoplasmic reticulum (ER). Hypothetical pathways are indicated with
dashed arrows. Adapted from [63]. Figure 6. Proposed pathway for the hybrid protein β-Gal-fragment C. Once the hybrid
protein is injected intramuscularly, it is found in large uncoated vesicles and then transported
retrogradely to the endoplasmic reticulum (ER). Hypothetical pathways are indicated with
dashed arrows. Adapted from [63]. Additionally, BDNF-fragment C and GDNF-fragment C hybrid proteins exert neuroprotective
effects in vitro and in vivo. For example, GDNF-fragment C promotes neuronal survival and neurite
outgrowth in animal models of Parkinson’s disease [66] and amyotrophic lateral sclerosis (ALS) [67,68],
and BDNF-fragment C is protective in a mouse model of ALS, although no synergistic effect of this Additionally, BDNF-fragment C and GDNF-fragment C hybrid proteins exert neuroprotective
effects in vitro and in vivo. For example, GDNF-fragment C promotes neuronal survival and neurite
outgrowth in animal models of Parkinson’s disease [66] and amyotrophic lateral sclerosis (ALS) [67,68],
and BDNF-fragment C is protective in a mouse model of ALS, although no synergistic effect of this
recombinant molecule was found [69]. Additionally, BDNF-fragment C and GDNF-fragment C hybrid proteins exert neuroprotective
effects in vitro and in vivo. For example, GDNF-fragment C promotes neuronal survival and neurite
outgrowth in animal models of Parkinson’s disease [66] and amyotrophic lateral sclerosis (ALS) [67,68],
and BDNF-fragment C is protective in a mouse model of ALS, although no synergistic effect of this
recombinant molecule was found [69]. Similarly, the hybrid protein that contains cardiotrophin-1 and fragment C promoted motor neuron
survival in vitro in a dose-dependent manner [70], and the combination of the anti-apoptotic molecule
Bcl-XL and fragment C improved cell survival and decreased apoptosis in the glutamate-mediated
excitotoxicity of SH-SY5Y neuronal cells [71]. Furthermore, fragment C of tetanus toxin and NGF share the same retrograde transport organelles,
suggesting neurotrophin receptor p75NTR as the first membrane marker of the retrograde endocytic
pathway used by fragment C of tetanus toxin [72]. p75NTR is a transmembrane receptor for neurotrophic
factors of the neurotrophin family, which comprises NGF, BDNF and neurotrophin-3 and -4/5. In
addition to p75NTR, neurotrophins signal, via the tropomyosin-related kinase (Trk) family of receptors,
tyrosine kinases Trk A, B and C. 2.1.2. Retrograde Axonal Transport One of the unique characteristics of tetanus toxin is that it can be transported retrogradely to the
central nervous system from the circulatory system. The first question was whether fragment C itself
could be transported by neurons similar to the native toxin while avoiding its toxicity. The trans-synaptic
transport of fragment C was intensively studied in one of the best-characterized systems, the primary
visual pathway [57,58], confirming its capacity as a carrier once it was injected intramuscularly [59]. Furthermore, the possibility of constructing hybrid molecules with fragment C has opened the door to an
interesting research field, the discovery of neuro-anatomical tracers, whose main purpose is to map
synaptic connections between neuronal cells. One of the most well-known hybrid proteins that has been used for this purpose is the hybrid
protein encoded by lacZ-fragment C. This protein has been tested in vitro and in vivo to determine its
activity in the hypoglossal system, and the detection of the labeled motor neurons was dependent on
time post-injection [60–62]. Since neuronal integrity is crucial for fragment C internalization, the
transneuronal molecular pathway at neuromuscular junctions was intensively studied using this hybrid
protein [63]. The protein was detected not only in the neuromuscular junction postsynaptic side but
also the soma of the motor neuron, away from the active zones in large uncoated vesicles (Figure 6). Other hybrid molecules form multicomponent proteins by recombining fragment C, the translocation
domain of diphtheria toxin, and the DNA-binding fragment of the GAL4 transcription factor. This
system was particularly effective in PC12 cells [64]. The advances in the understanding of these hybrid proteins have paved the way for new therapeutic
approaches using fragment C as a carrier of neurotrophic factors to ameliorate the disease process of
motor neuron diseases, neuropathies and pain. Moreover, fragment C represents a potential non-viral
vector for delivering exogenous biomolecules, such as proteins and DNA, to neurons in vitro and
in vivo [60]. Among the wide range of neurotrophic factors, such as nerve growth factor (NGF),
neurotrophin-3, insulin-like growth factors and vascular epithelial growth factor, the factors most used
to construct hybrid proteins with fragment C are brain-derived neurotrophic factor (BDNF) and
glial-derived neurotrophic factor (GDNF). More recently, a novel multi-component nanoparticle
system using polyethylene imine (PEI) has been evaluated to elicit the expression of BDNF in
neuronal cell lines [65]. Int. J. Mol. Sci. 2012, 13 6892 Figure 6. 3. Possible Signaling Pathways for Fragment C of Tetanus Toxin The molecular pathway involving the activation of the Trk receptors is closely shared by
neurotrophic factors and fragment C. The neurotrophin family has been shown to regulate survival,
development and functional aspects of neurons in the central and peripheral nervous systems through
the activation of one or more of the three members of the receptor tyrosine kinases (TrkA, TrkB, and
TrkC) in cooperation with p75NTR [75,76]. NGF can bind to the TrkA receptor or a complex of TrkA
and p75NTR [75], BDNF and neurotrophin-4/5 can bind to TrkB, and neurotrophin-3 binds to TrkC. Furthermore, similarly to the specialized internalization of fragment C in a clathrin-dependent process,
Schiavo and co-workers proposed that NGF can trigger the recruitment of a pool of p75NTR to
clathrin-coated pits to be delivered into the soma of motor neurons, which highlights the regulation of
p75NTR signaling in response to neurotrophins [30,77]. In addition, p75NTR can also interact with
pro-neurotrophins and, therefore, it serves as a signaling component of the receptor complex for
growth-inhibitory molecules of CNS, such as Nogo, prompting Nogo receptor-mediated signaling [78,79]. Aguilera and co-workers described an increase in serotonin synthesis in the central nervous system
induced by tetanus toxin, suggesting that the toxin-affected serotonergic innervation in the perinatal rat
brain triggered the translocation of calcium-phosphatidylserine-dependent protein kinase C (PKC) [80]. In particular, they found that tetanus toxin, but not botulinum toxin (BoNT/A), produced a specific
time- and dose-dependent inhibition of serotonin uptake in rat central nervous system synaptosomes. In fact, tetanus toxin altered a component involving inositol phospholipid hydrolysis, which is
associated with PKC activity translocation [81,82]. In addition to this translocation, an enhancement of
the tyrosine phosphorylation of the tyrosine receptor TrkA, phospholipase C (PLCγ-1) and ERK-1/2 was
also observed [83]. Because fragment C stimulated the PLC-mediated hydrolysis of phosphoinositides in
rat brain neurons, this fragment appeared to modulate some signaling pathways involving the transport
of serotonin [84]. Furthermore, the activation of intracellular pathways related to the PLCγ-1 phosphorylation and
activation of PKC isoforms and the kinases Akt (at Ser 473 and Thr 308) and ERK-1/2 (at Thr 202/Tyr
204) was induced by fragment C in rat brain synaptosomes and cultured cortical neurons. This signal
pathway activation was dependent on time and concentration, suggesting that fragment C could exert
neuroprotective effects, activating TrkA and TrkB receptors in a similar manner as do NGF and BDNF
or neurotrophin-4/5 [85,86]. 2.1.2. Retrograde Axonal Transport The two receptor systems can function synergistically, antagonistically
or independently of each other in different cell types [73]. The retrograde pathway of fragment C of
tetanus toxin is shared by p75NTR, TrkB and BDNF, which is strongly dependent on the activities of the
small GTPases Rab5 and Rab7 [56], which suggest that at least a portion of p75NTR is transported toward
the soma without undergoing proteolytic cleavage. In particular, an impairment of Rab7 activity inhibits Int. J. Mol. Sci. 2012, 13
6893 Int. J. Mol. Sci. 2012, 13 Int. J. Mol. Sci. 2012, 13 6893 the trafficking of fragment C-, p75NTR- and TrkB-containing carriers. However, Rab7 impairment in
PC12 cells led to an increase in TrkA and extracellular-regulated kinases (ERK-1/2) phosphorylation
and the stimulation of NGF [56]. Considering the potential use of fragment C as a non-viral vector and its likely sharing a common
molecular pathway as the one described for the neurotrophic factor BDNF [74], there is speculation
about its possible neuroprotective properties. Does fragment C have neuroprotective properties? 3. Possible Signaling Pathways for Fragment C of Tetanus Toxin Fragment C also protected cerebellar granular cells against potassium deprivation-induced apoptotic
death [87] and acted as a neuroprotector in a model of 1-methyl-4-phenylpyridinium (MPP+)-triggered
apoptosis, enhancing the survival pathways in rats with a dopaminergic lesion and improving different
motor behaviors. In this study, fragment C induced Ser 112 and Ser 136 BAD phosphorylation, Int. J. Mol. Sci. 2012, 13 Int. J. Mol. Sci. 2012, 13 6894 activated the transcription factor NF-κB, which prevents neuronal death, and induced a decrease in the
release of cytochrome c and, consequently, a reduction in the activation of procaspase-3 and chromatin
condensation [88,89] (Figure 7). Figure 7. Proposed signaling pathway for fragment C. Based on the results obtained by
Aguilera and co-workers, fragment C can induce cerebellar granular cell survival under
stress conditions, activating signaling pathways associated with Trk receptors that include
the activation of PLC, the Ras/MAPK pathway and the PI3 pathway, leading to the
survival of the cell [88,89]. Another possible retrograde pathway of fragment C of tetanus
toxin is shared by p75NTR, TrkB and BDNF, which is strongly dependent on the activities
of the small GTPases Rab5 and Rab7 [56]. of the small GTPases Rab5 and Rab7 [56]. Consequently, the main advantage of using fragment C as a potential therapeutic agent in non-viral
gene therapy is particularly interesting in amyotrophic lateral sclerosis (ALS). The failure of standard
treatments in ALS could rely on the inappropriate route of administration and/or the poor bioavailability
of molecules to the target cell [90]. The subcutaneous and intrathecal delivery of neurotrophic factors
can cause adverse side effects such as weight loss, fever, cough, fatigue and behavioral changes [91],
whereas viral gene therapy based on the use of an adeno-associated virus or lentivirus vectors is more
efficient than the neurotrophic factor delivery but can induce several inherent hazards [92]. An
alternative strategy that effectively reaches motor neurons, can exert neuroprotective properties and
does not show such adverse side effects implies the use of fragment C. Osta and co-workers found in a
mouse model of ALS, which carries the mutation G93A in human superoxide dismutase 1 (SOD1),
transgenic SOD1G93A mice, an amelioration of the decline in hind-limb muscle innervation in the animals Consequently, the main advantage of using fragment C as a potential therapeutic agent in non-viral
gene therapy is particularly interesting in amyotrophic lateral sclerosis (ALS). 3. Possible Signaling Pathways for Fragment C of Tetanus Toxin The failure of standard
treatments in ALS could rely on the inappropriate route of administration and/or the poor bioavailability
of molecules to the target cell [90]. The subcutaneous and intrathecal delivery of neurotrophic factors
can cause adverse side effects such as weight loss, fever, cough, fatigue and behavioral changes [91],
whereas viral gene therapy based on the use of an adeno-associated virus or lentivirus vectors is more
efficient than the neurotrophic factor delivery but can induce several inherent hazards [92]. An
alternative strategy that effectively reaches motor neurons, can exert neuroprotective properties and
does not show such adverse side effects implies the use of fragment C. Osta and co-workers found in a
mouse model of ALS, which carries the mutation G93A in human superoxide dismutase 1 (SOD1),
transgenic SOD1G93A mice, an amelioration of the decline in hind-limb muscle innervation in the animals Int. J. Mol. Sci. 2012, 13 6895 that were injected with either naked DNA-encoding fragment C (TTC) or naked DNA, encoding the
recombinant molecule fragment C and BDNF (BDNF-TTC). In addition, a significant delay in the onset
of symptoms and functional deficits, an improvement in the spinal motor neuron survival
(down-regulation of caspase-1 and caspase-3 levels and a significant phosphorylation of serine/threonine
protein kinase Akt) and a prolonged lifespan under both treatments was observed [69,93]. Although no
significant differences were found between TTC and BDNF-TTC treatments, recombinant plasmid
BDNF-TTC was detected in skeletal muscle and the corresponding recombinant protein reached the
spinal cord tissue of transgenic SOD1G93A mice, reinforcing on the one hand the carrier properties of
fragment C. The active state of BDNF in the recombinant molecule could suggest that BDNF could
exert an autocrine and neuroprotective role together with TTC to a similar extent as TTC alone;
however this effect could not be sufficient enough to prompt a synergistic effect. As a consequence,
BDNF-TTC molecule could mainly use the same pathway that mimics a neurotrophic secretion route,
prompting survival signals in the spinal cord of transgenic SOD1G93A mice [66]. Despite all these contributions to the understanding of the signaling pathway of fragment C, further
studies should be performed to elucidate its neuroprotective effect. 4. Conclusions Fragment C is considered to be a useful and valuable tool to carry therapeutic molecules due to its
efficient retroaxonal transport. This is the main reason why this particular property has been exploited
as a therapeutic strategy in the central nervous system to ameliorate the disease process of well-known
neurodegenerative diseases, such as Parkinson’s and ALS. However, recent in vitro and in vivo studies
have shown that fragment C can enhance cell survival by itself. This new property of fragment C has
opened the door to the understanding of its possible, although not yet well-characterized, molecular
pathways. Because the characterization of the protein-protein interaction at the molecular level is of
high importance and the plasma membrane of each nervous cell contains a particular composition, a
messenger, such as fragment C, can exert different effects depending on the signaling pathway it
modulates, which is directly related to the type and distribution of different receptors in the plasma
membrane. Thus, a messenger is not the molecule that carries the message; many results can be
obtained when studying the same effector molecule under different experimental conditions. Regarding fragment C, new studies will reveal the precise molecular mechanism by which it can
induce its neuroprotective properties. This mechanism could shed light on using fragment C as an
alternative therapeutic strategy for more neurodegenerative diseases in the near future. Acknowledgments This work was supported by grants from the Spanish Ministerio de Educación y Ciencia
SAF2009-13626 to José Aguilera, and also by grants from Caja Navarra: “Tú eliges, tú decides”;
PI10/0178 from the Fondo de Investigación Sanitaria of Spain; ALS Association N° S54406 and
the Ministerio de Ciencia e Innovacion INNPACTO IPT-2011-1091-900000 to Rosario Osta. This
manuscript has been proofread by Chuck Simmons, a native, English-speaking University Instructor
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transport. Traffic 2007, 8, 1736–1749. 78. Wang, K.C.; Kim, J.A.; Sivasankaran, R.; Segal, R.; He, Z. p75 interacts with the Nogo receptor
as a co-receptor for Nogo, MAG and OMgp. Nature 2002, 420, 74–78. 79. Twiss, J.L.; Chang, J.H.; Schanen, N.C. Pathophysiological mechanisms for actions of the
neurotrophins. Brain Pathol. 2006, 16, 320–332. 80. Aguilera, J.; Lopez, L.A.; Yavin, E. Tetanus toxin-induced protein kinase C activation and
elevated serotonin levels in the perinatal rat brain. FEBS 1990, 263, 61–65. 81. Gil, C.; Ruiz-Meana, M.; Álava, M.; Yavin, E.; Aguilera, J. Tetanus toxin enhances protein kinase
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TrkA, PLCγ-1, PKC isoforms and ERK-1/2 by tetanus toxin. FEBS Lett. 2000, 481, 177–182. 84. Pelliccioni, P.; Gil, C.; Najib, A.; Sarri, E.; Picatoste, F.; Aguilera, J. Tetanus toxin modulates
serotonin transport in rat-brain neuronal cultures. J. Mol. Neurosci. 2001, 17, 303–310. 85. Gil, C.; Chaib-Oukadour, I.; Blasi, J.; Aguilera, J. HC fragment (C-terminal portion of the heavy
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distributed under the terms and conditions of the Creative Commons Attribution license
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Zaragoza, P.; Aguilera, J.; Navarro, X.; et al. Fragment C of tetanus toxin, more than a carrier. Novel perspectives in non-viral ALS gene therapy. J. Mol. Med. 2010, 88, 297–308. © 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).
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A felnőttkori hipnotikus fogékonyság fejlődési és szocializációs meghatározói
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E-mail: andras.kolto@nuigalway.ie Beérkezett: 2018. december 4. – Elfogadva: 2019. január 27. KÖLTŐ ANDRÁS1,2 – JÓZSA EMESE1 – BÁNYAI ÉVA1 KÖLTŐ ANDRÁS1,2 – JÓZSA EMESE1 – BÁNYAI ÉVA1 1ELTE Eötvös Loránd Tudományegyetem Pszichológiai Intézet
2National University of Ireland Galway, Health Promotion Research Centre Kulcsszavak: szülői nevelési stílus, szülői viselkedés, hipnábilitás, hipnotikus fogékonyság Magyar Pszichológiai Szemle, 2019, 74.1/5. 65–80
DOI: 10.1556/0016.2019.74.1.5 Magyar Pszichológiai Szemle, 2019, 74.1/5. 65–80
DOI: 10.1556/0016.2019.74.1.5 BEVEZETÉS A hipnotikus interakció résztvevői – az alany és a hipnotizőr – között még a látszólag
semleges, laboratóriumi környezetben és kutatási céllal végzett, kötött forgatóköny-
vű hipnózisban is olyan kapcsolat jöhet létre, amely sok vonatkozásban hasonlít a
gyermek–szülő viszonyhoz. Ez a hasonlóság megjelenhet mind a viselkedés, mind a
hipnotikus élmények és az áttételi-viszontáttételi érzelmek, mind a pszichofiziológiai
változások szintjén. Ennek ellenére kevés kutatást végeztek annak feltárására, hogy a
szülőkkel kapcsolatos gyermekkori emlékek milyen kapcsolatban állnak a felnőttkori
hipnotikus válasz dimenzióval, s az ezzel kapcsolatos empirikus bizonyítékok java ré-
sze is egy öt évtizede elvégzett, nagy elemszámú kvalitatív kutatásból (J. R. Hilgard,
1979) származik. Olyan longitudinális vizsgálat pedig tudomásunk szerint egyáltalán
nincs, amely a vizsgálati személyeket és szüleiket a személyek születésétől kezdve tanul-
mányozná, hogy a gyermek–szülő interakció jellegzetességeit összevesse a személyek
felnőttkori hipnotikus reakcióival. Az eddig ismert determinánsok – pszichogenetikai
tényezők, személyiségvonások, figyelmi kapacitás és egyes felnőtt kötődési mintáza-
tok – mellett a szülői nevelés is hozzájárulhat a felnőttkori hipnábilitásban megfigyelt
változatossághoz. A hipnoterapeuta munkáját is segítheti, ha felméri a kliens szülők-
kel kapcsolatos emlékeit és élményeit, mert ezek befolyásolhatják a terápiás munka
minőségét. Ebben a tanulmányban áttekintjük azokat az idegtudományi eredményeket és (el-
sősorban kvalitatív) kutatásokat, amelyekben a szülői viselkedés felnőtt hipnotikus fo-
gékonyságra gyakorolt befolyását vizsgálták. Ezután összefoglaljuk saját, standard kér-
dőíveket és hipnábilitási skálákat alkalmazó vizsgálatainkat, amelyek megerősítették,
hogy a szülői nevelésre vonatkozó emlékek előrejelzik a felnőttkori hipnotikus válasz
dimenzióit, főleg bizonyos hipnózisbeli élményeket és áttételi érzelmeket. Végül ki-
térünk a jövőbeli lehetséges kutatási irányokra és eredményeink gyakorlati felhasznál-
hatóságára. Beérkezett: 2018. december 4. – Elfogadva: 2019. január 27. A kliens és a terapeuta közötti hipnoterápiás kapcsolat sok szempontból hasonlít a gyermek–szülő viszonyra. A hipnózis szociál-pszichobiológiai megközelítésében a felnőttkori hipnotikus fogékonyság mértékét az ideg-
rendszer fejlődése és a szocializációs folyamatok is befolyásolják. Ennek ellenére eddig nagyon kevés kutatás
foglalkozott a felnőttkori hipnotikus fogékonyság fejlődési előzményeivel. Szintén kevés vizsgálatot végeztek a
szocializációs hatások feltárására; a gyermekkori emlékeket ezekben is inkább kvalitatív módszerekkel tanul-
mányozták. Ebben a cikkben összefoglaljuk e kutatások főbb eredményeit, majd bemutatjuk kutatócsoportunk
2008 óta végzett vizsgálatait, amelyekben a szülői nevelési stílusra vonatkozó emlékek és a felnőttkori hip-
notikus válasz összefüggéseit kerestük. Standardizált, kvantitatív mérőeszközöket alkalmazó keresztmetszeti
vizsgálataink tanulsága, hogy a hideg-büntető szülői nevelési stílus előrejelzi a laboratóriumi hipnózisban
átélt negatív érzelmeket és a hipnotizőr rosszallásától való félelmet. Ezt a kapcsolatot részben az alexitímiás
érzelemfeldolgozás mediálja. Ezek az eredmények segítséget adhatnak a hipnoterápia megtervezéséhez és a
teherbíró pszichoterápiás szövetség kialakításához. Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 66 Költő András és mtsai HIPNÓZISSTÍLUSOK ÉS ARCHAIKUS BEVONÓDÁS Már maga Freud leírta a hipnoterápiás kapcsolat és a szülő–gyerek viszony hasonló-
ságát (Bachner-Melman és Lichtenberg, 2001), Ferenczi (1909/2002) pedig „anyai”
és „apai” hipnózistípusokat különített el. Korábbi vizsgálataink igazolták, hogy ezeket
a hipnotizőrök tudattalanul még a látszólag semleges, hipnotikus fogékonyság stan-
dard körülmények között történő felmérésére végzett hipnózisok alatt is alkalmazzák
(Bányai , 1998, 2002). A hipnózis pszichoanalitikus megközelítése a hipnotizált személy és a hipnotizőr
közötti áttételi–viszontáttételi érzelmi kapcsolatot az archaikus bevonódás fogalmával
írja le. Az alanyban a hipnotizőrről kialakuló reprezentáció gyakran leképezi azokról
a tekintélyszemélyekről – leggyakrabban a szülőkről – alkotott modelleket, akik fon-
tos szerepet játszottak korai életében. Shor (1962) ezt tekinti az archaikus bevonódás
alapjának. Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 67 A felnőttkori hipnotikus fogékonyság fejlődési és szocializációs meghatározói A korai tárgykapcsolatokban kialakított kötődési munkamodell (Bowlby, 1979/2005)
és a szülők viselkedésével kapcsolatos élmények tehát akár késő felnőttkorban is be-
folyásolhatják a hipnotikus bevonódást. Emiatt a hipnotikus interakció működésének
jobb megértéséhez figyelembe kell vennünk a személyek gyermekkori élményeit. A CSECSEMŐKORI KÖTŐDÉS ÉS AZ IDEGRENDSZERI FEJLŐDÉS SZEREPE A korai kapcsolatok befolyásolják a szelf fejlődését és az énreprezentációk kialakulá-
sát. A csecsemő- és kisgyermekkorban átélt összehangolódási élmények szolgáltatnak
idegrendszeri alapot az interszubjektivitáshoz, az önszabályozáshoz és a későbbi – fel-
nőttkori – interakciós szinkronitáshoz (Bányai, 2008). A dajkálás, ringatás, a csecse-
mő és a kisgyermek érzelmi-mentális állapotainak jelölése, visszatükrözése (Gergely
és Watson, 1996) magas interakciós szinkronitásra hangolja a központi idegrendszert
(Bányai, 2008). Így a csecsemő- és kisgyermekkori élmények áttételes módon a hipno-
tikus interakcióba való bevonódás képességét is befolyásolhatják. p
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A csecsemő kötődési mintázatai és a felnőttkori hipnotikus válasz közötti kapcsola-
tot írja le Posner és Rothbart (2011) elmélete. Szerintük a csecsemőkor során kiala-
kuló érzelmi és kognitív (ön)kontroll egyértelműen összefügg a szülők viselkedésével,
és a kapcsolatot a központi idegrendszer orientációs folyamatai jelentik. Gyakori szü-
lői stratégia a nyugtalan, síró csecsemő megnyugtatására egy addig ismeretlen tárgy
mutatása (Harman, Rothbart és Posner, 1997). Ez a csecsemő agyában aktiválja az
anterior gyrus cingulumot (ACC) és más, a végrehajtó figyelmi hálózatban szerepet
játszó agyterületeket, amelyek egymással szoros kölcsönhatásban fejlődnek (Posner,
Rothbart és Voelker, 2016). Az ACC és a végrehajtó figyelmi rendszerek szerepe a hip-
nózisban többszörösen bizonyított. A hipnózisbeli módosult tudatállapot, sőt még a
hipnábilitás is összefügg az ACC módosult aktivitásával (Faymonville, Boly és Laureys ,
2006; Gruzelier, 2006; Hoeft és mtsai, 2012). Úgy tűnik, ezt az aktivitásváltozást maga a
hipnotikus szuggesztió okozza (Raz, Fan és Posner, 2005; Raz, Shapiro, Fan és Posner ,
2002). A hipnózis erős hatást gyakorol a korábban kifejlődött orientációs hálózatra, de
gyengébbet a később kifejlődő végrehajtó hálózatra, ennek eredményeképpen „a fo-
gékony személy [hipnózisban] könnyebben kerül »külső kontroll« hatása alá. Ahogy
a korai életszakaszban a gyermek kontrollja a szülő kezében van, hipnózis során a fel-
nőtt személy kontrollját a hipnotizőr veszi át” (Posner és Rothbart, 2011, 3.). A meg-
nyugtatásra alkalmazott figyelmi reorientációs „trükk” révén pedig a csecsemő agya
nagyobb eséllyel fejlődik úgy, hogy a figyelmi hálózat felnőttkorban is aktívan reagál-
jon a külső ingerekre, így a hipnotizőrtől érkező szuggesztiókra. Mindez arra utal, hogy a meleg és szeretetteljes szülői viselkedés, a csecsemő igé-
nyeire és érzelmi állapotára való odafigyelés és a megnyugtatására tett kísérletek
(amelyek a biztonságos kötődési stílus jelzői) magasabb felnőttkori hipnábilitáshoz
vezethetnek. A bizonytalanul kötődő csecsemőknél – a biztonságos kötődési stílussal
jellemezhető csecsemőkhöz képest – alacsonyabb bal agyféltekei frontális aktivitást
mutattak ki (Lakatos és Gervai, 2003), ami megalapozhatja az alacsony felnőttkori
hipnotikus fogékonyságot (Bányai, 2015). A CSECSEMŐKORI KÖTŐDÉS ÉS AZ IDEGRENDSZERI FEJLŐDÉS SZEREPE Igaz, ezeket a kapcsolatokat a szocializáció Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 68 Költő András és mtsai és a későbbi életesemények, például traumatikus élmények (Butler, Duran, Jasiukai-
tis, Koopman és Spiegel, 1996) vagy a korrektív pszichoterápia (Beauregard, 2014)
elfedhetik vagy akár meg is szüntethetik. FEJLŐDÉSI „ÖSVÉNYEK” A modell alapvetése, hogy a gyermekkorban átélt bevonó- Unauthenticated | Downloaded 10/24/24 05:46 AM UTC Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 69 A felnőttkori hipnotikus fogékonyság fejlődési és szocializációs meghatározói dás magasabb felnőttkori hipnábilitáshoz vezet. A bevonódás mélységét és intenzitását
pedig nagy részben a szülői viselkedés, és a szeretetteljes szülőkkel való azonosulás,
vagy a hideg-büntető szülőktől való félelem alapozza meg. Elméletével összhangban,
az interjúk alapján kiszámított pontszámokkal mért szülői melegség, illetve büntetés
kb. r = 0,20–0,30 erősségű korrelációt mutatott az SHSS:C hipnábilitási pontszámmal. Érdekes ugyanakkor, hogy több, gyakori büntetést és hideg-elutasító szülőket felidéző
vizsgálati személy is kifejezte az előzetes interjúk során, hogy szeretne jó „hipnotikus
alany” lenni. Ez felveti, hogy a viselkedéses hipnábilitás mellett az is fontos lehet, hogy
az alanyoknak milyen elvárásaik vannak a hipnózissal kapcsolatban, milyen élményeket
élnek át hipnózisban, és milyen személyközi kapcsolatot alakítanak ki a hipnotizőrrel. FEJLŐDÉSI „ÖSVÉNYEK” A szülőkkel kapcsolatos emlékek és a felnőttkori hipnábilitás kapcsolatára vonatkozó
empirikus bizonyítékok alapja J. R. Hilgard (1979) és munkatársai kevert módszerű
kutatása. Ők összesen 839 személlyel készítettek félig strukturált interjút, mielőtt a
Stanford Hipnotikus Szuszceptibilitási Skála C változatával (SHSS:C) (Weitzenhoffer
és Hilgard, 1962) felmérték hipnábilitásukat, 146 személlyel pedig a skála alkalmazása
után készítették el az interjút. Utóbbiakat hipnózisbeli élményeikről is kikérdezték. J. Hilgard feltételezte, hogy a gyermekkorban átélt, erős képzeleti bevonódással
járó helyzetek magas felnőttkori hipnábilitáshoz vezetnek. Az interjúban rákérdeztek
a vizsgálati személyek gyermekkori családjának összetételére; az apával és anyával való
kapcsolatra; a szülők jutalmazó és büntető módszereire; a gyermekkori szabadidős te-
vékenységekre. Azok a személyek, akik gyermekkorukban gyakran merültek el erős képzeleti bevo-
nódást igénylő tevékenységekben (pl. olvasás, fantáziálás, képzeletbeli barátok, mese-
vagy regényhősökkel való azonosulás stb.), nagyobb eséllyel voltak közepesen vagy
erősen hipnábilisak. Azok, akik nem vagy csak ritkán éltek át képzeleti bevonódást
gyermekkorukban, inkább az alacsony hipnábilitási övezetbe estek. Két tipikus fejlődési „ösvény” határozta meg az erős képzeleti bevonódást. Azok,
akik arról számoltak be, hogy szüleik meleg és szeretetteljes kapcsolatban voltak velük,
gyakran megfigyelték, hogy maguk a szülők is erősen bevonódnak efféle tevékenysé-
gekbe (pl. olvasás, tévénézés, kertészkedés vagy más hobbi). Ezek a gyerekek az ér-
zelmi ragály és a szülőkkel való azonosulás révén lettek fogékonyak az erős képzeleti
bevonódásra. A másik jellegzetes mintázat azoké volt, akik érzelmileg hideg és elérhe-
tetlen, illetve gyakori és kemény büntetést (pl. testi fenyítést) alkalmazó szülőket idéz-
tek fel. Ők a képzeleti bevonódást igénylő tevékenységeket menekülési stratégiaként
használták. Felnőttkorukban is erősebb bevonódásról, élénkebb fantáziaképekről szá-
moltak be. Magasabb pontszámot értek el az SHSS:C skálán azok a személyek, akiknek gyer-
mekkorukban volt képzeletbeli barátja vagy segítőtársa. J. Hilgard a „mintha”-játékokat
is a hipnotikus jelenségek előképének tekinti. A képzeletbeli barátok vagy játszótársak
sokszor olyan funkciót látnak el, mint amit Winnicott (1953) az átmeneti tárgyaknak
tulajdonít. Lehetséges, hogy azok a gyerekek, akik ilyen belső átmeneti tárgyakat hasz-
nálnak, később nagyobb eséllyel képesek bevonódni a hipnózisba, amely pszichoana-
litikus szempontból szintén értelmezhető „átmeneti térként” (Baker, 2000). Ez teszi
lehetővé, hogy hipnoterápiás helyzetben a kliens a tanult viselkedés automatizmusá-
ból kilépve gondolatban más lehetőségeket, megoldási módokat is kipróbáljon, és így
gazdagítsa viselkedésrepertoárját (Bányai, 2015). g
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Ezeknek az eredményeknek az alapján J. Hilgard megfogalmazta a hipnózis „fejlő-
dési ösvények” elméletét. A GYERMEKI HIPNÁBILITÁS ÉS A SZÜLŐI STÍLUS HATÁSÁNAK
KVANTITATÍV VIZSGÁLATA A gyermekek fogékonyabbak a hipnózis iránt, mint a felnőttek (London, 1965; Mor-
gan és Hilgard, 1973), aminek a gyerekek mindennapi életében gyakran előfordu-
ló spontán fantáziajátékok és álmodozás lehet az alapja (Gardner, 1974; Gardner és
Olness , 1981). A fókuszált koncentráció, a korlátozott valóságellenőrzés, a konkrét (és
mágikus) gondolkodás, az érzelmi nyitottság az új élmények iránt – beleértve a reg-
resszív állapotokat is – és az érzelmi intenzitás miatt a gyerekek „természetes” magas
fogékonyságú hipnotikus alanyok. S ahogy azt J. Hilgard vizsgálatai és későbbi empiri-
kus munkák is mutatják, a felnőtt tekintélyszemélyek viselkedése, hipnózissal vagy más
disszociatív állapotokkal kapcsolatos attitűdje felerősítheti ezt a hatást (Rhue, 2004). p
p
j
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A kisgyermekkorban átélt hipnózis funkcionálisan és kognitív-érzelmi szabályozás
tekintetében is eltér a nagyobb gyerekek és felnőttek hipnózisától. Míg utóbbiak sa-
ját akaratukból, a hipnotizőrrel együttműködve vonódnak be a hipnotikus állapotba,
addig a kisebb gyerekeknél a bevonódás fő hatótényezője a tekintélyszemélyre irá-
nyuló figyelem és az általa gyakorolt kontroll szintje. Ezért a kisgyermekek hipnó-
zisát J. R. Hilgard és Morgan (1978, 286.) protohipnózisnak nevezi: „A nagyon fiatal
gyerekek inkább képesek arra, hogy figyelmüket egy történet mesélésével eltereljük,
mint hogy [a fájdalmas orvosi beavatkozástól, hipnoterápiás vagy önhipnotikus szug-
gesztiókkal] saját fantáziájukat használva távolítsák el magukat.” A protohipnózis és
az érett hipnózis közötti váltás kisiskoláskorban, 5–7 éves kor körül következik be. Vandenberg (2002) hangsúlyozza, hogy ebben a korban alakulnak ki a gyermekek
„metamentális” készségei, amelyek megfelelnek más szerzők, például Fonagy és mun-
katársai (Fonagy, Bateman és Bateman, 2011; Fonagy, Steele, Steele, Moran és Higgitt,
1991) mentalizáció- értelmezésének. Ide tartozik a saját gondolkodásunkra és a szociális
kapcsolatokra való reflektálás, a mentális aktivitás önszabályozása. Vanderberg szerint
ezekhez köthető, hogy az idősebb gyerekek és a felnőttek képesek a hipnotikus inter-
akcióban való tudatos részvételre. A fent említett példákat leszámítva a hipnóziskutatás irodalmában ritkák a fejlődési
háttérrel foglalkozó kutatások. A J. Hilgard szülői nevelésre vonatkozó kvalitatív ered-
ményeinek kvantitatív módszerekkel való megerősítésére az általunk ismert egyetlen
példa Rhue és Lynn vizsgálata (Lynn és Rhue, 1988; Rhue és Lynn, 1987). Ők a fan- Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 70 Költő András és mtsai táziálásra hajlamos személyiség fejlődési előzményeit keresték, valamint azt vizsgálták,
hogy ezek hogy függenek össze fiatal felnőttek hipnábilitásával. A fantáziálásra hajla-
mos személyek gyakoribb és keményebb gyerekkori büntetésről (testi fenyítésről) szá-
moltak be, mint azok, akiknek alacsony volt a képzeleti aktivitása. A gyakran büntetett
személyek – összhangban J. A GYERMEKI HIPNÁBILITÁS ÉS A SZÜLŐI STÍLUS HATÁSÁNAK
KVANTITATÍV VIZSGÁLATA Hilgard megfigyeléseivel – a képzeletet gyakran mene-
külési, megküzdési stratégiaként alkalmazták. Ugyanakkor a két csoport között nem
volt különbség abban a tekintetben, hogy szüleiket mennyire látták szeretetteljesnek,
ami rámutat arra, hogy a szülői büntetés és az érzelmi melegség nem feltétlenül zárja
ki egymást. A szerzők különböző mérőeszközök alkalmazásával megerősítették a szü-
lői büntetés, a fantáziakészség és a felnőttkori hipnábilitás kapcsolatát. Rhue (2004)
azonban az erős felnőttkori hipnábilitás fejlődési determinánsairól szóló tanulmányá-
ban maga is hangsúlyozza, hogy ezen a területen nagy szükség lenne további kvantita-
tív vizsgálatokra. A FEJLŐDÉS ÉS A SZOCIALIZÁCIÓ
A HIPNÓZIS SZOCIÁL-PSZICHOBIOLÓGIAI MODELLJÉBEN Az első olyan hipnóziselmélet, amely figyelembe vette a kora gyermekkori élményeket,
a fejlődési-interaktív modell volt. E szerint az elképzelés szerint a velünk született képes-
ségeket a fejlődés során élettapasztalatok – így a szocializáció – befolyásolják, így azokat
a hipnotikus válasz elemzésekor is figyelembe kell venni. Az interaktív jelleg pedig arra
vonatkozik, hogy a velünk született és fejlődés során szerzett hatások egymással köl-
csönhatásban alakítják a felnőttkori hipnotikus fogékonyságot (E. R. Hilgard , 1965). j
p
g
y
g
(
g
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Ezen az általános fejlődésalapú megközelítésen túl azonban az eddigi elméleti mo-
dellek közül egyedül a szociál-pszichobiológai megközelítés integrálja a fejlődés-ideg-
tudományi és szocializációs előzményeket a felnőttkori hipnábilitás magyarázatába. Bányai (2008; valamint Bányai tanulmánya a jelen tematikus számban, pp. 7–26.) meg-
közelítésében a hipnózis és a hozzá hasonló módosult tudatállapotok olyan, evolúci-
ós szempontból hasznosnak bizonyult személyközi interakciók, melyeknek kölcsönös
ingerbemenet-szabályozó szerepe révén mindkét résztvevőre jótékony hatása van. Az
interakcióban való részvétel azonban rugalmasságot kíván meg mind az alany, mind
a hipnotizőr részéről. Ezt a rugalmasságot a csecsemő és az elsődleges gondozó, első-
sorban az anya (később a kisgyermek és szülei, testvérei) közötti kötődésre lehet vissza-
vezetni. A csecsemő, kisgyermek arousalszintjének empatikus átvétele és modulációja
hozzásegíti őt ahhoz, hogy belső ritmusát fokozatosan a társas környezethez igazít-
sa. Az érzelemtükrözés és -jelölés (Fonagy, Gergely, Jurist és Target, 2002; Gergely és
Watson , 1996) pedig az érzelmek tudatosulását és az önkontrollt segítik elő, hozzájá-
rulnak ahhoz, hogy a szelf önszabályozó ágenssé váljon. A korai kapcsolatok minősége a
figyelmi orientációt, az ingerszelekciót, a stresszel való megküzdést is befolyásolja. A hipnózis olyan átmeneti helyzet, amely kedvez a résztvevők gyors egymásra han-
golódásának. A hipnotizőr az indukció során az alany aktivációs szintjének csökkenté-
sére vagy növelésére törekszik, s figyelmét a környezet figyelemelvonó ingereiről saját
testi folyamataira, belső történéseire irányítja (Bányai, 2008). Ebben az „ingercsök-
kentett” helyzetben a hipnotizőr és az alany figyelme egyre jobban egymásra irányul, Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 71 A felnőttkori hipnotikus fogékonyság fejlődési és szocializációs meghatározói az interakciós partnertől érkező szociális ingerek egyre nagyobb szerepet kapnak a
helyzet irányításában. A hipnotizőr folyamatosan visszajelzi az alany kívülről megfi-
gyelhető viselkedéses megnyilvánulásait és a pszichofiziológiai változások (pl. a rela-
xált állapot) jeleit, amivel tulajdonképpen ugyanazt a funkciót tölti be, mint a szülői,
érzelemtükröző és -jelölő „szociális biofeedback” (Gergely és Watson, 1996). A FEJLŐDÉS ÉS A SZOCIALIZÁCIÓ
A HIPNÓZIS SZOCIÁL-PSZICHOBIOLÓGIAI MODELLJÉBEN A hipnotikus fogékonyság ebben az elméletben annak a rugalmasságnak a mutató-
ja, hogy milyen könnyen lép be az alany a szoros személyközi interakcióba, amelyben
a kölcsönös szabályozó mechanizmusok révén hipnotikus módosult tudatállapotba
kerül. A felnőttkori hipnotikus fogékonyság Bányai (2008) szerint a szocializációs fo-
lyamatban alakul ki, így a szülőkkel való kapcsolat minősége, a szülők viselkedése és
kommunikációs stílusa befolyásolja annak szintjét. Ezt J. R. Hilgard (1979) eredmé-
nyei mellett saját, kvantitatív módszereket alkalmazó vizsgálataink is megerősítették. KÉRDŐÍVES VIZSGÁLATOK AZ ELTE HIPNÓZISKUTATÓ
LABORATÓRIUMÁBAN Kutatócsoportunk 2008 óta szisztematikus vizsgálatokat végez annak ellenőrzésére,
hogy a hipnábilitás multidimenzionális magyarázatához hozzájárul-e a szülői nevelési
stílus. A hipnózis többdimenziós jellegének eddigi legátfogóbb kutatását Lichtenberg,
Bachner-Melman, Ebstein és Crawford (2004) végezte. Ők többszörös regresszióelem-
zéssel megállapították, hogy a kitartás személyiségvonás, a fókuszált figyelem, az ab-
szorpció és a katekol-O-metiltranszferáz enzim genetikai polimorfizmusa az SHSS:C
skálával mért hipnábilitás 33,6%-át magyarázza. Az itt összefoglalt vizsgálatokban arra
kerestük a választ, hogy a szülőkre vonatkozó emlékek hozzátesznek-e a hipnotikus
fogékonyság magyarázatához. A fogékonyság alatt – a szociál-pszichobiológiai elmélet-
tel összhangban – nemcsak a hipnózismélység-mérő viselkedéses skálákon elért hip-
nábilitási pontszámot, de az alany által átélt archaikus bevonódás (Bányai, Varga és
Gősiné Greguss, 2001) és a tudatállapot módosulásával járó jellegzetes élménymintá-
zat (Varga , 2017) erősségét is értjük. Szülői nevelési stílus és hipnotikus fogékonyság Összhangban J. R. Hilgard (1979) „fejlődési ösvények” elméletével, feltételeztük, hogy
a szülői melegségre és hideg-büntető viselkedésre vonatkozó emlékek alacsony-köze-
pes mértékben, de szignifikánsan előrejelzik a felnőttkori hipnábilitást. Kutatócsopor-
tunk többdimenziós hipnózisfelfogására és korábbi eredményeire alapozva (ld. Bá-
nyai, Józsa és Költő, pp. 45–61., illetve Varga tanulmányait a jelen tematikus számban,
pp. 95–112.) feltételeztük, hogy az alany által felidézett szülői nevelési stílus összefügg
a hipnózisban mutatott archaikus bevonódással és a módosult tudatállapot jellegzetes
élményeivel. Egyes eredmények (pl. Varga és Kekecs, 2014), illetve Kasos és munkatár-
sai jelen tematikus számban (pp. 79–93.) bemutatott pszichoendokrin vizsgálatai is azt
sugallják, hogy e két dimenzió erősebben összefügg a szülőkre vonatkozó emlékekkel,
mint a viselkedéses hipnábilitás. Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 72 Költő András és mtsai A viselkedéses hipnábilitást a Shor és Orne (1962) által kifejlesztett Harvard Hipná-
bilitási Csoportskálával (HCsS, magyar változat: Költő, Gősi-Greguss, Varga és Bányai,
2015) és Bowers (1993) Waterloo-Stanford Hipnábilitási Csoportskálájával (WSGC)
(magyar változat: Gősiné Greguss, 1999) mértük. A hipnotizőrhöz fűződő érzelmi kap-
csolatot az Archaikus Bevonódási Skálával (ABS, l. a jelen tematikus számban Bányai
és mtsai tanulmányát, pp. 45–61.), a módosult tudatállapotra jellemző élményeket a
Tudat Fenomenológiája Kérdőívvel (PCI, l. a jelen tematikus számban Józsa és mtsai
tanulmányát, pp. 27–43.) vizsgáltuk. A szülői nevelési stílus mérésére az eredetileg
Svédországban kifejlesztett Emlékeim a Szülői Nevelésről (EMBU) kérdőív rövidített,
23 tételes változatát (Arrindell és mtsai, 1999) alkamaztuk. Az EMBU tételeivel való
egyetértést az apára és az anyára vonatkozóan négyfokú Likert-skálán jelölik a sze-
mélyek. A tételek három faktorba rendeződnek, amelyet az anyára és az apára vonat-
kozóan külön számolunk ki: 1. Elutasítás, 2. Érzelmi melegség, 3. Túlvédés. Hogy a J. R. Hilgard (1979) által leírt, büntetéssel kapcsolatos összefüggéseket vizsgálni tudjuk, az
EMBU megfelelő tételeiből (amelyek a testi fenyítésre, a gyermek megszégyenítésére,
egyes tevékenységek tiltására vonatkoztak) kialakítottunk egy kiegészítő, operacioná-
lis skálát: 4. Büntetés (Költő, 2008). A kérdőívhez csatoltunk néhány, a családi struktú-
rára vonatkozó kérdést (Költő, 2015). (
)
A 2008 és 2015 közötti időszakban két vizsgálatban összesen 438 egészséges fel-
nőtt személyt vizsgáltunk meg (Költő, Józsa és Bányai, in press). Az első vizsgálatban
részt vevő 196 személy átlagéletkora 23,93 ± 4,96 év, a nők aránya 65%. A második
vizsgálatban 246 személy szerepelt (átlagéletkoruk 28, 26 ± 9,63 év), a nők aránya
55% volt. A személyek kitöltötték az EMBU kérdőívet, illetve szakemberek által veze-
tett, standard laboratóriumi csoporthipnózison vettek részt. Szülői nevelési stílus és hipnotikus fogékonyság Az egyik vizsgálatban a
WSGC-t, a másikban a HCSs-t alkalmaztuk a hipnábilitás mérésére. A hipnózist köve-
tően a személyek kitöltötték az ABS és PCI kérdőívet. Az összefüggések ellenőrzésére
korrelációs vizsgálatokat és többszörös lineáris regresszióelemzést végeztünk. Az ösz-
szehasonlítások nagy száma miatt a korrelációs együtthatók kiszámításakor bootstrap-
ping technikát alkalmaztunk, amely az elsőfajú hiba felhalmozódását is kiküszöböli. A vizsgálatok részletes módszerét, valamint az eredményeket Költő és munkatársai (in
press) közlik. Az 1–2. táblázatban összefoglaljuk a két vizsgálatban lineáris regresszió-
elemzéssel nyert eredményeket. A táblázatokról leolvasható, hogy a szülői nevelési stílusra – elsősorban a bünte-
tő-kontrolláló viselkedésre és az érzelmi melegség (hiányára) – vonatkozó emlékek
szignifikáns előrejelzői a hipnotikus válasznak, bár a szülői nevelés által megmagya-
rázott változatosság szintje alacsony (3–22% közötti). Különbségeket találtunk a két
vizsgálat között, illetve a vizsgálati személyek neme szerint. Általánosságban azonban
elmondható, hogy a szülők büntető-elutasító viselkedése összefügg a hipnózisban át-
élt negatív érzelmekkel (az ide tartozó PCI-tételek magukba foglalják a feszültséget,
dühöt, félelmet és szomorúságot), és a hipnotizőr iránt érzett (negatív vagy pozitív
színezetű) áttétellel. Érdemes kiemelni, hogy az első vizsgálatban az anyai büntetés
a férfiak negatív archaikus bevonódásának 17,7%-át, a szülők büntető viselkedése a
hipnotizőr haragjától, rosszallásától való félelem 21,9%-át, míg a második vizsgálat-
ban a szülőkre vonatkozó emlékek (elsősorban az anya viselkedése) a nők hipnózis
alatti negatív érzelmeinek 19,3%-át megmagyarázta úgy is, hogy más magyarázó- vagy Unauthenticated | Downloaded 10/24/24 05:46 AM UTC Unauthenticated | Downloaded 10/24/24 05:46 AM UTC A felnőttkori hipnotikus fogékonyság fejlődési és szocializációs meghatározói 73 1. táblázat. Szülői nevelési stílus és hipnotikus fogékonyság A felidézett szülői nevelési stílus összefüggései a hipnotikus fogékonyság
viselkedéses, fenomenológiai és érzelmi mutatóival az első vizsgálatban:
többszörös lineáris regresszióelemzés (N = 196)
Kimeneti változó
Prediktor változó(k) (β)
F (df)
R2
Férfiak (n = 69)
WSGC Szubjektív pontszám
Anyai túlvédés (0,261)
4,914* (1, 67)
0,054
PCI Disszociatív kontroll
Anyai büntetés (0,304)
6,808* (1, 67)
0,079
PCI Pozitív érzelem
Anyai büntetés (0,274)
5,419* (1, 67)
0,061
PCI Negatív érzelem
Anyai elutasítás (0,247)
4,348* (1, 67)
0,047
PCI Vizuális képzelet
Anyai büntetés (0,276)
5,523* (1, 67)
0,062
PCI Belső folyamatokra irányuló figyelem
Anyai büntetés (0,239)
8,950* (1, 67)
0,105
ABS Pozitív
Anyai büntetés (0,239)
4,077* (1, 67)
0,043
ABS Negatív
Anyai elutasítás (0,243)
4,010* (1, 67)
0,044
ABS Csodálat és kötődés
Anyai büntetés (0,248)
4,113* (1, 63)
0,046
ABS Félelem a negatív megítéléstől
–
ABS Függőségigény
–
Nők (n = 127)
WSGC Szubjektív pontszám
–
PCI Disszociatív kontroll
–
PCI Pozitív érzelmek
–
PCI Negatív érzelmek
Apai érzelmi melegség
(−0,381)
Anyai érzelmi melegség
(0,268)
Anyai büntetés (0,807)
Anyai elutasítás (−0,666)
Végső modell
8,577* (4, 122)
0,193
PCI Vizuális képzelet
–
PCI Belső folyamatokra irányuló figyelem
–
ABS Pozitív
–
ABS Negatív
–
ABS Csodálat és kötődés
Anyai túlvédés (−0,188)
4,496* (1, 123) 0,027
ABS Félelem a negatív megítéléstől
–
ABS Függőségigény
–
Megjegyzés: WSGC = Waterloo-Stanford Hipnábilitási Csoportskála. PCI = Tudat Fenomenológiája Kérdőív. ABS = Archaikus Bevonódási Skála. *p < 0,049. Az International Journal of Clinical and Experimental Hypnosis
engedélyével újraközölve. Megjegyzés: WSGC = Waterloo-Stanford Hipnábilitási Csoportskála. PCI = Tudat Fenomenológiája Kérdőív. ABS = Archaikus Bevonódási Skála. *p < 0,049. Az International Journal of Clinical and Experimental Hypnosis
engedélyével újraközölve. Megjegyzés: WSGC = Waterloo-Stanford Hipnábilitási Csoportskála. PCI = Tudat Fenomenológiája Kérdőív. ABS = Archaikus Bevonódási Skála. *p < 0,049. Az International Journal of Clinical and Experimental Hypnosis
engedélyével újraközölve. kontrollváltozót nem vontunk be az elemzésbe. A második vizsgálatban azt tapasz-
taltuk, hogy a vizsgálati személyek korának és foglalkozásának kontrollálása növeli a
modellek magyarázóerejét (Költő, 2015). Ezek az eredmények részben megerősítik
J. R. Hilgard (1979) „fejlődési ösvények” elméletét. Ugyancsak alátámasztják kutató-
csoportunk tapasztalatait, hogy a viselkedéses hipnábilitási pontszám mellett érdemes
figyelembe venni az alanyok hipnózis alatti tudatmódosulásának mértékét, valamint a
hipnotizőr iránti áttétel érzelmi színezetét és intenzitását is. Néhány esetben látszólag Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 74 Költő András és mtsai 2. táblázat. Szülői nevelési stílus és hipnotikus fogékonyság A felidézett szülői nevelési stílus összefüggései a hipnotikus fogékonyság
viselkedéses, fenomenológiai és érzelmi mutatóival a második vizsgálatban:
többszörös lineáris regresszióelemzés (N = 249)
Kimeneti változó
Prediktor változó(k) (β)
F (df)
R2
Férfiak (n = 105)
HCsS Szubjektív pontszám
–
PCI Disszociatív kontroll
–
PCI Pozitív érzelmek
–
PCI Negatív érzelmek
Apai büntetés (0,225)
5,517* (1, 103)
0,042
PCI Vizuális képzelet
Anyai büntetés (−0,215)
5,008* (1, 103)
PCI Belső folyamatokra irányuló figyelem
–
ABS Pozitív
Apai büntetés (0,292)
9,614* (1, 103)
0,076
ABS Negatív
Anyai büntetés (0,430)
23,359* (1, 103) 0,177
ABS Csodálat és kötődés
–
ABS Félelem a negatív megítéléstől
Anyai büntetés (0,305)
Apai büntetés (0,239)
Végső modell
15,574* (2, 102) 0,219
ABS Függőségigény
Apai túlvédés (0,242)
6,382* (1, 103)
0,049
Nők (n = 124)
HCsS Szubjektív pontszám
Apai érzelmi melegség
(−0,206)
5,388* (1, 122)
0,034
PCI Disszociatív kontroll
–
PCI Pozitív érzelmek
Apai érzelmi melegség
(−0,248)
8,021* (1, 122)
0,054
PCI Negatív érzelmek
Anyai büntetés (0,218)
6,080* (1, 122)
0,040
PCI Vizuális képzelet
Anyai túlvédés (0,348)
Apai túlvédés (−0,260)
Végső modell
5,913* (2, 121)
0,074
PCI Belső folyamatokra irányuló figyelem
Anyai túlvédés (0,177)
3,955* (1, 122)
0,023
ABS Pozitív
Anyai túlvédés (0,179)
4,059* (1, 122)
0,024
ABS Negatív
–
ABS Csodálat és kötődés
–
ABS Félelem a negatív megítéléstől
–
ABS Függőségigény
Apai túlvédés (0,198)
4,960* (1,122)
0,031
Megjegyzés: HCsS = Harvard Hipnábilitási Csoportskála. PCI = Tudat Fenomenológiája Kérdőív. ABS = Ar-
* Megjegyzés: HCsS = Harvard Hipnábilitási Csoportskála. PCI = Tudat Fenomenológiája Kérdőív. ABS = Ar-
chaikus Bevonódási Skála. *p < 0,046. Az International Journal of Clinical and Experimental Hypnosis engedé-
lyével újraközölve. ellentmondásos összefüggéseket találtunk, például a férfiak esetében a szülők büntető
viselkedése előre jelezte a pozitív archaikus bevonódást. Véleményünk szerint ez an-
nak tulajdonítható, hogy – még laboratóriumi helyzetben is – a hipnotizőr inkább a
gondoskodó, „jó szülő” tudattalan reprezentációját mozgósítja a vizsgálati személyek-
ben. Ennek önmagában korrektív jellege lehet, főleg akkor, ha az alany valódi szülei
részéről a gyakori büntetést és az érzelmi melegség hiányát idézi föl. Ugyanakkor az, ellentmondásos összefüggéseket találtunk, például a férfiak esetében a szülők büntető
viselkedése előre jelezte a pozitív archaikus bevonódást. Véleményünk szerint ez an-
nak tulajdonítható, hogy – még laboratóriumi helyzetben is – a hipnotizőr inkább a
gondoskodó, „jó szülő” tudattalan reprezentációját mozgósítja a vizsgálati személyek-
ben. A szülői viselkedés és a hipnotikus bevonódás kapcsolatát az alexitímiás
érzelemfeldolgozás mediálja A biztonságos kötődés és a szülőkhöz fűződő meleg, szeretetteljes kapcsolat jó felnőtt-
kori mentalizációs készségekkel jár együtt, míg a zavart kötődési stílus, a szülők elha-
nyagoló-bántalmazó viselkedése a mentalizáció későbbi zavaraihoz vezethet (Bateman
és Fonagy, 2012). A mentalizációs készségek károsodásának egyik kifejeződése az ale-
xitímia, a saját érzelmi állapotok felismerésének, azonosításának és kifejezésének zava-
ra (Taylor, Bagby és Parker, 1997). Az alexitímia bizonyítottan összefügg a szülők hi-
deg, elhanyagoló, bántalmazó viselkedésével (Lumley, Mader, Gramzow és Papineau,
1996), valamint a pszichoterapeutától való félelemmel (Mallinckrodt, King és Coble,
1998). Ezek alapján feltételeztük, hogy a hideg-büntető szülői viselkedés az alexitími-
ás érzelemfeldolgozáson keresztül befolyásolhatja a hipnózisba való bevonódást, és a
hipnotikus fogékonysággal negatívan függ össze (Költő és Bányai, 2015). A Torontói
Alexitímia Skála (Cserjési, Luminet és Lénárd, 2007) alkalmazásával végzett korábbi
vizsgálatunk eredménye mindezt alátámasztja: az EMBU-val mért apai és anyai bünte-
tés összefügg a hipnózisbeli negatív érzelmekkel és a félelemmel a hipnotizőr haragjá-
tól, rosszallásától, de ezt a kapcsolatot részben vagy egészben a saját érzelmek azonosí-
tásának nehézsége mediálja (a mediáció mértéke 16,9–61,6% közötti) (Költő, 2015). Szülői nevelési stílus és hipnotikus fogékonyság Ennek önmagában korrektív jellege lehet, főleg akkor, ha az alany valódi szülei
részéről a gyakori büntetést és az érzelmi melegség hiányát idézi föl. Ugyanakkor az, Unauthenticated | Downloaded 10/24/24 05:46 AM UTC A felnőttkori hipnotikus fogékonyság fejlődési és szocializációs meghatározói 75 hogy a hipnózisbeli érzelmek és érzelmi viszonyulás egyes dimenziói a szülők meleg
és büntető viselkedésével egyszerre összefüggenek, arra utal, hogy a hipnotizőrrel való
kapcsolatban a szülők iránti ambivalens érzelmek is feléledhetnek. TOVÁBBI KUTATÁSI IRÁNYOK:
A HIPNÓZIS MINT KÖLCSÖNÖS MENTALIZÁCIÓ Az elmeteóriát és mentalizációt eddig kevéssé vették figyelembe a hipnózis fejlődési
szempontú értelmezésében (Kihlstrom, 2008), noha a korábbiakban említett kutatási
eredmények és elméleti megfontolások is arra utalnak, hogy a jobb mentalizációs kész-
ség erősebb hipnotikus fogékonysággal jár együtt (Bonshtein, 2012; Költő, 2015; Van-
denberg, 2002; valamint l. Varga tanulmányát a jelen tematikus számban, pp. 95–112.). Azok a személyek, akik jó mentalizációs készségekkel rendelkeznek, a személyközi
helyzetekben hatékonyabban képesek kiolvasni a partner mentális tartalmait, például
aktuális lelkiállapotát, érzelmeit, a helyzettel kapcsolatos hiedelmeit, elvárásait. Ez a
hipnózis kontextusában azt jelenti, hogy az alany pontosabban észleli, mit „vár el” tőle
a hipnotizőr; nem feltétlenül abban az értelemben, hogy az alany erősen hipnábilis
legyen, inkább hogy a hipnózisban való részvétel szokásosan milyen viselkedésjegyek-
kel társul. A jól mentalizáló hipnotizőr pedig az interakciós szinkronitás folyamatában
pontosabban észleli az alany pillanatnyi bevonódásának mértékét, és saját viselkedésé-
nek módosításával vagy gondosan megválasztott szuggesztiókkal befolyásolni, irányíta-
ni tudja azt. A hipnotizőr mentalizációs készségére vonatkozó adatokkal egyelőre nem
rendelkezünk. Viszont, ahogy korábban is említettük, az alexitímiás alanyok – akiknek Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 76 Költő András és mtsai saját érzelmi állapotaik felismerése és azonosítása, tehát önmentalizációja okoz nehéz-
séget – több negatív érzelmet (pl. félelmet és zavarodottságot) élnek át hipnózisban,
és jobban félnek a hipnotizőr negatív megítélésétől. Ennek az összefüggésnek a hát-
terében hideg-büntető szülői nevelésre vonatkozó emlékek álltak (Költő, 2015). Fontos, hogy további vizsgálatoknak vessük alá a mentalizációs készségek és a hip-
nózisba való bevonódás összefüggését (mind az alany, mind a hipnotizőr részéről). Ha
további eredmények is igazolják, hogy a mentalizáció összefügg a hipnózisba való erő-
sebb bevonódással, ennek a hipnoterápiára nézve is nagy jelentősége lehet. SZÜLŐKKEL KAPCSOLATOS EMLÉKEK ÉS HIPNOTERÁPIA Az az eredményünk, hogy a szülői büntetés és a hipnózisbeli negatív érzelmek, illet-
ve a hipnotizőrtől való félelem közötti összefüggést az alexitímiás érzelemfeldolgozás
mediálja, érzelmezhető úgy, hogy a gyakran büntetett gyermek a (szülő–gyermek kap-
csolatot felidéző) hipnózishelyzetben nagyobb ambivalenciát vagy félelmet él át, ami-
nek oka érzelmi zavarodottság és a hipnotizőr (vélt) elvárásainak való megfelelés le-
het. Azonban azt is láttuk, hogy szülői hidegség-büntetés a pozitív színezetű archaikus
bevonódással is összefügg, ami felveti annak lehetőségét, hogy az alany tudattalanul is
„jó szülőként” látja a hipnotizőrt. Talán épp ez teremt módot arra, hogy a hipnoterá-
piában a valódi szülőkkel kapcsolatos negatív emlékeket és a kötődési munkamodell
zavarait korrigálni tudjuk. Mély hipnózisban a két agyfélteke közötti munkamegosztás rugalmasabbá válik,
a agyféltekék működése jobban integrálódik és a váratlan, megszokottól eltérő inge-
rek automatikus feldolgozása a bal (verbális) agyféltekébe tevődik át. Erre vonatkozó
pszicho fiziológiai bizonyítékokat közöl a jelen tematikus számban Kasos és munkatársai
tanulmánya (pp. 79–93.). Ez az agyféltekei dominanciaeltolódás teszi lehetővé, hogy a
korábban kimondhatatlan, megfogalmazhatatlan – gyakran pszichoszomatikus tünetek-
be vagy betegségbe átfordított – élményeket a hipnoterápia folyamatában a kliens ver-
balizálni tudja (Bányai, 2015), ami révén a hipnózis az alexitímia oldásának eszköze is. j
y
p
Az alany által felidézett szülőkkel kapcsolatos emlékek, úgy tűnik, a hipnotizőrt
is „megérintik”. Varga és Kekecs (2014), illetve Kasos és munkatársai (a jelen tema-
tikus számban, pp. 79–93.) erős negatív korrelációt talált az alanyok által felidézett
(EMBU-val mért) szülői melegség és a hipnotizőrben a hipnózis során mért oxito-
cinfelszabadulás között. Azaz minél kevesebb melegséget idéz fel szülei visekedésével
kapcsolatban az alany, annál több oxitocin termelődik a hipnózis során a hipnotizőr
szervezetében. A szerzők ezt az eredményt a szociál-pszichobiológiai elméletbe illesz-
tik, és úgy értelmezik, hogy az alany implicit módon „magával hozza” korai kötődé-
si mintázatait a hipnózisba, a hipnotizőrben megfigyelt oxitocintermelődés pedig a
kapcsolatteremtés, stresszcsökkentés biológiai mutatója, ami szintén hozzájárulhat a
hipnoterápia korrektív szerepéhez. Az EMBU-faktorok és a hipnotizőr iránti archai-
kus bevonódás közötti összefüggések mintázata megerősíti ezt a feltételezést. gg
g
Láttuk azonban, hogy a gyakran büntetetett, hideg és szigorú légkörben felnevelt
gyerekek számára a hipnózis félelmetes vagy negatív élmény lehet. Kezdeti eredmé-
nyeink alátámasztják, hogy a hideg-büntető szülők gyermekei félelemtelibb attitűddel Unauthenticated | Downloaded 10/24/24 05:46 AM UTC A felnőttkori hipnotikus fogékonyság fejlődési és szocializációs meghatározói 77 viseltetnek a hipnózis iránt, és a meleg-szeretetteljes szülőket felidéző vizsgálati szemé-
lyeknél kevésbé hajlamosak arra, hogy önként jelentkezzenek hipnózisban való rész-
vételre (Költő, 2017). IRODALOM Arrindell, W. A., Sanavio, E., Aguilar, G., Sica, C., Hatzichristou, C., Eisemann, M., Recinos,
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Alto, CA: Consulting Psychologists Press. Winnicott, D. W. (1953). Transitional objects and transitional phenomena: A study of the fi rst
not-me possession. IRODALOM International Journal of Psychoanalysis, 34(2), 89–97. Unauthenticated | Downloaded 10/24/24 05:46 AM UTC 80 Költő András és mtsai DEVELOPMENTAL AND SOCIALISATIONAL DETERMINANTS
OF ADULT HYPNOTIC SUSCEPTIBILITY KÖLTŐ, ANDRÁS – JÓZSA, EMESE – BÁNYAI, ÉVA The hypnotherapeutic relationship between client and therapist bear many resemblances to the relationship
between child and parent. According to the social-psychobiological theory of hypnosis, adult hypnotic suscep-
tibility is infl uenced by neural development and socialization. Despite this fact, so far just a few studies in-
vestigated the developmental antecedents of adult hypnotic susceptibility. Similarly, the socialisation effects
were scarcely studied; the few studies in this area rather aimed to map childhood memories in a qualitative
manner. In this paper we summarise the results of these studies, then we present the investigations carried
out by our research team since 2008 in order to unfold the associations between recalled parental rearing
style and adult hypnotic response. The main conclusion of our cross-sectional studies that employed stan-
dardised quantitative measures is that cold-punishing parental style predicts negative emotions and fear
of the hypnotist’s negative appraisal in laboratory hypnosis sessions. This association is partially mediated
by alexithymic affective processing. These results inform the planning of hypnotherapy and reinforce the
therapeutic alliance. Keywords: parental rearing style, parental behaviour, hypnotizability, hypnotic susceptibility A cikk a Creative Commons Attribution 4.0 International License (https://
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A Robust Method for Automatic Panoramic UAV Image Mosaic
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Sensors
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Received: 1 February 2019; Accepted: 16 April 2019; Published: 22 April 2019 Abstract: This paper introduces a robust method for panoramic unmanned aerial vehicle (UAV) image
mosaic. In the traditional automatic panoramic image stitching method (Autostitch), it assumes that
the camera rotates about its optical centre and the group of transformations the source images may
undergo is a special group of homographies. It is rare to get such ideal data in reality. In particular,
remote sensing images obtained by UAV do not satisfy such an ideal situation, where the images
may not be on a plane yet and even may suffer from nonrigid changes, leading to poor mosaic
results. To overcome the above mentioned challenges, in this paper a nonrigid matching algorithm is
introduced to the mosaic system to generate accurate feature matching on remote sensing images. We also propose a new strategy for bundle adjustment to make the mosaic system suitable for the
UAV image panoramic mosaic effect. Experimental results show that our method outperforms the
traditional method and some of the latest methods in terms of visual effect. Keywords: image mosaic; nonrigid deformation; bundle adjustment; feature matching
Received: 1 February 2019; Accepted: 16 April 2019; Published: 22 April 2019 A Robust Method for Automatic Panoramic UAV
Image Mosaic Jun Chen 1,2,3
, Quan Xu 4, Linbo Luo 4,∗, Yongtao Wang 1,2 and Shuchun Wang 1,2
1
School of Automation, China University of Geosciences, Wuhan 430074, China;
chenjun71983@163.com (J.C.); ytwang@cug.edu.cn (Y.W.); wangscgt@126.com (S.W.)
2
Hubei Key Laboratory of Advanced Control and Intelligent Automation for Complex Syste
Wuhan 430074, China 3
Hubei Provincial Key Laboratory of Intelligent Robot, Wuhan Institute of Technology, Wuhan 430073, C
4
School of Mechanical Engineering and Electronic Information, China University of Geosciences,
Wuhan 430074, China; 1816312309@163.com 3
Hubei Provincial Key Laboratory of Intelligent Robot, Wuhan Institute of Technology, Wuhan 430073, China
4
School of Mechanical Engineering and Electronic Information, China University of Geosciences,
Wuhan 430074, China; 1816312309@163.com sensors sensors sensors Sensors 2019, 19, 1898; doi:10.3390/s19081898 www.mdpi.com/journal/sensors 1. Introduction Unmanned aerial vehicle (UAV) remote sensing is a low-altitude technology, which has become
an important means of information acquisition. UAV has been widely used in various ground survey
applications due to its advantages of high acquisition speed, convenient operation, good safety, and low
investment. However, a UAV image has a small view of scene due to the low flying altitude and
limited focal length of the camera; thus, capturing a relatively complete target area becomes difficult. Multiple images of the same target should be combined by technical means to obtain a complete scene
of the desired target. The purpose of the image mosaic is to combine multiple images with overlapping
fields to form a panoramic image [1–3]. Traditional image stitching [4] includes feature matching, image matching, bundle adjustment,
automatic panorama straightening, gain compensation, and multiband blending [5,6]. This system
may appear perfect, but several errors emerge when images contain a large amount of nonideal
data. For example, when UAV images are stitched using this system, ghosting and even incorrect
matches may arise. Several efforts have been exerted to reduce these errors, for example, seam cutting
methods [7,8] optimize pixel selection among the overlapping images to minimize visible seams,
whereas Laplacian pyramid blending [4,5] and Poisson image blending [9] minimize blurring due
to misalignments or exposure differences. Nevertheless, the effect remains unsatisfactory, and the
problem may be even further severe when scenes are captured by UAVs involving nonrigid changes. Sensors 2019, 19, 1898; doi:10.3390/s19081898 www.mdpi.com/journal/sensors Sensors 2019, 19, 1898 2 of 17 Two major challenges exist in the traditional stitching system when applying to UAV image mosaics. On the one hand, the geometrical relationship between UAV images is often complex due to variations
in ground relief, changes in imaging viewpoints, and shooting at low altitudes, where image pairs
cannot be accurately matched using a parametric transformation model (e.g., affine or homography)
as in most existing methods. Image matching is a critical prerequisite of image mosaic, which aims to
overlay two images of the same scene geometrically [10–13]. On the basis of the type of data given,
image matching can be divided into rigid and nonrigid. In traditional mosaic matching, the implicit
constraint is that the given image is rigid. However, for UAV images, nonrigid matching is crucial
because these images often contain local deformation, which cannot be solved by rigid matching. 1. Introduction One the other hand, remote sensing images captured by UAVs are from a low altitude, and the resulting
images cannot be approximated as a plane. In the traditional stitching system, single planar perspective
transform (homography) [14] is used with bundle adjustment for optimization, and this usage leads to
ghosting errors. To overcome such limitation, Chin et al. proposed as-projective-as-possible (APAP)
image stitching [15], which meshes images, and each grid is aligned with a homography, which is
suitable for UAV image mosaic. However, the method has a prerequisite that the images have been
accurately aligned. y
g
To address the aforementioned challenges, we introduce a nonrigid matching algorithm on
the basis of motion field interpolation, namely, vector field consensus (VFC) [16], to the mosaic
system for generating accurate feature matching on remote sensing images. In bundle adjustment [17],
we propose a new strategy that improves the original relationship of homography conversion and
use the homography response to perform bundle adjustment, which is robust to our image mosaic. The experiments on different sets of UAV images reveal the superiority of our method. 2. Related Work This section briefly reviews the background material on which our work is based, including
nonrigid matching algorithms, local transformation descriptions, bundle adjustment, and aerial
image mosaic. 2.1. Nonrigid Image Matching The image matching phase is a key step in image stitching and many other computer vision
tasks [18–21]. In the matching phase, the capability to minimize registration errors plays an important
role in the subsequent steps of the system. Here, the matching problem aims to align the overlapping
regions of two images pixel by pixel. Obtaining a good stitching effect requires a dense pixel-by-pixel
matching of the overlapping regions. When the unknown complex relationship between images cannot
be accurately modeled by a specific model, especially when a nonrigid change occurs in the image,
achieving accurate pixel-by-pixel registration is difficult. In general, the SIFT [22] feature points are extracted to register the relationship between images. Sparse feature point matching is used to guide dense pixel-by-pixel registration. Coping with a large
proportion of feature points is possible with a general linear model, such as RANSAC [23], and its
variants, including MLESAC [24], LO-RANSAC [25], and PROSAC [26], to estimate the spatial
transformation between images. This approach seems to be a good solution. However, this series of
algorithms relies on specific geometric parameter models. Images with nonrigid changes can no longer
be applied because the transformation cannot be modeled parametrically. Li and Hu [27] proposed an identifying correspondence function (ICF) algorithm based on
nonparametric models to achieve nonrigid matching, but its matching precision drops sharply when
many outliers are present Another strategy for solving the matching problem is to estimate the
corresponding matrix of the two point sets in combination with the parameter or nonparametric
geometric constraints. The method based on estimating the corresponding matrix jointly estimates the
correspondence between the point sets and transformation, compared with the previous independent
estimated point correspondence and transformation. Representative methods for finding inter-image Sensors 2019, 19, 1898 3 of 17 correspondence matrices, such as iterated closest point (ICP) [28], coherence point drift (CPD) [29] and
its variant [30], and locality preserving matching (LPM) [31,32], have also been proposed to match
nonrigid cases, but such methods generally cannot tolerate excessive outliers. Moreover, for robustness
purposes, such algorithms typically impose penalties on points that are not matched. The point set
matching problem can also be solved by solving a graph matching problem [33]. Such methods
construct an affinity matrix between point sets and then perform spectral analysis to obtain ordered
features of the point set. 2.2. Local Transformation Adaptive as natural as possible [41]
also uses global similarity transformations to correct shapes, but it can adaptively determine angles to
correct image shapes efficiently. As a homography cannot efficiently align the pixels of the overlapping part, Liu et al. [37]
proposed a content-preserving warp that minimizes the registration error and maintains the stiffness
of the scene. However, in image stitching, large differences are observed in the rotation and translation
between different views. The interpolation of this method is not flexible enough due to the constraint
of stiffness. Gao et al. [38] divided a scene into distant and ground planes that sweep out from
the camera’s location; they aligned the two planes with two homographies and then blended these
to align the images. This method is more flexible than using a single homography, but it remains
unsuitable for complex scenarios. Lin et al. [39] used a smoothly varying affine warp to align the
image. The local deformability and alignment capability were strong and the method can handle
parallax. However, the use of affine regularization is appropriate for interpolation, which may not be
optimal for extrapolation, and the affinity is insufficient for completing the perspective transformation. Chin et al. [15] proposed a transformation that is as projected as possible by using an algorithm that
is as transformable as possible. The purpose of the deformation is to perform a global perspective
transformation and allow local non-projection transformations by sampling the local transformation
model for image mosaic. The local model has high degree of freedom and is flexible, which can handle
local transformation and reduce ghosting problems. Subsequent methods use global similarity to
mitigate projection distortion in nonoverlapping regions, such as shape-preserving half-projective [40]. This approach adds constraints, combined with homography and similarity transformations, corrects
the shape of the stitched image, and reduces projection distortion. Adaptive as natural as possible [41]
also uses global similarity transformations to correct shapes, but it can adaptively determine angles to
correct image shapes efficiently. 2.2. Local Transformation The conversion relationship of the overlapping image alignment is estimated after acquiring the
image matching relationship. Then, the aligned images are combined into a common plane. In the
traditional stitching method, a homography is used to represent this conversion relationship. The goal
is to minimize alignment errors between overlapping pixels by uniform variations. A homography is
a commonly used transformation because it maintains the most flexible transformation of all lines,
and the resulting panorama does not produce considerable distortion. The homography has excellent
effect for all images in a plane. However, for the UAV images tackled in this study, this ideal condition
typically does not hold. yp
y
As a homography cannot efficiently align the pixels of the overlapping part, Liu et al. [37]
proposed a content-preserving warp that minimizes the registration error and maintains the stiffness
of the scene. However, in image stitching, large differences are observed in the rotation and translation
between different views. The interpolation of this method is not flexible enough due to the constraint
of stiffness. Gao et al. [38] divided a scene into distant and ground planes that sweep out from
the camera’s location; they aligned the two planes with two homographies and then blended these
to align the images. This method is more flexible than using a single homography, but it remains
unsuitable for complex scenarios. Lin et al. [39] used a smoothly varying affine warp to align the
image. The local deformability and alignment capability were strong and the method can handle
parallax. However, the use of affine regularization is appropriate for interpolation, which may not be
optimal for extrapolation, and the affinity is insufficient for completing the perspective transformation. Chin et al. [15] proposed a transformation that is as projected as possible by using an algorithm that
is as transformable as possible. The purpose of the deformation is to perform a global perspective
transformation and allow local non-projection transformations by sampling the local transformation
model for image mosaic. The local model has high degree of freedom and is flexible, which can handle
local transformation and reduce ghosting problems. Subsequent methods use global similarity to
mitigate projection distortion in nonoverlapping regions, such as shape-preserving half-projective [40]. This approach adds constraints, combined with homography and similarity transformations, corrects
the shape of the stitched image, and reduces projection distortion. 2.1. Nonrigid Image Matching These methods include dual decomposition (DD) [34], spectral matching
(SM) [35], and graph shift (GS) [36]. However, the computational complexity of these methods is
typically high, which cannot be applied to address large-scale real-time matching tasks. The VFC
algorithm proposed by Ma et al. is also based on the nonparametric model [16]. This algorithm
converts the point set matching problem into vector field interpolation and can efficiently generalize
sparse matching to dense matching, which is suitable for our UAV image matching. 2.4. Aerial Image Mosaic The theme of this study is the stitching of UAV images. In this part, we introduce the related work
in aerial image mosaic. Park et al. [44] proposed an algorithm for hierarchical multi-level image mosaicing for the
autonomous navigation of UAV. The algorithm prevents the error accumulation propagated along the
frames by incrementally building a long-duration mosaic on the fly, which is hierarchically composed
of short-duration mosaics. The real-time processing requirements in autonomous navigation are
fulfilled. In particular, the system can automatically adapt to the scene changes of the images
to be spliced and can automatically select suitable methods for feature selection. This point is
important in the autonomous navigation of UAV. The proposed system is a causal system and is
suitable for real-time application. Ghosh et al. [45] proposed a spatial domain-based super-resolution
mosaicing system and presented its evaluation results. This algorithm combines image mosaicing
and super-resolution-reconstruction algorithms and has the characteristics of robustness and simple
calculation. The authors used spatial domain-based super resolution and made it practical in real-time
applications, such as surveillance and remote sensing. Ghosh et al. also introduced indicators for the
quantitative evaluation of image mosaicing in multiple scene categories [46], which contributed to the
defects in this aspect. 2.3. Bundle Adjustment Given a set of overlapping images, we aim to project all images onto a common surface. This process will inevitably accumulate errors. To obtain an improved stitching effect, we must
simultaneously refine these errors. The current method [4,42] optimizes the focal length of all views
and the relative rotation of the camera pose through bundle adjustment and then aligns the series
of images. The bundle adjustment can be based on the projection of all points in the image while Sensors 2019, 19, 1898 4 of 17 extracting the 3D point coordinates describing the scene structure, the relative motion parameters,
and the optical parameters of the camera. Bundle adjustment is often used in feature-based 3D scene reconstruction algorithms as the
final step. This method is based on 3D structure and perspective parameters (e.g., camera position,
orientation, inherent calibration, and radial distortion). Providing an initial estimate, the bundle
adjustment simultaneously refines motion and structural parameters by minimizing projection errors
between observed and predicted image points. The best reconstruction effect is obtained under the
assumption that the obtained image features contain noises. The ultimate goal of the bundle adjustment is to reduce the positional projection transformation
(re-projection) error between the points of the observed and reference images (predicted image) by
using a least squares algorithm. The most successful strategy is Levenberg-Marquardt algorithm [43],
which is easy to implement and can quickly converge on a wide range of initial estimates. 3. Methodology Our task is to place a set of UAV images together in a system to obtain a mosaic panorama. The previous section indicates that the traditional stitching system cannot produce good results
because of the particularities of UAV images. Hence, we perform modifications to the system to make
it robust to UAV images. 3.1. Nonrigid Feature Matching Using Vector Field Consensus We can write the
complete-date log posterior as follows [48]: Q(θ, θold) = −1
2σ2 ∑N
n=1 pn∥yn −f(xn)∥2 + ln γ ∑N
n=1 pn
+ ln(1 −γ) ∑N
n=1(1 −pn) −D
2 ln σ2 ∑N
n=1 pn −λ
2 ∥f∥2,
(4) (4) where pn = P(zn = 1|xn, yn, θold). The value may be maximized by regarding zn as missing data from
the mixture model. E-step: Denote P = diag(p1, . . . , pN), where the responsibility pn can be computed by applying
the Bayes rule: pn =
γe−∥yn−f(xn)∥2
2σ2
γe−∥yn−f(xn)∥2
2σ2
+ (2πσ2)
D
2 (1 −γ)/a
,
(5) (5) The posterior probability pn is a soft decision, which indicates that the degree of sample n agrees
with the current estimated f. ld The posterior probability pn is a soft decision, which indicates that the degree of sample n agrees
with the current estimated f. ld M-step: We determine the revised parameter estimate θnew as follows: θnew = arg maxθ Q(θ, θold). By using the derivative of Q(θ) with respect to variance σ2 and the mixing coefficient γ and setting
them to zero, we obtain σ2 = ∑N
n=1 pn∥yn −f(xn)∥2
D ∑N
n=1 pn
,
(6)
∑N σ2 = ∑N
n=1 pn∥yn −f(xn)∥2
D ∑N
n=1 pn
,
(6)
γ = ∑N
n=1 pn
N
. (7) (6) D ∑n=1 pn
γ = ∑N
n=1 pn
N
. (7) γ = ∑N
n=1 pn
N
. (7) (7) To complete the EM algorithm, the vector field f should be estimated in the M-step, which can be
obtained by solving a regularization problem [16]. 3.2. Local Wrap and Bundle Adjustment 3.1. Nonrigid Feature Matching Using Vector Field Consensus In the feature matching stage, the SIFT descriptor is used to conduct initial feature matching
and obtain the corresponding relationship of feature points between images. UAV images may have
nonrigid changes. Thus, we introduce a nonrigid matching algorithm based on VFC [16]. Given a set of putative SIFT matches S = {(xn, yn)}N
n=1, our purpose is to distinguish false
matches (outliers) from the correct matches (inliers) and estimate the transformation f to fit the inliers
efficiently, where f ∈H. We assume that f is nonrigid and H is a reproducing kernel Hilbert space
(RKHS) [47]. We assume that the noise of inliers is Gaussian with zero mean and uniform standard deviation
σ. The outliers are uniformly distributed in the image domain, and the distribution is assumed to be
uniform 1/a, where a is a constant. Let γ be the percentage of inliers that we do not know in advance. Thus, the likelihood is a mixture model of distributions for inliers and outliers: Sensors 2019, 19, 1898
5 of 17 Sensors 2019, 19, 1898 5 of 17 p(Y | X, θ) = ∏N
n=1 p(yn, zn|xn, θ) = ∏N
n=1(
γ
(2πσ2)D/2 e−∥yn−f(xn)∥2
2σ2
+ 1−γ
a ),
(1) (1) where θ = {f, σ2, γ} is the set of unknown parameters. We associate sample n with a latent variable
zn ∈{0, 1}, where zn = 1 represents Gaussian distribution and zn = 0 represents uniform distribution. Considering the smoothness constraint, the prior of f can be written as p(f) ∝e−λ
2 ∥f∥2
H. (2) (2) By applying Bayes rule, p(θ|X, Y) ∝p(Y|X, θ)P(f), we estimate θ as an MAP solution, i.e., θ∗:
θ∗= arg max
θ
p(Y|X, θ)p(f),
(3) By applying Bayes rule, p(θ|X, Y) ∝p(Y|X, θ)P(f), we estimate θ as an MAP solution, i.e., θ∗: y pp y
g
y
, p( | ,
)
p( | , ) ( ),
,
,
θ∗= arg max
θ
p(Y|X, θ)p(f),
(3) θ∗= arg max
θ
p(Y|X, θ)p(f),
(3) (3) where θ∗corresponds to the estimate of the true θ. Thus, the transformation f can be obtained. We introduced and solved a latent variable zn by using the EM algorithm. We can write the
complete-date log posterior as follows [48]: where θ∗corresponds to the estimate of the true θ. Thus, the transformation f can be obtained. We introduced and solved a latent variable zn by using the EM algorithm. 3.2. Local Wrap and Bundle Adjustment After matching two images, the source image can be projected onto the target image through
homography. However, the errors accumulate and magnify when stitching multiple images, especially
in multiple overlapping regions [49]. We can simultaneously optimize projection functions to
register panoramic images under the framework of minimum mean square error and reduce the
cumulative error. The first step when stitching multiple images is to find the reference plane [50,51] to which
all images are projected through a basic homographic warp. We select an image from the input Sensors 2019, 19, 1898 6 of 17 image as the reference plane, as described in [4]. All overlapped images are identified through the
feature matching of the panoramic image. We select the image with the most overlapped parts as
the reference plane so that other images can be projected to the reference plane by a homographic
warp. The traditional method uses direct linear transformation (DLT) [52] to calculate the single
response between images (which requires f our pairs of matched points). The registration of UAV
images cannot be changed simply by a single response. Inspired by Chin et al. [15], the registration
error caused by a single response was effectively reduced by differentiating the image, and each
differential corresponds to a basic homographic matrix. Given two overlapping images I and I
′ and their matched points xi = (xi, yi)T, yi = (x
′
i, y
′
i)T,
i = 1, . . . , n, the global transformation between two images can be estimated by ey ∼Hex,
(8) ey ∼Hex, (8) where ex denotes x in homogeneous coordinates, ∼indicates the equality up to scale, and H is the
global homography that is a 3 × 3 matrix. In 2D projective warp, DLT is a basic method for estimating H from a set of noisy point matches. We vectorize H into a vector h. Let ai be the two rows of the matrix for two matched points. Given an
estimate h, the quantity ∥aih∥is the algebraic error. DLT minimizes the sum of squared algebraic errors: ˆh = arg minh ∑N
i=1 ∥aih∥2. (9) (9) By stacking vertically ai for all i into matrix A of size 2N × 9, the problem can be rewritten as By stacking vertically ai for all i into matrix A of size 2N × 9, the problem can be rewritten as ˆh = arg minh ∑N
i=1 ∥Ah∥2. 3.2. Local Wrap and Bundle Adjustment (10) (10) The solution is the least significant right singular vector of A. Given the estimated H (reconstructed
from ˆh), in the 2D projective warp, an arbitrary pixel x∗in the source image I is warped to the position
y∗in the target image I
′ by ey∗∼Hex∗,
(11) (11) For UAV images, global homography cannot efficiently match two images. In this case,
we introduce a location-dependent homography. ey∗∼H∗ex∗. (12) (12) Each pixel x∗corresponds to a location-dependent homography H∗, where x∗is estimated from
the weighted problem
N
i h∗= arg minh ∑N
i=1 ∥wi∗aih∥2. (13) (13) We can define the scalar weights {wi∗}N
i=1 as wi
∗= exp(−∥x∗−xi∥2/δ2),
(14) (14) where δ is a scale parameter. The exact solution steps are the same as before. k
K where δ is a scale parameter. The exact solution steps are the same as before. k
K
k When we have a set of location-dependent homographies {Hk∗}K
k=1, where each Hk∗corresponds
to a pair of matching points in two images, we should consider the cumulative error during image
mosaic. We minimize the cost to minimize the transfer error of all correspondences: E∗(Θ) =
N
∑
i=1
wi∗
∑K
k=1 ξik
K
∑
k=1
ξik∥xk
i −f (pi, Hk
∗)∥2,
(15) (15) where Θ = [Hk
1, . . . , Hk∗, p1, . . . , pN] and f (p, H) is the project warp defined as Sensors 2019, 19, 1898 7 of 17 f (p, H) =
r1[pT1]T
r3[pT1]T
r2[pT1]T
r3[pT1]T
T
,
(16) (16) where r1, r2 and r3 are the three rows of homography H. We introduce a parameter ξik ∈{0, 1},
where ξik = 1 indicates that correspondence exists; otherwise, ξik = 0. where r1, r2 and r3 are the three rows of homography H. We introduce a parameter ξik ∈{0, 1},
where ξik = 1 indicates that correspondence exists; otherwise, ξik = 0. 4. Experimental Results We compare our method with the traditional image mosaic method. In the experiment, the UAV
images have nonrigid changes, which cannot be treated as a plane. In the following, we initially report
the feature matching results and then provide the image mosaic results. 4.1. Remove Mismatches on Nonrigid Image Pairs In stitching remote sensing images, a nonrigid matching algorithm must be added because of
the high mismatching rate of remote sensing images. The false matching points of UAV images can
be removed effectively by adding the VFC algorithm. To evaluate the feature matching performance
of VFC, we use three other widely used methods for comparison, namely, RANSAC [23], ICF [27],
and GS [36]. We select two representational image pairs [32] that have local nonrigid distortion
for evaluation, where the ground truth feature matches are supplied by the dataset. Two common
evaluation indicators, namely, precision and recall, are used to measure the performance of our
algorithm [53]. The precision rate is defined as the ratio of the number of retained inlier points to the
total number of initial feature points, and the recall rate is defined as the ratio of the number of retained
inlier points to the number of initial inlier points. Experimental results show that this method is better
than other methods when performing nonrigid registration. Figure 1 and Table 1 shows the results. VFC can achieve better matching performance on the UAV images than other state-of-the-art methods. Figure 1. Experimental results on two typical image pairs of nonrigid objects. The lines indicate
mismatch removal results (blue = true positive, green = false negative, red = false positive). For visibility,
in the image pairs, only 50 randomly selected correspondences are presented, and the true negatives
are not shown. Figure 1. Experimental results on two typical image pairs of nonrigid objects. The lines indicate
mismatch removal results (blue = true positive, green = false negative, red = false positive). For visibility,
in the image pairs, only 50 randomly selected correspondences are presented, and the true negatives
are not shown. Figure 1. Experimental results on two typical image pairs of nonrigid objects. The lines indicate
mismatch removal results (blue = true positive, green = false negative, red = false positive). For visibility,
in the image pairs, only 50 randomly selected correspondences are presented, and the true negatives
are not shown. Figure 1. Experimental results on two typical image pairs of nonrigid objects. The lines indicate
mismatch removal results (blue = true positive, green = false negative, red = false positive). For visibility,
in the image pairs, only 50 randomly selected correspondences are presented, and the true negatives
are not shown. Table 1. 4.2. Overall Differentiation and Local Wrap In projection transformations, the traditional method (Autostitch) [4] completes the transformation
by using a homography matrix. Parallax changes in the image will bring negative effects. To solve
these problems, we adopt the idea of differentiation. This method can tolerate the parallax in the image. We present a simple example in Figure 2 to illustrate the role of this idea in projection transformation. Local and global warps will have different effects in the stitching, especially when the two images
are not on one plane (the two views differ by a rotation and translation). In Figure 2, a homography
matrix can efficiently retain the rigid change of the image, but it is bound to leave matching points. Instead, our differentiation method can include most (even all) of the match points. Although the
image stiffness may be sacrificed, the effect is acceptable. We also perform the experiment, and Figure 3
shows the results. In Figure 3, strictly aligning these images through the traditional method is difficult. Ghosts in the mosaic image are evident, because a homography matrix fails to match the objects in the
two images efficiently. Our method can greatly improve the situation. The image in the third row of Figure 3 is
our mosaic result with evidently few ghosts. White boxes are used to highlight the ghostly parts. Moreover, we compare the difference in the effects of the two strategies. Our method is improved with
local homography. Figure 2. To generate a 1D analogy of image stitching, a set of 1D matches {xn, yn}N
n=1 are generated
by projecting a 2D point cloud onto two 1D image “planes”. Here, the two views differ by a rotation
and translation. The first indicates that the match points are warped by a global homography and it is
unable to model the local deviations of the data. The second indicates that the match points are warped
by some local homography and it interpolates the local deviations flexibly. Figure 2. To generate a 1D analogy of image stitching, a set of 1D matches {xn, yn}N
n=1 are generated
by projecting a 2D point cloud onto two 1D image “planes”. Here, the two views differ by a rotation
and translation. The first indicates that the match points are warped by a global homography and it is
unable to model the local deviations of the data. 4.1. Remove Mismatches on Nonrigid Image Pairs The precision (P) and recall (R) results of different methods on the image pairs in Figure 1. The initial inlier percentage is 37.27% and 42.39% on the two image pairs, respectively. RANSAC [23]
ICF [27]
GS [36]
VFC
(P, R)
(99.66%, 97.67%)
(98.98%, 91.76%)
(99.59%, 81.51%)
(99.67%, 98.02%)
(P, R)
(100.0%, 97.07%)
(99.32%, 92.86%)
(100.0%, 94.06%)
(100.0%, 97.62%) Sensors 2019, 19, 1898 8 of 17 4.2. Overall Differentiation and Local Wrap The second indicates that the match points are warped
by some local homography and it interpolates the local deviations flexibly. Figure 2. To generate a 1D analogy of image stitching, a set of 1D matches {xn, yn}N
n=1 are generated
by projecting a 2D point cloud onto two 1D image “planes”. Here, the two views differ by a rotation
and translation. The first indicates that the match points are warped by a global homography and it is
unable to model the local deviations of the data. The second indicates that the match points are warped
by some local homography and it interpolates the local deviations flexibly. Differentiation is robust to local nonrigid transformation of images and can solve the problem
that an image is not on a plane. In this way, the problem of local projection mismatch can be reduced
(reducing the ghosting errors in image mosaic), and the performance of the subsequent bundle
adjustment can be improved. We also conduct a series of experiments to verify the effect of our method. We compare our method not only with the traditional method (Autostitch) [4] but also with other
state-of-the-art methods, including AANAP [41], SPHP [40], and parallax tolerant (PT) [54]. 9 of 17 9 of 17 Sensors 2019, 19, 1898 Figure 3. Mosaic results. The first row is the two images to be stitched together. The second row is two
stitched images by global homography and local homography, respectively. White boxes highlight the
ghostly parts. Figure 3. Mosaic results. The first row is the two images to be stitched together. The second row is two
stitched images by global homography and local homography, respectively. White boxes highlight the
ghostly parts. The results are reported in Figures 4–6. Red boxes highlight the obvious parts of the ghost effect
in the mosaic results. We enlarge the area in red boxes to make results clearer. Results show that our
method is robust for stitching UAV images and can get better results than the competitors. The mosaic
of panoramic image is based on the mosaic of two images. We can achieve improved results here, which
alleviate the pressure of subsequent panorama stitching. However, our results still have shortcomings,
and the ghosting of some parts cannot be completely eliminated, which can be further improved
during bundle adjustment. Figure 4. Qualitative comparison on UAV image mosaic. The first row is the two images to be
stitched. 4.2. Overall Differentiation and Local Wrap The second and third rows are the stitching results of Autostitch [4], AANAP [41], SPHP [40],
and PT [54]. Our result is shown in the last row. Red boxes highlight the ghostly parts. Figure 4. Qualitative comparison on UAV image mosaic. The first row is the two images to be
stitched. The second and third rows are the stitching results of Autostitch [4], AANAP [41], SPHP [40],
and PT [54]. Our result is shown in the last row. Red boxes highlight the ghostly parts. 10 of 17 10 of 17 Sensors 2019, 19, 1898 Figure 5. Qualitative comparison on UAV image mosaic. The first row is the two images to be
stitched. The second and third rows are the stitching results of Autostitch [4], AANAP [41], SPHP [40],
and PT [54]. Our result is shown in the last row. Red boxes highlight the ghostly parts. Figure 5. Qualitative comparison on UAV image mosaic. The first row is the two images to be
stitched. The second and third rows are the stitching results of Autostitch [4], AANAP [41], SPHP [40],
and PT [54]. Our result is shown in the last row. Red boxes highlight the ghostly parts. Figure 6. Qualitative comparison on UAV image mosaic. The first row is the two images to be
stitched. The second and third rows are the stitching results of Autostitch [4], AANAP [41], SPHP [40],
and PT [54]. Our result is shown in the last row. Red boxes highlight the ghostly parts. The Effect of Nonrigid Changes and Parallax
In this section, we will analyze how the mentioned challenges (such as nonrigid changes cause
ground relief, and parallax caused by changes in imaging viewpoints) affect the mosaic result. Figure 5. Qualitative comparison on UAV image mosaic. The first row is the two images to be
stitched. The second and third rows are the stitching results of Autostitch [4], AANAP [41], SPHP [40],
and PT [54]. Our result is shown in the last row. Red boxes highlight the ghostly parts. Figure 6. Qualitative comparison on UAV image mosaic. The first row is the two images to be
stitched. The second and third rows are the stitching results of Autostitch [4], AANAP [41], SPHP [40],
and PT [54]. Our result is shown in the last row. Red boxes highlight the ghostly parts. Figure 6. Qualitative comparison on UAV image mosaic. 4.2. Overall Differentiation and Local Wrap The first row is the two images to be
stitched. The second and third rows are the stitching results of Autostitch [4], AANAP [41], SPHP [40],
and PT [54]. Our result is shown in the last row. Red boxes highlight the ghostly parts. 4.3. The Effect of Nonrigid Changes and Parallax 4.3. The Effect of Nonrigid Changes and Parallax In this section, we will analyze how the mentioned challenges (such as nonrigid changes caused
by ground relief, and parallax caused by changes in imaging viewpoints) affect the mosaic result. Sensors 2019, 19, 1898 11 of 17 Some experiments show the effect of those challenges to the mosaicing results. In Figure 4,
nonrigid changes are observed in the ground. In the registration stage, the traditional method cannot
efficiently align these nonrigid changes and it is easy to form many mismatches. The large number of
small stones in the scene has formed a ghost because they are not efficiently matched. We enlarged
the scene in the red boxes. Results are displayed next to the mosaicing results. Our method can
improve the ghost effect. Figures 5 and 6 show the images obtained in two different scenes, but they
have a certain parallax. In Autostitch, the overlapping areas will be misaligned when the parallax
image is projected onto a common plane. There are some unsatisfactory areas in the mosaicing result. In Figure 5, the ground lines in the red boxes are clearly wrong in Autostitch. Such a mosaic result is
not what we want to see. Other advanced methods can almost alleviate these ghosts. Our methods can
acquire better results than these methods. Our results are clear and the line is still correct. In Figure 6,
there are duplicate parts about cars and roads in the red boxes. The stitching results in the last row are
better than the rest images. Our method is more suitable for such parallax scene. At the same time, we choose some sequential images without parallax and nonrigid changes to
carry out the experiment. Experimental results are shown in Figure 7. We can see that the results of
Autostitch are almost identical to the results of our approach. In the Figure 8, these source images
have some parallax and nonrigid changes. The result of Autostitch has errors, where the challenges
mentioned above lead to these errors. Figure 7. Experimental results of panoramic stitching. 4.2. Overall Differentiation and Local Wrap The source images are sequential images
captured from left to right without nonrigid changes and parallax. The first row is the results of
Autostitch. The second row is the results of our method. Figure 7. Experimental results of panoramic stitching. The source images are sequential images
captured from left to right without nonrigid changes and parallax. The first row is the results of
Autostitch. The second row is the results of our method. 4.4. The Effect of Noise and Blurriness When we acquire UAV images, introducing noise and blur into the images is inevitable. Therefore,
a good system must resist the intrusion of noise and blur. We also conduct experiments on this aspect. Each of these images are convolved with a Gaussian blurring kernel of SD = 1.2 (standard deviation). The images are also contaminated by additive white Gaussian noise with zero mean and variance
0.0004. We use these two strategies to evaluate the behavior of our mosaicing algorithm in the presence
of blur or noise. Figure 9 shows the results. Noise and blur in general have no effect on our system, because the
ghost in the mosaic image is not enhanced or enlarged. Thus, the algorithm can still work efficiently
and remove mismatched feature points further accurately in the stage of removing mismatches. In the
registration stage, the image is unaffected by noise and blur. The algorithm can precisely align the
pixels and then project the image to a common plane. Sensors 2019, 19, 1898 12 of 17 Figure 8. Experimental results of panoramic stitching. The first row is the image to be stitched together. The second row is the results of Autostitch and our method. Red boxes highlight the ghostly parts. The last two rows are the details of the red boxes. Figure 8. Experimental results of panoramic stitching. The first row is the image to be stitched together. The second row is the results of Autostitch and our method. Red boxes highlight the ghostly parts. The last two rows are the details of the red boxes. 4.5. Bundle Adjustment and Panoramic Stitching 4.5. Bundle Adjustment and Panoramic Stitching At this stage, our method has the same capability as Autostitch and can achieve the mosaic in
a group of arbitrary images. Hence, the source image can be any image of a scene. In Autostitch,
only one global homography is used for stitching. Thus, the bundle adjustment optimizes the relative
rotation and homographies between sets of overlapping images. This case only requires average
intensity blending of the aligned image. Results show that our stitching system has a better effect than
the traditional stitching system, and the ghosting effect is considerably reduced. Figure 8 presents
the results. As shown in Figures 3 and 8, when the traditional method (Autostitch) [4] mosaics the panorama
image, the error is cumulatively enlarged. When Autostitch mosaics two images, the ghosting effect is
not evident. When images are added to the system, the bundle adjustment cannot efficiently remove
the accumulated error. By contrast, our method can efficiently remove the accumulated error during
bundle adjustment. In Figures 10 and 11 [55], there are some images of arbitrary viewpoints and we
also compare the splicing results. In Figure 10, errors are displayed when Autostitch stitches source
images. This indicates that the method cannot process these images, so we do not report the result
of Autostitch, and only give the splicing result of our method. In Figure 11, The Autostitch stitching
results still have some misaligned areas. We have highlighted it in red boxes and zoomed in on the side. 13 of 17 13 of 17 Sensors 2019, 19, 1898 Figure 9. The first line is the blurred source image. The second line is the mosaic result of the unblurred
source image and the mosaic result of the blurred source image. The third line is the noisy source
image. The fourth line is the mosaic result of the source image without noise and the mosaic result of
the source image with noise. White boxes highlight the ghostly parts. Figure 10. Experimental results of panoramic stitching. The first and second rows are the images to be
stitched together. The third row is the result of our method. Autostitch cannot process these images. Our method sacrifices the stiffness of the stitching image. The effect is good when stitchi
o images. However, distortion is observed in the result when stitching multiple images. T Figure 9. The first line is the blurred source image. 4.5. Bundle Adjustment and Panoramic Stitching The second line is the mosaic result of the unblurred
source image and the mosaic result of the blurred source image. The third line is the noisy source
image. The fourth line is the mosaic result of the source image without noise and the mosaic result of
the source image with noise. White boxes highlight the ghostly parts. Figure 9. The first line is the blurred source image. The second line is the mosaic result of the unblurred
source image and the mosaic result of the blurred source image. The third line is the noisy source
image. The fourth line is the mosaic result of the source image without noise and the mosaic result of
the source image with noise. White boxes highlight the ghostly parts. Figure 9. The first line is the blurred source image. The second line is the mosaic result of the unblurred
source image and the mosaic result of the blurred source image. The third line is the noisy source
image. The fourth line is the mosaic result of the source image without noise and the mosaic result of
the source image with noise. White boxes highlight the ghostly parts. Figure 10. Experimental results of panoramic stitching. The first and second rows are the images to be
stitched together. The third row is the result of our method. Autostitch cannot process these images. Our method sacrifices the stiffness of the stitching image. The effect is good when stitching
wo images. However, distortion is observed in the result when stitching multiple images. Thi Figure 10. Experimental results of panoramic stitching. The first and second rows are the images to be
stitched together. The third row is the result of our method. Autostitch cannot process these images. Our method sacrifices the stiffness of the stitching image. The effect is good when stitching
two images. However, distortion is observed in the result when stitching multiple images. This Our method sacrifices the stiffness of the stitching image. The effect is good when stitching
two images. However, distortion is observed in the result when stitching multiple images. This 14 of 17 Sensors 2019, 19, 1898 situation can be seen from our stitching results in Figures 7–11, but the overall effect is acceptable. For
most UAV images, the mosaic results obtained by our system are acceptable. 4.5. Bundle Adjustment and Panoramic Stitching The distortion will affect
the image effect only when the parallax of the source image is particularly large. This case can be
improved later, i.e., how to obtain a good mosaic effect on a large-parallax image. Figure 11. Experimental results on panoramic stitching. The first and second rows are the images to be
stitched together. The third and fourth rows are the results of Autostitch and our method. Red boxes
highlight the ghostly parts. Figure 11. Experimental results on panoramic stitching. The first and second rows are the images to be
stitched together. The third and fourth rows are the results of Autostitch and our method. Red boxes
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seam-cut methods into stitching framework. 15 of 17 15 of 17 Sensors 2019, 19, 1898 Author Contributions: All authors have made great contributions to the work. L.L. and J.C. designed the research
and analyzed the results. Q.X. performed the experiments and wrote the manuscript. J.C., Y.W. and S.W. gave
insightful suggestions to the work and revised the manuscript. Funding: This work was supported in part by the National Natural Science Foundation of China under grant
no. 61603354, in part by the Fundamental Research Funds for the Central Universities grant nos. CUGL170221
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Evidence for a Resting State Network Abnormality in Adults Who Stutter
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ORIGINAL RESEARCH
published: 27 April 2018
doi: 10.3389/fnint.2018.00016
Evidence for a Resting State Network
Abnormality in Adults Who Stutter
Amir H. Ghaderi 1,2* , Masoud N. Andevari 2,3 and Paul F. Sowman 4,5
1
Cognitive Neuroscience Laboratory, University of Tabriz, Tabriz, Iran, 2 Iranian Neuro-wave Laboratory, Center of Isfahan,
Isfahan, Iran, 3 Department of Physics, School of Basic Science, Babol Noshirvani University of Technology, Babol, Iran,
4
Department of Cognitive Science, Faculty of Human Sciences, Macquarie University, Sydney, NSW, Australia, 5 ARC Centre
of Excellence in Cognition and its Disorders, Macquarie University, Sydney, NSW, Australia
Edited by:
James Danckert,
University of Waterloo, Canada
Reviewed by:
Gennady Knyazev,
State Scientific-Research Institute of
Physiology & Basic Medicine, Russia
Irene Messina,
Università degli Studi di Padova, Italy
*Correspondence:
Amir H. Ghaderi
a_ghaderi@tabrizu.ac.ir;
amirhoseinghaderi@gmail.com
Received: 24 January 2018
Accepted: 10 April 2018
Published: 27 April 2018
Citation:
Ghaderi AH, Andevari MN and
Sowman PF (2018) Evidence
for a Resting State Network
Abnormality in Adults Who Stutter.
Front. Integr. Neurosci. 12:16.
doi: 10.3389/fnint.2018.00016
Neural network-based investigations of stuttering have begun to provide a possible
integrative account for the large number of brain-based anomalies associated with
stuttering. Here we used resting-state EEG to investigate functional brain networks
in adults who stutter (AWS). Participants were 19 AWS and 52 age-, and gendermatched normally fluent speakers. EEGs were recorded and connectivity matrices were
generated by LORETA in the theta (4–8 Hz), alpha (8–12 Hz), beta1 (12–20 Hz), and
beta2 (20–30 Hz) bands. Small-world propensity (SWP), shortest path, and clustering
coefficients were computed for weighted graphs. Minimum spanning tree analysis was
also performed and measures were compared by non-parametric permutation test. The
results show that small-world topology was evident in the functional networks of all
participants. Three graph indices (diameter, clustering coefficient, and shortest path)
exhibited significant differences between groups in the theta band and one [maximum
betweenness centrality (BC)] measure was significantly different between groups in the
beta2 band. AWS show higher BC than control in right temporal and inferior frontal
areas and lower BC in the right primary motor cortex. Abnormal functional networks
during rest state suggest an anomaly of DMN activity in AWS. Furthermore, functional
segregation/integration deficits in the theta network are evident in AWS. These deficits
reinforce the hypothesis that there is a neural basis for abnormal executive function
in AWS. Increased beta2 BC in the right speech–motor related areas confirms previous
evidence that right audio–speech areas are over-activated in AWS. Decreased beta2 BC
in the right primary motor cortex is discussed in relation to abnormal neural mechanisms
associated with time perception in AWS.
Keywords: stuttering, functional brain networks, minimum spanning tree, executive function, time perception
INTRODUCTION
Stuttering is a developmental disorder of speech fluency that affects 1% of all adults (Craig et al.,
2002). The behavioral manifestations of stuttering include unplanned sound prolongations, blocks
in speech, and syllabic repetitions at the start of words and sentences. Stuttering is also associated
with abnormalities in complex cognitive functions such as language (Weber-Fox and Hampton,
2008), motor preparation (Mersov et al., 2016), time perception (Ezrati-Vinacour and Levin, 2001),
and also attention (Kamhi and McOsker, 1982).
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Ghaderi et al.
Resting State Network in Stuttering
et al., 2005; Mizuhara et al., 2005). Low-resolution brain
electromagnetic tomography (LORETA) is an approach to solve
inverse electromagnetic problem which transforms the EEG
scalp current topography into a gross approximation of the
EEG sources in brain space (Pascual-Marqui et al., 1994, 2011;
Pascual-Marqui, 2002). Using LORETA, one can obtain a highly
temporally-resolved neural signal that is mapped onto brain
space.
Graph theoretical analysis (GTA) has become an important
method for the study of complex systems in the field of
neuroscience as well as in physics, astronomy, genetics, and
engineering (Boccaletti et al., 2006; Bullmore and Sporns, 2009).
GTA has been used to reveal the topological properties of
structural brain networks and functional associations between
brain regions. Important properties of neural information
propagation and processing such as segregation and integration,
and modularity and efficiency have been characterized by GTA.
Graph theoretical indices such as the clustering coefficient, global
efficiency, and small-worldness are meaningful neurobiological
measures that can be calculated quickly (Rubinov and Sporns,
2010).
Small-world topography (Watts and Strogatz, 1998) provides
an optimal balance between segregation and integration
(Rubinov and Sporns, 2010). In 1998, Watts and Strogatz
introduced the concept of small-world graphs based on
Stanley Milgram’s works in the late 1960s (Watts and
Strogatz, 1998; Boccaletti et al., 2006). Small-world graphs
are simultaneously highly integrated and also highly segregated.
Dynamically, these graphs exist in a specific state between
random and regular graphs (Watts and Strogatz, 1998). Studies
indicate that the functional and structural topography of
the human brain, as well as other self-organizing systems,
exhibit small-world properties (Watts and Strogatz, 1998;
Achard et al., 2006; Bassett and Bullmore, 2006; Wang J.
et al., 2009), the conformations of which are affected by
developmental disorders (Wang L. et al., 2009; Barttfeld et al.,
2011).
A more recently developed approach, minimum spanning
tree (MST) analysis, is a powerful technique that can clarify
emergent properties of functional brain networks (Stam, 2014).
In weighted graphs there are many loops, which consist of
sets of edges that connect a node to itself. The presence of
loops in a graph increases the connectivity cost (the number
of routes and edges between nodes). Spanning trees are subsets
of graphs that cover all nodes without any loop (Stam, 2014;
Tewarie et al., 2015). The MST is the tree with the lowest total
cost (Graham and Hell, 1985); the unique, unweighted, binary
graph that is made by the shortest path between all pairs of
nodes without the occurrence of a loop. Therefore, the minimum
connectivity cost involved in spanning all nodes is recovered
by MST analysis (Tewarie et al., 2015). MST has been widely
applied in the investigation of functional brain connectivity
during tasks and rest (Stam et al., 2014; van Lutterveld et al.,
2017).
This study represents the first attempt to use quantitative
EEG (QEEG), LORETA, and graph theory in the field of
language-related disorders. Graph theoretical analysis is applied
Over the last two decades, a body of neuroimaging research
has amassed which suggests that stuttering likely emerges
from deficiencies in the brain mechanisms that support fluent
speech production (e.g., Chang et al., 2009, 2017). Structurally,
stuttering is related to several abnormalities in cortical and
subcortical brain areas such as the Broca’s area (BA 44, 45),
the basal ganglia, supplementary motor area, and parasylvian
cortex (Gordon, 2002; Büchel and Sommer, 2004; Chang
et al., 2009; Loucks et al., 2011; Sowman et al., 2017)
which can be linked to mechanistic explanations proposed
to account for stuttering such as auditory–speech dysfunction
(Liotti et al., 2010; Jansson-Verkasalo et al., 2014) and also
a speech–motor impairment (Neilson and Neilson, 1987).
More recently, neural network-based investigations of stuttering
have begun to provide a possible integrative account that
might account for the large number of brain-based anomalies
(for review see: Brown et al., 2005; Budde et al., 2014;
Belyk et al., 2015; Etchell et al., 2017) now associated with
stuttering.
Studies using connectivity analyses and graph theoretical
methods have demonstrated network abnormalities in stuttering
during resting state (Xuan et al., 2012; Ghaderi et al., 2016;
Chang et al., 2017) that may involve anomalies in the default
mode network (DMN) and affect attentional and executive
functions (Chang et al., 2017). Such studies provide an
insight into the neural correlates of psychological dysfunctions,
particularly anxiety, that have long been associated with
stuttering (Craig, 1990; Menzies et al., 1999; Iverach et al.,
2011). Recent studies such as that by Yang and colleagues
provide evidence that some of the hitherto unexplained neural
abnormalities evident in stuttering might be attributable to the
close association between stuttering and anxiety (Yang et al.,
2017). Indeed, their results bear considerable concordance with
other investigations that suggest emotional states and disorders
(e.g., depression and anxiety) are associated with functional
deficits in DMN activity (Coutinho et al., 2016; Messina et al.,
2016).
To date, investigations of brain networks in stuttering
have largely relied on functional magnetic resonance imaging
(fMRI; Luc et al., 2008; Chang et al., 2009; Loucks et al.,
2011). fMRI remains the gold standard for defining the
topographic nature of network dysfunction in stuttering due
to its excellent spatial resolution; however, as it has been
suggested that stuttering depends on abnormal timing of brief
durations and deficits in rapid movement control, planning,
and preparation (e.g., Etchell et al., 2014, 2015; Wieland
et al., 2015), neurophysiological methods that provide higher
temporal resolution than fMRI may provide complementary
information about the nature of neural connectivity in stuttering.
Electroencephalography (EEG) can be used to acquire an ongoing
record of the electrical activity of the brain with excellent
temporal resolution, but since the origin of brain waves is
a combination of post-synaptic potentials in the pyramidal
cortical neurons, the source of EEG waves is not generally
well reflected by the current distribution on the scalp. This
means that inter-electrode connectivity analyses cannot easily
be reconciled with fMRI network measurements (Babiloni
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Resting State Network in Stuttering
shielded faraday cage. EEG was digitized at 250 Hz and a low pass
filter (40 Hz cutoff) was applied to avoid aliasing effects. Eight
minutes of eyes open resting state EEG was recorded from each
participant (Wu et al., 2010). Participants were instructed to avoid
body or eye movements during the recording.
After recording, signals were screened for artifacts visually by
an expert and then submitted to a z-score based artifact rejection
algorithm implemented in the NeuroGuide software1 . Twentyfive artifact-free segments (each segment was between 4 and
5 s in duration) were selected and exported for LORETA source
localization.
here to investigate the brain’s topological network properties
in adults who stutter (AWS). Further, we investigate possible
relationships between the centrality of candidate brain regions
and stuttering. MST analysis is used as the GTA approach.
Based on previous reports that suggest that oscillatory brain
activity in the beta band is abnormal in stuttering (Salmelin
et al., 2000; Etchell et al., 2016; Mersov et al., 2016),
we hypothesize that networks connected by coherent beta
oscillations in stuttering will be compromised compared to
those in controls. Since abnormal attention and executive
network abnormalities have been observed in AWS (Chang
et al., 2017) we also predict that language network abnormalities
will be evident in AWS alongside abnormalities in the default
mode and executive networks. This hypothesis is in line with
previous findings that suggest stuttering is a disorder related to
impaired working memory (Kaganovich et al., 2010), attention
(Kaganovich et al., 2010), self-control (Eggers et al., 2013),
and linguistic processing speed (Anderson and Wagovich,
2010).
LORETA Analysis
Functional connectivity between 84 regions of interests (ROIs)
was obtained by LORETA software version 201702202 . LORETA
estimates cortical sources of brain waves based on the
distribution of scalp-recorded potentials (Pascual-Marqui, 2002).
This algorithm works as an inverse solution method and use
a smoothness matrix that optimizes the solution (see PascualMarqui et al., 1994 for details). Recent versions of LORETA
provide a connectivity utility (Pascual-Marqui et al., 2011).
Functional dynamic connectivity of cortical regions with high
temporal resolution can be calculated by LORETA. Although the
number of EEG electrodes has a relationship to the precision of
source estimation, a number of previous studies indicate that
a reliable LORETA estimation can be achieved with only 19
channels (e.g., Thatcher et al., 2014; Aoki et al., 2015; Emory
et al., 2015; Alahmadi et al., 2016; Clemens et al., 2016; Hata
et al., 2016; Mohan et al., 2016b; Mumtaz et al., 2017). Timeresolved activity in all Brodmann areas excepting areas 12, 14, 15,
16, and 26 (localization of these regions is not implemented in
the LORETA software) was estimated and the lagged coherences
between 84 ROIs (42 Brodmann areas in the left hemisphere and
42 Brodmann areas in the right side) were computed for four
frequency bands [theta (4–8 Hz), alpha (8–12 Hz), beta1 (12–
20 Hz), and beta2 (20–30 Hz)]. The length of selected segments
was at least 4 s and the sampling rate was 250 Hz (at least 1000
samples for each segment). The number of time frames per epoch
selected in the LORETA software was 1024.
MATERIALS AND METHODS
Participants
Nineteen AWS [74% male, aged between 19 and 31 years, mean
age (SD) 24.02 (3.65) years] and 52 age-, and gender-matched
normally fluent speakers [fluent; 71% male, aged between 19 and
32 years mean age (SD) 24.47 (4.78) years] participated in this
study. AWS participants were recruited from the Aftab Clinic
in the city of Isfahan. Fluent speakers (controls) were recruited
via an online announcement. All participants were native Persian
speakers with no reported history of psychiatric/neurological
disorders/diseases or use of medications that might affect
neural function (e.g., medication for depression or seizure). All
participants had normal hearing and normal or corrected-tonormal vision. At the time of testing, the Stuttering Severity
Instrument for Adults—Fourth Edition (SSI–4) was administered
by a speech therapist to each of the AWS and their stuttering
severities were rated to be between mild and severe (Riley,
1972). A consent form was signed by all participants after the
aim and procedure of the study was explained to them. The
study conformed to the Helsinki declaration obligations and
was approved by central ethical committee at Islamic Azad
University.
Connectivity Measure and Adjacency
Matrix
Non-instantaneous or lagged coherence is a methodological
approach to frequency domain connectivity that removes the
effects of volume conduction in EEG co-spectra (Pascual-Marqui,
2007; Pascual-Marqui et al., 2011). Lagged coherence has been
used to investigate functional connectivity in resting-state brain
networks in several disorders, e.g., Olbrich et al. (2013), Mohan
et al. (2016a), Schwartz et al. (2016). However, to our knowledge,
brain connectivity in AWS has not yet been investigated with
lagged coherence.
The 84 by 84 adjacency matrices were calculated separately
in the theta, alpha, beta1, and beta2 bands. In the adjacency
matrix, each row and each column represents a Brodmann area,
EEG Recording, Technical Setup, and
Signal Pre-processing
EEG was recorded from 19 scalp electrodes (Fp1, Fp2, F3, F4,
C3, C4, P3, P4, O1, O2, F7, F8, T3, T4, T5, T6, Fz, Cz, and Oz)
positioned according to the 10–20 standard systems. A further
two electrodes were positioned at left and right preauricular
points (A1 and A2). EEGs were recorded using a Brain Master
Discovery 24 amplifier and Electro-cap (eci). A linked-ear
reference, commonly used in QEEG studies (Rotondi et al., 2016)
was used. EEG cancelation is minimized by this montage (Sanei
and Chambers, 2013). Impedance was kept under 5 k during
recording. Recordings were performed in an electromagnetically
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2
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http://www.appliedneuroscience.com
http://www.uzh.ch/keyinst/loreta.htm
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Resting State Network in Stuttering
there is only information pertaining to the existence or not of
connections; connectivity strength between nodes is not visible.
Commonly, an adjacency brain connectivity matrix contains
connectivity measures between nodes (electrodes or brain
regions) that are not binary (e. g. coherence is a scalar that lies
in the range 0 to 1; phase lag falls between −1 and +1, etc.).
Therefore, the original adjacency brain connectivity matrix is a
weighted matrix. For simplification, thresholding can be applied
to transform weighted matrices to binary forms (Rubinov and
Sporns, 2010; Mohan et al., 2016b; Ghaderi et al., 2017). In this
approach, all of the matrix arrays with values higher than the
threshold are replaced by 1 and all other array values set to
zero. Although this approach has been used widely, e.g., Achard
et al. (2006), Mohan et al. (2016b), Ghaderi et al. (2017), two
basic problems are inherent in this approach. First, there is no
specific threshold that must be used to make binary matrices,
and therefore the choice of threshold can greatly influence
the resulting structure of the graph (Tewarie et al., 2015). By
systematically investigating the threshold “space,” any bias may
be countered, but a second problem then arises in the statistical
analysis where the use of multiple thresholds greatly increases the
number of comparisons being made. To avoid these problems,
analysis of weighted graphs has been proposed. Recently, a new
measure, Small-World Propensity (SWP), has been proposed for
identification of small-world properties of weighted networks
(Muldoon et al., 2016). SWP compares clustering coefficient and
and the lagged coherences between pairs of Brodmann areas
are quantified at their intersections. Weighted and undirected
adjacency matrices for the two groups (AWS and controls) are
presented in Figure 1.
Small-World Properties, Shortest Path,
and Clustering Coefficient of Weighted
Graph
Before 1998, graphs were classified generally as either random
or regular (Boccaletti et al., 2006). In a random graph, edges
are connected randomly to nodes and a node’s degree (the
number of edges connected to each node) conforms to a Gaussian
distribution (Boccaletti et al., 2006). Since the shortest path
between nodes is typically small, random graphs are highly
integrated. However, at the local level, there are no significant
clusters between nodes and therefore random graphs are not
segregated. Conversely, in a regular graph, all nodes have the
same degree. In these graphs, the shortest path is long, and
therefore, integration is minimal and segregation high.
In the majority of previous studies utilizing GTA analysis,
small-world topography has been calculated for binary, nonweighted, and undirected graphs. This represents the simplest
form of graph that requires the least computation and
programing for calculation of small-worldness (Boccaletti et al.,
2006; Humphries and Gurney, 2008). However, in a binary graph,
FIGURE 1 | Weighted and sparse (MST) adjacency matrices for the two groups (AWS and control) in the beta2 band. (A) Averaged weighted adjacency matrix for
controls. (B) Averaged weighted adjacency matrix for AWS. (C) Average MST adjacency matrix for controls. (D) Average MST adjacency matrix for AWS. Different
patterns are visually observable between the MST graphs.
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Ghaderi et al.
Resting State Network in Stuttering
poorly integrated (Rubinov and Sporns, 2010; Stam et al., 2014;
van Lutterveld et al., 2017). Here, MST analysis on the 84 by
84 adjacency matrices was performed using MATLAB R2016a
and the biograph toolbox. The measures of tree (e.g., BC, degree,
LF, diameter, and eccentricity) were obtained via the brain
connectivity toolbox developed by Rubinov and Sporns (2010)3 .
characteristic shortest paths of a given network using lattice and
random graphs:
q
12C +12L
φ =1−
2
Where, 1C is Clattice −Cgiven divided by Clattice −Crandom and 1L is
Lgiven −Lrandom divided byLlattice −Lrandom . In this equation,
Lattice and random graphs have the same size (number of nodes)
and the same distribution of degree (probability distribution of
degrees over all nodes) within the given network (Muldoon et al.,
2016).
As indicated in the above equation, SWP is related to the
characteristic shortest path (L) and clustering coefficient (C).
L is the average minimum number of edges between all pairs
of nodes. The clustering coefficient is defined by the number
of triangles around a given node relative to the number of
all neighbors (Boccaletti et al., 2006) and is closely related to
brain modularity and segregation (Bullmore and Sporns, 2009;
Rubinov and Sporns, 2010).
Here, random graphs with the same degree distribution and
same size were made by permutation of adjacency matrices. Then
L, C, and SWP were calculated for the stuttering group and
control using a MATLAB toolbox at http://www.seas.upenn.edu/
~dsb/ developed by Muldoon et al. (2016). For assessing SWP
measures for the two groups, the SWP of 50 randomly permuted
graphs was calculated and compared to the adjacency matrices.
Statistical Analysis
Non-parametric permutation tests (Maris and Oostenveld, 2007)
were applied to compare the between-subject measures of graph
indices within frequency bands. Each permutation contained
5000 random shuffles. Seven graph indices (SWP, L, C, maximum
BC, LF, diameter, and average eccentricity) were compared in
four frequency bands (theta, alpha, beta1, and beta2) and then
28 (7 indices × 4 frequencies) independent permutation tests
were performed. To minimize the possibility of false positives
resulting from multiple comparisons, False Discovery Rate (FDR)
correction was applied. The resulting q-values (corrected p-values
in FDR) less than 0.05 were accepted as indicating statistically
significant differences.
To evaluate the local corporation of cortical regions, BC
and eccentricity of all Brodmann areas was investigated using
a separate non-parametric permutation test. This latter analysis
was performed only at the frequencies that maximum BC or/and
average eccentricity was significant. FDR correction was also
applied to minimize the likelihood of type I errors that might
arise through comparison of 84 Brodmann areas over multiple
frequencies. Non-parametric statistical tests were performed in
MATLAB R2016a.
Minimum Spanning Tree (MST) and
Integration Measures
The aforementioned problems involved thresholding the
connectivity matrix are overcome by transforming the original
weighted matrix into a unique sparse matrix. Functional brain
connectivity using the MST approach can be quantified by the
derived measures of maximum BC, nodal degree, leaf fraction
(LF), diameter, and eccentricity (van Lutterveld et al., 2017).
BC is calculated by the counting all of the shortest paths
that pass through a given node. Nodal degree is comparable
to BC (it is simpler than BC). Degree is equal to the number
of edges that are connected to a node. Nodes with a higher
degree or BC play an important role in information processing
within a graph (Boccaletti et al., 2006). It is suggested that a
graph with higher maximum BC is more integrated (Bullmore
and Sporns, 2009; Stam et al., 2014). LF relates to the integrative
properties of the brain network (Stam et al., 2014; van Lutterveld
et al., 2017). In a MST, the LF is equal to the number of
nodes with degree 1 divided by N − 1, where N is the number
of nodes in the graph. Therefore, a tree with a central node
connected to all other nodes has maximum LF (equal to 1) and
is highly integrated. Conversely, a tree with a series of one-to-one
connected nodes exhibits the lowest possible LF (near zero) and
also minimal integration (Stam et al., 2014). On the other hand,
the maximum path length in a tree is defined as its diameter.
Higher diameter is negatively associated with brain integration
(Bullmore and Sporns, 2009; Stam, 2014). Eccentricity of a node
is related to nodal isolation and average nodal eccentricity shows
the tendency of nodes within the network to be isolated and
Frontiers in Integrative Neuroscience | www.frontiersin.org
RESULTS
Figure 2 shows that both the controls and AWS exhibit SWP
values over 0.6 in all frequency bands. Conversely, randomly
permuted graphs show SWP less than 0.6. As suggested by
Muldoon et al. (2016), SWP values over 0.6 are indicative
of small-world networks. Therefore, all of the brain-based
graphs (in both groups) exhibit small-world properties that are
significantly different from random graphs (Figure 2).
After FDR correction, three graph indices exhibited significant
differences between groups in the theta band and one measure
was significantly different between groups in the beta2 band.
In the theta band a significant difference between groups
occurred in the diameter of MST (t = −2.83, P = 0.0001). In this
band, the AWS group had a smaller diameter (mean = 13.894,
SD = 2.051) than the fluent control group (mean = 15.750,
SD = 2.573). Significant differences were also observed between
the two groups in the measures of characteristic shortest path
(t = 2.71, q = 0.003) and clustering coefficient (t = −2.81,
q = 0.002). The fluent group had a lower characteristic
shortest path and a higher clustering coefficient (respectively:
mean = 1.061, SD = 0.026; mean = 0.937, SD = 0.019) than
the AWS group (respectively: mean = 1.080, SD = 0.029;
mean = 0.922, SD = 0.023).
3
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Resting State Network in Stuttering
FIGURE 2 | SWP measures for all samples in permuted random graphs (group 1), the control group (group 2), and the AWS group (group 3). The control and AWS
groups exhibit values higher than 0.6, indicating that they have small-world connectivity. Permuted random graphs exhibit values less than 0.6.
In the beta2 band, a significant difference was seen in the
maximum BC of MST (t = 3.04, q = 0.007). The AWS group
(mean = 5023.684, SD = 574.688) had a higher maximum BC than
did the control group (mean = 4659.608, SD = 398.141). There
were no significant differences in any measures in the alpha and
beta1 bands between groups.
Since there was a significant difference between groups in the
maximum BC in the beta2 band, BC of MST was investigated
for the 84 Brodmann areas. After FDR multiple comparison
correction, significant differences between the stuttering group
and controls were observed in the right hemisphere; primary
motor cortex (BA4; t = −2.03, q = 0.001), inferior temporal lobe
(BA 20; t = 3.02, q = 0.0001) as a part of the DMN, and a part
of the inferior frontal gyrus (BA 47; F = 2.84, q = 0.0001). There
was no significant difference in the BC in the left hemisphere. The
normally fluent group exhibited higher BC in BA 4 than the AWS
group. However, they showed lower values of BC in BA 20 and
47 than the AWS group. Significant differences in BC are visually
presented in the Figure 3.
Weighted and sparse MST graphs for the beta2 band are
presented in Figure 1. While the weighted graphs are visually
similar between groups, differences are evident in the sparse MST
matrices. Topological MSTs are presented in Figure 3.
two sections with regard to the functional meaning of brain
oscillations and the regions involved in the observed functional
connectivity deficits in AWS.
DISCUSSION
In the theta band, AWS have a higher value of characteristic
shortest path and a lower value of clustering coefficient than
controls, suggesting that the theta network is disrupted at both
local and global levels in AWS. Theta-mediated networks in AWS
are less integrated and also less segregated.
Theta activity is closely associated with executive functions
such as problem solving, planning, working memory, and
also attention (Sauseng et al., 2005; Mizuhara and Yamaguchi,
2007). Further, functional connectivity in the theta band is
related to activity in central executive networks (Sauseng et al.,
2004, 2005) and abnormal theta connectivity during rest state
may suggest impaired DMN function (Scheeringa et al., 2008).
Abnormal functional connectivity in the theta network is
reported in attention-related disorders such as attention deficit
hyper activity disorder (ADHD) during rest (Ghaderi et al.,
2017) and during tasks (Sauseng et al., 2007). Similar to
our current findings in AWS, in ADHD impaired functional
segregation and integration in the theta network is evident
(Ghaderi et al., 2017). Such similarities reinforce the hypothesis
Alpha Wave: The Role of Emotion in
Stuttering
The role of alpha activity in emotional states such as anxiety
(Boutcher and Landers, 1988; Knyazev et al., 2006) and
depression (Gotlib, 1998; Fingelkurts et al., 2007) has been widely
investigated. Several studies suggest that those who stutter exhibit
higher levels of anxiety (e.g., social anxiety or social phobia)
than people whose speech is fluent (Mahr and Torosiana, 1999;
Messenger et al., 2004; Iverach and Rapee, 2014). However, our
results indicate that, at least during resting conditions, AWS
exhibit alpha connectivity that is not different from that seen in
control subjects. This result is consistent with previous studies
that explain anxiety in stuttering as a secondary reaction (Alm,
2004). In the context of a possible alpha difference in AWS that
might be based on reactive anxiety, then, in the current study
where the AWS were at rest, it might not be expected that alpha
network differences between groups would be evident.
Theta Wave: The Role of Executive
Network in Stuttering
These results indicate that alterations in very fast fluctuations
and synchronization of post synaptic dipole arrangements, in
various brain regions involved in generating EEG coherence,
are associated with stuttering. Both groups studied here show
small-world networks in the functional brain connectivity, and
all participants exhibit SWP higher than 0.6. Since a graph with
small-world topology exhibits an optimized and enhanced signalpropagation speed and synchronizability (Watts and Strogatz,
1998), optimized information transformation and propagation
occurs in adult who stutter (AWS) as well as in controls. However,
functional deficits in weighted graphs and MST analysis were
observed in the theta and beta2 bands in AWS. Significant
differences between controls and AWS in BC are also observable
in the right primary motor area, inferior temporal lobe, and
inferior frontal cortex. AWS show higher BC than controls in
right temporal and inferior frontal areas and lower BC in right
primary motor cortex. We discuss these results in the following
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Ghaderi et al.
Resting State Network in Stuttering
FIGURE 3 | Minimum spanning tree in the beta2 band. Significant differences of BC (red nodes) were observed for right primary motor cortex (4R), right inferior
temporal lobe (20R) and right inferior frontal gyrus (47R). The stuttering group (A) shows higher BC than the fluent group (B) in 20R and right 47R. The AWS exhibit
lower BC in 4R. The figure was generated using BrainNet viewer toolbox version 1.53 (Xia et al., 2013).
However, the present results were obtained during resting state
and could therefore fit with an explanation that posits DMN
deficits in stuttering (Xuan et al., 2012; Chang et al., 2017).
Recently it has been suggested by Etchell et al. (2014, 2016)
that stuttering is a timing deficit disorder underpinned by
abnormal functioning of beta-mediated timing networks (see
also Alm, 2004). According to the broader literature in this area
(Buhusi and Meck, 2005; Fujioka et al., 2009; Kononowicz and
van Rijn, 2015; Merchant and Bartolo, 2017), beta activity in the
basal ganglia-thalamocortical circuits (measured by EEG/MEG
from central brain locations) can be considered in relation to
interval timing. Behaviorally, deficits in response timing tasks
are frequently reported in people who stutter (Ezrati-Vinacour
and Levin, 2001; Olander et al., 2010; Falk et al., 2015) and
recently it has been suggested that impairment of resting state
functional connectivity is involved in time discrimination deficits
in stuttering (Chang et al., 2016). Since beta activity in primary
and supplementary motor cortices is associated with activity of
basal ganglia-thalamocortical circuit (Kropotov, 2010), decreased
BC of primary motor cortex in resting state beta2 adds further,
albeit indirect, neural evidence for a deficit in interval timing
networks in stuttering that involve the striatum.
AWS exhibit increased beta2 BC in the right speech–motor
related areas during resting state. This result suggests that extra
information is transferred via right audio–speech regions and
that hyper propagation of beta signals occurs in this region.
Increased activity of right audio–speech areas has previously
been observed in studies using fMRI (Luc et al., 2008; Chang
et al., 2009). Therefore, as suggested by other authors (Luc et al.,
2008; Chang et al., 2009), AWS may require more neural activity
in right audio–speech regions to compensate for deficiencies
elsewhere. It could be posited that decreased signal propagation
in the right primary cortex causes an imbalance in the neural
network and hence shortest paths are transferred from right
audio–speech regions.
that there is a neural basis for abnormal executive functions
among the stuttering group that may explain previous findings
that indicate impairment of working memory (Kaganovich et al.,
2010), attention (Kaganovich et al., 2010), self-control (Eggers
et al., 2013), and also linguistic processing speed (Anderson and
Wagovich, 2010) in stuttering. This result may also help explain
the reported treatment effects of attentional training (Nejati et al.,
2013) and also mindfulness (Boyle, 2011) in stuttering.
MST analysis indicates that there is no regional thetamediated abnormality in AWS. This finding, in combination
with the aforementioned local and global anomalies of the theta
network, may suggest that attentional deficits in AWS are related
to the functional connectivity of the whole brain rather than those
within a specific module (e.g., middle frontal lobe as a hub in
executive network).
Beta Wave: The Role of Motor Timing
and Audio–Speech Regions
Many studies have investigated the role of motor, speech and
auditory related impairments in stuttering, e.g., (Büchel and
Sommer, 2004; Watkins et al., 2008; Chang et al., 2009). These
confirm motor–speech (Watkins et al., 2008) and audio–speech
(Luc et al., 2008; Chang et al., 2009) deficits exist, at least at
the neural level, in stuttering. The current results reaffirm that
functional brain differences in AWS occur in both primary motor
related regions and also audio-speech areas. We show that the
BC of beta2 mediated connections is decreased in the right
primary motor cortex in AWS. A node with high BC lies on a
large number of shortest paths. Nodes with significant association
in information transfer often have high BC while BC is zero
for a dead-end node (Barthelemy, 2004; Rubinov and Sporns,
2010). Decreased BC in right primary motor cortex suggest that
this area plays a reduced role in neural communication and
information propagation within the cortico-cortical networks
in AWS. This result is comparable with previous findings that
show hypo-activity of the cortical motor and premotor areas in
stuttering (Salmelin et al., 2000; Watkins et al., 2008). Previous
findings suggest that abnormal motor and pre-motor activity
during speech tasks may be causal in stuttering (Packman, 2012).
Frontiers in Integrative Neuroscience | www.frontiersin.org
Study Limitations
Two limitations that affect this study should be considered.
Firstly, as LORETA accuracy is dependent to an extent on
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Ghaderi et al.
Resting State Network in Stuttering
also show impairment in the beta network in primary motor
cortex and audio-speech areas. We suggest that abnormal activity
in the beta network may relate to timing deficits and hypoactivation of motor related areas.
the EEG montage density, the relatively small number of
electrodes we were limited to suggest that some caution regarding
interpreting absolute source localization accuracy should be
exercised. However, this is always the case with EEG source
analysis, and the fact that the results presented here are
both physiologically plausible and in strong concordance with
previous studies mitigate this concern. Furthermore, whilst
there is some evidence that suggests montage density positively
correlates with deep source reconstruction accuracy, a clear
relationship to reconstruction of superficial sources is less clear
(Liu et al., 2018). Secondly, whilst not an absolute limitation,
our choice of referencing scheme, the linked ear montage, whilst
widely used in similar studies e.g., Hata et al. (2016), is not
universally accepted as the best option for EEG studies in source
space. Future studies would be advised to systematically address
the implications of the choice of reference scheme on possible
functional connectivity deficits in AWS.
AUTHOR CONTRIBUTIONS
AG performed the main analysis, data recording, and wrote the
main body of the manuscript. MA performed a part of graph
analysis and he collaborated in data recording. PS managed
the methodology, edited the manuscript, and also wrote a
considerable part of the Section “Discussion.”
FUNDING
PS is supported by the National Health and Medical Research
Council, Australia (#1003760), the Australian Research Council
(DE130100868 and DP170103148) and the Australian Research
Council Centre of Excellence for Cognition and its Disorders
(http://www.ccd.edu.au; CE110001021).
CONCLUSION
Our results reinforce previous findings that DMN deficits occur
in stuttering (Xuan et al., 2012; Chang et al., 2017). Altered
networks found in AWS include attentional circuits, primary
motor regions and also audio–speech related areas. We found
decreased functional integration and segregation comparable to
that seen in other developmental disorders (Ghaderi et al., 2017)
but no local impairments in specific regions were evident. AWS
ACKNOWLEDGMENTS
We are very thankful to Professor Warren H. Meck for great
comments on our presentation at Duke University.
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Conflict of Interest Statement: The authors declare that the research was
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Social responsibility: The changing role of business in South Africa
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South African journal of business management
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C.F. Wagenaar C.F. Wagenaar A report on a pilot study which preceded this survey, ap-
peared in the South African Journal of Business Manage-
ment, Volume 9 Number 4, pp 43-44, 1978. In the light of the political, social and economic problems
currently experienced in South Africa, it is not surprising
that growing demands are imposed on all institutions of
society. Business is also expected to contribute, to a great
extent, to the solving of these problems. Typical of these
demands is that business is expected to confront not only
economic but also social problems of South Africa. In
this respect one may refer to a remark made by Dr N. Motlana, former member of the now defunct Urban Ban-
tu Council of Soweto, that business is the only institution
that can save the South African society from social and
economic breakdown. 1 The United States ambassador in
South Africa recently referred to the role of businessmen
in South Africa: 'One of this country's growing assets, I
believe, is the social awareness of its business leaders'. 2 Business in South Africa is becoming increasingly in-
volved in the solution of the social problems of the
country, and business managers face growing demands
concerning the corporate social responsibility of the
private sector. The author investigated the nature and
scope of corporate social responsibility in South Africa,
considering both the viewpoint of business managers,
and the expectations of the public. Both questionnaires
and interviews were used in the investigation. The at-
titudes of managers and the expectations of society
are set out in detail, and are then compared, showing
that society is significantly more in favour of corporate
social responsibility than the business community. There is, however, also a growing social awareness
among South African business leaders. S. Afr. J. Bus. Mgmt 1979, 10:93-100 The fact that business is expected to be involved in the
betterment of the social welfare of society has important
implications as far as the division of responsibilities be-
tween government and private enterprise is concerned. Traditionally, it used to be the sole responsibility of the
government to take care of the social welfare of its socie-
ty. The view that business has a social responsibility,
however, results in uncertainty as to what the respective
roles of government and private enterprise are. C.F. Wagenaar The pur-
pose of this article is to discuss the results of research into
the nature and scope of corporate social responsibility in
South Africa. Sake-ondernemings in Suid-Afrika word toenemend
betrek by die oplossing van die sosiale probleme van
die land, en bestuurders is blootgestel aan toenemende
eise wat betref die sosiale verantwoordelikheid van die
private onderneming. Die skrywer het die aard en tref-
wydte van die sosiale verantwoordelikheid van Suid-
Afrikaanse ondernemings ondersoek, met inagneming
van sowel die standpunt van sakebestuurders as die
verwagtings van die publiek. Vraelyste en personlike
onderhoude is in die ondersoek gebruik. Die houdings
van bestuurders en die verwagtings van die gemeen-
skap word volledig uiteengesit, en word dan vergelyk. Dit blyk dat die gemeenskap betekenisvol meer ten
gunste is van sosiaal-verantwoordelike optrede deur
ondernemings, as die sakelui. Daar is egter ook 'n
groeiende bewustheid van sosiale verantwoordelikheid
onder Suid-Afrikaanse sakeleiers. S.-Afr. Tydskr. Bedryfsl. 1979, 10:93-100 Scope and method of the research During May 1978 questionnaires were sent to the
managing directors or chief executive officers of 510
listed companies. One hundred-and-twenty-three of the
510 questionnaires were completed, which represents a
response rate of 24070. It had been ascertained that these
companies, as far as different types of industries are con-
cerned, are representative of all listed companies. In 61070
of the cases the questionnaire was completed by a manag-
ing director or chief executive officer. Thirty-nine per
cent of the questionnaires were completed by other ex-
ecutives, such as personnel managers, administrative
managers and company secretaries. Table 1 · Reliability coefficients of the different
scales Kuder
No. of items
Scale
Richardson 14
in scale
Management scale
Society scale
Scale developed on the com-
bined responses of management
and society
0,77
0,83
0,86
16
23
24
These coefficients are satisfactory, particularly if it is taken into
account that the respective scales consist of relatively few items. The questionnaire was also completed by an area-
stratified probability sample (N = 924) which was drawn
from the total white population of South Africa. It was
ascertained that the sample was satisfactorily represen-
tative as far as sex, age, income level, nature of employ-
ment and education qualifications are concerned. These coefficients are satisfactory, particularly if it is taken into
account that the respective scales consist of relatively few items. Apart from the questions contained in the question-
naire, the group of managers was also requested to in-
dicate the extent to which business may possibly con-
tribute to the solution of specific social problems current-
ly experienced in South Africa. In order to test the construct validity• (as explained in
the Appendix), of the attitude scale the responses of each
group were factor analyzed. b To be able to measure a
possible difference in attitude between society on the one
hand, and managers on the other hand, a third scale was
developed by combining the responses of the two groups. The combined responses were subjected to a separate fac-
tor analysis. In order to evaluate the quantitative measurement of
the attitude of managers and the expectations of society
qualitatively, the most important results of the research
were discussed during personal interviews with top ex-
ecutives of 22 companies. All of these executives agreed
to a personal interview in their answers to the question-
naire. Scope and method of the research Scope a d
et od o t e esea c
In order to obtain a comprehensive view of corporate
social responsibility in South Africa, both the public
policy and the attitude of businessmen in this respect
were measured in a country-wide survey. An indication
of the public policy concerning corporate social respon-
sibility was obtained by measuring the extent to which
society expects business to accept a social responsibility. 3
Only the expectations of the white community were
measured. The assumption which underlies this decision
is the fact that the other communities in South Africa
(blacks, coloureds and Asians) expect to a greater extent
than whites, business to be socially responsible. This was
assumed because blacks, coloureds and Asians generally
have a lower income than whites and therefore expect C.F. Wagenaar•
Department of Business Economics,
Rand Afrikaans University,
P.O. Box 524, Johannestiurg 2000
•New Address: Group Marketing Division,
Standard Bank of SA Investment Corporation,
P.O. Box 3124, Johannesburg 2000 S.-Afr. Tydskr. Bcdryfsl. 1979, 10(3) 94 social responsibility. Responses were measured by means
of a five point Likert-type summated rating scale. more from their employers. Should it therefore appear
from the research that whites in fact do expect business to
accept a social responsibility, it is most likely that the
other population groups expect even more in this respect. The five possible response categories -
'to a very great
extent', 'to a great extent', 'to an average extent', 'to a
lesser extent' and 'not al all' -
were scored 5, 4, 3, 2, l,
respectively. As far as the measurement of the attitude of
businessmen was concerned, the research was limited to
companies listed on the Johannesburg Stock Exchange,
because of the fact that the separation between capital
and management in these firms is the greatest. As a result
of this, executives would also take the interests of
shareholders into account in their decision-making con-
cerning the social responsibility of the company. The reliability of the measurements obtained was
tested by means of an item analysisd of the questions
which were found to contribute to the construct validity
of the respective scales. The item analysis revealed that
some of the items were unreliable. After these items had
been omitted from the different scales, the Kuder
Richardson 14 reliability coefficient• for each scale was
calculated. These coefficients are shown in Table I. Scope and method of the research As the interviews were merely supplementary to the
measurements obtained by means of the attitude scale, it
was felt that no effort need be made to interview a
representative sample of executives. y
In each case, the factor analytical procedure involved
an initial first-order factor analysis. By means of this fac-
tor analysis a number of factors or dimensions were iden-
tified. On the basis of these factors being intercorrelated,
a second-order factor analysis was carried out in order to
determine whether the first-order factors could be com-
bined into a single second-order factor which could be
identified as the 'attitude towards corporate social
responsibility'.• The second-order factor analysis involv-
ed that the scores for the items forming each of the first-
order factors, were algebraically summed for each first-
order factor to obtain factor scores. These factor scores
were intercorrelated and subjected to a further factor
analysis. By means of this factor analysis it was determin-
ed that 21 of the original pool of questions answered by
the group of managers measured a single dimension
which could be identified as the 'attitude towards cor-
porate social responsibility'. The factor analysis of the
responses of the sample from society and the combined
responses of society and managers revealed that 28 ques-
tions in the case of society, and 27 questions in the case of
managers and society combined measured the 'attitude
towards corporate social responsibility'. The attitude of managers towards specific aspects
of corporate social responsibility
The managers' responses to the individual items of the
scale are shown in Table 3. The attitude of managers towards specific aspects
of corporate social responsibility
The managers' responses to the individual items of the
scale are shown in Table 3. The managers' responses to the individual items of the
scale are shown in Table 3. In the interpretation of the managers' responses to the
individual items, responses to those items that, according
to the first-order factor analysis, are related to a specific
factor, should be viewed together. The first-order factor
analysis showed that four factors or dimensions were
measured by the scale. These factors were identified as
'corporate social responsibility in respect of aspects not
related to the economic activity of the firm', 'the rela-
tionship between government and private enterprise as
far as the solution of social problems is concerned', 'the
firm's internal activities regarding corporate social
responsibility' and 'responsibility towards employees'. From the responses to those items related to the factor
'corporate social responsibility in respect of aspects not
related to the economic activity of the firm', (Items 1, 3,
9, 10 & 15) it appears that the majority of managers is on-
ly to an average and lesser extent of the opinion that the
firm should make a contribution to the promotion of In the interpretation of the managers' responses to the
individual items, responses to those items that, according
to the first-order factor analysis, are related to a specific
factor, should be viewed together. The first-order factor
analysis showed that four factors or dimensions were
measured by the scale. These factors were identified as
'corporate social responsibility in respect of aspects not
related to the economic activity of the firm', 'the rela-
tionship between government and private enterprise as
far as the solution of social problems is concerned', 'the
firm's internal activities regarding corporate social
responsibility' and 'responsibility towards employees'. The general attitude of managers *This percentage is the average score expressed as a percentage
of the maximum score. that the neutral attitude of managers can be attributed to
uncertainty among managers about the scope and nature
of corporate social responsibility. Because it was deemed possible that the attitude of
managers could be related to the characteristics of the
respondents themselves or to their companies, these
characteristics -
job title and age of executives, racial
composition of company's work force, number of
employees in the firm, the assets of the company and the
industrial classification of the firm -
were cross-
tabulated with the respondents' attitude scores. The chi
square test indicated that no statistically significant varia-
tion at the 0,05 level in executive attitudes could be at-
tributed to the effects of the following moderator
variables: job title and age of the executive who com-
pleted the questionnaires, racial composition of com-
pany's work force, number of employees in the firm and
the amount of assets of the company. It was, however,
found that executives in companies in the building sector
and in financial institutions had a more positive attitude
than executives in manufacturing companies. Concerning the attitude of managers that business is in
a better position than government to identify social pro-
blems, it was stated in the personal interviews that this at-
titude may be attributed to the fact that managers are in
daily contact with workers and for that reason are in a
more favourable position to form an impression of the
social problems that exist in the workers' communities. During the interview it was opinioned that the South
African government is not sensitive enough to especially
those social problems experienced by the urban black
communities. In commenting on the role of business in
the identification of social problems, some managers ex-
pressed the opinion that, in the past, business tended to
hide behind the argument that they were prevented by law
from playing any role at all in this respect. The finding that
managers regard business as being in a more favourable
position to identify social problems, is seen by them as a
reflection of a new realization on the part of business that in
spite of existing legislation the opportunity does exist to
make a contribution to the solution of social problems. The general attitude of managers g
p
An indication of the attitude of executives towards
specific aspects of corporate social responsibility was ob-
tained by analyzing the responses on the individual items
of the scale. The attitude of managers towards specific aspects
of corporate social responsibility p
The next factor that was identified by means of the fac-
tor analysis, concerns 'specific internal activities by the
firm regarding corporate social responsibility' (Items 5,
11, 12, 13 & 16). Managers seem to think to an average
and lesser extent that the image of business will improve
if advertisements depict the contribution of business to
the solution of social problems (Item 5). The vast majori-
ty of mnagers are not in favour of creating a special divi-
sion in the organization structure of the firm to manage
its social involvement (Item 11). As far as socially respon-
sible action towards employees is concerned, the majority
of managers deem it important that work should be in-
teresting and challenging (Item 12). It seems, however, as
if managers in general are not in favour of workers' par-
ticipation in management decisions, especially in cases
where it affects profitability (Item 13). The general attitude of managers The general attitude of managers The general attitude of managers was measured by means
of the management scale. A high score on the scale (the
maximum score being 80) indicates a positive attitude
towards corporate social responsibility. A low score (the
minimum score being 16) indicates a negative attitude
towards
corporate
social
responsibility. The
measurements obtained appear in Table 2. The average score of 58070 indicates that managers are
neither extremely in favour of, nor extremely against cor-
porate social responsibility. The reaction during the per-
sonal interviews was that acceptance of social respon-
sibility by business is considered as a condition for max-
imizing profitability. Because business has a responsibili-
ty
towards
other interest groups,
for
example
shareholders, it is not possible to overemphasize cor-
porate social responsibility. It was further pointed out The questionnaire consisted of a nunlber of questions
or items representing a variety of aspects of corporate S. Afr. J. Bus. Mngmnt 1979, 10(3) 95 Table 2 The measurement of executives' general
attitude towards corporate social responsibility
Measurement
Score
Highest score
67
Lowest score
29
Average score
46
Median score
45
Standard deviation
7,71
Average percentage•
58
*This percentage is the average score expressed as a percentage
of the maximum score. Table 2 The measurement of executives' general
attitude towards corporate social responsibility cultural activities and charities (Items 1, 3 & 15). Although the majority feels that social involvement
should not be undertaken at the cost of profitability
(Item 9) it has to be pointed out that 380Jo only support
this point of view to an average extent, while 180Jo feel
that social involvement should be undertaken even at the
cost of profits. The majority of executives deem it impor-
tant that business should contribute to social progress,
for example by donating funds to educational institutions
(Item 10). Concerning the
business-government
relationship
(Items 2, 4 & 6) the minority feels that the likelihood to
maximize profitability increases in the long run if
business plays a leading role in the solution of social pro-
blems (Item 4). It seems as if managers view the involve-
ment of business in the solution of social problems main-
ly in terms of the first phase of social problem solving,
namely problem identification (Item 6). However, as far
as the solution of social problems is concerned, the ma-
jority feels that these problems are the responsibility of
the government (Item 2). The attitude of managers towards specific aspects
of corporate social responsibility
The managers' responses to the individual items of the
scale are shown in Table 3. From the responses to those items related to the factor
'corporate social responsibility in respect of aspects not
related to the economic activity of the firm', (Items 1, 3,
9, 10 & 15) it appears that the majority of managers is on-
ly to an average and lesser extent of the opinion that the
firm should make a contribution to the promotion of From responses to items concerning the company's
responsibility to look after the general well-being of
employees (Items 7, 8 & 14), it appears that the majority
of managers is either to an average or to a great extent of
the opinion that business has this responsibility. The vast
majority of the respondents is of the opinion that, to a S.-Afr. Tydskr. Bedryfsl. 1979, 10(3) S.-Afr. Tydskr. Bedryfsl. 1979, 10(3) 96 S.-Afr. Tydskr. Bedryfsl. 1979, 10(3)
96
Table 3 The responses of managers to the individual items of the scale (N = 123)
.,, respondents .,, respondents '!, respondents
answering to a
answering to a
answering to
Item
no. very great or
an average
lesser extent
or not at all
Average Standard
deviation
Wording
great extent
extent
score
To what extent should it be one of the responsibilities
of business to make financial contributions to pro-
mote culture in the community? 2 To what extent is business in a better position than
government to solve social problems? 3 To what extent should business, apart from its
responsibility to make profits, also give financial
assistance to charities? 4 To what extent does business, by playing a leading
role in the solution of serious social problems, im-
prove the possibility of making large profits in the
long run? 5 To what extent will the image of business greatly be
enhanced if the contributions of business to solve
social problems are shown in advertisements? 6 To what extent is business in a better position than
government to identify the problems of the com-
munity because it is not hampered by politics? 7 To what extent should business take care of its
employees' after their retirement? 8 To what extent should business do more for
employees than is required by law? 9 To what extent should business help solve social pro-
blems whether or not it helps to create those pro-
blems even though doing so may reduce short-run
profits and long-run returns? The attitude of managers towards specific aspects
of corporate social responsibility
The managers' responses to the individual items of the
scale are shown in Table 3. 10 How important is it that business should contribute
voluntarily to community development, e.g. by mak-
ing donations to educational institutions? 11 To what extent should companies have a separate
department (similar to sales, production) which at-
tends to the role of business in the solution of social
problems? 12 How important is it that jobs performed by
employees should be made challenging and in-
teresting by companies, even at the expense of max-
imum profits? 13 How important is it that employees should be able to
participate in decision-making, even if it is at the ex-
pense of profitability? 14 How important is it that business concerns itself with
the welfare of the families of its employees? IS How important is it that business voluntarily donates
money to people and organizations who are in need
of it? 16 To what extent can the profits made by business be
better utilized if it is used to solve social problems? great extent, business has the responsibility to look after
6
15
16
34
21
47
69
59
18
40
9
46
15
45
22
11
28
28
37
29
33
25
18
34
38
37
21
28
26
41
39
17
66
57
47
37
46
28
13
7
44
23
70
26
59
14
39
72
2,24
2,49
2,66
2,93
2,69
3,20
3,86
3,71
2,66
3,28
1,96
3,30
2,33
3,43
2,78
2,13
0,82
0,96
0,83
1,18
1,07
1,21
1,04
0,87
0,92
l,OS
1,03
1,15
1,06
0,95
1,03
0,99
investigating the extent to which managers feel that Table 3 The responses of managers to the individual items of the scale (N = 123 development. In view of the fact that, according to official govern-
ment policy, homeland development is an important
aspect of the social, economic and political development
of South Africa, managers were requested during the in-
terviews to comment on the finding that businessmen feel
that they can do little in respect of homeland develop-
ment. Managers pointed out that the homelands are not
regarded as economically viable by the business com-
munity and therefore do not attract business. In a
number of cases reference was made to unfortunate ex-
periences as far as location in a homeland was concerned,
for example in terms of poor infra-structure and an insuf •
ficiently qualified labour force. It is regarded that
government concessions to encourage location in
homeland areas are not sufficient. Specific aspects of the expectations of society can be
examined by analysing the responses to the individual
items of the scale for society. The expectations of society concerning specific
aspects of corporate social responsiblllty
The responses of society to the individual items of the
society scale are shown in Table 6. The expectations of society concerning specific
aspects of corporate social responsiblllty The responses of society to the individual items of the
society scale are shown in Table 6. The first order factor analysis of the responses of the
sample from society produced three factors, which were
identified as 'the expectation to accept a social respon-
sibility even at the cost of profit maximization', 'the
responsibility towards customers or the consumer', and
'the relationship between government and private enter-
prise as far as the solution of social problems is concern-
ed'. Furthermore, managers pointed out that business
prefers to be involved in the solution of the problems ex-
perienced by the black urban communities. This is given a
higher priority than homeland development because
blacks in urban areas are becoming more important as a
source of qualified labour and as a consumer market. An analysis of the responses to the items related to the
factor, 'the expectation to accept a social responsibility
even at the cost of profit maximization' (Items 11, 13, 14,
20 & 21), shows that more importance is attached to The expectations of society
General expectations of society
The extent to which society expects business to accept a
social responsibility was measured by means of the socie-
ty scale. Item
no. Wording Wording 7 To what extent should business take care of its
employees' after their retirement? investigating the extent to which managers feel that
business may contribute to the solution of specific social
problems. The managers' responses in this respect are
shown in Table 4. great extent, business has the responsibility to look after
the well-being of workers even after retirement (Item 7). The analysis of the responses to the individual items of
the scale indicates that managers are more positively in-
clined towards certain facets of corporate social respon-
sibility than towards others. It appears from Table 4 that the majority of the
managers feels that business may contribute substantially
to the training and education of blacks, conservation of
the environment, energy conservation, a better standard
of living for blacks in urban areas, and the military
preparedness of the country. In contrast to this,
managers generally feel that business can do little regar-
ding a lowering of population growth and homeland The extent to which business may contribute to the
solution of specific social problems which are cur-
rently experienced In South Africa An indication of the nature of possible business involve-
ment in the solution of social problems was obtained by S. Afr. J. Bus. Mngmnt 1979, 10(3) 97 Table4 The extent to which managers feel business may contribute to the solution of specific social pro-
blems
0/o respondents 0/o respondents 0/o respondents
answering to a
answering to
answering to a
very great or
an average
lesser extent
Average
Standard
Social problems
great extent
extent
or not at all
score
deviation
N
Military preparedness
47
Lower population growth
II
Training and education of blacks
79
Conservation of the environment
61
Better quality of life for blacks in
urban areas
SI
Homeland development
27
Energy conservation
S2
Combatting inflation
41
Decreasing unemployment
30
31
22
16
29
34
34
32
30
39
22
3,35
1,11
121
67
2,16
1,02
121
s
4,07
0,89
122
10
3,67
0,92
121
IS
3,49
0,98
121
39
2,92
1,06
122
16
3,SS
0,96
122
29
3,20
1,10
122
31
3,01
1,03
122 Table4 The extent to which managers feel business may contribute to the solution of specific social pro-
blems social responsibility than non-shareholders. Item
no. People living
in urban areas, as well as those in the lower income
bracket, were found to be significantly more positively
oriented towards corporate social responsibility than
those living in rural areas and earning a higher income. development. development. A high score on the scale (the maximum score
being l 15) indicates that, to a great extent, business is ex-
pected to be socially responsible while a low score is an
indication of the opposite point of view. The
measurements obtained appear in Table 5. The expectations of society
General expectations of society The expectations of society
General expectations of society Table 5 The measurement of general expecta-
tions of society as far as corporate social respon-
sibility is concerned Measurement
Score
Highest score
IIS
Lowest score
32
Average score
81
Median score
82
Standard deviation
12,19
Average percentage•
70
1This percentage is the average score expressed as a percentage
of the maximum score. The average percentage of 700/o indicates that society
to a great extent expects business to be socially responsi-
ble. In order to examine possible patterns as far as the at-
titude towards corporate social responsibility is concern-
ed, the individual scores on the scale were cross-tabulated
with the following moderator variables: shareholding in a
public company, type of community (rural or urban) and
income level. The chi square test of significance was used
at the O,OS level. It was found that shareholders in public
companies were significantly less in favour of corporate 1This percentage is the average score expressed as a percentage
of the maximum score. S.-Afr. Tydskr. Bedryfsl. 1979, 10(3) S.-Afr. Tydskr. Bedryfsl. 1979, 10(3) S.-Afr. Tydskr. Bedryfsl. 1979, 10(3)
of the scale for society (N = 924)
nts
o a
or
nt
% respondents
answering to
an average
extent
40
32
36
30
26
37
10
32
17
37
33
32
34
35
10
16
19
28
30
37
41
%respondents
answering to a
lesser extent
or not at all
21
31
19
15
20
23
6
34
18
35
21
17
16
29
6
3
9
25
14
10
20
Average Standard
score
deviation
3,28
3,05
3,38
3,61
3,51
3,23
4,40
2,98
3,89
2,87
3,38
3,50
3,52
3,08
4,42
4,27
4,05
3,36
3,67
3,65
3,28
1,10
1,23
1,12
1, 14
1,20
1,16
0,96
1,30
1,36
1, 17
l, 18
1,09
1,12
1,19
0,95
0,88
1,04
1,29
1,16
1,04
1,05 98 Table 8 The responses of society to the individual items of the scale for society (N = 924)
Item
no. Wording
To what extent should it be one of the responsibilities
of business to make financial contributions to pro-
mote culture in the community? 2 To what extent is business in a better position than
government to solve social problems? 3 To what extent does business, by playing a leading
role in the solution of serious social problems, im-
prove the possibility of making large profits in the
long-run? The expectations of society
General expectations of society 4 To what extent will the image of business greatly be
enhanced if the contributions of business to solve
social problems are shown in advertisements? 5 To what extent is business in a better position than
government to identify the problems of the com-
munity because it is not hampered by politics? 6 To what extent does business have to play a role in
the solution of the social problems in the communi-
ty? 7 To what extent should business take care of its
employees after their retirement? 8 How important is it that government should have
more say in the affairs of business to ensure a just
distribution of profits? 9 To what extent should business, even if a product or
service is very profitable, stop selling it if it is harmful
to the community in some way or another? 10 To what extent should shareholders and employees
have equal say in the management of business? 11 To what extent is the present economic situation the
result of the fact that business only aimed at max-
imum profits in the past? 12 To what extent should business do more for the com-
munity than merely paying tax to the government? 13 To what extent should business concentrate more on
the welfare of the community than on maximizing
profits? 14 How important is it that employees should be able to
participate in decision-making, even if it is at the ex-
pense of profitability? 15 To what extent does business, and not the govern-
ment, have the responsibility to see to it that
dishonest advertising does not take place? 16 How important is it that business should pay more
attention to the interests of customers than to new
ways to increase profits? 17 How important is it that business concerns itself with
the welfare of the families of its employees? 18 To what extent should business provide information
about its activities to the general public? 19 How important is it that business voluntarily donates
money to people and organizations who are in need
f i ? The expectations of society
General expectations of society O!o respondents
answering to a
very great or
great extent
39
37
45
55
54
40
84
34
65
28
46
51
50
36
84
81
72
47
56
% respondents
answering to
an average
extent
40
32
36
30
26
37
10
32
17
37
33
32
34
35
10
16
19
28
%respondents
answering to a
lesser extent
or not at all
21
31
19
15
20
23
6
34
18
35
21
17
16
29
6
3
9
25
Average Standard
score
deviation
3,28
3,05
3,38
3,61
3,51
3,23
4,40
2,98
3,89
2,87
3,38
3,50
3,52
3,08
4,42
4,27
4,05
3,36
1,10
1,23
1,12
1, 14
1,20
1,16
0,96
1,30
1,36
1, 17
l, 18
1,09
1,12
1,19
0,95
0,88
1,04
1,29 of society to the individual items of the scale for society (N = 924) Item
no. Wording Wording To what extent should it be one of the responsibilities
of business to make financial contributions to pro-
mote culture in the community? 2 To what extent is business in a better position than
government to solve social problems? 7 To what extent should business take care of its
employees after their retirement? S. Afr. J. Bus. Mngmnt 1979, 10(3) 99 social responsibility than to profit maximization. The
responses to items 11, 20 and 22 are an indication of
either a lack of trust in, or a lack of knowledge about, the
free enterprise system. The majority of the respondents,
for example, were of the opinion that business makes ex-
cessively high profits (Item 20) and that present economic
conditions may be ascribed to the excessively high profits
made by the business community in the past (Item 11). social responsibility than to profit maximization. The
responses to items 11, 20 and 22 are an indication of
either a lack of trust in, or a lack of knowledge about, the
free enterprise system. The majority of the respondents,
for example, were of the opinion that business makes ex-
cessively high profits (Item 20) and that present economic
conditions may be ascribed to the excessively high profits
made by the business community in the past (Item 11). towards corporate social responsibility, while a low score
· is indicative of a negative attitude. The maximum ob-
tainable score is 120 and the minimum score is 24. The student's t-test was used to test the significance of
the difference between the mean scores of managers and
society respectively. The nil hypothesis was that no dif-
ference existed between the attitude of managers and the
expectations of society as far as corporate social respon-
sibility was concerned. As alternative hypothesis it was
stated that society was more in favour of corporate social
responsibility than the business community. The result of
the hypothesis testing appears in Table 7. An analysis of responses in respect of the responsibility
of business towards customers or consumers (especially
Items IS & 16) reveals that society attaches great impor-
tance to the responsibility of business towards customers
and consumers in general. Item
no. Furthermore, it appears that it
is expected that this responsibility, which, among other
things, includes the prevention of dishonest advertising
practices (Item IS), should be assumed by the business
community itself and should not be imposed by govern-
ment action. Table 7 The difference between the attitude of
managers and expectation of society as far as cor-
porate social responsibility is concerned Managers
Society
Mean
Standard
Mean
Standard
score
deviation
score
deviation
pt'
66
11,01
83,12
12,79
0,00
'Probability of difference between mean scores. The 0,05 level
of significance was used. Concerning the division of responsibilities between
government and business (Items 2, 3 & 5) the view is held
that business is in a better position than the government
to identify social problems as well as to solve these pro-
blems (Items 2 & 5). Society is also of the opinion that
business improves its chances to maximize profitability in
the long run by playing a leading role in the solution of
social problems (Item 3). It would thus seem that, on the
one hand, society has great confidence in the ability of
the business community to solve social problems, while a
lack of confidence in business is reflected in the opinion
that business is responsible for the fact that the rich are
becoming richer and the poor are becoming poorer. 'Probability of difference between mean scores. The 0,05 level
of significance was used. It appears from Table 7 that society is significantly more
in favour of corporate social responsibility than
managers. During the personal interviews managers
pointed out that increased pressure on business to accept
a greater extent of social responsibility is already being
experienced. The majority of managers referred to a
growing tendency towards a socialist mentality in the
sense that society is expecting a more equal distribution
of income. It was expected that pressure on business to
accept a social responsibility would take different forms. In this respect reference was made to the role of con-
sumer organizations, organized labour, government ac-
tion and public opinion as expressed through mass
media. It was also pointed out during the interviews that
pressure is not only expected to be exerted by society, but
that companies may influence one another mutually, for
example by means of shareholding or in the case of a
company being an important supplier or client. Item
no. Although no factor related to corporate responsibility
towards employees was determined by means of the f ac-
tor analysis, the responses to items 7, 17 and 23 reveal
that society to a great extent expects business to take care
of employees. Society seems to be in favour of workers'
participation in managerial decision-making (Item 14). Equal representation of workers and owners is, however,
not particularly favoured. It appeared from the personal interviews that managers
view the abovementioned results as indicative of a lack of
insight on the part of society. Special reference in this
respect was made to the finding that social responsibility
is considered as being more important than profit max-
imization. This lack of insight was attributed to an un-
willingness among members of the public to lower their
standard of living during an economic recession. As a
result, it is increasingly expected that the employers take
care of the social welfare of employees. Furthermore,
managers felt that society is not well-informed about the
nature of the free enterprise system. A number of ex-
ecutives stressed the fact that the business community has
a responsibility to inform employees, as well as the public
in general, about the free enterprise system. Conclusions
Th
fi di The findings of the research indicate a growing social
awareness among South African business leaders. Managers' attitudes that business is in a better position
than government to identify social problems and the fact
that managers feel that business can make a contribution
to the solution of social problems, for example training
and education of blacks and energy conservation, are of
special importance. In order to facilitate a coordinated approach towards
the solution of the social problems of South Africa, it is
of great importance that some form of dialogue between
government and business should take place. The need for
this communication is reflected in the finding that
managers are sceptical about the economic viability of
the homelands, while according to government policy the
development of the homelands is of cardinal importance
for the political survival of South Africa. The extent of agreement between the attitude of
managers and the expectation of soci,ty as far as
corporate social responsibility Is concerned
· In order to determine the extent of agreement between
the attitude of managers and the expectation of society,
the scale which had been developed on the combined
responses of the two groups, was used. This scale consists
of 24 items. A high score indicates a positive attitude The extent of agreement between the attitude of
managers and the expectation of soci,ty as far as
corporate social responsibility Is concerned S.-Afr. Tydskr. Bedryfsl. 1979, 10(3) S.-Afr. Tydskr. Bedryfsl. 1979, 10(3) S.-Afr. Tydskr. Bedryfsl. 1979, 10(3) 100 Appendix In order to facilitate this dialogue between government
and business, the finding that business is in a superior
position to identify social problems should be taken as
starting point. The participation of business in this
respect is essential in the creation of a plan of action to
find solutions to the social problems of South Africa. "The construct validity of a scale is the extent to which
the scale may be said to measure a theoretical construct
-
in this case, the attitude towards corporate social
responsibility. As far as the difference between the attitude of
managers and the expectations of society are concerned,
it is important that some form of agreement should be
reached. On the one hand, society will have to accept that
the most important objective of business in a free enter-
prise system is to maximize profits. In order to establish
this acceptance it is of great importance that society
should be informed about the system of free enterprise. On the other hand, the business community should be
sensitive to the expectations of society and try to meet
these expectations within the framework of profit max-
imization. bBy means of the technique of factor analysis the
number of questions in terms of which the attitude of
an individual is measured, is reduced to a smaller
number of variables or 'factors'. A factor then is
representative of a group of questions which measures
a common characteristic. A Principal Factor Analysis
Model was used. The rotation of axes was carried out
according to the 'Direct Oblimin' criterion. 'This technique is an innovation of Professor J.M. Schepers of the Department of Psychology at the Rand
Afrikaans University. The author wishes to express his
thanks to Professor Schepers for his assistance in ap-
plying this technique. Acknowledgements The author is indebted to Professors J. Poolman and
A.J.B. Wiid for their assistance in conducting the
research which is described in this paper. The author also
acknowledges the financial assistance for the research
provided by the Human Sciences Research Council. dBy means of item analysis the reliability of individual
questions can be determined by investigating the stan-
dard deviation for each question, as well as the correla-
tion between the score for each question and the total
score for the questionnaire. References
I THE STAR, 23 May 1978.
2 EDMONDSON, W.B. Speech at Johannesburg Chamber of Com-
merce, 8 August 1978.
3 PRESTON, I.E. & POST, J.E. In: Private Management and Public
Policy -
the Principle of Public Responsibility, 1st Ed., Prentice-
Hall Inc., Englewood Cliffs, 1975, p. 106, proposed this method of
measurement of the public policy on a specific matter. 2 EDMONDSON, W.B. Speech at Johannesburg Chamber of Com-
merce, 8 August 1978. References I THE STAR, 23 May 1978. 'The Kuder Richardson 14 reliability coefficient is one
of the measurements that can be used to measure the
reliability of a scale. This coefficient is a quantification
in the form of a correlation coefficient of the con-
sistency of subjects' responses to all questions in the
scale.
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Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity
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BMC Microbiology
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cc-by
| 7,343
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RESEARCH ARTICLE Open Access Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Keywords: Mycobacterium tuberculosis, Cell wall, Rv1096, Peptidoglycan deacetylase, Lysozyme M. tuberculosis has a distinctive cell wall structure, which
is called the “core” mycolyl arabinogalactan-peptidoglycan
(mAGP) complex. It is well-known that the bacterial cell
wall is a reservoir for many essential biomolecules that
interact with the surrounding environment. Peptidoglycan
(PG) the skeletal structure of the cell wall, enables bac-
teria to resist osmotic pressure. The nucleotide-binding
oligomerization domain (Nods) proteins in host cells,
which have been identified as unique intracellular pattern-
recognition receptors of PG and PG-derived muropeptides,
are potential virulence factors [3,4]. Therefore, bacteria
may have developed PG modification properties to modu-
late Nods-mediated host surveillance [3]. This is evidenced
from the role PG plays in the pathogenesis of Streptococcus
pneumoniae [5], Listeria monocytogenes [6] and Helicobac-
ter pylori [7]. © 2014 Yang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Shufeng Yang1,2, Fei Zhang1, Jian Kang1, Wenli Zhang1, Guoying Deng1,2, Yi Xin3 and Yufang Ma1,4* hufeng Yang1,2, Fei Zhang1, Jian Kang1, Wenli Zhang1, Guoying Deng1,2, Yi Xin3 and Yufang Ma1 Abstract Background: Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG)
deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis
have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been
identified. Results: In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096
protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed
metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG
deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin−1;
and Kcat = 0.099 ± 0.007 (S−1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096
protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light
microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein,
M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that
Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion: We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of
Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG
may be involved in evasion of host immune defenses by M. tuberculosis. * Correspondence: yufang_ma@hotmail.com
1Department of Biochemistry and Molecular Biology, Dalian Medical
University, 9 W Lushun South Road, Dalian 116044, China
4Liaoning Provincial Core Lab of Glycobiology and Glycoengineering, Dalian
116044, China
Full list of author information is available at the end of the article Mycobacterium tuberculosis Rv1096 protein: gene
cloning, protein expression, and peptidoglycan
deacetylase activity Shufeng Yang1,2, Fei Zhang1, Jian Kang1, Wenli Zhang1, Guoying Deng1,2, Yi Xin3 and Yufang Ma1,4* Expression and purification of Rv1096 protein The pColdII-Rv1096 plasmid was transformed into E. coli
ER2566 cells (Novagen) by a chemical transformation
method [15]. E. coli ER2566 harboring the pColdII-
Rv1096 plasmid (ER2566/Rv1096, Table 1) was grown in
300 ml of LB broth containing ampicillin (100 μg/ml) at
37°C. Isopropyl-D-thiogalactopyranoside at a final con-
centration of 1 mM was added to the culture when the
OD600 reached 0.5, after which the culture was incu-
bated at 16°C for 24 h. Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Page 2 of 9 Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 italics) and Rv1096 primer 2 (5′ GGCAAGCTTTACG
CACCGTTATTTGGC 3′; the HindIII site is italics). The 876 bp PCR product was ligated to a pJET1.2 blunt
vector to generate a pJET-Rv1096 plasmid, the presence
of which was confirmed by DNA sequencing. Rv1096
was ligated to the NdeI and HindIII sites of pColdII
(Takara, Dalian, China) to generate the pColdII-Rv1096
plasmid for expression in E. coli. Rv1096 was also ligated
to the NdeI and HindIII sites of pVV2 (Colorado State
University, USA) to obtain the pVV2-Rv1096 M. smeg-
matis expression plasmid (Table 1). Deacetylation of PG in several bacterial species, such
as S. pneumonia, L. monocytogenes and Lactococcus lac-
tis, prevents fusion of the phagosome with macrophage
lysozyme [5,8-13]. Although peptidoglycan deacetylase
has been identified in some bacteria [5-8], it has not yet
been identified in M. tuberculosis. M. smegmatis is commonly used as a model for studying
gene function in M. tuberculosis because it proliferates
rapidly and is non-pathogenic. M. smegmatis and M. tu-
berculosis have the same basic cell wall structure [14]. Therefore, M. smegmatis peptidoglycan can be used as a
substrate to investigate peptidoglycan deacetylase activity. In this study, we cloned M. tuberculosis Rv1096 and
expressed Rv1096 protein in Escherichia coli and M. smegmatis. We determined the peptidoglycan deacety-
lase activity of purified Rv1096 and its biochemical char-
acteristics. We also investigated whether the Rv1096
protein in M. smegmatis was lysozyme resistant. M. smegmatis is commonly used as a model for studying
gene function in M. tuberculosis because it proliferates
rapidly and is non-pathogenic. M. smegmatis and M. tu-
berculosis have the same basic cell wall structure [14]. Therefore, M. smegmatis peptidoglycan can be used as a
substrate to investigate peptidoglycan deacetylase activity. In this study, we cloned M. tuberculosis Rv1096 and
expressed Rv1096 protein in Escherichia coli and M. smegmatis. We determined the peptidoglycan deacety-
lase activity of purified Rv1096 and its biochemical char-
acteristics. We also investigated whether the Rv1096
protein in M. smegmatis was lysozyme resistant. Rv1096 cloning and expression vector construction v1096 cloning and expression vector construction The Rv1096 was amplified from M. tuberculosis H37Rv
genomic DNA (Colorado State University, USA) using
Pfu DNA polymerase with Rv1096 primer 1 (5′ TTCA-
TATGCCGAAGCGACCCGACAAC 3′; the NdeI site is Table 1 Bacteria and plasmids
Bacteria and plasmids
Relevant characteristic(s)
Resource
Strains
E. coli NovaBlue
Used for cloning and propagation of plasmids
Novagen
E. coli ER2566
Used for expression of Rv1096 protein
Novagen
M. smegmatis
mc2155 strain, used for expression of Rv1096 protein and preparation of peptidoglycan
ATCC
E. coli ER2566/Rv1096
E. coli ER2566 carrying pColdII-Rv1096 plasmid
This work
M. smegmatis/Rv1096
M. smegmatis mc2155 carrying pVV2-Rv1096 plasmid
This work
Plasmids
pJET1.2/blunt vector
Carries ampR gene; used for cloning PCR product
Fermentas
pColdII-Rv1096
Carries ampR gene; used for expression Rv1096 protein in E. coli ER2566
This work
pVV2-Rv1096
Carries kanR gene; used for expression of Rv1096 protein in M. smegmatis mc2155
This work Bacterial strains and growth conditions Bacterial strains and growth conditions
E. coli NovaBlue (Novagen, Madison, WI) and ER2566
(Novagen) strains were routinely grown in Luria-Bertani
media (LB, Invitrogen, Carlsbad, CA). The M. smegmatis
mc2155 (ATCC, USA) strain was grown in LB broth
containing 0.05% (v/v) Tween 80 (LBT) or LB agar at
37°C. Antibiotics were added at appropriate concentra-
tions if needed. To prepare PG, M. smegmatis mc2155
was grown in M9 minimal glucose medium (12.8 g so-
dium phosphate heptahydrate, 3 g potassium phosphate
monobasic, 0.5 g sodium chloride, 1 g ammonium chlor-
ide, 0.24 g magnesium sulfate, 4 g glucose and 11.1 mg
calcium chloride per L). The pVV2-Rv1096 plasmid was transformed into M. smegmatis mc2155 using an electroporation method [15]. M. smegmatis mc2155 harboring the pVV2-Rv1096 plas-
mid (M. smegmatis/Rv1096, Table 1) was grown in 300 ml
of LBT broth with kanamycin at 50 μg/ml at 37°C for
24 h. The cultures were centrifuged at 5000 × g for 15 min
and the cell pellets were resuspended in 5 ml of lysis
buffer (500 mM Tris-HCl, pH 8.0, 20 mM NaCl and
20% glycerol) with 1 mM phenylmethyl sulfonyl fluoride. After sonication, the lysates were centrifuged at 15000 ×
g for 20 min and the supernatant fraction was loaded
onto a Ni-NTA column (Qiagen, Hilden, Germany) by
gravity flow. The column was washed with 20 ml of
wash buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl,
20% glycerol and 30 mM imidazole). The purified pro-
tein was eluted with 10 ml of elution buffer (20 mM Background Mycobacterium tuberculosis, the agent of tuberculosis, is
associated with greater morbidity and longer dormancy in-
fection times in humans than any other type of bacterial
illness. Approximately one third of the population world-
wide are infected with M. tuberculosis, which causes nearly
two million deaths each year [1]. The chronic state and
dormancy of tuberculosis implies that M. tuberculosis has
developed sophisticated strategies to modify and evade the
innate and adaptive immune surveillance mechanisms of
humans [2]. * Correspondence: yufang_ma@hotmail.com
1Department of Biochemistry and Molecular Biology, Dalian Medical
University, 9 W Lushun South Road, Dalian 116044, China
4Liaoning Provincial Core Lab of Glycobiology and Glycoengineering, Dalian
116044, China
Full list of author information is available at the end of the article * Correspondence: yufang_ma@hotmail.com
1Department of Biochemistry and Molecular Biology, Dalian Medical
University, 9 W Lushun South Road, Dalian 116044, China
4Liaoning Provincial Core Lab of Glycobiology and Glycoengineering, Dalian
116044, China
Full list of author information is available at the end of the article Lysozyme susceptibility assays y
y
y
y
To investigate whether the Rv10196 protein contributed to
lysozyme resistance in M. smegmatis, wild-type M. smeg-
matis or M. smegmatis/Rv1096 with over-expressed Rv1096
protein were treated with lysozyme. Both bacterial strains
were incubated in LBT medium at 37°C. When the OD600
reached ~0.2, the cultures were divided into two equal
volumes parts. One part was treated with lysozyme (Sigma-
Aldrich) at a final concentration of 200 μg/ml; the other
was not given this treatment. Bacterial growth was moni-
tored by measuring the optical density at 600 nm. Bacterial
viability was evaluated by counting the number of colony
forming units (CFU) per milliliter on LB agar [23]. Table 1 Bacteria and plasmids Table 1 Bacteria and plasmids ds
Relevant characteristic(s)
Resource
Used for cloning and propagation of plasmids
Novagen
Used for expression of Rv1096 protein
Novagen
mc2155 strain, used for expression of Rv1096 protein and preparation of peptidoglycan
ATCC
E. coli ER2566 carrying pColdII-Rv1096 plasmid
This work
M. smegmatis mc2155 carrying pVV2-Rv1096 plasmid
This work
Carries ampR gene; used for cloning PCR product
Fermentas
Carries ampR gene; used for expression Rv1096 protein in E. coli ER2566
This work
Carries kanR gene; used for expression of Rv1096 protein in M. smegmatis mc2155
This work Page 3 of 9 Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Yang et al. BMC Microbiology 2014, 14:174
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http://www.biomedcentral.com/1471-2180/14/174 Tris-HCl, pH 8.0, 500 mM NaCl and 200 mM imidazole),
and the first 3 ml was collected for sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and west-
ern blotting, as well as deacetylase activity detection. The
purified protein (1.25 μg) was subjected to 12% SDS-PAGE
and then transferred to a nitrocellulose membrane (PALL,
NY, USA) in blotting buffer (20 mM Tris-base, 150 mM
glycine and 20% methanol, pH 8.3). After blocking with
10% non-fat dry milk in TBST buffer (10 mM Tris-HCl,
pH 8.0, 150 mM NaCl and 0.05% Tween 20), the mem-
brane was incubated with a monoclonal (anti)-polyhistidine
His-1 antibody (1:5000; Sigma-Aldrich). The membrane
was washed with TBST buffer three times and then incu-
bated with alkaline-phosphatase conjugated anti-mouse-
IgG (1:2500, Sigma-Aldrich). The His6-tagged-protein band
was visualized with 5-bromo-4-chloro-3-indolyl phosphate
and nitro blue tetrazolium (Sigma-Aldrich) solution. temperature centrifugation at 27,000 × g for 30 min and
then washed as described above. The PG was dissolved in
50 mM HEPS buffer (pH 7.0) at 1 mg/ml until further use. Preparation of M. smegmatis PG Preparation of M. smegmatis PG
M. smegmatis PG was prepared from cell wall fractions as
described previously [16-18]. Briefly, a 500 ml culture of
M. smegmatis mc2155 in M9 minimal glucose medium
was harvested when the OD600 reached 0.6, after which
the cells were washed three times with pre-cooled phos-
phate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 2 mM KH2PO4, pH 7.0). The pellets
were resuspended in distilled water to 0.2 g/ml, mixed
with an equal volume of boiling 8% SDS added drop-wise
with continuous boiling for 30 min. A cell-wall-enriched
fraction was obtained by centrifugation at 100,000 × g at
20°C for 60 min, followed by three washes with pre-cooled
PBS. The pellet was washed with distilled water at least six
times to remove the SDS. The sample was resuspended in
5 ml of buffer (10 mM Tris-HCl and 10 mM NaCl,
pH 7.0) and then sonicated for 5 min. α-amylase and imid-
azole were added to the sample at final concentrations of
100 μg/ml and 0.32 M, respectively, and the solution was
incubated at 37°C for 2 h to remove glycogen. Afterwards,
proteinase K was added to the sample at a final concentra-
tion of 100 μg/ml, followed by incubation at 37°C for 1.5 h
to remove lipoprotein. The proteinase K solution was then
inactivated by addition of an equal volume of boiling 8%
SDS with vigorous stirring for 15 min. The mixture was
ultracentrifuged at 100,000 × g at 20°C for 30 min. The
pelleted material was washed as described above. The
resulting mAGP (mycolyl-arabinogalactan-peptidoglycan)
complex was washed with acetone and dried under a
vacuum. Mycolic acids were removed with 1% potassium
hydroxide in methanol at 37°C for 72 h. After room
temperature centrifugation at 27,000 × g for 30 min, the pel-
leted arabinogalactan-PG was washed with distilled water
twice and dried under a vacuum. Arabinogalactan was re-
moved by washing with 49% hydrofluoridic acid at 4°C for
120 h with stirring. The resulting PG was pelleted by room Deacetylase activity assays Deacetylase activity assays
The acetyl group released from the PG was measured
using an acetic acid detection kit (Roche, Darmstadt,
Germany). Briefly, Rv1096 protein (2.88 μg/ml) prepared
from ER2566/Rv1096 and M. smegmatis/Rv1096 were
separately incubated with M. smegmatis PG. The reactions
were performed at 37°C for 30 min and stopped by
10 min boiling. After room temperature centrifugation at
5000 × g for 10 min, the supernatant was collected for
acetic acid measurement using a spectrophotometric assay
accordingly to the kit instructions. The kinetic properties of the Rv1096 protein toward
M. smegmatis PG were determined as described previ-
ously [19]. The molarity of M. smegmatis PG was calcu-
lated based the assumption that M. smegmatis PG is
primarily composed of repeat units of GlcNAc-MurNAc
(MurNGlyc)-L-Ala-D-Glu-A2pm, MW 868.8 [20-22]. First,
the initial velocity was evaluated according to the duration
of each reaction (5, 10, 15, 30 or 45 min) and the Rv1096
concentration (1.22, 2.88 or 3.65 μg/ml) curves. Then, the
optimal conditions for the enzymatic reactions were deter-
mined. Based on the initial velocity and the optimal condi-
tions that we identified, the steady-state kinetic parameters
were determined by a Lineweaver-Burke plot. Results (Olympus CHB, Japan). The cells for electron microscopic
analysis were fixed with 2.5% glutaraldehyde, followed by
post-fixation at room temperature for 2 h with 1% os-
mium tetroxide. The samples were dehydrated with etha-
nol, which was replaced with liquid carbon dioxide by
critical point drying. The dried samples were applied to a
silicon wafer slide and sputter-coated with gold before
examination by an electronic microscope (JSM-6360 scan-
ning electron, JEOL, Japan). gy
y
The amino acid sequences of the Rv1096 protein and other
known polysaccharide deacetylases [5,8-12] were com-
pared by Multalin analysis. The S. pneumoniae PgdA pro-
tein (spPdgA), L. monocytogenes PgdA (lmo0415) and L. lactis PgdA (XynD) were identified as N-acetylglucosamine
deacetylase proteins containing CE-4 NodB domains
[10,25,26]. Rv1096 also contained a CE-4 NodB domain. Rv1096 shared 31.6% sequence identity with the S. pneu-
moniae PgdA protein, whose deacetylase domain has
recently been defined as a crystal structure [10,25]. The
catalytic core of the amino acids involved in deacetylase
activity is highly conserved between Rv1096 and S. pneu-
moniae PgdA proteins (Figure 1). Morphology of the M. smegmatis strains after lysozyme
treatment Light microscopy and electron microscopy were used to
investigate whether the Rv1096 protein affected the
morphology of M. smegmatis in the presence of lyso-
zyme. Bacteria that were treated with lysozyme for 9 h
were harvested by centrifugation at 4,500 × g at 4°C for
10 min, after which the pellets were washed with ste-
rilized 1 M PBS (pH 7.0), three times. Samples were pre-
pared for Ziehl-Neelsen acid-fast staining as described
previously [24], and observed under a light microscope Page 4 of 9 Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Rv1096 exhibits peptidoglycan deacetylase activity Figure 2 Rv1096 protein analysis. SDS-PAGE (A) and western blot
(B) analysis of purified Rv1096 protein. Lane 1, purified Rv1096
protein over-expressed in M. smegmatis; Lane 2, purified Rv1096
protein over-expressed in E. coli. M, PageRuler™Prestained Protein
Ladder (MBI Fermentas, Lithuania). Rv1096 exhibits peptidoglycan deacetylase activity
To assess its deacetylase activity, Rv1096 protein at 1.22,
2.88, 3.65 or 4.74 μg/ml was incubated with M. smegmatis
PG at 1 mg/ml. The acetyl group released from PG was
measured using an acetic acid detection kit (Roche Diag-
nostics, Germany). The results revealed that the purified
Rv1096 protein over-expressed in both E. coli and M. smegmatis exhibited peptidoglycan deacetylase activity
(Figure 3A). There was no significant difference between
the Rv1096 proteins prepared from either bacterium in
terms of their specific enzymatic activities (p > 0.05). Therefore, the Rv1096 protein prepared from E. coli was
used for the following enzyme kinetics experiments as it
was easier to prepare and produced a greater yield than
that produced in M. smegmatis. Figure 2 Rv1096 protein analysis. SDS-PAGE (A) and western blot
(B) analysis of purified Rv1096 protein. Lane 1, purified Rv1096
protein over-expressed in M. smegmatis; Lane 2, purified Rv1096
protein over-expressed in E. coli. M, PageRuler™Prestained Protein
Ladder (MBI Fermentas, Lithuania). According to the time versus concentration curve
(Figure 3B), when the Rv1096 protein concentration was
2.88 μg/ml, acetic acid was released at a constant rate over
a 30 min period. Therefore, the initial velocity range fell
within 30 min, and the optimal concentration for Rv1096
was 2.88 μg/ml. The optimal deacetylation reaction condi-
tions were determined by changing the pH and temperature
of the reaction. From this, the optimal pH was found
to be 7.0 and the optimal temperature 37°C (data not
shown). The kinetic parameters were calculated by a Rv1096 overexpressed in E. coli and M. smegmatis is a
soluble protein Rv1096 overexpressed in E. coli and M. smegmatis is a
soluble protein Soluble Rv1096 protein, over-expressed in both E. coli and
M. smegmatis, was purified by Ni-NTA affinity chromatog-
raphy. The purified Rv196 protein was analyzed by SDS-
PAGE and western blotting (Figure 2). The results showed
that purified Rv1096 had a molecular weight of 35 kDa. Figure 3 PG deacetylase activity of purified Rv1096 protein. A) Acetic acid released by the Rv1096 protein over-expressed in E. coli and M. smegmatis. PG (1 mg/ml) from wild-type M. Rv1096 exhibits peptidoglycan deacetylase activity smegmatis was used as a substrate and mixed with different concentrations of purified Rv1096
(1.22, 2.88, 3.65 or 4.74 μg/ml). After incubation at 37°C for 30 min, acetyl group release was detected using an acetic acid kit. Squares indicate
Rv1096 protein over-expressed in E. coli and triangles indicate Rv1096 protein over-expressed in M. smegmatis. Values are means ± SD. B) Time
course and concentration curve for Rv1096. Purified Rv1096 protein at 1.22, 2.88 or 3.65 μg/ml was incubated with M. smegmatis PG (1 mg/ml)
substrate at 37°C for 5, 10, 15, 30 and 50 min. Plotted values are means ± SD. C) Km and Vmax values for Rv1096 PG deacetylase activity. Kinetic
parameters were calculated by a double reciprocal plot. D) Rv1096 protein exhibited a metallo dependent enzymatic activity. Various divalent
cations (Mg2+, Mn2+, Co2+, Ca2+or Zn2+) were added to a final concentration of 0.5 μM. Values are mean ± SD. Figure 3 PG deacetylase activity of purified Rv1096 protein. A) Acetic acid released by the Rv1096 protein over-expressed in E. coli and M. smegmatis. PG (1 mg/ml) from wild-type M. smegmatis was used as a substrate and mixed with different concentrations of purified Rv1096
(1.22, 2.88, 3.65 or 4.74 μg/ml). After incubation at 37°C for 30 min, acetyl group release was detected using an acetic acid kit. Squares indicate
Rv1096 protein over-expressed in E. coli and triangles indicate Rv1096 protein over-expressed in M. smegmatis. Values are means ± SD. B) Time
course and concentration curve for Rv1096. Purified Rv1096 protein at 1.22, 2.88 or 3.65 μg/ml was incubated with M. smegmatis PG (1 mg/ml)
substrate at 37°C for 5, 10, 15, 30 and 50 min. Plotted values are means ± SD. C) Km and Vmax values for Rv1096 PG deacetylase activity. Kinetic
parameters were calculated by a double reciprocal plot. D) Rv1096 protein exhibited a metallo dependent enzymatic activity. Various divalent
cations (Mg2+, Mn2+, Co2+, Ca2+or Zn2+) were added to a final concentration of 0.5 μM. Values are mean ± SD. Figure 3 PG deacetylase activity of purified Rv1096 protein. A) Acetic acid released by the Rv1096 protein over-expressed in E. coli and M. smegmatis. PG (1 mg/ml) from wild-type M. smegmatis was used as a substrate and mixed with different concentrations of purified Rv1096
(1.22, 2.88, 3.65 or 4.74 μg/ml). After incubation at 37°C for 30 min, acetyl group release was detected using an acetic acid kit. Statistical analysis Data are summarized as mean value ± standard deviation
(SD). Data were assessed by two-tailed unpaired t tests. A
p value of <0.05 was considered statistically significant. Figure 1 Multiple sequence alignment of Rv1096, spPgdA, lmo0415 and XynD proteins. spPgdA, S. pneumoniae peptidoglycan GlcNAc deacetylase
(gi:14972969); lmo0415, L. monocytogenes peptidoglycan GlcNAc deacetylase (gi:16409792); XynD, L. Lactis peptidoglycan GlcNAc deacetylase
(gi:281490824). Black regions indicate identical residues in the four proteins, while residues conserved between at least two of the proteins are marked by
boxes. Two catalytic histidine residues (H-326 and H-330) are conserved among Rv1096 and the other three deacetylases [10]. Rv1096 contains the metal
ligand sites, Asp (D-275), Arg (A-295), Asp (D-391) and His (H-417) residues, which were identified in the S. pneumonia PgdA protein. Figure 1 Multiple sequence alignment of Rv1096, spPgdA, lmo0415 and XynD proteins. spPgdA, S. pneumoniae peptidoglycan GlcNAc deacetylase
(gi:14972969); lmo0415, L. monocytogenes peptidoglycan GlcNAc deacetylase (gi:16409792); XynD, L. Lactis peptidoglycan GlcNAc deacetylase
(gi:281490824). Black regions indicate identical residues in the four proteins, while residues conserved between at least two of the proteins are marked by
boxes. Two catalytic histidine residues (H-326 and H-330) are conserved among Rv1096 and the other three deacetylases [10]. Rv1096 contains the metal
ligand sites, Asp (D-275), Arg (A-295), Asp (D-391) and His (H-417) residues, which were identified in the S. pneumonia PgdA protein. Page 5 of 9 Page 5 of 9 Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Rv1096 exhibits peptidoglycan deacetylase activity M. smegmatis/Rv1096 exhibits lysozyme resistance To determine the contribution of Rv10196 protein to M. smegmatis resistance to lysozyme, M. smegmatis/Rv1096
and wild-type M. smegmatis cultures were divided into
two parts at the beginning of the exponential growth
phase. Test samples received 200 μg/ml lysozyme, unlike
the control samples. As shown in Figure 4A, the wild-type
M. smegmatis culture suspension treated with lysozyme
lost its opaque, hazy appearance, becoming transparent at
the end of the exponential growth phase, or shortly after
reaching stationery phase. Its OD600 and CFU values de-
creased, indicating that cell lysis took place in the wild-
type lysozyme-treated M. smegmatis. The M. smegmatis/
Rv1096 growth curves for lysozyme treatment showed al-
most no difference to the lysozyme-untreated group, sug-
gesting that Rv10196 protein contributed to M. smegmatis
resistance to lysozyme degradation. There was also no sig-
nificant difference between the M. smegmatis/Rv1096 and
wild-type M. smegmatis treated with lysozyme in terms of
bacterial viability within the initial four hours of growth
(Figure 4B). However, viable wild-type M. smegmatis bac-
teria decreased rapidly after lysozyme treatment for 4 h. A
significant difference (P < 0.01) in viability was observed
between M. smegmatis/Rv1096 and wild-type M. smegma-
tis after lysozyme treatment for 9 h. About 107 wild-type
M. smegmatis cells survived, whereas only 1016 M. smeg-
matis/Rv1096 cells survived. Figure 4 Lysozyme susceptibility assay. A) Lysozyme treatment
growth curves for M. smegmatis/Rv1096 and wild-type M. smegmatis. M. smegmatis/Rv1096 (square) and wild-type M. smegmatis (triangle) were
grown in LBT medium at 37°C to an OD600 of 0.2; the cultures were then
divided into two parts. One part (closed symbol) was treated with
lysozyme, the other part was not. Three microliter samples from each
culture were collected at 1 h intervals for OD600 measurements. M. smegmatis/Rv1096 showed significantly greater resistance to lysozyme
than did wild-type M. smegmatis (**P < 0.01). Values are means ± SD. B) Cell survival curves for M. smegmatis/Rv1096 and wild-type M. smegmatis
under lysozyme treatment. M. smegmatis/Rv1096 (square) and wild-type
M. smegmatis (triangle) were each grown in LBT medium at 37°C to an
OD600 of 0.2, then the cultures were divided into two parts. One part
(closed symbol) was treated with lysozyme, the other part was not. Three
microliter culture samples were collected at 1 h intervals to measure
CFU/ml. M. smegmatis/Rv1096 exhibited greater cell survival than that of
the wild-type bacterium (**P < 0.01). Values are means ± SD. wild-type M. Rv1096 exhibits peptidoglycan deacetylase activity Squares indicate
Rv1096 protein over-expressed in E. coli and triangles indicate Rv1096 protein over-expressed in M. smegmatis. Values are means ± SD. B) Time
course and concentration curve for Rv1096. Purified Rv1096 protein at 1.22, 2.88 or 3.65 μg/ml was incubated with M. smegmatis PG (1 mg/ml)
substrate at 37°C for 5, 10, 15, 30 and 50 min. Plotted values are means ± SD. C) Km and Vmax values for Rv1096 PG deacetylase activity. Kinetic
parameters were calculated by a double reciprocal plot. D) Rv1096 protein exhibited a metallo dependent enzymatic activity. Various divalent
cations (Mg2+, Mn2+, Co2+, Ca2+or Zn2+) were added to a final concentration of 0.5 μM. Values are mean ± SD. Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Page 6 of 9 Figure 4 Lysozyme susceptibility assay. A) Lysozyme treatment
growth curves for M. smegmatis/Rv1096 and wild-type M. smegmatis. M. smegmatis/Rv1096 (square) and wild-type M. smegmatis (triangle) were
grown in LBT medium at 37°C to an OD600 of 0.2; the cultures were then
divided into two parts. One part (closed symbol) was treated with
lysozyme, the other part was not. Three microliter samples from each
culture were collected at 1 h intervals for OD600 measurements. M. smegmatis/Rv1096 showed significantly greater resistance to lysozyme
than did wild-type M. smegmatis (**P < 0.01). Values are means ± SD. B) Cell survival curves for M. smegmatis/Rv1096 and wild-type M. smegmatis
under lysozyme treatment. M. smegmatis/Rv1096 (square) and wild-type
M. smegmatis (triangle) were each grown in LBT medium at 37°C to an
OD600 of 0.2, then the cultures were divided into two parts. One part
(closed symbol) was treated with lysozyme, the other part was not. Three
microliter culture samples were collected at 1 h intervals to measure
CFU/ml. M. smegmatis/Rv1096 exhibited greater cell survival than that of
the wild-type bacterium (**P < 0.01). Values are means ± SD. double reciprocal plot (Figure 3C): Km = 0.910 ± 0.007 mM;
Vmax = 0.514 ± 0.038 μM min−1; and Kcat = 0.099 ± 0.007
(S−1). As shown in Figure 1, Rv1096 contained the same Asp-
His-His conserved residues known to interact with Co2+
in S. pneumoniae PgdA. To ensure that Rv1096 was also a
metallo-dependent deacetylase, various divalent cations
(Mg2+, Mn2+, Co2+, Ca2+ or Zn2+) were added to the reac-
tion buffer, each at a final concentration of 0.5 μM; EDTA
at 50 μM served as a control. M. smegmatis/Rv1096 exhibits lysozyme resistance smegmatis had an irregular appearance (en-
larged shape, destructed cell wall and wrinkled surface) in
the presence of lysozyme, whereas M. smegmatis/Rv1096
had a regular shape, undamaged cell wall and smooth sur-
face after 9 h lysozyme treatment (Figure 6). Rv1096 exhibits peptidoglycan deacetylase activity The results showed that the
enzymatic reactivity reached the highest level in the pres-
ence of Co2+; however, enzymatic activity was lost in the
presence of EDTA (Figure 3D). Therefore, we determined
that Rv1096 is a metallo-dependent PG deacetylase. The M. smegmatis/Rv1096 cell wall was undamaged by
9 h of lysozyme treatment M. tuberculosis Rv1096 protein, S. pneumoniae PgdA
protein (spPdgA), L. monocytogenes PgdA (lmo0415),
and L. lactis PgdA (XynD) are carbohydrate esterase 4
(CE-4) superfamily members. The CE-4 superfamily in-
cludes peptidoglycan GlcNAc deacetylases, rhizobial
NodB chito-oligosaccharide deacetylases, chitin deace-
tylases, acetyl xylan esterases, and xylanases [27]. The
substrates of these enzymes are polymers or basic struc-
tures that assemble PG backbone glycan strands. In this
study, Rv1096 protein, over-expressed in both E. coli Because the most apparent differences in bacterial growth
and viability were observed (Figures 4A and B) after treat-
ment with lysozyme for 9 h, morphological observations
were performed at this time point. The results of the Ziehl-
Neelsen acid-fast staining showed that wild-type M. smeg-
matis lost its acid-fastness and became blue dyed, whereas
M. smegmatis/Rv1096 retained its acid-fastness (Figure 5). Scanning electronic microscopy (SEM) showed that the Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Page 7 of 9 Page 7 of 9 Figure 5 Acid-fast staining of M. smegmatis/Rv1096 and wild-type cells. A) Wild-type M. smegmatis without lysozyme treatment,
B) wild-type M. smegmatis with lysozyme treatment, C) M. smegmatis/Rv1096 without lysozyme treatment and, D) M. smegmatis/Rv1096 with
lysozyme treatment (×1000). Lysozyme treatment was for 9 h. Figure 5 Acid-fast staining of M. smegmatis/Rv1096 and wild-type cells. A) Wild-type M. smegmatis without lysozyme treatment,
B) wild-type M. smegmatis with lysozyme treatment, C) M. smegmatis/Rv1096 without lysozyme treatment and, D) M. smegmatis/Rv1096 with
lysozyme treatment (×1000). Lysozyme treatment was for 9 h. residues (H-326 and H-330) [10]. The metal ligand sites,
including Asp (D-275), Arg (A-295), Asp (D-391) and His
(H-417) residues, which were identified in the S. pneumo-
nia PgdA protein [5,10,28], are all present in the Rv1096
protein. These highly conserved sequences in Rv1096 and M. smegmatis, was able to deacetylate M. smegmatis
peptidoglycan. Therefore, M. tuberculosis Rv1096 pro-
tein is a peptidoglycan deacetylase. As shown in Figure 1, Rv1096 and three other deacety-
lases share sequence conservation at two catalytic histidine Figure 6 Scanning electron micrographs of M. smegmatis/Rv1096 and wild-type M. smegmatis. A) Wild-type M. smegmatis without lysozyme
treatment, B) wild-type M. smegmatis with lysozyme treatment, C) M. smegmatis/Rv1096 without lysozyme treatment and, D) M. smegmatis/Rv1096
with lysozyme treatment. Lysozyme treatment was for 9 h. Figure 6 Scanning electron micrographs of M. smegmatis/Rv1096 and wild-type M. smegmatis. A) Wild-type M. smegmatis without lysozyme
treatment, B) wild-type M. smegmatis with lysozyme treatment, C) M. Competing interests Competing interests
The authors declare that they have no competing interests. Co
pet
g
te ests
The authors declare that they have no competing interests. 1.
Watts G: WHO annual report finds world at a crossroad on tuberculosis.
BMJ 2012, 345:e7051–e7061. 1. Watts G: WHO annual report finds world at a crossroad on tuberculosis. BMJ 2012, 345:e7051–e7061. 2. Behar SM, Divangahi M, Remold HG: Evasion of innate immunity by
Mycobacterium tuberculosis: is death an exit strategy? Nat Rev Microbiol
2010, 8(9):668–674. 2. Behar SM, Divangahi M, Remold HG: Evasion of innate immunity by
Mycobacterium tuberculosis: is death an exit strategy? Nat Rev Microbiol
2010, 8(9):668–674. 3. Boneca IG: The role of peptidoglycan in pathogenesis. Curr Opin Microbiol
2005, 8(1):46–53. 3. Boneca IG: The role of peptidoglycan in pathogenesis. Curr Opin Microbiol
2005, 8(1):46–53. 4. Girardin SE, Travassos LH, Herve M, Blanot D, Boneca IG, Philpott DJ, Sansonetti
PJ, Mengin-Lecreulx D: Peptidoglycan molecular requirements allowing
detection by Nod1 and Nod2. J Biol Chem 2003, 278(43):41702–41708. 5. Vollmer W, Tomasz A: Peptidoglycan N-acetylglucosamine deacetylase, a
putative virulence factor in Streptococcus pneumoniae. Infect Immun 2002,
70(12):7176–7178. 6. Lenz LL, Mohammadi S, Geissler A, Portnoy DA: SecA2-dependent
secretion of autolytic enzymes promotes Listeria monocytogenes
pathogenesis. Proc Natl Acad Sci U S A 2003, 100(21):12432–12437. Acknowledgements
h
k This work was supported by the National Basic Research Program of China
(No. 2012CB518803) and Research Fund for the Doctoral Program of Higher
Education of China (No. 20112105110002). Authors’ contributions SY constructed expression vectors, prepared Rv1096 protein and conducted
lysozyme susceptibility assays, deacetylase activity assays, as well as prepared
this manuscript. FZ purified Rv1096 protein and determined kinetic parameters
of PG deacetylase. JK performed bioinformatic analyses of Rv1096 with known
PG deacetylases. WZ performed bioinformatic analysis of Rv1096 and the
statistical analyses. GD prepared samples for acid-fast staining and SEM. YX
participated in designing experiments of the study. YM proposed this project,
designed most of experiments and prepared this manuscript. All authors read
and approved the final manuscript. It has been reported that PG deacetylase contributes to
lysozyme resistance in some bacterial species, such as
Bacillus cereus [29], S. pneumonia [10] , L. monocytogenes
[6] and Shigella flexneri [28]. Generally, pdgA mutants are
more sensitive to lysozyme degradation in the stationary
phase. Similarly, M. smegmatis over-expressing Rv1096
protein showed remarkable resistance to lysozyme at the
end of log phase growth. In the present study, the viability
of M. smegmatis/Rv1096 was 109-fold higher than that of
wild-type M. smegmatis after lysozyme treatment, indicat-
ing that PG deacetylation by the Rv1096 deacetylase had
increased lysozyme resistance. The morphological changes
observed between wild-type M. smegmatis and M. smeg-
matis/Rv1096 provides strong evidence that Rv1096 activ-
ity helped to preserve the integrity of the cell wall during
lysozyme treatment. Wild-type M. smegmatis lost its
acid-fastness because of the increased cell wall perme-
ability caused by lysozyme treatment. SEM observations
showed that wild-type M. smegmatis had a wrinkled cell
surface with outward spilling of its cell contents, while
M. smegmatis/Rv1096 maintained its cell wall integrity
and acid fastness. Therefore, it is likely that the func-
tionality of the Rv1096 protein of M. smegmatis/Rv1096
contributed to its cell wall integrity. Author details
1D
f 1Department of Biochemistry and Molecular Biology, Dalian Medical
University, 9 W Lushun South Road, Dalian 116044, China. 2Department of
Microbiology, Dalian Medical University, Dalian 116044, China. 3Department
of Biotechnology, Dalian Medical University, Dalian 116044, China. 4Liaoning
Provincial Core Lab of Glycobiology and Glycoengineering, Dalian 116044,
China. Received: 17 January 2014 Accepted: 25 June 2014
Published: 30 June 2014 Received: 17 January 2014 Accepted: 25 June 2014
Published: 30 June 2014 Authors’ information Shufeng Yang (M.S.) and Guoying Deng (M.S.):Department of Microbiology,
Dalian Medical University Dalian 116044, China; Fei Zhang (B.S.), Jian Kang
(Ph.D.), Wenli Zhang (Ph.D.) and Yufang Ma (Ph.D.): Department of
Biochemistry and Molecular Biology, Dalian Medical University Dalian 116044,
China. Yi Xin (Ph.D.), Department of Biotechnology, Dalian Medical University
Dalian 116044, China. Shufeng Yang (M.S.) and Guoying Deng (M.S.):Department of Microbiology,
Dalian Medical University Dalian 116044, China; Fei Zhang (B.S.), Jian Kang
(Ph.D.), Wenli Zhang (Ph.D.) and Yufang Ma (Ph.D.): Department of
Bi
h
i
d M l
l
Bi l
D li
M di
l U i
i
D li
60 Biochemistry and Molecular Biology, Dalian Medical University Dalian 116044,
China. Yi Xin (Ph.D.), Department of Biotechnology, Dalian Medical University
Dalian 116044, China. Conclusion We identified M. tuberculosis Rv1096 as a PG deacetylase
and found that the PG deacetylase activity of this protein
contributed to lysozyme resistance in M. smegmatis. Our
findings suggest that PG deacetylation may be involved in
immune evasion by M. tuberculosis in its host. 7. Wang G, Maier SE, Lo LF, Maier G, Dosi S, Maier RJ: Peptidoglycan
deacetylation in Helicobacter pylori contributes to bacterial survival by
mitigating host immune responses. Infect Immun 2010, 78(11):4660–4666. 8. Vollmer W, Tomasz A: The pgdA gene encodes for a peptidoglycan N-
acetylglucosamine deacetylase in Streptococcus pneumoniae. J Biol Chem
2000, 275(27):20496–20501. 9. Inês Crisóstomo M, Vollmer W, AS K o, Gehre F, Buckenmaier S, Tomasz A:
Attenuation of penicillin resistance in a peptidoglycan O-acetyl
transferase mutant of Streptococcus pneumoniae. Molecular Microbiol
2006, 61(6):1497–1509. 9. Inês Crisóstomo M, Vollmer W, AS K o, Gehre F, Buckenmaier S, Tomasz A:
Attenuation of penicillin resistance in a peptidoglycan O-acetyl
transferase mutant of Streptococcus pneumoniae. Molecular Microbiol
2006, 61(6):1497–1509. PG: Peptidoglycan; mAGP: Mycolyl-arabinogalactan-peptidoglycan;
Nod: Nucleotide-binding oligomerization domain; IPTG: Isopropyl-D-
thiogalactopyranoside; PMSF: Phenylmethyl-sulphonyl fluoride; BCIP/NBT:
5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium; SDS: Sodium
dodecyl sulfate; CFU: Colony forming unit; OsO4: Omium tetroxide;
SEM: Scanning electronic microscopy; CE-4: Carbohydrate esterase 4. The M. smegmatis/Rv1096 cell wall was undamaged by
9 h of lysozyme treatment smegmatis/Rv1096 without lysozyme treatment and, D) M. smegmatis/Rv1096
with lysozyme treatment. Lysozyme treatment was for 9 h. Yang et al. BMC Microbiology 2014, 14:174
http://www.biomedcentral.com/1471-2180/14/174 Page 8 of 9 Page 8 of 9 Page 8 of 9 suggest that it may have metallo-dependence. Indeed,
our results show that the enzymatic activity of Rv1096
increased after supplementation with divalent cations,
especially Co2+. Taken together, our results suggest that
Rv1096 may use similar catalytic mechanisms as the S. pneumoniae PgdA protein to deacetylate PG. Competing interests
The authors declare that they have no competing interests. Competing interests Competing interests References In fact, PG N-deacetylase has been shown to be a viru-
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gene cloning, protein expression, and peptidoglycan deacetylase activity.
BMC Microbiology 2014 14:174. Abbreviations
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Submit your manuscript at
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KM, Bouriotis V: Peptidoglycan N-acetylglucosamine deacetylases from
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http://www.biomedcentral.com/1471-2180/14/174 Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 30. Milani CJ, Aziz RK, Locke JB, Dahesh S, Nizet V, Buchanan JT: The novel
polysaccharide deacetylase homologue Pdi contributes to virulence of the
aquatic pathogen Streptococcus iniae. Microbiology 2010, 156(Pt 2):543–554. • Convenient online submission doi:10.1186/1471-2180-14-174
Cite this article as: Yang et al.: Mycobacterium tuberculosis Rv1096 protein:
gene cloning, protein expression, and peptidoglycan deacetylase activity. BMC Microbiology 2014 14:174. Submit your manuscript at
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https://openalex.org/W3165669090
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https://hal.archives-ouvertes.fr/hal-03253302/document
|
English
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Hydrated Peridotite – Basaltic Melt Interaction Part I: Planetary Felsic Crust Formation at Shallow Depth
|
Frontiers in earth science
| 2,021
|
cc-by
| 19,325
|
Hydrated Peridotite – Basaltic Melt Interaction Part I:
Planetary Felsic Crust Formation at Shallow Depth
Anastassia Yu. Borisova, Nail R Zagrtdenov, Michael J Toplis, Wendy A
Bohrson, Anne Nédélec, Oleg G Safonov, Gleb S Pokrovski, Georges
Ceuleneer, Ilya N Bindeman, Oleg E Melnik, et al. To cite this version: Anastassia Yu. Borisova, Nail R Zagrtdenov, Michael J Toplis, Wendy A Bohrson, Anne Nédélec,
et al.. Hydrated Peridotite – Basaltic Melt Interaction Part I: Planetary Felsic Crust Formation at
Shallow Depth. Frontiers in Earth Science, 2021, 9, pp.640464. 10.3389/feart.2021.640464. hal-
03253302 Hydrated Peridotite – Basaltic Melt
Interaction Part I: Planetary Felsic
Crust Formation at Shallow Depth Anastassia Y. Borisova 1,2*, Nail R. Zagrtdenov 1, Michael J. Toplis 3, Wendy A. Bohrson 4,
Anne Nédélec 1, Oleg G. Safonov 2,5,6, Gleb S. Pokrovski 1, Georges Ceuleneer 1,
Ilya N. Bindeman 7,8, Oleg E. Melnik 9, Klaus Peter Jochum 10, Brigitte Stoll 10, Ulrike Weis 10,
Andrew Y. Bychkov 2, Andrey A. Gurenko 11, Svyatoslav Shcheka 12, Artem Terehin 5,
Vladimir M. Polukeev 5, Dmitry A. Varlamov 5, Kouassi Chariteiro 1, Sophie Gouy 1 and
Philippe de Parseval 1 Borisova
anastassia.borisova@get.omp.eu Specialty section:
This article was submitted to
Petrology,
a section of the journal
Frontiers in Earth Science Specialty section:
This article was submitted to
Petrology, Received: 11 December 2020
Accepted: 26 January 2021
Published: 28 May 2021 Keywords: experiment, basaltic melt-serpentinite rock interaction, TTG, ophiolites, Hadean eon, Noachian, Mars,
protocrust 1Géosciences Environnement Toulouse, GET/OMP (CNRS, UT3, IRD, CNES), Toulouse, France, 2Geological Department,
Lomonosov Moscow State University, Vorobievy Gory, Moscow, Russia, 3Institut de Recherche en Astrophysique et Planétologie
(IRAP) UT3, CNRS, Toulouse, France, 4Department of Geology and Geological Engineering, Colorado School of Mines, Golden
CO, United States, 5Korzhinskii Institute of Experimental Mineralogy, Chernogolovka, Moscow Region, Russia, 6Department of
Geology, University of Johannesburg, Auckland Park, 2006, Johannesburg, South Africa, 7Department of Sciences, University of
Oregon, Eugene, OR, United States, 8Fersman Mineralogical Museum, Leninsky Prospect 18, Moscow, Russia, 9Institute of
Mechanics, Moscow State University, 1- Michurinskii Prosp, Moscow, Russia, 10Climate Geochemistry Department, Max Planck
Institute for Chemistry, Mainz, Germany, 11Centre de Recherches Pétrographiques et Géochimiques, UMR 7358, Université de
Lorraine, 54501 Vandœuvre-lès-Nancy, France, 12Bavarian Research Institute of Experimental Geochemistry and Geophysics
(BGI), University of Bayreuth, Bayreuth, Germany Edited by:
Scott Andrew Whattam,
King Fahd University of Petroleum and
Minerals, Saudi Arabia Reviewed by:
Kent Condie,
New Mexico Institute of Mining and
Technology, United States
Elena Sizova,
University of Graz, Austria Current theories suggest that the first continental crust on Earth, and possibly on other
terrestrial planets, may have been produced early in their history by direct melting of
hydrated peridotite. However, the conditions, mechanisms and necessary ingredients for
this crustal formation remain elusive. To fill this gap, we conducted time-series experiments
to investigate the reaction of serpentinite with variable proportions (from 0 to 87 wt%) of
basaltic melt at temperatures of 1,250–1,300°C and pressures of 0.2–1.0 GPa
(corresponding to lithostatic depths of ∼5–30 km). The experiments at 0.2 GPa reveal
the formation of forsterite-rich olivine (Fo90–94) and chromite coexisting with silica-rich
liquids (57–71 wt% SiO2). These melts share geochemical similarities with tonalite-
trondhjemite-granodiorite rocks (TTG) identified in modern terrestrial oceanic mantle
settings. By contrast, liquids formed at pressures of 1.0 GPa are poorer in silica
(∼50 wt% SiO2). Our results suggest a new mechanism for the formation of the
embryonic continental crust via aqueous fluid-assisted partial melting of peridotite at
relatively low pressures (∼0.2 GPa). We hypothesize that such a mechanism of felsic crust
formation may have been widespread on the early Earth and, possibly on Mars as well,
before the onset of modern plate tectonics and just after solidification of the first ultramafic-
mafic magma ocean and alteration of this primitive protocrust by seawater at depths of less
than 10 km. *Correspondence:
Anastassia Y. HAL Id: hal-03253302
https://hal.science/hal-03253302v1
Submitted on 8 Jun 2021 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. ORIGINAL RESEARCH
published: 28 May 2021
doi: 10.3389/feart.2021.640464 INTRODUCTION For example, the early terrestrial crust of intermediate to
felsic composition might have been created by direct melting of
a serpentinized (e.g., hydrated) peridotite at shallow depths
(Rudnick, 1995; Rudnick and Gao, 2003). A possible present-
day analogue is the formation of tonalites-trondhjemites in the
shallow mantle beneath oceanic spreading centers (called
oceanic
plagiogranites
in
this
context)
(Coleman
and
Peterman, 1975; Amri et al., 1996; Amri et al., 2007). Trondhjemite-tonalite veins are common features throughout
the cores in the few sites of the Deep Sea Drilling Project, and
Ocean Drilling, Integrated Ocean Drilling and International
Ocean Discovery Programs (DSDP, ODP, and IODP) where the
deep oceanic crust has been drilled along present-day spreading
ridges. These rocks have been ascribed to local remelting
processes in the presence of hydrothermal fluids along high-
temperature normal shear zones. These rocks are not abundant
(no more than 1% of the volume of the sections), but their
abundance might be underestimated due to poor core recovery
in the fault zones (e.g., Pietranik et al., 2017). Their origin is
enigmatic, although understanding the origin of TTG rocks
situated in modern oceanic lithosphere may
open new
perspectives on the formation of the first planetary felsic crust. Rare low-pressure melting experiments (≤1.0 GPa) involving
peridotite and low water contents (up to 0.5 wt% in the bulk As an alternative, or complement to direct hydrous melting
of peridotite, a currently untreated question is to what extent
the presence of basaltic melt may have an impact on the
production of felsic crust. This impact may be direct
(through chemical reactions with the host peridotite) or
indirect (by providing a heat source). In this respect,
experiments that reproduce basaltic melt-peridotite reaction
at low pressures (<0.8 GPa) are currently limited to systems
undersaturated in aqueous fluid (e.g., Fisk, 1986; Kelemen
et al., 1990; Morgan, and Liang, 2003; Van den Bleeken
et al., 2010; 2011). Although the experiments show the
presence
of
dry
basaltic
to
andesitic-basaltic
melts
in
association with olivine, no direct data exist concerning the
partial melting of hydrated (serpentinized) peridotite (or
serpentinite) triggered by the emplacement of basaltic melts
at shallow crustal depths (5–15 km). INTRODUCTION source) indicate that peridotite-derived partial melts may become
enriched in SiO2 (up to 57 wt%) and other lithophile elements
(Al,
alkalis),
with
abundances
approaching
those
of
continental crustal rocks (Hirschmann et al., 1998; Ulmer,
2001). The increase in the silica contents of partial melts at low
pressures is generally considered to be due to the combined
effects of water, alkalis, and low pressure on the structure of the
aluminosilicate melt, shifting the olivine-pyroxene cotectics to
higher SiO2 contents through a decrease in the silica activity
coefficient
(Kushiro,
1968;
Hirschmann
et
al.,
1998;
Lundstrom, 2000). The conditions and mechanisms that led to the production of
the earliest intermediate to felsic (Si- and Al-enriched) crust on
Earth and Mars are the subject of intense debate (Rudnick and
Gao, 2003; Harrison, 2009; Reimink et al., 2014, 2016; Sautter
et al., 2015, 2016; Burnham and Berry, 2017). There are some
indications that this crust was composed of igneous rocks of
granitic composition (I-type) and may have been formed in a
tonalite-trondhjemite-granodiorite (TTG)-like environment,
i.e., by partial melting of a garnet-bearing lower crust, as far
back as the geological record goes (e.g., the parental melt of the
4.37–4.02 Ga Jack Hills detrital zircon crystals, Burnham and
Berry, 2017). Ancient granodiorite rocks have also been
identified on Mars (Sautter et al., 2015; Sautter et al., 2016),
indicating that formation of felsic crust occurred on other
terrestrial
planets
even
without
plate
tectonics
and
subduction. In this context, melting of dry peridotite at
shallow depths does not produce appropriate intermediate to
felsic melt compositions comparable to that of bulk continental
crust (Hirschmann et al., 1998; Rudnick and Gao, 2003;
Harrison, 2009; Reimink et al., 2014, 2016; Burnham and
Berry, 2017). As a result, most currently accepted models for
the generation of felsic crust on the Earth or Mars consider a
complex multi-stage process at different depths, which involves
extraction of basaltic magma from peridotitic mantle followed
by fractional crystallization (Reimink et al., 2014, 2016; Udry
et al., 2018) and/or re-melting of hydrated mafic rocks at
lithospheric conditions of ≥0.6 GPa (Harrison, 2009; O’Neil
and Carlson, 2017). However, specific conditions during the
Hadean lead us to envisage petrological mechanisms that are not
typical of present-day felsic magma generation (i.e., not
involving subduction; see Herzberg et al., 2010; Sautter et al.,
2016). Citation: Borisova AY, Zagrtdenov NR,
Toplis MJ, Bohrson WA, Nédélec A,
Safonov OG, Pokrovski GS,
Ceuleneer G, Bindeman IN, Melnik OE,
Jochum KP, Stoll B, Weis U,
Bychkov AY, Gurenko AA, Shcheka S,
Terehin A, Polukeev VM, Varlamov DA,
Chariteiro K, Gouy S and de Parseval P
(2021) Hydrated Peridotite – Basaltic
Melt Interaction Part I: Planetary Felsic
Crust Formation at Shallow Depth. Front. Earth Sci. 9:640464. doi: 10.3389/feart.2021.640464 May 2021 | Volume 9 | Article 640464 1 Frontiers in Earth Science | www.frontiersin.org Felsic Crust Formation at Shallow Depth Borisova et al. Frontiers in Earth Science | www.frontiersin.org INTRODUCTION Ol, high-Mg olivine; Opx, orthopyroxene; Cpx, clinopyroxene; Chr, chromite or
chromiferous magnetite; felsic glass, intermediate to felsic glasses and fluid, aqueous fluid. Lower panel: Backscattered electron images of the hybrid and mixed runs at
0.2–1.0 GPa pressures. (A) mixed run SB7 at 0.2 GPa; (B) zoom on the SB7 with magnification of 1,100; hybrid runs: (C) P32 at 0.2 GPa; (D) P1 at 0.5 GPa; (E) P3
at 1.0 GPa. Mixed samples (SB7) is homogeneous sample with high-Mg olivine and interstitial felsic glasses. Hybrid samples (P32, P1, and P3) contain two zones:
olivine-rich zone (the former serpentinite zone) with interstitial felsic melt (Lint) and zone of reacting basalt (Lbas). Ol, high-Mg olivine; Opx, orthopyroxene; Chr, chromite,
and F, aqueous fluid. It should be noted that the homogeneous olivine-rich zone containing high-Mg olivine, chromite, interstitial felsic melt, and aqueous fluid is formed in
the mixed run SB7 (R 0.25) at an early stage (in less than 2 h), suggesting early attainment of steady state in this system with a high starting proportion of serpentinite
(80 wt%). All details may be found in Table 1 and Supplementary Table S2. GURE 1 | Upper panel: Scheme of time-series runs at 0.2 GPa, showing starting configurations and final products containing felsic glasses. Three type
experiment are described: serpentinite dehydration, and mixed and hybrid runs. Ol, high-Mg olivine; Opx, orthopyroxene; Cpx, clinopyroxene; Chr, chrom
omiferous magnetite; felsic glass, intermediate to felsic glasses and fluid, aqueous fluid. Lower panel: Backscattered electron images of the hybrid and mixed
–1.0 GPa pressures. (A) mixed run SB7 at 0.2 GPa; (B) zoom on the SB7 with magnification of 1,100; hybrid runs: (C) P32 at 0.2 GPa; (D) P1 at 0.5 GPa; (E) P
.0 GPa. Mixed samples (SB7) is homogeneous sample with high-Mg olivine and interstitial felsic glasses. Hybrid samples (P32, P1, and P3) contain two
ine-rich zone (the former serpentinite zone) with interstitial felsic melt (Lint) and zone of reacting basalt (Lbas). Ol, high-Mg olivine; Opx, orthopyroxene; Chr, ch
d F, aqueous fluid. INTRODUCTION This latter scenario is of
primary interest in early Earth history (and possibly also for
Mars), because the ultramafic-mafic protocrust formed from
the crystallization of a magma ocean was most likely hydrated,
either by volatiles released by the magma ocean itself or
through interactions with liquid water available on the
planetary surface (Albarède and Blichert-Toft, 2007; Elkins-
Tanton, 2012). A present-day analogue can be observed at the
level of the mantle/petrologic Moho boundary in the oceanic
lithosphere (at depths of 6 km), where interactions of basaltic
magmas
with
peridotites
produce
chromitite-dunite
associations (Kelemen et al., 1995; Arai, 1997; Borisova
et al., 2012; Zagrtdenov et al., 2018; Rospabé et al., 2019)
and serpentinized peridotite (hydrated residual peridotite)
undergoes
partial
melting
(Benoit
et
al.,
1999). Furthermore,
such
shallow
conditions
of
serpentinite
melting in the presence of basaltic melt might also occur in
mantle plumes (Bindeman, 2008; Reimink et al., 2014, 2016;
Borisova et al., 2020a), in Hadean heat-pipe volcanoes (Moore
and Webb, 2013) or Hadean proto-rift volcanoes (Capitanio
et al., 2020) and/or during meteorite impacts (Marchi et al.,
2014). The above considerations provide a strong motivation for the
present study, which aims to directly investigate the generation of
intermediate to felsic crust on young terrestrial planets. For this
purpose, we have conducted a set of time-series experiments in
serpentinite-basalt
systems
under
relatively
low
pressures
(≤1 GPa),
corresponding
to
depths
of
≤30 km. Modern
(i.e., post-Archean) magmatism near the petrologic Moho in
the oceanic lithosphere can then be used as a ground-truth
test within its limits. Rare low-pressure melting experiments (≤1.0 GPa) involving
peridotite and low water contents (up to 0.5 wt% in the bulk May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 2 Felsic Crust Formation at Shallow Depth Borisova et al. : Scheme of time-series runs at 0.2 GPa, showing starting configurations and final products containing felsic glas
ed: serpentinite dehydration, and mixed and hybrid runs. Ol, high-Mg olivine; Opx, orthopyroxene; Cpx, clinopyro
sic glass, intermediate to felsic glasses and fluid, aqueous fluid. Lower panel: Backscattered electron images of the h FIGURE 1 | Upper panel: Scheme of time-series runs at 0.2 GPa, showing starting configurations and final products containing felsic glasses. Three types
of experiment are described: serpentinite dehydration, and mixed and hybrid runs. Experimental Strategy To investigate the serpentinite rock-basaltic melt reactions, we
have conceived three types of time-series experiments: (i) pure
serpentinite dehydration in a basalt-free system, and so-called (ii)
“mixed” and iii) “hybrid” experiments in the serpentinite-basalt
system as a function of pressure and basalt-to-serpentinite ratio. (Figures 1A–E; Table 1; Supplementary Tables S1, S2). The
“mixed” experiments were performed on well-homogenized
serpentinite
and
basaltic
powders
compressed
together,
whereas the “hybrid” experiments were performed using
cylindrical pieces of serpentinite rock placed in the upper part
of the capsule and a basaltic powder in the lower part of the
capsule in contact with the base of the serpentinite cylinder
(Figures 1A-E). These two distinct experimental setups were
designed to simulate two principal geological scenarios of basaltic
melt emplacement. The mixed experiments (with variable basalt-
to-serpentinite mass ratios, Rbas/serp or R from 0.25 to 4) simulate
the case of efficient fertilization of peridotite in order to achieve a
closer approach to equilibrium and obtain stable associations of the
minerals and melts produced by the reactions on the relatively
short time scales that are accessible experimentally. The hybrid
experiments (with generally higher R, from ∼3 to ∼7) were
designed to simulate the process of basaltic melt infiltration into
serpentinite rock, that does not necessarily reach equilibrium. The use of Au80Pd20 alloy for the experimental capsule
material avoids Fe diffusion into the capsule because of the
negligibly small iron solubility at the temperature-pressure-
redox conditions of our experiments (Balta et al., 2011). Therefore, the capsules were not pre-doped with Fe. The
redox conditions in our experiments were buffered by the
initial Fe2+/Fe3+ ratios imposed by the starting basaltic glass
and serpentinite in the capsule. Note that our run durations
(≤48 h) are too short to allow use of a double-capsule technique
with common mineral buffers of oxygen fugacity that require
longer times to attain equilibrium (Matjuschkin et al., 2015). The
redox conditions (i.e., oxygen fugacity, fO2) during the runs are
estimated from the compositions of co-existing olivine and
chromite using equations of Ballhaus et al. (1991) that yield
ΔQFM values of +1.8 to +4.6 (where ΔQFM denotes the logfO2
value
relative
to
the
quartz-fayalite-magnetite
buffer,
Supplementary Table S2). Additionally, the Fe2+/Fe3+ ratios
in several glass run products were derived from XANES (X-
ray absorption near edge structure) spectroscopy using the Fe
K-edge at the European Synchrotron Radiation Facility (ESRF) in
Grenoble, France (Supplementary Figure S1). INTRODUCTION It should be noted that the homogeneous olivine-rich zone containing high-Mg olivine, chromite, interstitial felsic melt, and aqueous fluid is fo
mixed run SB7 (R 0.25) at an early stage (in less than 2 h), suggesting early attainment of steady state in this system with a high starting proportion of serp FIGURE 1 | Upper panel: Scheme of time-series runs at 0.2 GPa, showing starting configurations and final products containing felsic glasses. Three types
of experiment are described: serpentinite dehydration, and mixed and hybrid runs. Ol, high-Mg olivine; Opx, orthopyroxene; Cpx, clinopyroxene; Chr, chromite or
chromiferous magnetite; felsic glass, intermediate to felsic glasses and fluid, aqueous fluid. Lower panel: Backscattered electron images of the hybrid and mixed runs at
0.2–1.0 GPa pressures. (A) mixed run SB7 at 0.2 GPa; (B) zoom on the SB7 with magnification of 1,100; hybrid runs: (C) P32 at 0.2 GPa; (D) P1 at 0.5 GPa; (E) P3
at 1.0 GPa. Mixed samples (SB7) is homogeneous sample with high-Mg olivine and interstitial felsic glasses. Hybrid samples (P32, P1, and P3) contain two zones:
olivine-rich zone (the former serpentinite zone) with interstitial felsic melt (Lint) and zone of reacting basalt (Lbas). Ol, high-Mg olivine; Opx, orthopyroxene; Chr, chromite,
and F, aqueous fluid. It should be noted that the homogeneous olivine-rich zone containing high-Mg olivine, chromite, interstitial felsic melt, and aqueous fluid is formed in
the mixed run SB7 (R 0.25) at an early stage (in less than 2 h), suggesting early attainment of steady state in this system with a high starting proportion of serpentinite
(80 wt%). All details may be found in Table 1 and Supplementary Table S2. Frontiers in Earth Science | www.frontiersin.org May 2021 | Volume 9 | Article 640464 3 Felsic Crust Formation at Shallow Depth Borisova et al. Experimental Strategy The Fe2+/Fe3+
ratios obtained by XANES correspond to ΔQFM (+1.8 to +3.6)
based on model of Borisov et al. (2018) (Supplementary Table
S2), in excellent agreement with the values estimated from phase
composition. The relatively oxidized conditions inferred for our
runs are principally the result of the presence of water, although
partial H2 loss through the capsule wall cannot be fully excluded. However, redox potential does not have a significant influence on
the concentrations of the major redox-insensitive elements of the
felsic crust (predominantly Si, and Al). All experiments were conducted using two types of piston
cylinder and an internally heated pressure vessel for durations
ranging from 1 min to 120 h at pressures of 0.2, 0.5, and 1.0 GPa
and temperatures of 1,250 and 1,300°C (see Supplementary
Materials
for
details). In
modern
oceanic
settings,
temperatures of ∼1,050°C are considered to be sufficient to
initiate the reaction of serpentinized peridotite with basaltic
magma at 0.2 GPa (Borisova et al., 2012). In the present
experimental
study,
however,
we
chose
to
use
higher
temperatures (1,250–1,300°C) since these provide a closer
analogue of the thermal conditions of the Hadean and early
Martian upper mantle (Herzberg et al., 2010; Sautter et al., 2016). Redox conditions expressed relative to the conventional quartz-
fayalite-magnetite
fO2
buffer
[ΔQFM
log10
fO2
(experiment)–log10fO2 (QFM) +1.8 to +4.6] established in
our experiments are comparable to those characteristic of the
Jack Hills zircon crystals in equilibrium with the mantle (Trail
et al., 2011). In addition, higher temperatures should substantially
increase the rates of serpentinite-basaltic melt reactions. On the
time scale of laboratory experiments, such conditions correspond
to the complete melting of basalt, which is consistent with most
existing models of basaltic melt extraction from the mantle and its
emplacement in the upper crust (Fisk, 1986; Hirschmann et al.,
1998; Ulmer, 2001; Morgan and Liang, 2003). We used the law of mass conservation and the minimization
code of Oliphant (2006) (version 1.17). Two steps were applied to
calculate phase proportions obtained in hybrid runs. In the first
step, the initial MORB glass composition was introduced for the
compositions of Lbas (basaltic liquid) and Lint (interstitial felsic
liquid) (Supplementary Tables S1, S2). In the second step, the EXPERIMENTAL STRATEGY AND
METHODS experiments varied from 1 min to 120 h. The experiments were
performed
in
two
laboratories:
Korzhinskii
Institute
of
Experimental
Mineralogy
(Russia)
and
Bavarian
Research
Institute
of
Experimental
Geochemistry
and
Geophysics
(Germany)
(Table
1;
Supplementary
Table
S2). The
dehydration runs and the mixed runs were performed for
durations of 0.5, 2, 5, and 48 h. The starting basalt used in the
hybrid and mixed experiments is an enriched mid-ocean ridge
basaltic glass containing 8.2 wt% MgO and 0.7 wt% H2O
(Supplementary Table S1) from the Knipovich ridge of the
Mid Atlantic Ridge sampled by dredging during the 38th
expedition of R/V Akademik Mstislav Keldysh (number 3786/
3, Sushchevskaya et al., 2000). The serpentinite (11.9 wt% H2O)
(Supplementary Table S1) used as a starting material is from a
homogeneous antigorite-dominated sample from Zildat, in the
Ladakh area, northwest Himalayas (TSL-19) (Deschamps et al.,
2010). This sample has accessory magnetite but no trace of
chromite, olivine or orthopyroxene. The serpentinite was
prepared either as doubly polished ∼1 mm high and ∼2.6 mm-
diameter cylinders (for hybrid runs) or as serpentinite powder
(<100 μm; for mixed runs). Experimental Conditions and Starting
Materials To describe mechanisms of the serpentinite dehydration and the
serpentinite interaction with basaltic melt, the duration of the May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 4 Felsic Crust Formation at Shallow Depth Borisova et al. part
are
reported
in
Supplementary
Material
and
Supplementary Tables S5–S7. basaltic and interstitial glasses (Lbas and Lint, respectively) were
distinguished as different phases. The run products (glasses and
minerals)
were
analyzed
for
chemical
compositions
(Supplementary Tables S3, S4; Supplementary Material) to
make reasonable comparison with natural felsic melts and
magmas. Furthermore,
thermodynamic
modelling
was
performed to constrain the equilibrium compositions of the
liquids, minerals as well as fluid produced during serpentinite
dehydration and responsible for the felsic melt production
(Supplementary Tables S5–S7; Supplementary Material). Dehydration Experiments at 0.2–0.5 GPa y
p
Three experiments on pure serpentinite dehydration (100 wt%
serpentinite) at 0.2–0.5 GPa and 1,250°C were performed with
different run durations. In experiment P29 (0.5 h duration), we
observe
a
very
porous
aggregate
consisting
of
enstatite
[92–95 Mg# 100 Mg/(Mg + Fe2+)], high-Mg olivine (Fo91–92,
mol% forsterite) and chromite [69–73 Mg#, and 68–95 Cr# 100
Cr/(Cr + Al), Figures 1, 2, Supplementary Tables S2, S3]. Similarly, in the long-duration experiments P27 and P28
(48 h), the products are represented by a re-crystallized
powder containing enstatite, high-Mg olivine and chromite. No trace of silicate glass was found in the run products. In all
dehydration experiments at 0.2 and 0.5 GPa, fluid was present,
likely produced by serpentinite decomposition. Mixed Experiments at 0.2 GPa Based on textural and compositional characteristics, three types
of mineral-glass assemblage can be distinguished in nine samples
from the mixed experiments on serpentinite-basaltic melt
reactions with different proportions of serpentinite (R
0.25–4.0) in the starting material. RESULTS Experimental Sample Description
In this section, we describe the results of 23 experimental runs
performed at 0.2–1.0 GPa and 1,250–1,300°C to investigate pure
serpentinite dehydration and serpentinite-basalt interaction in
the mixed and hybrid systems with different durations
(1 min–120 h),
different
proportions
of
serpentinite
(13–100 wt%) and variable basalt-to-serpentinite initial ratios
(R 0.25–6.5) in the starting material (Table 1; Supplementary
Table S2). nalytical and Microanalytical Methods Analytical and Microanalytical Methods
The bulk-rock chemical compositions of the starting serpentinite
and basalt samples have been analyzed by inductively coupled
plasma-mass spectrometry (ICP-MS) and inductively coupled
plasma-optical
emission
spectroscopy
(ICP-OES),
using
a
method developed at the SARM (Nancy, France). Scanning
electron microscope (SEM) and electron probe microanalysis
and mapping (EPMA) of minerals and glasses were performed
at the Géosciences Environnement Toulouse (GET, Toulouse,
France) laboratory and at the Centre de Microcaractérisation
Raimond Castaing (Toulouse, France). Secondary ion mass
spectrometry (SIMS) was applied to measure the H2O contents
at CRPG-CNRS (Nancy, France). Laser ablation-inductively
coupled plasma-mass spectrometry (LA-ICP-MS) was applied
to analyze major and trace element contents at the Max Planck
Institute
for
Chemistry,
(Mainz,
Germany). XANES
spectroscopy was used to estimate iron redox state in
selected
experimental
glass
products
from
Fe
K-edge
XANES spectra acquired at the FAME beamline of the
European Synchrotron Radiation Facility (ESRF, France). All details of the applied methods may be found in the
Supplementary Material. Modelling Approaches g
pp
To interpret the experimental and analytical data obtained in
this study and to enable comparisons with natural systems, we
employed complementary modelling approach. Note that there
is no currently available thermodynamic model and associated
software tools allowing to directly simulate the aqueous fluid-
assisted melting of serpentinite in the presence of basaltic melt
at pressures below 1 GPa, that could predict, simultaneously
and
within
the
same
thermodynamic
framework,
the
compositions of fluid, melts and minerals. By contrast,
robust thermodynamic models currently exist for i) hydrous
melts and ii) aqueous fluid phase. Therefore, we conducted two
types of thermodynamic modelling to better constrain the
compositions
of
the
different
phases
produced
in
the
experiments: i) calculations of liquid and mineral phase
proportions and compositions formed at different physical
chemical
conditions
(temperature,
pressure,
T-P)
using
MELTS and Magma Chamber Simulator, MCS software and
ii) calculations of the equilibrium aqueous fluid composition
and major element speciation in the fluid phase to constrain the
capacity of the aqueous phase to mobilize and fractionate major
elements at the experimental T-P conditions from the initial
materials (basalt and serpentinite). All details of the modelling 1)
Most samples of the SB (Serpentinite-Basalt) series with
high initial proportions of serpentinite (R 0.25–1.0) are
characterized by a homogeneous zone of polyhedral
unzoned crystals of high-Mg olivine (Fo90–94 mol%)
associated
with
interstitial
felsic
glasses
(SiO2
62–71 wt%) as well as fluid bubbles (Figures 1A,B, 2). Chromite (Cr# 68–89; Mg# 56–73) is associated with the
euhedral olivine grains, and crystals of clinopyroxene
(Mg# 58–68) are characteristic of the runs with R 1. Figures 1A,B, 2 show that samples SB7, SBbis2, SBter3
with high initial proportions of serpentinite (R 0.25) are
characterized by a single zone composed of polyhedral
unzoned olivine (Fo90–94) and chromite crystals associated
with interstitial felsic glasses (Lint) and interstitial crystals
of
chromite
and
fluid
bubbles. Remarkably,
the
proportions of interstitial melt are high (33–40 wt%). 2)
Experiments SB1, SB4, SBbis1, and SBter2, with lower
initial
proportions of
serpentinite
(R
1–4), are
characterized
by
polyhedral
unzoned
olivine May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 5 Felsic Crust Formation at Shallow Depth Borisova et al. TABLE 1 | Experimental run conditions and the resulting phases with the phase proportions. Modelling Approaches Serpentinite dehydration experiments *P, pressure; T, temperature; SERP, serpentinite; BAS, basalt; L int, interstitial glass; L bas, hydrous basaltic glass; Serp, serpentine minerals
ferrosilite; Cpx, diopside; Amph, amphibole; Chr, chromite; ChrMgt, chromiferous magnetite. enstatite (Mg# 96.5 ± 2.1) and high-Mg olivine (Fo93±1),
and a periphery (80–280 μm thick) consisting of olivine and
interstitial felsic glass. In sample P32, which was kept for 2.5 h at
1,250°C and 0.2 GPa, the former serpentinite zone has a
harzburgitic core (olivine Fo89±1 and orthopyroxene Mg# 95 ±
3) and an outer thick rim composed of olivine and interstitial
glass. Chromite is localized exclusively in the former serpentinite
zone (Figures 1, 2). In sample P35, which was produced after 5 h at
1,250 °C and 0.2 GPa, a large (∼200 × 290 μm) aggregate of
chromite is observed in the former serpentinite zone at the
contact with the hydrous basalt glass. Moving away from the
contact to the center of the former-serpentinite zone, pockets of
interstitial glass can be found with rare crystals of amphibole and
zoned olivine (more magnesian in the core and more ferric at the
rim). The abundance of interstitial glass is lower in the olivine-rich
area consisting of olivine and enstatite (olivine Fo80±7 and
orthopyroxene Mg# 92 ± 2). For the experiment conducted
for 48 h under these conditions (run P33), the olivine-rich zone
comprises euhedral zoned olivine crystals with highly magnesian
cores and more ferrous rims associated with interstitial felsic glasses
and interstitial crystals of clinopyroxene, amphibole and fluid phenocrystals. The matrix of these samples contains a
clinopyroxene-olivine assemblage with interstitial felsic
glasses
(Lint). Oxide
minerals
are
represented
by
chromite phenocrysts. p
y
3)
Finally, the longest run sample SBter1 (48 h) with the
lowest initial proportion of serpentinite (20 wt%) is
represented by homogeneous basaltic glass formed by
complete hybridization of the starting basaltic liquid
with
serpentinite. By
contrast,
the
intermediate-
duration sample SBbis3 (5 h) is characterized by a
residual olivine-rich zone, even though the starting
material contains the same proportion of serpentinite
as SBter1 (20 wt%). Modelling Approaches №
Run
P
T
Time
Percentage of
the starting
components
(wt%)
R
Resulting experimental phases
(GPa)
(°C)
(h)
SERP
BAS*
BAS-to-SERP
mass
ratio
(Phase proportion in wt%)
Serpentinite dehydration experiments
1
P29
0.2
1250
0.5
100
-
0
Ol (54.1) + Opx (30.4) + Chr (2.6) + F(12.8)
2
P28
0.2
1250
48
100
-
0
Ol + Opx + Chr + F
3
P27
0.5
1250
48
100
-
0
Ol (55.8) + Opx (28.6) + Chr (2.7) + F(12.8)
Mixed experiments
4
SB1
0.2
1250
2
19.95
80.05
4.0
Ol (39.6) + Cpx (22.3) + Chr (6.8) + Lint (34.0) + F (3.3)
5
SB4
0.2
1250
2
49.89
50.11
1.0
Ol + Cpx + Chr + Lint + F
6
SB7
0.2
1250
2
80.01
19.99
0.25
Ol (59.2) + Chr (6.5) + Lint (33.5) + F (5.9)
7
SBbis3
0.2
1250
5
19.95
80.05
4.0
Ol + L bas + F
8
SBbis1
0.2
1250
5
49.89
50.11
1.0
Ol (37.9) + Cpx (19.3) + Chr (6.7) + Lint (39.9) + F (2.1)
9
SBbis2
0.2
1250
5
80.01
19.99
0.25
Ol (59.4) + Chr (6.0) + Lint (33.4) + F (6.1)
10
SBter1
0.2
1250
48
19.95
80.05
4.0
L bas + F
11
SBter2
0.2
1250
48
49.89
50.11
1.0
Ol (36.0) + Cpx (22.0) + Chr (7.1) + Lint (38.9) + F (2.1)
12
SBter3
0.2
1250
48
80.01
19.99
0.25
Ol (57.5) + Chr (6.0) + Lint (35.0) + F (6.5)
Hybrid experiments
13
P37
0.2
1250
0.5
28.2
71.8
2.5
L bas (72.8) + L int (0.4) + Ol (17.0) + Opx (6.8) + Cpx + ChrMgt (0.0) + F (3.0)
14
P32
0.2
1250
2.5
17.7
82.3
4.6
L bas (72.7)+ L int (8.7) + Ol (10) + Opx (7) + Chr (0.0) + F (1.6)
15
P35
0.2
1250
5
18.8
81.2
4.3
L bas + L int + Ol + Opx + Chr + ChrMgt + Amph + F
16
P33
0.2
1250
48
19.8
80.2
4.1
L bas (57.1) + L int (15.4) + Ol (13.1) + Opx* (0.0) + Chr (6.0) + Amph (6.6) + F (1.7)
17
P42
0.2
1250
120
17.6
82.4
4.7
L bas (∼58) + L int (∼27.8) + Ol (12.8) + ChrMgt (0.0) + F (1.4)
18
P1
0.5
1300
0.02
19.2
80.8
4.2
L bas + L int + Ol + Opx + Chr + F
19
P15
0.5
1300
0.5
15.7
84.3
5.4
L bas + L int + Ol + Opx + ChrMgt
20
P18
0.5
1300
5
13.4
86.6
6.5
L bas + L int + Ol + Opx + Mgt + Chr
21
P36
0.5
1250
8
14.6
85.4
5.8
L bas (+ F)
22
P3
1.0
1300
2.5
15.3
84.7
5.5
L bas + L int + Ol + Opx + ChrMgt
23
P7
1.0
1300
9
15.9
84.1
5.3
L bas
*P, pressure; T, temperature; SERP, serpentinite; BAS, basalt; L int, interstitial glass; L bas, hydrous basaltic glass; Serp, serpentine minerals; Ol, olivine; Opx, orthopyroxene; Opx*,
ferrosilite; Cpx diopside; Amph amphibole; Chr chromite; ChrMgt chromiferous magnetite TABLE 1 | Experimental run conditions and the resulting phases with the phase proportions. Hybrid Experiments at 0.2 GPa The samples from hybrid experiments with low starting
proportions of serpentinite (18–28 wt%; R 2.5–4.7) produced
at 0.2 GPa pressure show two distinct zones: a former serpentinite
(or olivine-rich) zone and a zone of quenched basaltic melt
(Figures 1C, 2; Table 1; Supplementary Tables S2, S3). The
olivine-rich zone in sample P37 shows a core composed of May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 6 Borisova et al. Felsic Crust Formation at Shallow Depth GURE 2 | Backscattered electron images and elemental X-ray maps of the hybrid and mixed runs to investigate aqueous fluid-assisted partial melting of peridotite
pressures of 0.2 and 0.5 GPa. The images show mineral, glass and fluid phase associations observed in the capsules, particularly the olivine-rich zones after
enching. Ol, Opx, Chr, Lint, Lbas, and F on the images stand for olivine, orthopyroxene, chromite, interstitial and basaltic glass, and fluid bubbles, respectively. The run
mbers correspond to those used in Table 1; Supplementary Table S2. (P29) The serpentinite dehydration sample produced at 0.2 GPa displays a single zone
presented by enstatite with high-Mg olivine and orthopyroxene. (SB7, SB bis2, and SB ter3) The mixed samples with highest initial proportions of serpentinite (80 wt%;
0.25) are characterized by a unique polyhedral olivine-rich domain of unzoned crystals of olivine associated with interstitial felsic glass (Lint) and fluid bubbles. hromite is associated with the euhedral olivine grains (high-Mg olivine Fo90–94). (P32) The hybrid run sample P32 lasting 2.5 h at 1,250°C and 0.2 GPa (R 4.6) shows a
mer-serpentinite zone with an inner harzburgite part and an outer olivine-rich part (olivine Fo89±1) composed of olivine and interstitial glass. Chromite is localized
(Continued) ackscattered electron images and elemental X-ray maps of the hybrid and mixed runs to investigate aqueous fluid-assisted partial melting of peridotite
of 0.2 and 0.5 GPa. The images show mineral, glass and fluid phase associations observed in the capsules, particularly the olivine-rich zones after
Opx, Chr, Lint, Lbas, and F on the images stand for olivine, orthopyroxene, chromite, interstitial and basaltic glass, and fluid bubbles, respectively. The run
espond to those used in Table 1; Supplementary Table S2. (P29) The serpentinite dehydration sample produced at 0.2 GPa displays a single zone
y enstatite with high-Mg olivine and orthopyroxene. Hybrid Experiments at 0.5 GPa The following group of samples with low initial proportions of
serpentinite (13–19 wt%; R 4.2–6.5) in the starting material was
produced at 0.5 GPa and 1,300°C (except P36 at 1,250°C) with run
durations from 1 min to 8 h (Table 1; Supplementary Tables S2,
S4). In sample P1 with the shortest run duration (1 min), the
quenched glass zone and former-serpentinite zone are both
present. The former-serpentinite zone contains fine-grained
aggregates (5–10 µm) of high-Mg olivine Fo95, enstatite with
Mg# 95, chromite (Cr# 89) and an interstitial glass of
basaltic andesite composition (Figures 1D, 2). Chromite
crystals (a few microns in size) are disseminated within this
zone. The bubbles in the quenched melt zone are evidence for the
presence of fluid water during quenching. Sample P15 (0.5 h) also
shows two distinct zones (quenched basaltic melt zone and
olivine-rich zone). The quenched basaltic melt zone consists of
hydrous basaltic glass characterized by a high magnesium content
(11 wt% MgO). The former serpentinite zone consists of 5–20 µm
aggregates of high-Mg olivine Fo93 and enstatite with Mg# 97. These aggregates are associated with an interstitial glass of
andesitic composition and chromiferous magnetite. The outer
part of this zone is characterized by a layer (∼120 μm thick),
consisting of olivine crystals and interstitial glass. Chromiferous
magnetite is concentrated in two large areas (up to 200 μm and
400 µm in size) or disseminated near these areas. Sample P18 (5 h
of interaction) is also characterized by a hydrous basaltic zone
and a former-serpentinite zone (Table 1; Supplementary Tables
S2, S4). However, in this case, the olivine-rich zone consists of
two areas; the outer area of nearly 200 μm width contains olivine
and basaltic interstitial glass with disseminated chromite (a few
microns in size), while the inner area is situated 200 μm from
quenched basaltic glass. This inner area consists of high-Mg
olivine Fo93, enstatite with Mg# 97 and contains aggregate
of magnetite (∼20 × 40 μm). For the longest experiment of this
series (P36, 8 h), serpentinite has completely dissolved into the
basalt melt (Table 1; Supplementary Tables S2, S4). Run
products show a homogeneous basaltic glass with high MgO Hybrid Experiments at 0.2 GPa (SB7, SB bis2, and SB ter3) The mixed samples with highest initial proportions of serpentinite (80 wt%
characterized by a unique polyhedral olivine-rich domain of unzoned crystals of olivine associated with interstitial felsic glass (Lint) and fluid bubbles. GURE 2 | Backscattered electron images and elemental X-ray maps of the hybrid and mixed runs to investigate aqueous fluid-assisted partial melting of peridotite
pressures of 0.2 and 0.5 GPa. The images show mineral, glass and fluid phase associations observed in the capsules, particularly the olivine-rich zones after
enching. Ol, Opx, Chr, Lint, Lbas, and F on the images stand for olivine, orthopyroxene, chromite, interstitial and basaltic glass, and fluid bubbles, respectively. The run
mbers correspond to those used in Table 1; Supplementary Table S2. (P29) The serpentinite dehydration sample produced at 0.2 GPa displays a single zone
presented by enstatite with high-Mg olivine and orthopyroxene. (SB7, SB bis2, and SB ter3) The mixed samples with highest initial proportions of serpentinite (80 wt%;
)
(
) FIGURE 2 | Backscattered electron images and elemental X-ray maps of the hybrid and mixed runs to investigate aqueous fluid-assisted partial melting of peridotite
at pressures of 0.2 and 0.5 GPa. The images show mineral, glass and fluid phase associations observed in the capsules, particularly the olivine-rich zones after
quenching. Ol, Opx, Chr, Lint, Lbas, and F on the images stand for olivine, orthopyroxene, chromite, interstitial and basaltic glass, and fluid bubbles, respectively. The run
numbers correspond to those used in Table 1; Supplementary Table S2. (P29) The serpentinite dehydration sample produced at 0.2 GPa displays a single zone
represented by enstatite with high-Mg olivine and orthopyroxene. (SB7, SB bis2, and SB ter3) The mixed samples with highest initial proportions of serpentinite (80 wt%;
R 0.25) are characterized by a unique polyhedral olivine-rich domain of unzoned crystals of olivine associated with interstitial felsic glass (Lint) and fluid bubbles. Chromite is associated with the euhedral olivine grains (high-Mg olivine Fo90–94). (P32) The hybrid run sample P32 lasting 2.5 h at 1,250°C and 0.2 GPa (R 4.6) shows a
former-serpentinite zone with an inner harzburgite part and an outer olivine-rich part (olivine Fo89±1) composed of olivine and interstitial glass. Chromite is localized
(Continued) May 2021 | Volume 9 | Article 640464 7 Frontiers in Earth Science | www.frontiersin.org Felsic Crust Formation at Shallow Depth Borisova et al. FIGURE 2 | exclusively in the former-serpentinite zone. Hybrid Experiments at 1.0 GPa y
p
Two experiments with low initial proportions of serpentinite
(∼15 wt%; R 5.3–5.5) in the starting material were performed at
1.0 GPa and 1,300°C. The run products from experiment P3
(2.5 h)
contain
a
basaltic
glass
zone
and
the
former-
serpentinite zone (Figure 1E; Table 1; Supplementary Tables
S2, S4). High-Mg olivine (Fo92), enstatite with Mg# 96 and
chromiferous magnetite are associated with interstitial glass of
basaltic composition (up to 51 wt% SiO2) in the former-
serpentinite zone. A longer-duration experiment (P7, 9 h)
yields products containing hydrous basaltic glass with 13.0 wt
% MgO (Supplementary Tables S2, S4). More details are
reported in Borisova et al. (2020a). Hybrid Experiments at 0.2 GPa (P33) The hybrid P33 run was performed at 0.2 GPa and 1,250°C for 48 h (R 4.1). The olivine-rich zone
comprises euhedral zoned crystals of olivine associated with interstitial felsic glasses and interstitial crystals of clinopyroxene, amphibole and fluid bubbles. Chromite and
chromiferous magnetite are located in the interstitial zones between the euhedral olivine grains (olivine Fo90±4) and are also associated with amphibole microcrystals
grown around the Cr-rich spinels. (P1) A quenched glass zone and former-serpentinite zone are present in the hybrid sample P1 produced at 0.5 GPa pressure in the
shortest duration experiment (1 min, R 4.2). The former-serpentinite zone contains fine-grained (5–10 µm) aggregates of olivine Fo95, enstatite, chromite, and interstitial
glass of basaltic andesite composition. Chromite crystals (few microns in size) are disseminated within this zone. The bubbles in the quenched melt zone are evidence for
the presence of water fluid during quenching. The textures demonstrate that the formation of the felsic liquids precedes the complete dissolution of orthopyroxene in the
olivine-rich zones of the shortest-duration runs at 0.5 GPa. All data on the hybrid and mixed samples may be found in the Supplementary Tables S2, S3. content (11.5 wt%). More details are reported in Borisova et al. (2020a). Numerous bubbles with dendritic amphibole aggregates
are present in the glass, suggesting amphibole growth upon
quenching. bubbles (Figure 2). Chromite and chromiferous magnetite are
located in the interstitial zones between the euhedral olivine
grains (high-Mg olivine Fo90±4) and are also associated with
amphibole microcrystals grown around the Cr-rich spinels. Only
one crystal of ferrous orthopyroxene (hypersthene) is recorded in
association with interstitial glass. In sample P42 (120 h), which
contains a quenched melt zone of hydrous basaltic glass with rare
euhedral olivine, the olivine-rich zone consists of euhedral olivine
crystals (Fo92.8±0.5), chromiferous magnetite and interstitial glass of
dacitic composition. Summary of the Major Findings From the
Whole Series of Experimental Data 1)
Highly magnesian olivine (Fo91–92, mol% forsterite),
orthopyroxene [92–95 Mg# 100 Mg/(Mg + Fe2+)] and
chromite [69–73 Mg#, and 68–95 Cr# 100 Cr/(Cr + Al)]
are found in the serpentinite dehydration runs containing
only serpentinite as starting material (R 0), but no
interstitial glass can be detected (Figure 3A). 1)
Highly magnesian olivine (Fo91–92, mol% forsterite),
orthopyroxene [92–95 Mg# 100 Mg/(Mg + Fe2+)] and
chromite [69–73 Mg#, and 68–95 Cr# 100 Cr/(Cr + Al)]
are found in the serpentinite dehydration runs containing
only serpentinite as starting material (R 0), but no
interstitial glass can be detected (Figure 3A). 2)
Bulk assimilation of the dehydrated serpentinite by the
basaltic melt and production of homogeneous crystal-free
hydrous basaltic melts (Lbas) are consistently observed in
the hybrid and mixed experiments with the highest
proportions of basalt and the lowest proportions of
serpentinite (R ≥4, Borisova et al., 2020a) in the
starting material and with durations longer than 5 h at
pressures
of
0.2–1.0 GPa
(Figure
3B;
Table
1;
Supplementary Tables S2–S4). 3) pp
y
3)
The shortest hybrid runs at pressures of 0.5–1.0 GPa,
and in all hybrid runs at 0.2 GPa lasting for 0.5–120 h
contain olivine-rich zone composed of high-Mg olivine,
Cr-rich spinels and interstitial glasses (Lint) (former-
serpentinite zone or olivine-rich zone) and zone of
hydrous
basaltic
glass
(Figures
1C–E;
Figure
2;
Figure 3B; Table 1; Supplementary Tables S3, S4). The infiltration of basaltic melt into serpentinite at high
basalt-to-serpentinite ratios (R 2.5–6.5), as simulated
in the hybrid experiments, is able to generate new
interstitial melts of intermediate to felsic composition May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 8 Felsic Crust Formation at Shallow Depth Borisova et al. FIGURE 3 | (A) Phase compositions plotted as serpentine (SERP)
proportion (in wt%) vs. run duration (h) for the pure serpentinite dehydration
experiments (R 0) and mixed serpentinite-basalt reaction experiments at
0.2 GPa and 1,250°C and basalt-to-serpentinite ratios R 0.25, 1 and 4. The diagram shows mineral, glass, and fluid phase associations observed
after quenching. Ol, Opx, Cpx, Lint, Lbas, and F stand for olivine,
(Continued) FIGURE 3 | orthopyroxene, clinopyroxene, interstitial glass, basaltic glass,
and fluid, respectively. All olivine-rich samples demonstrate the presence of
chromite and chromiferous magnetite. Summary of the Major Findings From the
Whole Series of Experimental Data The run numbers and corresponding
experimental products with their compositions are further detailed in Sup-
plementary Tables S2–S4. (B) Phase compositions plotted as pressure vs. run duration (h) for the hybrid serpentinite-basalt reaction experiments at
0.2–1.0 GPa and 1,250–1,300°C and R 2.5–6.5. The diagram shows
mineral, glass, and fluid phase associations observed in the olivine-rich zone
after quenching. Lbas is a unique zone of hydrous basaltic glass, while the
olivine-rich zone contains residual serpentine minerals (Serp), predominant
magnesian olivine (Ol), and orthopyroxene (Opx) in the interstitial glass (Lint), in
the presence of fluid (F) bubbles. All olivine-rich samples show the presence of
chromite or chromiferous magnetite. R is the ratio of starting basaltic melt to
serpentinite rock. (SiO2 contents ranging from 57 to 67 wt%) in most
hybrid 0.2 GPa experiments. Only two shortest (≤0.5 h)
of
the
0.5 GPa
runs
yield
interstitial
melts
of
intermediate
composition
(SiO2
56–60 wt%),
whereas the shortest (2.5 h) 1.0 GPa run generates
interstitial melt of mafic composition (SiO2 50 wt
%). These results are in line with the fact that the
combined effects of water, alkalis and low pressure
on the structure of the aluminosilicate melt shift the
equilibrium olivine-pyroxene cotectics to higher SiO2
contents (Kushiro, 1968; Hirschmann et al., 1998;
Lundstrom, 2000). 4)
Finally, a homogeneous and stable olivine-rich zone
containing mostly high-Mg olivine (Fo90–94, mol%),
chromite (56–73 Mg#, and 68–89 Cr#) and interstitial
felsic glass is systematically observed in all mixed (SB
series)
time-series
experiments
with
low
basalt-to-
serpentinite
ratios
(R
0.25–1.0)
in
the
starting
material, suggesting the formation of stable association
of olivine, chromite and felsic melts at 0.2 GPa (Figures
1A,B, 2, 3A, and 4; Supplementary Table S3). 4)
Finally, a homogeneous and stable olivine-rich zone
containing mostly high-Mg olivine (Fo90–94, mol%),
chromite (56–73 Mg#, and 68–89 Cr#) and interstitial
felsic glass is systematically observed in all mixed (SB
series)
time-series
experiments
with
low
basalt-to-
serpentinite
ratios
(R
0.25–1.0)
in
the
starting
material, suggesting the formation of stable association
of olivine, chromite and felsic melts at 0.2 GPa (Figures
1A,B, 2, 3A, and 4; Supplementary Table S3). 4) Interstitial Glass Composition and
Equilibration With Minerals We argue for near equilibrium (steady) state of the produced felsic
melts in assemblages with high-Mg olivine based on the mixed time-series
experiments. Constant concentrations are established very early (in less than
2 h) for R 0.25 and R 1.0 in the mixed runs, suggesting attainment of
steady state or thermodynamic equilibrium of the run products. X axis scale
breaks from 12 to 40. The major element composition of the glass produced in these
experiments is mainly controlled by pressure, but also depends on
the basalt-to-serpentinite initial ratio (R) in the mixed runs. The
glasses produced in the hybrid run lasting 2.5 h at 1.0 GPa are
mafic (50 wt% SiO2), with an average MgO content of 10 wt%,
similar to the glasses produced in previous studies of equilibrium
partial melting of hydrated peridotite at similar pressures (Ulmer,
2001). By
contrast,
generally
higher
SiO2
(57–71 wt%,
recalculated to 100% on a volatile-free basis) contents are
found in glasses from all hybrid runs at 0.2 GPa, and from the
mixed experiments at 0.2 GPa with the highest proportion of
serpentinite (50–80 wt%; R 0.25–1.0), as well as those produced
in short (≤0.5 h) runs at 0.5 GPa. For example, the mixed runs
with the highest proportion of serpentinite (80 wt%) and low
basalt-to-serpentinite initial ratios (R 0.25) produce dacite
glasses with up to 70–71 wt% SiO2 contents (Figures 4–6,
Supplementary Table S3). Furthermore, the 0.2 GPa glasses
are enriched in alkalis (up to 3.9 wt% Na2O+ K2O), aluminum
(up to 20.4 wt% Al2O3) and chromium (up to ∼710 Cr ppm)
(Figures 5, 6; Supplementary Tables S3, S4). The experimental glass composition obtained in the 0.2 GPa
mixed experiments is in an agreement with that obtained through
modeling using MELTS at condition of the melts saturation with
H2O fluid (Figures 5, 6; Supplementary Table S6). In the
modeling, we used mixture of serpentinite with basalt in
different proportions comparable to those observed in the
mixed
experiments
(see
Supplementary
Material). The
modeled phase proportions (e.g., melt proportions up to 35 wt
%, Supplementary Table S6) and the mineral compositions are
in agreement with those of the experimental ones (melt
proportion up to 40 wt%, Supplementary Table S2). Interstitial Glass Composition and
Equilibration With Minerals In the hybrid and mixed runs at 0.2–1.0 GPa, the olivine-rich
zones host interstitial pockets of glass ranging in size from 10 to
200 μm, showing enrichment in silica (up to 66–71 wt%),
aluminum, alkalis and water (Figures 4, 5, Supplementary
Tables
S2–S4). The
calculated
chromite-olivine
pair
equilibration
temperatures
range
from
1,222
to
1,261°C
(Supplementary Table S2), in accordance with the runs
temperature
of
1,250
°C,
confirming
an
attainment
of
thermodynamic
equilibrium
between
solid
phases
during
experiments even if Fe-Mg partition coefficients between
olivine and the associated felsic melt range from 0.19 to 0.24
in the hybrid runs and from 0.08 to 0.25 in the mixed runs
according to model of (Toplis, 2005), indicating a lack of Fe-Mg
equilibrium between phases involving the felsic liquid (although
such low values as 0.16 have been reported in alkali-rich systems). This variability may be at least partly due to quench artifacts. Indeed, the glass transition temperature of hydrous andesitic FIGURE 3 | (A) Phase compositions plotted as serpentine (SERP)
proportion (in wt%) vs. run duration (h) for the pure serpentinite dehydration
experiments (R 0) and mixed serpentinite-basalt reaction experiments at
0.2 GPa and 1,250°C and basalt-to-serpentinite ratios R 0.25, 1 and 4. The diagram shows mineral, glass, and fluid phase associations observed
after quenching. Ol, Opx, Cpx, Lint, Lbas, and F stand for olivine,
(Continued) May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 9 Felsic Crust Formation at Shallow Depth Borisova et al. yield olivine-felsic glass Fe-Mg partition coefficients of 0.24 ±
0.13, close to the theoretical range of 0.31 ± 0.05 (Toplis,
2005) (Supplementary Table S2), suggesting that the felsic
melts are close to Fe-Mg equilibrium with the associated olivine. FIGURE 4 | Weight proportion of interstitial felsic melt (in wt%),
magnesium index (100 Mg/(Mg + Fe), total alkalis, SiO2 and Al2O3 (in wt%) in
the interstitial felsic melts of the time-series experiments vs. run duration (h). The oxide contents are recalculated to 100% on a volatile-free basis. R is
the mass ratio of starting basaltic melt to serpentinite rock (basalt-to-
serpentinite ratio). The dotted lines indicate compositional tendencies for the
felsic melts of the mixed and hybrid runs. The mixed runs for given R values
(0.25 and 1.0) are characterized by homogeneous interstitial felsic melts with
constant Si, Al, and alkalis, while Lint proportions are independent of run
duration. Interstitial Glass Composition and
Equilibration With Minerals For
example,
high-Mg
olivine
(91–93 mol%
Fo),
chromite
(20–45 mol% of chromite molecule), aqueous fluid and Si-rich
liquid (up to 61 wt% SiO2 on anhydrous basis) appear in
equilibrium
assemblages
at
1,250–1,300°C,
0.2 GPa
and
ΔQFM+2 to +3 in the models using a mixture of 80–95 wt%
serpentinite and 20–5 wt% basalt, respectively. The felsic liquids
contain up to 67 wt% SiO2 if the composition of aqueous fluid is
taken
into
consideration
(Supplementary
Table
S6). Additionally, the MELTS liquids modelled at 0.2 GPa (up to
61 wt% SiO2 on anhydrous basis) are richer in SiO2 than those
modelled at 0.5–0.9 GPa (up to 55–59 wt% SiO2 on anhydrous
basis) (Supplementary Table S6), confirming our experimental
observations at 0.5 and 1.0 GPa. FIGURE 4 | Weight proportion of interstitial felsic melt (in wt%),
magnesium index (100 Mg/(Mg + Fe), total alkalis, SiO2 and Al2O3 (in wt%) in
the interstitial felsic melts of the time-series experiments vs. run duration (h). The oxide contents are recalculated to 100% on a volatile-free basis. R is
the mass ratio of starting basaltic melt to serpentinite rock (basalt-to-
serpentinite ratio). The dotted lines indicate compositional tendencies for the
felsic melts of the mixed and hybrid runs. The mixed runs for given R values
(0.25 and 1.0) are characterized by homogeneous interstitial felsic melts with
constant Si, Al, and alkalis, while Lint proportions are independent of run
duration. We argue for near equilibrium (steady) state of the produced felsic
melts in assemblages with high-Mg olivine based on the mixed time-series
experiments. Constant concentrations are established very early (in less than
2 h) for R 0.25 and R 1.0 in the mixed runs, suggesting attainment of
steady state or thermodynamic equilibrium of the run products. X axis scale
breaks from 12 to 40. FIGURE 4 | Weight proportion of interstitial felsic melt (in wt%),
magnesium index (100 Mg/(Mg + Fe), total alkalis, SiO2 and Al2O3 (in wt%) in
the interstitial felsic melts of the time-series experiments vs. run duration (h). The oxide contents are recalculated to 100% on a volatile-free basis. R is
the mass ratio of starting basaltic melt to serpentinite rock (basalt-to-
serpentinite ratio). The dotted lines indicate compositional tendencies for the
felsic melts of the mixed and hybrid runs. Interstitial Glass Composition and
Equilibration With Minerals The mixed runs for given R values
(0.25 and 1.0) are characterized by homogeneous interstitial felsic melts with
constant Si, Al, and alkalis, while Lint proportions are independent of run
duration. We argue for near equilibrium (steady) state of the produced felsic
melts in assemblages with high-Mg olivine based on the mixed time-series
experiments. Constant concentrations are established very early (in less than
2 h) for R 0.25 and R 1.0 in the mixed runs, suggesting attainment of
steady state or thermodynamic equilibrium of the run products. X axis scale
breaks from 12 to 40. To evaluate the result of energy constrained interaction
between serpentinite and basalt, we have performed Magma
Chamber Simulator (MCS, Bohrson et al., 2014; Bohrson
et al., 2020; Heinonen et al., 2020) modeling in the range from
0.1 to 0.5 GPa at condition of the magma saturation with H2O
fluid (Supplementary Table S7). The MCS modeled liquid
composition is in excellent agreement with that of the
experimental felsic melts (Figures 5, 6). The only exception is
Mg index, but this is likely related to the Fe-Mg re-equilibration of melts with ∼5 wt% H2O is very low, Tg ≈300°C (e.g., Deubener
et al., 2003). As a result, these melts are particularly sensitive to
modification during quenching, thus contributing to the observed
Fe-Mg disequilibrium. Hybrid run samples P32, P33, and P42 May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 10 Felsic Crust Formation at Shallow Depth Borisova et al. FIGURE 5 | Sum of alkali oxides (in wt%) vs. SiO2 (in wt%) in interstitial glasses produced during hybrid and mixed runs of serpentinite-basalt interaction plotted in
the classification diagram of Le Maitre (2002). The major element compositions of the measured (“meas. melts”, red dots are recalculated to 100% on a volatile-free
basis). The error bars reflect the felsic glass heterogeneity compared to the mean values. The glass compositions are compared with the starting basalt and serpentinites,
the anhydrous 0.1 MPa experimental glasses (Fisk, 1986), modern TTG or plagiogranites in mantle section of ophiolites (Amri et al., 1996, 2007; Shervais, 2008; Xu
et al., 2017), the bulk Earth continental crust (Wedepohl, 1995; Rudnick and Gao, 2003), Archaean TTG (Moyen and Martin, 2012), Acasta Gneiss Complex rocks
(Idiwhaa gneisses) (Reimink et al., 2016), Martian felsic rocks (Sautter et al., 2016). BA, basaltic andesite; A, andesite. Interstitial Glass Composition and
Equilibration With Minerals Red lines with black points correspond to the
thermodynamic modeling using MELTS of equilibrium liquids coexisting with mixture of 80–95 wt% serpentinite with 20–5 wt% basalt (R < 1) at 0.2 GPa and different
temperatures ranging from 1,500°C to 1,250°C and redox conditions of ΔQFM+2: 1) 80 wt% serpentinite and 20 wt% basalt (R 0.25); 2) 90 wt% serpentinite and
10 wt% basalt (R 0.11), and 3) 95 wt% serpentinite and 5 wt% basalt (R 0.05) (Supplementary Table S6). The compositions modeled at 1,250°C are corrected
according to SiO2 contents of 6 wt% SiO2 in the aqueous fluid (Supplementary Tables S5, S6). Pink crosses correspond to the Magma Chamber Simulator (MCS)
modeling results of equilibrium liquid appearing upon the interaction between the serpentinite and basalt at different basalt-to-serpentinite mass ratio, R, different
temperatures of the serpentinite rock (1,100–1,300°C) and pressures from 0.1 to 0.5 GPa at conditions of the magma saturation with H2O fluid (Supplementary
Table S7). FIGURE 5 | Sum of alkali oxides (in wt%) vs. SiO2 (in wt%) in interstitial glasses produced during hybrid and mixed runs of serpentinite-basalt interaction plotted in
the classification diagram of Le Maitre (2002). The major element compositions of the measured (“meas. melts”, red dots are recalculated to 100% on a volatile-free
basis). The error bars reflect the felsic glass heterogeneity compared to the mean values. The glass compositions are compared with the starting basalt and serpentinites,
the anhydrous 0.1 MPa experimental glasses (Fisk, 1986), modern TTG or plagiogranites in mantle section of ophiolites (Amri et al., 1996, 2007; Shervais, 2008; Xu
et al., 2017), the bulk Earth continental crust (Wedepohl, 1995; Rudnick and Gao, 2003), Archaean TTG (Moyen and Martin, 2012), Acasta Gneiss Complex rocks
(Idiwhaa gneisses) (Reimink et al., 2016), Martian felsic rocks (Sautter et al., 2016). BA, basaltic andesite; A, andesite. Red lines with black points correspond to the
thermodynamic modeling using MELTS of equilibrium liquids coexisting with mixture of 80–95 wt% serpentinite with 20–5 wt% basalt (R < 1) at 0.2 GPa and different
temperatures ranging from 1,500°C to 1,250°C and redox conditions of ΔQFM+2: 1) 80 wt% serpentinite and 20 wt% basalt (R 0.25); 2) 90 wt% serpentinite and
10 wt% basalt (R 0.11), and 3) 95 wt% serpentinite and 5 wt% basalt (R 0.05) (Supplementary Table S6). Interstitial Glass Composition and
Equilibration With Minerals The compositions modeled at 1,250°C are corrected
according to SiO2 contents of 6 wt% SiO2 in the aqueous fluid (Supplementary Tables S5, S6). Pink crosses correspond to the Magma Chamber Simulator (MCS)
modeling results of equilibrium liquid appearing upon the interaction between the serpentinite and basalt at different basalt-to-serpentinite mass ratio, R, different
temperatures of the serpentinite rock (1,100–1,300°C) and pressures from 0.1 to 0.5 GPa at conditions of the magma saturation with H2O fluid (Supplementary
Table S7). the experimental liquids with the predominant olivine upon
quenching. produced in our experiments are likely formed by the fertilization
of olivine-rich zones due to the presence of aqueous fluids
produced by the interaction of serpentinite and basaltic melt
at 0.2–1.0 GPa. At lower pressures, this process is associated with
the incongruent melting of harzburgite producing olivine at the
expense of orthopyroxene. Indeed, according to Kushiro (1968),
Shaw (1999), Shaw et al. (1998), Lundstrom (2000), Shaw and
Dingwell (2008), and Borisova et al. (2020a) silica-rich liquids
may be formed via orthopyroxene dissolution during melt-
peridotite interactions. The presence of polyhedral and mainly
unzoned
olivine
crystals,
co-existing
with
homogeneous
interstitial felsic melt in the olivine-rich zone of the longest
runs (Figure 2; Table 1; Supplementary Table S2), supports
the growth of new olivine that attains equilibrium with the melt at
1,250°C and 0.2 GPa. Felsic Melt Formation The measured glass compositions vary from basaltic andesite to
dacite, which is consistent with the general tendency of olivine-
saturated partial melts to become progressively richer in SiO2
with decreasing pressure and increasing water content (e.g.,
Hirschmann et al., 1998). Additionally, it is widely known that
water fluid expands the thermodynamic stability of forsterite-rich
olivine (e.g., Green, 1973; Nicholls et al., 1973) and chromite
(Veksler and Hou, 2020). Thus, it is not surprising that high-Mg
olivine in association with chromite is recorded in equilibrium
with felsic melts of dacitic composition in our low-pressure
experiments (0.2 GPa). Most glasses produced at 0.2 GPa are
richer in Si, Al, and alkalis than dry glasses produced at 0.1 MPa
(Fisk, 1986) as well as nearly dry glasses produced at 0.5 GPa
(Kelemen et al., 1995). The hydrous intermediate to felsic melts A steady state is established within less than 2 h after the start
of reaction in the mixed runs (Figures 3, 4). The fluid-present
reaction leads to the formation of high-Mg olivine + chromite +
felsic melt. Note that the composition of the interstitial felsic melt
produced in this way is a function of the basalt-to-serpentinite
mass ratio (R). High and constant SiO2 contents (70–71 wt%, May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 11 Felsic Crust Formation at Shallow Depth Borisova et al. FIGURE 6 | Magnesium index (100 Mg/(Mg + Fe)), major and minor Ti, Al, P oxides (in wt%), and Cr (in ppm) vs. SiO2 (in wt%) in interstitial glasses produced at
0.2–1.0 GPa during serpentinite-basalt interaction of hybrid and mixed runs. The major element compositions of the measured (meas.) melts are recalculated to 100%
on a volatile-free basis. The error bars reflect the felsic glass inter-sample heterogeneity compared to the mean values (Supplementary Tables S3, S4). The glass
composition is compared to the starting basalt (BAS) and serpentinite (SERP), the anhydrous 0.1 MPa experimental glasses (Fisk, 1986), modern TTG
(plagiogranites) in mantle sections of ophiolites (Amri et al., 1996, 2007; Shervais, 2008; Xu et al., 2017), the bulk Earth continental crust (Bulk CC) (Wedepohl, 1995;
(Continued) FIGURE 6 | Magnesium index (100 Mg/(Mg + Fe)), major and minor Ti, Al, P oxides (in wt%), and Cr (in ppm) vs. SiO2 (in wt%) in interstitial glasses produced at
0.2–1.0 GPa during serpentinite-basalt interaction of hybrid and mixed runs. Felsic Melt Formation The compositions modeled at 1,250°C are corrected according to SiO2 contents of 6 wt% SiO2 in
the aqueous fluid (Supplementary Tables S5, S6). Pink crosses correspond to the Magma Chamber Simulator (MCS) modeling results of equilibrium liquid appearing
upon the interaction between the serpentinite and basalt at different basalt-to-serpentinite mass ratio, R, different temperatures of the serpentinite rock (1,100–1,300°C)
and pressures from 0.1 to 0.5 GPa at conditions of the magma saturation with H2O fluid (Supplementary Table S7). FIGURE 6 | Rudnick and Gao, 2003), Archaean TTG (Moyen and Martin, 2012), Acasta Gneiss Complex rocks (Idiwhaa gneisses) (Reimink et al., 2016) and Martian felsic
rocks (Sautter et al., 2016). Both EPMA and LA-ICP-MS data for the 0.2 GPa felsic melts are used to determine the Cr concentrations of hybrid melts, whereas only
EPMA data are used for the P contents (Supplementary Table S3). Red lines labelled 1, 2 and 3 with black points correspond to the thermodynamic modeling using
MELTS of equilibrium liquids coexisting with mixture of 80–95 wt% serpentinite with 20–5 wt% basalt (R < 1) at 0.2 GPa and different temperatures ranging from 1,500°C
to 1,250°C and redox conditions of ΔQFM+2: 1) 80 wt% serpentinite and 20 wt% basalt (R 0.25); 2) 90 wt% serpentinite and 10 wt% basalt (R 0.11) and 3) 95 wt%
serpentinite and 5 wt% basalt (R 0.05) (Supplementary Table S6). The compositions modeled at 1,250°C are corrected according to SiO2 contents of 6 wt% SiO2 in
the aqueous fluid (Supplementary Tables S5, S6). Pink crosses correspond to the Magma Chamber Simulator (MCS) modeling results of equilibrium liquid appearing
upon the interaction between the serpentinite and basalt at different basalt-to-serpentinite mass ratio, R, different temperatures of the serpentinite rock (1,100–1,300°C)
and pressures from 0.1 to 0.5 GPa at conditions of the magma saturation with H2O fluid (Supplementary Table S7). hybrid run products (Supplementary Table S3, S4), suggesting
local thermodynamic equilibrium was attained between the fluid
and these minerals. recalculated to 100% on a volatile-free basis) are observed in the
felsic melts at the lowest R of 0.25. For the run series with R 1.0,
clinopyroxene is an additional stable crystalline phase formed in
association with high-Mg olivine, chromite and felsic melt. Felsic Melt Formation Remarkably, the proportions of interstitial melt (33–40 wt%),
as well as the concentrations of SiO2, Al2O3, and alkalis (Na2O+
K2O wt%) in the melt, remain constant over a wide range of R
(0.25–1.0) throughout the whole run duration (Figure 4). The
Mg/(Mg + Fe) index of the melt may be affected by Fe-Mg
exchange with olivine and chromite during quenching. The aqueous fluid in mixed and hybrid experiments is
predicted to be enriched in Si (up to 6 wt%), Na (up to 4 wt%),
K (up to 0.4 wt%), and Al (up to 0.2 wt%), with other elements
being much less abundant (Mg, Ca, Fe typically < ∼10 ppm). Increasing the pressure from 0.2 to 1.0 GPa generally results in an
increase of all element concentrations in the fluid, in agreement
with the increase of water solvent density and water solvation
power (e.g., Pokrovski et al., 2013). A similar systematic order of
decreasing concentration in the fluid is maintained over the whole
range of pressures and basalt-to-serpentinite initial ratios: Si > Na >
K > Al >> Fe > Ca > Mg. This finding strongly suggests that
intrinsic uncertainties in the thermodynamic model are unlikely to
obscure
the large
amplitude
of general
trends. The
low
concentrations of Fe and Ca (typically 100 to 10,000 times less
abundant than Si, Na, K or Al), and in particular of Mg (106–108
less abundant than Si, Supplementary Table S5), are in agreement
with the typical order of elemental abundance in natural
hydrothermal-magmatic fluids of relatively low salinity as
inferred from fluid inclusions (e.g., Kouzmanov and Pokrovski,
2012; references therein). This is corroborated by our experimental
results, providing evidence for major transfer of Si, Na, K, and Al
from basalt and serpentinite to felsic melts. Remarkably, the Si/Mg
ratio (∼106–1010) in the fluid phase predicted by calculations
systematically increases by almost 1,000 times with decreasing
pressure from 1 to 0.2 GPa (Supplementary Table S5). Even
though our calculations are semi-quantitative at such extreme
conditions for the fluid phase (see Supplementary Material),
this large relative change is an additional indication that
relatively low pressures should favor the enrichment of both
fluid and melt phases in Si relative to “mafic” elements (Mg) in
water-present systems, thus promoting the formation of felsic
(i.e. Si-rich) melts quenched to glasses, as observed in our direct
experiments. Aqueous Fluid Composition Aqueous Fluid Composition
In all hybrid and mixed experiments at 0.2 and 0.5 GPa, fluid
bubbles
are
present,
likely
produced
by
serpentinite
decomposition. Therefore,
we
conducted
thermodynamic
modelling of the element solubility in the aqueous fluid phase
(Supplementary Material), which provides an independent
qualitative support for our conclusions drawn from the
analysis of the run products described above (e.g., interstitial
felsic glasses). Modeling shows that the aqueous fluid generated
by serpentinite dehydration in the presence of basaltic melt is
systematically enriched in Si, Al, Na, and K (Supplementary
Table S5) compared to both the initial and reacted serpentinite
(olivine-rich zone, see above). The major mineral phases
predicted
to
form
at
equilibrium
are
olivine
and
orthopyroxene, together with minor amounts of clinopyroxene
and feldspar-like glass phases, are in good agreement with those
observed in most hybrid and mixed experiments. The calculations
predict high-Mg olivine (Fo93–96 mol%) and orthopyroxene (Mg#
94–96) compositions, which are within the range analyzed in the Felsic Melt Formation The major element compositions of the measured (meas.) melts are recalculated to 100%
on a volatile-free basis. The error bars reflect the felsic glass inter-sample heterogeneity compared to the mean values (Supplementary Tables S3, S4). The glass
composition is compared to the starting basalt (BAS) and serpentinite (SERP), the anhydrous 0.1 MPa experimental glasses (Fisk, 1986), modern TTG
(plagiogranites) in mantle sections of ophiolites (Amri et al., 1996, 2007; Shervais, 2008; Xu et al., 2017), the bulk Earth continental crust (Bulk CC) (Wedepohl, 1995;
(Continued) FIGURE 6 | Magnesium index (100 Mg/(Mg + Fe)), major and minor Ti, Al, P oxides (in wt%), and Cr (in ppm) vs. SiO2 (in wt%) in interstitial glasses produced at
0.2–1.0 GPa during serpentinite-basalt interaction of hybrid and mixed runs. The major element compositions of the measured (meas.) melts are recalculated to 100%
on a volatile-free basis. The error bars reflect the felsic glass inter-sample heterogeneity compared to the mean values (Supplementary Tables S3, S4). The glass
composition is compared to the starting basalt (BAS) and serpentinite (SERP), the anhydrous 0.1 MPa experimental glasses (Fisk, 1986), modern TTG
(plagiogranites) in mantle sections of ophiolites (Amri et al., 1996, 2007; Shervais, 2008; Xu et al., 2017), the bulk Earth continental crust (Bulk CC) (Wedepohl, 1995;
(Continued) May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 12 Felsic Crust Formation at Shallow Depth Borisova et al. FIGURE 6 | Rudnick and Gao, 2003), Archaean TTG (Moyen and Martin, 2012), Acasta Gneiss Complex rocks (Idiwhaa gneisses) (Reimink et al., 2016) and Martian felsic
rocks (Sautter et al., 2016). Both EPMA and LA-ICP-MS data for the 0.2 GPa felsic melts are used to determine the Cr concentrations of hybrid melts, whereas only
EPMA data are used for the P contents (Supplementary Table S3). Red lines labelled 1, 2 and 3 with black points correspond to the thermodynamic modeling using
MELTS of equilibrium liquids coexisting with mixture of 80–95 wt% serpentinite with 20–5 wt% basalt (R < 1) at 0.2 GPa and different temperatures ranging from 1,500°C
to 1,250°C and redox conditions of ΔQFM+2: 1) 80 wt% serpentinite and 20 wt% basalt (R 0.25); 2) 90 wt% serpentinite and 10 wt% basalt (R 0.11) and 3) 95 wt%
serpentinite and 5 wt% basalt (R 0.05) (Supplementary Table S6). Felsic Melt Formation The MCS modeling data demonstrate that i) after the
serpentinite addition, SiO2 content in the equilibrated liquid
increases with decreasing pressures (Supplementary Table
S7). ii)
The
lower
basalt-to-serpentinite
(R)
mass
ratio
increases the SiO2 content in the equilibrated melt (Figures 5,
6). In MCS results there is a massive crystallization event
following the reaction with serpentinite, but the following
crystallization will be discussed in a future paper. iii) The
lower the temperature of the serpentinite, the higher the SiO2
increase in the equilibrated liquid. This is a good outcome since
the experiments cannot really realistically reproduce the lower
temperature of the serpentinite. iv) Having Cr in the initial
serpentinite rock also increases the SiO2 in the equilibrated
liquid. Both thermodynamic
models
(MELTS
and
MCS)
suggest that the mineral association observed in the 0.2 GPa
mixed experiments is near thermodynamic equilibrium. GEOLOGICAL SIGNIFICANCE The incompatible
trace element patterns of the felsic glasses are nearly identical to
those of modern TTG (plagiogranite) rocks found in the mantle
sections of ophiolites (Amri et al., 1996; 2007; Shervais, 2008; Xu
et al., 2017). The felsic glasses produced in runs at 0.2 GPa have
major and trace element contents (Si, Ti, Al, Mg, Ca, Na, K, P, and
Cr, REE), as well as Mg/(Mg + Fe), that share some similarities
with modern TTG situated in the mantle sections of ophiolites
(Figures 5–7). The 0.2 GPa intermediate to felsic melts are
characterized by lower SiO2 and Na2O + K2O contents and
higher Cr contents compared with Archaean TTG rocks
(Figures 5, 6). The slight enrichment in LREE of the felsic
glasses produced in our experiments differs from the strongly
fractionated
patterns
of
Archaean
tonalite-trondhjemite-
granodiorite (TTG) suites (Moyen and Martin, 2012). This is
in line with the conditions of shallow felsic crust formation by a
mechanism distinct from the “sagduction” or plate subduction
operating during the Archaean (Moyen and Martin, 2012). At
similar SiO2 contents, the higher Mg index and Cr contents (up to
1,400 Cr ppm) in the experimental felsic glasses produced at
0.2 GPa contrast strongly with the 4.02 Ga Idiwhaa gneisses,
northwest Canada (Figure 6), suggesting that the Hadean
Acasta Gneiss Complex magmas did not form due to reaction
of serpentinite with basaltic melts. Except for fluid-mobile B and
Pb, the trace element compositions of the 0.2 GPa interstitial
felsic glasses are similar to those of the starting basaltic melt
(Figure 7). Additionally, hafnium contents (0.5–4.0 ppm Hf) and
Lu/Hf ratios [LuN/HfN 0.11–2.2, normalized to the primitive
mantle composition of Sun and McDonough (1989)] in the
0.2 GPa felsic melts are similar to those of the starting basaltic
melt (2.7 ppm and 0.7, respectively), suggesting that the
experimental melts might have inherited their trace element
and Hf isotope signatures from a precursor basaltic reservoir
(Supplementary Table S3). Shallow Felsic Crust Production by Melting
of Serpentinized Peridotite GEOLOGICAL SIGNIFICANCE elements (LREE) observed in felsic glasses is an important
signature of typical continental rocks compared to mantle-
derived magmas (Rudnick and Gao, 2003). The incompatible
trace element patterns of the felsic glasses are nearly identical to
those of modern TTG (plagiogranite) rocks found in the mantle
sections of ophiolites (Amri et al., 1996; 2007; Shervais, 2008; Xu
et al., 2017). The felsic glasses produced in runs at 0.2 GPa have
major and trace element contents (Si, Ti, Al, Mg, Ca, Na, K, P, and
Cr, REE), as well as Mg/(Mg + Fe), that share some similarities
with modern TTG situated in the mantle sections of ophiolites
(Figures 5–7). The 0.2 GPa intermediate to felsic melts are
characterized by lower SiO2 and Na2O + K2O contents and
higher Cr contents compared with Archaean TTG rocks
(Figures 5, 6). The slight enrichment in LREE of the felsic
glasses produced in our experiments differs from the strongly
fractionated
patterns
of
Archaean
tonalite-trondhjemite-
granodiorite (TTG) suites (Moyen and Martin, 2012). This is
in line with the conditions of shallow felsic crust formation by a
mechanism distinct from the “sagduction” or plate subduction
operating during the Archaean (Moyen and Martin, 2012). At
similar SiO2 contents, the higher Mg index and Cr contents (up to
1,400 Cr ppm) in the experimental felsic glasses produced at
0.2 GPa contrast strongly with the 4.02 Ga Idiwhaa gneisses,
northwest Canada (Figure 6), suggesting that the Hadean
Acasta Gneiss Complex magmas did not form due to reaction
of serpentinite with basaltic melts. Except for fluid-mobile B and
Pb, the trace element compositions of the 0.2 GPa interstitial
felsic glasses are similar to those of the starting basaltic melt
(Figure 7). Additionally, hafnium contents (0.5–4.0 ppm Hf) and
Lu/Hf ratios [LuN/HfN 0.11–2.2, normalized to the primitive
mantle composition of Sun and McDonough (1989)] in the
0.2 GPa felsic melts are similar to those of the starting basaltic
melt (2.7 ppm and 0.7, respectively), suggesting that the
experimental melts might have inherited their trace element
and Hf isotope signatures from a precursor basaltic reservoir
(Supplementary Table S3). elements (LREE) observed in felsic glasses is an important
signature of typical continental rocks compared to mantle-
derived magmas (Rudnick and Gao, 2003). Compositional Features of Interstitial
Glasses The intermediate to felsic glasses from the hybrid and mixed
runs were analyzed not only for major and minor (Cr, Ni) but also
for
trace
elements
(Supplementary
Material). Figure
7
demonstrates
incompatible
trace
element
compositions
(normalized to the primitive mantle) of the interstitial felsic
glasses. The positive Pb anomaly relative to light rare earth May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 13 Borisova et al. Felsic Crust Formation at Shallow Depth FIGURE 7 | Primitive mantle-normalized trace element abundance patterns of felsic glasses produced at 0.2 GPa during serpentinite-basalt interaction. The
primitive mantle composition is after (Sun and McDonough, 1989) and (Lyubetskaya and Korenaga, 2007) for B only. The felsic glass composition is compared to basalt
(starting MORB) and serpentinite (starting SERP), modern TTG (Amri et al., 1996, 2007; Shervais, 2008; Xu et al., 2017), the bulk continental crust after Wedepohl (1995). FIGURE 7 | Primitive mantle-normalized trace element abundance patterns of felsic glasses produced at 0.2 GPa during serpentinite-basalt interaction. The
primitive mantle composition is after (Sun and McDonough, 1989) and (Lyubetskaya and Korenaga, 2007) for B only. The felsic glass composition is compared to basalt
(starting MORB) and serpentinite (starting SERP), modern TTG (Amri et al., 1996, 2007; Shervais, 2008; Xu et al., 2017), the bulk continental crust after Wedepohl (1995). FIGURE 7 | Primitive mantle-normalized trace element abundance patterns of felsic glasses produced at 0.2 GPa during serpentinite-basalt interaction. The
primitive mantle composition is after (Sun and McDonough, 1989) and (Lyubetskaya and Korenaga, 2007) for B only. The felsic glass composition is compared to basalt
(starting MORB) and serpentinite (starting SERP), modern TTG (Amri et al., 1996, 2007; Shervais, 2008; Xu et al., 2017), the bulk continental crust after Wedepohl (1995). Shallow Felsic Crust Production by Melting
of Serpentinized Peridotite Our study provides new insight into potential mechanisms of
intermediate to felsic crust formation. Serpentinized peridotites
and mafic cumulates are the most abundant constituents of the
modern slow-spreading oceanic lithosphere at the level of the
Moho mantle-crust boundary (Cannat, 1993). Cyclic magmatic
and hydrothermal processes in the modern Earth likely lead to the
generation of intermediate to felsic material within the peridotitic
lithosphere below spreading centers at high temperature and
shallow (<10 km) depths. These processes include repeated
intrusions of mantle-derived basaltic magmas (O’Hara and
Mathews, 1981; Amri et al., 1996; 2007; Borisova et al., 2012),
where reactions are possible between hot basaltic melts and a
previously formed cold hydrated ultramafic lithosphere during
periods of low magma supply and oceanic water circulation at
shallow depth (Figure 8). The melting of serpentinized peridotite
produced in our experiments leads to the formation of dunite-
wehrlite- or chromitite-type rocks (Figures 1–3A,B) similar to
their natural analogues described in ophiolites (e.g., Dick, 1977;
Benoit et al., 1999; Borisova et al., 2020c; Zagrtdenov et al., 2018;
Rospabé et al., 2019; Borisova et al., 2020b). Our experimental
data thus support an empirical model for the reaction between
hydrated mantle and basaltic magma inferred from natural data
on
chromite-hosted
inclusions
from
the
Oman
ophiolite
(Borisova et al., 2012). The olivine-rich aggregates containing
interstitial hydrous silica-saturated liquids are gravitationally
unstable, like those obtained in our experiments. This is
because of the high density contrast between hydrous felsic
melts (<2.4 g/cm3, Supplementary Table S6) and mafic silicate
minerals (3.2–3.3 g/cm3) and chromite (∼5.0 g/cm3), while melt
mobility is also favored by low viscosity. With reference to
processes occurring at oceanic spreading centers (Amri et al.,
1996, 2007; Benoit et al., 1999), which lead to the formation of May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 14 Felsic Crust Formation at Shallow Depth Borisova et al. FIGURE 8 | marked as “t”. The generated melts forms intrusive bodies in
the host peridotite and overlying crust at shallow depths <10 km. Black
dotted lines correspond to isotherms. The horizontal scale of the model is
∼30 km. The depths on the vertical scale are calculated assuming a
serpentinite density of 3 g/cm3. Middle panel: Model for the formation of
the Hadean felsic crust due to activity of either a heat-pipe or a proto-rift
volcanoes. Dehydration is initiated by the intrusion of mantle-derived
magmas into (ultra-)mafic protocrust. Shallow Felsic Crust Production by Melting
of Serpentinized Peridotite The released fluids promote partial
melting of the peridotite, which produces felsic (e.g., tonalite) magmas
marked as “t”. The generated melts form intrusive bodies in the host
peridotite protocrust at shallow depths <10 km. Black dotted lines cor-
respond to isotherms. The horizontal scale of the model is ∼30 km. The
depths on the vertical scale are calculated assuming a serpentinite density
of 3 g/cm3. Lower panel: Model of impact formation of felsic crust on the
early Earth and Mars. Impact-induced melting of the hydrated ultramafic-
mafic protocrust promotes fertilization and partial melting of the peri-
dotite. The (ultra-) mafic protocrust is hydrated through hydrothermal
circulation due to contact with seawater. The partial melting produces
intermediate to felsic (tonalite and/or granodiorite) magmas marked as “t”. The production of intermediate to felsic melts may occur when the
predominant proportion of serpentinite in the hybrid system is above
50 wt% (R ≤1). The generated melts form an impact body at shallow
depths <10 km. Blue dotted lines correspond to isotherms. The horizontal
scale of the model is ∼30 km. FIGURE 8 | Upper panel: Model for the formation of shallow felsic
crust in mantle peridotites of the oceanic lithosphere. Dehydration is
initiated by the intrusion of mantle-derived magmas into hydrated
(serpentinized) mantle. The released fluid promotes fertilization and
partial melting of the peridotite, which leads to production of TTG magm
(Continue
| ophiolites, low-density hydrous felsic melts generated below and/
or at the Moho transition zone are collected in decimeter to meter
sized pods and veins which then progressively coalesce into larger
intrusions in the shallow oceanic lithosphere. Alternatively, the
felsic melts produced by aqueous fluid-assisted partial melting of
peridotite may remain scattered in peridotites at shallow depths
<10 km, as shown by olivine-hosted melt inclusions in peridotite
xenoliths (Hirschmann et al., 1998). If segregation into intrusions
takes place, cooling (e.g., due to hydrothermal circulation) will
result in crystallization of these hydrous felsic liquids. In addition
to the generation of low-temperature plagiogranite magmas in
the oceanic crust due to partial melting of hydrated gabbros
(Koepke et al., 2004), we suggest that high-temperature
plagiogranites melts and magmas (or high-temperature TTG)
may be formed through aqueous fluid-assisted partial melting of
serpentinized peridotite. Implications for the Early Earth Hydrated peridotite, and its isochemical analogue serpentinite,
has been suggested to be a major shallow component of the
Hadean and Noachian protocrusts on Earth and Mars (Albarède
and Blichert-Toft, 2007; Elkins-Tanton, 2012), being the result of
the interaction of seawater-derived fluids with peridotites (Guillot
and Hattori, 2013). Indeed, planets like Earth and Mars were
massive enough to allow the formation of an early ultramafic
(peridotite-like, silica-poor) magma ocean, while the distance
from the Sun and the surface temperature were appropriate for
the existence of an ocean of liquid water (Valley et al., 2002;
Albarède and Blichert-Toft, 2007; Müntener, 2010). It is widely
recognized (Albarède and Blichert-Toft, 2007; Elkins-Tanton,
2012)
that
magma
ocean(s)
underwent
solidification
to
produce
shallow
ultramafic-mafic
protocrust
during
the
earliest stages of planetary evolution. However, it remains
unclear how early Hadean crustal rocks could have generated
quartz-normative to quartz-saturated magmas of intermediate to FIGURE 8 | Upper panel: Model for the formation of shallow felsic
crust in mantle peridotites of the oceanic lithosphere. Dehydration is
initiated by the intrusion of mantle-derived magmas into hydrated
(serpentinized) mantle. The released fluid promotes fertilization and
partial melting of the peridotite, which leads to production of TTG magmas
(Continued ) May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 15 Felsic Crust Formation at Shallow Depth Borisova et al. 4.43–4.13 Ga-old
granodioritic
and/or
tonalitic
crustal
component on early Mars (Sautter et al., 2015; Sautter et al.,
2016). These quartz-normative rocks, which are believed to
represent the early Martian continental crust, cannot be
produced from partial melting of the mantle and subsequent
fractional crystallization (Sautter et al., 2015; Sautter et al., 2016). By contrast, recent modeling using the MELTS software (Udry
et al., 2018) suggests that the felsic rocks of Gale crater were
produced by the accumulation/fractionation of feldspar from
basaltic melt; however, these authors make no comparison
with
modern
TTG
(plagiogranite)
rocks. Our
data
demonstrated on Figures 5, 6 are also in line with our model
and suggest that the Noachian granodiorite (felsic) rocks on Mars
may have been produced by aqueous fluid-assisted partial melting
of peridotite induced by reaction with basaltic melts. Survival of
the primary intermediate to felsic liquids and formation of a solid
crust may have been possible if the effects of gravitational
overturn were limited (Elkins-Tanton, 2012; Bouvier et al.,
2018). Implications for the Early Earth Thus, the primordial environment of the peridotite
protocrust in the presence of mantle-derived (e.g., basaltic)
melt at shallow depth is favorable for the production of silica-
enriched melts provided liquid water is present in sufficient
amounts. The mechanism suggested in our study may thus
have led to formation of large volumes of the earliest felsic crust. felsic composition. Direct natural evidence for the precursor of
Hadean (≥4 Ga) felsic crust is scarce, and remains the subject of
intense debate (Wedepohl, 1995; Rudnick and Gao, 2003;
Harrison, 2009; Reimink et al., 2014, 2016; Burnham and
Berry,
2017;
Bell
et
al.,
2018). Investigations
of
the
4.37–4.02 Ga Jack Hills detrital zircon crystals (JHZ), which
are the oldest remnants of primordial felsic magmas on Earth,
support the existence of a felsic crust during the earliest stages of
the Hadean eon (Cavosie et al., 2006; Harrison, 2009; Burnham
and Berry, 2017). Either reworking into younger crust or
recycling of the Hadean crust into the mantle have been
proposed to explain the lack of remnants of the earliest felsic
crust on Earth (O’Neil and Carlson, 2017; Nédélec et al., 2017). However, studies of the Jack Hills zircon crystals allow to infer the
presence of an igneous protolith that had experienced low-to-
moderate temperature alteration by aqueous fluids, thereby
ruling out a sedimentary source (Burnham and Berry, 2017;
Whitehouse et al., 2017), although alternative opinions still
exist (Harrison, 2009; Bell et al., 2018). We propose that basaltic melts were introduced in the
primitive
ultramafic
protocrust
at
the
very
end
of
the
crystallization of the magma ocean, and/or by heat-pipe
volcanoes through a stagnant lid (Moore and Webb 2013),
during the earliest stages of Earth evolution, or as proto-rifts
at the end of the Hadean (Capitanio et al., 2020). Unfortunately,
no traces of Hadean tectonic regimes survived so far. Thus, we
propose here that the basaltic melts were intruded into an
ultramafic protocrust, initiating dehydration processes. The
released fluids promoted fertilization and partial melting of the
peridotite, which in turn led to the production of tonalite-
granodiorite
magmas
forming
intrusive
bodies
in
the
peridotite protocrust at shallow depths <10 km (Figure 8). The segregation of felsic melts in the upper protocrust was
favored by lithospheric hydraulic fracturing and the low
density of the hydrous melts, with high melt proportions (up
to 33–40 wt%, Supplementary Table S2) corresponding to high
liquid volume fractions (φ) of ∼37–45 vol%. Implications for Early Mars The proposed mechanism of the aqueous fluid-assisted partial
melting of peridotite may have led to the formation of a shallow
felsic (continental) crust on other rocky planets such as Mars,
provided liquid water or a volatile element-rich magma ocean was
present at an early stage of their evolution. The finding of ancient
serpentinites at the surface of Mars (Ehlmann et al., 2010)
suggests that serpentinite formation and aqueous fluid-assisted
melting of serpentinized peridotite may be envisaged during the
earliest history of this planet. There is also evidence for a Implications for the Early Earth Alternatively, the
generation of such intermediate to felsic melts and magmas at
shallow depths within the early ultramafic-mafic protocrust may
also have been possible due to impact-induced melting (Marchi
et al., 2014), particularly if the proportion of serpentinite was
above 50 wt% in the hybrid system (R ≤1). Such melting would
have favored intense mixing of shallow hydrated ultramafic and
mafic components at high temperature (as modelled in our mixed
experiments), well above the liquidus of basalt before the onset of
modern plate tectonics (Figure 8). CONCLUSION The proposed mechanism of aqueous fluid-assisted partial melting
of peridotite induced by reaction with basaltic melt supports the
possibility of felsic crust formation at depths of ≤10 km. The
shallow depths of generation of felsic magma proposed in this
study imply that the formation of silica-rich crust does not require
convergent plate tectonics, in agreement with geodynamic
simulations of the hotter mantle on early Earth and Mars
(Herzberg et al., 2010; Sautter et al., 2016). The most favorable
conditions for felsic crust formation are high-temperature
interactions between serpentinized peridotite and basaltic melts
at 0.2 GPa and low basalt-to-serpentinite mass ratio (≤1). Such
conditions may have occurred in various geologic settings that do
not require modern plate tectonics. Such conditions are highly
probable at spreading centers, hot spots and Hadean heat-pipe
volcanoes or proto-rifts. A felsic crust may also have been formed
in a more transient way during intensive impact-induced melting
of the ultramafic-mafic protocrust in the presence of a liquid water
ocean on early Earth and Mars. Even though the geodynamic and
thermal conditions on both planets have changed, small volumes of
felsic crust are still forming by this process at modern spreading
centers. REFERENCES Bohrson, W. A., Spera, F. J., Ghiorso, M. S., Brown, G. A., Creamer, J., and
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microanalyses of chromite-hosted inclusions. J. Petrology 53, 2411–2440. doi:10.1093/petrology/egs054 Balta, J. B., Beckett, J. R., and Asimow, P. D. (2011). Thermodynamic properties of
alloys of gold-74/palladium-26 with variable amounts of iron and the use of Au-
Pd-Fe alloys as containers for experimental petrology. Am. Mineral. 96,
1467–1474. doi:10.2138/am.2011.3637 Borisova, A. Y., Gurenko, A. A., Martel, C., Kouzmanov, K., Cathala, A.,
Bohrson, W. A., et al. (2016). DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and
accession number can be found in the article/Supplementary
Material, Supplementary Tables S1–S7. May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 16 Felsic Crust Formation at Shallow Depth Borisova et al. FUNDING This
work
is
funded
by
the
Institut
Carnot
ISIFoR
“TRACENEO”, AST “Planets” in Toulouse (France)
and
Deutsche Forschungsgemeinschaft (DFG, Germany) to ABo
and ATUPS (Université Paul Sabatier, Toulouse, France) for
providing a travel grant in 2015–2016 to NZ. This research
is
fulfilled
under
the
Research
Program
AAAA–A18–118020590148–3 of the Korzhinskii Institute of
Experimental Mineralogy RAS and partially supported by the
Russian Scientific Foundation (project 18-17-00206 to OS) and
(grant 19–17–00200 to ABy). This work was supported by the
French
National
Research
Agency
(project
RadicalS
ANR–16–CE31–0017 to GP and AG) and by the Institut ACKNOWLEDGMENTS This study is in tribute to E.Y. Borisova, E.V. Bibikova and Y.V. Bychkova for their works on zircon-based geochronology and
Precambrian geology. The first author ABo dedicates this work to
the memory of her brother, I.Y. Borisov. We thank Editors SW
and M. Pistone, and eight anonymous reviewers for comments
and suggestions, that helped to improve the manuscript. F. Spera,
E. Sizova, J. Reimink, M. Belosevic, D. Baratoux and A.V. Sobolev
are thanked for helpful discussions. We acknowledge the
European Synchrotron Radiation Facility for providing access
to beamtime, and O. Proux and M. Muñoz for help with XANES
data acquisition and processing. C. McCammon and H. Keppler
are thanked for offering access to the Bavarian Research Institute
of Experimental Geochemistry and Geophysics (Germany). The
English style and grammar were post-edited by M.S.N. Carpenter. AUTHOR CONTRIBUTIONS Carnot ISIFoR (project OrPet to GP). IB and OM thank RNF
Grant (RNF19–17–00241) for support. Funding from the US
National Science Foundation to WB supported this work. ABo, AN, MT, and OS developed the conceptual idea of the study;
ABo and NZ, SS prepared and conducted high T-P runs at BGI;
NZ, OS, ABy, AT, VP, DV prepared and performed experiments at
IEM; NZ, GP, WB, OM conducted thermodynamic modeling; GP,
NZ, and AG carried out XAS and SIMS measurements,
respectively; KJ, BS, and UW performed LA-ICP-MS analyses. IB performed bulk-rock analyses of serpentinite. ABo, NZ, SG, PP,
and KC performed microanalytical measurements and mapping
using EPMA; ABo, AN, GC developed geological applications; all
authors contributed to data interpretation and manuscript writing. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/feart.2021.640464/
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Parseval. This is an open-access article distributed under the terms of the
Creative Commons Attribution License (CC BY). The use, distribution or
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the copyright owner(s) are credited and that the original publication in this journal is
cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms. Turek, A., Riddle, C., Cozens, B. J., Tetley, N. W., Udry, A., Gazel, E., et al. (1976). Determination of chemical water in rock analysis by Karl Fischer titration. Chem. Geol. J. Geophys. Res. Planets 17, 261–267. doi:10.1029/2018JE005602 Udry, A., Gazel, E., and McSween, H. Y. (2018). Formation of evolved rocks at Gale
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215–232. doi:10.1016/S0031-9201(01)00229-1 May 2021 | Volume 9 | Article 640464 Frontiers in Earth Science | www.frontiersin.org 20
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Natural Selection beyond Life? A Workshop Report
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Natural Selection beyond Life? A Workshop Report
Sylvain Charlat, André Ariew, Pierrick Bourrat, María Ferreira Ruiz, Thomas
Heams, Philippe Huneman, Sandeep Krishna, Michael Lachmann, Nicolas
Lartillot, Louis Le Sergeant d’Hendecourt, et al. To cite this version: Sylvain Charlat, André Ariew, Pierrick Bourrat, María Ferreira Ruiz, Thomas Heams, et al.. Natural
Selection beyond Life? A Workshop Report. Life, 2021, 11 (10), pp.1051. 10.3390/life11101051. hal-03418757 Distributed under a Creative Commons Attribution 4.0 International License Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7,
Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10,
Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and
Zorana Zeravcic 15 Natural Selection beyond Life? A
Workshop Report. Life 2021, 11,
1051. https://doi.org/10.3390/
life11101051 Citation: Charlat, S.; Ariew, A.;
Bourrat, P.; Ferreira Ruiz, M.;
Heams, T.; Huneman, P.; Krishna, S.;
Lachmann, M.; Lartillot, N.;
Le Sergeant d'Hendecourt, L.; et al. Natural Selection beyond Life? A
Workshop Report. Life 2021, 11,
1051. https://doi.org/10.3390/
life11101051
Academic Editor: David A. Baum
Received: 24 August 2021
Accepted: 29 September 2021
Published: 7 October 2021 g
9 Santa Fe Institute, Santa Fe, NM 87501, USA; lachmann@santafe.edu 10 Centre de St-Jérôme, Laboratoire de Physique des Interactions Ioniques et Moléculaires,
Aix-Marseille Université, CNRS, UMR 7345, 13013 Marseille, France; ldh@univ-amu.fr 11 Centre de Recherche Interuniversitaire sur la Science et la Technologie (CIRST), Département de
Philosophie, Université du Québec à Montréal (UQAM), 455 Boulevard René-Lévesque Est, Montréal,
QC H3C 3P8, Canada; malaterre.christophe@uqam.ca p
q
12 Laboratoire Biophysique et Evolution, CNRS UMR Chimie Biologie Innovation 8231, ESPCI Paris, p
q
12 Laboratoire Biophysique et Evolution, CNRS UMR Chimie Biologie Innovation 8231, ESPCI Paris,
Université PSL, 10 Rue Vauquelin, 75005 Paris, France; philippe.nghe@espci.fr Université PSL, 10 Rue Vauquelin, 75005 Paris, France; philippe.nghe@espci.fr 0 Rue Vauquelin, 75005 Paris, France; philippe.nghe@espci.f 13 Center for Interdisciplinary Research in Biology (CIRB), Collège de France, CNRS, INSERM, Université PSL,
75005 Paris, France; olivier.rivoire@college-de-france.fr 13 Center for Interdisciplinary Research in Biology (CIRB), C
75005 Paris, France; olivier.rivoire@college-de-france.fr 75005 Paris, France; olivier.rivoire@college-de-france.fr 14 Max Planck Institute for Mathematics in the Sciences, Inselstrasse 22, 04103 Leipzig, Germany;
msmerlak@perimeterinstitute.ca p
15 Gulliver Lab, CNRS UMR 7083, ESPCI Paris, PSL University, 75005 Paris, France;
zorana.zeravcic@espci.fr Received: 24 August 2021
Accepted: 29 September 2021
Published: 7 October 2021 * Correspondence: sylvain.charlat@univ-lyon1.fr Abstract: Natural selection is commonly seen not just as an explanation for adaptive evolution, but
as the inevitable consequence of “heritable variation in fitness among individuals”. Although it re-
mains embedded in biological concepts, such a formalisation makes it tempting to explore whether
this precondition may be met not only in life as we know it, but also in other physical systems. This
would imply that these systems are subject to natural selection and may perhaps be investigated in
a biological framework, where properties are typically examined in light of their putative functions. Here we relate the major questions that were debated during a three-day workshop devoted to
discussing whether natural selection may take place in non-living physical systems. HAL Id: hal-03418757
https://univ-lyon1.hal.science/hal-03418757v1
Submitted on 8 Nov 2021 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Perspective Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7,
Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10,
Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and
Zorana Zeravcic 15 Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7,
Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10,
Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and
Zorana Zeravcic 15 1 Laboratoire de Biométrie et Biologie Évolutive, Université de Lyon, Université Lyon 1, CNRS, UMR 5558,
43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, France; nicolas.lartillot@univ-lyon1.fr (N.L.);
Etienne.Rajon@univ-lyon1.fr (E.R.) 1 Laboratoire de Biométrie et Biologie Évolutive, Université de Lyon, Université Lyon 1, CNRS, UMR 5558,
43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, France; nicolas.lartillot@univ-lyon1.fr (N.L.);
Etienne.Rajon@univ-lyon1.fr (E.R.) j
y
2 Department of Philosophy, University of Missouri, 438 Strickland Hall, Columbia, MO 65211, USA;
ariewa@missouri.edu 3 Department of Philosophy, Macquarie University, Balaclava Road, North Ryde, NSW 2109, Australia;
pierrick.bourrat@mq.edu.au 4 Charles Perkins Centre, Department of Philosophy, The University of Sydney, Camperdown, NSW 2006,
Australia 5 Department of Philosophy, University of Bielefeld, 33615 Bielefeld, Germany; mariaferreiraruiz@gmail.com p
p y
y
y
g
6 INRAE, Domaine de Vilvert Bâtiment 211, 78352 Jouy-en-Josas, France; thomas.heams@agroparistech.fr 6 INRAE, Domaine de Vilvert Bâtiment 211, 78352 Jouy-en-Josas, France; thomas.heams@agroparistech.fr
7 Institut d’Histoire et de Philosophie des Sciences et des Techniques, CNRS (Centre National de la Recherche
Scientifique), Université Paris I Sorbonne, 13 Rue du Four, 75006 Paris, France;
philippe.huneman@gmail.com 7 Institut d’Histoire et de Philosophie des Sciences et des Techniques, CNRS (Centre National de la Recherche
Scientifique), Université Paris I Sorbonne, 13 Rue du Four, 75006 Paris, France;
philippe.huneman@gmail.com 8 Simons Centre for the Study of Living Machines, National Centre for Biological Sciences,
Tata Institute of Fundamental Research, Bangalore 560065, India; sandeep@ncbs.res.in Citation: Charlat, S.; Ariew, A.;
Bourrat, P.; Ferreira Ruiz, M.;
Heams, T.; Huneman, P.; Krishna, S.;
Lachmann, M.; Lartillot, N.;
Le Sergeant d'Hendecourt, L.; et al. Natural Selection beyond Life? A
Workshop Report. Life 2021, 11,
1051. https://doi.org/10.3390/
life11101051
Academic Editor: David A. Baum
Received: 24 August 2021
Accepted: 29 September 2021
Published: 7 October 2021 Citation: Charlat, S.; Ariew, A.;
Bourrat, P.; Ferreira Ruiz, M.;
Heams, T.; Huneman, P.; Krishna, S.;
Lachmann, M.; Lartillot, N.;
Le Sergeant d'Hendecourt, L.; et al. Natural Selection beyond Life? A
Workshop Report. Life 2021, 11,
1051. https://doi.org/10.3390/
life11101051 Citation: Charlat, S.; Ariew, A.;
Bourrat, P.; Ferreira Ruiz, M.;
Heams, T.; Huneman, P.; Krishna, S.;
Lachmann, M.; Lartillot, N.;
Le Sergeant d'Hendecourt, L.; et al. 1. Introduction: Why Investigate “Natural Selection Beyond Life”? The principle of natural selection occupies a central role in biology: explaining why
living organisms harbour properties apparently fitted to particular functions, and thus
denoted as “adaptive”. In doing so, it provides a non-finalistic justification for “functional
thinking” [1,2]; a typically biological mode of inquiry where structures, or more generally
features, are investigated in light of their observed or putative effects, in interrelations
with others, with which they form a functioning “whole”, the organism. Within the stand-
ard evolutionary framework, the process of natural selection is commonly conceived as
the inevitable consequence of necessary and sufficient preconditions, namely “heritable
variation in fitness related traits” [3] (provided it is not overwhelmed by random events). Such a formulation naturally leads one to wonder whether non biological systems may
also fulfil these conditions. In turn, such an interrogation constitutes an occasion to revisit
whether evolution by natural selection necessarily produces features that can be qualified
as functions, that is, whether functional thinking becomes a relevant mode of inquiry
whenever the conditions for natural selection are fulfilled. Life-derived objects not typically seen as “living”, such as words, ideas or computer
programs, are nevertheless endowed with some kind of self-replicating ability, and thus
stand as obvious candidates for “evolution by natural selection” outside of biology. Ac-
cordingly, the principle of natural selection made its way into linguistics, cultural evolu-
tion and computer sciences [4,5]. The potential relevance of natural selection to physical
sciences is a priori less obvious: since living entities are part of the physical world, con-
cepts from the physical sciences commonly flow into biology, but the reverse flow would
be unusual. Perhaps unusual but not logically impossible insofar as concepts may flow
between scientific disciplines without respecting the natural hierarchy of their objects. If
the a priori objection that natural selection cannot be relevant to physical sciences is thus
ruled out, the question of whether it is remains largely unexplored. This issue was at the centre stage of a workshop held in November 2019, that gath-
ered a group of evolutionary biologists, chemists, physicists, and philosophers of science. Here we relate the major questions that were debated in this context. The article is struc-
tured as follows. We first briefly describe the research objects of connected fields, from
synthetic biology to the origins of life, that we take as starting points for considering nat-
ural selection at the edge of biology. Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7,
Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10,
Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and
Zorana Zeravcic 15 Overall, these discussions thus lead us to conjecture that understanding, in physico-chemical
terms, how individuality emerges and how it can be recognised, will be essential in the search for
instances of evolution by natural selection outside of living systems. Keywords: natural selection; individuality; levels of selection; evolutionary biology; physics; phi-
losophy of biology; exobiology; origins of life Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7,
Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10,
Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and
Zorana Zeravcic 15 We start this
report with a brief overview of research fields dealing with “life-like” or “proto-biotic” systems,
where mimicking evolution by natural selection in test tubes stands as a major objective. We con-
tend the challenge may be as much conceptual as technical. Taking the problem from a physical
angle, we then discuss the framework of dissipative structures. Although life is viewed in this con-
text as a particular case within a larger ensemble of physical phenomena, this approach does not
provide general principles from which natural selection can be derived. Turning back to evolution-
ary biology, we ask to what extent the most general formulations of the necessary conditions or Abstract: Natural selection is commonly seen not just as an explanation for adaptive evolution, but
as the inevitable consequence of “heritable variation in fitness among individuals”. Although it re-
mains embedded in biological concepts, such a formalisation makes it tempting to explore whether
this precondition may be met not only in life as we know it, but also in other physical systems. This
would imply that these systems are subject to natural selection and may perhaps be investigated in
a biological framework, where properties are typically examined in light of their putative functions. Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and institu-
tional affiliations. Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (http://crea-
tivecommons.org/licenses/by/4.0/). www.mdpi.com/journal/life Life 2021, 11, 1051. https://doi.org/10.3390/life11101051 Life 2021, 11, 1051 2 of 12 2 of 12 signatures of natural selection may be applicable beyond biology. In our view, such a cross-discipli-
nary jump is impeded by reliance on individuality as a central yet implicit and loosely defined con-
cept. Overall, these discussions thus lead us to conjecture that understanding, in physico-chemical
terms, how individuality emerges and how it can be recognised, will be essential in the search for
instances of evolution by natural selection outside of living systems. signatures of natural selection may be applicable beyond biology. In our view, such a cross-discipli-
nary jump is impeded by reliance on individuality as a central yet implicit and loosely defined con-
cept. 2. Natural Selection in Protobiotic Systems? Although not always stated in such terms, the very possibility of applying the prin-
ciple of natural selection to physico-chemical systems is a common theme of several re-
search areas lying on the fringe of physical and life sciences: synthetic biology, exobiology,
and the origins of life. While pursuing distinct objectives, these fields share a common
vast technical and conceptual challenge: bridging the gap between physico-chemical and
biological systems, explaining the transition from inanimate to living matter. Darwinian
evolution being recognised as an important component of such a transition, many exper-
imental and theoretical systems have been designed with the objective of mimicking this
process. Such setups originally implemented some form of experimenter-assisted natural
selection in the laboratory [6]. This approach led to a now well-established process of “in
vitro” or “directed” evolution [7,8]. Yet such settings take for granted recurrent human
interventions to handle the core feature of replication, while our focus here is on systems
that replicate autonomously and are left to evolve by themselves. Many potential candidates have been designed, falling into partially overlapping
broad groups. Some are directly inspired by the template-based replication of nucleic ac-
ids seen in extant organisms. Such systems typically follow up from the discovery of nat-
urally occurring ribozymes [9,10] and fit in a model where RNA occupies a central role in
the emergence of life. The continued search for an RNA-replicase has uncovered increas-
ingly powerful ribozymes capable of ligating up to a hundred nucleotides in emulsions,
though still short of exhibiting complete self-replication capability [11,12], for a review,
see [13]. Other systems take the form of autocatalytic networks and are thus more centred
on metabolism as a central feature. Some are based on peptides [14,15] but others involve
RNA and remain tightly connected with the RNA-world hypothesis [16–18]. Yet another
group of protobiotic replicating systems involves more physical or self-organizing entities
such as vesicles, crystals, colloids, or nanotubes [19–21]. Objects from these different cat-
egories may also be merged to combine their respective advantages, e.g., [13]. g
y
g
p
g
g
Many of these systems arguably display some form of self-replication. Some varia-
tions among replicating entities may also exist but only a limited fraction of them is herit-
able. As a result, it remains currently unclear whether such systems should be considered
as evolvable by natural selection. 1. Introduction: Why Investigate “Natural Selection Beyond Life”? Experimentally mimicking evolution by natural se-
lection in these fields stands as a key objective, that seems hindered not only by technical
difficulties, but also by the conceptual challenge of tracking this process, identifying its
conditions, and expected outcomes. We then discuss whether natural selection can be sit-
uated in the framework of far-from-equilibrium physics, a field that is explicitly aimed at
encompassing living systems. Our tentative conclusion is that it cannot, perhaps because
natural selection, at least as currently formalised, is at odds with the epistemic status and
usage of physical principles. Next, we conversely examine whether some physical systems
may be situated in the framework of evolutionary biology and argue that this will require
further formalisation of the natural selection principle, to make it portable across disci-
plines. In particular, we highlight that natural selection requires “individuals” (i.e., well-
identified units) as a precondition although, paradoxically, biological individuals are also Life 2021, 11, 1051 3 of 12 3 of 12 considered as outcomes of this process. We end this report with the conjecture that solving
this paradox may be a necessary first step toward the search for natural selection beyond
life. This implies understanding how individuality may emerge and perhaps be reinforced
in the course of evolution. 3. Natural Selection in the Context of Physical Phenomena Assessing the possibility that natural selection takes place in non-living physical sys-
tems first implies positioning living systems, where natural selection is recognized to hap-
pen, in the range of physical phenomena. This question has been predominantly exam-
ined from a thermodynamic perspective, starting in the 1940s with Schrödinger’s influen-
tial book [30]. We discuss more specifically the approach developed in the 1970s by the
Brussels school of thermodynamics [31–33], which continues to this date to inspire new
works [34,35]. Within this approach, as explained in the following, living systems are
viewed as belonging to a larger class of open and far-from-equilibrium systems called
dissipative structures, and one seeks a general framework in the form of a variational
principle (as defined below), of which natural selection could be a particular case. p
p
p
The term “dissipative structures” designates steady states that display spatial and/or
temporal patterns (e.g., inhomogeneous distribution of chemical species, or sustained os-
cillations) which typically occur due to an instability from a homogeneous, patternless
steady state subject to a small perturbation. Dissipative structures commonly occur in sys-
tems which are open (i.e., can exchange matter and energy with the environment), non-
linear (in terms of the governing dynamics equations) and far from equilibrium. A canon-
ical example arises from the hydrodynamic instability known as the Rayleigh–Bénard
convection [36,37]. This instability is observed in a horizontal layer of fluid heated from
below. As the temperature difference between the bottom and the top is raised, a thresh-
old is reached at which the fluid loses its spatial homogeneity and shows motions orga-
nized in patterns. This transition formally corresponds to an instability of the homogene-
ous state upon fluctuations (small random variations in density for instance). Depending
on the difference of temperatures, the initial and boundary conditions (i.e., spatial and
temporal constraints set by the environment), and the protocol followed to raise the tem-
perature, one can observe patterns of many different forms, from simple convection cells
and rolls to more complicated spatio-temporal structures. Dissipative structures are also
found in reaction-diffusion systems in chemistry (Turing patterns, Belousov–Zhabotinsky
oscillations), in fluid mechanics (Faraday waves) and in nonlinear optics (light beam mod-
ulations). Several biological phenomena have also been studied from the standpoint of
dissipative structures, including gene regulation and biological rhythms [38]. 2. Natural Selection in Protobiotic Systems? This may reflect the technical difficulty of designing
systems that will effectively display a larger diversity of heritable states. Strikingly, the
challenge is also conceptual, to the point that it appears difficult to even imagine theoret-
ical systems that would radically differ from the biological paradigm of self-replicating
nucleic acids and could yet be considered as evolvable by natural selection [22–24]. Ad-
dressing this challenge may require shifting away from a categorical to a continuous per-
spective, as previously advocated in the context of the origin of “lifeness” [25–29]: asking,
not if these systems are evolvable through natural selection, but to what extent they are,
on a quantitative scale that remains to be formalised in cross-disciplinary terms. In the
next section, we discuss whether the physical approach to far-from-equilibrium systems
may take us closer to that objective. Life 2021, 11, 1051 4 of 12 3. Natural Selection in the Context of Physical Phenomena The over-
arching theme in all these examples is that structures can emerge from fluctuations
through instabilities when a system is maintained out of equilibrium, for instance by a
gradient of temperature or an influx of chemical compounds. From a thermodynamic perspective, living organisms constitute far-from-equilib-
rium open systems, but populations of organisms can also be conceived this way, and
their evolution through natural selection may then be framed in terms of instability [39]. In particular, introducing a mutant in a previously stable population can be seen as pro-
voking an instability in the population dynamics, eventually leading to a new steady state
with a completely different structure, that is, a different composition of the population. Furthermore, in numerical simulations of artificial chemistries aiming at modelling the
emergence of evolutionary dynamics from elementary physical rules [40,41] one observes
instabilities giving rise to various dissipative structures, such as competing catalytic cy-
cles. From this standpoint, one may therefore consider biological populations subject to
natural selection as particular dissipative structures. A major goal of the Brussels school was to identify a variational principle that gener-
ally applies to non-equilibrium steady states, and therefore to dissipative structures. In
physics, variational principles take the form of mathematical functions of one or more
physical quantities whose minimisation allows one to predict the final state of a system,
without reference to its initial conditions or particular dynamics. For example, in equilib-
rium thermodynamics, this is achieved by minimizing free energy. Far from equilibrium,
however, in systems such as dissipative structures, no such principle has been found [42]. In fact, it is well-established through explicit counter examples that the most likely steady
state of a non-equilibrium system can depend on parameters that cannot be estimated Life 2021, 11, 1051 5 of 12 5 of 12 from the immediate vicinity of stable steady states only [43]. Hence, a state-function
whose optima (that is, whose minima) indicates which non-equilibrium steady states are
favoured cannot be derived. In other words, dissipative structures cannot be generally
predicted from summary descriptors of the steady states. A general framework exists for
rationalizing a variety of patterns observed in non-equilibrium systems, but it is limited
to local stability analyses [42]. In short, one can recognize that a particular steady state is
unstable but not generally predict which final state it will reach. From this standpoint, natural selection is not different. 3. Natural Selection in the Context of Physical Phenomena In particular cases, a fitness
function can be defined in which the maximum describes the steady state of population
dynamics. Many other examples exist, however, where such functions cannot be defined,
for example in cases where optimal trait values depend on the composition of the popu-
lations, e.g., in systems following dynamics analogous to those of the rock-paper-scissors
game [44]. This is unsurprising from the standpoint of general dynamical systems, where
no steady state is even generally guaranteed. What non-equilibrium thermodynamics
teaches us is that even describing biological systems in a physical framework, as subclasses
of dynamical systems (e.g., by invoking physical constraints), would not be sufficient to
solve this problem, that is, to understand them using a general variational principle. From the standpoint of its epistemic status and usage, the principle of natural selec-
tion is, however, markedly different from variational principles in physics: unlike varia-
tional principles in physics, the principle of natural selection is used even in the absence
of a precise quantitative description or a well-defined optimum. Trying to subsume natu-
ral selection into a variational principle, or reciprocally, may therefore be inappropriate. One may nevertheless wonder if the principle of natural selection, which is so fruitful in
biology despite not constituting a valid variational principle from a physical standpoint,
can provide comparable insights into physical but non-biological systems. In the follow-
ing section, we discuss whether the conditions and signatures of natural selection, as de-
picted in the framework of evolutionary biology, can provide hints on what such systems
could be. 4. Natural Selection as a Framework We take here two complementary approaches to try and characterise non-living
physical systems that may be subject to natural selection. One is to look for the causes of
natural selection, its necessary and sufficient conditions. The other is to look for its conse-
quences, the patterns it generates. The former may thus be labelled as “causal” or “mech-
anistic”, while the latter is more correlative or phenomenological, and will only provide
strong evidence for natural selection if it relies on specific and unambiguous signatures. 4.1. The Conditions of Natural Selection If we turn to the notion of “fitness-related traits”, perhaps not encouraging is the
acknowledgment that even within biology, reaching a consensual definition of this ex-
pression is probably impossible, for fitness is a notoriously slippery term [51–53]. Yet most
biologists would probably agree that it relates to survival and/or reproduction, that con-
dition one’s own persistence and the number of offspring one may produce. Taking only
the “survival” component, the fitness of an object may be regarded as equivalent to its
“stability”, a concept that seems readily applicable to any physical entity. Including the
“reproduction” component breaks this equivalence. Fitness may then be related to an ex-
tended notion of stability that would apply to dynamic structures maintained although
they are made of unstable components, that is, to steady states. Yet, proposals to define
fitness in physical terms along those lines, including for instance dynamic kinetic stability
[34] are subject to the limitations reviewed in Section 3: they cannot provide a general
criterion for specifying the steady state that a system will adopt unless their scope of ap-
plication is precisely and rigorously circumscribed. The third component of the above-defined Darwinian recipe, individuality, is prob-
ably the most central although it is generally kept implicit and thus goes unnoticed: her-
itable differences and fitness related traits just cannot be conceived without referring to
individuals. Applying the recipe approach to non-biological systems thus requires defin-
ing individuality without referring to biological concepts. Clarifying the meaning of this
term within biology would probably be a good start, but this task in itself is acknowledged
as very challenging [54]. Although central to virtually any biological reasoning, “individ-
uals” are perhaps sufficiently evident in many contexts to let biologists live well without
defining the underlying concept. Yet many problematic situations can also be found
within biology. Vegetative propagation through cutting, or more generally clonal repro-
duction, is a situation where many individuals may be seen as a single individual of a
higher level [45,51,55]. Obligate symbiotic associations pose similar difficulties. These bor-
der-line cases have prompted the development of more rigorous approaches to individu-
ality [56–58]. 4.1. The Conditions of Natural Selection Following Lewontin [3] and many subsequent authors, natural selection is often pre-
sented as necessarily resulting from heritable differences in fitness-related traits among
individuals. This Darwinian recipe provides a starting point for the causal approach, alt-
hough, as abstract and general as it may sound to most biologists, it remains very much
dependent upon biological concepts. Making it portable across disciplines requires more
formal definitions of its components: “heritable differences”, “fitness-related traits” and
“individuals”. “Heritable differences”, first, refer to parent-offspring resemblance, and more specif-
ically to the fraction of differences seen among individuals that stem from differences
among their parents. The concept of inheritance is tightly connected to that of reproduc-
tion, although they are not strictly equivalent. Specifically, reproduction may take place
without heritable differences if all individuals are identical, but heritable differences, in-
sofar as they refer to parents and offspring, imply reproduction. It has been argued that
reproduction should not be seen as a necessary ingredient of natural selection [45–48] be-
cause mere differences in “survival” among different kinds of entities, if they are persis-
tent enough, suffice to induce deterministic changes in their relative abundances over Life 2021, 11, 1051 6 of 12 6 of 12 time. Under this broadened view, natural selection is the biological name for the sorting
process taking place, with or without reproduction, in any dynamical systems composed
of entities differing in their inherent stability or emergence rates. As discussed in Box 1
using a toy mathematical model (and illustrated in Figure 1), reproduction nevertheless
introduces radical changes in the efficiency of this sorting process, opening the possibility
of otherwise unlikely cumulative changes. It may thus be recognised that reproduction is
a necessary component, if not of natural selection in its most general sense, at least of
“cumulative changes through natural selection”, which we take as an equivalent to “evo-
lution by natural selection”, and to which we happily restrict our focus. The biological
concept of reproduction is equivalent to that of autocatalysis in chemistry [49,50] and can
also be related to exponential growth. The latter is essentially a mathematical concept but
captures the dynamics of processes involving self-amplification. Yet we note that many
physical systems, such as nuclear chain reactions, display exponential dynamics without
being reducible to self-amplification of particular entities. In that sense, reproduction and
auto-catalysis constitute, not a general equivalent of exponential growth, but rather a par-
ticular case. 4.1. The Conditions of Natural Selection In particular, the research fields of “major evolutionary transitions” and
“levels of selection” provide us with useful conceptual tools to apprehend this problem
in an evolutionary perspective, by describing how new levels of individuality may emerge
through increased cooperation and decreased conflict among lower-level entities, turning
higher levels into more effective targets of natural selection (e.g., see [56,59–62]). In this
framework, individuals are thus depicted as the product of an evolutionary process,
which comes as a paradox if individuals are also recognised as an essential ingredient of
evolution by natural selection. Life 2021, 11, 1051 7 of 12 Figure 1. Efficiency of a sorting process with or without reproduction. Without reproduction (full
bullet points), the fittest entities (in green) dominate the system at equilibrium, but the blue ones
still constitute a fifth of the population. In contrast, with reproduction (empty bullet points), the
blue entities are just not present at equilibrium if one assumes a finite population size (1000 in these
simulations). Figure 1. Efficiency of a sorting process with or without reproduction. Without reproduction (full
bullet points), the fittest entities (in green) dominate the system at equilibrium, but the blue ones
still constitute a fifth of the population. In contrast, with reproduction (empty bullet points), the
blue entities are just not present at equilibrium if one assumes a finite population size (1000 in these
simulations). 4.2. The Signatures of Natural Selection When it comes to describing the outcomes of natural selection, one conceptual tool is
frequently invoked: the Price equation [63]. This equation formalizes in statistical terms
the notion that the change in the mean value of a biological trait across two time points
(typically, between two generations) can be partitioned into an “individual-level term”
(how much individuals themselves or their offspring have changed) and a “population-
level term” (how much the relative abundances of the different classes of individuals have
changed). Technically, both terms are defined at the population level, but the first refers
to individual changes, hence the terminology used here. The population term captures the
outcome of natural selection as a covariance between trait value and abundance, although
this covariance may also be inflated by pure chance [64]. The Price equation is most re-
nowned for its abstractness (thus its generality) and its robustness to particular assump-
tions, making it a versatile tool adapted to a wide diversity of evolutionary questions,
from epidemiology to non-genetic inheritance and social or cultural evolution [65–68]. Yet, also because of its abstractness and generality, this equation remains purely descrip-
tive and may be judged of limited utility unless it is used in a biologically well-delimited
context [69]. While initially derived in reference to genetics and evolutionary biology, the Price
equation was also explicitly perceived by its own author as a possible first step toward “a
general selection theory” that may be used beyond its original field [70]. In principle, it
may indeed apply to any dynamical system where sets of individual entities can be
mapped over different time points, for example through parent-offspring relationships,
or simply through conserved “identities” in non-reproducing systems. It then formalizes
the notion that the average change over time in any property can be partitioned into some
individual level changes and some higher-level changes, where the latter is a covariance
between the property value and its relative abundance. If, under this framework, the ob-
servation of a systematic (that is, non-random) non-zero covariance, for a given property,
is to be taken as a signature of natural selection on this property, one may conclude that
natural selection is just everywhere, or more specifically, in any system where some form
of stability is deterministically associated with some property. Box 1. Mere sorting versus evolution by natural selection. Box 1. Mere sorting versus evolution by natural selection. One way to discuss whether natural selection should be seen as equivalent to
mere sorting is to assess the efficiency of a sorting process with or without reproduc-
tion. This can be performed with a simple mathematical model (equivalent to those
previously used by Earnshaw-Whyte [79] and Bourrat [80]) simulating the dynamics
of a system composed of two or more types of entities, only differing in their rates of
decay, the equivalent of “survival” in biology. Specifically, let us consider a system of
green and blue entities, produced at the same rate but differing in their respective sta-
bilities, e.g., with 80% chance of being maintained at each time step for the green kind,
and only 40% for the blue kind. We assume no transition between the two types, that
is, the colour (and thus the degree of stability) of the entities does not change. (
g
y)
g
Starting from an equal proportion of the two, Figure 1 shows what would then
happen in two situations: one without reproduction (small points), where the lost en-
tities are replaced with green or blue ones with equal chance, versus one with repro-
duction (open circles) where the lost entities are more often replaced by the most
abundant type, with a probability that equals its frequency (equivalent to choosing a
random entity to reproduce). Without reproduction, green entities dominate the sys-
tem at equilibrium, but the blue ones still constitute a fifth of the population. In con-
trast, with reproduction, the blue entities are just not present at equilibrium if one as-
sumes a finite population size (1000 in these simulations). These radically different
dynamics also mean that reproduction increases the chances of cumulative changes,
where each new step is facilitated by the very high abundance of the fittest type. In
sum, it remains theoretically possible to envisage a process akin to natural selection
without reproduction, but reproduction so radically changes the dynamics that it in-
troduces the possibility of otherwise very improbable cumulative changes. In that
sense, reproduction may be considered an essential ingredient, perhaps not of natural
selection in its broadest sense, but of evolution by natural selection, implying cumula-
tive changes. Life 2021, 11, 1051 8 of 12 8 of 12 4.2. The Signatures of Natural Selection 4.2. The Signatures of Natural Selection In that sense, using the
Price equation to detect natural selection appears as an excessively permissive approach,
that fails at limiting our focus on what we defined above as evolution by natural selection,
which implies reproduction. p
p
Another difficulty lies in finding the appropriate level of description. If the individ-
ual- versus population-level partitioning is always possible, the equation in itself does not
help to define those levels, because of the so-called problem of “cross-level by-products”
[56,71]: natural selection acting at one level will also give rise to a non-zero covariance at
higher organisational levels, provided an appropriate grouping scheme. Notably, this dif-
ficulty may also occur in standard applications of the Price equation, that is, in biological
systems, if different levels may be perceived as “individuals”. To account for these cases,
some sophistications have been added to produce a “multi-level Price equation”. Yet, even
then, any arbitrary choice may reveal elevated covariance terms at higher levels than those
where fitness differences effectively take place, so that the choice of the description level
requires some other kind of knowledge [56,72]. This problem is obviously inflated when
it comes to considering non biological candidates for evolution by natural selection, where
intuition is of no help to circumscribe individuals and populations. p
p p
In principle, multiplying the features to be measured and scanning various grains of
description may provide a means to circumvent this problem: if individuals correspond
to levels of organisation where selection is indeed effective, they should be identifiable as
grains of description where most of the observed change can be attributed to the covari-
ance term, and simultaneously so for a diversity of features (that is, in biological terms,
for a diversity of “traits”). To our knowledge, such an approach has not been developed
yet (though see discussions in [73,74]), perhaps because it implies that the features to be
measured must be defined and measurable at any granularity, a constraint that does not Life 2021, 11, 1051 9 of 12 9 of 12 readily fit with the intuition that individuals should display “emergent properties” that
is, features that precisely cannot be defined at all levels of description. The information
theory may offer a possible way out of this difficulty, by providing a means to “measure”
individuality in non-biological terms [75], but the very feasibility of this approach is also
questioned [73]. 5. Conclusion: Individuality Beyond Life? Building on the conception that natural selection should follow from some necessary
and sufficient conditions, our discussions aimed at exploring the possibility that this pro-
cess could take place beyond life as we know it, that is, in other physical systems where
these conditions would be met. A short survey of protobiotic systems revealed how much
evolvability through natural selection is perceived in this research field as an important
yet unattained objective, perhaps because of an excessively categorical scheme, as op-
posed to continuous, which we take as evidence that the challenge is not only technical,
but also conceptual. Considering the problem from a physical perspective, we discussed
the possibility of placing natural selection within the context of dissipative structures. While this framework makes it possible to situate life among other physical phenomena,
it has not produced general principles, of which natural selection might have been a par-
ticular case, reflecting that current physics does not include a readily usable equivalent to
natural selection. Turning to evolutionary biology, we asked whether the conditions for
natural selection, or its signatures, were defined with sufficient formalism to be identified
outside of their original context; we contend they are not. Most strikingly, the implicit but
essential notion of individuality stands as a major conceptual obstacle. How can individuals
be recognised without a priori knowledge of the appropriate level of description? How can
individuals be at the same time considered as essential ingredients and outcomes of evolu-
tion by natural selection? Addressing these paradoxical questions appears as an essential
prerequisite for further investigating where and how natural selection may take place. In fact, the question of how individuality may emerge resembles one that has at-
tracted much attention in the field of major evolutionary transitions: how and why, in the
history of life, have individuals merged into higher organisational levels, such as procar-
yotic symbionts into large eucaryotic cells, or clonal cells into multicellular organisms
[54,56,59,71]? Yet in the case of the very first emergence of individuality, low level indi-
viduals cannot be part of the initial conditions. In other words, a general theory for the
emergence of individuality cannot, by definition, rely on individuals. In biology, explain-
ing jumps in levels of individuality implies identifying conditions, such as limited spatial
diffusion or relatedness, that generate interdependence among the fitness of various enti-
ties, setting the stage for the evolution of cooperative traits [77]. 4.2. The Signatures of Natural Selection More generally, we note a connection between this problem and the field
of coarse graining, where one aims to determine the optimal levels of description to char-
acterise a system [76]. If natural selection is taking place, this objective may be much akin
to that of defining individuals and populations. 5. Conclusion: Individuality Beyond Life? This mode of reasoning
has recently been applied to very early stages of life evolution [78] and may also help in
explaining the very origin of individuality. Yet at that stage it remains unclear how to
even model these questions without assuming the existence of some kind of well-delim-
ited self-replicators, that is, without starting from individuals of lower levels. Future work
will hopefully clarify whether the biological concept of individuality, and the biological
principle of natural selection, can be grounded in physico-chemical roots, to perhaps ex-
tend the breadth of their applicability. Life 2021, 11, 1051 10 of 12 10 of 12 Author Contributions: This paper is the culmination of discussions that involved all the authors. S.C. initiated the workshop, produced the first draft, and integrated the diverse contributions to the
manuscript. P.B., C.M., O.R. and Z.Z. contributed from early writing stages. E.R. performed the
simulations behind Figure 1. All authors further contributed to improving the manuscript. Authors
other than S.C. are listed in alphabetical order. All authors have read and agreed to the published
version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We are most grateful to the scientific committee of the “Institut d’Etudes Sci-
entifiques de Cargèse” (IESC), the “Mission pour les Initiatives Transverses” (MITI) of the CNRS,
and the “Groupement de Recherche” Sapienv (GDR CNRS 3770) for supporting this workshop. Acknowledgments: We are most grateful to the scientific committee of the “Institut d’Etudes Sci-
entifiques de Cargèse” (IESC), the “Mission pour les Initiatives Transverses” (MITI) of the CNRS,
and the “Groupement de Recherche” Sapienv (GDR CNRS 3770) for supporting this workshop. Conflicts of Interest: The authors declare no conflict of interest. References 1. Neander,
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https://hal.archives-ouvertes.fr/hal-03511543/document
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Photocatalytic recovery of metals by Lindqvist-type polyoxometalate–porphyrin copolymer
|
Comptes rendus. Chimie
| 2,021
|
cc-by
| 7,116
|
Photocatalytic recovery of metals by Lindqvist-type
polyoxometalate–porphyrin copolymer
Zhaohui Huo, Vasilica-Adriana Badets, Antoine Bonnefont, Corinne Boudon,
Laurent Ruhlmann
To cite this version:
Zhaohui Huo, Vasilica-Adriana Badets, Antoine Bonnefont, Corinne Boudon, Laurent Ruhlmann.
Photocatalytic recovery of metals by Lindqvist-type polyoxometalate–porphyrin copolymer. Comptes
Rendus. Chimie, 2021, 24 (S3), pp.141-155. 10.5802/crchim.120. hal-03511543 Zhaohui Huo, Vasilica-Adriana Badets, Antoine Bonnefont, Corinne Boudon,
Laurent Ruhlmann Zhaohui Huo, Vasilica-Adriana Badets, Antoine Bonnefont, Corinne Boudon,
Laurent Ruhlmann To cite this version: Zhaohui Huo, Vasilica-Adriana Badets, Antoine Bonnefont, Corinne Boudon, Laurent Ruhlmann. Photocatalytic recovery of metals by Lindqvist-type polyoxometalate–porphyrin copolymer. Comptes
Rendus. Chimie, 2021, 24 (S3), pp.141-155. 10.5802/crchim.120. hal-03511543 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03511543
https://hal.science/hal-03511543v1
Submitted on 4 Jan 2022 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Zhaohui Huo, Vasilica Badets, Antoine Bonnefont, Corinne Boudon
and Laurent Ruhlmann Zhaohui Huo, Vasilica Badets, Antoine Bonnefont, Corinne Boudon
and Laurent Ruhlmann ∗Corresponding author. Photocatalytic recovery of metals by Lindqvist-type
polyoxometalate–porphyrin copolymer Volume 24, issue S3 (2021), p. 141-155 Les Comptes Rendus. Chimie sont membres du
Centre Mersenne pour l’édition scientifique ouverte
www.centre-mersenne.org Les Comptes Rendus. Chimie sont membres du
Centre Mersenne pour l’édition scientifique ouverte
www.centre-mersenne.org Comptes Rendus
Chimie
2021, 24, nS3, p. 141-155
https://doi.org/10.5802/crchim.120 MAPYRO: the French Fellowship of the Pyrrolic Macrocyclic Ring / MAPYRO: la
communauté française des macrocycles pyrroliques MAPYRO: the French Fellowship of the Pyrrolic Macrocyclic Ring / MAPYRO: la
communauté française des macrocycles pyrroliques Récupération photocatalytique des métaux par un copolymère
polyoxométallate-porphyrine de type Lindqvist Zhaohui Huoa, b, Vasilica Badets
b, Antoine Bonnefont
b, Corinne Boudonb
and Laurent Ruhlmann
∗, b https://comptes-rendus.academie-sciences.fr/chimie/ 1. Introduction Recovery of metallic ions from aqueous solutions
can be obtained through a homogeneous photocat-
alytic process in the presence of a sacrificial electron
donor such as propan-2-ol that undergoes photoly-
sis under UV illumination. In these conditions, POMs
are quantitatively reduced and this leads to the re-
duction of metallic ions. This procedure is an use-
ful alternative for synthesis and recovery of metallic
nanoparticles [12,13]. Polyoxometalates (POMs), a large family of tran-
sition metal oxygen anion clusters with d0 elec-
tronic configurations, exhibit interesting photocat-
alytic properties. Upon light irradiation, electrons
are promoted from an oxygen-centered 2p orbital to
an empty metallic d-orbital, generating a highly re-
active charge-separated state [1]. The impregnation
of polyvinylidene fluoride-based solid polymer elec-
trolyte by POM was also shown to improve the per-
formance of dye sensitized solar cell. The fabricated
cell generated high open circuit voltage of 426 mV
and short circuit current of 3.90 mA upon illumi-
nation with visible light [2]. The above-mentioned
excellent properties and pioneering studies indicate
that POMs are promising candidates to optimize the
photocurrent generation efficiency. To expand the practical application range, their
association to a light-harvesting antenna is so far
necessary, since POMs themselves are mainly pho-
toactive only in the UV part of the solar spectrum. Among photosensitizers, porphyrins offer the advan-
tages of strong absorption coefficients in the visi-
ble domain. Synthesis of covalently bonded POM–
porphyrin hybrid system [14,15] can be proposed in
order to obtain efficient photoinduced intramolec-
ular electron transfer from the porphyrin ring to
the POM cluster. Formation of covalently bonded
POM–porphyrin copolymers can be similarly pro-
posed. Then the reduced POMs can be used for
the reduction of metallic ions. The electrochemical
synthesis of the POM–porphyrin copolymers uses
the previously published nucleophilic substitution
on porphyrins via an E(ECNECB)nE process [16–21]. A polarization of a working electrode at the first
ring-oxidation potential of porphyrins in the pres-
ence of pyridine induces a nucleophilic attack and
leads to the attachment of the pyridyl nitrogen to
the meso positions of the porphyrin. If the applied
potential corresponds to the second ring-oxidation
potential of porphyrins in the presence of bipyri-
dine, copolymer with viologen spacers can be ob-
tained. In the present work, the nucleophile pyri-
dine groups came from the functionalized POM
(Py-POM-Py) [22–24], resulting in the formation of
{POM–porphyrin}n copolymers. Zhaohui Huoa, b, Vasilica Badets
b, Antoine Bonnefont
b, Corinne Boudonb
and Laurent Ruhlmann
∗, b a Department of Chemistry, Guangdong University of Education, Guangzhou 510303,
PR China b Université de Strasbourg, Institut de Chimie, UMR CNRS 7177, Laboratoire
d’Electrochimie et de Chimie Physique du Corps Solide, 4 rue Blaise Pascal, CS 90032,
67081 Strasbourg cedex, France E-mails: zhaohuihuo@hotmail.com (Z. Huo), badets@unistra.fr (V. Badets), E-mails: zhaohuihuo@hotmail.com (Z. Huo), badets@unistra.fr (V. Badets)
bonnefont@unistra.fr (A. Bonnefont), cboudon@unistra.fr (C. Boudon),
lruhlmann@unistra.fr (L. Ruhlmann) E-mails: zhaohuihuo@hotmail.com (Z. Huo), badets@unistra.fr (V. Badets
bonnefont@unistra.fr (A. Bonnefont), cboudon@unistra.fr (C. Boudon),
lruhlmann@unistra.fr (L. Ruhlmann) mails:
ao u
uo@ ot
a .co
( . uo), badets@u
st a. ( . adets
bonnefont@unistra.fr (A. Bonnefont), cboudon@unistra.fr (C. Boudon),
lruhlmann@unistra fr (L Ruhlmann) bonnefont@unistra.fr (A. Bonnefont), cboudon@unistra.fr (C. Boudon), Abstract. Hybrid polyoxometalate–porphyrin copolymeric films can be obtained by the electro-
oxidation of 5,15-ditolyl porphyrin (H2T2P) and zinc-β-octaethylporphyrin (ZnOEP) in the presence
of the Lindqvist-type polyoxovanadates TBA2[V6O13{(OCH2)3CNHCO(4-C5H4N)}2] (Py-V6O13-Py). The photocatalytic properties of these films have been studied for the reduction of silver and plat-
inum ions. In these hybrid materials, porphyrins can be excited by visible light and then play the role
of photosensitizers able to give electrons to the polyoxovanadates catalysts. Résumé. Des films copolymères hybrides polyoxométalate–porphyrine peuvent être obtenus par
électrooxydation de la 5,15-ditolyl porphyrine (H2T2P) et du zinc-β-octaéthylporphyrine (ZnOEP)
en présence des polyoxovanadates de type Lindqvist TBA2[V6O13{(OCH2)3CNHCO(4-C5H4N)}2]
(Py-V6O13-Py). Les propriétés photocatalytiques de ces films ont été étudiées pour la réduction des
ions argent et platine. Dans ces matériaux hybrides, les porphyrines peuvent être excitées par la lu-
mière visible et jouer alors le rôle de photosensibilisateurs capables de donner des électrons aux cata-
lyseurs polyoxovanadates. Keywords. Porphyrin, Polyoxometalate, Photoreduction hybrid copolymer, Metal remediation, Silver
and platinum nanoparticles. Mots-clés. Porphyrine, Polyoxométallate, Photoréduction, Copolymère hybride, Dépollution mé-
tallique, Nanoparticules de platine et d’argent. Available online 30th September 2021 ISSN (electronic) : 1878-1543 https://comptes-rendus.academie-sciences.fr/chimie/ https://comptes-rendus.academie-sciences.fr/chimie/ 142 Zhaohui Huo et al. 1. Introduction Furthermore, POMs are also of interest for oxida-
tive photodegradation of organic compounds in wa-
ter purification technology [3]. Metal recovery is also a topic of great concern from
economic and environmental aspect. Since many
metals are either valuable or toxic, the development
of methods that aim to recover them could con-
tribute to save the limited resources and resolve the
environmental problems. Metallic nanoparticles can be produced using var-
ious methods, such as thermal decomposition [4],
electrochemical techniques [5,6], sonochemical syn-
thesis [7,8] radiolysis [9], microwave irradiation [10]
and photocatalysis [11]. In particular, polyoxometalates (POMs) can be
used in photocatalytic processes for the recovery
of metals or the synthesis of nanoparticles. Indeed,
POMs are also excellent electron reservoirs as they
exhibit variable oxidation states and the possibility
of multiple reductions making the POMs an excel-
lent candidate as photocatalysts for the reduction of
metal ions. In the process, illumination at the O →
M charge-transfer band (UV region), renders POM
strong oxidants able to extract electrons from organic
electron donors. POMs display remarkable activity
and selectivity which can be adjusted by the choice
of POM with suitable redox potential, while opera-
tional parameters such as the concentration of POM,
organic substrate (sacrificial electron donor such as
propan-2-ol), and metal ions should affect the pho-
tocatalytic efficiency. Preliminary
study
concerning
the
use
of
photosensitized
systems
coupled
with
POMs
for the elaboration of metallic silver nanosheets
and
nanowires
(heterogeneous
photocatalysis)
has
been
reported. In
this
case,
the
copoly-
mers were composed of porphyrin and Anderson-
type
POM
subunits
[22]. Precisely,
the
hybrid
copolymer
was
obtained
by
electropolymeriza-
tion
of
zinc
β-octaethylporphyrin
(ZnOEP)
or
zinc
5,15-dipyridinium-octaethylporphyrin
(5,15-
ZnOEP(py)2+
2 )
in
the
presence
of
a
function- C. R. Chimie — 2021, 24, nS3, 141-155 143 Zhaohui Huo et al. Scheme 1. Top:
representation
of
(A)
zinc-β-octaethylporphyrin
ZnOEP,
(B)
free
base
5,15-
ditolylporphyrin H2T2P and (C) Lindqvist-type polyoxometalate [V6O13{(OCH2)3CNHCO(4-C5H4N)}2]2−
(Py-V6 O13-Py). Bottom: electropolymerization scheme of H2T2P in the presence of Py-V6O13-Py giving
the copolymer poly-V6O13-H2T2P. Scheme 1. Top:
representation
of
(A)
zinc-β-octaethylporphyrin
ZnOEP,
(B)
free
base
5,15-
ditolylporphyrin H2T2P and (C) Lindqvist-type polyoxometalate [V6O13{(OCH2)3CNHCO(4-C5H4N)}2]2−
(Py-V6 O13-Py). Bottom: electropolymerization scheme of H2T2P in the presence of Py-V6O13-Py giving
the copolymer poly-V6O13-H2T2P. 2. Results and discussion alized
Anderson-type
polyoxometalate
bearing
two
pyridyl
groups
[MnMo6O18{(OCH2)3CNHCO
(4-C5H4N)}2]3−(Py-MnMo6O18-Py) [1,24]. The pho-
tocatalytic reduction of AgI
2SO4 using this copolymer
was conducted under visible light illumination and
aerobic conditions in the presence of propan-2-ol. Quantitative formation of metallic Ag0 nanowires as
well as triangular nanosheets was observed. 2.1. Electrochemical synthesis of the copolymers The syntheses of the copolymers were achieved us-
ing our electropolymerization method, as reported
earlier [25,26]. It corresponds to the addition of
the dipyridyl-substituted Lindqvist-type polyoxo-
vanadate
[V6O13{(OCH2)3CNHCO(4-C5H4N)}2]2−
(Py-V6O13-Py) to an electrogenerated dicationic
5,15-ditolylporphyrin
(H2T2P)
or
zinc-β-
octaethylporphyrin
(ZnOEP)
obtained
by
itera-
tive scans between 0 V and +1.60 V versus SCE
(Scheme 1) [27]. The main goal of this work is to demonstrate
that other hybrid porphyrin–POM copolymers are
still working as photocatalysts for the reduction
of metallic ions. By changing the nature of the
porphyrin and of the polyoxometalate, a change
in the photoreduction kinetics as well as in the
shape of the nanoparticles is expected. In the
present paper, we use copolymers obtained by elec-
tropolymerization of metalloporphyrin-type zinc-
β-octaethylporphyrin
(ZnOEP)
or
free-base-type
5,15-ditolyl porphyrin (H2T2P) with the functional-
ized Lindqvist polyoxovanadate bearing two pyridyl
groups [V6O13{(OCH2)3CNHCO(4-C5H4N)}2]2−(Py-
V6O13-Py) (Scheme 1). The photocatalytic reduction
of AgI
2SO4 as well as the H2PtIVCl6 is studied under
visible irradiation in the presence of propan-2-ol,
acting as sacrificial donor, at the 2D interface be-
tween water and the copolymeric films deposited on
quartz substrate. Using this method, the poly-Py-V6O13-Py-H2T2P
and the poly-Py-V6O13-Py-ZnOEP hybrid copoly-
mers were prepared as described previously [27]. Note
that
the
free
base
meso-5,15-
ditolylporphyrin (H2T2P) presents only two meso
positions
occupied
by
one
substitutable
pro-
ton at positions C10 and C20 while the zinc-β-
octaethylporphyrin (ZnOEP) presents four substi-
tutable meso positions at C5, C10, C15 and C20 (top
of Scheme 1). The described electrochemical synthe-
sis of the copolymers uses the previously reported
E(ECNECB)nE process of nucleophilic substitution
on porphyrins [17–19,26–29]. As soon as the iterative
scans were performed at an anodic potential suffi- C. R. Chimie — 2021, 24, nS3, 141-155 144 Zhaohui Huo et al. Figure 1. (A) UV–Vis absorption spectra of poly-V6O13-ZnOEP and poly-V6O13-H2T2P obtained after 20
iterative scans between 0.00 and +1.60 V versus SCE on ITO. (B) Thickness of poly-V6O13-ZnOEP and
poly-V6O13-H2T2P measured by AFM versus different numbers of iterative scans. Tapping mode AFM
topography and section analysis of the aggregate marked by a blue line of (C) and (C′) poly-V6O13-H2T2P,
and of (D) and (D′) poly-V6O13-ZnOEP (film obtained after 20 scans, between 0.00 V and +1.60 V). Figure 1. (A) UV–Vis absorption spectra of poly-V6O13-ZnOEP and poly-V6O13-H2T2P obtained after 20
iterative scans between 0.00 and +1.60 V versus SCE on ITO. 2.1. Electrochemical synthesis of the copolymers (B) Thickness of poly-V6O13-ZnOEP and
poly-V6O13-H2T2P measured by AFM versus different numbers of iterative scans. Tapping mode AFM
topography and section analysis of the aggregate marked by a blue line of (C) and (C′) poly-V6O13-H2T2P,
and of (D) and (D′) poly-V6O13-ZnOEP (film obtained after 20 scans, between 0.00 V and +1.60 V). ciently high to allow the formation of the porphyrin
dication, the formation of a copolymer coating the
working electrode was observed. pyridyl group which is still active for nucleophilic
attack. This pendant pyridyl group can further at-
tack oxidized porphyrin continuing the growth of
the copolymer. Thus, the polarization of a working
electrode at the porphyrin’s second ring-oxidation
potential in the presence of Py-V6O13-Py, leads to
the formation of the two hybrid copolymer films
with general formula [Py+-V6O2−
13 -Py+-porphyrin]n
(bottom of Scheme 1), namely poly-V6O13-H2T2P
and poly-V6O13-ZnOEP. The mechanism of the formation of the copoly-
mer can be described such as: first, the por-
phyrin (abbreviated Porph) radical cation (Porph•+,
electrochemical step E) and dication (Porph2+,
electrochemical
step
E)
are
electrogenerated. Then, the dication porphyrin Porph2+ can be at-
tacked by a two pendant isonicotinate groups of
the
pyridyl-substituted
Lindqvist-type
polyoxo-
vanadate
[V6O13{(OCH2)3CNHCO(4-C5H4N)}2]2−
(abbreviated Py-V6O13-Py) at meso-carbon position
to yield an isoporphyrin (chemical step CNmeso). This
later intermediate can be oxidized (electrochemical
step E) and the hydrogen atom originally situated on
the meso-carbon is released inducing the rearoma-
tization of the porphyrin (chemical step CB) which
leads to the monosubstituted porphyrin Porph-
meso-Py+-V6O13-Py. At this stage, monosubstituted
porphyrin is obtained with one pyridinium cova-
lently connected to the porphyrin and one pendant UV–visible spectra of both copolymers on ITO
electrodes presented similar characteristics [27]. They exhibit a large Soret absorption band (Fig-
ure 1A), which was red shifted by 15 and 20 nm
compared to the ZnOEP and H2T2P monomer re-
spectively [27]. It can be explained by intra- or inter-
molecular excitonic interactions between the por-
phyrin subunits [17,30]. The red shifts observed are
in agreement with the presence of the disubstituted
porphyrin ring by two pyridinium groups [31] in the
copolymer. C. R. Chimie — 2021, 24, nS3, 141-155 145 Zhaohui Huo et al. observed for one week indicating the good stabil-
ity of the silver nanoparticles. Moreover, it has been
found that the slides of quartz can be reused at least
five times with almost no change in the efficiency
of the photocatalysis. 2.2. Photocatalytic recovery of silver The electrochemically deposited poly-V6O13-ZnOEP
or poly-V6O13-H2T2P copolymers were dissolved
and removed from ITO with dimethyl formamide
(DMF). Subsequently, the copolymer in DMF solu-
tion was deposited on a quartz slide, and the DMF
solvent was evaporated in air. Then, the quartz slide
was plunged in an optical cell containing a deaerated
aqueous solution with 8 × 10−5 mol·L−1 Ag2SO4 and
0.13 mol·L−1 propan-2-ol. The whole sample was
illuminated under visible light with a 385 nm cutoff
filter to prevent POM photoexcitation allowing only
the excitation of the porphyrin. Figure 2 presents the
absorption spectra recorded during the experiment
performed with the poly-V6O13-ZnOEP (Figure 2A)
or poly-V6O13-H2T2P (Figure 2B) films. The ab-
sorbance increases in the whole visible domain dur-
ing illumination and the solution becomes slightly
yellow which reveals the formation of silver nanopar-
ticles [22,32]. The initial rate of Ag+ reduction is
3.4×10−5 mol·L−1·min−1 for poly-V6O13-ZnOEP and
3.1 × 10−5 mol·L−1·min−1 for poly-V6O13-H2T2P. After almost 95 min (for poly-V6O13-ZnOEP) and
105 min (for poly-V6O13-H2T2P) of visible light ir-
radiation, the absorption spectrum does not evolve
anymore which indicates the end of the reaction. The 6-fold rotational symmetry displayed by the
diffraction spots implied that the faces represented
the {111} planes. The first set of spots could be in-
dexed to the formally forbidden 1/3 {422} reflec-
tions of face-centered cubic (fcc) silver with a cor-
responding lattice spacing of 2.48 Å. The second
set corresponded to Bragg diffraction from the
{220} planes of fcc silver with a lattice spacing of
1.44 Å (1.445 Å in JCPDS file 04-0783). These obser-
vations were in agreement with the usual published
indexes for silver nanosheets [33,34]. We explain the
observation of 1/3 {422} reflections that are formally
forbidden for a perfect fcc structure by the presence
of stacking faults [35]. These stacking faults could be
caused by bending, which explains the presence of
Moiré pattern in TEM images [36]. The EDS spec-
trum in Figure 4F reveals the presence of metallic Ag
in the sample, the C and Cu detected belonging to
the TEM grid. Conversely, the nanostructures obtained with
poly-V6O13-H2T2P exhibited larger dispersion in
size and shape, because the samples presented not
only spherical, elongated particles but also long lin-
ear silver nanowires (thickness ca. 20–40 nm and
length ca. 200–500 nm) and large silver nanosheets
(Figure 5). 2.1. Electrochemical synthesis of the copolymers Silver ions Ag(I) are reduced
quantitatively at the interface between water and the
copolymeric films without poisoning the surface (as
checked by AFM analysis, data not shown). The films were also examined by scanning atomic
force microscopy (AFM) showing tightly packed coils
with average diameters of 40–60 nm and a height of
5 nm in the case of poly-V6O13-H2T2P (Figures 1C
and C′). The rms surface roughness of the film was
3.5 nm for 1 mm2 area. For poly-V6O13-ZnOEP the
diameter and the height of the coils were almost two
times larger (Figures 1D and D′) [27]. Thickness of the deposited copolymeric film was
measured using AFM by scratching the film with a
metallic tip. The thickness increased upon the num-
ber of iterative scans as shown in Figure 1B. The
values obtained after 20 scans between 0.00 V and
+1.60 V were about 23 nm for poly-V6O13-ZnOEP
and 56 nm for poly-V6O13-H2T2P. The TEM micrographs confirmed the formation of
silver particles (Figures 4 and 5). The nanoparticles
obtained with poly-V6O13-ZnOEP have an average
diameter of 20 nm but are agglomerated (Figure 4). The presence of Moiré pattern in TEM images
of silver particles is due to the presence of metal-
lic silver crystal planes with different spacing and/or
orientations. Each silver nanoparticles seems to be
crystalline as shown by electron diffraction analysis
(Figure 4E). C. R. Chimie — 2021, 24, nS3, 141-155 2.2. Photocatalytic recovery of silver The EDS spectrum in Figure 5C reveals the
presence of metallic Ag in the sample. After removal of the quartz slide covered with the
film, the UV–visible spectrum of the solution exhibits
a large plasmon band in the whole visible domain
with a maximum around 410 nm (Figure 3). This
can be attributed to the presence of silver nanopar-
ticles in the solution. No change in the spectrum is Two possible mechanisms can be involved to de-
scribe the formation of AgNPs. As shown in Fig-
ure 6A, the first mechanism consists in the reduc-
tion of the excited porphyrins by propan-2-ol, fol- C. R. Chimie — 2021, 24, nS3, 141-155 146 Zhaohui Huo et al. Figure 2. Change in the UV–Vis absorption spectra of a deaerated aqueous solution of 8.0 ×10−5 mol·L−1
Ag2SO4 and 0.13 mol·L−1 propan-2-ol containing a slide of quartz modified with (A) poly-V6O13-ZnOEP,
(B) poly-V6O13-H2T2P film under illumination. Inset: plot of the absorbance at (A) λ = 421 and (B) λ =
429 nm versus the time of irradiation. Figure 2. Change in the UV–Vis absorption spectra of a deaerated aqueous solution of 8.0 ×10−5 mol·L−1
Ag2SO4 and 0.13 mol·L−1 propan-2-ol containing a slide of quartz modified with (A) poly-V6O13-ZnOEP,
(B) poly-V6O13-H2T2P film under illumination. Inset: plot of the absorbance at (A) λ = 421 and (B) λ =
429 nm versus the time of irradiation. Figure 3. UV–Vis absorption spectra of the sil-
ver nanoparticles solution before and after the
photocatalysis using the poly-V6O13-ZnOEP
(red curve) or the poly-V6O13-H2T2P (blue
curve) films. Figure 3. UV–Vis absorption spectra of the sil-
ver nanoparticles solution before and after the
photocatalysis using the poly-V6O13-ZnOEP
(red curve) or the poly-V6O13-H2T2P (blue
curve) films. The photoreduction processes should involve
the reduction of the excited porphyrin (Porph∗) by
propan-2-ol leading to the formation of the reduced
porphyrin (Porph•−) and the alcohol radical accord-
ing to the global reaction (1): Porph∗+(CH3)2CHOH →Porph•−
+(CH3)2C•OH+H+
(1) (1) This reaction is thermodynamically favorable. In-
deed, the reduction potentials of the excited por-
phyrins are evaluated according to the relation: E(Porph∗/Porph•−) = E(Porph/Porph•−)+ES0→S1
(2) E(Porph∗/Porph•−) = E(Porph/Porph•−)+ES0→S1
(2) E(Porph∗/Porph•−) = E(Porph/Porph•−)+ES0→S1
(2) Figure 3. UV–Vis absorption spectra of the sil-
ver nanoparticles solution before and after the
photocatalysis using the poly-V6O13-ZnOEP
(red curve) or the poly-V6O13-H2T2P (blue
curve) films. 2.2. Photocatalytic recovery of silver (2) where E(Porph/Porph•−) is the first reduction poten-
tial of the porphyrin and ES0→S1 corresponds to the
energy of the lowest electronic transition determined
from the absorbance spectrum. The calculated E(Porph∗/Porph•−) values are
higher than the reported values for (CH3)2C•OH/
(CH3)2CHOH (E = 0.80 V versus NHE [37]). lowed by the cascade electron transfer via the pyri-
dinium to the POM subunit {V6O13}2−giving the re-
duced {V6O13}3−. Then, {V6O13}3−can in turn reduce
silver ions. A complexation step between alcohol rad-
ical and silver ions can initiate the formation of silver
clusters (reactions (4)–(7)) before a thermodynami-
cally possible direct reduction with the reduced POM
({V6O13}3−) or with the alcohol radical (CH3)2C•OH
(when the nuclearity of initial silver aggregates is suf-
ficient). The second mechanism (Figure 6B) corresponds
to a direct intramolecular electron transfer from the
excited porphyrins to polyoxovanadate via the reduc-
tion of the pyridinium subunit which acts as relay
of electron followed by the reduction of silver ions
by {V6O13}3−. A fast intramolecular electron transfer
agrees with fluorescence quenching of the films ob-
served and is also thermodynamically favorable. C. R. Chimie — 2021, 24, nS3, 141-155 147 Zhaohui Huo et al. gure 4. (A–D) TEM images of the silver nanoparticles obtained with the poly-V6O13-ZnOEP film in
eaerated solution of 8.0×10−5 mol·L−1 Ag2SO4. (E) Selected-area electron diffraction pattern of the silver
anoparticles. The inner spots (circled) corresponded to the formally forbidden 1/3 {422} reflections. The
cond spots (squared) could be indexed to the {220} reflections. (F) EDS spectrum showing the presence
Ag in the sample. Figure 4. (A–D) TEM images of the silver nanoparticles obtained with the poly-V6O13-ZnOEP film in
deaerated solution of 8.0×10−5 mol·L−1 Ag2SO4. (E) Selected-area electron diffraction pattern of the silver
nanoparticles. The inner spots (circled) corresponded to the formally forbidden 1/3 {422} reflections. The
second spots (squared) could be indexed to the {220} reflections. (F) EDS spectrum showing the presence
of Ag in the sample. Indeed, the reduction potentials of the excited
porphyrins are evaluated according to the relation: It should be mentioned that in the two proposed
mechanisms, the simple consideration of the redox
potential of the radical alcohol (CH3)2C•OH), the
reduced porphyrin or the reduced {V6O13}3−does
not allow to explain the subsequent photoreduc-
tion of Ag+ ions. 2.2. Photocatalytic recovery of silver Indeed, the redox potential of the
(Ag+/Ag1) couple is too low (E° = −1.75 V versus
NHE [38]) to allow the direct reduction of Ag+ to E(Porph+•/Porph∗) = E(Porph+•/Porph)−ES0→S1
(3) E(Porph+•/Porph∗) = E(Porph+•/Porph)−ES0→S1
(3) E(Porph+•/Porph∗) = E(Porph+•/Porph)−ES0→S1
(3) where E(Porph+•/Porph) is the first oxidation po-
tential of the porphyrin and ES0→S1 corresponds
to the energy of the lowest electronic transition
determined from the absorbance spectrum. C. R. Chimie — 2021, 24, nS3, 141-155 148 Zhaohui Huo et al. igure 5. (A) TEM images of the silver nanomaterials obtained with the poly-V6O13-H2T2P film in
eaerated solution of 8.0×10−5 mol·L−1 Ag2SO4. (B) Selected-area electron diffraction pattern of the silver
nanoparticles. The inner spots (circled) corresponded to the formally forbidden 1/3 {422} reflections. The
econd spots (squared) could be indexed to the {220} reflections. (C) EDS spectrum showing the presence
f Ag in the sample. Figure 5. (A) TEM images of the silver nanomaterials obtained with the poly-V6O13-H2T2P film in
deaerated solution of 8.0×10−5 mol·L−1 Ag2SO4. (B) Selected-area electron diffraction pattern of the silver
nanoparticles. The inner spots (circled) corresponded to the formally forbidden 1/3 {422} reflections. The
second spots (squared) could be indexed to the {220} reflections. (C) EDS spectrum showing the presence
of Ag in the sample. NHE [40]) is slightly higher than the one the couple
Ag+/Ag1. This process cannot be excluded. a single Ag1 atom either by the reduced porphyrin
(E(H2T2P∗/H2T2P−•) = 1.60 V versus NHE) or the re-
duced {V6O13}3−(E({V6O13}2−/{V6O13}3−) = −0.58 V
versus NHE). Finally, as the redox potential of the silver cluster,
E(Agn+/Agn), increases with the nuclearity n, the di-
rect reduction of large clusters by the reduced por-
phyrin or the alcohol radical becomes thermody-
namically feasible. Referring to radiolytic studies, the reduction of
the Ag+ might be enabled by a complexation step
between alcohol radical and silver ions that initiates
the formation of silver clusters (4)–(7) [13,38,39]: Note that the absence of reduced polyoxovana-
date at the end of the reaction is in favor of the mech-
anism (B). It can be explained by an intramolecular
back electron transfer between the reduced {Py+-
V6O3−
13 -Py} and the porphyrins oxidized Porph+•
which is thermodynamically favorable. C. R. Chimie — 2021, 24, nS3, 141-155 2.2. Photocatalytic recovery of silver (CH3)2C•OH+Ag+ →Ag(CH3)2C•OH+
(4)
Ag(CH3)2C•OH+ +Ag+ →Ag+
2 +(CH3)2C = O+H+
(5)
2Ag+
2 →Ag2+
4
(6)
Agx+
m +Agy+
p →Ag(x+y)+
(m+p)
(7) (CH3)2C•OH+Ag+ →Ag(CH3)2C•OH+
(4)
Ag(CH3)2C•OH+ +Ag+ →Ag+
2 +(CH3)2C = O+H+
(5) (4) 2Ag+
2 →Ag2+
4
(6
Agx+
m +Agy+
p →Ag(x+y)+
(m+p)
(7 To justify this electron transfer in the case of
the mechanism B (Figure 6B), we can calculate the
∆rG of this process using the Rehm–Weller equa-
tion: ∆G = E(Porph•+/Porph) −E(POM/POMred) −
ES0–S1
where
E(Porph+•/Porph)
corresponds
to Another reasonable explanation is the forma-
tion of Ag0
1 by reaction between Ag+ and the
alcohol radical (CH3)2C•OH even if the poten-
tial (E((CH3)2CO/(CH3)2C•OH) = −1.71 V versus C. R. Chimie — 2021, 24, nS3, 141-155 149 Zhaohui Huo et al. Figure 6. (A) and (B) Two possible mechanisms for the photoreduction of silver ions by the use of the
poly-V6O13-H2T2P copolymer. Similar mechanism can be proposed for poly-V6O13-ZnOEP. Bottom:
scale of apparent standard redox potentials of couples involved in this mechanism in the case of H2T2P. ZnOEP gives similar tendencies. Note that the reduction of the pyridinium group is achieved at a redox
potential very close to that of the reduction of {V6O13}. Figure 6. (A) and (B) Two possible mechanisms for the photoreduction of silver ions by the use of the
poly-V6O13-H2T2P copolymer. Similar mechanism can be proposed for poly-V6O13-ZnOEP. Bottom:
scale of apparent standard redox potentials of couples involved in this mechanism in the case of H2T2P. ZnOEP gives similar tendencies. Note that the reduction of the pyridinium group is achieved at a redox
potential very close to that of the reduction of {V6O13}. namely {V6O13}3−) via probably a relay including
the reduction of the pyridium. Then, oxidized por-
phyrins porph+• are regenerated by reaction with
propan-2-ol which leads to the formation of alco-
hol radicals (CH3)2C•OH. This reaction is also really
plausible, since the redox potential of the couple
(CH3)2C•OH/(CH3)2CHOH is estimated to 0.80 V
versus NHE [37], that is lower than the oxidation
potential of the porphyrin. the
first
oxidation
potential
of
the
porphyrin
(E(Porph+•/Porph) = +1.25 V versus NHE for H2T2P)
and E(POM/POMred) corresponds to the first reduc-
tion potential of the POM (E({V6O13}2−/{V6O13}3−) =
−0.58 V versus NHE) [27]. ES0–S1 corresponds to
the energy of the lowest electronic transition of
the porphyrin estimated from its absorbance spec-
trum in solution. 2.2. Photocatalytic recovery of silver This value leads to negative ∆G
(−61.5 kJ·mol−1), indicating that the electron trans-
fers between the excited porphyrins and the POMs
subunit {V6O13}2−are thermodynamically possi-
ble. These electronic transfers lead to oxidized por-
phyrins (porph+•) and reduced POMs (POMred, However, the potential of the couple {V6O13}2−/
{V6O13}3−) is too high to permit the reduction of sil-
ver ions (E(Ag+/Ag1) = −1.75 V versus NHE), cor-
responding to the redox potential of a single Ag(0) C. R. Chimie — 2021, 24, nS3, 141-155 150 Zhaohui Huo et al. Figure 7. (A) Change in the UV–Vis absorption spectra of a deaerated aqueous solution of 1.6 ×
10−4 mol·L−1 H2PtCl6 and 0.13 mol·L−1 propan-2-ol containing a slide of quartz modified with poly-
V6O13-ZnOEP film under illumination. Inset: plot of the intensity of the absorbance at λ = 201 and
262 nm versus the time of irradiation. (B) Spectrum of the platinum solution before and after photo-
catalysis. Inset: photo of the obtained solution in the cuvette after the irradiation. Figure 7. (A) Change in the UV–Vis absorption spectra of a deaerated aqueous solution of 1.6 ×
10−4 mol·L−1 H2PtCl6 and 0.13 mol·L−1 propan-2-ol containing a slide of quartz modified with poly-
V6O13-ZnOEP film under illumination. Inset: plot of the intensity of the absorbance at λ = 201 and
262 nm versus the time of irradiation. (B) Spectrum of the platinum solution before and after photo-
catalysis. Inset: photo of the obtained solution in the cuvette after the irradiation. atom, different from the one corresponding to metal-
lic silver which is 0.799 V versus NHE. We can also
exclude the possibility of a photoinduced electron
transfer from the excited porphyrins to the silver
ions, because the ∆G value of such transfer is posi-
tive and is estimated to +77.2 kJ·mol−1. The TEM micrographs confirm the formation
of the Pt nanoparticles. The diameter of the basic
nanoparticle subunit is 1.0–1.5 nm which forms the
closed-packed nanoparticles with a diameter of 10–
20 nm (Figure 8). The EDS spectrum in Figure 8E in-
dicates the presence of merely pure Pt in the sample. Figure 9 presents the absorption spectra of a
deaerated
aqueous
solution
containing
1.6 ×
10−4 mol·L−1 H2PtCl6 and 0.13 mol·L−1 propan-2-ol
with poly-V6O13-H2T2P covered quartz slide dur-
ing visible light irradiation. The initial absorbance
of PtIVCl2−
6
anions disappeared completely after
110 min which is a little slower than for poly-V6O13-
ZnOEP. 2.2. Photocatalytic recovery of silver After 24 h of illumination, we obtained a
uniform and faint yellow solution which is different
than the sediment obtained when using poly-V6O13-
H2T2P. The TEM micrographs of the obtained Pt
nanostructures are presented in Figure 10. The Pt
nanoparticles showed again closed-packed spherical
structure similar to the ones obtained using poly-
V6O13-ZnOEP film. Nevertheless, such structures are
also mixed with large nanosheets. The obtained Pt
nanomaterial is in this case inhomogeneous in size
and shape. The EDS spectrum in Figure 10E shows
the presence of Pt nanoparticles in the sample. C. R. Chimie — 2021, 24, nS3, 141-155 2.3. Photocatalytic recovery of platinum To extend the photocatalytic application, we have
explored the photocatalysis of the reduction of
PtIVCl2−
6
anions using the two covalent porphyrin–
Lindqvist-type POM copolymers. The study is con-
ducted under deaerated medium in the presence
of 1.6 × 10−4 mol·L−1 H2PtIVCl6 (to keep the same
concentration of metal ions) and 0.13 mol·L−1
propan-2-ol. Figure 7 illustrates the change in the
UV–Vis absorption spectrum recorded during the
visible light irradiation of a quartz slide covered
with poly-V6O13-ZnOEP. The initial absorbance of
PtIVCl2−
6
anions decreased during illumination and
disappeared completely after 100 min, however,
because the Pt nanoparticles do not possess plas-
mon band or other type of UV–visible absorbance,
it is impossible to monitor the creation of the Pt
nanoparticles from the UV–visible spectra. After almost 3 h for poly-V6O13-ZnOEP and 4 h
for poly-V6O13-H2T2P of visible light irradiation, the
absorption spectrum does not evolve anymore which
indicates the end of the reaction. The duration of
the photocatalytic reduction is longer in comparison After 24 h of illumination, a yellow sediment on
the bottom of the cuvette is obtained, and after stir-
ring, the absorbance in the whole domain increases. C. R. Chimie — 2021, 24, nS3, 141-155 151 Zhaohui Huo et al. Figure 8. (A–D) TEM images of the platinum nanomaterial obtained with the poly-V6O13-ZnOEP in a
deaerated solution of 1.6 × 10−4 mol·L−1 H2PtCl6. (E) EDS spectrum showing the presence of Pt in the
sample. Figure 8. (A–D) TEM images of the platinum nanomaterial obtained with the poly-V6O13-ZnOEP in a
deaerated solution of 1.6 × 10−4 mol·L−1 H2PtCl6. (E) EDS spectrum showing the presence of Pt in the
sample. to the silver recovery which can be explained by the
number of electron implied: 4 for the Pt(IV) versus 1
in the case of the Ag(I). that is the electron transfer from the excited por-
phyrin via the pyridinium to the polyoxovanadates
(mechanism B). Last but not least, the heterogeneous photocataly-
sis can also be repeated at least five times while keep-
ing the same efficiency. Platinum ions are reduced
quantitatively and no deposition at the surface of the
slide was observed (as checked by AFM analysis, data
not shown). C. R. Chimie — 2021, 24, nS3, 141-155 2.3. Photocatalytic recovery of platinum We assume that the mechanism for the photo-
catalysis Pt(IV) is much more complicated than the
one proposed for the reduction of Ag(I) ions probably
due to the fact that 4 electrons must be transferred
between the catalysts and the metallic ion. We will
therefore not provide a mechanism in this present
case although there is a good chance that the first
step is similar to that of the reduction of Ag(I) ions, C. R. Chimie — 2021, 24, nS3, 141-155 152 Zhaohui Huo et al. Figure 9. (A) Change in the UV–Vis absorption spectra of a deaerated aqueous solution of 1.6 ×
10−4 mol·L−1 H2PtCl6 and 0.13 mol·L−1 propan-2-ol containing a slide of quartz modified with poly-
V6O13-H2T2P film under illumination. Inset: plot of the intensity of the absorbance at λ = 201 and 262 nm
versus the time of irradiation. (B) Spectrum of the platinum solution before and after photocatalysis. In-
set: photo of the obtained solution in the cuvette after the irradiation. Figure 9. (A) Change in the UV–Vis absorption spectra of a deaerated aqueous solution of 1.6 ×
10−4 mol·L−1 H2PtCl6 and 0.13 mol·L−1 propan-2-ol containing a slide of quartz modified with poly-
V6O13-H2T2P film under illumination. Inset: plot of the intensity of the absorbance at λ = 201 and 262 nm
versus the time of irradiation. (B) Spectrum of the platinum solution before and after photocatalysis. In-
set: photo of the obtained solution in the cuvette after the irradiation. 3. Conclusion Sigma-Aldrich. Lindqvist-type
polyoxovanadate
TBA2[V6O13{(OCH2)3CNHCO(4-C5H4N)}2] (abbrevi-
ated Py-V6O13-Py) was synthesized according to the
previous publications [24,41]. We
demonstrated
the
efficiency
of
hybrid
polyoxovanadate–porphyrin
copolymers
(poly-
V6O13-H2T2P and poly-V6O13-ZnOEP) in the pho-
tocatalytic reduction of Ag(I) and Pt(IV) using vis-
ible light. Under ambient conditions, AgI
2SO4 or
H2PtIVCl6 were reduced quantitatively at the inter-
face between water and the copolymeric films with-
out poisoning the surface. The heterogeneous catal-
ysis can be repeated at least five times while keeping
the same efficiency at room temperature and under
mild reaction conditions. The corresponding copolymers poly-Py-V6O13-
Py-H2T2P and poly-Py-V6O13-Py-ZnOEP were pre-
pared as described previously [27]. Electropolymerization have been carried out un-
der an argon atmosphere using a 0.1 mol·L−1 solution
of 0.1 mol·L−1 TBAPF6 in 1,2-C2H4Cl2/CH3CN (7/3)
containing 0.25 mmol·L−1 of ZnOEP or H2T2P and
0.25 mmol·L−1·mmol·L−1 of Py-V6O13-Py (Scheme 1)
between 0 and 1.6 V versus SCE. ITO electrodes, with
a surface of 1 cm2, were used as working electrode. For each copolymer, the number of iterative scans
(n) was 20. After electropolymerization, the modified
working electrodes were washed with CH3CN and
then with CH2Cl2 in order to remove the monomers
and the conducting salt present on the deposited
films. This process verifies our starting hypothesis that
the copolymerization of photosensitizers such as
porphyrin and POMs yields photoactive materi-
als that are useful for photocatalytic reduction in
general. The obtained silver nanoparticles form
aggregates while the Pt nanoparticles showed closed-
packed spherical structures and some large sheets. C. R. Chimie — 2021, 24, nS3, 141-155 4. Experimental section The
electrochemically
deposited
poly-V6O13-
ZnOEP or poly-V6O13-H2T2P films were dissolved
and removed from ITO electrode with DMF. The op-
eration is repeated six times. Subsequently, the DMF
solution containing the copolymer was deposited on
a quartz slide, and the DMF solvent was evaporated
in air. Most common laboratory chemicals were reagent
grade, purchased from commercial sources and used
without further purification. The
5,15-ditolylporphyrin
(H2T2P)
was
pur-
chased
from
SAS
PorphyChem®
and
zinc-β-
octaethylporphyrin (ZnOEP) was purchased from C. R. Chimie — 2021, 24, nS3, 141-155 153 Zhaohui Huo et al. igure 10. (A–D) TEM images of the platinum nanostructures obtained with the poly-V6O13-H2T2P fi
n a deaerated solution of 1.6×10−4 mol·L−1 H2PtCl6. (E) EDS spectrum showing the presence of Pt in t
ample. Figure 10. (A–D) TEM images of the platinum nanostructures obtained with the poly-V6O13-H2T2P film
in a deaerated solution of 1.6×10−4 mol·L−1 H2PtCl6. (E) EDS spectrum showing the presence of Pt in the
sample. Water was obtained by passing through a Milli-
RO4 unit and subsequently through a Millipore Q
water purification set. dispersion and to produce a beam converging to the
photochemical cell. According to the supplier, the ir-
radiance of the lamp from 320 to 790 nm was around
50 mW·m−2·nm−1. The samples consisted of 4 mL of
aqueous solutions with propan-2-ol, the quartz slide
covered by the copolymer Py-V6O13-Py-H2T2P and
poly-Py-V6O13-Py-ZnOEP and, Ag2SO4 or H2PtCl6
contained in a spectrophotometer quartz cell of 1 cm
path length. Irradiation was performed using a 300 W Xe arc
lamp (Lot, Quantum design) with intense focused
output beams (50 mm beam diameter) equipped
with a water cell filter to absorb the IR radiation. A
spherical reflector collects the output from the rear
of the lamp and focuses it on or near the arc for
collection by the condenser. One condenser is posi-
tioned for compensating focal length change due to Deaerated solutions were obtained by bubbling
with argon (Ar–U, from Air Liquide) before illumina- 154 Zhaohui Huo et al. tion. All experiments were carried out at room tem-
perature. [7] Y. Nagata, Y. Watananabe, S. Fujita, T. Dohmaru, S. Taniguchi,
J. Chem. Soc. Chem. Commun., 1992, 21, 1620-1622. [8] T. Fujimoto, S. Y. Terauchi, H. Umehara, I. Kojima, W. Hender-
son, Chem. Mater., 2001, 13, 1057-1060. We checked that the temperature of the solution
did not increase by more than 1 degree during light
illumination. [9] J. Belloni, M. The authors declare no conflicts of interest. [22] D. Schaming, C. Allain, R. Farha, M. Goldmann, S. Lobstein,
A. Giraudeau, B. Hasenknopf, L. Ruhlmann, Langmuir, 2010,
26, 5101-5109. 4. Experimental section Mostafavi, H. Remita, J. L. Marignier, M. O. Delcourt, New J. Chem., 1998, 11, 1239-1255. UV–Vis absorption spectra were recorded with
a single beam Hewlett-Packard HP 8453 diode ar-
ray spectrophotometer operated at a resolution of
2 nm. [10] H. Yin, T. Yamamoto, Y. Wada, S. Yanagida, Mater. Chem. Phys.,
2004, 83, 66-70. [11] B. Ohtani, M. Kakimoto, H. Miyadzu, S. Nishimoto, T. Kagiya,
J. Phys. Chem., 1988, 92, 5773-5777. [12] A. Troupis, A. Hiskia, E. Papaconstantinou, Appl. Catal. B:
Environ., 2003, 42, 305-315. Atomic force micrographs (AFM) measurements
have been conducted directly on the ITO surfaces us-
ing a Veeco Dimension 3100 apparatus in the tap-
ping mode under ambient conditions. Silicon can-
tilevers (Veeco probes) with a spring constant of
300 N/m and a resonance frequency in the range of
120–139 kHz have been used. The scanning rate was
1.0 Hz. [13] C. Costa-Coquelard, D. Schaming, I. Lampre, L. Ruhlmann,
Appl. Catal. B: Environ., 2008, 84, 835-842. [14] C. Allain, D. Schaming, S. Sorgues, J.-P. Gisselbrecht, I. Lam-
pre, L. Ruhlmann, B. Hasenknopf, Dalton Trans., 2013, 42,
2745-2754. [15] Y. Zhu, Y. Huang, Q. Li, D. Zang, J. Gu, Y. Tanf, Y. Wei, Inorg. Chem., 2020, 59, 2575-2583. [16] L. El Kahef, M. Gross, A. Giraudeau, J. Chem. Soc. Chem. Com-
mun., 1989, 49, 963-963. Transmission electronic microscopy (TEM) obser-
vations were performed with a JEOL 100 CXII TEM
instrument operated at an accelerating voltage of
100 kV. Samples for TEM analysis were prepared by
dropping the solution on carbon-coated copper TEM
grids. [17] A. Giraudeau, L. Ruhlmann, L. El Kahef, M. Gross, J. Am. Chem. Soc., 1996, 118, 2969-2679. [18] L. Ruhlmann, S. Lobstein, M. Gross, A. Giraudeau, J. Org. Chem., 1999, 64, 1352-1355. [19] A. Giraudeau, S. Lobstein, L. Ruhlmann, D. Melamed, K. M. Barkigia, J. Fajer, J. Porphyrins Phthalocyanines, 2001, 05, 793-
797. [20] L. Ruhlmann, A. Giraudeau, Chem. Commun., 1996, 17, 2007-
2008. Acknowledgments [23] S. Favette, B. Hasenknopf, J. Vaissermann, P. Gouzerh,
C. Roux, Chem. Commun., 2003, 21, 2664-2665. [24] C. Allain,
S. Favette,
L. Chamoreau,
J. Vaissermann,
L. Ruhlmann, B. Hasenknopf, Eur. J. Inorg. Chem., 2008,
3433-3441. The authors are grateful to the University of Stras-
bourg and the CNRS for constant financial support. The authors also thank the Overseas Study Program
of Guangzhou Elite Project (GEP) for the Ph.D. grant
of Zhaohui Huo. [25] L. Ruhlmann, A. Schulz, A. Giraudeau, C. Messerschmidt, J.-
H. Fuhrop, J. Am. Chem. Soc., 1999, 121, 6664-6667. [26] L. Ruhlmann, J. Hao, Z. Ping, A. Giraudeau, J. Electroanal. Chem., 2008, 621, 22-30. [27] Z. Huo, I. Azcarate, R. Farha, M. Goldmann, H. Xu, B. Hasen-
knopf, E. Lacôte, L. Ruhlmann, J. Solid State Electrochem.,
2015, 19, 2611-2621. Conflicts of interest [21] L. Ruhlmann, A. Giraudeau, Eur. J. Inorg. Chem., 2001, 659-
668. The authors declare no conflicts of interest. C. R. Chimie — 2021, 24, nS3, 141-155 References [28] A. Giraudeau, D. Schaming, J. Hao, R. Farha, M. Goldmann,
L. Ruhlmann, J. Electroanal. Chem., 2010, 638, 70-75. 8] A. Giraudeau, D. Schaming, J. Hao, R. Farha, M. Gol [1] D.-L. Long, R. Tsunashima, L. Cronin, Angew. Chem. Int. Ed. Engl., 2010, 49, 1736-1758. [29] D. Schaming, I. Ahmed, J. Hao, V. Alain-Rizzo, R. Farha,
M. Goldmann, H. Xu, A. Giraudeau, P. Audebert, L. Ruhlmann,
Electrochim. Acta, 2011, 56, 10454-10463. [2] S. Anandan,
S. Pitchumani,
B. Muthuraaman,
P. Maruthamuthu, Sol. Energy Mater. Sol. Cells, 2006, 90,
1715-1720. [30] J. L. Sessler, M. R. Johnson, S. E. Creager, J. C. Fettinger, J. Ibers,
J. Am. Chem. Soc., 1990, 112, 9310-9329. [3] A. Mylonas, A. Hiskia, E. Papaconstantinou, J. Mol. Catal. A
Chem., 1996, 114, 191-200. [31] D. Schaming, Y. Xia, R. Thouvenot, L. Ruhlmann, Chem. Eur. J., 2013, 19, 1712-1719. [4] Y. H. Kim, D. K. Lee, Y. S. Kang, Colloids Surf. A: Physicochem. Eng. Asp., 2005, 273, 257-258. [32] I. Ahmed, X. Wang, N. Boualili, H. Xu, R. Farha, M. Goldmann,
L. Ruhlmann, Appl. Catal. A: Gen., 2012, 447–448, 89-99. [5] M. Reetz, W. Helbig, J. Am. Chem. Soc., 1994, 116, 7401-7402. [6] M. Starowicz, B. Stypuła, J. Bana´s, Electrochem. Commun.,
2006, 8, 227-230. C. R. Chimie — 2021, 24, nS3, 141-155 155 Zhaohui Huo et al. [33] R. Jin, Y. Cao, C. A. Mirkin, K. L. Kelly, G. C. Schatz, J. G. Zheng,
Science, 2001, 294, 1901-1903. [37] E. Gachard, H. Remita, J. Khatouri, B. Keita, L. Nadjo, J. Bel-
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J. Phys. Chem. B, 2006, 110, 11796-11799. [41] J. W. Han, K. I. Hardcastle, C. L. Hill, Eur. J. Inorg. Chem., 2006,
2598-2603. C. R. Chimie — 2021, 24, nS3, 141-155
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28
Raumforschung und Raumordnung
1989
Heft 1
ERNST-JÜRGEN SCHRÖDER
Auswirkungen wirtschaftsstruktureller Änderungen
auf den Güterverkehr
in der Bundesrepublik Deutschland seit 197 4
Kurzfassung
Im Güterverkehr in der Bundesrepublik Deutschland ist seit
den 50er Jahren eine kontinuierliche Abnahme des Marktanteils
der Eisenbahn zu erkennen, die sich unter dem Einfluß des durch
die Stahlkrise im Jahre 1974 beschleunigten Strukturwandels
verstiirkt hat. Diese für die Bahn einschneidende Entwicklung,
in deren Gefolge sie als Verkehrstriiger auf den zweiten Platz
verwiesen wurde, trat selbst unter dem Schutzschild der sie
begünstigenden deutschen M arktordnungfür den Straßengüterverkehr ein. Die Bundesbahn hat in jüngerer Zeit mehrere
erfolgversprechende Offensiven zur Gewinnung neuer Miirkte
im Güterverkehr unternommen, doch stehen ihr die eigentlichen
Herausforderungen im Zusammenhang mit der für 1993 vorgesehenen Liberalisierung des Europiiischen Verkehrsmarktes noch
bevor. Die Grundlagen der seitherigen Entwicklung, ihre Ursachen und ihren Ablauf im Blick auf den gesamten Güterverkehr
darzustellen, ist der Zweck dieses Beitrages.
1989
Heft 1
Ernst-Jürgen Schröder: Auswirkungen wirtschaftsstruktureller Änderungen ...
1. Die Güterverkehrstr,iger und ihre Anteile an der
Güterverkehrsleistung (modal split)
Die Träger des Güterverkehrs sind in der Bundesrepublik
die Eisenbahn, die Binnenschiffahrt, der Straßen-, der Rohrleitungs- und der Luftverkehr.
Als Vorteile der Eisenbahn sind Sicherheit, Pünktlichkeit,
Schnelligkeit und eine automatisierte, im Falle der Elektrifizierung noch verstärkt umweltfreundliche und energiesparende Betriebsführung zu nennen. Aufgrund der hohen Fixkostenbelastung und der damit verbundenen Kostendegression
pro beförderte Einheit mit zunehmender Auslastung des
Laderaumes und zunehmender Entfernung ist die Eisenbahn
prädestiniert für den Knotenpunktverkehr über mittlere und
weite Strecken. Eine hohe, dem Straßenverkehr entsprechende Netzdichte ist damit unter Effizienzgesichtspunkten
nicht angezeigt. Die Eisenbahn kann somit im Sinne von
Voppel als "begrenzt dezentralisierend" angesprochen werden, wobei ihrer historischen Entwicklung eine standortkonzentrierende und Ballungen verursachende Wirkung zugeschrieben wird (Voppel, G., 1975, S. 177).
Der Straßenverkehr hat neben dem Vorteil der intensivsten
Raumerschließung unter allen Verkehrsträgern noch den der
Individualität. Schnelligkeit, Pünktlichkeit und Sicherheit
hängen stark von den Witterungsverhältnissen und dem Verkehrsaufkommen ab. Gravierende Nachteile sind die durch
ihn verursachte Schadstoff- und Lärmbelastung der Umwelt
und der vergleichsweise hohe Flächen- und Energieverbrauch.
Zum Schutze der Bahn, aber auch vor ruinöser Konkurrenz
innerhalb des Kraftverkehrsgewerbes sieht die im Güterkraftverkehrsgesetz für den gewerblichen Straßengüterfernverkehr
festgelegte Marktordnung staatliche Maßnahmen zur Kapazitätsbeschränkung und zur Tarifgestaltung vor. Die Ausübung
des gewerblichen Güterfernverkehrs wird durch staatliche
Konzessionen reguliert, die vom Bundesverkehrsminister für
den allgemeinen Güterfernverkehr, den Bezirksgüterfernverkehr (bis 150 km) und den grenzüberschreitenden Verkehr
nach dem Verkehrsbedarf kontingentiert werden. Ausge-
29
nommen von der Kontingentierung sind der Güternahverkehr, der zur Eisenbahn eher in einem komplementären als in
einem konkurrierenden Verhältnis steht, und der W erkverkehr, d.h. der von den Unternehmen für eigene Zwecke betriebene Verkehr.
Bei der Binnenschiffahrt stehen den Vorteilen der im Vergleich zu anderen Verkehrsträgern niedrigeren Transportkosten und des geringeren Energieaufwands die Nachteile der
Langsamkeit, der Witterungsabhängigkeit und der Weitmaschigkeit des Netzes gegenüber. Der Luftverkehr kommt
aufgrund seiner Schnelligkeit, aber auch seiner hohen Transportkosten nur für hochwertige und verderbliche Güter in
Betracht. Der billigste Verkehrsträger für flüssige, gasförmige
und auch manche feste Stoffe ist bei großem, stets gleichbleibendem Aufkommen zwischen wenigen festgelegten Punkten
und damit geringer räumlicher Erschließung der Rohrleitungsverkehr (Schliephake, K., 1982, S. 69 f., 96).
Einen zusammenfassenden, nach den jeweiligen Verkehrsträgern getrennten Überblick über die Entwicklung des
Güterverkehrs in der Bundesrepublik seit 1974 sowohl hinsichtlich der Höhe des in Tonnen gemessenen Güteraufkommens als auch der in T ariftonnenkilometern ausgedrückten
Güterverkehrsleistung vermittelt die Tabelle 1.
Die im ganzen stagnierende Entwicklung des Güterverkehrsmarktes im Zeitraum 1974-1986 läßt erkennen, daß die
Verkoppelung von Wirtschafts- und Transportwachstum
infolge des ökonomischen Strukturwandels weithin entfallen
ist. Hierbei kam es für die Bahn im Güterverkehr trotz des
ordnungspolitischen Flankenschutzes, dessen Wirkung durch
die in jenem Zeitraum starke Zunahme des von der Kontingentierung ausgenommenen Werkverkehrs und durch den
starken Ausbau des Straßen- und des Wasserstraßennetzes
eingeschränkt wurde, aufgrund veränderter Transportanforderungen zu empfindlichen Marktanteilsverlusten in erster
Linie zugunsten des Straßengüterfernverkehrs. Die Auswirkungen auf den Rohrleitungsverkehr bleiben hier angesichts
seines zur Zeit im wesentlichen auf den Mineralölbereich
begrenzten Einsatzfeldes außer Betracht.
Tabelle 1:
Güterverkehrsau/kommen (in Mio. t) und -Leistung (in Mrd. tkm) und Anteile der Verkehrstriiger in der Bundesrepublik Deutschland /974-/986
Verkehrsträger
1974
1986
abs.
%
abs.
%
Eisenbahn Mio. t
Mrd. tkm
389,7
69,3
40,6
35,4
305,3
60,6
31,9
27,7
Binnenschiffahrt Mio. t
Mrd. tkm
252,1
51,0
26,3
26,0
229,5
52,2
24,0
23,9
Straßengüterfernverkehr Mio. t
Mrd. tkm
224,6
58,5
23,4
29,9
351,2
95,9
36,7
43,9
Rohrfernleitungen Mio. t
Mrd. tkm
92,5
16,9
9,6
8,6
70,5
9,7
7,3
4,4
Luftverkehr Mio. t
Mrd. tkm
0,5
0,2
0,1
0,1
0,9
0,3
0,1
0,1
959,4
195,9
100,0
100,0
957,4
218,7
100,0
100,0
Binnenländischer Verkehr
(ohne Straßengüternahverkehr) Mio. t
Mrd. tkm
Qrn..- lll·: Vt.•rkchr in Zahk·n. 1987, S. 194 ff
30
Raumforschung und Raumordnung
2. Ursachen der Anteilsverschiebungen im Güterverkehr
a) Folgen des wirtschaftlichen Strukturwandels
Auch für die Bundesrepublik Deutschland ist die für Länder
mit hohem Entwicklungsstand der Volkswinschaft bekannte
Gesetzmäßigkeit einer nur noch unterproportionalen Zunahme bzw. einer Stagnation der Güterverkehrsnachfrage im
Verhältnis zum Wirtschaftswachstum zu bestätigen. Wichtige transportintensive Branchen gehören heute zu den stagnierenden oder schrumpfenden Winschaftsbereichen, während dagegen der tertiäre Sektor mit geringem Bedarf an
Güterverkehrsleistungen expandiert (Hamm, W., 1987,
S. 422). Insbesondere der Steinkohlenbergbau, die Stahlindustrie, der Hochbau und die Landwirtschaft sind seit 1974,
vornehmlich aufgrund einer allgemeinen Marktsättigung, von
strukturellen Einbrüchen betroffen, und diesen zufolge verloren u.a. der mit dem Montansektor verbundene Massengüterbereich sowie der der Düngemittel und der Baustoffe größere
Anteile am Güterverkehrsaufkommen. Einen überdurchschnittlichen Zuwachs erfuhr durch die zunehmende Spezialisierung der Produktion der Transport chemischer Erzeugnisse und hochwertiger Halb-und Fertigprodukte.
Generell ist seit einiger Zeit eine Umschichtung des Sozialproduktes von schweren, geringwertigen Massengütern zu
leichten, hochwertigen Produkten sowie ein insgesamt sinkender Rohstoffeinsatz je Produkteinheit mit ebenfalls stagnierender Wirkung für das Güterverkehrsvolumen zu beobachten (Bretzke, W .-R., 1988, S. 18). Die Verwendung der
Maßeinheiten Tonne bzw. Tonnenkilometer für die Verkehrsleistung läßt sich damit zumeist nur noch aus Gründen
der zeitlichen Vergleichbarkeit rechtfertigen.
Von diesen mit dem Wirtschaftswachstum einhergehenden
Güterstruktureffekten und den damit verbundenen Änderungen der Transportanforderungen wurden die Verkehrsträger
verschieden stark betroffen. Die Bahn und auch - in abgeschwächtem Maße - die Binnenschiffahrt hatten durch ihre
starke Position im Massengutverkehr (insbesondere im Montanbereich) und die daraus resultierenden Abhängigkeiten
erhebliche Verluste hinzunehmen, ohne eine nennenswerte
Stellung auf den wachsenden Teilverkehrsmärkten für höherwertige Güter erreichen zu können, bei denen erfahrungsgemäß eine hohe Straßen-, aber auch Luftfahrtaffinität gegeben
ist.
1989
Heft 1
Lozierung der Aktivitäten der Unternehmen auf ihre jeweiligen Standorte, wobei auf die regional politisch unerwünschten
Ergebnisse einer Tendenz zur interregionalen funktionalen
Arbeitsteilung hingewiesen wurde (Bade, F.-j. , 1986, S. 695 f.).
Dieser Entwicklung entsprechend konzentrieren sich an den
Firmensitzen in den Agglomerationen die in bezug auf Einkommens- und Aufstiegsmöglichkeiten höherwenigen Funktionen, insbesondere die im Rahmen der "Tertiärisierung" der
Produktion stark vermehnen produktionsorientierten Dienstleistungen (u.a. Planung, Kontrolle, Forschung und Entwicklung) sowie hochspezialisierte und technologieintensive Prozesse, während die peripheren Gebiete mit andersartiger
Struktur des Arbeitsmarktes eher die standardisierte Massenproduktion an sich ziehen (Griiber, H. u.a., 1986, S. 681).
Eine weitere Zunahme der internationalen Arbeitsteilung
und damit der Verkehrsnachfrage ist im Zusammenhang mit
der bereits von mehreren Unternehmen zur Erhaltung ihrer
Wettbewerbsfähigkeit und/oder zur Erschließung neuer Absatzmärkte angekündigten teilweisen Verlagerung der Produktion ins Ausland zu erwarten. Generell geht damit, unterstützt durch den Einsatz der modernen Kommunikationstechnik, eine tendenzielle Dezentralisierung der Produktion
einher.
Verbunden war dieser räumliche Anpassungsprozeß der
Wirtschaft mit einer steigenden Nachfrage nach schneller und
terminierter Beförderung kleiner und mittlerer Sendungen,
die den Straßengüterfernverkehr durch seine Systemvorteile
in der größeren Flächenerschließung und der Beweglichkeit
begünstigte. Systembedingt konnte die Bahn mit ihrer unbestrittenen Leistungskompetenz im Ganzzugverkehr in diesem
wachsenden Segment des Teilladungsmarktes mit extremer
Feinverteilung nicht mithalten. Auch im grenzüberschreitenden Verkehr, der durch den Ausbau der internationalen Wirtschaftsbeziehungen stärker angestiegen war als der Binnenverkehr und ebenfalls eine zunehmende Bedeutung der kleinen
und mittleren Sendungen verzeichnen konnte, vermochte die
Bahn kaum Erfolge zu erzielen. Auf diesem besonderen
Wachstumsfeld des Güterverkehrsmarktes steht sie vor der
Aufgabe, durch Einrichtung neuer Direktzugverbindungen
zwischen den nationalen und internationalen Wirtschaftszentren unter Abbau ihrer bisherigen Leistungsmängel ihre
Systemstärken zur Geltung kommen zu lassen.
c) Neue Formen der Produktion
b) Zunahme der nationalen und internationalen Arbeitsteilung
Während die Entwicklung des Güterverkehrsaufkommens
den sektoralen Umschichtungen zufolge insgesamt eine stagnierende Tendenz aufwies, nahm die Güterverkehrsleistung
dennoch leicht zu. Aus der mit der generellen Ausweitung der
Handelsbeziehungen verbundenen Intensivierung der nationalen und internationalen Arbeitsteilung resultierten räumliche Verflechtungen und damit zusätzliche Güterverkehrsleistungen, die das fehlende Mengenwachstum bislang mehr als
ausgleichen konnten. Die mit der räumlichen Arbeitsteilung
u.a. durch Spezialisierungs- und Rationalisierungsvorteile
ersparten Produktionskosten werden um so weniger durch
den zusätzlichen Raumüberwindungsaufwand relativiert, je
leistungsfähiger das Verkehrsnetz ist (Ihde, G.B., 1984, S. 102 f.,
108). Kritische Beachtung fand hierbei die seit etwa 1974 vor
allem von Mehrbetriebsunternehmen, aber auch von horizontal gegliederten Konzernen im Inland verstärkt angewandte
interne Arbeitsteilung und Spezialisierung durch optimale
Der Trend zu kleineren und mittleren Sendungen mit
garantierten Lieferfristen ergibt sich auch aus der vor allem in
der Maschinenbau- und der Automobilindustrie zunehmenden Einführung eines flexiblen Produktionsverfahrens, der
"Just-in-time-Produktion", die einem weitgehend lagerlosen
Produktionsablauf dienen soll. Entscheidende Bedeutung
kommt hier der durch die Telekommunikation unterstützten
modernen Logistik zu, worunter die Ausrichtung aller operativer und dispositiver Tätigkeiten auf" . .. die bedarfsgerechte,
nach Art, Menge, Raum und Zeit abgestimmte Bereitstellung
von Realgütern .. . " für die Produktion, aber auch für die
Distribution zu verstehen ist (lhde, G .B. , 1984, S. 25). Zu den
Zielen der modernen Logistik gehört eine Reduzierung der
inner- und zwischenbetrieblichen Transportzeiten durch kürzere Lieferrhythmen zwischen Auslieferungs- und Empfangsort, um das Kostensenkungspotential in der Lagerhaltung
auszunutzen, das nach den weitgehend ausgeschöpften Rationalisierungsmöglichkeiten in der Produktion stärker in den
1989
Heft 1
Ernst-Jürgen Schröder: Auswirkungen wirtschaftsstruktureller Änderungen ...
Vordergrund getreten ist (Hamm, W., 1987, S. 422 f.). Den
logistischen Anstrengungen der verladenden Wirtschaft zufolge ergab sich wiederum eine Modal-split-Verschiebung zugunsten desjenigen Verkehrsträgers, der am ehesten den veränderten Qualitätsanforderungen entsprechen konnte. Wenngleich auch hier zunächst eine weitgehend straßenaffine Entwicklung eintrat, ist es doch fraglich, ob das Güterkraftverkehrsgewerbe mit seiner derzeitig mittelständischen Struktur
als Logistikpartner der Wirtschaft die über den reinen Transportvorgang hinausgehende Verpflichtung zur Übernahme
von Termingarantien und/oder zu Lagerhaltungsfunktionen
- typischer Aufgaben einer Spedition nach § 408 HGB ohne verstärkte Konzentration erfüllen kann.
3. Bisherige Anpassungsstrategien der Deutschen Bundesbahn
Um zusätzliche Anteile auf den wachsenden hochwertigen
Verkehrsmärkten zum Ausgleich der strukturell und konjunkturell bedingten Transportverluste im Massenguterkehr
zu gewinnen, entwickelte die Bundesbahn 1984 in voller Ausnutzung ihrer Systemvorteile, der schnellen, sicheren und
kostengünstigen Beförderung großer Gütermengen über
weite Entfernungen, neue Angebotsformen. Insbesondere
kam es darauf an, für Kaufmannsgüter eine dem Straßengüterfernverkehr in bezug auf die Schnelligkeit vergleichbare, aber
um den Vorteil der Zuverlässigkeit erweiterte und insgesamt
billigere T ransportalternative anzubieten.
Da einerseits schnelle Beförderungszeiten zwischen zwei
Orten sich nur über fahrplanmäßige Direktverbindungen
erzielen lassen, andererseits der hohen Fixkosten wegen die
Bündelung von Wagenladungen zu Ganzzügen notwendig ist,
kam das 1984 unter der Bezeichnung "lnterCargo" eingeführte System nur für Gebiete mit entsprechend großem
regelmäßigen Aufkommen in Betracht. Nach diesem Kriterium wurden 11 Gebiete abgegrenzt: Hamburg, Bremen,
Hannover, Östliche Ruhr, Westliche Ruhr, Rhein, RheinMain, Rhein-Neckar-Saar, Stuttgart, Franken und München.
Die entsprechenden lnterCargo-Zugbildungs- bzw. -auflösungsbahnhöfe, durch 90 direkt verkehrende Güterzüge im
Nachtsprung (in der Zeit zwischen 21.00 und 5.00 Uhr) bei
einer Mindestentfernung von 200 km untereinander verknüpft, sind Maschen, Bremen Hbf/Bremen Rbf, HannoverLinden, Wanne-Eickel/Hagen Gbf, Duisburg, Köln-Eifeltor,
Frankfurt(M) Hgbf/Frankfurt(M) Ost, Karlsruhe/Saarbrücken/
Heidelberg, Stuttgart-Untertürkheim, Nürnberg Hgbf/Nürnberg Rbf/Nürnberg-Dutzenteich/Fürth und München-Süd/
München-Laim (Pohl, H., 1984, S. 240 ff.). Die Wahl der
Knotenbahnhöfe im lnterCargo-System orientierte sich ausschließlich an den Aufkommensgebieten hochwertiger Kaufmannsgüter, Halbfertigwaren und Vorprodukten, die im Vorlauf von 16.00 Uhr an bei den Versendern gesammelt und im
Nachlauf bis 8.00 Uhr den Empfängern zugestellt werden.
Durch die später hinzugekommene Garantie des zugesagten
Liefertermins sowie durch rechnerunterstützte Transportinformationssysteme konnte lnterCargo zu einem wesentlichen
Bestandteil der unternehmerischen Logistikkonzeptionen
werden.
Andere Angebote für den stark wachsenden und sehr anspruchsvollen Teilladungsmarkt, nicht zuletzt im Hinblick
auf die weiter abnehmende Sendungsgröße, sind die "Partiefracht", die schnelle und ebenfalls mit Termingarantien versehene Beförderung mehrerer, in einer Wagenladung gebündelter Ladungspartien, und der speziell auf den Kleingutsektor
31
zugeschnittene Termin- und der IC-Kurierdienst. Auch auf
dem Gebiet des in mehreren Varianten durchgeführten Kombinierten Ladungsverkehrs zwischen Schiene und Straße, dessen Vorteil in der Verknüpfung der Systemvorteile dieser
Verkehrsträger in der Form des Knotenpunktverkehrs im
Nachtsprung auf der Schiene und des Verkehrs zu bzw. von
den Umschlagbahnhöfen auf der Straße liegt, sind erhebliche
Verbesserungen durchgeführt worden. Die Zunahme des
Verkehrs mit Containern um 4,6 %, mit Wechselbehältern um
6,5 %, mit Sattelaufliegern um 1,4 % und der "rollenden
Landstraße" um 24 % allein 1985/86 signalisiert der Bahn in
diesem wachsenden Segment des Güterverkehrsmarktes große
Chancen.
Da diese hochwertigen Angebote im Schienengüterverkehr
vorwiegend den Verdichtungsräumen und ihren Randzonen
gelten, ist der weitere Rückzug der Schiene aus der Fläche
vorprogrammiert.
Zusammenfassung und Ausblick
Der 1974 eingeleitete Prozeß der sektoralen und räumlichen
Anpassung der westdeutschen Wirtschaft an die veränderten
ökonomischen Rahmenbedingungen hat in seinen Auswirkungen dazu geführt, daß die Bahn, obwohl von der Verkehrsordnungspolitik begünstigt, ihre beherrschende Stellung im
Güterverkehr verlor. Entscheidend war hierbei der durch die
Stahlkrise ausgelöste strukturelle Rückgang des Transports
der Massengüter Kohle und Eisen innerhalb des gesamten
Verkehrsaufkommens, der die Bahn in ihrer traditionell engen
Verflechtung mit der Montanwirtschaft ungleich stärker traf
als die Binnenschiffahrt. Auch gelang es ihr nicht, in den
wachsenden Teilverkehrsmarkt der höherwertigen, eilbedürftigen Kaufmannsgüter einzudringen, die aufgrund ihrer
Eigenschaften und Transportanforderungen eine hohe Affinität zum Straßen- und zum Luftverkehr aufweisen. Dasselbe
gilt für den infolge der internationalen Arbeitsteilung vor
allem innerhalb der EG stark zugenommenen grenzüberschreitenden Verkehr, den herausragenden Wachstumsträger
auf dem Güterverkehrsmarkt, dessen Entwicklung die Bahn
weithin überging. Insgesamt, vor allem im Zusammenhang
mit den Logistikanstrengungen der verladenden Wirtschaft,
ist der Trend zu kleineren und mittleren, mit Termingarantien versehenen Sendungen unverkennbar, der die Bahn
infolge ihrer besonderen Ausrichtung auf den Massengütertransport tendenziell benachteiligt. Um jedoch auf diesem
anspruchsvollen Verkehrsmarkt für eilige und hochwertige
Güter zusätzliches Terrain zu gewinnen, entwickelte sie unter
Ausnutzung ihrer unverwechselbaren Vorzüge der Schnelligkeit, der Zuverlässigkeit und der Termingarantie neue Angebotsformen, die bislang vom Markt durchaus aufgenommen
wurden.
In Rechnung zu stellen sind auch die Folgen der für das Jahr
1993 geplanten Schaffung des EG-Binnenmarktes und der
damit verbundenen Liberalisierung des grenzüberschreitenden Verkehrs einschließlich der Zulassung von ausländischen
Unternehmen zu Transporten im Inland, worauf zur Sicherung der im EWG-Vertrag vorgesehenen Dienstleistungsfreiheit auch auf dem Gebiet des internationalen Verkehrs keinesfalls verzichtet werden kann (Bürgel, H., 1988, S. 240). Erwartet wird eine erhebliche Zunahme des grenzüberschreitenden
Verkehrs innerhalb der EG durch verstärkte Handelsbeziehungen und zugleich - dem Wegfall der Marktzugangsregelungen und der freien Preisbildung zufolge - eine Intensivie-
32
Raumforschung und Raumordnung
rung des Wettbewerbs zwischen den Verkehrsträgern mit
einer gravierenden Modal-split-Verschiebung zu Lasten der
Bahn und eine weitere Konzentration ihres Leistungsangebots
auf die Hauptkorridore zwischen den Agglomerationen.
Literaturverzeichnis
Bade, F. -]. : Funktionale Aspekte der regionalen Wirtschaftsstruktur.
In: Raumforschung und Raumordnung, 37. Jg. (1979) H. 6,
s. 253-268.
Bade, F.-J.: Funktionale Arbeitsteilung und regionale Beschäftigungsentwicklung. In: Informationen zur Raumentwicklung (1986)
H. 9/10, S. 695-713.
Bretzke, W .-R.: Strukturwandel im Güterverkehr als verkehrspolitischer Handlungsbedarf. In: Internationales Verkehrswesen, 40. Jg.
(1988) H. 1, S. 18-25.
Bürgel, H.: Die deutsche Marktordnung für den Straßengüterverkehr
und das europäische Recht. In: Internationales Verkehrswesen, 40.
Jg. (1988) H . 4, S. 240-243.
Der Bundesminister für Verkehr (Hrsg.): Verkehr in Zahlen 1987. Bonn 1987.
Fischer, R.: Eisenbahnverkehr 1986. In: Wirtschaft und Statistik, 7,
1987, s. 546-549.
1989
Heft 1
Griiber, H. u.a.: Zur Bedeutung der externen Kontrolle für die regionale Wirtschaftssentwicklung. In: Informationen zur Raumentwicklung (1986) H . 9/10, S. 679-694.
Hamm, W.: Strukturwandel im Güterverkehr als Grund für verkehrspolitischen Handlungsbedarf. In: Internationales Verkehrswesen (1987) H . 6, S. 422-426.
Ihde, G.B.: Transport, Verkehr, Logistik. - München 1984.
Piillmann, W .: Aktuelle Angebotsstrategien der Deutschen Bundesbahn. In: Informationen zur Raumentwicklung (1986) H. 4/5,
s. 265-272.
Pohl, H. : lnterCargo - Konzeption, Planung und Projektorganisation. In: Die Deutsche Bundesbahn, 60. Jg. (1984), S. 239-246.
Sehliebe, K.: Qualitätsstrukturen der regionalen Schienenerreichbarkeit. In: Informationen zur Raumentwicklung (1986) H. 4/5,
s. 249-263.
Schliephake, K. : Verkehrsgeographie. In: Sozial- und Wirtschaftsgeographie, Bd. 2. - München 1982, S. 39-156.
Schliephake, K.: Verkehrsgeographie. In: Geographische Rundschau,
Jg. 39 (1987) H . 4, S. 200-212.
Statistisches Bundesamt Wiesbaden (Hrsg.): Güterverkehr der Verkehrszweige 1986 (= Verkehr, Fachserie 8, Reihe 1). - Stuttgart,
Mainz 1987.
van Suntum, V.: Verkehrspolitik. - München 1986.
Voppel, G.: Wirtschaftsgeographie. - Stuttgart, Düsseldorf 1975.
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Constructions of “otherness” and the role of education: The case of Ethiopia
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Constructions of “otherness” and the
role of education: The case of Ethiopia Sisay Mengstie
mengstiesisay7@gmail.com 7 Journal of Education Culture and Society No. 2_2011 Abstract This article attempts to discuss the concept of constructing “otherness”, the techniques
to be used and the role of educational systems. Ethiopia as a multilingual and multicultural
country is the basis of discussion for this concept. Hence, the writer pays due attention
to answer the question: How far the Ethiopian educational policy is designed to reflect
diversified group interests fairly, if not equally? Therefore, major theoretical assumptions
on construction of “otherness” and some practical experiences of the Ethiopian educational
systems are thoroughly examined in this paper. Key words: construction, “otherness”, education, Ethiopia. Introduction “Otherness” is the way of defining one’s own “self” or one’s own “identity” in
relation to others. It is mainly a result of social, political, cultural and other kinds of
constructions through different approaches. Education as one of the major agents
can construct or deconstruct “otherness”. Here in Ethiopia, as in many countries
in the world, education (it may be traditional, religious, or modern) has played
a pivotal role in constructing “otherness”. Indeed, history has prevailed; education has also been serving to construct
“otherness” among diversified linguistic, cultural, ethnic, religious, gender, etc. groups for the benefit of the dominant groups. Therefore, in this article the writer
tries to explain the concepts of “otherness”, its relationship with stereotypes and
prejudices and finally analyzes the disadvantages and the possible roles of educa-
tion in Ethiopia in relation to construction of “otherness”. DOI: 10.15503/jecs20112-7-15 DOI: 10.15503/jecs20112-7-15 The concept of “otherness” The term “otherness” simply means a quality of being not alike; being distinct
or different from that which is otherwise experienced or known. Most of the time,
otherness is interpreted by referring to two or more different groups’ distinct
features or by referring to special qualities of each group that makes them diffe-
rent or unique in relation to another. This experience of being other can be expres-
sed in many ways. Usually age, ethnicity, sex, physical ability, race, sexual orien-
tation, social-economic class, and other demographic factors are the most common 8 Experience factors for an individual or a society to be leveled or identified as being of a certain
kind (Gallos, Ramsey 1997, p. 45). (
y
p
)
According to Selcen Dogan’s explanation, although the sources of otherness
are numerous and they are extremely different in their types, it is mainly rela-
ted with the “terms of identity and difference” (Dogan 2000, p. 16). He further
argued that: in the fields of feminism, cultural studies and sociology, “difference”
increasingly replaces the concept of “otherness”. This explanation leads us to the
essential meaning making process of human beings based on their differences. For instance, to talk about male identity, it is first essential to know about “fema-
leness”. Or to judge about the identity or, sometimes, about the qualities of certain
ethnic groups they must be compared with other groups. It is mainly this know-
ledge of difference that helps us to create meanings. Without the knowledge of
difference meaning could not exist.i g
“…there are two general points to note here, first, from many different direc-
tions, and within many different disciplines, this question of »difference« and
»otherness« has come to play an increasingly significant role. Secondly, »difference«
is ambivalent. It can be both, positive or negative. It is necessary for the production
of meaning, the formation of language and culture and for social identities - and at
the same time, threatens, a site of danger, of negative feelings, of splitting, hostility
and aggression towards the »Other«” (Hall 1997, p. 238 in Dogan 2000, p. 17). gg
(
p
g
p
)
Therefore, the central idea of otherness lies just on the divide, like normal and
abnormal, insiders and outsiders, and it is generally the issue of “Us” and “Them”. This division usually leads to Stereotyping, which is part of the maintenance of
social and symbolic order. The concept of “otherness” As illustrated by S. Dogan, stereotype “sets up a sym-
bolic frontier between the normal and the deviant, the normal and the patholo-
gical, the acceptable and the unacceptable, what belongs and what does not or is
Other, between insiders and outsiders, »Us« and »Them«” (Dogan 2000, p. 18). Possible Agents and Aspects of constructing “otherness” As explained, in many ways, otherness is the result of constructions of identi-
ties through continuous interactions of human beings. In this case it is more rela-
ted with constructions of individual and/or groups’ identities. Hence, identity can
be constructed or reconstructed through social interactions, cultural practices and
value exchanges, political setups or decisions, and educational processes. g
In addition, there are many agents for the process of constructing otherness. The
major agents include: social interactions, education, Media, literature, art (music,
drama, theater, and film), folklore, etc. In the process of constructing otherness edu-
cation plays the dominant role in many ways. Because education has the ability to
construct or deconstruct one’s own identity at individual and/or group levels. Stereotypes and “otherness” The concept of stereotype is highly related with that of “othering” and “other-
ness”. Like “otherness” stereotype is dominantly about “Us” and “Them”. So exa-
mining stereotype helps us to understand how “otherness” can be constructed 9 Journal of Education Culture and Society No. 2_2011 and it is very important to know the very notion of the term. Thus, Stereotype
refers to a fixed mental impression of human beings. Or as described by Gordon
Allport, stereotype can also be defined as an exaggerated belief associated with
a category. Its function is to justify or to rationalize our behavior in relation to that
category. This definition implies a discrepancy between an objectively ascertaina-
ble reality and a subjective perception of that reality (Van den Berghe 1996, p. 354
in Dogan 2000, p. 9). Characteristics of stereotypes So we can characterize Stereotype as over generalizations of a whole group
in to one and the same. It simplifies and ignores social, cultural, and other types
of diversities. All comments or group characteristics are presented as if they are
universal to the whole group and to each individual member of that group, often
as specific group or national characters that are inherent, natural, and therefore
unchangeable. These characteristics are very often couched in terms of an implicit
moralizing dichotomy, which draws boundaries between “Them” and “Us”. But
these boundaries are not merely passively descriptive; they incorporate a value
judgment of the group that is embedded in the power differential between the
various groups within that society. Stereotypes are thus highly emotionally char-
ged (Breger, Hill 1998, p. 11). “We are told about the world before we see it. We imagine most things before
we experience them. And those preconceptions... govern deeply the whole process
of perception. They mark out certain objects as familiar or strange, emphasizing
the difference, so that the slightly familiar is seen as very familiar, and somewhat
strange as sharply alien... they are aroused by small signs... aroused, they flood
fresh vision with older images and project into the world what has resurrected in
memory” (Dogan 2000, p. 8). Ethnic stereotypes As Roza N. Ismagilova explained it (ethnic stereotypes) prejudices and biases
significantly and affects inter-ethnic relations and the ongoing processes of natio-
nal intelligence (Ismagilova 1986).i g
g
Indeed, he once defined Ethnic prejudice as “a negative, unfavorable attitude
towards a group or its individual members; it is characterized by stereotyped beliefs;
the attitude results from processes within the bearer of the attitude rather than from
reality testing of the attributes of the group in question” (Ismagilova 1986, p. 107).i Certain stereotypes are prevalent in respect of specific ethnic groups, and what
is really important is that an ethnic stereotype is not a generalized image of the
real features inherent to a nationality or race, rather it is a derivative of the respec-
tive social situation and historical survivals. Here, if we take the case of “Gurage” (one of the various ethnic groups in
Ethiopia) ethnic group as an example, this group is believed to be, on one side,
a people who have the tendency for cooperating or helping each other, as smart
minded, successful and very fast in businesses areas. On the other side, they are
also considered as “people” who are mischievous and unfaithful in business acti-
vities, very passive in politics, etc. Consequences of stereotypes Stereotype naturally does not only mean a negative attitude, a belief, or a prior
knowledge of persons towards others. It can be either positively or negatively
perceived images of groups or individuals towards “others”. But most of the time
the stereotyped description of groups is closer to prejudice, a mostly negative atti-
tude. So, one of the dangers of stereotypes is that they are very close to prejudice. “Even that we do not know enough about a group Giddens says that prejudice
refers to opinions and attitudes held by members of one group towards another. A prejudiced person’s preconceived views are often based on hearsay rather than
on direct evidence, and are resistant to change even in the face of new informa-
tion” (Giddens, p. 212 in Dogan 2000, p. 9)i Hence, it is not difficult to recognize how stereotypes and/or prejudices are
practiced. It is not about their representation of reality. But it is believed that if
someone needs to clearly understand how ethnic, gender, racial or any other kinds
of representation actually works, the set of representational practices or stereoty-
ping should also be critically examined. According to Hall’s explanation, Stereoty- 10 Experience ping reduces people to a few, simple and essential characteristics, which are repre-
sented as fixed by Nature (Hall 1997, p. 257 in Dogan 2000). Although stereotypes
have different identities and characteristics based on their types, the dominant
types of stereotypes result from cultural or ethnic differences, linguistic, and racial
diversities. Likewise, the major causes for stereotypes here in Ethiopia stems from
ethnic diversity. Next we will see how ethnic diversity would be a cause for ste-
reotypes and the disadvantages of negative stereotypes. Disadvantages of ethnic stereotypes A specific ethnos is often characterized as “aggressive”, such preconceived
judgment affects human relationship and may lead to a conflict situation if an
emotional hostility and general negative attitudes are prevalent in respect of this
ethnic group.i Needless to mention, a specific ethnic stereotype is a product of historical deve-
lopment, economic conditions and social structure. However, many sociologists’
studies of racism and ethnic prejudices indicate that, the greatest intolerance is
characteristic of those social strata that fear economic competition and are uncer-
tain of their tomorrow. This instability causes fears and a continuous quest for
potential enemies and competitors. This is one major disadvantage of ethnic ste-
reotyping (Ismagilova 1986, p. 108). In fact, every human being belongs to a certain people. From infancy a person
develops love for his/her own language, culture, traditions or identity in general. He or she perceives and appreciates the culture and customs of other nationalities
against the background of possessed cultural values. “If a desire hereby creeps in
to contrast and oppose one’s own as alien it gives rise to a disdainful and psycho- 11 Journal of Education Culture and Society No. 2_2011 logically hostile attitude towards other peoples, to problems of inert-ethnic rela-
tions, discrimination, etc. resulting in ethnic prejudice” (Ismagilova 1986, p. 108). This phenomenon is related not only to theories which justify the policy of racial
discrimination, but also to the concepts of exclusiveness and original development
(Ismagilova 1986, p. 108). “When we look at the field of race and ethnic relations,
a »stereotype« is often defined as an overgeneralization about the behavior or other
characteristics of members of particular groups. Ethnic and racial stereotypes can be
positive or negative, although they are more frequently negative. Even ostensibly
positive stereotypes can often imply a negative evaluation. Thus, to say that blacks
are musical and have a good sense of rhythm comes close to the more openly nega-
tive stereotype that they are childish, and happy-go-lucky” (Dogan 2000, p. 9). The possible roles of education As explained in the above sections of this paper, education is one of the most
important agents in constructing “otherness”. For example, in and through edu-
cation “otherness” can be constructed or reconstructed in many ways. School
environments, social interactions of teachers and students in and out of school,
teaching learning processes, student-teacher relationships, the nature and con-
tents of subjects, text books (regarding contents, naming, exemplification, histo-
rical and cultural contents or their representations for certain groups), language
usage, etc. are important factors in the process of constructing otherness. g
p
p
g
Therefore, in multicultural societies like Ethiopia, education is expected to be
designed in such a way that all diversity groups have equal attention and treatment
in the construction process and the contents or approaches of educational systems
should be designed as the right representative of each group. As it has been said
by many scholars, in diversified societies, education should not only be structured
to sustain the power, history, culture, values, beliefs, worldviews, etc. of dominant
groups only. Thus, it is only through multicultural educational approaches that
peaceful coexistence can be achieved. That is why Patricia G. Ramsey and Leslie R. Williams puts this idea as one of the major goals of multicultural education. “Its [multicultural education] primary goal is to design systems of education
that are culturally relevant and are inclusive, rather than exclusive, to ensurethat
all students have a school possessing the skill for social, academic, political and
economic successes to teach students to relate respectively both differences and
commonalities; and to recognize, investigate and actively challenge injustice”
(Ramsey, Williams 2003, p. 260). By using education as a tool; Ethnic, racial, gender, and other difference can be
treated equally or at least fairly. Through education, some conflicts due to such dif-
ferences would be resolved, peaceful coexistence and respecting each other among
diversified groups would be achieved. Building healthy relationships among diver-
sified groups through education, on the other hand needs a well organized and well
planed educational curriculum and appropriate school systems in the context of
diversified groups. As Harriett D. Romo suggested, there are many ways of impro-
ving relationships of diversified groups other than focusing on academic issues only. 12 Experience “Consequently, for schools to focus on academia, they must make efforts to
prevent ethnic and racial clashes. The possible roles of education Recognizing common values (all students want
to feel that they belong) and differential power (some groups »belong« more than
others) is essentially for maintaining stability and positive relationships in mul-
tiethnic classrooms. Interventions to reduce prejudice and discrimination are also
essential” (Romo 2001, p. 17). (
p
)
Educational approaches that have equal or at least fair consideration for all
diversified groups are believed to be basic strategies especially to avoid discrimi-
nation, stereotypes or prejudices among group members in and outside the school
community. y
“Educational approaches expose students and teachers to accurate information
about other group similarities and differences. When individuals have accurate
information, they are less likely to accept stereotypes and adopt prejudices. As
students and educators gain knowledge about other groups and their histories,
they become more likely to respect members of those groups and cooperate with
them. Drawing attention to the process of discrimination, engaging actively in
team building, and consulting continuously with students all help develop a new
culture of tolerance and understanding” (Romo 2001, p. 17). g (
p
)
Yet, in the process of construction or reconstruction of “otherness”, education
does not only refer to the formal type of teaching learning process. Rather, it inc-
ludes different kinds of approaches and teaching learning processes such as: mass
education, cooperative education, and other informal educational systems. As
H.D.Romo stated it, sometimes instead of teaching facts about different groups
to students, “vicarious experience approaches can be inert group educational pro-
grams that use films, plays, biographies, novels, and other methods to present
members of all groups in a respectful way” (Romo 2001, p. 17) using these mate-
rials is very important for students or any target groups for the lessons to under-
stand and recognize the commonalities of all groups and reduce their tendency to
draw sharp boundaries between “Them” and “Us”. p
Educating people and presenting different groups by using the aforementio-
ned approaches may also have negative consequences that might aggravate or
be causes for ethnic conflicts, prejudices, and other kinds of group tensions. For
instance, if the presentations of such approaches are poor or misleading, or if the
images, histories, cultures, values, etc. of certain groups are wrongly presented
(whether it is intentional or not) that might lead to disastrous results. Educational policies and construction of “other-
ness” in Ethiopia The historical development of Ethiopia’s educational policy is mainly related to
the introduction of modern education in the country during the first decades of the
20th century. By then, when modern education was introduced, educational policy
making was almost a strange phenomenon for Ethiopia Tefera Seyoum properly puts
in the right way how modern education was introduced in our country as follows: g
y
y
“Interestingly enough, it [modern education] was an imported item like the car
or the radio, with hardly any input from the society. In fact it was a foreign bag-
gage, with foreign teachers, with foreign language as its medium of instruction,
with imported curriculums and text books…” (Seyoum 2006).i p
(
y
)
In relation to educational policy making, fascist Italy, during its five years stay,
was the first in Ethiopia to make an attempt. Let alone the other side of the story,
the fascist regime introduced educational policy in Ethiopia for the first time in
its history. This policy was proposed in two different ways as: “for Italian type
schools and schools for colonial subjects”. By the same token, this attempt can also
be considered as a pioneer in constructing “otherness” through modern education
in Ethiopia. p
Although, its aim was to make and create a favorable condition for their divide
and rule strategy, this educational policy was characterized by multilingual lan-
guage policy. It allows some majority languages of the country to be used as
a medium of instruction in education. This can also be taken as a historical coinci-
dence to consider cultural diversity or cultural pluralism in the country’s educa-
tional policy for the first time (Getachew, Derib 2006, p. 45). p
yi
(
p
)
Moreover, in the history of the Ethiopian educational policy making process,
two major attempts were made almost after 47 years of the first educational policy
made by the Italian fascists. During those periods the Ethiopian modern educa-
tional system was unable to achieve any significant development in any aspect. Rather, scholars criticized the situation as - limited in its access, unable to treat
all societies as equal city urban, male, and class biased of the country, as elitist
that was accessible for the few, and they were wasteful in general (Seyoum 2006). Both education policies of Emperor Hailesilase and that of the Derg. The possible roles of education (
)
g
Because of the above confusing contributions of education, multicultural and
multilingual states have been very much concerned in their educational polices
to serve all linguistic, cultural, gender, racial, and other groups equally as far as
possible. Consequently, such countries have developed their own educational
approaches and designed teaching strategies that are suitable for their societies. pp
g
g
g
Approaches such as assimilation, cultural relativism, multiculturalism, ethno-
centrism, particularism etc. are also the results of these controversies and yet they
have been applied and tested in educational sectors of different countries. Ethio-
pia as a multicultural nation has also been applying different approaches in her
educational programs since the introduction of modern education in the country. 13 Journal of Education Culture and Society No. 2_2011 Journal of Education Culture and Society No. 2_2011 Educational policies and construction of “other-
ness” in Ethiopia The educa-
tion sector review (ERS) and Evaluative research of the general education system
in Ethiopia (ERGESE) respectively were criticized as policies of assimilation that
reflected the cultures, values, beliefs, religion of the northern highlands of Ethio-
pia specifically that of Amhara and Tigray regions. This was primarily aimed at
achieving the building of an educational system that would contribute to a strong
national identity and to survive a strong unified nation that seems the major goal
of the two regimes (Getachew, Derib 2006, p. 45-47). g
(
p
)
Be that as it may, the current government of Ethiopia tried to design a multilin-
gual language policy in the education sector. EPRDF at first tried to introduce its
“ethnic federalism strategy” by providing equal recognition for different ethnic, 14 Experience linguistic or cultural groups in the country. For instance Article 39, 2 in the con-
stitution provides several rights for Nations, Nationalities and peoples as follows: p
g
p
p
“Every Nation, Nationality and People in Ethiopia has the right to speak, to
write and to develop its own language; to express, to develop and to promote its
culture; and to preserve its history” (The Constitution of the Federal Democratic Repu-
blic of Ethiopia 1995, p. 96). f
p
p
)
This constitution also gave a special emphasis for cultural and linguistic diver-
sity in relation to language choice and language use for different activities. In
article 5 sub articles 1 and 3 are the best examples of this: p
“1. All Ethiopian languages shall enjoy equal state of recognition p
“1. All Ethiopian languages shall enjoy equal state of recognition 3. Members of the Federation may determine their respective language 3. Members of the Federation may determine their respective languages”. As a result of such rights in the constitution, regional states have chosen their
respective official languages for various purposes. Several languages have the
opportunity to be used for official, education, medium of communication, media
and other activities in different parts of the country. Following the above rights
under the constitution, the 1994. Ethiopian education and training policy also introduced a new trend in relation
to language use in education. Educational policies and construction of “other-
ness” in Ethiopia Section 3.5.1 of the Education and Training Policy (1994)
reads: “Cognizant of the pedagogical advantage to the child in learning in mother
tongue and the rights of nationalities to promote the use of their languages, primary
education will be given in nationality languages” (Education and Training Policy 1994). g
y
g
g
(
g
y
)
Because of these covert policies and strategies given by the government, diffe-
rent linguistic groups and regional sates started enjoying their languages for edu-
cational purposes at different levels. In principle it is important for these groups to
design their local or regional education policies to serve as a means of expressing
their own cultural, ethnic, social, and historical identities. But the new multilin-
gual education policies of regional states also started to face new forms of challen-
ges by the time of implementation. g
y
For example, language as one of the dominant factors in designing education poli-
cies at any level and as a major contributor in the processes of constructing “otherness”
is a good example to show the challenges of these new multicultural and multilingual
policies in Ethiopia. The statuses of many languages to be used for educational activi-
ties were low. The presence of different dialects and language groups in specific areas
was also becoming a cause for conflicts and a problem for choice of one language for
educational purposes. The lack of skilled manpower, scarcity of teaching materials,
and in the ability to produce new books and written documents in most of the mino-
rity languages were also some of he challenges to implementation of the policies. Bibliography (1986), Education and society in contemporary Ethiopia, “Proceedings of the ninth interna-
tional congress of Ethiopian studies Moscow”, Vol. 3. g
p
The Constitution of the Federal Democratic Republic of Ethiopia, 1995 1st year No. 1., Addis Ababa. Yeo F. (2001), The Barriers of Diversity: Multicultural education and rural schools, [in:] Schultz F. (ed.),
Multicultural Education, McGraw-Hill/Dushkin, Connecticut. Yeo F. (2001), The Barriers of Diversity: Multicultural education and ru
Multicultural Education, McGraw-Hill/Dushkin, Connecticut. Concluding Remarks It is a widely accepted fact that, education plays a vital role in the overall deve-
lopment of human activities. It has been serving as a source of power in intellec-
tual, social, material, psychological, economic and other areas of development that
human beings have been trying to succeed in for a long period of time. However,
in this article it is neither possible to mention or beyond its scope to discuss and 15 Journal of Education Culture and Society No. 2_2011 illustrate the possible roles of education in Ethiopia in every aspect. Rather, the
writer tried to focus on particular issues in the meaning and process of construc-
tion of “otherness”, and the possible roles of education in Ethiopia. illustrate the possible roles of education in Ethiopia in every aspect. Rather, the
writer tried to focus on particular issues in the meaning and process of construc-
tion of “otherness”, and the possible roles of education in Ethiopia. p
p
Education as one of the major agents for constructing and/or deconstructing
“otherness” in diversified societies and has a significant position in the social,
historical, cultural, developments, and the past experiences of Ethiopia just as in
many other countries, are results of modern and/or traditional educational sys-
tems. The remarkable achievements of the past (in different fields), social interac-
tions, traditional ways of life and world views, traditional laws or judicial/legal
systems, folklore, folk medicines etc. are highly related to education.i y
g
y
These things have also played significant roles for the well being of the people
and to living peacefully within their groups and with other groups and it helps
these groups to understand the identity of members of others and to define them-
selves. This is one important essence of constructing “otherness” in and through
education. Nowadays, in the age of globalization, education continues as one of
the leading factors in the process of constructing and deconstructing “otherness”. Bibliography Akintunde O. (2001), White racism, white supremacy, white privilege, & the social construction of race:
Moving from modernist to postmodernist multiculturalism, [in:] Schultz F. (ed.), Multicultural Education,
McGraw-Hill/Dushkin, Connecticut. Akintunde O. (2001), White racism, white supremacy, white privilege, & the social construction of race:
Moving from modernist to postmodernist multiculturalism, [in:] Schultz F. (ed.), Multicultural Education,
McGraw-Hill/Dushkin, Connecticut. /
Ayalew E. (ed.) (2006), Proceedings of the conference on Teacher Education for sustainable Development in
Ethiopia, Addis Ababa University, Addis Ababa. p
y
Areaya S. (2006), The process of policy formulation and curriculum Soul, speaking from the Heart, Jossey-Bass
Publishers, San Francisco. Education and Training policy (1994), TGE (Transitional Government of Ethiopia), Addis Ababa. Gallos J.V., Ramsey V.J. (1997), Teaching Diversity: Listing to the soul, speaking from the heart, Jossey-Bass
Publishers, San Francisco. Getachew A., Derib A. (2006), Language policy in Ethiopia, Jimma University Publishers, Jimma. Ismagilova R.N. (1986), Ethnic Stereotypes and Problems of National Integration in Contemporary Tropica
Africa, “Proceedings of the ninth international congress of Ethiopian studies Moscow”, Vol. 3. Ismagilova R.N. (1986), Ethnic Stereotypes and Problems of National Integration in Contemporary
Africa, “Proceedings of the ninth international congress of Ethiopian studies Moscow”, Vol. 3 Negash T. (2006), Education in Ethiopia: From crisis to the brink of collapse, Nordiska Afrikainstitute,
Uppsala. pp
Ramsey P.G., Williams L.R. (2003), Multicultural Education, Routledgefaimer, New York, London. R
H D (2001) I
i
h i
d
i l
l i
i
h
h
l
[i
] S h l
F ( d ) M l i
l
l pp
Ramsey P.G., Williams L.R. (2003), Multicultural Education, Routledgefaimer, New York, London. Romo H.D. (2001), Improving ethnic and racial relations in the schools, [in:] Schultz F. (ed.), Multicultural Ramsey P.G., Williams L.R. (2003), Multicultural Education, Routledgefaimer, New York, London. Romo H.D. (2001), Improving ethnic and racial relations in the schools, [in:] Schultz F. (ed.), Multicultural
Education, McGraw-Hill/Dushkin, Connecticut. Romo H.D. (2001), Improving ethnic and racial relations in the schools, [in:] Schultz F. (ed.), Multicultural
Education, McGraw-Hill/Dushkin, Connecticut. Schultz F. (ed.) (2001), Multicultural Education, McGraw-Hill/Dushkin, Connecticut. Sleeter Ch.E. (1996), Multicultural Education as Social Activism, State University of New York Press,
Albany. y
Seyoum T. (2006), Educational Policy and Development: The Ethiopian Case, [in:] Ayalew E. (ed.), Proce-
edings of the conference on Teacher Education for sustainable Development in Ethiopia, Addis Ababa
University, Addis Ababa. y
Teshome G.W. Netography Dogan S. (2000), Turkey, as “other” and being “othered”, http://www.selcendogan.com, retrieved 21.03.2
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Molecular Dynamics Numerical and Experimental Analysis of Fretting Wear Mechanism of Variable Gauge Bogie
|
Research Square (Research Square)
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cc-by
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Molecular Dynamics Numerical and Experimental
Analysis of Fretting Wear Mechanism of Variable
Gauge Bogie China University of Petroleum Huadong - Qingdao Campus
https://orcid.org/0000-0002-1349-6864
He Sheng Research Article Keywords: Molecular Dynamics, Fretting Wear, Variable Gauge Bogie, MoS2 Coating, Ion Nitriding
Posted Date: March 17th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1348199/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/17 Abstract Under the "One belt, One road" strategy, the trade in goods is more frequent in diverse countries. The
difference in train gauge between countries often leads to low efficiency of freight transportation. In
several schemes, the variable gauge bogie can efficiently solve this problem. The fretting wear between
the axles and wheels of train gauge changeable bogies may cause the connection relaxation and failure
in the actual working process, resulting in potential safety hazards. Diverse experimental researches
show that surface treatment technology can effectively reduce the friction and wear between axles and
wheels. However, its micro wear reduction mechanism is not clear, to date. Therefore, the three-
dimensional fretting wear model of bogie axle and wheel is established by molecular dynamics method
in this paper. The wear reduction mechanism of surface treatment technology is then clarified from the
micro point of view, and also verified by experiments. The results show that there is micro convex contact
between the two surfaces under the contact stress. The micro convex is extruded and deformed, hence
forming an adhesion. Whereas, the convex adhesion is desorbed by the shear force during separation,
resulting in fretting wear. Since the wear reduction is caused by the reduction of transverse force, good
lubrication and wear reduction effect can be achieved by MoS2 coating and nitriding. The surface
treatment of MoS2 coating also reduces the friction coefficient between axles and wheels along with
improving load bearing. Whereas, the friction coefficient can be decreased by increasing the nitriding
amount. 1 Introduction With the vigorous development of the global trade integration, transnational cargo transportation is
becoming ever more frequent. The railway’s development has turned into a crucial goal of various
countries due to its low cost and huge transportation capacity [1, 2]. However, due to the differences in
train gauges in various countries, it takes a lot of time to replace any identical device during the
transportation, which leads to a great inconvenience of transnational cargo transportation under the
strategy of "One Belt and One Road" [3]. Variable gauge bogie wheelsets can realize migration on
different gauge tracks, and have the advantages of low cost and high efficiency [4, 5]. Meanwhile, the
axle and wheel mating mode make it easy to produce axial slip, impact and vibration between the axles
and wheels, resulting in wear of key components [6]. Since the available research on wear mechanism is
not very comprehensive [7–13], the present study of fretting wear is focused on macroscopic experiment. The proposal of the "Hard Ball" model and the successful development of multi-body Embedded-Atom
Method (EAM) [14–16] ake molecular dynamics become a significant method to study the properties of
metal materials at the nanoscale level [17]. Various researchers analyzed the fretting on the surface of
metal Zr and alloy AZ91, while others have studied the tribological properties of coated materials such as
MoS2 based on molecular dynamics method [18–23]. Their results show that the surface treatment can
reduce wear by changing the fretting area. However, in the present microscopic fretting wear mechanism
analysis, the research focus is mainly on the single-atom metal or simple metal compounds. Furthermore,
the molecular dynamics analysis of complex alloy structure is rarely seen [24]. Page 2/17 Page 2/17 To solve the above-mentioned problems, a variable gauge bogie model with complex alloy structure has
been established by molecular dynamics method in this paper. The microscopic wear mechanism and
wear reduction mechanism of surface treatment of bogie have also been revealed by combining the
simulation and experiment. The results are highly significant for the wear resistance design of variable
gauge bogie and the microcosmic modeling of complex alloy. 2 Configuration Of The Exoskeleton Arm (1) [14]: E =
1
2 ∑
N
i=1∑j =1≠iφij(rij) + ∑
N
i=1Fi(ρi) 1 Where E represents the sum of potential energy between atoms, φij represents the pair potential of
interatomic interaction, rij represents the distance between atoms, Fi is the energy required when atom i is
embedded into the position of electron density unit 1, and ρi shows the local electron density generated
by all atoms in the system at the position of atom i. Lennard-Jones (LJ) potential consists of a simple structure with high calculation efficiency. It can
simultaneously calculate the interaction force between the two interfaces with high calculation
efficiency[28]. Hence, LJ potential has been used in this paper to represent the interaction force between
wheel and axle. The potential function form of LJ method is shown in Eq. (2) [28]: ULJ(r) = 4ε[(
σ
r )12 −(
σ
r )6] ULJ(r) = 4ε[(
σ
r )12 −(
σ
r )6] 2 where, ULJ represents the sum of potential energy between atoms, ε is the potential well depth that
reflects the strength of the interaction between two atoms, σ is the fixed distance between atoms when
the interaction potential is equal to zero, and r is the distance between two atoms at any instance. where, ULJ represents the sum of potential energy between atoms, ε is the potential well depth that
reflects the strength of the interaction between two atoms, σ is the fixed distance between atoms when
the interaction potential is equal to zero, and r is the distance between two atoms at any instance. According to the addition and subtraction properties of Eq. (2) regarding r, an increase in r to a certain
value leads the potential energy of LJ to zero. The distance at this point is known as truncation radius rc. This phenomenon occurs when the distance between the analyzed atom and the observed atom is
greater than rc. Due to the relatively greater spacing between atoms as compared to the truncation radius,
the interaction force becomes insignificant [29]. Hence, the force between the two atoms is not
calculated. The truncation radius rc is calculated to be 11 Å, according to the atomic parameters of wheel
and axle material [29]. 2 Configuration Of The Exoskeleton Arm Previous researches [25, 26] demonstrate that DZ2 steel of axle material and D2 steel of wheel material
belong to face-centered cubic lattice (FCC), and their lattice constants are a = b = c = 3.43 mm and α = β =
γ = 90°. Therefore, Fe matrix is extended based on the face-centered lattice under this parameter. Fe
atoms have been randomly replaced in the matrix according to the material composition in Table 1 to
complete the doping in DZ2 and D2 steel alloys. The final molecular dynamics model is shown in Fig. 1. Table 1
Material compositions
Material
DZ2
D2
C
Si
Mn
P
S
Cr
Mo
Ni
0.24 ~ 0.32
0.20 ~ 0.40
0.60 ~ 0.90
≤ 0.01
≤ 0.01
0.90 ~ 1.20
0.20 ~ 0.30
0.50 ~ 1.50
0.48 ~ 0.58
0.65 ~ 0.80
≤ 0.015
≤ 0.015
≤ 0.030
≤ 0.08
≤ 0.030
≤ 0.016 The whole model is divided into three parts: fixed layer, thermostat and free layer, as demonstrated in Fig. 1. In the present model, the boundary layer fixed model does not move in the y direction. Therefore, the y
direction is an aperiodic boundary, and the x and z directions are periodic boundaries. The thermostat is
used to adjust the ambient temperature of the system, whereas the initial temperature is set at 300 K. The
free layer moves in accordance with Newton's law of motion, and the data needed for the micro-friction of
wheel and axle are then obtained. A rough peak with height of 20.58 Å was established in the free layer to
simulate the fretting wear process. The micro-canonical ensemble, also known as NVE ensemble, is
selected for the simulation of the working condition between the wheel and axle. This way, the whole
system has no energy and particle exchange with the outside world. EAM is suitable for intermetallic molecular dynamics simulation [27]. In this paper, EAM is used for
relaxation processing to accurately characterize the interactions between atoms in the alloy under stable Page 3/17 Page 3/17 Page 3/17 state. The EAM potential function is shown in Eq. (1) [14]: state. The EAM potential function is shown in Eq. 3 Fretting Wear Numerical Results And Analysis Due to the influence
of atomic bond formation and contact point growth, the peak friction coefficient is found to be greater
than one [26]. After that, the shear force results in the breaking of the atomic bond, and atoms are
desorbed from the matrix. Meanwhile, the friction coefficient keeps on decreasing, simultaneously. The
subsequent cyclic friction did not form a large extrusion deformation as with the first contact. Hence, the
friction coefficient oscillated at a smaller value with an average single-peak friction coefficient of 0.225. 3 Fretting Wear Numerical Results And Analysis The research has been conducted using Large-scale Atomic/Molecular Massively Parallel Simulator
(LAMMPS) for calculating and using Visual Merchandising (VMD) for post-processing. The timestep and
the output interval τ is set as 0.0025 fs and 1.25 fs, respectively. The wheel DZ2 steel is fixed, and the
axle DZ2 steel moves horizontally at a constant speed of 2.5 m/s. Figure 2 demonstrates the fretting
wear phenomenon between wheels and axles. The two rough peaks completely contact and overlap at 8τ
(Fig. 2(a)). Whereas, the two rough peaks are separated at 15τ (Fig. 2(b)). The two rough peaks contact
for the second time at 45τ (Fig. 2(c)). The residual heights of the two rough peaks are about 17.15 Å, and
The system begins to stabilize at 200τ. Page 4/17 Page 4/17 Figure 3 depicts a detailed representation of the deformation of the rough peak during the fretting wear. The contact of the two rough peaks resulted in obvious extrusion deformation, as shown in Fig. 3(a). This
lead to an interatomic adhesion (Fig. 3(b)), causing some atoms to be desorbed under the shear force
(Fig. 3(c), top view). Therefore, the general process of fretting wear can be deduced as following steps: Figure 3 depicts a detailed representation of the deformation of the rough peak during the fretting wear. The contact of the two rough peaks resulted in obvious extrusion deformation, as shown in Fig. 3(a). This
lead to an interatomic adhesion (Fig. 3(b)), causing some atoms to be desorbed under the shear force
(Fig. 3(c), top view). Therefore, the general process of fretting wear can be deduced as following steps: 1. The contact stress makes the micro-bumps contact, resulting into extrusion deformation and adhesion. 1. The contact stress makes the micro-bumps contact, resulting into extrusion deformation and adhesion. 2. During the separation, the protruding adhesive is desorbed by the shear force that leads to the fretting
wear. 2. During the separation, the protruding adhesive is desorbed by the shear force that leads to the fretting
wear. 2. During the separation, the protruding adhesive is desorbed by the shear force that leads to the fretting
wear. Figure 4 shows the varying regulation of friction coefficient with time in fretting friction process. It can be
visualized that the friction coefficient surges during the contact extrusion deformation and reaches a
peak value at the maximum contact, which is the maximum extrusion deformation. 4 Fretting Wear Reduction Mechanism Of Surface Treatment
Technology And Experimental Analysis The damage and failure of materials mostly begin with the surface damage, for which there is a
modeling lubrication surface treatment technology for materials [30]. In the surface treatment process,
nitriding and coating MoS2 can achieve good anti fretting wear effect. Therefore, this chapter explores its
wear reduction mechanism [31]. 4.1 Fretting Wear reduction mechanism and experimental
analysis of MoS2 model Various researches have demonstrated that the solid lubrication coating of MoS2 bonded on LZ50 steel
has good resistance to radial fretting wear [6–9]. Therefore, the surface MoS2 coating was added on the
basis of the model to analyze the influence of surface treatment technology on fretting wear, as
presented in Fig. 5. The revised model is depicted in Fig. 5 (a), and the simulation results obtained are shown in Fig. 5 (c). It
can be seen that the surface treated by MoS2 produces fretting wear behaviors, such as extrusion
deformation, adhesion and desorption fretting. The varying regulation of friction coefficient with time
(Fig. 5 (b)) shows that the average friction coefficient is 0.218, which is lower than the corresponding
friction coefficient of the untreated model. The results exhibit that the coating MoS2 can significantly
reduce the wear, and protect the metal from fretting friction. Page 5/17 Page 5/17 To further optimize the ideal conditions of MoS2 coating, simulation analysis method was applied to
observe the number of coating layers, temperature and load, and the obtained varying regulation of
friction coefficient with time, as shown in Fig. 6. It has been observed that the friction coefficient has no
obvious change trend with the coating thickness and temperature changing (Fig. 6(a) and 6(b)). Whereas,
the friction coefficient values have been found to decrease with the increase of load (Fig. 6(c)). This
shows that the friction coefficient of MoS2 is inversely correlated with the load. Several researchers have
conducted in-depth studies on MoS2 lubrication characteristics, and found that the friction coefficient
decreases with the increase of load [32–34]. The simulation results are in line with those published
above. Therefore, it is concluded that MoS2 coating can play a crucial wear reduction lubrication effect at
both high and low temperatures. However, the wear reduction effect is mainly influenced by the load, and
the lubrication effect and the pressure are in direct relation with each other. The surface of DZ2 steel was coated with MoS2 with bonding solid lubricant layer. The workpiece
obtained by this method has been found to have good fretting wear resistance [35]. WTM-2E friction and
wear tester machine is specially used to test the friction coefficient and wear resistance of various
coatings or solid lubricating materials, with an advantage of high testing accuracy and simple
operational process. 4.1 Fretting Wear reduction mechanism and experimental
analysis of MoS2 model Hence, the friction coefficient of the workpiece coated with MoS2 was measured by
WTM-2E under diverse loading. In the experiments, the speed was set as 400 rpm, a loading radius of 3
mm and a cylindrical fixture with a probe diameter of 2.4 mm was used for testing. Three groups of
friction and wear experiments were carried out under different pressures of 0.043 MPa (20 grams), 0.108
MPa (50 grams) and 0.152 MPa (70 grams). The average and maximum friction coefficients obtained are
presented in Table 2. The average friction coefficient of each group is approximately 0.1, which is lower
than that of the untreated workpiece (0.225). The decline rate was 55.6%, as shown in Fig. 4. This
indicates that the surface coating MoS2 significantly reduces the wear. Furthermore, the average friction
coefficient gradually decreases with the increase of load, which is also in line with the numerical findings. Table 2
Material compositions
Load
0.043 MPa
0.108 MPa
0.152 MPa
favg
fmax
0.111
0.172
0.103
0.184
0.990
0.264
4.2 Fretting Wear reduction mechanism and experimental
analysis of nitriding model 4.2 Fretting Wear reduction mechanism and experimental
analysis of nitriding model 4.2 Fretting Wear reduction mechanism and experimental
analysis of nitriding model The surface chemical composition and organization considerably alter after the nitriding treatment of the
workpiece. As shown in Fig. 7 (a), Nitrogen atoms were swapped, randomly, and potential functions were
reasonably chosen to characterize the nitriding model after relaxation [36]. The varying regulation of Page 6/17 friction coefficient with time after simulation has been shown in Fig. 7 (b). Statistically, the maximum
friction coefficient of the nitriding model is 6.83, and the average friction coefficient is 0.194. Compared
with the prior untreated model group, the friction coefficient of the two groups is decreased, indicating
that the nitriding treatment can greatly reduce fretting wear. This can be attributed to the formation of
high hardness value of nitride Fe4N between nitrogen and metal after nitriding. Hence, the nitriding
treatment also enhances the hardness and wear resistance of the steel surface. Varying the nature of nitriding process will lead to the modification of compound and nitrogen content in
bogie, thus affecting the lubrication effect [37]. Figure 8 compares the influence of different nitriding
depth, nitriding amount and load on the nitriding model's wear reduction effect. 4.1 Fretting Wear reduction mechanism and experimental
analysis of MoS2 model It can be seen that the
friction coefficient does not significantly alter with the depth and load of nitriding treatment. However, the
friction coefficient decreases with the increase of the nitriding amount. The results depict that a fine wear
reduction effect can be attained by increasing the nitriding amount. However, the increase of nitriding
amount in the alloy will also lead to low plasticity and toughness of the wheel [38]. Therefore, relevant
factors need to be comprehensively addressed to choose the optimized amount of nitriding for the
industrial application of the process. The ion nitriding workpiece can be generated by the application of three-stage nitriding method with the
best anti-fretting wear effect [39]. Therefore, to verify the accuracy of the simulation analysis, the current
study adopts the three-stage nitriding method to treat the workpiece surface. WTM-2E was used to carry
out the experimental study under the pressure values of 0.043 MPa, 0.108 MPa and 0.152 MPa,
respectively. The remaining experimental conditions are consistent with the ones described in the section
4.1. All other parameters were consistent with the experimental setup of coated MoS2. The average and
maximum friction coefficients obtained under different loads are presented in Table 3. Experimental data
show that the value of friction coefficient of nitriding workpiece is less than that of the untreated
workpiece under various load conditions. This indicates that the nitriding treatment method is far
effective in reducing the wear. Furthermore, it can be observed that the average friction coefficient of
nitriding workpiece is not influenced by the variation of load, when the analysis of the average friction
coefficient has been performed under each load. The ion nitriding workpiece can be generated by the application of three-stage nitriding method with the
best anti-fretting wear effect [39]. Therefore, to verify the accuracy of the simulation analysis, the current
study adopts the three-stage nitriding method to treat the workpiece surface. WTM-2E was used to carry
out the experimental study under the pressure values of 0.043 MPa, 0.108 MPa and 0.152 MPa, respectively. The remaining experimental conditions are consistent with the ones described in the section
4.1. All other parameters were consistent with the experimental setup of coated MoS2. The average and
maximum friction coefficients obtained under different loads are presented in Table 3. 4.1 Fretting Wear reduction mechanism and experimental
analysis of MoS2 model Experimental data
show that the value of friction coefficient of nitriding workpiece is less than that of the untreated
workpiece under various load conditions. This indicates that the nitriding treatment method is far
effective in reducing the wear. Furthermore, it can be observed that the average friction coefficient of
nitriding workpiece is not influenced by the variation of load, when the analysis of the average friction
coefficient has been performed under each load. Table 3
The friction coefficient of nitriding workpiece under
different loads
Load
0.043 MPa
0.108 MPa
0.152 MPa
favg
fmax
0.206
0.274
0.215
0.275
0.208
0.291
4.3 Fretting wear reduction mechanism analysis 4.3 Fretting wear reduction mechanism analysis 4.3 Fretting wear reduction mechanism analysis The wear reduction mechanism of the surface treatment process was studied by the untreated models,
i.e. nitriding model (20%) and MoS2 model (100 Kcal/mole.Å). The varying regulation of positive pressure Page 7/17 Page 7/17 (Y direction) and friction (X direction) of the three output models in fretting wear are shown in Fig. 9 It can
be visualised that the positive pressure of all models has a similar trend over time (Fig. 9 (a)). The
oscillation becomes obvious when the contact and the detachment occur, and the oscillation starts to
weaken after the contact. The friction coefficient of MoS2 model is found to be the lowest followed by the
nitriding model, and the untreated model is the largest, as shown in Fig. 9 (b). The discussed results are
consistent with the simulation results of friction coefficient. Therefore, by comprehensive analysis of the
discussed results, it can be concluded that the decrease of friction coefficient after surface treatment is
not due to the change of positive pressure, but mainly due to the decrease of transverse friction force
(bond-breaking shear force) during the desorption fracture. 5 Conclusion Aiming at the fretting wear behavior, the fretting wear model between bogie wheel and axle was
established from the microscopic point of view by using molecular dynamics method. The fretting wear
phenomenon and the changing regulation of friction coefficient with time were analyzed. The results
demonstrate that the general process of fretting wear can be presented as follows: 1. The micro-bumps contact under stress, then the micro-bumps extrude and form adhesion. 2. The adhesion is desorbed by the shear force during the separation to form fretting wear. On the basis of fretting wear model, two molecular dynamics models of coated MoS2 and ion nitriding
surface treatment were established, respectively. Simulation and experimental results conclude that the
surface treatment technology can play a vital role in effectively reducing the wear effect. It is found that
the friction coefficient of coated MoS2 model significantly decreases by increasing the load. Whereas, a
considerable decrease of the friction coeffocoent of nitriding model has been observed with the increase
of nitriding amount. By analyzing the varying regulation of transverse and longitudinal force with time, it
is found that the reduction of transverse force is the principal reason for reducing the wear of surface
treatment applications. Acknowledgements The authors sincerely thanks to Professor Cai Zhenbing of Southwest Jiaotong University for his critical
discussion and reading during manuscript preparation. Competing interests The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper. Availability of data and materials The datasets supporting the conclusions of this article are included within the article. Authors’ contributions The author’ contributions are as follows: Ji Jiaxin was in charge of the whole trial; He Sheng wrote the
manuscript; Ren Yanping assisted with sampling and laboratory analyses. Funding This research was funded by the National Natural Science Foundation of China (NSFC) (51805547), the
Shandong Provincial Natural Science Foundation (ZR2017LEE016), and Fundamental Research Funds of
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Metal Hotworking Technology. doi: 10.3969/j.issn.1001-3814.2007.06.022 Page 11/17 Page 11/17 Figures Figure 1 Molecular dynamics simulation model between wheels and axles Molecular dynamics simulation model between wheels and axles Page 12/17 Page 12/17 Page 12/17 Page 12/17 Figure 2 Fretting friction-wear process for single rough peak. (a) For 8τ Wear during operation; (b) For 15τ Wear
during operation; (c) 45τ Wear during operation. Fretting friction-wear process for single rough peak. (a) For 8τ Wear during operation; (b) For 15τ Wear
during operation; (c) 45τ Wear during operation. Figure 3
Characteristics of fretting friction behavior: (a) extrusion, (b) adhesion, (c) desorption. Characteristics of fretting friction behavior: (a) extrusion, (b) adhesion, (c) desorption. Characteristics of fretting friction behavior: (a) extrusion, (b) adhesion, (c) desorption. Page 13/17 Page 13/17 Figure 7 Figure 7 (a) Molecular dynamics simulation model of nitriding model, (b) Changing regulation of friction
coefficient of nitriding model and untreated model. Figure 8
Changing regulation of friction coefficient with (a) nitriding depth variation, (b) load fluctuation and (c)
nitriding quantity variation. Figure 4 Figure 4
(a) Single. (b) Double rough peak’s changing regulation of friction coefficient. (a) Single. (b) Double rough peak’s changing regulation of friction coefficient. Page 14/17
Figure 5 Figure 5 Page 14/17 ) Molecular dynamics simulation model of MoS2 surface coating, (b) Characteristics of fretting friction
ehavior of MoS2 model, (c) Changing regulation of friction coefficient of coated MoS2 model. (a) Molecular dynamics simulation model of MoS2 surface coating, (b) Characteristics of fretting friction
behavior of MoS2 model, (c) Changing regulation of friction coefficient of coated MoS2 model. (a) Molecular dynamics simulation model of MoS2 surface coating, (b) Characteristics of fretting friction
behavior of MoS2 model, (c) Changing regulation of friction coefficient of coated MoS2 model. 2
( ) Figure 6 Page 15/17 Varying regulation of friction coefficient with (a) number of coating layer variation, (b) temperature
variation and (c) load fluctuation Varying regulation of friction coefficient with (a) number of coating layer variation, (b) temperature
variation and (c) load fluctuation Varying regulation of friction coefficient with (a) number of coating layer variation, (b) temperature
variation and (c) load fluctuation ng regulation of friction coefficient with (a) number of coating layer variation, (b) temperature
ion and (c) load fluctuation
e 7
olecular dynamics simulation model of nitriding model, (b) Changing regulation of friction
cient of nitriding model and untreated model. e 8
ging regulation of friction coefficient with (a) nitriding depth variation, (b) load fluctuation and (c)
ng quantity variation. Figure 7
(a) Molecular dynamics simulation model of nitriding model, (b) Changing regulation of friction
coefficient of nitriding model and untreated model. Figure 8 Figure 8 Changing regulation of friction coefficient with (a) nitriding depth variation, (b) load fluctuation and (c)
nitriding quantity variation. Page 16/17 Figure 9
Changing regulation of (a) positive pressure and (b) frictional force with the three model. Figure 9 Changing regulation of (a) positive pressure and (b) frictional force with the three model. Page 17/17
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Ecophysiology of coral reef primary producers across an upwelling gradient in the tropical central Pacific
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Maggie D. JohnsonID*¤a, Michael D. Fox¤b, Emily L. A. Kelly¤c, Brian J. Zgliczynski, Stuart
A. Sandin, Jennifer E. Smith Maggie D. JohnsonID*¤a, Michael D. Fox¤b, Emily L. A. Kelly¤c, Brian J. Zgliczynski, Stuart
A. Sandin, Jennifer E. Smith Maggie D. JohnsonID*¤a, Michael D. Fox¤b, Emily L. A. Kelly¤c, Brian J. Zgliczynski, Stuart
A. Sandin, Jennifer E. Smith Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography, University of California
San Diego, San Diego, California, United States of America Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography, University of Califor
San Diego, San Diego, California, United States of America ¤a Current address: Biology Department, Woods Hole Oceanographic Institution, Massachusetts, United
States of America
¤b Current address: Geology and Geophysics Department, Woods Hole Oceanographic Institution,
Massachusetts, United States of America
¤c Current address: Center for Ocean Solutions, Stanford University, California, United States of America
* magjohnson@whoi.edu ¤a Current address: Biology Department, Woods Hole Oceanographic Institution, Massachusetts, United
States of America
¤b Current address: Geology and Geophysics Department, Woods Hole Oceanographic Institution,
Massachusetts, United States of America
¤c Current address: Center for Ocean Solutions, Stanford University, California, United States of America
* magjohnson@whoi.edu a1111111111
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a1111111111 RESEARCH ARTICLE Abstract Patterson, Australian Bureau of
Agricultural and Resource Economics and
Sciences, AUSTRALIA
Received: September 15, 2019
Accepted: January 15, 2020
Published: February 4, 2020
Copyright: © 2020 Johnson et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Editor: Heather M. Patterson, Australian Bureau of
Agricultural and Resource Economics and
Sciences, AUSTRALIA Copyright: © 2020 Johnson et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Abstract Upwelling is an important source of inorganic nutrients in marine systems, yet little is known
about how gradients in upwelling affect primary producers on coral reefs. The Southern Line
Islands span a natural gradient of inorganic nutrient concentrations across the equatorial
upwelling region in the central Pacific. We used this gradient to test the hypothesis that ben-
thic autotroph ecophysiology is enhanced on nutrient-enriched reefs. We measured metab-
olism and photophysiology of common benthic taxa, including the algae Porolithon,
Avrainvillea, and Halimeda, and the corals Pocillopora and Montipora. We found that tem-
perature (27.2–28.7˚C) was inversely related to dissolved inorganic nitrogen (0.46–4.63
μM) and surface chlorophyll a concentrations (0.108–0.147 mg m-3), which increased near
the equator. Contrary to our prediction, ecophysiology did not consistently track these pat-
terns in all taxa. Though metabolic rates were generally variable, Porolithon and Avrainvillea
photosynthesis was highest at the most productive and equatorial island (northernmost). Porolithon photosynthetic rates also generally increased with proximity to the equator. Photophysiology (maximum quantum yield) increased near the equator and was highest at
northern islands in all taxa. Photosynthetic pigments also were variable, but chlorophyll a
and carotenoids in Avrainvillea and Montipora were highest at the northern islands. Phycobi-
lin pigments of Porolithon responded most consistently across the upwelling gradient, with
higher phycoerythrin concentrations closer to the equator. Our findings demonstrate that the
effects of in situ nutrient enrichment on benthic autotrophs may be more complex than labo-
ratory experiments indicate. While upwelling is an important feature in some reef ecosys-
tems, ancillary factors may regulate the associated consequences of nutrient enrichment on
benthic reef organisms. OPEN ACCESS
Citation: Johnson MD, Fox MD, Kelly ELA,
Zgliczynski BJ, Sandin SA, Smith JE (2020)
Ecophysiology of coral reef primary producers
across an upwelling gradient in the tropical central
Pacific. PLoS ONE 15(2): e0228448. https://doi. org/10.1371/journal.pone.0228448 org/10.1371/journal.pone.0228448
Editor: Heather M. Patterson, Australian Bureau of
Agricultural and Resource Economics and
Sciences, AUSTRALIA
Received: September 15, 2019
Accepted: January 15, 2020
Published: February 4, 2020
Copyright: © 2020 Johnson et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Editor: Heather M. Introduction The availability of inorganic nutrients in the environment influences organismal physiology
and can have cascading effects on ecosystem-scale processes [1]. Both natural and anthropo-
genic sources of nutrients can change community structure and function by facilitating growth
of some species over others, in addition to increasing an ecosystem’s energetic foundation,
capacity to support greater biomass [2], and trophic complexity [3]. In marine systems, the
majority of research on nutrient enrichment has focused on the effects of anthropogenic
inputs, including the process of eutrophication, which can overwhelm and fundamentally alter
coastal habitats [4]. However, natural inputs are also important in nearshore systems, and can
be linked to large-scale oceanographic processes such as upwelling, eddies, and internal tides
[5]. Though the effects of coastal upwelling have been relatively well-studied in temperate eco-
systems [6], less is known about how inorganic nutrient input from upwelling affects classically
oligotrophic systems such as tropical coral reefs. Persistent upwelling caused by long-standing currents [7], or periodic upwelling caused by
episodic processes (e.g., internal tides) [8], deliver subthermocline water enriched in dissolved
inorganic nitrate, phosphate, and carbon to the ocean surface [5]. These deep, cooler waters
mix with warmer surface waters and replenish depleted inorganic nutrients that are important
for regulating biological activity [9,10]. Such interactions of environmental variables with
organismal function are referred to as ecophysiology. Large-scale oceanographic processes,
like upwelling, can influence the ecophysiology of resident organisms, particularly those that
require inorganic nutrients for essential biological processes. For example, nutrient availability
directly influences autotroph photosynthesis and respiration. Alterations to the nutrient land-
scape resulting from natural or anthropogenic enrichment can have cascading effects on
broader ecosystem processes by altering primary production [11,12], by favoring growth of
some species over others [13], and, ultimately, by affecting how much energy is available to
higher trophic levels [3]. Thus, understanding how the delivery of essential nutrients by
upwelling can influence autotroph ecophysiology will shed light on how natural oceanographic
processes shape ecosystem structure and function. Autotrophs are particularly sensitive to availability of inorganic nutrients, because dissolved
inorganic nitrogen (DIN) and phosphorous (DIP) are requisite in the synthesis and use of
photosynthetic machinery [11,14]. Though marine photosynthesis is generally not limited by
the availability of carbon, due to the abundant supply of inorganic carbon in seawater and car-
bon-concentrating mechanisms in many autotrophs [15], it is often limited by the availability
of inorganic nutrients [11]. Upwelling effects on coral and algal ecophysiology Competing interests: The authors have declared
that no competing interests exist. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Funding: This work was supported by funding
from the Moore Family Foundation, the Gordon
and Betty Moore Foundation, the Scripps family,
and anonymous donors. The funders had no role in
study design, data collection and analysis, or
preparation of the manuscript. 1 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Introduction For example, nitrate or phosphate limitation decreases productiv-
ity and growth in numerous autotrophic taxa [1,16] including phytoplankton [17], microalgae
[18], and macroalgae [19]. Accordingly, greater availability of DIN and DIP enhances photo-
synthesis and generally increases marine primary production [20], and the input of inorganic
nutrients from upwelling consistently increases productivity of phytoplankton communities in
the surface ocean [10,21]. Though a positive relationship between autotroph physiology and
nutrient availability is well established, there are instances where ancillary abiotic factors, such
as iron limitation, can constrain photosynthesis and primary production of algae in nutrient
replete conditions [22,23]. Given the fundamental role of nutrients in marine photosynthesis and autotroph physiol-
ogy, ecosystems depleted in nutrients and dominated by autotrophs have the potential to
respond strongly to nutrient inputs. Tropical coral reefs are typically oligotrophic, with the
benthos dominated by highly productive macroalgae and corals that provide the primary
source of carbon fixation for the ecosystem [24]. Corals are considered mixotrophs, because
their energy comes from consuming organic particulates (i.e., heterotrophy) and from utilizing PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 2 / 22 Upwelling effects on coral and algal ecophysiology carbon fixed by photosynthesis of endosymbiotic microalgae in the family Symbiodiniaceae
(i.e., autotrophy). Yet, at an ecosystem scale, they typically function as autotrophs, because the
majority (up to 100%) of the daily energetic requirements of most corals is derived from pho-
tosynthesis [25, 26]. Hereafter, we consider corals from an autotrophic perspective, grouping
them (and their endosymbiotic microalgae) with algal taxa common on coral reefs. In tropical
ecosystems with an abundance of autotrophs, such as coral reefs, the availability of DIN and
DIP is perhaps the most important factor constraining photosynthesis and primary produc-
tion, because light is abundant and temperatures are relatively stable [27]. Coral reefs are, thus,
an ideal study system to explore the effects of natural nutrient inputs from upwelling in a typi-
cally low nutrient ecosystem. Upwelling has been documented on coral reefs across ocean basins, ranging from the Flor-
ida Keys [8] and the Colombian Caribbean [28] to the Seychelles [29] and the Great Barrier
Reef [30]. The tropical central Pacific, where coral reefs exist around remote islands and atolls,
is another region where upwelling likely plays an important role [31,32]. Introduction Equatorial upwelling
is a persistent oceanographic process, caused by divergence of Ekman transport to the equator
[7], that creates a region from ~8˚N to 8˚S of the equator [33] where surface-ocean inorganic
nutrient concentrations are higher than oligotrophic tropical gyres [34]. The nutrient input
from upwelling supports high levels of primary production in equatorial Pacific surface waters,
as evidenced by increased surface-ocean productivity closer to the equator [10,21,35]. While
patterns of phytoplankton productivity in the equatorial Pacific are well established, less is
known about how the associated increase in inorganic nutrients effects the ecophysiology of
benthic autotrophs. We used a natural gradient of surface ocean productivity and nutrient availability across
the Southern Line Islands (SLI) to explore how upwelling-driven nutrient enrichment influ-
enced the ecophysiology of benthic autotrophs common on coral reefs (Fig 1). The goals of
this study were to, 1) characterize key environmental characteristics associated with upwelling
across the SLI archipelago and, 2) to quantify corresponding changes in the ecophysiology of
common benthic autotrophs. We hypothesized that metabolism (i.e., photosynthesis and res-
piration) and photophysiology (i.e., maximum quantum yield and photosynthetic pigment
content) of abundant coral and algal genera would increase across the SLI with greater expo-
sure to upwelling. We further predicted that metabolic rates and pigment concentrations
would increase (indicating enhancement), with increasing proximity to the equator. By under-
standing patterns in ecophysiology of key benthic autotrophs across the SLI, we can consider
how patterns in surface-ocean phytoplankton productivity relate to patterns in benthic pro-
ductivity on coral reefs. PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Materials and methods
Site description This study was conducted on a 2013 expedition to the SLI aboard the M/Y Hanse Explorer
under a Scientific Research Permit granted to SAS from the Republic of Kiribati, Environment
and Conservation Division (permit #021113). The five uninhabited islands of the SLI span
~900 km from Malden Island in the north to Flint Island in the south (S1 Table). At 4.0˚ and
5.6˚ south of the equator, Malden and Starbuck Islands, respectively, are situated within the
equatorial upwelling region that spans ~8˚N to 8˚S of the equator [33]. The remaining atoll
and two islands (Millennium Atoll, Vostok Island, Flint Island) lie at increasing distances from
the upwelling region (S1 Table), with Flint Island at the southernmost end of the archipelago. The coral reefs of the SLI are characterized by high biomass of herbivorous fish and top preda-
tors [36] and are dominated by reef-building coralline algae and corals [37]. PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 3 / 22 Upwelling effects on coral and algal ecophysiology Fig 1. Coral and algae of the Southern Line Islands. Common genera of corals and algae across the Southern Line Islands
in the Republic of Kiribati, central Pacific. (a) The crustose coralline alga, Porolithon sp. and (b) the corals Pocillopora
meandrina and (c) Montipora aequituberculata were collected from all five islands. (d) The fleshy green alga Avrainvillea
amadelpha was found only at Vostok and Malden, and (e) the calcareous green alga, Halimeda sp., was found at Flint,
Millennium and Starbuck. Fig 1. Coral and algae of the Southern Line Islands. Common genera of corals and algae across the Southern Line Islands
in the Republic of Kiribati, central Pacific. (a) The crustose coralline alga, Porolithon sp. and (b) the corals Pocillopora
meandrina and (c) Montipora aequituberculata were collected from all five islands. (d) The fleshy green alga Avrainvillea
amadelpha was found only at Vostok and Malden, and (e) the calcareous green alga, Halimeda sp., was found at Flint,
Millennium and Starbuck. https://doi.org/10.1371/journal.pone.0228448.g001 Environmental parameters Water samples were collected for nutrient analyses at each island over three separate sampling
intervals. During a cruise to the SLI in 2009, water samples were collected in triplicate from ~ 1 m
above the benthos at 1–3 sites per island (referred to as 2009 in situ) [31]. During the 2013 cruise,
three water samples were collected from ~1 m above the benthos every 5 m from 5–25 m depth
(referred to as 2013 in situ) [40]. There were no differences in nutrient concentrations across
depths in 2013, thus all depths were pooled for a more robust estimate of environmental conditions
across the reef slope of an island on a given day (n = 9–12, per island). Additionally, triplicate
water samples were collected from the bulk seawater used during metabolic incubations at each
island (referred to as 2013 incubation). Seawater samples were filtered through 1.2 μM GF/C filters
(Whatman) and frozen at -20˚C. Samples from 2009 and 2013 in situ were analyzed at the Univer-
sity of Hawaii Hilo EPSCoR analytical laboratory and 2013 incubation samples were analyzed at
the University of California Santa Barbara Marine Science Institute Analytical lab. Seawater was
analyzed for total DIN, which includes nitrate (NO3
-), nitrite (NO2
-) and ammonium (NH4
+), and
for DIP. Replicate samples were averaged by island for each sampling period. To assess in situ temperature we deployed 6–8 autonomous sensors (Manta 2, Eureka Environ-
mental Engineering) at 10 m depth for 3–4 days per island (5 min sampling interval). Daily mean
temperatures were calculated across sensors to determine daily and overall site means. Photosyn-
thetically active radiation (PAR) was measured adjacent to the instrument with a 4π quantum sen-
sor (LI-193) attached to a LiCor LI-1400 meter in an underwater housing. We measured PAR
every minute for 2–3 days and calculated an average light intensity during the period of maximum
irradiance and peak photosynthesis per day (10:00–14:00) [43], and then a daily average per island. All deployments occurred on sunny days, except for one cloudy day at Vostok. every minute for 2–3 days and calculated an average light intensity during the period of maximum
irradiance and peak photosynthesis per day (10:00–14:00) [43], and then a daily average per island. All deployments occurred on sunny days, except for one cloudy day at Vostok. Remote sensing To estimate surface-ocean primary production across the SLI, we used the eight-day 0.0417˚ (4
km) spatial resolution product of chlorophyll a (chl a, mg mg-3) derived from the Moderate
Resolution Imaging Spectroradiometer (https://modis.gsfc.nasa.gov/about/) (sensu Gove et al. (2013)[38]). Monthly mean chl a data were generated for each island and averaged over the
period of September–December 2013 to provide an integrated estimate of surface-ocean pri-
mary production around each island over the period of the cruise. The oceanographic condi-
tions during this time are most relevant to the ecophysiology of the organisms studied [39]
and provide a reliable estimate of annual conditions across the SLI [40]. While these estimates PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 4 / 22 Upwelling effects on coral and algal ecophysiology of surface-ocean production are measured in offshore waters adjacent to the SLI reefs, they
can be used to reliably infer reef-scale patterns such as benthic community structure [41] and
ecosystem calcification [42]. To confirm that large-scale patterns of surface primary produc-
tion track in situ nutrient environments across the SLI, we also collected discrete water sam-
ples from above the reef benthos as described below. Maximum quantum yield Maximum quantum yield is a useful proxy for photosynthetic efficiency because it is propor-
tional to the rate of electron transport, and it provides a convenient, non-invasive, and instan-
taneous estimate of the performance of the photosynthetic machinery in both algae and corals
[49]. Maximum quantum yield was measured with a red Pulse Amplitude Modulation Fluo-
rometer (Diving PAM, Walz). Samples were dark adapted for 2 hours after respiration mea-
surements. To account for variation in fluorescence-based maximum quantum yield (Fv/Fm)
measurements within individuals, we calculated an average of three haphazardly selected
points on each individual from approximately the same region (~ 2 cm from the tip/edge). Measuring light intensity was minimized to avoid actinic effects (F0 = 300–500) and gain was
minimized to avoid amplifying noise [50]. Saturation intensity (8) and saturation pulse width
(0.8) were constant for all measurements. Due to logistical difficulties, no measurements were
taken at Flint. Specimen collection Two species of coral and up to three genera of algae were collected at 10 m depth from
unshaded microhabitats at each island (adjacent to sensors), with four individuals of each taxa
(Fig 1 and S1 Table). After collection, samples were held in buckets with ambient seawater
under shaded, ambient light until physiological incubations. Water was regularly replenished
with new ambient seawater. In general, we sampled the most abundant coral and algal taxa on
a given island, but due to biogeographic differences we were unable to collect the same species
at all islands (S1 Table). At all islands, we collected similar sized fragments of the branching
coral Pocillopora meandrina, the plating coral Montipora aequituberculata, and the crustose
coralline alga (CCA) Porolithon sp. (5–8 cm diameter/height). Calcareous algae in the genus
Halimeda were present only at Flint, Starbuck, and Millennium. We collected 8 cm long frag-
ments of the most abundant species at each of the three islands (Flint: H. opuntia, Starbuck: H. micronesica, Millennium: H. taenicola). We consider Halimeda at the genus level because
photophysiology and response to higher nutrient concentrations are similar across species
within the genus [44,45]. Abundance of fleshy macroalgae is exceedingly low across the SLI
[37], and the fleshy green alga Avrainvillea amadelpha was present only at Malden and
Vostok. 5 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Upwelling effects on coral and algal ecophysiology Metabolism Physiological studies were conducted aboard the M/Y Hanse Explorer over 3–4 days at each
island, with incubations commencing within 1–2 hrs after sample collection. Photosynthesis
and respiration rates were determined by measuring oxygen exchange under saturating irradi-
ance and total darkness, respectively, using sealed metabolic incubation chambers and optical
dissolved oxygen (DO) probes (Hach Intellical LDO101) following the exact methods of John-
son et al. (2017) [46]. All incubations of a given species (n = 4, per species), and a blank con-
trol, were run simultaneously in a temperature controlled bath. Each chamber contained one
specimen (or seawater control) and a magnetic stir bar. No more than 2 taxa were incubated
on a given day. Samples were kept in ambient conditions for 1–3 hrs before the start of incuba-
tions, and were acclimated to incubation chambers for 5–10 min before the start of measure-
ments. Saturating irradiance (~700 μM quanta m2 s-1) was supplied by two LED light fixtures
(Aqua Illumination, Hydra) suspended directly above the incubation chambers. Light levels
inside incubation chambers were measured with a submerged 4π quantum sensor (LI-193)
and a LiCor LI-1400 meter. Due to a probe malfunction, no oxygen production data were col-
lected for Porolithon at Starbuck. Photosynthesis and respiration were calculated as the linear slope of oxygen concentration
over the duration of the incubation (45 min), and the blank for each set of incubations was
subtracted from the rates of respective replicates to account for background changes in oxygen. Oxygen production in the light was assumed to represent net photosynthesis and oxygen con-
sumption in the dark to represent respiration. To estimate total oxygen produced during incu-
bations, or gross photosynthesis, respiration rates were added to net photosynthesis. For
consistency, rates across all taxa were normalized to surface area and are expressed as μmol O2
cm-2 hr-1. Surface area was determined for coralline fragments by foil-wrapping following
March (1970) [47], for corals by wax dipping following Stimson and Kinzie (1991) [48], and
for algae by planar image analysis using ImageJ. Photosynthetic pigments Samples were frozen at -20˚C after physiological assessments and transported to Scripps Insti-
tution of Oceanography for pigment analyses. Extractions of chl a and carotenoids followed
the methods and equations of Moran and Porath (1980) [51] and Wellburn (1994) [52]. In
brief, chl a and carotenoids were extracted from three intact subsamples of each Halimeda and
Avrainvillea individual in N,N-Dimethylformamide (DMF). Pigments were extracted in total
darkness at 4˚C for 24 hrs. The extract was then centrifuged and the supernatant was analyzed PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 6 / 22 Upwelling effects on coral and algal ecophysiology spectrophotometrically with a diode array spectrophotometer (Agilent, UV-vis 8453) with 1
nm resolution. For the coral samples, coral tissue was first removed from the skeleton with an
airbush, and the blastate was homogenized. The endosymbiotic algal fraction was isolated
from the host animal tissue, and chl a and carotenoids were extracted from the algal fraction
in DMF as described above. Two subsamples from each coral individual were analyzed. Pigments were extracted from the coralline alga, Porolithon, with procedures adapted from
Payri et al. (2001) [53] and Kursar and Alberte (1983) [54]. Water soluble phycobilin pigments
were extracted first using 0.01 M phosphate buffer. Two 1-cm2 punches were collected from
each coralline algal sample and then each subsample was ground separately in the dark and
over ice using a mortar and pestle. Pigments were extracted in darkness at 4˚C for 24 hrs. Sam-
ples were then centrifuged and the supernatant was analyzed for allophycocyanin, phycocya-
nin, and phycoerythrin content based on the equations of Kursar and Alberte (1983) [54]. The
remaining pellets were then processed for chl a and carotenoids in DMF following the proce-
dure above. Subsamples from algal pigment extractions were averaged for each individual. Por-
olithon and coral pigments were normalized to subsample surface area and macroalgal
pigments were normalized to subsample wet weight. spectrophotometrically with a diode array spectrophotometer (Agilent, UV-vis 8453) with 1
nm resolution. For the coral samples, coral tissue was first removed from the skeleton with an
airbush, and the blastate was homogenized. The endosymbiotic algal fraction was isolated
from the host animal tissue, and chl a and carotenoids were extracted from the algal fraction
in DMF as described above. Two subsamples from each coral individual were analyzed. Pigments were extracted from the coralline alga, Porolithon, with procedures adapted from
Payri et al. Photosynthetic pigments (2001) [53] and Kursar and Alberte (1983) [54]. Water soluble phycobilin pigments
were extracted first using 0.01 M phosphate buffer. Two 1-cm2 punches were collected from
each coralline algal sample and then each subsample was ground separately in the dark and
over ice using a mortar and pestle. Pigments were extracted in darkness at 4˚C for 24 hrs. Sam-
ples were then centrifuged and the supernatant was analyzed for allophycocyanin, phycocya-
nin, and phycoerythrin content based on the equations of Kursar and Alberte (1983) [54]. The
remaining pellets were then processed for chl a and carotenoids in DMF following the proce-
dure above. Subsamples from algal pigment extractions were averaged for each individual. Por-
olithon and coral pigments were normalized to subsample surface area and macroalgal
pigments were normalized to subsample wet weight. Statistical analyses All variables met assumptions of normality and homogeneity of variances, determined by
visual inspection of residuals using q-q plots and Shapiro-Wilks tests and Levene’s test, respec-
tively. Mean environmental parameters were analyzed across islands with a one-way ANOVA
with island as a fixed factor. In situ temperatures and remotely sensed chl a were compared
using daily and 8-day mean values, respectively. Inorganic nutrient concentrations were ana-
lyzed separately for each sampling interval (2009 in situ, 2013 in situ, 2013 incubation). Daily
mean light intensity was compared using values between 10:00–14:00. Each response variable
was analyzed separately by genus with a one-way ANOVA with island as a fixed factor. Where
significant differences were detected, Tukey’s HSD identified differences among islands. All
statistical analyses were conducted in R [55]. PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Environmental parameters https://doi.org/10.1371/journal.pone.0228448.g002 https://doi.org/10.1371/journal.pone.0228448.g002 https://doi.org/10.1371/journal.pone.0228448.g002 incubations (S2 Table). DIN was ~350% higher at Malden (4.63 ± 0.13 μM) versus Flint
(0.46 ± 0.01 μM) in 2013, and ~200% higher than at either Vostok (0.93 ± 0.05 μM) or Millen-
nium (0.95 ± 0.08 μM) (Table 1). Concentration of DIP was 75–150% higher at the northern
islands than at the three southern islands (Table 1). incubations (S2 Table). DIN was ~350% higher at Malden (4.63 ± 0.13 μM) versus Flint
(0.46 ± 0.01 μM) in 2013, and ~200% higher than at either Vostok (0.93 ± 0.05 μM) or Millen-
nium (0.95 ± 0.08 μM) (Table 1). Concentration of DIP was 75–150% higher at the northern
islands than at the three southern islands (Table 1). Environmental parameters Surface chl a concentrations were higher near the equator, and lower farther from the equator
(Fig 2 and S2 Table). From Sept—Dec 2013, mean surface chl a was highest at Malden, lowest
at Flint, and similar across the three middle islands. Malden surface water chl a concentrations
were 10% higher than Starbuck, while Starbuck, Millennium and Vostok were 15–20% higher
than Flint (Table 1). Mean daily in situ temperatures decreased significantly across the SLI based on latitude (S2
Table), with coolest temperatures closest to the equator. Temperatures were similarly warm at
Vostok and Millennium, at 28.70 ± 0.01˚C and 28.70 ± 0.02˚C, respectively, and decreased to
27.20 ± 0.05˚C at Malden (Table 1). Average daily PAR levels did not vary significantly across
islands (S2 Table), though the lowest daily average intensity between the hours of 10:00–14:00
was at Vostok on a cloudy day (428 ± 43) and the highest was at Malden (884 ± 1) (Table 1). In situ water samples from 2009 and 2013, and bulk water samples from incubations in
2013, showed a consistent pattern of increasing ambient DIN and DIP from south to north
that mirrors the larger surface chl a and temperature gradients across the SLI (Table 1). Mal-
den and Starbuck were significantly enriched in DIN and DIP compared to Flint, Vostok and
Millennium, regardless of whether the water was sampled from ~1 m above the benthos in
2009, throughout the water column in 2013, or from the nearshore surface water used in PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 7 / 22 Upwelling effects on coral and algal ecophysiology Fig 2. Surface-ocean chl a during cruise. Average surface-ocean chl a concentrations increased from south to north
across the Southern Line Islands over four months encompassing the research expedition. Surface chl a (mg mg-3) was
estimated from the eight-day 0.0417˚ (4 km) spatial resolution product derived from the Moderate Resolution Imaging
Spectroradiometer, and concentrations ranged from 0.01 (blue) to 0.17 mg mg-3 (red) across the archipelago. Fig 2. Surface-ocean chl a during cruise. Average surface-ocean chl a concentrations increased from south to north
across the Southern Line Islands over four months encompassing the research expedition. Surface chl a (mg mg-3) was
estimated from the eight-day 0.0417˚ (4 km) spatial resolution product derived from the Moderate Resolution Imaging
Spectroradiometer, and concentrations ranged from 0.01 (blue) to 0.17 mg mg-3 (red) across the archipelago. Upwelling effects on coral and algal ecophysiology Table 1. Environmental parameters across the Southern Line Islands, with islands listed from south to north. Island
Year
Temp (˚C)
PAR§
Chl a (mg m-3)
DIN (μM)
DIP (μM)
Flint
2013 in situ
28.50 ± 0.04
608 ± 3
0.108 ± 0.003
0.46 ± 0.01
0.13 ± 0.003
2013 incubation
2009 in situ
1.16 ± 0.16
0.78 ± 0.02
0.20 ± 0.005
0.15 ± 0.003
Vostok
2013 in situ
28.70 ± 0.01
428 ± 43
0.128 ± 0.001
0.93 ± 0.05
0.16 ± 0.01
2013 incubation
2009 in situ
1.62 ± 0.33
1.51 ± 0.35
0.23 ± 0.02
0.16 ± 0.003
Millennium
2013 in situ
28.70 ± 0.02
647 ± 4
0.125 ± 0.004
0.95 ± 0.08
0.16 ± 0.01
2013 incubation
2009 in situ
1.18 ± 0.13
2.03 ± 0.06
0.22 ± 0.02
0.21 ± 0.01
Starbuck
2013 in situ
28.30 ± 0.01
798 ± 12
0.133 ± 0.001
3.86 ± 0.05
0.42 ± 0.03
2013 incubation
2009 in situ
4.50 ± 0.04
3.59 ± 0.38
0.35 ± 0.02
0.23 ± 0.01
Malden
2013 in situ
27.20 ± 0.05
884 ± 1
0.147 ± 0.003
4.63 ± 0.13
0.44 ± 0.01
2013 incubation
2009 in situ
5.15 ± 0.06
3.87 ± 0.32
0.51 ± 0.07
0.27 ± 0.02
I l
d
l
(
SE)
i
t l
t
th S
th
Li
I l
d
I
it t
t
d h t
th ti
ll
ti
di ti
(PAR)
d Table 1. Environmental parameters across the Southern Line Islands, with islands listed from south to north. Island-scale mean (± SE) environmental parameters across the Southern Line Islands. In situ temperature and photosynthetically active radiation (PAR) were measured
with autonomous sensors for 2–4 days per island. Average PAR at peak irradiance (between 1000–1400) is presented, as these values most closely resemble intensities
used in lab incubations. Chl a concentrations were derived from satellite data spanning a four-month window around the expedition (Sept—Dec 2013). In situ water
samples were collected for analysis of dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorous (DIP) from one site per island in 2013 (n = 9–12), and in
triplicate from 3–6 sites per island during a research cruise in 2009. §μmol quanta-1 m-2 s-1. Island-scale mean (± SE) environmental parameters across the Southern Line Islands. Metabolism Metabolic rates of all genera were variable across the SLI archipelago, and although there was
some evidence of enhancement in ecophysiology with increasing proximity to the equator, the
trend was not consistent across all taxa (Fig 3 and Table 2). PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 8 / 22 In situ temperature and photosynthetically active radiation (PAR) were measured
with autonomous sensors for 2–4 days per island. Average PAR at peak irradiance (between 1000–1400) is presented, as these values most closely resemble intensities
used in lab incubations. Chl a concentrations were derived from satellite data spanning a four-month window around the expedition (Sept—Dec 2013). In situ water
samples were collected for analysis of dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorous (DIP) from one site per island in 2013 (n = 9–12), and in
triplicate from 3–6 sites per island during a research cruise in 2009. §μmol quanta-1 m-2 s-1. https://doi.org/10.1371/journal.pone.0228448.t001 Photosynthesis (net and gross) and respiration of the CCA Porolithon were highest at Mal-
den near the equator (Fig 3A and Table 2), but did not vary significantly across the three
southern islands, Flint, Vostok, and Millennium (S3 Table). Some of the variability in gross
photosynthesis is related to different patterns of response in net photosynthesis versus respira-
tion, because gross photosynthesis is the sum of both. In Porolithon, the significant increase in
gross photosynthesis at Malden is linked to a 211% increase in net photosynthesis from Flint
to Malden, but only a 24% increase in respiration. In Halimeda, gross photosynthesis was con-
sistent at the three islands where it was found (Flint, Millennium, Starbuck) (Fig 3C). How-
ever, this pattern obscures a marginally significant 45% increase in net photosynthesis at the
southernmost island (Flint) (S3 Table), and a significant 173% increase in respiration rates at
the northern islands (Table 2). These opposing trends in net photosynthesis and respiration
across the islands are negated in the estimates of gross photosynthesis. In Avrainvillea, gross
photosynthesis was 54% higher at Malden than Vostok (Fig 3E and Table 2), which was driven
by significantly higher net photosynthesis (131%) but no difference in respiration from Vostok
to Malden (S3 Table). Coral metabolism was highly variable across the SLI. Gross photosynthesis of Pocillopora
varied among islands but did not clearly relate to proximity to the equator and the associated
increase in nutrient concentrations (Fig 3G and Table 2). These differences were associated
with variable, but not significantly different, rates of net photosynthesis and respiration (S3
Table). The plating coral, Montipora, also had variable photosynthesis (net and gross) and res-
piration rates across the archipelago (Fig 3I and S3 Table), with highest rates at Vostok and
lowest rates at Millennium (Table 2). PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Island-scale mean (± SE) environmental parameters across the Southern Line Islands. In situ temperature and photosynthetically active radiation (PAR) were measured
with autonomous sensors for 2–4 days per island. Average PAR at peak irradiance (between 1000–1400) is presented, as these values most closely resemble intensities
used in lab incubations. Chl a concentrations were derived from satellite data spanning a four-month window around the expedition (Sept—Dec 2013). In situ water
samples were collected for analysis of dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorous (DIP) from one site per island in 2013 (n = 9–12), and in
triplicate from 3–6 sites per island during a research cruise in 2009.
§μmol quanta-1 m-2 s-1. 9 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Upwelling effects on coral and algal ecophysiology Fig 3. Coral and algal metabolism and photosynthetic efficiency. Box plots of gross photosynthesis and maximum
quantum yield of (a-b) Porolithon sp., (c-d) Halimeda sp., (e-f) Avrainvillea amadelpha, (g-h) Pocillopora meandrina, and
(i-j) Montipora aequituberculata. Islands are ordered from south to north across the x-axis (from left to right), and
missing bars indicate where a given species was not present at an island. Due to logistical difficulties, there were no
maximum quantum yield measurements at Flint or photosynthesis measurements from Porolithon at Starbuck. Significant differences between islands were determined by Tukey’s HSD, different letters represent significant
differences at p < 0.05. Gross photosynthetic rates for Porolithon and corals were normalized to surface area, and are
expressed as μmol O2 cm-2 hr-1. Rates for the algae, Halimeda and Avrainvillea, were normalized to fresh weight, thus
for those taxa the units are μmol O2 mg-2 hr-1. Fig 3. Coral and algal metabolism and photosynthetic efficiency. Box plots of gross photosynthesis and maximum
quantum yield of (a-b) Porolithon sp., (c-d) Halimeda sp., (e-f) Avrainvillea amadelpha, (g-h) Pocillopora meandrina, and
(i-j) Montipora aequituberculata. Islands are ordered from south to north across the x-axis (from left to right), and
missing bars indicate where a given species was not present at an island. Due to logistical difficulties, there were no
maximum quantum yield measurements at Flint or photosynthesis measurements from Porolithon at Starbuck. Significant differences between islands were determined by Tukey’s HSD, different letters represent significant
differences at p < 0.05. Gross photosynthetic rates for Porolithon and corals were normalized to surface area, and are
expressed as μmol O2 cm-2 hr-1. Rates for the algae, Halimeda and Avrainvillea, were normalized to fresh weight, thus
for those taxa the units are μmol O2 mg-2 hr-1. https://doi.org/10.1371/journal.pone.0228448.g003 Upwelling effects on coral and algal ecophysiology Table 2. Photosynthesis and respiration rates. Genus
Island
Net Photosynthesis
(μmol O2 cm-2 hr-1)
Respiration
(μmol O2 cm-2 hr-1)
Porolithon
Flint
0.19 ± 0.16
0.91 ± 0.23
Vostok
0.05 ± 0.13
0.94 ± 0.04
Millennium
0.40 ± 0.15
0.96 ± 0.09
Starbuck
-
0.44 ± 0.06
Malden
0.59 ± 0.13
1.13 ± 0.05
Halimeda
Flint
0.61 ± 0.07
0.11 ± 0.03
Millennium
0.43 ± 0.05
0.28 ± 0.01
Starbuck
0.42 ± 0.03
0.30 ± 0.04
Avrainvillea
Vostok
0.15 ± 0.01
0.32 ± 0.03
Malden
0.35 ± 0.05
0.36 ± 0.05
Pocillopora
Flint
0.69 ± 0.10
0.49 ± 0.03
Vostok
0.34 ± 0.23
0.52 ± 0.06
Millennium
0.66 ± 0.09
0.62 ± 0.04
Starbuck
0.43 ± 0.13
0.49 ± 0.03
Malden
0.49 ± 0.09
0.55 ± 0.02
Montipora
Flint
0.68 ± 0.17
0.62 ± 0.12
Vostok
0.80 ± 0.09
0.90 ± 0.10
Millennium
0.58 ± 0.05
0.45 ± 0.02
Starbuck
0.93 ± 0.19
0.58 ± 0.02
Malden
0.77 ± 0.08
0.60 ± 0.03
Mean (± SE) net photosynthesis and dark respiration rates of corals and algae surveyed across the SLI, n = 4 per genus at each island. Rates for Porolithon and corals
were normalized to surface area. Rates for the algae, Halimeda and Avrainvillea, were normalized to fresh weight, thus, the units for those taxa are μmol O2 mg-2 hr-1. Table 2. Photosynthesis and respiration rates. Mean (± SE) net photosynthesis and dark respiration rates of corals and algae surveyed across the SLI, n = 4 per genus at each island. Rates for Porolithon and corals
were normalized to surface area. Rates for the algae Halimeda and Avrainvillea were normalized to fresh weight thus the units for those taxa are μmol O mg-2 hr-1 quantum yield did not differ across islands for Halimeda (Fig 3D and S3 Table), and was sig-
nificantly higher at Malden than at Vostok for Avrainvillea (Fig 3F and S3 Table). Maximum quantum yield of the coral Pocillopora was significantly higher at Malden than at
the southern islands, which did not differ from each other (Fig 3H and S3 Table). In Monti-
pora, maximum quantum yield was significantly higher at Starbuck than at Malden, and
higher at Malden than at Vostok and Millennium (Fig 3J and S3 Table). quantum yield did not differ across islands for Halimeda (Fig 3D and S3 Table), and was sig-
nificantly higher at Malden than at Vostok for Avrainvillea (Fig 3F and S3 Table). Maximum quantum yield of the coral Pocillopora was significantly higher at Malden than at
the southern islands, which did not differ from each other (Fig 3H and S3 Table). In Monti-
pora, maximum quantum yield was significantly higher at Starbuck than at Malden, and
higher at Malden than at Vostok and Millennium (Fig 3J and S3 Table). Maximum quantum yield Maximum quantum yield increased across the SLI with increasing proximity to the equator
for all taxa, though the magnitude of change varied by genus. Porolithon maximum quantum
yield was up to 65% higher at Malden than at the southern islands (Fig 3B), where yield did
not differ significantly from Vostok and Millennium to Starbuck (S3 Table). Maximum 10 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Photosynthetic pigments Porolithon and coral pigment
concentrations were normalized to surface area, and rates are expressed as μg cm-2. Halimeda and Avrainvillea
pigment concentrations were normalized to fresh weight, thus units for those taxa are μg mg-2. Fig 4. Photosynthetic pigments of corals and algae. Box plots of chl a and carotenoid concentrations in (a-b) https://doi.org/10.1371/journal.pone.0228448.g004 Photosynthetic pigments Photosynthetic pigment concentrations generally followed the predicted pattern of increasing
with proximity to the equator (Fig 4 and S3 Table). Chl a concentrations tended to increase
from south to north for all taxa but Porolithon and Halimeda. However, in Pocillopora, chl a
concentrations were lower than expected at Starbuck, but higher than expected at Millennium
(Fig 4G). In Montipora, chl a concentrations increased by 115–198% from Flint to Malden
(Fig 4I). Carotenoids showed little pattern across taxa or islands due to high levels of variation
among individuals (Fig 4). Phycobilin pigment concentrations, including allophycocyanin, phycocyanin, and phycoer-
ythrin of the coralline red alga Porolithon were variable across the SLI (Fig 5), with island-spe-
cific significant differences for phycoerythrin only (S3 Table). Phycoerythrin concentrations
increased by 150% from Flint to Malden (Fig 5). Though there were similar trends for phyco-
cyanin, including a 185% increase from Flint to Malden, these differences were not significant,
likely due to high variability among individuals within an island. 11 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Upwelling effects on coral and algal ecophysiology Fig 4. Photosynthetic pigments of corals and algae. Box plots of chl a and carotenoid concentrations in (a-b)
Porolithon sp., (c-d) Halimeda sp., (e-f) Avrainvillea amadelpha, (g-h) Pocillopora meandrina, and (i-j) Montipora
aequituberculata. Islands are ordered from south to north across the x-axis (from left to right), and missing bars
indicate where a given species was not present at an island. Significant differences between islands were determined by
Tukey’s HSD, different letters represent significant differences at p < 0.05. Porolithon and coral pigment
concentrations were normalized to surface area, and rates are expressed as μg cm-2. Halimeda and Avrainvillea
pigment concentrations were normalized to fresh weight, thus units for those taxa are μg mg-2. https://doi.org/10.1371/journal.pone.0228448.g004
Upwelling effects on coral and algal ecophysiology Fig 4. Photosynthetic pigments of corals and algae. Box plots of chl a and carotenoid concentrations in (a-b) Fig 4. Photosynthetic pigments of corals and algae. Box plots of chl a and carotenoid concentrations in (a-b)
Porolithon sp., (c-d) Halimeda sp., (e-f) Avrainvillea amadelpha, (g-h) Pocillopora meandrina, and (i-j) Montipora
aequituberculata. Islands are ordered from south to north across the x-axis (from left to right), and missing bars
indicate where a given species was not present at an island. Significant differences between islands were determined by
Tukey’s HSD, different letters represent significant differences at p < 0.05. Discussion The isolated and remote SLI represent a unique study system to evaluate how variability in
exposure to equatorial upwelling, and thus availability of inorganic nutrients, influences coral
and algal ecophysiology. Results of our in situ measurements, discrete water samples, and
remote sensing of surface chl a illustrate that the SLI are increasingly exposed to high levels of
surface primary production and inorganic nutrients with proximity to the equator. We
hypothesized that the ecophysiology of benthic autotrophs (i.e., metabolism, maximum PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 12 / 22 Upwelling effects on coral and algal ecophysiology Fig 5. Phycobilin pigment concentrations. Mean (± SE) phycobilin pigment concentrations normalized to surface
area for the crustose coralline alga Porolithon sp. across the Southern Line Islands. Islands are ordered from south to
north. Allophycocyanin (light pink), phycocyanin (pink), and phycoerythrin (dark pink) were measured on 2
subsamples of each individual (n = 4) per island. Data points are means of subsamples per individual. Significant
differences in pigment concentrations across islands were determined for each pigment with Tukey’s HSD. Different
letters represent significant differences at p < 0.05. https://doi.org/10.1371/journal.pone.0228448.g005 Fig 5. Phycobilin pigment concentrations. Mean (± SE) phycobilin pigment concentrations normalized to surface
area for the crustose coralline alga Porolithon sp. across the Southern Line Islands. Islands are ordered from south to
north. Allophycocyanin (light pink), phycocyanin (pink), and phycoerythrin (dark pink) were measured on 2
subsamples of each individual (n = 4) per island. Data points are means of subsamples per individual. Significant
differences in pigment concentrations across islands were determined for each pigment with Tukey’s HSD. Different
letters represent significant differences at p < 0.05. https://doi.org/10.1371/journal.pone.0228448.g005 https://doi.org/10.1371/journal.pone.0228448.g005 quantum yield, pigment concentrations) would reflect this pattern of nutrient enrichment. Although in some instances photophysiology was enhanced at the more nutrient-enriched
islands, the overall patterns were variable across taxa and did not indicate a clear trend of phys-
iological enhancement. The lack of a clear relationship between exposure to upwelling and the ecophysiology of pri-
mary producers is notable because greater availability of inorganic nutrients generally has a
predictable, positive effect on autotrophs. While our findings corroborate well-established pat-
terns of increasing surface ocean primary production near the equator [10,56,57], they indicate
that provisioning of nutrients from equatorial upwelling does not necessarily enhance auto-
troph physiology on the benthos, as we observed in the SLI. Discussion The ambiguous relationship
between nutrient availability and ecophysiology suggests that other abiotic or biotic factors
may be contributing to benthic biological processes in the central Pacific. These results indi-
cate that nutrients may not be the primary drivers of benthic primary production on some
coral reefs. Environmental parameters The equatorial islands of Starbuck and Malden were cooler and more nutrient-rich than the
southern islands of Flint, Vostok, and Millennium, which became incrementally warmer and
depleted in nutrients with proximity to the equator. Our measurements of environmental con-
ditions were constrained to discrete samples, short-duration in situ measurements, and short-
term remote sensing of chl a. With these data, alone, our scope for interpretation is temporally
limited. However, our findings corroborate a plethora of existing long-term data sets and a PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 13 / 22 Upwelling effects on coral and algal ecophysiology rich history of oceanographic research that, collectively, improve our understanding of long-
term upwelling patterns in the remote equatorial Pacific. Decades of research have character-
ized equatorial upwelling as a persistent and predictable oceanographic feature driven by the
Equatorial Undercurrent (EUC) [7,10]. The EUC delivers cool, nutrient rich water to the
ocean surface on a west-to-east pathway [33], and causes net accumulation and temporal per-
sistence of high nitrate and phosphate around the equator [34]. Higher DIN and DIP fuels
phytoplankton growth and, thus, increases surface chl a concentrations. This trend is evident
in our 4-month chl a data, and is even more conspicuous in the climatology from 2004–2015
[40]. Further, the same patterns have been described in numerous other studies in the same
region [21,35,58]. The coupling of upwelling with oceanic production is well established, but
much less is known about potential cascading effects on primary production of the benthos,
particularly in coral reef habitats. Coral reefs are typically considered to exist in oligotrophic waters that are often likened to
marine deserts [24], and on average DIN and DIP concentrations in the Caribbean and Pacific
are estimated to be ~0.4 μM and ~0.2 μM, respectively [59]. However, our findings demon-
strate that nutrient concentrations on some reefs can well exceed these global averages. At the
most equatorial site, Malden, the conditions are more akin to the nutrient regime of a temper-
ate kelp forest in southern California, where nitrate concentrations can increase up to 5 μM
during wind-driven upwelling season [60], than the archetypal oligotrophic coral reef. Environmental parameters Similar
patterns of natural enrichment have been documented in other upwelling-influenced coral
reef systems, such as on Conch Reef in the Florida Keys, where episodic pulses driven by inter-
nal tidal bores can increase ambient DIN to 1–4 μM and DIP to 0.1–0.3 μM [61]. Elevated lev-
els of DIN and DIP often have negative implications for overall community structure on coral
reefs, because excess nutrients can fuel the dominance of fleshy macroalgae [4]. Yet, the ben-
thos on nutrient-enriched islands of Malden and Starbuck is dominated by reef-building corals
and calcifying algae, where reef builders cover up to ~80% of the benthos (as opposed to fleshy
and turf algae that account for ~15%) [37]. Our findings indicate that coral reef ecosystems in
the central Pacific can thrive in nutrient conditions typically considered detrimental to reef-
building corals. Indeed, coral cover is also high on Jarvis Island, which is just north of Malden
and at the epicenter of equatorial upwelling, where DIN concentrations can naturally fluctuate
up to 16 μmol [62]. Though inorganic nutrients can influence benthic dynamics, the relative
dominance of reef-builders on these islands likely is also linked to high biomass of herbivorous
fishes [36]. Given the persistence of an extreme gradient in inorganic nutrient concentrations
across coral reefs of the central Pacific, there is a clear need to explore the role of naturally
nutrient variability in shaping biological processes and the resulting implications for the asso-
ciated benthic reef communities. Metabolism Evaluating the relationship between autotroph metabolism and inorganic nutrient availability
provides insight into the potential effects of equatorial upwelling on benthic biological pro-
cesses. Metabolic rates indicate the physiological status of corals and algae, and photosynthetic
rates are directly proportional to rates of primary production. Given the potential for nutrient
availability to influence rates of primary production in microalgae [63] and macrophytes [64],
we hypothesized that photosynthesis of corals and algae would generally increase across the
SLI corresponding to DIN and DIP availability. However, our measurements of coral and algal
metabolism did not show a strong pattern of enhancement across the SLI, except in the CCA,
Porolithon. PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 14 / 22 Upwelling effects on coral and algal ecophysiology Porolithon metabolic rates generally tracked with the nutrient gradient. Of the benthic
macroalgae, metabolism of the green alga, Avrainvillea, was higher at the site closer to the
equator, while Halimeda was largely unchanged across sites. These data should be interpreted
cautiously as each macroalgal genera was found at only 2 and 3 of the 5 islands, respectively. Metabolic rates of the corals, Pocillopora and Montipora, were variable across all islands with
no particular pattern. The lack of a clear metabolic response to nutrient enrichment across all
taxa in this study may indicate that other environmental factors influence metabolism, or may
be a result of high variability associated with the methodology. Further, gross metabolic rates
may not fully capture the capacity for autotrophs to respond to nutrient availability at the
time-scale of the present study. In contrast to photosynthetic efficiency (maximum quantum yield) and photosynthetic pig-
ment content, which quantify the functioning of cellular light-harvesting machinery, meta-
bolic rates constitute a net organismal response. Comparing and contrasting the metabolic
responses of corals and macroalgae across the same gradient may thus be encumbered by
inherent differences between strictly autotrophic algae and mixotrophic corals [65]. While
algal metabolism represents cellular processes (e.g., respiration and photosynthesis) of only the
autotrophic alga, coral metabolism is a net response of the coral holobiont, which encompasses
the heterotrophic coral host, the autotrophic endosymbionts and the associated microbial
community [66]. Estimating photosynthesis through oxygen production, particularly with cor-
als, may be too coarse a metric to resolve finer-scale trends underlying genera-specific
responses. Metabolism The physiological effects of nutrient enrichment on corals may thus be more effec-
tively assessed by pigment content and maximum quantum yield [67]. PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Photosynthetic pigments Pigment concentrations in algae and corals are flexible and highly responsive to prevailing
environmental conditions [73], particularly to irradiance and nutrient concentrations [74]. A
positive photosynthetic pigment-nutrient relationship has been demonstrated in a suite of
photosynthetic taxa and across a range of environmental conditions. This relationship gener-
ally manifests as an increase in chl a and carotenoid concentrations in response to excess avail-
ability of DIN and DIP [14,75]. The predictability of this relationship makes algal pigment
content a useful metric in understanding the abiotic conditions of an ecosystem because pig-
ments in coral reef algae [76] and corals [67,77] can be used as bioindicators for nutrient pollu-
tion and water quality. Given the strength and consistency of this relationship, we expected
that pigment concentrations would increase across the SLI with increasing proximity to the
equator. In most taxa studied, the primary photosynthetic pigments, chl a and carotenoids, were
highest at the most northern islands, with some variability across the middle islands of the
archipelago. Chl a generally increased with greater exposure to nutrient availability in the
green algae and the corals, and patterns of carotenoids mirrored those of chl a, but with more
variability. Porolithon showed the least consistent pattern with respect to the primary pig-
ments, but the accessory phycobilin pigments demonstrated the strongest response to the
nutrient gradient. There was a positive, incremental increase in phycoerythrin and phycocya-
nin concentrations at islands that corresponded to increasing availability of ambient DIN and
DIP, though this trend was significant only for phycoerythrin. The phycobilin complexes in
crustose corallines, and other red algae, act as accessory pigments that broaden the spectrum
of light available for photosynthesis [78]. Phycobilins are also particularly responsive to DIN,
in part because algae can use phycobilin pigment complexes to store excess nitrogen [54]. Nitrogen storage for luxury consumption by algae is an ecological adaptation to nutrient limi-
tation and provides a source of DIN that can be later metabolized to fuel growth when nutri-
ents are limiting [79]. The increase in Porolithon phycobilins with greater exposure to
inorganic nutrient availability may indicate that these algae can store the excess DIN delivered
by equatorial upwelling in pigment-protein complexes. Maximum quantum yield Maximum quantum yield is a direct measure of the functioning of chl a pigment molecules,
and provides a proxy for the photosynthetic efficiency of autotrophs. At the organismal scale, a
higher photosynthetic efficiency indicates that, when all reaction centers are engaged, the auto-
troph can fix more carbon per quanta through the light-dependent reactions of photosynthesis
[68]. Because photosynthetic efficiency is directly coupled with pigment content, and there is a
predictable and well-established relationship between pigment content and nutrients, we
hypothesized that maximum quantum yield would increase with increasing availability of DIN
and DIP. Maximum quantum yield followed the predicted latitudinal pattern for corals and
algae across the SLI, though the magnitude of response was taxon-specific. Fluorescence measurements of photosystem II provide valuable insight into the photosyn-
thetic performance of autotrophs and their response to varying nutrient regimes [69,70]. Indeed, photosynthetic efficiency of coral and algal taxa was highest at most northern islands
sampled, and there was an overall trend of increasing photosynthetic efficiency with increasing
exposure to equatorial upwelling. These results indicate that long-term exposure to ample DIN
and DIP availability may increase the number, function, or efficiency of photosystem reaction
centers. Our data corroborate a wide body of work demonstrating that DIN and DIP availabil-
ity directly impacts the ability of algae to build and maintain properly functioning photosystem
complexes that are essential for photosynthesis [20]. Enhanced photosynthetic efficiency could
have ecological ramifications because efficiency is positively correlated with primary produc-
tion [71,72]. Thus, greater exposure to inorganic nutrient availability may indicate higher
potential for primary production and a potential increase in the capacity of benthic autotrophs
to channel more energy to higher trophic levels. However, future experiments should confirm
the consistency of these patterns by incorporating larger sample sizes and greater replication
across natural nutrient regimes. PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 15 / 22 Upwelling effects on coral and algal ecophysiology Photosynthetic pigments The increase in some of the photosyn-
thetic pigments with increased availability of DIN and DIP indicates that equatorial upwelling
could fuel primary production in benthic autotrophs by facilitating the development of more
pigments, which supports greater capacity to harvest light and generate energy. PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Effects of equatorial upwelling on ecophysiology The lack of a strong relationship between benthic autotroph ecophysiology and nutrient avail-
ability in the SLI is an unexpected result, and indicates that DIN and DIP may not have been
the only limiting factor for metabolism and photophysiology in the taxa studied. Other poten-
tial abiotic drivers of autotroph ecophysiology across islands could include temperature, light,
and other nutrients. For many algae, maximum photosynthesis occurs over a range of temper-
atures, thus the 1.5˚C change in temperature from south to north did not likely contribute to
metabolic rates [80]. Similarly, light can be ruled out as a confounding factor because there
was no significant difference in the amount of available light across the SLI, despite the 4˚
change in latitude. Biotic factors could also have influenced ecophysiology as well, such as
prior damage from grazing or exposure to competition or disease, though we attempted to
minimize these effects by selecting individuals that appeared healthy. The limited temporal scope of both our physiological and environmental measurements are
also important to consider. Due to the logistical constraints associated with field work at
remote islands, physiological measurements could only be taken once, and thus represent a PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 16 / 22 Upwelling effects on coral and algal ecophysiology snapshot of ecophysiology at one point in time. The inorganic nutrient gradient resulting from
upwelling is persistent through time [40], but could be magnified at different times of the year,
depending on the strength of prevailing winds. Our environmental measurements are likewise
temporally limited, though we have bolstered our discrete samples with historical perspective
and satellite data that encompasses several months around the expedition. Furthermore, we
quantified only DIN and DIP in our water samples, using discrete samples at one time point
over two different expeditions. Thus, we cannot conclude what other nutrients may have influ-
enced autotroph ecophysiology. We can speculate that iron limitation of photosynthesis may
have contributed to some of the observed patterns in photosynthesis (and photophysiology). In areas of high nitrate and low surface chl a, iron availability can limit photosynthesis and
growth of phytoplankton [81]. However, our satellite data illustrate that there was high sur-
face-ocean production at the equatorial islands, and, thus, do not provide evidence that phyto-
plankton primary production was iron limited. Effects of equatorial upwelling on ecophysiology Future work should explore the relationship of
coral and algal ecophysiology in response to equatorial upwelling in more detail, the potential
for seasonal responses to inorganic nutrient availability, and the role of additional abiotic fac-
tors in constraining benthic photosynthesis in nutrient replete conditions. These data provide an interesting perspective on the role of inorganic nutrients and upwell-
ing in shaping benthic biological processes. Despite the potential for nutrient availability to
elicit strong photosynthetic responses, we found only moderate, and sometimes inconsistent,
physiological enhancement with increasing exposure to equatorial upwelling in the taxa stud-
ied. This lack of a clear pattern contrasts with the conspicuous gradient in surface ocean pro-
ductivity and inorganic nutrient availability. Our findings agree with one other study to
explore ecophysiology of coral reef benthic taxa in response to upwelling. Eidens et al. (2014)
similarly documented substantial variation in benthic autotroph metabolic responses across
an upwelling gradient in Colombia [82]. Using comparable methodologies, they found that
upwelling increased photosynthetic rates in turf algae, decreased rates in corals and inconsis-
tently affected photosynthesis in macroalgae and coralline algae [82]. Though surface ocean
productivity is key in the biophysical coupling of large-scale oceanography with benthic com-
munities, it does not appear to be directly linked to patterns in benthic autotroph ecophysiol-
ogy, at least in short-term physiological assessments. Future experiments should incorporate
growth rates, net community metabolism, and other more integrated measures of benthic pro-
duction to better understand the relationship between surface ocean production and benthic
ecosystem processes. PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Conclusions Here we document, for the first time, the ecophysiology of abundant benthic autotrophs on
coral reefs of the SLI in the remote central Pacific. We illustrate that the SLI span a conspicu-
ous gradient of increasing surface ocean production and inorganic nutrient availability (DIN,
DIP) that indicate the SLI become increasingly exposed to upwelling with decreasing distance
to the equator (i.e., latitude). Though we predicted that coral and algal metabolism (photosyn-
thesis, respiration) and photophysiology (maximum quantum yield, photosynthetic pigments)
would increase with greater exposure to upwelling, we found evidence of this in only some
parameters. The CCA, Porolithon, generally responded positively with greater exposure to
nutrient availability, particularly with respect to metabolism and phycobilin pigment content. The green algae and corals also showed some indications of photophysiological enhancement,
but with more variability. The lack of a strong and consistent physiological response in all taxa
across this upwelling gradient indicates that other factors may be limiting photosynthesis and
photophysiology of benthic autotrophs in the SLI. PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 17 / 22 Upwelling effects on coral and algal ecophysiology The effects of nutrients on organismal physiology may have broader ecosystem implica-
tions, if physiological enhancement (or lack thereof) is indicative of primary production. Our
results would then suggest that benthic primary production may not always track with patterns
in surface ocean productivity. A limited number of studies on upwelling in coral reefs indicate
that inorganic nutrient delivery via upwelling or internal tides can increase surface ocean pro-
ductivity of oceanic islands and atolls [32], biomass of fish [2], and heterotrophy in corals [40]. However, the link between increased surface productivity due to upwelling and benthic pro-
ductivity remains unclear. Unraveling the role of natural nutrient sources in shaping coral
reefs may be one key to improving our understanding of how coral reef community structure
and function will change as humans continue to alter natural nutrient landscapes. Supporting information S1 Table. Coordinates, land area, and benthic taxa sampled at the five Southern Line
Islands in the Republic of Kiribati. Islands closest to the equator have higher inorganic nutri-
ent concentrations due to equatorial upwelling. In all tables and figures, islands are listed from
south to north in order of increasing proximity to the equator. (DOCX) S3 Table. ANOVA table of island effects on ecophysiology by genera. Significance at
p < 0.05 is noted in bold.
(DOCX) S4 Table. Raw physiological data of corals and algae across the Southern Line Islands. Raw
physiological data of coral and algae replicates from incubations, quantum yield measure-
ments, and pigment content. (XLSX) Acknowledgments We are grateful to the Environment and Conservation Division of the Republic of Kiribati for
allowing us to conduct research in the Southern Line Islands. We thank the captain and crew
of the M/Y Hanse Explorer for logistical support and field assist. We also thank U. Abeysekera,
A. Emanuel, E. Jacobs, E. Juhlin, J. Le, and A. Vawter for assistance in the lab, as well as A. Andersson, J. Kohn, and J. Leichter for feedback on an earlier draft of the manuscript. We are
grateful to the support and assistance of F. Rohwer during the cruise and for his contributions
to an earlier draft of the manuscript. We thank the Moore Family Foundation, the Gordon
and Betty Moore Foundation, the Scripps family, and the anonymous donors who helped
make this research possible with their financial support. Author Contributions Conceptualization: Maggie D. Johnson, Michael D. Fox, Emily L. A. Kelly, Jennifer E. Smith. Data curation: Maggie D. Johnson, Michael D. Fox. Formal analysis: Maggie D. Johnson, Michael D. Fox. Funding acquisition: Stuart A. Sandin, Jennifer E. Smith. 18 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0228448
February 4, 2020 Upwelling effects on coral and algal ecophysiology Investigation: Maggie D. Johnson, Michael D. Fox, Emily L. A. Kelly, Brian J. Zgliczynski, Stu-
art A. Sandin, Jennifer E. Smith. Methodology: Maggie D. Johnson, Michael D. Fox, Jennifer E. Smith. Project administration: Brian J. Zgliczynski, Stuart A. Sandin, Jennifer E. Smith. Resources: Brian J. Zgliczynski, Stuart A. Sandin, Jennifer E. Smith. Resources: Brian J. Zgliczynski, Stuart A. Sandin, Jennifer E. Smith. Supervision: Jennifer E. Smith. Supervision: Jennifer E. Smith. Validation: Maggie D. Johnson. Validation: Maggie D. Johnson. Visualization: Maggie D. Johnson, Michael D. Fox. Visualization: Maggie D. Johnson, Michael D. Fox. Writing – original draft: Maggie D. Johnson, Michael D. Fox, Emily L. A. Kelly, Jennifer E. Smith. Writing – review & editing: Maggie D. Johnson, Michael D. Fox, Emily L. A. Kelly, Brian J. Zgliczynski, Stuart A. Sandin, Jennifer E. Smith. References 1. Elser JJ, Bracken MES, Cleland EE, Gruner DS, Harpole WS, Hillebrand H, et al. Global analysis of
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Тестування інформаційно-керуючої системи з різносенсорними каналами інформаційної взаємодії на основі методу прямого моделювання із зовнішнім еталоном
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Received: August 2, 2023 | Revised: August 19, 2023 | Accepted: August 31, 2023 Received: August 2, 2023 | Revised: August 19, 2023 | Accepted: August 31, 2023 DOI: 10.33445/sds.2023.13.4.5 Теоретична цінність: основними результатами дослідження за
тематикою статті є: синтез аналітичної моделі процесу
тестування класу інформаційно-керуючих систем з
різносенсорними каналами інформаційної взаємодії в
умовах впливу негаусових завад. Paper type: theoretical (computational and analytical). Paper type: theoretical (computational and analytical). Paper type: theoretical (computational and analytical). Тип статті: теоретична (розрахунково-аналітичний). DOI: 10.33445/sds.2023.13.4.5 Мета роботи: полягає в обґрунтуванні аналітичного підходу до
моделювання процесу тестування класу інформаційно-
керуючих
систем
з
різносенсорними
каналами
інформаційної взаємодії в умовах впливу негаусових
завад. Purpose: the purpose of the article is to substantiate the analytical
approach to modeling the process of testing a class of
information-control systems with multi-sensory channels of
information interaction under the influence of non-Gaussian
disturbances. Мета роботи: полягає в обґрунтуванні аналітичного підходу до
моделювання процесу тестування класу інформаційно-
керуючих
систем
з
різносенсорними
каналами
інформаційної взаємодії в умовах впливу негаусових
завад. Method: the main research method is the method of direct modeling
based on the orthogonality theorem for the case of a
compatible system with an external standard. Метод дослідження: основним методом дослідження є метод
прямого
моделювання
на
основі
теореми
ортогональності для випадку сумісної системи із
зовнішнім еталоном. Findings: the paper presents the results of modeling the process of
testing a class of information-control systems with multi-
sensory channels of information interaction under the
influence of non-Gaussian interference for the case of a
compatible system with an external standard based on
synthesized analytical expressions. Findings: the paper presents the results of modeling the process of
testing a class of information-control systems with multi-
sensory channels of information interaction under the
influence of non-Gaussian interference for the case of a
compatible system with an external standard based on
synthesized analytical expressions. Результати дослідження: в роботі представлено результати
моделювання процесу тестування класу інформаційно-
керуючих
систем
з
різносенсорними
каналами
інформаційної взаємодії в умовах впливу негаусових
завад для випадку сумісної системи із зовнішнім
еталоном на основі синтезованих аналітичних виразів. Theoretical implications: the main results of the research on the topic
of the article are: synthesis of an analytical model of the
process of testing a class of information-control systems
with multi-sensory channels of information interaction
under the influence of non-Gaussian disturbances. Theoretical implications: the main results of the research on the topic
of the article are: synthesis of an analytical model of the
process of testing a class of information-control systems
with multi-sensory channels of information interaction
under the influence of non-Gaussian disturbances. Theoretical implications: the main results of the research on the topic
of the article are: synthesis of an analytical model of the
process of testing a class of information-control systems
with multi-sensory channels of information interaction
under the influence of non-Gaussian disturbances. ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 Ключові слова: інформаційна система, Негаусова завада, тестування
системи, адаптація, аналітичне моделювання.
Key words: information system, non-Gaussian interference, system
testing, adaptation, analytical modeling. Тестування інформаційно-керуючої системи з
різносенсорними каналами інформаційної взаємодії
на основі методу прямого моделювання із зовнішнім
еталоном Testing of the information control system with
multi-sensory channels of information
interaction based on the method of direct
modeling with an external reference Денис Котов А
Denys Kotov А
Corresponding author: ад’юнкт, e-mail: zvyagel.zt@ukr.net, ORCID:
0000-0002-6775-5593
Corresponding author: PhD student, e-mail: zvyagel.zt@ukr.net,
ORCID: 0000-0002-6775-5593
Віктор Клименко A
Viktor Klymenko A
к. тех. н.,
старший
науковий
співробітник,
e-mail:
viktorklymenko1971@gmail.com, ORCID: 0000-0002-8073-4404
Candidate of Technical Sciences, Senior Researcher, e-mail:
viktorklymenko1971@gmail.com, ORCID: 0000-0002-8073-4404
А Військова академія м. Одеса, Україна
А Military Academy, Odesa, Ukraine А Military Academy, Odesa, Ukraine А Military Academy, Odesa, Ukraine А Військова академія м. Одеса, Україна ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 -9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 У відповідності зі стандартом [1], впровадження таких систем відповідає різним рівням
автоматизації автомобіля. Як правило, ці системи відносяться до типу систем Advanced Driver
Assistance Systems (ADAS), призначені для надання допомоги водію у керуванні транспортним
засобом. Системи допомоги водію ADAS можливо віднести до інформаційно-керуючих систем
(ІКС), в яких управлінське рішення залишається за водієм. Але для випадку безпілотного режиму
руху автомобіля, ІКС типу ADAS потребують удосконалення в частині забезпечення їх стійкого
функціонування в умовах впливів дестабілізуючих факторів. 1. Вступ Розвиток сучасних зразків автомобільної техніки характеризується високим рівнем
автоматизації процесів моніторингу стану систем та механізмів автомобіля, умов його руху та
визначення стану водія. Впровадження таких систем у першу чергу направлене на підвищення
безпеки автомобіля як механічного транспортного засобу, що приймає участь у дорожньому
русі та є об’єктом небезпеки. Наявність систем, що автоматизують функції водія, забезпечують
підвищення безпекового рівня у керуванні автомобілем у пілотному його режимі шляхом
надання водію більшого об’єму інформації для прийняття управлінського рішення. Engineering and Technology Engineering and Technology 48 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 2. Теоретичні основи дослідження У наш час у світі розроблено і широко використовуються робототехнічні системи (РС) у різних
сферах життєдіяльності. Роботизація замінила людей на небезпечних, для життя та здоров’я,
сферах та у важких умовах праці. Розглянемо деяку активну робототехнічну систему, що містить інформаційні пристрої
та системи (ІПС) і яка взаємодіє із зовнішнім середовищем (Рис.1). Інтелектуальні здібності в таких РС визначаються керуючою та інформаційно-
вимірювальною підсистемами, а виконавчі та комунікативні здібності реалізуються під
системами зв’язку та керування [2]. Відмінними рисами РС є їх універсальність, здатність до навчання та адаптації в процесі
сприйняття вхідної інформації та вплив на навколишнє середовище (за допомогою виконавчих
механізмів), а також багатоцільове призначення, пов'язане з автоматизацією фізичної та
інтелектуальної діяльності оператора [2]. Залежно від розвиненості сенсорної системи та
системи управління РС діляться на чотири покоління [1-3]. Рисунок 1 – Узагальнена структурно-функціональна схема робототехнічної системи,
що взаємодіє з навколишнім середовищем
Система зв’язку
«Мова»
Інформаційно-
вимірювальна система
«Сенсорика»
Керуюча система
«Мозок»
Виконавча система
«Моторика»
Зовнішнє середовище
Робототехнічна система Система зв’язку
«Мова» Інформаційно-
вимірювальна система
«Сенсорика» Керуюча система
«Мозок» Зовнішнє середовище Рисунок 1 – Узагальнена структурно-функціональна схема робототехнічної системи,
що взаємодіє з навколишнім середовищем Припустимо, що ця активна робототехнічна система має апріорну інформацію про
середовище та в процесі функціонування отримує поточну інформацію як про зовнішнє
середовище, так і про власний стан. Метою активної робототехнічної системи є прийняття
рішень, пов’язаних із перетворенням чи аналізом інформації щодо навколишнього
середовища. Найчастіше інформація буває неповною, тому робототехнічна система
функціонує за умов невизначеності. У зв’язку з цим під адаптацією розумітимемо здатність
активної робототехнічної системи досягати заданих цілей в умовах невизначеності на основі Engineering and Technology Engineering and Technology 49 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 використання поточної інформації про власний стан та стан середовища з об’єктами, При
цьому можуть змінюватися параметри робототехнічної системи, її структура та алгоритм
функціонування [4]. Залежно від рівня автоматизації та сукупності функцій керування автомобілем
ускладнюється сам процес формування та обробки первинної інформації про навколишнє
середовище і, безпосередньо, про автомобіль як технічний пристрій, щоб розробити варіанти
його поведінки в безпілотному режим роботи (руху) [4]. Таким чином, для вирішення проблеми автономного керування, починаючи з третього-
четвертого рівнів автоматизації, ключовим завданням є створення ІКС, модулі якої мали б
можливість реалізації алгоритмів (моделей) багатосенсорного інформаційного масиву із
заданим ступенем якості при апріорній невизначеності щодо структури вхідного
інформаційного масиву, який апріорі є випадковим [4-5]. 2. Теоретичні основи дослідження Функціонально такого типу ІКС повинні мати можливість реалізації стійкого процесу
формування та фільтрації інформаційних процесів незалежно від моделі корисного сигналу та
моделей шумових завад [4-5]. Таким чином структурно та функціонально інформаційно-керуюча система автомобіля
починаючи з четвертого рівня автоматизації повинна забезпечувати вирішення ряду підзадач:
сприйняття (реєстрацію) первинної інформації – вхідного інформаційного процесу від
підсистеми різносенсорних датчиків, формування моделі ситуації (зображення об’єкта ) на
основі вхідного інформаційного процесу з прийнятним рівнем якості, пошук та класифікацію
об’єктів середовища функціонування, визначення цих об’єктів в робочій зоні, визначення
орієнтації об’єктів у просторі та вимірювання їх характерних параметрів. Тому в контексті постановки мети дослідження системного значення набуває питання
формалізації предметної області функціонування роботизованого автомобіля, інформаційна
система якого являє собою інформаційно-керуючою систему з різносенсорними каналами
інформаційної взаємодії, яка функціонує в умовах впливу дестабілізуючих факторів (рис. 2). Система формалізації передбачає визначення процесної моделі спостережень та
застосування мови просторово-часового опису умов виконання операції 𝑤∈𝑊, стану об’єкта
роботизації та впливу на нього зовнішніх і внутрішніх збурень. Прийнятий в процесній моделі
підхід виходить з вибраного критерію ефективності 𝐽𝐺 або заданої цільової функції в наступній
інформаційно-ймовірнісній інтерпретації вихідних даних. Зокрема: − сукупність апріорно невідомих параметрів процесу технологічної взаємодії
різносенсорної системи роботизованого автомобіля з електромагнітними джерелами
інформації середовища експлуатації представляється 𝑚-мірним вектором 𝑋= 𝑋𝑚×1. В
прив’язці до реальної ситуації припускається існування зовнішніх перешкод 𝜂(𝑡) і носія
інформаційних параметрів, так званого корисного сигналу 𝑆(𝑡), які описуються k -мірним
вектором 𝜂= 𝜂𝑘×1 з 𝑚𝜂= 𝑀(𝜂) = 0 і 𝐴𝜂= 𝑀(𝜂 𝜂т) та k -мірним вектором 𝑆= 𝑆𝑘×1 з 𝑚𝑆=
𝑀(𝑆) і 𝐴𝑆= 𝑀(𝑆𝑆т), відповідно; − результат спостереження процесу взаємодії різносенсорної системи незалежних
датчиків 𝐷𝑋 об’єкта роботизації з сукупністю вхідних реалізацій 𝑋 описується за правилами
векторно-матричних перетворень: (1) 𝑈= 𝐷𝑋(𝑆+ 𝜂 ) + 𝑛,
(1) де
𝐷𝑋= 𝐷𝑋(𝑚×𝑘)
– матриця передаточних функцій системи різносенсорних датчиків;
𝑛= 𝑛𝑚×1
– 𝑚-мірний вектор внутрішніх шумів системи датчиків (незалежних
помилок
вимірювання),
який
має
нульове
математичне
сподівання 𝑚𝑛= 𝑀(𝑛) = 0 та одиничну матрицю кореляції 𝐴𝑛=
𝑀(𝑛𝑛т) = 𝐼; триця передаточних функцій системи різносенсорних датчиків; 50 Engineering and Technology Engineering and Technology Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 − передбачається, що вектори корисного сигналу 𝑆, зовнішніх перешкод 𝜂 та
внутрішніх шумів системи 𝑛 некорельовані між собою, тобто: 𝑀[𝜂𝑛т]
= 0; 𝑀[𝑆𝑛т]
= 0;
𝑀[𝑆т𝜂]
= 0, де 0 = 0𝑘×𝑚 – прямокутна нульова (
)
m
k
-мірна матриця. 2. Теоретичні основи дослідження − передбачається, що вектори корисного сигналу 𝑆, зовнішніх перешкод 𝜂 та
внутрішніх шумів системи 𝑛 некорельовані між собою, тобто: 𝑀[𝜂𝑛т]
= 0; 𝑀[𝑆𝑛т]
= 0;
𝑀[𝑆т𝜂]
= 0, де 0 = 0𝑘×𝑚 – прямокутна нульова (
)
m
k
-мірна матриця. Рисунок 2 – Приклад функціональної схеми інформаційної системи РА
Інформаційно-
вимірювальні
пристрої
Датчики зовнішнього
середовища
(інформація про місцевість,
дорожню обстановку,
маршрут руху)
Вимірювачі
положення та стану
транспортного
засобу
Пристрій введення
зовнішньої
інформації
ІНФОРМАЦІЙНО-КЕРУЮЧА СИСТЕМА АВТОМОБІЛЯ З
РІЗНОСЕНСОРНИМИ КАНАЛАМИ ІНФОРМАЦІЙНОЇ ВЗАЄМОДІЇ
Планування руху
(маршруту, вироблення локальних цілей, орієнтування, вибір напрямку
руху, прогнозування дорожньої обстановки)
Вироблення керуючих впливів
(команд на зміну швидкості та
напряму руху)
Система безпеки
руху
Керуючий
пристрій
Підсилювально-
привідні
пристрої
Керування
швидкістю руху
Керування
напрямом руху
Керування
двигуном,
трансмісією
Керування
гальмами
Вузли шасі
Двигун
Трансмісія
Гальма
Рульовий механізм Датчики зовнішнього
середовища
(інформація про місцевість,
дорожню обстановку,
маршрут руху) Вимірювачі
положення та стану
транспортного
засобу Інформаційно-
вимірювальні
пристрої Пристрій введення
зовнішньої
інформації ІНФОРМАЦІЙНО-КЕРУЮЧА СИСТЕМА АВТОМОБІЛЯ З
РІЗНОСЕНСОРНИМИ КАНАЛАМИ ІНФОРМАЦІЙНОЇ ВЗАЄМОДІЇ Планування руху
(маршруту, вироблення локальних цілей, орієнтування, вибір напрямку
руху, прогнозування дорожньої обстановки) Керуючий
пристрій Вироблення керуючих впливів
(команд на зміну швидкості та
напряму руху) Система безпеки
руху Керування
напрямом руху Керування
швидкістю руху Підсилювально-
привідні
пристрої Керування
двигуном,
трансмісією Керування
гальмами Вузли шасі Рисунок 2 – Приклад функціональної схеми інформаційної системи РА Зазначена інформаційно-ймовірнісна інтерпретація моделі даних враховує власну
надлишковість модельного варіанту роботизованого автомобіля та надмірність середовища
його експлуатації. Інформаційна надлишковість вводиться в результат спостереження 𝑈 таким
чином, щоб оцінка параметрів 𝑆̃ невідомого вектору 𝑆 задовольняла критерію оптимальності
в евклідовій метриці, зокрема, мінімуму середньоквадратичній похибки наближення: 𝜀= 𝑀{‖𝑆−𝑆̃‖
2} = 𝑚𝑖𝑛,
(2) (2) де ‖•‖ – норма вектору, що описує параметри корисного сигналу. Оцінка параметрів 𝑆̃ знаходиться у класі лінійних перетворень шляхом об’єднання
(комплексування)
результатів
спостереження
𝑈
на
виході
системи
незалежних
різносенсорних датчиків за наступним алгоритмом Engineering and Technology 51 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 𝑆̃ = 𝐹𝐷
−1(𝑈) = 𝐹𝐷
−1[𝐹(𝑋)] = 𝑊𝑋𝑈, (3) 𝑊𝑋= 𝑊𝑋(𝑘×𝑚)
– матрична передавальна характеристика системи об’єднання
результатів спостереження 𝑈, яка оптимальна за критерієм (2). 𝑊𝑋= 𝑊𝑋(𝑘×𝑚)
– матрична передавальна характеристика системи об’єднання
результатів спостереження 𝑈, яка оптимальна за критерієм (2). Схематична інтерпретація алгоритмів (1) і (3) в узагальненому вигляді наведена на рис. 5. 2. Теоретичні основи дослідження Схематична інтерпретація алгоритмів (1) і (3) в узагальненому вигляді наведена на рис. 5. Узагальненість даної схеми полягає у тому, що в ній, поряд із зовнішніми факторами 𝜂, Схематична інтерпретація алгоритмів (1) і (3) в узагальненому вигляді наведена на рис. 5. Узагальненість даної схеми полягає у тому, що в ній, поряд із зовнішніми факторами 𝜂,
відображуються і внутрісистемні фактори дестабілізації 𝛥𝑊, які в алгоритмах (1) і (3) на даний
момент відсутні. Узагальненість даної схеми полягає у тому, що в ній, поряд із зовнішніми факторами 𝜂,
відображуються і внутрісистемні фактори дестабілізації 𝛥𝑊, які в алгоритмах (1) і (3) на даний
момент відсутні. Рисунок 3 – Схема формування оцінки параметрів корисного сигналу
𝑋= 𝑆+ 𝜂
𝐷𝑋
𝛴
𝑈
𝑛
𝛥𝑊
𝑆̃
𝑊𝑋 Рисунок 3 – Схема формування оцінки параметрів корисного сигналу Визначення передавальної характеристики системи 𝑊𝑋 базується на умові
ортогональності вектору результату спостереження 𝑈 та вектору незалежних похибок
вимірювання 𝐸= 𝑆−𝑆̃: 𝑀{[𝑆𝑘×1 −𝑆̃𝑘×1] 𝑈1×𝑚
т
}
= 0𝑘×𝑚,
(4) (4) Підставляючи до (4) значення оцінки (3), отримуємо систему рівнянь для визначення
оптимальної передавальної характеристики 𝑊𝑋: Підставляючи до (4) значення оцінки (3), отримуємо систему рівнянь для визначення
оптимальної передавальної характеристики 𝑊𝑋: 𝑀{[𝑆−𝑆̃] 𝑈т}
= 𝑊𝑋 𝑀{𝑈𝑈т}. (5) (5) Рішення зазначеної системи рівнянь являє собою класичну обернену задачу, за
результатами розв’язання якої визначається передавальна характеристика системи
об’єднання різносенсорних інформаційних каналів, зокрема: 𝑊𝑋(𝑘×𝑚) = 𝛼𝑘×𝑚 𝐴𝑚×𝑚
−1
,
(6) (6) де 𝐴𝑚×𝑚= 𝑀{𝑈𝑚×1𝑈1×𝑚
т
} – кореляційна матриця процесу спостереження;
𝛼𝑘×𝑚
– прямокутна матриця еталонних векторів у вигляді 𝛼𝑘×𝑚=
(
𝛼1,(1×𝑚)
т
𝛼2,(1×𝑚)
т
⋮
𝛼𝑘,(1×𝑚)
т
)
. (7) (7) Підставивши вираз (6) до (3), отримаємо значення лінійної незміщеної оцінки вектору
корисного сигналу: 52
Engineering and Technology 52 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 𝑆̃ = 𝑆̃𝑘×1 = 𝛼𝑘×𝑚 𝐴𝑚×𝑚
−1
𝑈𝑚×1,
(8) 𝑆̃ = 𝑆̃𝑘×1 = 𝛼𝑘×𝑚 𝐴𝑚×𝑚
−1
𝑈𝑚×1, 𝑆̃ = 𝑆̃𝑘×1 = 𝛼𝑘×𝑚 𝐴𝑚×𝑚
−1
𝑈𝑚×1,
(8) (8) з мінімальною похибкою вимірювання
U
A
α
S
S
S
E
1
min
~
−
−
=
−
=
. з мінімальною похибкою вимірювання
U
A
α
S
S
S
E
1
min
~
−
−
=
−
=
. з мінімальною похибкою вимірювання
U
A
α
S
S
S
E
1
min
~
−
−
=
−
=
. Engineering and Technology 2. Теоретичні основи дослідження 4, – 2023 Для першої ситуації, коли надлишковість системи датчиків відсутня 𝑚< 𝑘, оцінка
середньоквадратичної похибки визначається за формулою: Для першої ситуації, коли надлишковість системи датчиків відсутня 𝑚< 𝑘, оцінка
середньоквадратичної похибки визначається за формулою: 𝜀̃|(𝑚< 𝑘)(𝑘−𝑚) 1
𝑞𝑆𝑡𝑟[𝐷𝑋(𝑚×𝑘) 𝐷𝑋(𝑘×𝑚)
т
]
−1
𝑚𝑖𝑛
,
(11) (11) де
S
q – співвідношення сигнал/шум в інформаційному каналі. де
S
q – співвідношення сигнал/шум в інформаційному каналі. За альтернативної ситуації, коли має місце надлишковість системи датчиків 𝑚> 𝑘,
оцінка середньоквадратичної похибки набуває такого значення 𝜀̃|(𝑚> 𝑘) 1
𝑞𝑆𝑡𝑟[𝐷𝑋(𝑘×𝑚)
т
𝐷𝑋(𝑚×𝑘)]
−1
𝑚𝑖𝑛
. (12) (12) Для ситуації 𝑚= 𝑘 оцінка середньоквадратичної похибки складає 𝜀̃|(𝑚= 𝑘) 1
𝑞𝑆𝑡𝑟[𝐷𝑋(𝑚×𝑚)
-1
𝐷𝑋(𝑚×𝑚)
-1
]
1
𝑞𝑆‖𝐷𝑋(𝑚×𝑚)
-1
‖
2
𝑚𝑖𝑛
. (13) (13) Виконавши ряд стандартних перетворень і трансформацій з (11) – (13), отримаємо
систему рівнянь для ситуаційного аналізу оцінки середньоквадратичної похибки в умовах
впливу дестабілізуючих факторів зовнішнього і внутрісистемного походження. Зокрема: 𝜀̃|(𝑚< 𝑘)(𝑘−𝑚)
𝑞𝜂
𝑞𝑆𝑡𝑟[𝐷𝑋(𝑚×𝑘)𝐷𝑋(𝑘×𝑚)
т
]
−1
𝑊𝑋
2
(𝑚, 𝑘)|(𝑚< 𝑘)
𝑚𝑖𝑛
,
(14)
𝜀̃|(𝑚> 𝑘)
𝑞𝜂
𝑞𝑆𝑡𝑟[𝐷𝑋(𝑘×𝑚)
т
𝐷𝑋(𝑚×𝑘)]
−1
𝑊𝑋
2
(𝑚, 𝑘)|(𝑚> 𝑘)
𝑚𝑖𝑛
,
(15)
𝜀̃|(𝑚= 𝑘)
𝑞𝜂
𝑞𝑆‖𝐷𝑋(𝑚×𝑚)
-1
‖
2
𝑊𝑋
2
(𝑚, 𝑘)|(𝑚= 𝑘)
𝑚𝑖𝑛
,
(16) |(𝑚< 𝑘)(𝑘−𝑚)
𝑞𝜂
𝑞𝑆𝑡𝑟[𝐷𝑋(𝑚×𝑘)𝐷𝑋(𝑘×𝑚)
т
]
−1
𝑊𝑋
2
(𝑚, 𝑘)|(𝑚< 𝑘)
𝑚𝑖𝑛
,
(14) (14) 𝜀̃|(𝑚> 𝑘)
𝑞𝜂
𝑞𝑆𝑡𝑟[𝐷𝑋(𝑘×𝑚)
т
𝐷𝑋(𝑚×𝑘)]
−1
𝑊𝑋
2
(𝑚, 𝑘)|(𝑚> 𝑘)
𝑚𝑖𝑛
,
(15)
𝜀̃|(𝑚= 𝑘)
𝑞𝜂
𝑞𝑆‖𝐷𝑋(𝑚×𝑚)
-1
‖
2
𝑊𝑋
2
(𝑚, 𝑘)|(𝑚= 𝑘)
𝑚𝑖𝑛
,
(16) 𝜀̃|(𝑚> 𝑘)
𝑞𝜂
𝑞𝑆𝑡𝑟[𝐷𝑋(𝑘×𝑚)
т
𝐷𝑋(𝑚×𝑘)]
−1
𝑊𝑋
2
(𝑚, 𝑘)|(𝑚> 𝑘)
𝑚𝑖𝑛
,
(15) (15) 𝜀̃|(𝑚= 𝑘)
𝑞𝜂
𝑞𝑆‖𝐷𝑋(𝑚×𝑚)
-1
‖
2
𝑊𝑋
2
(𝑚, 𝑘)|(𝑚= 𝑘)
𝑚𝑖𝑛
,
(16) (16) де
𝑞𝜼
– перевищення потужності перешкоди зовнішнього середовища над
рівнем потужності внутрішніх шумів системи датчиків;
𝜎𝑾𝑿
2
– відносний рівень дестабілізуючих внутрісистемних збурень;
𝑓(𝑚, 𝑘) – функція чутливості системи інформаційних датчиків до дестабілізуючих
внутрісистемних збурень за умовах (𝑚< 𝑘), (𝑚> 𝑘) або (𝑚= 𝑘). 𝑞𝜼
– перевищення потужності перешкоди зовнішнього середовища над
рівнем потужності внутрішніх шумів системи датчиків;
𝜎2
відносний рівень дестабілізуючих внутрісистемних збурень; де р
у
у р
у
д
;
𝑾𝑿
2
– відносний рівень дестабілізуючих внутрісистемних збурень; 2. Теоретичні основи дослідження Кореляційна матриця похибки вимірювання 𝐸 з урахуванням раніше визначених
статистичних моментів має наступний вигляд Кореляційна матриця похибки вимірювання 𝐸 з урахуванням раніше визначених
статистичних моментів має наступний вигляд 𝑀(𝐸𝐸т) = 𝐴𝑆−𝛼 𝐴−1𝛼 т. (9) (9) Слід кореляційної матриці 𝑡𝑟{𝑀(𝐸𝐸т)} виражає найменше з можливих значень
середньоквадратичної похибки: 𝜀= 𝑡𝑟{𝑀(𝐸𝐸т)} . Слід кореляційної матриці 𝑡𝑟{𝑀(𝐸𝐸т)} виражає найменше з можливих значень
середньоквадратичної похибки: 𝜀= 𝑡𝑟{𝑀(𝐸𝐸т)} . Модельний варіант об’єднання за алгоритмом (9) різносенсорних каналів
інформаційної системи роботизованого автомобіля в умовах апріорної невизначеності
середовища його перебування, представлено на рис. 3. Відображуючи оптимальну структуру об’єднання даних, модельний варіант алгоритму
об’єднання (рис. 4) передбачає операцію «відбілювання» 𝐴𝑚×𝑚
−1
зашумлених реалізацій на
виході системи датчиків 𝑈𝑚×1 та операцію порівняння отриманого результату з вектором
еталонів 𝛼𝑚×𝑘 (апріорних образів). За наслідками виконання цих операцій формується
оптимальна оцінка 𝑆̃𝑘×1 вектору стану зовнішнього середовища. Оптимальність оцінки вектору 𝑆̃𝑘×1 проаналізуємо у відповідності до трьох ситуацій: 𝑆̃𝑘×1 проаналізуємо у відповідності до трьох ситуацій: − перша ситуація – кількість вимірювань 𝑚 (датчиків інформації) менше кількості
вимірювальних параметрів 𝑘, тобто 𝑚< 𝑘; − перша ситуація – кількість вимірювань 𝑚 (датчиків інформації) менше кількості
вимірювальних параметрів 𝑘, тобто 𝑚< 𝑘; − друга ситуація – кількість вимірювань 𝑚 (датчиків інформації) більше кількості
вимірювальних параметрів k , тобто
k
m
; − третя ситуація – кількості вимірювань m (датчиків інформації) і дорівнює кількості
вимірювальних параметрів k , тобто
k
m =
. − третя ситуація – кількості вимірювань m (датчиків інформації) і дорівнює кількості
вимірювальних параметрів k , тобто
k
m =
. Дослідження
зазначених
ситуацій проводяться
на
основі
відносної
оцінки
середньоквадратичної похибки параметрів вектору 𝑆𝑘×1: (
)
2
2
2
т
min
~
tr
~
S
S
S
S
E
E
−
=
=
M
M
. Рисунок 4 – Схема оптимальної системи об’єднання різносенсорних каналів РА (
)
2
2
2
т
min
~
tr
~
S
S
S
S
E
E
−
=
=
M
M
. (10) (10) Рисунок 4 – Схема оптимальної системи об’єднання різносенсорних каналів РА Engineering and Technology 53 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023
Для першої ситуації, коли надлишковість системи датчиків відсутня 𝑚< 𝑘, оцінка
середньоквадратичної похибки визначається за формулою: 2
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 3. Постановка проблеми Дослідження проблематики завдань стійкого функціонування інформаційно-керуючих систем
із різносенсорними каналами інформаційної взаємодії, як підкласу інформаційних систем які
функціонують в умовах впливу дестабілізуючих факторів, лежить в площині удосконалення
існуючих і синтезу нових методів та алгоритмів якісної фільтрації інформаційних процесів на
тлі широкого спектру моделей завад. Існуючі моделі та алгоритми фільтрації інформаційних процесів в інформаційно-
керуючих системах, як правило, базуються на рішеннях задач з гаусовою формою моделей
завад. При цьому, ефективність роботи таких систем по критерію співвідношення Engineering and Technology 54 Engineering and Technology 42
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 сигнал/завада+шум, де модель завади має негаусову форму, а в моделі шуму адитивно
об’єднані внутрішній шум та внутрішньосистемні збурення є не ефективною. Тому дослідження підходів, на основі методів теорії лінійної алгебри, щодо синтезу
аналітичних моделей обробки (фільтрації) інформаційних процесів в інформаційно-керуючих
системах з різносенсорними каналами інформаційної взаємодії є актуальним науковим
завданням, в сенсі розробки методів тестування такого класу систем в умовах впливу
негаусових завад на етапі їх синтезу. 4. Результати 4, – 2023 дестабілізуючих факторів в процесі передачі сформованого вихідного інформаційного
процесу на пункт приймання для подальшого прийняття управлінського рішення оператором д
у
ф
р
р ц
р д
ф р
д
ф р
ц
процесу на пункт приймання для подальшого прийняття управлінського рішення оператором
В цьому випадку вихідний процес 𝑌(𝑛, 1) можна інтерпретувати моделлю виду (1) а
його оцінку моделлю (3) за схемою на рис. 3. В цьому випадку вихідний процес 𝑌(𝑛, 1) можна інтерпретувати моделлю виду (1) а
його оцінку моделлю (3) за схемою на рис. 3. Отже, для нашого варіанту узагальненої задачі результат фільтрації процесу: 𝑋(𝑚, 1):
буде мати вид: 𝑋̂(𝑚, 1) = 𝑊(𝑚, 𝑛)𝑌(𝑛, 1) =
= 𝑊(𝑚, 𝑛)𝐻(𝑛, 𝑚)𝑋(𝑚, 1) + 𝑊(𝑚, 𝑛)𝑛(𝑛, 1) =
= 𝑄(𝑚, 𝑚)𝑋(𝑚, 1) + 𝑊(𝑚, 𝑛)𝑛(𝑛, 1)
(18) (18) Що відповідає моделі оцінки результатів фільтрації в (3) за схемою на рис.3 без
врахування внутрісистемних дестабілізуючих факторів. Що відповідає моделі оцінки результатів фільтрації в (3) за схемою на рис.3 без
врахування внутрісистемних дестабілізуючих факторів. Але реальні умови фільтрації вхідного процесу далекі від ідеальних умов, тому необхідно
проводити фільтрацію з врахуванням впливу всіх дестабілізуючих факторів: впливу зовнішніх
факторів 𝜂, внутрісистемних факторів дестабілізації 𝛥𝑊 та фактору внутрішніх шумів 𝑛. ф
р
𝜂
у р
ф
р
ф
ру
у р
у
В такому випадку результат фільтрація за схемою (рис.5,б) в умовах внутрішньо-
системних збурень параметричної матриці буде мати вид (19): 𝑋̃(𝑚, 1) = [𝑊(𝑚, 𝑛) + 𝛥𝑊(𝑚, 𝑛)]𝐻(𝑛, 𝑚)𝑋(𝑚, 1) +
+[𝑊(𝑚, 𝑛) + 𝛥𝑊(𝑚, 𝑛)]𝑛(𝑛, 1)
(19) (19) При цьому оцінка якості фільтрації буде проводитися через визначення помилки
фільтрації в умовах внутрішньої невизначеності за виразом 𝜀≜𝑋−𝑋̃ = 𝑋−(𝑊+ 𝛥𝑊)𝐻𝑋−(𝑊+ 𝛥𝑊)𝑛=
= [𝐼−(𝑊+ 𝛥𝑊)𝐻]𝑋−(𝑊+ 𝛥𝑊)𝑛
(20) (20) Випадкові процеси 𝛥𝑊, 𝑛 мають наступні статистичні властивості 𝛥𝑊:
Статистичні властивості 𝛥𝑊: Випадкові процеси 𝛥𝑊, 𝑛 мають наступні статистичні властивості 𝛥𝑊:
Статистичні властивості 𝛥𝑊: 1. 𝛥𝑊(𝑚, 𝑛) = 𝑂(𝑚, 𝑛);
2. 𝛥𝑊𝑇(𝑛, 𝑚)𝛥𝑊(𝑚, 𝑛) = 𝜎𝑤
2𝐼(𝑛, 𝑛); 1. 𝛥𝑊(𝑚, 𝑛) = 𝑂(𝑚, 𝑛);
2. 𝛥𝑊𝑇(𝑛, 𝑚)𝛥𝑊(𝑚, 𝑛) = 𝜎𝑤
2𝐼(𝑛, 𝑛); . 𝛥𝑊𝑇(𝑛, 𝑚)𝛥𝑊(𝑚, 𝑛) = 𝜎𝑤
2𝐼(𝑛, 𝑛); Статистичні властивості 𝑛⃗ : Статистичні властивості 𝑛⃗ : 1. 𝑛(𝑛, 1) = 𝑂(𝑛, 1); 𝑛𝑇(1, 𝑛)𝑛(𝑛, 1) = ‖𝑛(𝑛, 1)‖2 = 𝑃ш;
2. 𝑛(𝑛,1)𝑛𝑇(1,𝑛)
‖𝑛(1,𝑛)‖2
= 𝐼(𝑛, 𝑛) – “білий” шум Зауважимо, що вектори корисного сигналу, зовнішніх перешкод та внутрішніх шумів
системи некорельовані між собою, тобто статистичні зв’язки між процесами 𝛥𝑊, 𝑋 та 𝑛
відсутні. 4. Результати Для перевірки викладених теоретичних положень розглянемо варіант узагальненої задачі
фільтрації інформаційних процесів в умовах внутрішньо системних збурень (внутрішньо-
системної невизначеності) що циркулюють в інформаційно-керуючій системі роботизованого
автомобіля з різносенсорними каналами інформаційної взаємодії Тут 𝑋(𝑚, 1)– вхідний (тестовий) процес, що об’єднує в собі апріорну інформацію про
навколишнє середовище з об’єктами, яка поступає в інформаційно-керуючу систему по
різносенсорним каналам інформаційної взаємодії та об’єднується в єдиний інформаційний
вхідний процес за схемою представленою на рис. 4. На рисунку 5 схематично представлено процедури фільтрації вхідних процесів в
залежності від умов впливу дестабілізуючих факторів. Тут вихідний процес 𝑌(𝑛, 1), який являє собою результат обробки вхідного (тестового)
процесу 𝑋(𝑚, 1), формується за схемою представленою на рис. 5а,. у відповідності з виразом
(17) 𝑌(𝑛, 1) = 𝐻(𝑛, 𝑚)𝑋(𝑚, 1) + 𝑛(𝑛, 1)
(17) (17) де 𝑛(𝑛, 1) – адитивна шумова складова, яка ілюструє внутрішні шуми системи 𝐻(𝑛, 𝑚), що
формує випадковий процес 𝑌(𝑛, 1). де 𝑛(𝑛, 1) – адитивна шумова складова, яка ілюструє внутрішні шуми системи 𝐻(𝑛, 𝑚), що
формує випадковий процес 𝑌(𝑛, 1). а)
б)
Рисунок 5 – Схеми фільтрації вхідних процесів в залежності від умов впливу
дестабілізуючих факторів. б) а) б) а) Рисунок 5 – Схеми фільтрації вхідних процесів в залежності від умов впливу
дестабілізуючих факторів. Якість вихідного випадкового процесу 𝑌(𝑛, 1) буде залежати від передавальної
характеристики системи 𝐻(𝑛, 𝑚), адитивної шумової складової 𝑛(𝑛, 1) та внутрішньо-
системних збурень, що об’єктивно присутні в інформаційних системах [6-10]. Для випадку ідеальної системи формування вихідного випадкового процесу 𝑌(𝑛, 1)
результат буде максимально близьким до вхідного процесу 𝑋(𝑚, 1) [7]. В такому випадку вплив апріорної невизначеності щодо передавальної характеристики
системи 𝐻(𝑛, 𝑚) та дестабілізуючих факторів буде мінімальним і якість отриманого вихідного
процесу за критерієм (10) для ситуації сумісної системи за виразом (16) буде достатньою для
вироблення управлінського рішення. Тобто співвідношення сигнал/завада+шум буде
більшим за одиницю. У випадку недостатньої якості отриманого вихідного процесу 𝑌⃗ (𝑛, 1) виникає
необхідність фільтрації такого сигналу. Така необхідність виникає і у випадку впливу Engineering and Technology Engineering and Technology 55 Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. де 𝐴(𝑛, 𝑛) = 𝐻(𝑛, 𝑚)𝐻𝑇(𝑚, 𝑛). 3. Визначена система: 𝑚= 𝑛→ 4. Результати Тут відносна оцінка (21) середньоквадратичної похибки параметрів вектору корисного
сигналу буде мати вид: Тут відносна оцінка (21) середньоквадратичної похибки параметрів вектору корисного
сигналу буде мати вид: д
‖𝜀‖2 = 𝑃ш𝑡𝑟𝐴−1(𝑚, 𝑚) + 𝜎𝐻
2{(𝑛−𝑚)‖𝐴−1(𝑚, 𝑚)‖2𝑃ш +
+𝑡𝑟𝐴−1(𝑚, 𝑚)[𝑃ш𝑡𝑟𝐴−1(𝑚, 𝑚) + ‖𝑋(𝑚, 1)‖2]};
(22)
𝑇(𝑚, 𝑛)𝐻(𝑛, 𝑚) (22) де 𝐴(𝑚, 𝑚) = 𝐻𝑇(𝑚, 𝑛)𝐻(𝑛, 𝑚) де 𝐴(𝑚, 𝑚) = 𝐻𝑇(𝑚, 𝑛)𝐻(𝑛, 𝑚) де 𝐴(𝑚, 𝑚) = 𝐻𝑇(𝑚, 𝑛)𝐻(𝑛, 𝑚) 2. Перевизначена система: 𝑛> 𝑚; 𝑟𝑎𝑛𝑘𝐻(𝑛, 𝑚) = 𝑚
У цьому випадку матриця 𝑊(𝑚𝑛) буде рівна: 2. Перевизначена система: 𝑛> 𝑚; 𝑟𝑎𝑛𝑘𝐻(𝑛, 𝑚) = 𝑚 2. Перевизначена система: 𝑛> 𝑚; 𝑟𝑎𝑛𝑘𝐻(𝑛, 𝑚) = 𝑚 2. Перевизначена система: 𝑛> 𝑚; 𝑟𝑎𝑛𝑘𝐻(𝑛, 𝑚) = 𝑚 У цьому випадку матриця 𝑊(𝑚, 𝑛) буде рівна: У цьому випадку матриця 𝑊(𝑚, 𝑛) буде рівна: 𝑊(𝑚, 𝑛) = [𝐻𝑇(𝑚, 𝑛)𝐻(𝑛, 𝑚)]−1𝐻𝑇(𝑚, 𝑛). 𝑊(𝑚, 𝑛) = [𝐻𝑇(𝑚, 𝑛)𝐻(𝑛, 𝑚)]−1𝐻𝑇(𝑚, 𝑛). Тут відносна оцінка (21) середньоквадратичної похибки параметрів вектору корисного
сигналу буде мати вид: Тут відносна оцінка (21) середньоквадратичної похибки параметрів вектору корисного
сигналу буде мати вид: ‖𝜀‖2 = ‖𝑋(𝑚, 1)‖2 −‖𝑋̂(𝑚, 1)‖
2 + 𝑃ш𝑡𝑟𝐴−1(𝑛, 𝑛) +
+𝜎𝐻
2{(𝑚−𝑛)[𝑃ш‖𝐴−1(𝑛, 𝑛)‖2 + ‖𝐴−1(𝑛, 𝑛)𝐻(𝑛, 𝑚)𝑋(𝑚, 1)‖2] +
+𝑡𝑟𝐴−1(𝑛, 𝑛) [𝑃ш𝑡𝑟𝐴−1(𝑛, 𝑛) + ‖𝑋̂(𝑚, 1)‖
2]} ;
(23) (23) 4. Результати Як зазначається в (6, 7) рішення задачі (19) залежить від узгодженості розмірності
системи формування вихідного процесу𝑌(𝑛, 1) (схема рис. 5,а) або системи фільтрації (схема
рис. 5, 6) з розмірністю вхідного випадкового процесу 𝑋(𝑚, 1). Іншими словами на рішення
буде впливати визначеність системи в сенсі її узгодженості по розмірності з вхідним
випадковим процесом. Отже, подальше дослідження можливих ситуацій щодо визначеності інформаційно-
керуючої системи проводяться на основі відносної оцінки (21) середньоквадратичної похибки
параметрів вектору корисного сигналу. Engineering and Technology Engineering and Technology 56 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 ‖𝜀‖2 = 𝑋𝑇[𝐼−𝐻𝑇(𝑊𝑇+ 𝛥𝑊𝑇)][𝐼−(𝑊+ 𝛥𝑊)𝐻]𝑋+ 𝑛𝑇(𝑊𝑇+ 𝛥𝑊𝑇)(𝑊+ 𝛥𝑊)𝑛=
= 𝑋𝑇[𝐼−𝐻𝑇𝑊𝑇−𝑊𝐻+ 𝐻𝑇(𝑊𝑇𝑊+ 𝛥𝑊𝑇𝛥𝑊)𝐻]𝑋+
+𝑛⃗ 𝑇(𝑊𝑇𝑊+ 𝛥𝑊𝑇𝛥𝑊)𝑛= 𝑋𝑇(𝐼−𝐻𝑇𝑊𝑇−𝑊𝐻+ 𝐻𝑇𝑊𝑇𝑊𝐻)𝑋+ 𝑛𝑇𝑊𝑇𝑊𝑛+
+𝑋𝑇𝐻𝑇(𝛥𝑊𝑇𝛥𝑊) 𝐻𝑋+ 𝑛𝑇(𝛥𝑊𝑇𝛥𝑊)𝑛;
(21) (21) В результаті застосування процедури усереднення квадратичних форм для складових
у виразі (21) отримаємо наступні результати: 1. 𝑛𝑇𝑊𝑇𝑊𝑛= 𝑡𝑟(𝑊𝑇𝑊𝑛𝑛𝑇) = 𝑃ш‖𝑊‖2;
2. 𝑛𝑇𝛥𝑊𝑇𝛥𝑊𝑛 𝑡𝑟(𝛥𝑊𝑇𝑊𝑛𝑛𝑇) = 𝑃ш‖𝛥𝑊‖2;
3. 𝑋𝑇𝐻𝑇𝛥𝑊𝑇𝛥𝑊𝐻𝑋= 𝑡𝑟(𝐻𝑇𝛥𝑊𝑇𝛥𝑊𝐻𝑋𝑋𝑇)
4 1. 𝑛𝑇𝑊𝑇𝑊𝑛= 𝑡𝑟(𝑊𝑇𝑊𝑛𝑛𝑇) = 𝑃ш‖𝑊‖2;
2. 𝑛𝑇𝛥𝑊𝑇𝛥𝑊𝑛 𝑡𝑟(𝛥𝑊𝑇𝑊𝑛𝑛𝑇) = 𝑃ш‖𝛥𝑊‖2;
3. 𝑋𝑇𝐻𝑇𝛥𝑊𝑇𝛥𝑊𝐻𝑋= 𝑡𝑟(𝐻𝑇𝛥𝑊𝑇𝛥𝑊𝐻𝑋𝑋𝑇). 4. (
)
4. Отже, з врахуванням вище викладеного, необхідно зазначити, що система, яка
досліджується в першому варіанті (схема рис. 5, а), тобто 𝑌(𝑛, 1) = 𝐻(𝑛, 𝑚)𝑋(𝑚, 1) + 𝑛(𝑛, 1)
або система в другому варіанті (схема рис. 5, б), тобто
𝑋̂(𝑚, 1) = 𝑊(𝑚, 𝑛)𝑌(𝑛, 1)
можуть мати три ситуації щодо визначеності (11,12) або система в другому варіанті (схема рис. 5, б), тобто можуть мати три ситуації щодо визначеності (11,12) 1. Недовизначена система: 𝑛< 𝑚; 𝑟𝑎𝑛𝑘𝐻(𝑛, 𝑚) = 𝑛. 1. Недовизначена система: 𝑛< 𝑚; 𝑟𝑎
У цьому випадку матриця 𝑊(𝑚, 𝑛) буде рівна: 1. Недовизначена система: 𝑛< 𝑚; 𝑟𝑎𝑛𝑘𝐻(𝑛, 𝑚) = 𝑛. У цьому випадку матриця 𝑊(𝑚, 𝑛) буде рівна: д
;
У цьому випадку матриця 𝑊(𝑚, 𝑛) буде рівна: (
)
У цьому випадку матриця 𝑊(𝑚, 𝑛) буде рівна: У цьому випадку матриця 𝑊(𝑚, 𝑛) буде рівна: 𝑊(𝑚, 𝑛) ≙𝐻𝑇(𝑚, 𝑛)[𝐻(𝑛, 𝑚)𝐻𝑇(𝑚, 𝑛)]−1. 3.
Визначена система: 𝑚= 𝑛→ У цьому випадку формули 𝐴(𝑛, 𝑛) = 𝐻(𝑛, 𝑚)𝐻𝑇(𝑚, 𝑛) та 𝐴(𝑚, 𝑚) = 𝐻𝑇(𝑚, 𝑛)𝐻(𝑛, 𝑚)
сходяться до вигляду 𝐻𝑇= 𝐻, а відносна оцінка (21) середньоквадратичної похибки
параметрів вектору корисного сигналу буде мати вид: ‖𝜀‖2 = 𝑃ш‖𝐻−1‖2 + 𝜎𝐻
2‖𝐻−1‖2[𝑃ш‖𝐻−1‖2 + ‖𝑋‖2];
(24) (24) Engineering and Technology 57 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 За виразом (24) було проведено статистичне моделювання в середовищі Mathlab
процесу обробки ряду вхідних процесів у формі кольорових зображень різної розмірності та
роздільної здатності для випадку сумісної системи за схемою рис.5,б . За виразом (24) було проведено статистичне моделювання в середовищі Mathlab
процесу обробки ряду вхідних процесів у формі кольорових зображень різної розмірності та
роздільної здатності для випадку сумісної системи за схемою рис.5,б . оздільної здатності для випадку сумісної системи за схемою рис.5,б . На рис. 6 представлено результат моделювання процесу відновлення вихідного
випадкового інформаційного процесу 𝑌(𝑛, 1) (рис. 6 б, 7 б, 8 б), який являє собою результат
впливу дестабілізуючих факторів на вхідний тестовий сигнал 𝑋(𝑚, 1) представлений на рис
(рис. 6 а, 7 а) і сформований за схемою на рис. 5.а. Вплив дестабілізуючих факторів моделювався завадами з різною щільністю розподілу
ймовірностей (рис. 6 г, 7 г, 8 г ) та різною структурою просторового спектру при однаковому
рівні потужності завад (рис. 6 д, 7 д, 8 д) за виразом (25): Noise=(Sgrt(3)) ∙𝑃∙(2 ∙𝑟𝑎𝑛𝑑𝑛(5, 𝐿) −𝑜𝑛𝑒𝑠(5, 𝐿)) + 𝑝𝑑𝑑∙𝑃∙𝑟𝑎𝑛𝑑𝑛(5, 𝐿) (25)
де 𝑝𝑑𝑑 – коефіцієнт зміни щільності розподілу ймовірностей, 𝐿 – розмірність вхідного процесу (25) е 𝑝𝑑𝑑 – коефіцієнт зміни щільності розподілу ймовірностей, 𝐿 – розмірність вхідного процесу
а)
г)
б)
д)
в)
е)
Рисунок 6 – Результати моделювання процесу відновлення зображення в умовах шумів
(щільність розподілу ймовірностей з коефіцієнтом pdd = 0) г) а) а) а) г) д) б) б) д) е) в) в) е) Рисунок 6 – Результати моделювання процесу відновлення зображення в умовах шумів
(щільність розподілу ймовірностей з коефіцієнтом pdd = 0) Engineering and Technology Engineering and Technology 58 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 6. Фінансування Це дослідження не отримало конкретної фінансової підтримки. 3.
Визначена система: 𝑚= 𝑛→ 4, – 2023 f
f
p
p
y ,
,
,
а)
г)
б)
д)
в)
е)
Рисунок 7 – Результати моделювання процесу відновлення зображення в умовах шумів
( щільність розподілу ймовірностей з коефіцієнтом pdd = 0,5) а) г) 59
а)
г)
б)
д)
в)
е)
Рисунок 7 – Результати моделювання процесу відновлення зображення в умовах шумів
( щільність розподілу ймовірностей з коефіцієнтом pdd = 0,5)
а)
г)
Engineering and Technology а) г) д) б) б)
д)
в)
е)
Рисунок 7 – Результати моделювання процесу відновлення зображення в умовах шумів
( щільність розподілу ймовірностей з коефіцієнтом pdd = 0,5)
а)
г) б) д) е) в) в) Рисунок 7 – Результати моделювання процесу відновлення зображення в умовах шумів
( щільність розподілу ймовірностей з коефіцієнтом pdd = 0,5) г) а)
г) а) а) Engineering and Technology Engineering and Technology 59 ISSN 2522-9842
Journal of Scientific Papers “Social Development and Security”, Vol. 13, No. 4, – 2023 б)
д)
в)
е)
Рисунок 8 – Результати моделювання процесу відновлення зображення в умовах шумів
(щільність розподілу ймовірностей з коефіцієнтом pdd = 1) д) б) б) е) в) в) Рисунок 8 – Результати моделювання процесу відновлення зображення в умовах шумів
(щільність розподілу ймовірностей з коефіцієнтом pdd = 1) Як видно з рисунків 6е, 7е, 8е передавальна характеристика системи фільтрації 𝑊 в
умовах внутрішньосистемної невизначеності (внутрісистемних факторів дестабілізації) 𝛥𝑊
адаптивно налаштовується на корисний (тестовий) сигнал в просторовому спектрі
радіовипромінювання як для варіанту гаусовської завади так і для негаусовських завад з
довільною щільністю розподілу ймовірностей. Результат фільтрації (відновлення) вихідного випадкового сигналу 𝑌(𝑛, 1) за схемою
зображеною на рис. 5б по вхідному тестовому сигналу, який надходив до системи фільтрації
𝑊 з напрямку 00 просторового спектру радіовипромінювання (рис. 6 д), представлено на рис. 6 в, 7 в, 8 в. 5. Висновки Таким чином, в даній статті розглянуто аналітичний підхід до моделювання процесу
відновлення (фільтрації) вихідного випадкового сигналу 𝑌(𝑛, 1) в умовах впливу
дестабілізуючих факторів з різною щільністю розподілу ймовірностей. Адекватність
приведеного
аналітичного
апарату
підтверджена
результатами
статистичного моделювання наведеного на рис. 6в, 7в, 8в за схемою рис. 5б. References 1. SAE: SAE J3016-201806 – Taxonomy and
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в
інформаційно-керованій
системі
безпілотного
автомобіля. Системи
озброєння і військова техніка: ХНУПС
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https://openalex.org/W2915283543
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0212539&type=printable
|
English
| null |
Apolipoprotein B correlates with intra-plaque necrotic core volume in stable coronary artery disease
|
PloS one
| 2,019
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cc-by
| 5,589
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Methods We assessed plaque composition in patients with stable coronary artery disease (SCD)
admitted to our hospital for percutaneous coronary intervention (PCI) between November 1,
2012, and March 10, 2015. Before PCI, fibrous (FI), fibrofatty (FF), necrotic core (NC), and
dense calcium (DC) regions were evaluated using VH-IVUS, and the contributions of each
to the culprit lesion volume were recorded. Plasma LDL-C, HDL-C, Apo-B, and Apo-A1 lev-
els were assessed before PCI. The relationship between the regions on VH-IVUS and
plasma lipid levels was assessed. Patients were categorized into low Apo-B (LAB) and high
Apo-B (HAB) groups, based on the overall cohort median Apo-B level. Editor: Xianwu Cheng, Nagoya University, JAPAN
Received: September 26, 2018
Accepted: February 5, 2019
Published: February 19, 2019 Editor: Xianwu Cheng, Nagoya University, JAPAN
Received: September 26, 2018
Accepted: February 5, 2019
Published: February 19, 2019
Copyright: © 2019 Ohwada et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Copyright: © 2019 Ohwada et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. OPEN ACCESS Citation: Ohwada T, Sakamoto T, Kanno Y,
Yokokawa S, Amami K, Nakazato K, et al. (2019)
Apolipoprotein B correlates with intra-plaque
necrotic core volume in stable coronary artery
disease. PLoS ONE 14(2): e0212539. https://doi. org/10.1371/journal.pone.0212539 Abstract a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 RESEARCH ARTICLE Apolipoprotein B correlates with intra-plaque
necrotic core volume in stable coronary artery
disease Takayuki OhwadaID1*, Takayuki Sakamoto1, Yuki Kanno2, Sayoko Yokokawa2,
Kazuaki Amami2, Kazuhiko Nakazato2, Yasuchika Takeishi2, Kenichi Watanabe1 1 Fukushima Red Cross Hospital, Department of Cardiology, Fukushima City, Japan, 2 Fukushima Medical
University, Department of Cardiology, Fukushima City, Japan * ikyoku18@fukushima-med-jrc.jp * ikyoku18@fukushima-med-jrc.jp Objective To determine the relationship between plaque composition and low-density lipoprotein cho-
lesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoprotein B (Apo-B),
and Apo-A1 using virtual-histology intravascular ultrasound (VH-IVUS). Results We enrolled 115 patients (median Apo-B, 91 mg/dL, male n = 88) with 57 and 58 patients in
the LAB (Apo-B 90 mg/dL) and HAB (Apo-B 91 mg/dL) groups, respectively. Vessel,
plaque, and %NC volumes were significantly greater in the HAB group than in the LAB
group. The %FI, %FF, and %DC volumes were similar in both groups. In all 115 patients,
the %NC volume correlated with LDL-C (r = 0.2353, P = 0.0114) and Apo-B (r = 0.2487, P =
0.0074) but not with HDL-C and Apo A-1. The high-sensitivity C-reactive protein level
tended to be higher in the HAB group than in the LAB group. Multiple regression analysis
showed that being male, Apo-A1, and Apo-B were significant predictors of %NC volume
extent. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Funding: The authors received no specific funding
for this work. Competing interests: The authors have declared
that no competing interests exist. 1 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 Apolipoprotein B and coronary artery disease Elevated Apo-B level was related to the %NC in target coronary artery lesions in SCD
patients, suggesting a role of Apo-B as a biomarker of unstable plaque in this population. Elevated Apo-B level was related to the %NC in target coronary artery lesions in SCD
patients, suggesting a role of Apo-B as a biomarker of unstable plaque in this population. Introduction Low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and high-density lipopro-
tein cholesterol (HDL-C) are standard biomarkers used to predict adverse cardiovascular
events in patients with ischemic heart disease. [1, 2] However, recent reports indicate that apo-
lipoprotein B (Apo-B) has a greater predictive value than LDL-C. [1–4] Using virtual-histology intravascular ultrasound (VH-IVUS), the PROSPECT study dem-
onstrated that the necrotic cores (NCs) of thin-cap fibroatheromas are sites at which processes
associated with adverse cardiovascular events occur. [5] Intensive lipid-lowering by statins can
lead to atheromatous plaque stabilization or regression through various mechanisms, such as
the reduction of inflammation and lipid accumulation in the core. Furthermore, statin therapy
alters the plaque composition in coronary arteries, although the mechanism for this change is
controversial. [6–8]To date, no study has used IVUS to evaluate the associations of LDL-C and
Apo-B with atheroma composition. Further, the effects of lipoproteins and apolipoproteins on
plaque composition in patients with stable coronary artery disease (SCD) are unclear and
whether these proteins affect plaque vulnerability to rupture is not known. Conventional gray-
scale IVUS images are generated using the amplitude of the radiofrequency (RF) signal. How-
ever, the frequency and power of the signal differ between tissues, regardless of similarities in
the amplitude. Therefore, gray-scale IVUS has limited value in the accurate identification of
specific plaque components. Analysis of IVUS radiofrequency backscatter enables a more
detailed characterization of plaque morphology and tissues, and provides insight into the fea-
tures of vulnerable plaque. [9] Therefore, we used VH-IVUS to investigate the relationship between Apo-B and atheroma
composition in patients with SCD. Our hypothesis was that Apo-B could be a potential bio-
marker for acute coronary syndrome. Materials and methods The Fukushima Red Cross Hospital Ethics Committee approved this study, and all study par-
ticipants provided written informed consent for participation before enrollment. PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 IVUS image acquisition IVUS examinations of the culprit lesions were performed before PCI. A phased-array,
20-MHz, 3.2-F IVUS catheter (Eagle Eye, Volcano Corporation, Rancho Cordova, CA, US)
was placed in the distal coronary artery and pulled back to the aorto-ostial junction using a
motorized catheter pull-back system set at 0.5 mm/s (Eagle Eye, Volcano Corporation). The
gray-scale IVUS and captured radiofrequency data were written onto a DVD-R. Study population We prospectively evaluated 334 consecutive patients with SCD that visited our hospital for per-
cutaneous coronary intervention (PCI) between November 1, 2012 and March 10, 2015, for
study eligibility. Forty patients selected medical therapy. A total of 294 patients who underwent
diagnostic coronary angiography (CAG) and had coronary artery stenosis greater than 75%
were eligible for the study. PCI indications were evaluated according to the Japanese Society of
Cardiology guidelines for elective PCI in patients with SCD. [10] We excluded patients who
did not consent to participate (63 patients) and those who had no significant stenosis (50
patients), chronic total occlusion (18 patients), and restenosis of a prior stent (20 patients). After obtaining consent, we enrolled 143 patients and performed PCI; IVUS was performed in
all enrolled patients. We excluded patients when IVUS could not be completed without PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 2 / 10 Apolipoprotein B and coronary artery disease balloon angioplasty because of a severely stenosed, tortuous, or heavily calcified culprit lesion
(16 patients). Patients with VH-IVUS images of inadequate quality for analysis were also
excluded (12 patients). The remaining 115 patients underwent plasma Apo-B measurements
and were categorized into the low Apo-B (LAB) or high Apo-B (HAB) groups on the basis of
their overall cohort median Apo-B level. Clinical laboratory measurements Laboratory evaluations of plasma lipid and apolipoprotein levels were performed within two
days before PCI. Commercial reagent kits (Determiner L TC II, Determiner L LDL-C II,
Determiner L HDL-C II, and Determiner L TG II; Kyowa Medex, Tokyo, Japan) were used to
analyze plasma TC, LDL-C, HDL-C, and triglyceride (TG) levels, and lipid concentrations
were measured using an automated chemical analyzer (Labospect 006, Hitachi, Tokyo, Japan). Apo-B and Apo-A1 were measured by immunonephelometry, and high-sensitivity C-reactive
protein (hs-CRP) levels were measured by nephelometry. Apolipoprotein levels were mea-
sured at a central clinical laboratory (SRL, Inc., Tokyo, Japan). Gray-scale and VH-IVUS analyses Off-line gray-scale and VH-IVUS analyses were performed using echoPlaque 4.0 software
(INDEC Systems, Inc., Los Altos, CA, USA). Corresponding proximal and distal reference
IVUS images were identified for each culprit lesion. These were analyzed to determine the pla-
que volume within the involved arterial segment. Gray-scale IVUS analysis was performed
according to the American College of Cardiology Clinical Expert Consensus Document on
Standards for Acquisition, Measurement and Reporting of Intravascular Ultrasound Studies. [10] In the gray-scale conventional IVUS analysis, images were assessed for lesion length,
lumen volume, plaque volume, and vessel volume. In the VH-IVUS analysis, fibrous, fibro-
fatty, NC, and dense calcium regions were color-coded, and estimates of the contribution of
each to the volume of the entire culprit lesion were reported as percentages of the plaque vol-
ume as %NC (= the percentage of NC volume). The same investigator along with another (T.S. and K.W.) subsequently reanalyzed the images to assess the intraobserver and interobserver
reproducibility of the measurements. Results The median Apo-B level in the 115 study patients was 91 mg/dL. There were 57 and 58 patients
in the LAB (Apo-B 90 mg/dL) and HAB (Apo-B 91 mg/dL) groups, respectively. Anti-
platelet and statin treatments were more frequent in the LAB group than in the HAB group
(Table 1). Compared with the HAB group, the LAB group had lower TC, TG, and LDL-C lev-
els and a higher HDL-C level (Table 1). Representative VH-IVUS images are shown in Fig 1. The intraobserver (r = 0.98, 0.98, and
0.99) and interobserver (r = 0.96, 0.97, and 0.98) measurement variabilities were acceptable. The vessel, plaque, and %NC volumes and lesion length were significantly greater in the HAB
group than in the LAB group (Table 2). In all patients, the %NC volume was significantly
correlated with the Apo-B (r = 0.2487, P = 0.0074, Fig 2A) and LDL-C (r = 0.2353, P = 0.0114,
Fig 2B) levels, but not with the HDL-C (r = -0.0019, P = 0.9837) and the Apo A-1 (r = 0.01,
P = 0.9169) levels. The hs-CRP level tended to be higher in the HAB group (1883.356 ±
424.334) than in the LAB group (1340.807 ± 427.359, P = 0.369). However, in all patients
(n = 115), the hs-CRP level was not correlated with the %NC volume (r = 0.0755, P = 0.4231). Table 3 displays a model summary of the multiple regression analysis of the value of Apo-B
as a biomarker. The value of the correlation coefficient adjusted R-square (0.9110) indicates
the presence of a strong correlation between the combinations of independent variables and
the %NC volume, as the level of significance is less than 0.05. In order to identify significant
independent variables, we examined the values of the regression coefficients. The analysis
indicated the existence of a positive relationship between male sex (coefficient: 2.2405,
beta = 0.112, P = 0.039), Apo-A1 (coefficient: 0.0615, beta = 0.480, P <0.001), and Apo-B
(coefficient: 0.0698, beta = 0.3866, P <0.001) and %NC volume. In order to rank the indepen-
dent variables, their beta values were examined. The significance ranking was 1: Apo-A1,
beta = 0.4800; 2: Apo-B, beta = 0.3866; and 3: male sex, beta = 0.1124. Table 4 displays a model summary of the multiple regression analysis for the value of
LDL-C as a biomarker. Results The value of the correlation coefficient adjusted R-square (0.0846)
indicates the presence of a weak significant correlation between the combinations of indepen-
dent variables and %NC. This indicated the existence of a positive relationship between age
(coefficient: -0.1256, beta = -0.2135, P = 0.0192) and LDL-C (coefficient: 0.0347, beta = 0.2205,
P = 0.0156). Apolipoprotein B and coronary artery disease performed. The statistical analysis indicated a significant relationship between %NC and the
1st model (model 1), which comprised seven predictive variables including Apo-B (age, male
sex, smoking, statin use, hs-CRP, Apo-A1, Apo-B); and the 2nd model (model 2), which com-
prised seven predictive variables including LDL-C (age, male sex, smoking, statin use, hs-CRP,
Apo-A1, LDL-C). All statistical analyses were performed using Ekuseru-Toukei for Windows,
version 1.02 (SSRI Co., Ltd., Tokyo, Japan). Statistical analysis Data are presented as means ± standard error. Categorical data are presented as numbers (n)
and percentages (%). Categorical and continuous variables were compared between the LAB
and HAB groups using the chi-square test, Welch’s t-test, and Wilcoxon’s signed rank test. All
statistical assessments were two-sided and evaluated at a significance level of 0.05. To investi-
gate the relationship between the NC and each lipid in all patients (not divided into LAB and
HAB groups), Pearson’s correlations between %NC and Apo-B, %NC and LDL-C, and %NC
and Apo-A1 were also evaluated. To detect the amount of shared variance and the strength of
the relationship between the variables of interest, multiple least square regression analysis was PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 3 / 10 Discussion In the present study, we demonstrated that the lesion length and vessel, plaque, and %NC vol-
umes were higher in patients with high plasma Apo-B levels than in those with low plasma
Apo-B levels. Interestingly, the Apo-B (r = 0.2483) and LDL-C (r = 0.2105) levels were posi-
tively correlated with %NC volume in the VH-IVUS volumetric analyses. In this study, Apo-B
and %NC appeared to be more closely correlated than were LDL-C and %NC. However, we
found no correlation between Apo-A1 and %NC. Furthermore, although hs-CRP tended to be
higher in the HAB group than in the LAB group, this difference was not statistically significant. On the other hand, multiple regression analysis (model 1) revealed that the male sex, Apo-A1, PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 4 / 10 Values are means ± standard error or numbers of patients (percentage). LAB, low apolipoprotein-B ( 90 mg/dL); HAB, high apolipoprotein B ( 91 mg/dL); PCI,
percutaneous coronary intervention; CCB, calcium-channel blocker; ACEI/ARB, angiotensin-converting enzyme inhibitor and angiotensin II receptor blocker; DM,
diabetes mellitus; RCA, right coronary artery; LAD, left anterior descending coronary artery; LCX, left circumflex coronary artery; TC, total cholesterol; HDL-C, high-
density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; Apo-A1, apolipoprotein A1; Apo-B, apolipoprotein B; HbA1c, hemoglobin A1c; hs-CRP,
high-sensitivity C-reactive protein.
❋P<0 05 Values are means ± standard error or numbers of patients (percentage). LAB, low apolipoprotein-B ( 90 mg/dL); HAB, high apolipoprotein B ( 91 mg/dL); PCI, mbers of patients (percentage). LAB, low apolipoprotein-B ( 90 mg/dL); HAB, high apolipoprotein B ( 91 mg/dL); PCI, PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 p
y
; CC ,
;
C
/
,
g
g
y
g
p
;
,
diabetes mellitus; RCA, right coronary artery; LAD, left anterior descending coronary artery; LCX, left circumflex coronary artery; TC, total cholesterol; HDL-C, high-
density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; Apo-A1, apolipoprotein A1; Apo-B, apolipoprotein B; HbA1c, hemoglobin A1c; hs-CRP,
high-sensitivity C-reactive protein. On the other hand, the value of the PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 5 / 10 Apolipoprotein B and coronary artery disease Fig 1. Representative virtual-histology intravascular ultrasound images. A. A cross-sectional image of the lesion in
a patient in the low apolipoprotein B (LAB) group. The composition of the entire lesion by volume percentage is
fibrous (green), 54.43%; fibrofatty (light green), 29.09%; necrotic core (red), 12.96%; and dense calcium (white), 3.52%
B. Longitudinal section of the lesion in the same patient in the LAB group. The yellow line indicates the location of the
cross-section shown in (A). C. A cross-sectional image of the lesion in a patient in the high apolipoprotein B (HAB)
group. The composition of the entire lesion by volume percentages are as follows: fibrous (green), 48.99%; fibrofatty
(light green), 20.24%; necrotic core (red), 22.36%; and dense calcium (white), 8.41%. D. Longitudinal section of the
lesion in the same patient in the HAB group. The yellow line indicates the location of the cross-section shown in (C). https://doi.org/10.1371/journal.pone.0212539.g001 Fig 1. Representative virtual-histology intravascular ultrasound images. A. A cross-sectional image of the lesion in
a patient in the low apolipoprotein B (LAB) group. The composition of the entire lesion by volume percentage is
fibrous (green), 54.43%; fibrofatty (light green), 29.09%; necrotic core (red), 12.96%; and dense calcium (white), 3.52%
B. Longitudinal section of the lesion in the same patient in the LAB group. The yellow line indicates the location of the
cross-section shown in (A). C. A cross-sectional image of the lesion in a patient in the high apolipoprotein B (HAB)
group. The composition of the entire lesion by volume percentages are as follows: fibrous (green), 48.99%; fibrofatty
(light green), 20.24%; necrotic core (red), 22.36%; and dense calcium (white), 8.41%. D. Longitudinal section of the
lesion in the same patient in the HAB group. The yellow line indicates the location of the cross-section shown in (C). https://doi.org/10.1371/journal.pone.0212539.g001 https://doi.org/10.1371/journal.pone.0212539.g001 https://doi.org/10.1371/journal.pone.0212539.g001 correlation coefficient adjusted R-square (0.9110) of model 1 including Apo-B was very high,
and %NC in the lesion plaque of SCD patients could be predicted by this model. Apolipoprotein B and coronary artery disease Table 1. Characteristics and laboratory data in patients with low and high plasma apolipoprotein B. Characteristic
LAB group
n = 57
HAB group
n = 58
P
Age (years)
72.2 ± 1.2
67.8 ± 1.1
0.0086❋
Male (%)
40 (70.2)
48 (81.4)
0.1595
Clinical Histories
Diabetes mellitus (%)
20 (35.1)
19 (32.2)
0.7423
Hypertension
40 (70.2)
45 (76.3)
0.4583
Dyslipidemia
33 (57.9)
36 (61.0)
0.7320
Smoking
14 (24.6)
26 (44.1)
0.0271❋
Previous PCI
29 (50.9)
23 (39.0)
0.1987
Previous Stroke
1 (1.8)
3 (5.1)
0.3257
Oral Medications
CCB
30 (52.6)
28 (47.5)
0.5774
ACEI/ ARB
27 (47.4)
25 (42.4)
0.5886
Antiplatelet drugs
47 (82.5)
37 (62.7)
0.0174❋
Anti-DM drugs
9 (15.8)
10 (16.9)
0.8660
Insulin
1 (1.8)
1 (1.7)
0.9804
Statin
34 (59.6)
17 (28.8)
0.0008❋
Culprit Vessel
RCA
22 (38.6)
20 (33.9)
0.5986
LAD
22 (38.6)
25 (42.4)
0.6787
LCX
13 (22.8)
14 (23.7)
0.9065
Laboratory data
TC (mg/dL)
167.0 ± 3.4
212.8 ± 4.6
<0.001❋
TG (mg/dL)
121.7 ± 7.0
205.3 ± 12.7
<0.001❋
HDL-C (mg/dL)
57.1 ± 1.8
51.0 ± 1.3
0.009❋
LDL-C (mg/dL)
85.6 ± 2.9
121.9 ± 4.2
<0.001❋
Apo-A1 (mg/dL)
134.9± 2.8
123.7± 2.4
0.5569
Apo-B (mg/dL)
75.8 ± 1.4
110.2 ± 2.6
<0.001❋
HbA1c (%)
6.0 ± 0.1
6.2 ± 0.1
0.2526
hs-CRP (ng/mL)
1340.8 ± 427.4
1903.9 ± 431.2
0.3556 Table 1. Characteristics and laboratory data in patients with low and high plasma apolipoprotein B. https://doi.org/10.1371/journal.pone.0212539.t001 and Apo-B were significant predictors of %NC volume. It is interesting that Apo-A1 was not
correlated with %NC volume on Pearson’s correlation analysis but affected it on the regression
analysis. This can be explained by the fact that the significance of Apo-A1 in the model is
dependent upon all the factors being in the model. Male sex and Apo-B were acting as a sort of
mediator of the relationship between Apo-A1 and %NC volume. Moreover, these results
showed that Apo-B induces plaque progression, vascular remodeling, and, especially, NC pro-
gression, the latter of which is also affected by male sex and Apo-A1. Additionally, Apo-B
might be involved in plaque vulnerability. Furthermore, in model 2 of the regression analysis,
age and LDL-C were significant predictors of %NC volume although the value of the correla-
tion coefficient adjusted R-square (0.0846) was very low. https://doi.org/10.1371/journal.pone.0212539.t002 Values are means ± standard error. LAB, low apolipoprotein B ( 90 mg/dL); HAB, high apolipoprotein B ( 91 mg/dL); VH-IVUS, virtual histology intravascular
ultrasound. A previous study demonstrated that the accuracy of VH-IVUS for ex vivo tissue characteri-
zation exceeds 93.5%.[11] When not influenced by calcium-induced acoustic attenuation, 45
MHz VH-IVUS technology allowed tissue-type identification in combined tissues with accu-
racy >88%, which was greater than the accuracy of the gold standard histologic assessment,
and maintained high interobserver and intraobserver reproducibility. [12] Moreover, the con-
cordance between the histological and VH-IVUS classifications of carotid plaques was 86.1%. [13] On the basis of these reports, the accuracy of VH-IVUS plaque characterization is
acceptable. Table 2. Gray scale- and VH-IVUS data in patients with low and high plasma apolipoprotein B. LAB group
n = 57
HAB group
n = 58
P
Gray scale-IVUS data
Vessel volume (mm3)
665.2 ± 59.1
1000.1 ± 79.7
0.0011❋
Plaque volume (mm3)
399.1 ± 37.1
621.3 ± 52.4
0.0008❋
Lumen volume (mm3)
266.1 ± 23.4
378.8 ± 31.3
0.0048❋
Lesion length (mm)
50.9 ± 3.8
72.5 ± 4.3
0.0003❋
VH-IVUS data
Fibrous volume (mm3)
140.602 ± 13.853
237.805 ± 23.030
0.0001❋
Fibrous volume (%)
54.400 ± 0.860
55.134 ± 0.764
0.5241
Fibrous fatty volume (mm3)
68.951 ± 9.570
92.804 ± 12.697
0.0347❋
Fibrous fatty volume (%)
23.648 ± 1.053
21.113 ± 1.083
0.0963
Necrotic core volume (mm3)
38.838 ± 3.830
74.304 ± 7.463
<0.00001❋
Necrotic core volume (%)
15.293 ± 0.608
17.622 ± 0.725
0.0157❋
Dense calcium volume (mm3)
14.702 ± 1.244
23.013 ± 2.206
0.0111❋
Dense calcium volume (%)
6.659 ± 0.496
6.132 ± 0.469
0.4414
Values are means ± standard error. LAB, low apolipoprotein B ( 90 mg/dL); HAB, high apolipoprotein B ( 91 mg/dL); VH-IVUS, virtual histology intravascular
ultrasound. P<0.05. Gray scale- and VH-IVUS data in patients with low and high plasma apolipoprotein B. 6 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 Apolipoprotein B and coronary artery disease Fig 2. Correlation between %NC volume and Apo-B level and LDL-C level in all patients (n = 115). A. Correlation between %NC volume and Apo-B level
in all patients. The %NC volume was significantly correlated with Apo-B level (r = 0.2487, P = 0.0074). B. Correlation between %NC volume and LDL-C level
in all patients. The %NC volume was significantly correlated with LDL-C level (r = 0.2353, P = 0.0114). %NC, the percentage of necrotic core volume; Apo-B,
apolipoprotein B; LDL-C, low-density lipoprotein cholesterol. h
//d i
/10 1371/j
l
0212539 002 Fig 2. https://doi.org/10.1371/journal.pone.0212539.t003 Apolipoprotein B and coronary artery disease Table 4. Model 2. Multiple regression analysis to analyze the contribution of each predictor including LDL-C in predicting the %NC (n = 115). Predictors
B
Beta
t
P
F
Age
-0.1256
-0.2135
-2.3765
0.0192
5.6478
LDL-C
0.0347
0.2205
2.4548
0.0156
6.0262
P<0.05 changes (r = -0.23, p < 0.05) and percent stenosis (r = 0.30, p < 0.01), respectively. While Gol-
shahi et al. [22] demonstrated that Apo-A1 was associated with mild (but not severe) coronary
atherosclerosis, they found that Apo-B was associated with severe (but not mild) coronary ath-
erosclerosis. Further, HDL-C, LDL-C, and TG were not significant risk factors in their study. Elevated hs-CRP is a surrogate marker of the inflammatory process that is reportedly
related to the plaque burden in non-culprit coronary arteries [23] and the NC volume in cross-
sections of culprit lesions in patients with stable angina. [16] In our study, hs-CRP was not cor-
related with %NC in the culprit lesion. Although hs-CRP levels in the HAB group tended to be
higher than those in the LAB group, this difference was not significant. This difference in hs-
CRP between previous studies and our own is likely attributable to the differences in method-
ology used. Our data were based on an estimate of the volume of the whole lesion, whereas pre-
vious studies assessed the cross-sectional area of the lesion with the smallest stenosis degree
that was most affected by inflammation. However, we found that Apo-B was significantly cor-
related with %NC volume, and affected %NC volume on multiple regression analysis. Our
data showed that, compared with hs-CRP, Apo-B was a more sensitive marker of unstable pla-
ques with a large NC. g
Fluorescein angioscopy and microscopy images of human coronary artery plaques [24]
showed that Apo-B begins to deposit before plaque formation, accumulates with plaque
growth, and disappears after NC formation. These results, taken together with our own, indi-
cate that Apo-B contributes to NC formation. The present study has some limitations worth noting. First, this was a single-center study,
and the study sample was relatively small. Therefore, our results need to be validated by larger
studies. Second, follow-up data were not available. Therefore, we could not assess the prognos-
tic implications of an elevated Apo-B level in patients with SCD. Supporting information S1 File. All individual patient data used in the study. (XLSX) S1 File. All individual patient data used in the study. (XLSX) Despite these limitations, our
study identified a correlation between the Apo-B level and plaque composition of the target
coronary artery lesion in patients with SCD. In conclusion, an elevated Apo-B level was related to the %NC of the target coronary artery
lesion in patients with SCD. Our findings suggest a potential role for Apo-B as a biomarker of
unstable plaques in this patient population. Moreover, our study showed the Apo-B more
strongly related to plaque advancement, and therefore is a more important contributing factor
to the development of acute coronary syndrome than LDL-C. Therefore, future studies should
further evaluate the role of Apo-B in cardiovascular disease pathogenesis. Correlation between %NC volume and Apo-B level and LDL-C level in all patients (n = 115). A. Correlation between %NC volume and Apo-B level
in all patients. The %NC volume was significantly correlated with Apo-B level (r = 0.2487, P = 0.0074). B. Correlation between %NC volume and LDL-C level
in all patients. The %NC volume was significantly correlated with LDL-C level (r = 0.2353, P = 0.0114). %NC, the percentage of necrotic core volume; Apo-B,
apolipoprotein B; LDL-C, low-density lipoprotein cholesterol. https://doi.org/10.1371/journal.pone.0212539.g002 https://doi.org/10.1371/journal.pone.0212539.g002 https://doi.org/10.1371/journal.pone.0212539.g002 Numerous previous studies have evaluated the use of plasma biomarkers for the identifica-
tion of high-risk lesions or vulnerable plaques in patients with SCD. Furthermore, VH-IVUS
has frequently been used to obtain detailed information about the composition and character-
istics of coronary atherosclerotic plaques. Consequently, several plasma biomarkers[14] and
the plaque composition assessed by VH-IVUS [15, 16] have been proposed as predictors of
plaque rupture. Compared with LDL-C, non-HDL-C and Apo-B more successfully predict cardiovascular
disease (CVD) risk. [17] The AMORIS study [18], which compared Apo-B and LDL-C, found
that Apo-B was a more accurate predictor of CVD risk in 175,553 healthy individuals. Our
finding that Apo-B is more strongly correlated with %NC than LDL-C is consistent with the
results of these previous clinical studies. Of note, Moss et al. [19] reported that Apo-B (hazard ratio, 1.82; 95% CI, 1.10–3.00) and
low levels of Apo-A1 (hazard ratio, 1.84; 95% CI, 1.10–3.08) were independently associated
with recurrent coronary events, whereas LDL-C was not. This is consistent with our finding of
a correlation between Apo-B and Apo-A1 levels and %NC. McGill et al. [20] were the first to describe the correlation between Apo-B and angiographic
lesion morphology in patients with premature coronary heart disease. They demonstrated that
the Apo-B level independently predicted the extent of angiographically defined coronary ath-
erosclerosis. Moreover, the BECAIT trial [21] reported that the HDL3 cholesterol and plasma
Apo-B concentrations were independent predictors of the mean minimum lumen diameter Table 3. Model 1. Multiple regression analysis to analyze the contribution of each predictor including Apo-B in predicting the %NC (n = 115). Predictors
B
Beta
T
P
F
male
2.2405
0.1124
2.0882
0.0390
4.3607
Apo-A1
0.0615
0.4800
4.7806
<0.001
22.8545
Apo-B
0.0698
0.3866
3.6008
<0.001
12.9659
P<0.05 PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 7 / 10 Acknowledgments The authors gratefully acknowledge the expert work of the staff members of our catheteriza-
tion laboratory, especially Toshimi Kobayashi, Yoko Sato, Noriko Sato, Akemi Hagiwara, PLOS ONE | https://doi.org/10.1371/journal.pone.0212539
February 19, 2019 8 / 10 Apolipoprotein B and coronary artery disease Noriko Kohata, Naoko Morita, Miyuki Konno, Murakami Futa, Shiozawa Shota, Emiko Sato,
Aya Nakayama, Michiko Hayasaka, and Kenichi Hashimoto as well as the staff of the catheteri-
zation laboratory at Fukushima Red Cross Hospital. Author Contributions Conceptualization: Kenichi Watanabe. Investigation: Kazuhiko Nakazato. Investigation: Kazuhiko Nakazato. Investigation: Kazuhiko Nakazato. Project administration: Takayuki Sakamoto. Supervision: Yasuchika Takeishi. Writing – original draft: Takayuki Ohwada. Writing – review & editing: Takayuki Ohwada. References 1. Contois JH, McConnell JP, Sethi AA, Csako G, Devaraj S, Hoefner DM, et al. Apolipoprotein B and car-
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74. PMID: 23676365 10 / 10
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0229475&type=printable
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English
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Characterization of transgenic mouse lines for selectively targeting glial cells in dorsal root ganglia
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bioRxiv (Cold Spring Harbor Laboratory)
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cc-by
| 11,323
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Characterization of transgenic mouse lines for
selectively targeting satellite glial cells and
macrophages in dorsal root ganglia Yasmine Rabah¤☯, Bruna RubinoID☯, Elsie Moukarzel☯, Cendra AgulhonID*
Integrative Neuroscience and Cognition Center (CNRS UMR8002), Glia-Glia & Glia-Neuron Interactions
Laboratory, Faculty of Basic and Biomedical Sciences, Paris Descartes University, Paris, France Yasmine Rabah¤☯, Bruna RubinoID☯, Elsie Moukarzel☯, Cendra AgulhonID*
Integrative Neuroscience and Cognition Center (CNRS UMR8002), Glia-Glia & Glia-Neuron Interactions
Laboratory, Faculty of Basic and Biomedical Sciences, Paris Descartes University, Paris, France ☯These authors contributed equally to this work. ¤ Current address: Energy and Memory Laboratory, Brain Plasticity Unit, CNRS, ESPCI Paris, PSL
Research University, Paris, France
* cendra.agulhon@parisdescartes.fr a1111111111
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a1111111111 Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Funding: This work was supported by a starting
grant (Chair of Excellence) from the Foundation
Ecole des Neuroscience de Paris (ENP), a
European Marie Skłodowska-Curie career
integration grant (# 334497), a DIM Cerveau &
Pense´e-Re´gion Ile-de-France grant, as well as
CNRS and Paris Descartes University financial
support to CA. YR and EM were recipients of PhD Abstract The importance of glial cells in the modulation of neuronal processes is now generally
accepted. In particular, enormous progress in our understanding of astrocytes and microglia
physiology in the central nervous system (CNS) has been made in recent years, due to the
development of genetic and molecular toolkits. However, the roles of satellite glial cells
(SGCs) and macrophages–the peripheral counterparts of astrocytes and microglia–remain
poorly studied despite their involvement in debilitating conditions, such as pain. Here, we
characterized in dorsal root ganglia (DRGs), different genetically-modified mouse lines pre-
viously used for studying astrocytes and microglia, with the goal to implement them for
investigating DRG SGC and macrophage functions. Although SGCs and astrocytes share
some molecular properties, most tested transgenic lines were found to not be suitable for
studying selectively a large number of SGCs within DRGs. Nevertheless, we identified and
validated two mouse lines: (i) a CreERT2 recombinase-based mouse line allowing trans-
gene expression almost exclusively in SGCs and in the vast majority of SGCs, and (ii) a
GFP-expressing line allowing the selective visualization of macrophages. In conclusion,
among the tools available for exploring astrocyte functions, a few can be used for studying
selectively a great proportion of SGCs. Thus, efforts remain to be made to characterize
other available mouse lines as well as to develop, rigorously characterize and validate new
molecular tools to investigate the roles of DRG SGCs, but also macrophages, in health and
disease. PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Rabah Y, Rubino B, Moukarzel E, Agulhon
C (2020) Characterization of transgenic mouse
lines for selectively targeting satellite glial cells and
macrophages in dorsal root ganglia. PLoS ONE
15(9): e0229475. https://doi.org/10.1371/journal. pone.0229475 Editor: Eliseo A. Eugenin, University of Texas
Medical Branch at Galveston, UNITED STATES Editor: Eliseo A. Eugenin, University of Texas
Medical Branch at Galveston, UNITED STATES Editor: Eliseo A. Eugenin, University of Texas
Medical Branch at Galveston, UNITED STATES
Received: February 5, 2020
Accepted: August 12, 2020
Published: September 11, 2020 Published: September 11, 2020 Copyright: © 2020 Rabah et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. PLOS ONE PLOS ONE Introduction Astrocytes and microglia serve essential support and immune functions, and contribute to dis-
eases of the CNS [1]. For a long time, their heterogeneity and roles in the CNS have remained
unclear due to the lack of tools to specifically identify them, and monitor or alter their activity. However, over the last fifteen years, new genetically-encoded tools to selectively visualize 1 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia fellowships from the French Ministry of Research,
and BR received a PhD fellowship from the
European Union Horizon 2020 research and
innovation program under the Marie Skłodowska-
Curie grant agreement (# 66585). YR was awarded
a master 2 fellowship from the Institute of
Neuroscience and Cognition. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript. astrocytes and microglia as well as read out or abolish astrocytic Ca2+ activity have been gener-
ated. They include (i) transgenic mice expressing green fluorescent protein (GFP) under the
control of astrocytic or microglial promoters [2–5], (ii) transgenic mice expressing genetically-
encoded Ca2+ indicators (GCaMP) to monitor astrocyte Ca2+ dynamics [6–8], or (iii) IP3R2
knockout mice to abolish global Gq protein-coupled receptor (Gq GPCR)-mediated Ca2+ ele-
vations in astrocyte cell bodies and large processes [9,10]. These tools have been extremely use-
ful for probing astrocyte and microglia functions in the CNS and are now extensively used in
the field. fellowships from the French Ministry of Research,
and BR received a PhD fellowship from the
European Union Horizon 2020 research and
innovation program under the Marie Skłodowska-
Curie grant agreement (# 66585). YR was awarded
a master 2 fellowship from the Institute of
Neuroscience and Cognition. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript. In the peripheral nervous system (PNS), SGCs in DRGs share several properties with astro-
cytes, including expression of cytosolic proteins [e.g. glial fibrillary acidic protein: GFAP [11];
S100 calcium-binding protein beta: S100β [12]; glutamine synthetase [13]], membrane neuro-
transmitter transporters [e.g. glutamate aspartate transporter: GLAST [13]], and channels [e.g. inwardly rectifying K+ channel 4.1 (Kir 4.1) [14]; connexin 43 (Cx43)-based gap junctions and
hemichannels [15]]. As astrocytes and many other cell types, SGCs use Ca2+ as a signaling mol-
ecule [16]. Introduction Additionally, there is substantial evidence for a SGC role in chronic pain wherein
SGCs undergo a reactive gliotic response accompanied with increased GFAP expression,
hypertrophy, proliferation and upregulation of Cx43 [17,18]. Furthermore, the counterparts of
CNS microglia in DRGs are the macrophages, and both express the ionized Ca2+-binding
adapter molecule 1 (Iba1) [19,20]. Emerging evidence has implicated the contribution of DRG
macrophages to neuropathic pain development and axonal repair in the context of nerve
injury [21–23]. Thus, understanding how SGC and macrophage morphology and function are
remodeled in physiology and pathology can help to find new therapeutic targets for pain-
related diseases [13,24–26]. However, SGC and macrophage heterogeneity and role remain
largely unknown in DRGs, mainly due to the unavailability of tools to specifically visualize
DRG glial cells and examine or manipulate their Ca2+ dynamics. Satellite glial cell or macro-
phage specific gene expression or deletion would therefore help to clarify the roles of those
cells in DRGs. Competing interests: The authors have declared
that no competing interests exist. To accomplish this, using immunohistochemistry or 2-photon Ca2+ imaging, we have char-
acterized different available genetically-modified mouse lines widely used to study astrocytes
or microglia. Our results showed that most lines used for examining astrocyte functions are
very inefficient for studying selectively a large proportion of SGCs in DRGs. However, we have
identified two mouse lines allowing either the selective expression of a Ca2+ biosensor in SGCs
or the labelling of macrophages. Competing interests: The authors have declared
that no competing interests exist. Immunohistochemistry, image acquisition and analysis Animals were transcardially perfused with 4% paraformaldehyde under ketamine/xylazine
(100 mg/kg– 10 mg/kg respectively, i.p.) anesthesia. Brains and lumbar L3, L4 and L5 DRGs
were removed, post-fixed for 24 h or 2 h in 4% paraformaldehyde, respectively. Then, tissues
were cryoprotected overnight at 4˚C in 0.02 M phosphate buffer saline (PBS, pH 7.4) contain-
ing 20% sucrose, and frozen in optimal cutting temperature compound. Sixteen or 14 μm
thick sections (brain or DRG, respectively) were cut using a cryostat (Leica), mounted on
Superfrost glass slides and stored at -80˚C. The day of the experiment, sections were washed 3
times for 15 min each in 0.02 M PBS. Sections were incubated overnight in 0.02 M PBS con-
taining 0.3% Triton X100, 0.02% sodium azide and primary antibodies (Table 1) at room tem-
perature in a humid chamber. In order to readily identify fine subcellular compartments
expressing GFP/eGFP or GCaMP6f and to not miss any transgene expression, GFP/eGFP/
GCaMP6f signal was amplified using an antibody directed against GFP. Of note, GFP/eGFP/
GCaMP6f signal was visible without such amplification. The following day, sections were
washed 3 times for 15 min each in 0.02 M PBS, and incubated for 2 h at room temperature
with secondary antibodies diluted in 0.02 M PBS containing 0.3% Triton X100 and 0.02%
sodium azide. Then, sections were washed 3 times for 15 min in 0.02 M PBS and mounted
between slide and coverslip using Vectashield medium containing DAPI (Vector Laborato-
ries). Negative controls, i.e. slices incubated with secondary antibodies only, were used to set
criteria (gain, exposure time) for image acquisition in each experiment. Image acquisition was
performed with an Axio Observer Z1 epifluorescence Zeiss microscope, an ORCA Flash 2.8
million pixel camera, and a PlanNeoFluar 20x/0.5NA objective. Images were extracted using
the ZEN 2011 blue edition software (Zeiss). Cell counting measurements were performed Table 1. List of primary and secondary antibodies with corresponding dilutions used in the current study. Antibodies
Species
Company/Cat. Animals Mice were housed in transparent plastic cages (5 mice/cage) and fed ad libitum. Illumination
was controlled automatically with a 12/12h light-dark cycle. S100β-eGFP [5], ALDH1L1-eGFP
[27] and CX3CR1-eGFP [3] transgenic lines were used. Furthermore, CAG-lox-STOP-lox-
GCaMP6f transgenic mouse line [7] was crossed with GLAST-CreERT2 [28], GFAP-Cre [29],
Cx30-CreERT2 [28] or Cx43-CreERT2 [30] mice. As a result, we obtained four new double
transgenic mouse lines that we named GLAST-CreERT2::GCaMP6f, GFAP-Cre::GCaMP6f,
Cx30-CreERT2::GCaMP6f and Cx43-CreERT2::GCaMP6f. To induce expression of the Ca2+
biosensor GCaMP6f, GLAST-CreERT2::GCaMP6f and Cx30-CreERT2::GCaMP6 mice were
injected intraperitoneally (i.p.) with tamoxifen (1 mg/day, Sigma) diluted in corn oil (Sigma)
during 5 consecutive days. To obtain optimum GCaMP6f expression in brain astrocytes of
Cx43-CreERT2::GCaMP6f mice, tamoxifen treatment lasted 10 days as previously described
[31]. Animals were then used 2 weeks after the first day of treatment. All lines used were kept PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 2 / 25 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia heterozygous for transgenes encoding GFP/eGFP, GCaMP6f, Cre or CreERT2. Experiments
were conducted in 2 to 3 month-old male and female mice from the C57BL/6JRj background. Animal care and procedures were carried out according to the guidelines set out in the Euro-
pean Community Council Directives. The protocol was approved by the Committee on the
Ethics of Animal Experiments of Paris Descartes University (Protocol Number:
2018061412588713). Calcium imaging Acute intact DRG preparations were prepared from GFAP-Cre::GCaMP6f mice. Briefly, verte-
bras and dura mater were removed and lumbar L4 and L5 DRGs were exposed and immedi-
ately covered with ice cold (slushy) incubation ACSF solution (95 mM NaCl, 1.9 mM KCl, 1.2
mM KH2PO4, 0.5 mM CaCl2, 7 mM MgSO4, 26 mM NaHCO3, 15 mM glucose, 50 mM
sucrose) and bubbled with 95% O2 and 5% CO2. DRGs were incubated for 30 min at 35˚C in
the incubation solution and then left to recover for 1 h 30 min at room temperature. A single
DRG was placed in the recording chamber of a custom-built 2-photon laser-scanning micro-
scope with a 20x water immersion objective (x20/0.95w XLMPlanFluor, Olympus). GCaMP6f
was excited at 920 nm with a Ti:Sapphire laser (Mai Tai HP; Spectra-Physics). DRGs were con-
tinuously superfused at a rate of 4 ml/min with recording solution (127 mM NaCl, 1.9 mM
KCl, 1.2 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4, 26 mM NaHCO3, 15 mM glucose) and
bubbled with 95% O2–5% CO2. To evoke intracellular Ca2+ elevations in DRG GCaMP6f-
expressing cells, a cocktail of agonists to endogenous Gq GPCRs containing 50 μM DHPG
(Abcam), 10 μM histamine (Sigma Aldrich), 10 μM carbachol (Abcam), and 50 μM ATP
(Sigma Aldrich) was bath applied for 30 sec. tification in DRG SGCs and V1 astrocytes from the mouse lines used in the current study. Table 2. Data quantification in DRG SGCs and V1 astrocytes from the mouse lines used in the current study. Immunohistochemistry, image acquisition and analysis #
Dilution (DRG & V1)
Anti-CD68
Rat
Serotec/MCA1957
1:2000
Anti-GFAP
Rabbit
Dako/Z0334
1:5000
Anti-GFAP
Chicken
Abcam/ab4674
1:1000
Anti-GFP
Chicken
Invitrogen/A10262
1:1000
Anti-GLAST
Rabbit
Frontier Institute/Af660
1:5000
Anti-Iba1
Rabbit
Wako/019-1974
1:2500 (DRG)
1:1000 (V1)
Anti-NeuN
Guinea Pig
Millipore/ABN90
1:1000
Anti-NF
Rabbit
Millipore/AB9568
1:1000
Anti-S100β
Rabbit
Abcam/ab52642
1:1000
Alexa Fluor1 488 anti-chicken
Goat
Invitrogen/A-11039
1:1000
Alexa Fluor1 546 anti-guinea pig
Goat
Invitrogen/A-11074
1:1000
Alexa Fluor1 546 anti-rabbit
Goat
Invitrogen/A-11035
1:1000
Alexa Fluor1 546 anti-rat
Goat
Invitrogen/A-11081
1:1000
https://doi.org/10.1371/journal.pone.0229475.t001 t of primary and secondary antibodies with corresponding dilutions used in the current study. Table 1. List of primary and secondary antibodies with corresponding dilutions used in the current study. PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 3 / 25 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia using manual cell counting plugin function in ImageJ software (National Institutes of Health,
USA). Cells were counted on 3–4 mice/genotype, 3 brain slices/mouse (positive control) and
8–15 DRG slices/mouse. The total numbers of counted cells for the 3–4 mice/genotype are pre-
sented in Tables 1 and 2. Because it was difficult to discriminate individual SGCs within the
several SGCs surrounding a single neuronal cell body, “rings” surrounding individual neuro-
nal cell bodies were quantified.. Abbreviations: G6, GCaMP6f; GFP, green fluorescent protein; Nbr, number; NF, neurofilament. https://doi.org/10.1371/journal.pone.0229475.t002 Transgenic mouse lines for investigating the distribution and morphology
of sgcs and macrophages S100β-eGFP mice exhibit eGFP expression in both SGCs and sensory neurons. S100β protein is a
commonly used astrocytic marker in the brain and spinal cord and has also been reported to
be a marker of SGCs in DRGs [12,32]. The S100β-eGFP transgenic mouse [5], which expresses
enhanced GFP (eGFP) under the control of the S100β promoter, has proved to be a useful tool
to selectively label almost all astrocytes [32,33]. In the primary visual cortex (V1), we found
that eGFP was expressed in 86% of astrocytes (Fig 1A; Table 2) and only marginally (0.02%)
in neurons (Fig 1B; Table 2), corroborating those previous studies. This mouse line appears
also to be valuable for labeling SGCs within DRGs, since eGFP immunoreactivity was detected
in 85.8% of SGCs as shown by the colocalization of eGFP with GLAST, a specific SGC marker
(Fig 2A; Table 2). In addition, some cells expressing eGFP, and exhibiting a typical feature of
non-myelinating Schwann cells, were found in nerves attached to DRGs (Fig 2C; Table 2);
this is expected for cells known to synthetize S100β [34,35]. However, 13.5% of sensory neuron
soma (Fig 2B; Table 2) and their corresponding axons (Fig 2C) were found to express eGFP. To test whether this unexpected neuronal eGFP expression reflected normal endogenous
S100β protein expression in DRGs of wildtype mice, we used an antibody directed against
S100β. S100β endogenous protein was detected in both DRG SGCs and neurons of wildtype
mice (S1 Fig), in agreement with the eGFP expression pattern observed in S100β-eGFP mice. Hence, the S100β promoter does not represent a valuable promoter to target transgene expres-
sion selectively in DRG SGCs. However, it does allow eGFP expression in the vast majority of
DRG SGCs. ALDH1L1-eGFP mice express eGFP in a subset of SGCs and some neurons. Another recently
discovered marker of astrocytes is the protein aldehyde dehydrogenase 1 family member L1
[aldh1l1; [2]]. The ALDH1L1 promoter has been used to generate several transgenic mouse
lines [2,8,36], including the ALDH1L1-eGFP mice that exhibit eGFP selectively in most astro-
cytes of the CNS [2]. In agreement, we found that eGFP is expressed in 82.1% of V1 astrocytes
(Fig 3A; Table 2) with no detectable expression in neurons (Fig 3B; Table 2). The distribution
of eGFP-expressing cells was then investigated in DRGs of ALDH1L1-eGFP mice. Calcium imaging DORSAL ROOT GANGLIA
Mouse lines
Nbr of mice, Nbr
of slices/mouse
Total NeuN+ or
NF+ neurons
Total GFP+ or
G6+ neurons
% of GFP+ or G6
+ neurons
Total GLAST
+SGCs
Total GFP+ or G6
+ SGCs
% of GFP+ or G6
+ SGCs
S100β-eGFP
3, 10
599
81
13.5
345
296
85.8
ALDH1L1-eGFP
3, 11
1002
72
7.2
571
322
56.4
GFAP-Cre::GCaMP6f
3, 14
1189
696
58.5
1250
22
1.8
GLAST-CreERT2::
GCaMP6f
4, 15
1148
44
3.8
892
48
5.4
Cx30-CreERT2::
GCaMP6f
3, 8
837
0
0
721
0
0
Cx43-CreERT2::
GCaMP6f
3, 9
433
31
4.0
363
336
92.6
VISUAL CORTEX
Mouse lines
Nbr of mice, Nbr
of slices/mouse
Total NeuN
+ neurons
Total GFP+ or
G6+ neurons
% of GFP+ or
G6+ neurons
Total S100β
+astrocytes
Total GFP+ or G6
+ astrocytes
% of GFP+ or G6
+ astrocytes
S100β-eGFP
3, 10
4737
1
0.02
1288
1108
86.0
ALDH1L1-eGFP
3, 8
7807
0
0
1360
1117
82.1
GLAST-CreERT2::
GCaMP6f
3, 9
9533
3
0.03
1267
713
56.3
Cx30-CreERT2::
GCaMP6f
3, 9
5343
5
0.1
1000
464
46.4
Cx43-CreERT2::
GCaMP6f
3, 9
5203
227
4.4
780
74
9.5
Abbreviations: G6, GCaMP6f; GFP, green fluorescent protein; Nbr, number; NF, neurofilament. Table 2. Data quantification in DRG SGCs and V1 astrocytes from the mouse lines used in the current study. PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 4 / 25 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 Transgenic mouse lines for investigating the distribution and morphology
of sgcs and macrophages Enhanced
GFP immunoreactivity was observed only in 56.4% of SGCs (Fig 4A; Table 2) as well as in a
low percentage (7.2%) of sensory neuron soma, making this transgenic line unsuitable for the
specific visualization of the majority of SGCs. CX3CR1-eGFP mice show specific eGFP expression in macrophages. The CX3C chemokine
receptor 1 (CX3CR1), known as the fractalkine receptor [3], is a marker of microglial cells
[37,38]. The CX3CR1-eGFP mouse line, expressing eGFP under the control of the CX3CR1
promoter [3], has been extremely useful to visualize microglial cells and dynamic changes in
their morphology. In support of this previous study, we found a selective eGFP expression in
99.5% of V1 microglial cells with no detectable expression in neurons (Fig 5A and 5B; CX3CR1-eGFP mice show specific eGFP expression in macrophages. The CX3C chemokine
receptor 1 (CX3CR1), known as the fractalkine receptor [3], is a marker of microglial cells
[37,38]. The CX3CR1-eGFP mouse line, expressing eGFP under the control of the CX3CR1
promoter [3], has been extremely useful to visualize microglial cells and dynamic changes in
their morphology. In support of this previous study, we found a selective eGFP expression in
99.5% of V1 microglial cells with no detectable expression in neurons (Fig 5A and 5B;
Table 3). Since microglial cells are the CNS resident macrophages, we reasoned that peripheral
macrophages should also express eGFP in DRGs from CX3CR1-GFP mice. Indeed, we
observed that eGFP was expressed in 90.9% of Iba1-expressing macrophages (Fig 6A;
Table 3), validating the use of the CX3CR1-eGFP mouse line for labeling a prominent number
of DRG macrophages and investigating their morphological remodeling. Note that a few
eGFP-positive elements did not express Iba1 (Fig 6A, arrow). Because Iba1-immunopositive
profiles are spotty and not found within the whole cytosol, but instead are localized in some
subcellular compartments of macrophages, such eGFP-positive elements might correspond to
Iba1-negative macrophage compartments. g
p
g
Table 3). Since microglial cells are the CNS resident macrophages, we reasoned that peripheral
macrophages should also express eGFP in DRGs from CX3CR1-GFP mice. Indeed, we
observed that eGFP was expressed in 90.9% of Iba1-expressing macrophages (Fig 6A;
Table 3), validating the use of the CX3CR1-eGFP mouse line for labeling a prominent number
of DRG macrophages and investigating their morphological remodeling. Note that a few
eGFP-positive elements did not express Iba1 (Fig 6A, arrow). Transgenic mouse lines for investigating the distribution and morphology
of sgcs and macrophages https://doi.org/10.1371/journal.pone.0229475.g001 6 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia 371/journal.pone.0229475
September 11, 2020
7 / 25 7 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Fig 2. Expression of eGFP in S100β-eGFP mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from
S100β-eGFP mice showing eGFP-expressing cells (A-C left, green), GLAST-expressing SGCs (A middle, red, arrowheads), and
neurofilament-expressing neurons (B middle, red, asterisks). C, Images of proximal DRG nerves showing eGFP staining (C left,
green), non-myelinating Schwann cells (C left, arrows) and neurofilament-expressing axons (C middle, arrowheads). A-C right
show superimposed pictures. In A-C, bottom panel images correspond to enlargements of boxed areas in top panel images. For
each row, scale bar in left picture applies to middle and right corresponding pictures. Fig 2. Expression of eGFP in S100β-eGFP mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from
S100β-eGFP mice showing eGFP-expressing cells (A-C left, green), GLAST-expressing SGCs (A middle, red, arrowheads), and
neurofilament-expressing neurons (B middle, red, asterisks). C, Images of proximal DRG nerves showing eGFP staining (C left,
green), non-myelinating Schwann cells (C left, arrows) and neurofilament-expressing axons (C middle, arrowheads). A-C right
show superimposed pictures. In A-C, bottom panel images correspond to enlargements of boxed areas in top panel images. For
each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g002 Transgenic mouse lines for investigating the distribution and morphology
of sgcs and macrophages Because Iba1-immunopositive
profiles are spotty and not found within the whole cytosol, but instead are localized in some
subcellular compartments of macrophages, such eGFP-positive elements might correspond to
Iba1-negative macrophage compartments. 5 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 Fig 1. Expression of eGFP in S100β-eGFP mouse brain. A, B, Representative images of immunohistochemistry in the primary visual cortex (V1) from S100β-
eGFP mice showing eGFP-expressing cells (A & B left, green), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing neurons (B
middle, red, asterisks). A & B right show superimposed pictures. In A & B, bottom panel images correspond to higher magnification images. For each row,
scale bar in left picture applies to middle and right corresponding pictures. Abbreviations: ec, external capsule; CA1, subfield 1 of Ammon’s horn; I-VI, layers
of V1. https://doi.org/10.1371/journal.pone.0229475.g001
S ONE
Mouse lines for targeting glial cells in dorsal root ganglia PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Fig 1. Expression of eGFP in S100β-eGFP mouse brain. A, B, Representative images of immunohistochemistry in the primary visual cortex (V1) from S100β-
eGFP mice showing eGFP-expressing cells (A & B left, green), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing neurons (B
middle, red, asterisks). A & B right show superimposed pictures. In A & B, bottom panel images correspond to higher magnification images. For each row,
scale bar in left picture applies to middle and right corresponding pictures. Abbreviations: ec, external capsule; CA1, subfield 1 of Ammon’s horn; I-VI, layers
of V1 Fig 1. Expression of eGFP in S100β-eGFP mouse brain. A, B, Representative images of immunohistochemistry in the primary visual cortex (V1) from S100β-
eGFP mice showing eGFP-expressing cells (A & B left, green), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing neurons (B
middle, red, asterisks). A & B right show superimposed pictures. In A & B, bottom panel images correspond to higher magnification images. For each row,
scale bar in left picture applies to middle and right corresponding pictures. Abbreviations: ec, external capsule; CA1, subfield 1 of Ammon’s horn; I-VI, layers
of V1. PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 Mouse lines for investigating the roles of SGC Ca2+ signaling A & B right show superimposed pictures. In A & B,
bottom panel images correspond to enlargements of boxed areas in top panel pictures. In A bottom row, boxed area has been
rotated by about 45˚ clockwise. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g004 Fig 4. Expression of eGFP in ALDH1L1-eGFP mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs
from ALDH1L1-eGFP mice showing eGFP-expressing cells (A & B left, green), GLAST-expressing SGCs (A middle, red,
arrowheads), and NeuN-expressing neurons (B middle, red, asterisks). A & B right show superimposed pictures. In A & B,
bottom panel images correspond to enlargements of boxed areas in top panel pictures. In A bottom row, boxed area has been
rotated by about 45˚ clockwise. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g004 Fig 4. Expression of eGFP in ALDH1L1-eGFP mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs
from ALDH1L1-eGFP mice showing eGFP-expressing cells (A & B left, green), GLAST-expressing SGCs (A middle, red,
arrowheads), and NeuN-expressing neurons (B middle, red, asterisks). A & B right show superimposed pictures. In A & B,
bottom panel images correspond to enlargements of boxed areas in top panel pictures. In A bottom row, boxed area has been
rotated by about 45˚ clockwise. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g004 https://doi.org/10.1371/journal.pone.0229475.g004 mice using two different antibodies directed against GFAP (Table 1) have revealed almost no
GFAP-expressing SGCs (data not shown). However, a large number of studies have shown
increases in SGC GFAP expression level under pathological conditions, including, peripheral
nerve injury, DRG compression and pain [17,24]. In the CNS, GFAP is known to be expressed in neural progenitors during developmental
stages, giving rise to neurons, astrocytes and oligodendrocytes, thus preventing the use of the
non-inducible GFAP-Cre mice and Cre-LoxP system to selectively study astrocytes [29,42–
44]. The observation that astrocytes, neurons, and neuropil from V1 GFAP-Cre::GCaMP6f
expressed GCaMP6f (Fig 7A and 7B) was consistent with those previous studies and the view
that recombination occurs during development. However, because SGCs derive from the neu-
ral crest, a different lineage than astrocytic lineage, there was a possibility that GFAP-Cre mice
could be useful to drive gene expression selectively in a great proportion of SGCs. Mouse lines for investigating the roles of SGC Ca2+ signaling GFAP-Cre::GCaMP6f mouse line expresses GCaMP6f Ca2+ biosensor mainly in DRG sensory
neurons. Emerging evidence showing that Ca2+ is an important signaling messenger in SGCs
[12,39,40] prompted us to search for molecular tools to probe SGC Ca2+ dynamics. To do so,
we took advantage of the powerful CAG-lox-STOP-lox-GCaMP6f transgenic mouse line [7]
that was recently developed to express the genetically-encoded Ca2+ indicator GCaMP6f in cell
types of interest. We first crossed CAG-lox-STOP-lox-GCaMP6f mice with GFAP-Cre mice
[29] to generate GFAP-Cre::GCaMP6f double transgenic mice. GFAP is indeed extensively
used as a gold standard astrocytic marker, which expression is enhanced in reactive astrocytes
during aging, CNS injury, pain, and diseases [41]. GFAP has also been reported as a marker of
DRG SGCs [11], although we have not found convincing immunohistochemical evidence in
the literature showing clear SGC GFAP expression under physiological conditions. In agree-
ment, in our hands, immunohistochemical experiments conducted on DRGs from wildtype Fig 3. Expression of eGFP in ALDH1L1-eGFP mouse brain. A, B, Representative images of immunohistochemistry in V1 from ALDH1L1-eGFP mice showing eGFP-
expressing cells (A & B left, green), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing neurons (B middle, red, asterisks). A & B right
show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi org/10 1371/journal pone 0229475 g003 Fig 3. Expression of eGFP in ALDH1L1-eGFP mouse brain. A, B, Representative images of immunohistochemistry in V1 from ALDH1L1-eGFP mice showing eGFP-
expressing cells (A & B left, green), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing neurons (B middle, red, asterisks). A & B right
show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g003 8 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Mouse lines for targeting glial cells in dorsal root ganglia
1371/journal.pone.0229475
September 11, 2020
9 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020
9 / 25 9 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Fig 4. Expression of eGFP in ALDH1L1-eGFP mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs
from ALDH1L1-eGFP mice showing eGFP-expressing cells (A & B left, green), GLAST-expressing SGCs (A middle, red,
arrowheads), and NeuN-expressing neurons (B middle, red, asterisks). PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Table 3. Data quantification in DRG macrophages and V1 microglial cells from the CX3CR1-eGFP mouse line. DORSAL ROOT GANGLIA
Mouse line
Nbr of mice, Nbr of
slices/mouse
Total NeuN
+ neurons
Total GFP
+ neurons
% of GFP
+ neurons
Total Iba1
+macrophages
Total GFP
+ macrophages
% of GFP
+ macrophages
CX3CR1-eGFP
3, 8
689
0
0
320
291
90.9
VISUAL CORTEX
Mouse line
Nbr of mice, Nbr of
slices/mouse
Total NeuN
+neurons
Total GFP
+ neurons
% of GFP
+ neurons
Total Iba1
+microglia
Total GFP
+microglia
% of GFP
+ microglia
CX3CR1-eGFP
3, 8
2488
0
0
851
847
99.5
Abbreviations: GFP, green fluorescent protein; Nbr, number. Data quantification in DRG macrophages and V1 microglial cells from the CX3CR1-eGFP mouse line. diameter neurons; Fig 8A; Table 2). To assess whether GCaMP6f was expressed under detect-
able levels in SGCs, SGC functional Ca2+ signals were registered using 2-photon imaging in
intact ex vivo DRGs. Bath application of an agonist cocktail to Gq GPCRs did not evoke any
Ca2+ elevations in cells morphologically identified as SGCs (i.e. ring-shaped cells surrounding
neuronal soma). However, 23.5% of GCaMP6f-expressing sensory neurons exhibited marked
intracellular Ca2+ elevations (Fig 8B). Taken together these results show that the GFAP-Cre::
GCaMP6f double transgenic mouse line is not an adequate tool for studying Ca2+ dynamics
selectively in DRG SGCs. y
GLAST-CreERT2::GCaMP6f and Cx30-CreERT2::GCaMP6f mice express GCaMP6f in a few
or no SGCs, respectively. GLAST has been conventionally used to identify a subset of astrocytes
and the majority of DRG SGCs [45–47], making the GLAST promoter a good candidate to tar-
get gene expression in a large number of SGCs. Additionally, connexin 30 (Cx30) has also
been used as a marker of a fraction of astrocytes [45], although its expression in DRG SGCs
has not yet been reported. To determine whether GLAST and Cx30 promoters could drive
GCaMP6f expression selectively in SGCs, we crossed both inducible GLAST-CreERT2 [28]
and Cx30-CreERT2 [28] mice with CAG-lox-STOP-lox-GCaMP6f mice. To induce GCaMP6f
expression in the resultant GLAST-CreERT2::GCaMP6f and Cx30-CreERT2::GCaMP6f dou-
ble transgenic lines, mice were treated with 1 mg tamoxifen per day during 5 consecutive days. As expected, in the CNS (V1), GCaMP6f was detected in 56.3% and 46.4% of astrocytes, in
these two lines respectively (Fig 9A; Fig 10A; Table 2) with an insignificant expression in neu-
rons (0.03% and 0.1%, respectively; Fig 9B; Fig 10B; Table 2). Mouse lines for investigating the roles of SGC Ca2+ signaling In DRGs
from GFAP-Cre::GCaMP6f mice, GCaMP6f was expressed in only 1.8% of SGCs while, to our
surprise, it was observed in 58.5% of sensory neurons (including small- and large-sized Fig 5. Expression of eGFP in CX3CR1-eGFP mouse brain. A, B, Representative images of immunohistochemistry in V1 from CX3CR1-eGFP mice showing eGFP-
expressing cells (A & B left, green), Iba1-expressing microglial cells (A middle, red, arrowheads), and NeuN-expressing neurons (B middle, red, asterisks). A & B
right show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi org/10 1371/journal pone 0229475 g005 Fig 5. Expression of eGFP in CX3CR1-eGFP mouse brain. A, B, Representative images of immunohistochemistry in V1 from CX3CR1-eGFP mice showing eGFP-
expressing cells (A & B left, green), Iba1-expressing microglial cells (A middle, red, arrowheads), and NeuN-expressing neurons (B middle, red, asterisks). A & B
right show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g005 https://doi.org/10.1371/journal.pone.0229475.g005 10 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 Mouse lines for targeting glial cells in dorsal root ganglia PLOS ONE In contrast, DRGs from
GLAST-CreERT2::GCaMP6f mice showed GCaMP6f immunoreactivity in only 5.4% of SGCs
and a low percentage (3.8%) of sensory neurons (Fig 11A and 1B; Table 2), invalidating this
mouse line for investigating Ca2+ dynamics selectively in SGCs. Furthermore, no GCaMP6f
expression at all was found in SGCs or sensory neurons of Cx30-CreERT2::GCaMP6f (Fig
12A and 12B; Table 2), making this mouse line unsuitable to study Ca2+ signaling in DRG
SGCs. Surprisingly though, GCaMP6f was observed in non-identified cells, which occasionally
expressed macrophage markers (Fig 12C), suggesting that a few of them are macrophages. Of
note, GCaMP6f was also found to be expressed at the DRG surface, possibly in tissues encapsu-
lating DRGs (Fig 12B). Cx43-CreERT2::GCaMP6f mouse line allows the detection of Ca2+ transients in the vast
majority of SGCs. Cx43 is a widely used specific marker of a large proportion of CNS astrocytes
and almost all DRG SGCs [13,48], making it particularly relevant to astrocyte and SGC
research. In another attempt to establish a tool for monitoring Ca2+ transients selectively in a
great number of SGCs, we generated the inducible Cx43-CreERT2::GCaMP6f double trans-
genic mouse line by crossing the tamoxifen-inducible Cx43-CreERT2 [30] with the CAG-lox-
STOP-lox-GCaMP6f mice [7]. Two weeks after tamoxifen treatment, V1 cortex and DRGs 11 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Fig 6. Expression of eGFP in CX3CR1-eGFP mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from CX3CR1-eGFP mice
showing eGFP staining (A & B left, green, arrowheads), Iba1 staining (A middle, red, arrowheads), and neurons identified by NeuN immunoreactivity (B Fig 6. Expression of eGFP in CX3CR1-eGFP mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from CX3CR1-eGFP mice
showing eGFP staining (A & B left, green, arrowheads), Iba1 staining (A middle, red, arrowheads), and neurons identified by NeuN immunoreactivity (B Fig 6. Expression of eGFP in CX3CR1-eGFP mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from CX3CR1-eGFP mice
showing eGFP staining (A & B left, green, arrowheads), Iba1 staining (A middle, red, arrowheads), and neurons identified by NeuN immunoreactivity (B Fig 6. Expression of eGFP in CX3CR1-eGFP mouse DRGs. PLOS ONE A, B, Representative images of immunohistochemistry in DRGs from CX3CR1-eGFP mice
showing eGFP staining (A & B left, green, arrowheads), Iba1 staining (A middle, red, arrowheads), and neurons identified by NeuN immunoreactivity (B 12 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia middle, red, asterisks). In A bottom panel, the arrow points to an eGFP-expressing element that does not colocalize with Iba1 staining. A & B right show
superimposed pictures. In A & B, bottom panel images correspond to higher magnification images. For each row, scale bar in left picture is applied to
middle and right corresponding pictures. middle, red, asterisks). In A bottom panel, the arrow points to an eGFP-expressing element that does not colocalize with Iba1 staining. A & B right show
superimposed pictures. In A & B, bottom panel images correspond to higher magnification images. For each row, scale bar in left picture is applied to
middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g006 https://doi.org/10.1371/journal.pone.0229475.g006 were analyzed. To our disappointment, GCaMP6f immunoreactivity was observed in only
9.5% of V1 astrocytes (Fig 13A; Table 2), a much lower percentage than previously reported
in hippocampal astrocytes (~70%; 31). Possible explanations for these discrepancies are
regional (V1 versus hippocampus) variability in transgene recombination efficiency, in addi-
tion to the fact that we tested our mice 2 weeks after tamoxifen treatment compared to 4 weeks
[31]. Furthermore, GCaMP6f was also detectable in a small subset (4.4%) of neurons (Fig 13B;
Table 2). These results suggest that Cx43-CreERT2 mouse line may be ineffective for driving
substantial GCamP6f expression specifically in V1 astrocytes, even when treating mice 10
times (10 x 1 mg/kg tamoxifen) as previously reported [31]. However, and importantly, cellular immunohistochemical characterization in DRGs
revealed that GCaMP6f was expressed in 92.6% of SGCs and a very small subset (4%) of neu-
rons (Fig 14A and 14B; Table 2). In conclusion, the inducible Cx43-CreERT2::GCaMP6f line
can be used to study the role of Ca2+ activity in the majority of DRG SGCs. We believe that
this mouse line will prove to be a valuable tool to examine DRG SGC functions. Fig 7. Expression of GCaMP6f in GFAP-Cre::GCaMP6f mouse brain. A, B, Representative images of immunohistochemistry in V1 from GFAP-Cre::GCaMP6f mice
showing GCaMP6f ubiquitous expression (A & B left, green), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing neurons (B middle, red,
asterisks). PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE A & B right show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g007 Fig 7. Expression of GCaMP6f in GFAP-Cre::GCaMP6f mouse brain. A, B, Representative images of immunohistochemistry in V1 from GFAP-Cre::GCaMP6f mice
showing GCaMP6f ubiquitous expression (A & B left, green), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing neurons (B middle, red,
asterisks). A & B right show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g007 13 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Fig 8. Cellular expression and functionality of GCaMP6f in GFAP-Cre::GCaMP6f mouse DRGs. A, Representative images of immunohistochemistry in DRGs from
GFAP-Cre::GCaMP6f mice showing GCaMP6f staining (top & bottom left panels, green), GLAST-expressing SGCs (top middle panel, red, arrowheads), and small
and large sensory neurons (bottom middle panel, red, asterisks). Top & bottom right panels show superimposed pictures. For each row, scale bar in left picture applies
to middle and right corresponding pictures. B, Representative images of 2-photon Ca2+ imaging experiment in ex vivo DRGs where neuronal GCaMP6f-expressing cell
bodies (outlined areas of interest, left panel) exhibit intracellular Ca2+ increases; ➀baseline, ➁Gq GPCR agonist cocktail (50μM ATP, 10μM Histamine, 10μM
Carbachol and 50μM DHPG) application, and ➂wash (right panel). https://doi org/10 1371/journal pone 0229475 g008 Fig 8. Cellular expression and functionality of GCaMP6f in GFAP-Cre::GCaMP6f mouse DRGs. A, Representative images of immunohistochemistry in DRGs from
GFAP-Cre::GCaMP6f mice showing GCaMP6f staining (top & bottom left panels, green), GLAST-expressing SGCs (top middle panel, red, arrowheads), and small
and large sensory neurons (bottom middle panel, red, asterisks). Top & bottom right panels show superimposed pictures. For each row, scale bar in left picture applies
to middle and right corresponding pictures. B, Representative images of 2-photon Ca2+ imaging experiment in ex vivo DRGs where neuronal GCaMP6f-expressing cell
bodies (outlined areas of interest, left panel) exhibit intracellular Ca2+ increases; ➀baseline, ➁Gq GPCR agonist cocktail (50μM ATP, 10μM Histamine, 10μM
Carbachol and 50μM DHPG) application, and ➂wash (right panel). https://doi.org/10.1371/journal.pone.0229475.g008 https://doi.org/10.1371/journal.pone.0229475.g008 Discussion In this study we report the characterization of several tools for investigating SGC and macro-
phage morphological changes as well as Ca2+ activity within DRGs. Our data show that most
tested transgenic mice widely used to investigate astrocyte morphology and function are not
suitable for studying DRG SGCs. Indeed, among these mice, some exhibit ectopic transgene
expression in small to large proportions of neurons while others show low to no transgene
expression in SGCs. PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 14 / 25 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Fig 9. Expression of GCaMP6f in GLAST-CreERT2::GCaMP6f mouse brain. A, B, Representative images of immunohistochemistry in V1 from GLAST-CreERT2::
GCaMP6f mice showing GCaMP6f-expressing cells (A & B left, green, arrowheads), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing
neurons (B middle, red, asterisks). A & B right show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g009 Fig 9. Expression of GCaMP6f in GLAST-CreERT2::GCaMP6f mouse brain. A, B, Representative images of immunohistochemistry in V1 from GLAST-CreERT2::
GCaMP6f mice showing GCaMP6f-expressing cells (A & B left, green, arrowheads), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing
neurons (B middle, red, asterisks). A & B right show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g009 https://doi.org/10.1371/journal.pone.0229475.g009 However, we generated and identified a double transgenic line, named Cx43-CreERT2::
GCaMP6f, allowing inducible GCaMP6f expression primarily in the vast majority of DRG
SGCs (92.6%) with only a very small percentage (4%) of neurons expressing GCaMP6f. Con-
sidering the high GCaMP6f expression level detected in SGCs, ectopic CreERT2-mediated
recombination in neurons might be easily reduced by simply decreasing the number of tamox-
ifen injections (< 10 injections) as well as time post-treatment (< 15 days). Although it would
be of interest to test whether GCaMP6f is detectable in other DRG cell types, including
Schwann cells, fibroblasts, pericytes and endothelial cells, this possibility seems unlikely based
on (i) the peculiar and readily identifiable SGC shape (ring surrounding round neurons), (ii)
the fact that no GCaMP6f expression was observed in nerves attached to DRG, and (iii) the
fact that drug-induced Ca2+ elevations were observed exclusively in ring-shaped cells sur-
rounding neuronal cell bodies from the Cx43-CreERT2::GCaMP6f (data not shown). PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 Discussion With the
emerging interest in SGC Ca2+ signaling in modulating nociceptive neuron activity [39,40,49],
this new line should be applicable for investigating a wide array of questions in pain research. Additionally, it is worth pointing out that Cx43 expression is very stable and even upregulated
in SGCs following PNS injury. Therefore we do not anticipate any compromised (downregu-
lated) transgene expression using the Cx43-CreERT2 mouse line, strengthening the likely
applicability of this mouse line for a wide array of questions in PNS neurobiology and pain
research. Interestingly, a publication from Valeria Cavalli’s laboratory was recently posted on
BioRxiv and reported a mouse line (BLBPCre-ERT2) allowing conditional expression of trans-
genes specifically in SGCs, complementing the use of the Cx43-CreERT2 mouse line. Further-
more, we identified a second mouse line, called CX3CR1-eGFP, displaying eGFP expression
selectively in most DRG macrophages. This line is likely to be useful for the study of spinal PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 15 / 25 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Fig 10. Expression of GCaMP6f in Cx30-CreERT2::GCaMP6f mouse brain. A, B, Representative images of immunohistochemistry in V1 from Cx30-CreERT2::
GCaMP6f mice showing GCaMP6f-expressing cells (A & B left, green, arrowheads), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing
neurons (B middle, red, asterisks). A & B right show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g010 Fig 10. Expression of GCaMP6f in Cx30-CreERT2::GCaMP6f mouse brain. A, B, Representative images of immunohistochemistry in V1 from Cx30-CreERT2::
GCaMP6f mice showing GCaMP6f-expressing cells (A & B left, green, arrowheads), S100β–expressing astrocytes (A middle, red, arrowheads), and NeuN-expressing
neurons (B middle, red, asterisks). A & B right show superimposed pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g010 https://doi.org/10.1371/journal.pone.0229475.g010 https://doi.org/10.1371/journal.pone.0229475.g010 cord injury in which abnormal pain strongly correlates with an increased number of DRG
macrophages [50]. Among the other mouse lines we characterized, S100β-eGFP line may be considered appli-
cable to some extent, even though eGFP was expressed in a substantial percentage of sensory
neurons (13.5%), but also in a great proportion of DRG SGCs (85.8%). The readily identifiable
ring-shaped SGCs can indeed be almost undoubtedly differentiated from the rounded neuro-
nal cell bodies. PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 Discussion Additionally, eGFP expression was found to be brighter in SGCs relatively to
neuronal cell bodies (Fig 2A and 2B), which helps ascertain the identity of both cell types. Of
note, we found that eGFP expression in SGCs and neurons reflects the endogenous S100β pro-
tein expression, showing that, in DRGs, S100β is not a glial selective promoter. These data
complement studies reporting that S100β promoter drives transgene expression in some
motor neurons within the brainstem and spinal cord [33,51]. The fact that the GFAP promoter drives merely no transgene expression in SGCs from
GFAP-Cre::GCaMP6f mouse line is consistent with our immunohistochemistry data showing
only rare GFAP-expressing SGCs. Furthermore, our data showing Cre-mediated recombina-
tion in 58.5% of sensory neurons in such GFAP-Cre::GCaMP6f mice suggest the possibility
that GFAP is expressed in DRG neuronal lineage during development. This possibility though
is not supported by the absence of evidence for a developmental GFAP expression in PNS neu-
ronal lineage, while such expression is well described in the CNS [52]. Thus, together our
results suggest that the GFAP promoter used in the GFAP-Cre::GCaMP6f mice is not PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 16 / 25 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Fig 11. Expression of GCaMP6f in GLAST-CreERT2::GCaMP6f mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from
GLAST-CreERT2::GCaMP6f mice showing GCaMP6f labeling (A & B left, green), GLAST-expressing SGCs (A bottom panel, red, arrowheads,), and Fig 11. Expression of GCaMP6f in GLAST-CreERT2::GCaMP6f mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from
GLAST-CreERT2::GCaMP6f mice showing GCaMP6f labeling (A & B left, green), GLAST-expressing SGCs (A bottom panel, red, arrowheads,), and Fig 11. Expression of GCaMP6f in GLAST-CreERT2::GCaMP6f mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from
GLAST-CreERT2::GCaMP6f mice showing GCaMP6f labeling (A & B left, green), GLAST-expressing SGCs (A bottom panel, red, arrowheads,), and 17 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia NeuN-expressing neurons (B bottom panel, red, asterisks). A & B right show superimposed pictures. In A & B, bottom panel images correspond to higher
magnification images. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g011 NeuN-expressing neurons (B bottom panel, red, asterisks). A & B right show superimposed pictures. Discussion In A & B, bottom panel images correspond to higher
magnification images. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g011 sufficient to drive a strong and selective expression of transgenes in a large number of SGCs
under physiological conditions. However, in disagreement of this hypothesis, the same pro-
moter (2.2kB human GFAP minimum promoter, 29) has been previously used successfully to
highly express a transgene selectively in the overwhelming majority of DRG SGCs with no Fig 12. Expression of GCaMP6f in Cx30-CreERT2::GCaMP6f mouse DRGs. A-C, Representative images of immunohistochemistry in DRGs from
Cx30-CreERT2::GCaMP6f mice showing GCaMP6f immunoreactivity (A–C left, green, arrowheads), GLAST-expressing SGCs (A middle, red), and NeuN-
expressing neurons (B middle, red, asterisks). In B left, empty arrows point to GCaMP6f expression at the surface of the DRG. In C left, white arrow points
to GCaMP6f staining that colocalizes with the macrophage marker CD68 immunoreactivity (C middle, red,). A-B right show superimposed pictures. For
each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi org/10 1371/journal pone 0229475 g012 Fig 12. Expression of GCaMP6f in Cx30-CreERT2::GCaMP6f mouse DRGs. A-C, Representative images of immunohistochemistry in DRGs from
Cx30-CreERT2::GCaMP6f mice showing GCaMP6f immunoreactivity (A–C left, green, arrowheads), GLAST-expressing SGCs (A middle, red), and NeuN-
expressing neurons (B middle, red, asterisks). In B left, empty arrows point to GCaMP6f expression at the surface of the DRG. In C left, white arrow points
to GCaMP6f staining that colocalizes with the macrophage marker CD68 immunoreactivity (C middle, red,). A-B right show superimposed pictures. For
h
l b
i l f
i
li
iddl
d i h
di
i Fig 12. Expression of GCaMP6f in Cx30-CreERT2::GCaMP6f mouse DRGs. A-C, Representative images of immunohistochemistry in DRGs from
Cx30-CreERT2::GCaMP6f mice showing GCaMP6f immunoreactivity (A–C left, green, arrowheads), GLAST-expressing SGCs (A middle, red), and NeuN-
expressing neurons (B middle, red, asterisks). In B left, empty arrows point to GCaMP6f expression at the surface of the DRG. In C left, white arrow points
to GCaMP6f staining that colocalizes with the macrophage marker CD68 immunoreactivity (C middle, red,). A-B right show superimposed pictures. For
each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g012 https://doi.org/10.1371/journal.pone.0229475.g012 18 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia Fig 13. Discussion Expression of GCaMP6f in Cx43-CreERT2::GCaMP6f mouse brain. A, B, Representative images of immunohistochemistry in V1 from Cx43-CreERT2::
GCaMP6f mice showing GCaMP6f immunoreactivity (A & B left, green), S100β–expressing astrocytes (A middle bottom panel, red, arrowheads) and NeuN-
expressing neurons (B bottom panel, red, asterisk). A & B right show superimposed pictures. In A & B, bottom panel pictures correspond to enlargements of top panel
pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g013 Fig 13. Expression of GCaMP6f in Cx43-CreERT2::GCaMP6f mouse brain. A, B, Representative images of immunohistochemistry in V1 from Cx43-CreERT2::
GCaMP6f mice showing GCaMP6f immunoreactivity (A & B left, green), S100β–expressing astrocytes (A middle bottom panel, red, arrowheads) and NeuN-
expressing neurons (B bottom panel, red, asterisk). A & B right show superimposed pictures. In A & B, bottom panel pictures correspond to enlargements of top panel
pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi org/10 1371/journal pone 0229475 g013 Fig 13. Expression of GCaMP6f in Cx43-CreERT2::GCaMP6f mouse brain. A, B, Representative images of immunohistochemistry in V1 from Cx43-CreERT2::
GCaMP6f mice showing GCaMP6f immunoreactivity (A & B left, green), S100β–expressing astrocytes (A middle bottom panel, red, arrowheads) and NeuN-
expressing neurons (B bottom panel, red, asterisk). A & B right show superimposed pictures. In A & B, bottom panel pictures correspond to enlargements of top panel
pictures. For each row, scale bar in left picture applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g013 https://doi.org/10.1371/journal.pone.0229475.g013 19 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Fig 14. Expression of GCaMP6f in Cx43-CreERT2::GCaMP6f mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from
Cx43-CreERT2::GCaMP6f mice showing GCaMP6f immunoreactivity (A & B left, green), GLAST-expressing SGCs (A middle, red, arrowheads), and NF-
expressing neuronal cell bodies (B middle, red, asterisks). Note: Yellow asterisk (A, top panel) shows a single neuron expressing GCaMP6f. A & B right show
superimposed pictures. In A & B, bottom panel pictures correspond to enlargements of boxed areas in top panel images. For each row, scale bar in left picture
applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g014
S ONE
Mouse lines for targeting glial cells in dorsal root ganglia Mouse lines for targeting glial cells in dorsal root ganglia Fig 14. Expression of GCaMP6f in Cx43-CreERT2::GCaMP6f mouse DRGs. Discussion A, B, Representative images of immunohistochemistry in DRGs from
Cx43-CreERT2::GCaMP6f mice showing GCaMP6f immunoreactivity (A & B left, green), GLAST-expressing SGCs (A middle, red, arrowheads), and NF-
expressing neuronal cell bodies (B middle, red, asterisks). Note: Yellow asterisk (A, top panel) shows a single neuron expressing GCaMP6f. A & B right show
superimposed pictures. In A & B, bottom panel pictures correspond to enlargements of boxed areas in top panel images. For each row, scale bar in left picture Fig 14. Expression of GCaMP6f in Cx43-CreERT2::GCaMP6f mouse DRGs. A, B, Representative images of immunohistochemistry in DRGs from
Cx43-CreERT2::GCaMP6f mice showing GCaMP6f immunoreactivity (A & B left, green), GLAST-expressing SGCs (A middle, red, arrowheads), and NF-
expressing neuronal cell bodies (B middle, red, asterisks). Note: Yellow asterisk (A, top panel) shows a single neuron expressing GCaMP6f. A & B right show
superimposed pictures. In A & B, bottom panel pictures correspond to enlargements of boxed areas in top panel images. For each row, scale bar in left picture
applies to middle and right corresponding pictures. https://doi.org/10.1371/journal.pone.0229475.g014 https://doi.org/10.1371/journal.pone.0229475.g014 20 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia ectopic sensory neuronal expression [53]. This implies that other factors (e.g. number of trans-
gene copies, gene microenvironment) may account for the variability of transgene expression
levels in SGCs and cell selectivity. Indeed, transgenes insert randomly into the genome and the
transgene expression cannot always mimic endogenous promoter activity. Thus, controlling
gene microenvironment represents a strategy to enhance the specificity of transgenic targeting. One approach is to insert transgenes into a cassette containing all introns, promoter regulatory
elements, exons and 5’ and 3’ flanking DNA of the GFAP gene [52]. This approach has been
used in a recent study showing specific transgene expression in SGCs of the trigeminal ganglia,
although it remains to be evaluated in DRG SGCs [49]. Another approach is to insert trans-
genes into bacterial artificial chromosomes (BACs) as they reduce the influence of chromo-
some position effects and allow more predictable transgene expression patterns. Despite these
advantages, BAC transgenic mice appear to suffer from some remaining expression variation. Discussion Indeed, using the BAC-based GLAST-CreERT2 mice [28] to generate the GLAST-CreERT2::
GCaMP6f mice, we obtained both (i) a marginal percentage of GLAST-driven CreERT2-me-
diated recombination in DRG SGCs, and (ii) a lack of cell specificity, while endogenous
GLAST protein is selectively and prominently expressed in essentially all SGCs [47]. Therefore,
it stands to reason that even though both, conventional and BAC-based transgenesis, can lead
to robust and cell specific transgene expression in the CNS glial cells, their expression pattern
may significantly differ in DRG glia (and vice versa). Knock-in technology may be considered
as a good alternative to traditional transgenic techniques; indeed it enables transgene insertion
at a specific glial gene locus of the mouse genome, which allows excellent control of gene
microenvironment, and thus is likely to better avoid chromosome position effects and circum-
vent the above-discussed drawbacks associated with conventional and BAC-based transgen-
esis. Our findings using the knock-in CX3CR1-eGFP mouse line does support the relevance of
this approach by showing specific eGFP expression in the vast majority of microglial cells and
macrophages in V1 and DRGs, respectively (Fig 5; Fig 6). Thus, to improve DRG glial cell spe-
cific targeting, it would be of interest in future studies to generate and/or use knock-in glial
mouse models. One of such mouse models, the knock-in GLAST-CreERT2 mouse line in
which CreERT2 transgene is inserted at the GLAST gene locus [54], appears to be a good can-
didate to examine next. A large number of in vivo studies have used the GFAP or S100β promoters to drive different
transgene expression in astrocytes. Results from these studies are routinely interpreted as due
to the expression of transgenes only in astrocytes. Our finding that the GFAP or S100β pro-
moters drive GCaMP6f or eGFP expression in 58.8% or 13.5% of DRG sensory neurons,
respectively, should be considered when interpreting in vivo results from such studies. In conclusion, most of the tools tested in the current study were found ineffective in study-
ing selectively the majority of SGCs in DRGs, although a lot of molecular and functional simi-
larities exist between DRG SGCs and astrocytes. Therefore, further work is required for
characterizing and identifying other already available tools as well as developing new geneti-
cally-modified mouse lines and adeno-associated viral tools to specifically target large propor-
tions of DRG SGCs, but also macrophages. Discussion Together with the two mouse lines validated here
(Cx43-CreERT2::GCaMP6f and CX3CR1-eGFP), these future molecular tools will be of prom-
inent interest in understanding better how DRG glia can modulate sensory information pro-
cessing under physiological and pathological conditions. Acknowledgments We gratefully acknowledge S. Antoine and S. Guinoiseau for animal care; S. Guinoiseau and P. Meriau for dissecting some tissues; C. Ayissi Sama and M. Tantouch for slicing some tissues; J. M. Andrieu, F. Charbonnier, B. Delhomme, P. Djian, and M. Oheim for sharing lab spaces
and pieces of equipment (cryostat, microscopes); J. Strinnakre for proof reading; and both the
BioMedTech imaging and mouse core facilities. Supporting information S1 Fig. S100β immunoreactivity signal (red) in DRGs from wildtype mice, showing that
endogenous S100β is expressed in both ring-shaped SGCs (arrowheads) and sensory 21 / 25 PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
September 11, 2020 PLOS ONE Mouse lines for targeting glial cells in dorsal root ganglia neuron cell bodies (asterisks). (PDF) Author Contributions Conceptualization: Yasmine Rabah, Elsie Moukarzel, Cendra Agulhon. Data curation: Yasmine Rabah, Bruna Rubino, Elsie Moukarzel, Cendra Agulhon. Formal analysis: Yasmine Rabah, Bruna Rubino, Elsie Moukarzel. Funding acquisition: Yasmine Rabah, Bruna Rubino, Elsie Moukarzel, Cendra Agulhon. Investigation: Yasmine Rabah, Bruna Rubino, Elsie Moukarzel. Methodology: Cendra Agulhon. Project administration: Cendra Agulhon. Project administration: Cendra Agulhon. Validation: Bruna Rubino, Elsie Moukarzel, Cendra Agulhon. Validation: Bruna Rubino, Elsie Moukarzel, Cendra Agulhon. Visualization: Yasmine Rabah, Bruna Rubino, Cendra Agulhon. Writing – original draft: Yasmine Rabah, Cendra Agulhon. Writing – review & editing: Bruna Rubino, Elsie Moukarzel, Cendra Agulhon. PLOS ONE | https://doi.org/10.1371/journal.pone.0229475
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Proof of Concept trial of diagnostic ultrasound in the orthopaedic triage setting.
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RESEARCH ARTICLE RESEARCH ARTICLE Abstract: Purpose: This study aims to investigate the benefits of Point of
Care (POC) ultrasound and Scheduled Ultrasound Clinics using
a Proof of Concept approach in the orthopaedic triage setting. Materials and Methods: The trial ran for a six week period
sourcing all patients referred to the orthopaedic triage service. The trial collected three measurable outcomes i.e. two Patient
Reported Outcome Measures: MSK-HQ and The Care Measure,
and a Proof of Concept clinician survey via Survey Monkey. These were completed by the clinicians and patient cohort
during the scheduled clinic and POC clinical contact. Results: A total of 135 patients received a diagnostic
ultrasound scan over the six week trial period. 34 patients
received a diagnostic ultrasound scan from the Extended Scope
Practitioner (ESP) Physiotherapist in a Scheduled Care setting. 101 patients received a diagnostic ultrasound scan from the ESP
Podiatrist in orthopaedic triage over the six week period (74 in
Scheduled care clinics and 27 at Point of Care). The outcome
measure suggest that patients recieved a rapid diagnosis (n.135),
implementation of appropriate treatment pathway (86.36%), a
positive impact on Referral to Treatment (88.55%), appropriate
implementation of conservative treatment (91.11%) and had
excellent co-production during the trial with an 88% average
CARE Measure Score. The MSK-HQ results suggested a wide
variety of muscuoskeletal conditions were asseessed during the
trial with a mean MSK-HQ score of 27.6 with a Standard
Deviation of 12. Purpose: This study aims to investigate the benefits of Point of
Care (POC) ultrasound and Scheduled Ultrasound Clinics using
a Proof of Concept approach in the orthopaedic triage setting. Materials and Methods: The trial ran for a six week period
sourcing all patients referred to the orthopaedic triage service. The trial collected three measurable outcomes i.e. two Patient
Reported Outcome Measures: MSK-HQ and The Care Measure,
and a Proof of Concept clinician survey via Survey Monkey. These were completed by the clinicians and patient cohort
during the scheduled clinic and POC clinical contact. Received: 18th September 2018
Accepted: 2nd February 2018
Published: 25th March 2018 These were completed by the clinicians and patient cohort
during the scheduled clinic and POC clinical contact. Results: A total of 135 patients received a diagnostic
ultrasound scan over the six week trial period. 34 patients
received a diagnostic ultrasound scan from the Extended Scope
Practitioner (ESP) Physiotherapist in a Scheduled Care setting. Competing Interests: The
author have declared that no
competing interests exists. Open Science Journal – March 2018 Funding: The author(s) received
no specific funding for this work Proof of Concept Trial of Diagnostic
Ultrasound in the Orthopeadic Triage
Setting Gafin Ericson Morgan1*, Helen Welch1, Denise Jenkins1, Lisa
Medhurst-Wroe1 1Cwm Taf University Health Board, United Kingdom *Corresponding author: Gafin Ericson Morgan: gafin.morgan@wales.nhs.uk *Corresponding author: Gafin Ericson Morgan: gafin.morgan@wales.nhs.uk Citation: Morgan G.E., Welch H.,
Jenkins D., Wroe L.M. (2018) Proof
of Concept Trial of Diagnostic
Ultrasound in the Orthopaedic
Triage Setting.Open Science
Journal 3(1) Introduction Diagnostic ultrasound has been thought of as a rapid, accurate, repeatable,
non-expensive, noninvasive and without the risk of radiation [1]. It has been used
to focus clinical questions, improve differential diagnosis and direct patients to
the most suitable treatment [2]. However there is a need to define the benefits of
appropriate use and, limit any unnecessary imaging and its consequences [3]. It is
becoming accepted practice for ultrasound diagnostics to expand beyond the
borders of Radiology departments due to the evolving nature of clinical practice
and the requirement of prudent healthcare principles [4,5]. The Clinical
Musculoskeletal Assessment Triage Service (CMATS) is an orthopaedic triage
service that assess primary care referrals into secondary care orthopaedic clinics. Assessment supported by appropriate imaging can often be utilised to ensure the
implementation of the right pathway for patients at the earliest opportunity. Historically, provision of MSK Ultrasound diagnostics has been via a referral
to Radiology. However, there is increasing evidence that assessment, investigation
and initiating treatment at the initial appointment are shown to be cost-effective
and increase patient satisfaction [6]. Utilising this approach also reduces repeated
hospital visits for further diagnostics and appointment times for results. The aim
of using Point of Care (POC) ultrasound is to enhance the patient experience
through instant access to diagnosis, timely implementation of most appropriate
clinical pathway and achievement of the optimal outcome in the shortest possible
time. Abstract: 101 patients received a diagnostic ultrasound scan from the ESP
Podiatrist in orthopaedic triage over the six week period (74 in
Scheduled care clinics and 27 at Point of Care). The outcome
measure suggest that patients recieved a rapid diagnosis (n.135),
implementation of appropriate treatment pathway (86.36%), a
positive impact on Referral to Treatment (88.55%), appropriate
implementation of conservative treatment (91.11%) and had
excellent co-production during the trial with an 88% average
CARE Measure Score. The MSK-HQ results suggested a wide
variety of muscuoskeletal conditions were asseessed during the
trial with a mean MSK-HQ score of 27.6 with a Standard
Deviation of 12. Copyright:© 2018 This is an
open access article under the terms
of the Creative Commons
Attribution License, which permits
unrestricted use, distribution, and
reproduction in any medium,
provided the original author and
source are credited. Funding: The author(s) received
no specific funding for this work Open Science Journal – March 2018 1 Open Science Journal Open Science Journal Research Article Conclusion: Results from clinician sourced Survey Monkey data,
The CARE Measure and MSK HQ suggest that POC ultrasound
and Scheduled care clinics offer a positive benefit for the patient’s
care pathway, are beneficial clinically and suggest adequate
demand for the service. Keywords: Point of Care, diagnostic ultrasound, service review,
orthopaedic triage Keywords: Point of Care, diagnostic ultrasound, service review,
orthopaedic triage Results The total number of patients who received a scan in this study were 135, of
which 45% were New Assessments and 55.55% Follow Ups. The total number
from ESP Physiotherapist planned scanning session was 34, and the total number
from ESP Podiatrist planned scanning session was 74. The total number of
patients who received a scan at Point of Care was 27. The average number of of
scans per week was 22.5. For the six week period 267 patient contacts were made
in the orthopaedic triage setting by the ESP Podiatrist and 101 of these recieved
an ultrasound scan. Results from each measure are indicated below. Materials and Methods Three clinicians attained a PGCert in Diagnostic Musculoskeletal Ultrasound
(Consortium for the Accreditation of Sonographic Education Accredited -
http://www.case-uk.org/) in 2015 and subsequently supported by Cwm Taf
University Health Board Radiology Department underwent a 12 month
mentorship programme. Two of these clinicians were directly involved in the
trial. An Extended Scope Practitioner (ESP) Physiotherapist participated in one
clinical session per week of a scheduled ultrasound clinic. An ESP Podiatrist
participated in two clinical scheduled scanning sessions per week and in addition Open Science Journal – March 2018 2 Open Science Journal Research Article to this utilised the ultrasound equipment in a POC setting. The patient caseload
was sourced from the orthopaedic triage pathway. Patients were allocated to the scheduled ultrasound scan clinics prior to the
trial and the scanning clinics were populated. The trial ran for a six week period. This study utilised three measures to assess the benefits of diagnostic
ultrasound service provision. The outcome measures are outlined in the following
section and include Clinician Input data on Survey Monkey (Fig.1), and patient
questionnaires (The CARE Measure (Fig2) and MSK-HQ (Fig.3)). Open Science Journal – March 2018 Survey Monkey clinician survey Results obtained from the Survey Monkey clinician survey for the period of
the study (Fig a). A total of 135 patients recieved a diagnostic ultrasound scan
over the six week period. 83.36% of scans resulted in a change of patient
pathway, suggesting the diagnostic ultrasound scan identified an appropriate
pathway for the patient. The largest amount of scans completed were of the foot
and ankle due to the ESP Podiatrist utilising the diagnostic ultrasound at POC
and the number of scheduled scanning clinics. A small number (8.15%) of
patients recieved a rapid referral to orthopaedics referral following their
ultrasound scan. A significant number of scans resulted in appropriate
implementation of conservative treatment, allowed the provision of specific
advice / empowerment and ensured better co-production, improved patient
decision making and a positive impact on referral to treatment (Fig a). Quantitative or
qualitative
Data Description
Data
Source
Total
Number
Quantitative
Total Number of US scans carried out
Survey
Monkey
135
Total number that would have been referred to Radiology (in
the absence of ultrasound equipment. Survey
Monkey
135
Percentage of additional scans with POCT
Calculation
20%
Number of patients whose pathway changed as a
Survey 3 Open Science Journal Open Science Journal Open Science Journal Research Article Research Article
consequence of the scan
Monkey
86.36%
Number of shoulders scanned
Survey
Monkey
25
Number of Hip scanned
Survey
Monkey
1
Number of Knees scanned
Survey
Monkey
2
Number of wrists/hand scanned
Survey
Monkey
5
Number of Foot and Ankle Scanned
Survey
Monkey
101
Number of Diagnostics
Survey
Monkey
139
Quantitative
Number of patients where the scan changed the patient care
pathway. Survey
Monkey
83.36%
Number of patients where the scan signposted for rapid
attendance at an alternative service. E.g. Rheumatology
Survey
Monkey
2.22%
Number of patients where the scan signposted for rapid
referral to Orthopaedics
Survey
Monkey
8.15%
Number of patients where the scan resulted in appropriate
implementation conservative treatment. Survey
Monkey
91.11%
Number of patients where the scan identified the need for
further investigation
Survey
Monkey
17.29%
Number of patients where the scan recognised a serious
pathology. Survey
Monkey
0%
Qualitative
Number of patients where the scan informed the diagnosis
and facilitated provision of specific advice / empowerment
and ensured better co-production
Survey
Monkey
99.21%
Clinician’s opinion that undertaking the scan as part of the
consultation changed the clinical decision for this patient. Survey
Monkey
100%
Clinicians opinion that delay in receiving the scan would
have resulted in poorer outcome
(See appendix 1 for details)
Survey
Monkey
57.78%
Clinician’s opinion that the scan had a positive impact on
the Referral to Treatment
Survey
Monkey
88.55%
Fig a: Survey Monkey data results. Open Science Journal – March 2018 4 Open Science Journal Research Article MSK-HQ Results The MSK-HQ is a validated musculoskeletal health questionnaire which
measures key health domains i.e. pain severity, physical function, work, fatigue,
emotional health, physical activity, independence, understanding, confidence to
self manage and overall impact. The highest possible outcome score for MSK-HQ
is 56, the least outcome being 0 [7]. The study population had a mean MSK-HQ score of 27.6 which is almost a
50% reduction in MSK health domains. This is indicative of a population of
significant MSK health issues affecting not only physical but emotional and
general wellbeing. The study population had a standard deviation (Standard Deviation P) of
MSK-HQ scores of 12. This is indicative that the sample scores have high
variance and the mean score results are an inaccurate representation of the
population scores. There is therefore great variability of the MSK-HQ scores
within the population sampled suggesting a wide variety of MSK condition
sampling within the study. (See Fig. b) 0
10
20
30
40
50
60
0
20
40
60
80
100
MSK HQ Scores
Patients
Fig b: MSK-HQ Scores Standard Deviation Fig b: MSK-HQ Scores Standard Deviation There was no significant correlation between MSK-HQ scores and amount of
physical activity measured as a component of the MSK-HQ questionnaire. The
Mean daily Physical Activity level of the measured population was 1.67 days
which is less than the recommended weekly physical activity recommendations
recommended by the World Health Organisation [7]. However, the standard
deviation of the population measured was 12 which is indicative of a varied
spread of physical activity within the population. The Care Measure Outcome The CARE measure (Consultation And Relational Empathy Measure) is a
person-centered process measure that was developed and researched at the Open Science Journal – March 2018 5 Open Science Journal Open Science Journal Research Article Departments of General Practice in Glasgow and Edinburgh Universities
supported by the Scottish Government. The CARE Measure is a quick (10
questions), clear and easy to complete patient-completed questionnaire. It
measures empathy in the context of the therapeutic relationship during a one-on-
one consultation between a clinician and a patient. Originally developed and
rigorously tested for use by General Medical Practitioners, it has since been
successfully used by other medical staff, allied health professionals and nurses. Question
Answers %
Poor
Fair
Good
Very Good
Excellent
Making you feel at ease
0
0
0
11
88.15
Letting you tell your story
0
0
0.74
14.81
84.44
Really Listening
0
0
1.53
12.21
86.26
Being Interested in you as a whole person
0
0
2.31
10
87.69
Fully understanding your concerns
0
0
2.27
7.58
90.15
Showing care and compassion
0
0
1.54
6.92
91.54
Being Positive
0
0
3
12
85
Explaining things clearly
Helping you take control
0
0
1.63
6.5
91.87
Helping you take control
0
0.78
0.78
10.94
85.94
Making a plan of action with you
0
0.74
0.74
8.15
89.63
Fig c: Care Measure Results Results of The Care Measure indicate an average “Excellent” score of 88% of
patients. Suggestive that an excellent therapeutic relationship was measured and
experienced by the patient. The authors declare that they have no conflict of interest. The authors declare that they have no conflict of interest. Conclusion It is recognised that appropriate use of ultrasound has the potential to
improve quality of care but robust clinical governance is required to ensure that
procurement and deployment of equipment is appropriate, service provision is
evidence based and delivered by qualified and competent clinicians. Patients are
the primary benefactors of POC ultrasound with improved diagnostic accuracy,
faster and appropriate implementation of treatment pathways and demonstrating
excellent co-production with the patient population. Radiology is also a co-
benefactor with the likely reduced referral rates to the service. The results of this study suggest that POC ultrasound in orthopaedic triage
has a positive outcome on the patient care pathway and experience. Open Science Journal – March 2018 6 Open Science Journal Research Article References: 1. Abu-Zidan F, 2012, Point-of-care ultrasound in critically ill patients: Where do we stand?, J Emerg
Trauma Shock,Jan-Mar; 5(1): 70–71 1. Abu-Zidan F, 2012, Point-of-care ultrasound in critically ill patients: Where do we stand?, J Emerg
Trauma Shock,Jan-Mar; 5(1): 70–71 2. Adhikari s, Amini R, Stolz L, Blaivas M, Imact of point of care ultrasound o quality of care in
clinical practice. Reports in Medical Imaging. 2014:7 81–93 2. Adhikari s, Amini R, Stolz L, Blaivas M, Imact of point of care ultrasound o quality of care in
clinical practice. Reports in Medical Imaging. 2014:7 81–93 3. Moore C, Copel J, 2011, Point of Care Ultrasonography, New England Journal of Medicine,
364:749-757, February 24 4. McKiernan S, Chiarelli P, Warren-Forward H. Diagnostic ultrasound use in physiotherapy,
emergency medicine, and anaesthesiology. Radiography, 2010 16, 154e159 5. Prudent Healthcare, http://www.prudenthealthcare.org.uk/principles/ 6. Sivan M, Brown J, Brennan S, Bhakta B, 2010, A one-stop approach to the management of soft
tissue
and
degenerative
musculoskeletal
conditions
using
clinic-based
ultrasonography. Musculoskeletal Care. 2011;9(2):63–8. 7. Hill JC, Kang S, Benedetto E, et al Development and initial cohort validation of the Arthritis
Research UK Musculoskeletal Health Questionnaire (MSK-HQ) for use across musculoskeletal care
pathways BMJ Open 2016;6:e012331. doi: 10.1136/bmjopen-2016-012331 8. World Health Organisation, 2010, Global Recommendations on Physical Activity for Health, WHO
Library Cataloguing-in-Publication Data Library Cataloguing-in-Publication Data Open Science Journal – March 2018 Appendix 1 Clarification of statement: “If the patient had to wait 6 months for this scan
would this have resulted in poor treatment outcomes?” Clinician Comments: •
Chronic tendinopathy
•
Synovitis acute joint damage
•
Patient concordance
•
Patient immobilising
•
Incorrect diagnosis
•
Patient in severe pain
•
High pain levels
•
Patient was immobilising and developing chronic pain
•
Inappropriate conservative intervention
•
Patient immobile
•
Patient doing inappropriate self Physio
•
Rapid diagnosis and referral
•
Definitive staging of tendinopathy
•
Off-loading for 6 months until diagnosis completed
•
Inappropriate treatment without scan
•
Chronic tenosynovitis •
Chronic tendinopathy •
Synovitis acute joint damage •
Patient concordance •
Patient immobilising Open Science Journal – March 2018 Open Science Journal Open Science Journal Research Article •
Patient treated for plantar fasciopathy and has back problem
•
Chronic pain
•
Possible need for surgical intervention
•
Risk of nerve compression and long term damage
•
Delayed diagnosis would delay appropriate treatment to restore
function
•
Poor function with previous treatment ineffective, Correct and
prompt diagnosis allows early treatment and restoration of function
•
Very poor function, would become chronic waiting for diagnosis
•
Inappropriate conservative management
•
Central Sensitisation
•
Irreversible changes to tendon
•
Increasing neovascularity
•
Assist mobilising in advanced rehab
•
Ongoing pain and disability. Chronic tendon changes
•
Patient was immobilising. Scan resulted in mobilising. •
Partial tear of Achilles
•
Needs surgical management of muscle tear. •
Reduced function due to pain
•
Poor function leading to chronic movement restriction
•
Delay in management of injection treatment and restoring function
•
Incorrect diagnosis: New pathway spinal ESP
•
Incorrect diagnosis from orthopaedics
•
Incorrect diagnosis from GP: Vague differential diagnosis
•
Diagnosis improved management of condition
•
Patient doing wrong exercises aggravating tendon
•
Chronic fasciopathy
•
Delayed care and implementation of treatment
•
Active synovitis could cause tendon damage
•
Attenuated tendon
•
Immediate implementation of conservative treatment
•
Complete attenuation of tibialis posterior with risk to integrity of foot
•
Acute on chronic tendinopathy
•
Pt being treated for tendinopathy but actually arthropathy
•
More appropriate pathway. Prudent. Open Science Journal – March 2018 Appendix 1 •
Chronic tendinopathy
•
Ongoing pain and discomfort
•
Severe mechanical foot changes
•
Patient now requires orthopaedic surgical management due to
dislocation of Long head of Biceps which would cause damge to
underlying muscle
•
Would have delayed injection and physio management causing further
stiffness and loss of function
•
Poor function and capsular restriction were the patients main
complaint and the ultrasound scan ruled out a diagnosis which would
have warranted ortho referral
•
Calcification was diagnosed initally and may have resulted in
barbotage procedure when not needed which may have resulted in •
Risk of nerve compression and long term damage •
Delayed diagnosis would delay appropriate treatment to restore
function •
Poor function with previous treatment ineffective, Correct and
prompt diagnosis allows early treatment and restoration of function •
Patient now requires orthopaedic surgical management due to
dislocation of Long head of Biceps which would cause damge to
underlying muscle •
Would have delayed injection and physio management causing further
stiffness and loss of function •
Poor function and capsular restriction were the patients main
complaint and the ultrasound scan ruled out a diagnosis which would
have warranted ortho referral •
Calcification was diagnosed initally and may have resulted in
barbotage procedure when not needed which may have resulted in
delay of physio management which was outcome of exam 8 Open Science Journal Open Science Journal Research Article •
Chronic pain
•
Patient currently frustrated with lack of diagnostics
•
Achilles Tendon rehab rupture
•
Immobile in boot
•
Upscaling of pain mechanisms
•
Chronic tendinopathy. suffering and distress of patient. •
Clearer management of chronic tendinopathy
•
Chronic pain. chronic tendinopathy
•
Chronic pain and deformity in STJ - change in foot position
•
Chronic Changes in tendon
•
Ruptured Achilles Tendon
•
Severely degenerative peroneal tendons. at point of rupture
•
Sensitisation poor rehab longer conservative management •
Ruptured Achilles Tendon •
Severely degenerative peroneal tendons. at point of rupture •
Sensitisation, poor rehab, longer conservative management Appendix 2 Individual Patient Feedback from Care Measure written in section “Do you
have any further comments”: “Felt very much at ease” “truly satisfied” “excellent support” “Very helpful & excellent treatment. Very good advice and putting my mind
at ease” “Excellent Service” “Very satisfied and happy with treatment” “Very understanding about the condition and giving me the confidence that a
plan is set in place to help with pain relief. Thank you. “ “Absolutely very helpful and happy to help, understands everything” “Very helpful and explained many things thoroughly” “Well looked after” “Excellent service received. Thank you very much” “Having the ultrasound equipment here was ideal. All done in one go!” “Very positive consultation. Many Thanks” Open Science Journal – March 2018 9
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IDENTIFICACION DEL CILINDRO NUDOSO EN IMÁGENES TC DE TROZAS PODADAS DE PINUS RADIATA UTILIZANDO REDES NEURONALES ARTIFICIALES
|
Maderas. Ciencia y tecnología
| 2,010
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cc-by
| 5,918
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ISSN impresa 0717-3644
ISSN online 0718-221X
maderas
CIENCIA Y TECNOLOGIA, 12(3):229-239, 2010
DOI 10.4067/S0718-221X2010000300007
IDENTIFICACION DEL CILINDRO NUDOSO EN IMÁGENES TC
DE TROZAS PODADAS DE PINUS RADIATA UTILIZANDO REDES
NEURONALES ARTIFICIALES
IDENTIFICATION OF KNOTTY CORE IN PINUS RADIATA LOGS FROM COMPUTED
TOMOGRAPHY IMAGES USING ARTIFICIAL NEURAL NETWORK
Gerson Rojas-Espinoza1 , Oscar Ortíz-Iribarren2
RESUMEN
La factibilidad de identificar el cilindro nudoso en imágenes de tomografía computarizada
de rayos X (TC) de trozas podadas de pino radiata (Pinus radiata D. Don), fue evaluada utilizando
un método de clasificación supervisada basado en Redes Neuronales Artificiales (RNA). El proceso
de clasificación consideró también la identificación de la zona libre de defectos y nudos. Treinta
trozas podadas de pino radiata fueron escaneadas en un escáner médico multi-slice de rayos X,
donde las imágenes TC resultantes fueron obtenidas cada 5 mm. Un total de 270 imágenes TC fueron
clasificadas utilizando la técnica Redes Neuronal Artificial y los mapas temáticos resultantes, fueron
filtrados con un filtro de mediana de 7 x 7. La precisión del proceso de clasificación de las imágenes
TC fue obtenida a partir de una matriz de confusión y el estadístico Kappa. Los resultados indicaron
que el cilindro nudoso puede ser identificado y separado con una precisión de 92.7%, mientras que
para la precisión global se obtuvo un valor de 85.0%. Tras filtrar los mapas temáticos, los valores de
precisión aumentaron a 96.3% y 92.3% para el cilindro nudoso y la precisión global, respectivamente.
Los valores Kappa fueron de 0.607 y 0.764 para los mapas temáticos y mapas temáticos filtrados,
respectivamente. Estos valores indicaron que existe un fuerte grado de conformidad entre los datos
de referencia y el proceso de clasificación. Los resultados sugieren que es factible aplicar RNA como
procedimiento de clasificación para identificar el cilindro nudoso en imágenes TC de trozas podadas
de pino radiata.
Palabras claves: Cilindro nudoso, tomografía computarizada, redes neuronales artificiales, matriz de
confusión, pino radiata
ABSTRACT
The feasibility of identifying Knotty core in images of X-ray computed tomography (CT)
of pruned radiata pine logs (Pinus radiata D. Don), was evaluated using a supervised classification
method based on artificial neural networks (ANN). The classification process also considers the
identification of the clear wood and knots. Thirty pruned radiata pine logs were scanned in a multislice scanner medical X-ray, where the resulting CT images were obtained every 5 mm. A total of
270 CT images were classified using the ANN, and the resulting thematic maps were filtered with a
median filter of 7 x 7. The accuracy of the classification process of the CT images was obtained from
a confusion matrix and Kappa statistics. The results indicated that the Knotty core can be identified
and separated with an accuracy of 92.7%, while for the overall accuracy was obtained a value of
85.0%. After filtering thematic maps, the precision values increased to 96.3% and 92.3% for the
defective core and overall accuracy, respectively. Kappa values were 0.607 and 0.764 for thematic
maps and thematic maps filtered, respectively. These values indicate that there is a strong degree of
agreement between reference data and classification process. The results suggest that it is feasible to
apply artificial neural networks as classification procedure to identify the Knotty core in CT images
of pruned radiata pine logs.
Keywords: Knotty core, computed tomography, artificial neural networks, confusion matrix, radiata
pine.
Académico (Ph.D) Departamento de Ingeniería en Maderas, Facultad de Ingeniería, Universidad del Bío-Bío, Concepción, Chile.
Magíster en Ciencia y Tecnología de la Madera, Departamento de Ingeniería en Maderas, Facultad de Ingeniería, Universidad del Bío-Bío, Concepción, Chile.
Autor para correspondencia: grojas@ubiobio.cl
Recibido: 23.06.2010 Aceptado: 14.10.2010
1
2
229
maderas
CIENCIA Y TECNOLOGIA, 12(3):229-239, 2010
Universidad del Bío-Bío
INTRODUCCION
En general, la eficiencia del proceso de aserrío se ve influenciada principalmente por la
calidad de la materia prima, donde la presencia de defectos externos e internos en las trozas tiene un
efecto significativo sobre la calidad final de la madera aserrada producida. La presencia de defectos en
la madera tales como nudos, grietas internas, bolsas de resina, bolsas de corteza, entre otros, castiga
drásticamente el precio final de la madera. En Chile, las principales empresas madereras realizan
grandes esfuerzos por mejorar la calidad de sus bosques, principalmente, de la especie pino radiata, la
cual sustenta la industria maderera debido a su disponibilidad y variadas aplicaciones de su madera.
En esta dirección, la poda es el principal tratamiento silvícola que las empresas están aplicando a
los bosques de pino radiata para obtener fustes de mejor calidad. Este tratamiento silvícola, es una
actividad que elimina las ramas vivas o muertas del árbol y permite la producción de madera libre
de nudos, una vez que el crecimiento en diámetro del fuste cubre la zona de defecto. De esta manera,
la parte interna de las ramas podadas queda confinada en una zona central, respecto al volumen del
fuste, zona denomina cilindro nudoso (CN). El diámetro del CN depende principalmente de la calidad
del sitio, el número de podas y edad del árbol en el momento de la poda. Así, la identificación del
CN antes de iniciar el proceso de aserrío se transforma en uno de los principales problemas de los
aserraderos. Normalmente los operadores utilizan las características de la superficie de la troza como
indicadores para predecir la localización y tipo de defectos internos
En general, la identificación de las características y defectos internos de las trozas requiere
de la utilización de técnicas no destructivas. En este sentido, diferentes técnicas, algunas de ellas
habitualmente utilizadas en la ciencia médica como una herramienta para examinar y evaluar de manera
no destructiva la estructura del cuerpo humano, han sido evaluadas con el objetivo de caracterizar
internamente las trozas. Los ultrasonidos (Birkeland y Han 1991), la resonancia magnética nuclear
(Chang et al. 1989; Coates et al. 1998) y Radiación gamma (Hagman 1993, Karsulovic et al. 2002 y
2005) son algunas de las técnicas utilizadas para este fin. Sin embargo, la Tomografía Computarizada
(TC) de rayos X, ha mostrado un gran potencial para detectar defectos internos y otras irregularidades
en trozos de coníferas y latifoliadas (Taylor et al. 1984; Funt y Bryant 1987; Zhu et al. 1991; Li et
al. 1996; Guddanti y Chang 1998; Bhandarkar et al. 1999; Schmoldt et al. 1995, 1998, 2000 and
Nordmark 2002, 2003).
Taylor et al. (1984) evaluaron un sistema automático de segmentación de imágenes TC,
basado en el nivel de gris obtenido a partir de histogramas de intensidad de frecuencia, para identificar
y localizar nudos. Funt y Bryant (1987) desarrollaron un programa que interpreta automáticamente
las imágenes TC y que utiliza la alta densidad y la forma elíptica de los nudos y la baja densidad,
junto con la textura rugosa de zonas con pudrición, para diferenciar la madera podrida de la madera
libre de defectos. Estos autores coinciden en que la variación del contenido de humedad en la troza
en algunos casos dificulta la identificación. Schmoldt et al. (1995) indicaron que el nivel de gris de
las imágenes TC está directamente relacionado con la densidad, variando dramáticamente con las
diferentes especies y contenido de humedad. Oja y Temnerud (1999) reportaron que en imágenes TC
de trozas verdes de Abeto Rojo (Picea abies (I.) Kars), el nivel de gris de la madera difiere mucho
entre duramen y albura, debido principalmente a la diferencia en el contenido de humedad. Una
situación similar fue observada en imágenes TC de trozas de Arce (Acer saccharum Marsh) por Rojas
et al. (2005).
En otra dirección, varios métodos de clasificación de imágenes TC, basados en el conocimiento
a priori del nivel de gris asociado a cada característica o defecto interno han sido evaluados, con el
fin de mejorar la capacidad de interpretación de este tipo de imágenes. Rojas et al. (2006, 2007)
presentaron un método de clasificación supervisada basado en el algoritmo de máxima verosimilitud
(CMV), para identificar y separar la albura del duramen, nudos y pudrición, a partir de imágenes
TC de trozas de Arce (Acer saccharum Marsh). Los resultados indicaron que la albura puede ser
identificada con una precisión de 97. 8 %, mientras que la precisión global fue de 82.6 %. Por su
parte, para esta especie y utilizando el algoritmo (CMV) y un análisis de textura de las imágenes TC,
230
Identificación del cilindro nudoso...:Rojas-Espinoza y Ortíz-Iribarren.
maderas
CIENCIA Y TECNOLOGIA, 12(3):229-239, 2010
Wei et al. (2008a) reportaron valores de precisión de 85.6 %, 72.1 % y 83% para la detección de la
albura, nudos y corteza respectivamente. Recientemente, Rojas y Ortiz (2009) utilizaron el CMV para
identificar y separar el CN en imágenes TC de trozas podadas de pino radiata. De acuerdo a estos
autores, el CN puede ser separado con una precisión de 98.5 %.
En esta dirección, la utilización de algoritmos de clasificación basados en redes
neuronales artificiales (RNA) también ha sido evaluada. Schmoldt et al. (2000) utilizaron RNA
para la identificación y clasificación de albura, corteza, nudos y grietas en roble rojo (Quercus
rubra L.), álamo amarillo (Liriodendron tulipifera L.) y cerezo negro (Prumus serotina Ehrh). Si
bien estas características internas fueron identificadas en las distintas especies, al igual que en los
estudios citados anteriormente, existía superposición entre el nivel de gris asociado a las diferentes
características o defectos internos. Nordmark (2002) también utilizó RNA, para identificar nudos en
trozas de pino silvestre (Pinus sylvestris L.). Si bien los nudos pudieron ser separados del resto de
la madera con una precisión de 95.9 %, existe un cierto grado de superposición del nivel de gris en
la zona límite entre los nudos y la madera libre de defectos. De igual manera, este autor utilizó esta
técnica para desarrollar modelos para predecir nudos y la geometría de las trozas de pino silvestre
(Nordmark2003), reportando un valor de precisión de 90%. En esta dirección, Wei et al. (2008b)
desarrollaron un método que utiliza RNA y análisis textural para identificar albura duramen, nudos
y corteza en imágenes TC de trozas de arce (Acer saccharum Marsh) y picea negra (Picea mariana
(Mill)). Los resultados de este estudio indicaron que una precisión de global de 89.5 % y 92.4 %
puede ser obtenida para arce y picea negra, respectivamente.
En general, de acuerdo a la literatura revisada, queda de manifiesto que la tomografía
computarizada de rayos X, es un método no destructivo que permite identificar los defectos y
características internas de las trozas. Sin embargo, el problema no está completamente resuelto. La
compleja estructura y variabilidad inherente a la madera, las variaciones en la densidad y contenido
de humedad limitan, en algunos casos, la precisión en la identificación de los defectos internos. En
este sentido el objetivo principal de este estudio es evaluar la factibilidad de utilizar redes neuronales
artificiales para identificar el cilindro nudoso en imágenes TC de trozas podadas de pino radiata.
MATERIALES Y METODOS
Materiales.
Treinta árboles podados de pino radiata (Pinus radiata D. Don) de 23 años de edad, fueron
seleccionados y talados en el predio El Espigado, localizado en la zona de Arauco, distante a 100 Km
de la ciudad de Concepción. La primera poda de estos árboles fue realizada a los 5 años de edad y a
una altura de 2.7 m. De cada árbol se obtuvo una troza de la zona podada del árbol, de largo comercial
equivalente a 4.3 m aproximadamente y enviados para su inmediato procesamiento en el pabellón de
tecnología de la madera de la Universidad del Bío-Bío. Cada rollizo fue trozado en tres partes, de un
metro de longitud cada una. Las trozas obtenidas fueron marcadas y luego selladas en cada extremo
con impermeabilizante Anchorseal, para evitar la pérdida de humedad. Un total de noventa trozas
fueron obtenidas y utilizadas como material de ensayo.
Las trozas fueron escaneadas en un escáner médico de rayos X (Tomografía computarizada
(CT)), de marca Philips, multi-Slice, perteneciente al laboratorio de imagenología del Hospital
del Trabajador, en Concepción. El proceso de escaneo fue realizado a 120 kVp y 249 mA, donde
las imágenes TC resultantes fueron obtenidas cada 5 mm, en formato de 512 x 512 píxeles y con
resolución de 8 bits. La resolución espacial de un píxel, varió entre 0.7 y 0.9 mm, dependiendo del
diámetro de cada troza. Luego, tres imágenes TC fueron seleccionadas por cada troza, obteniendo
finalmente una muestra de 270 imágenes, las que fueron analizadas en este estudio.
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Clasificación de imágenes TC usando redes neuronales artificiales
Las RNA son modelos computacionales que se desarrollaron originalmente para modelar
la forma en que el cerebro humano desarrolla una determinada tarea y actualmente se aplican en
la clasificación de imágenes (reconocimiento de patrones) y otros trabajos de análisis de imágenes
(Wei et al. 2008b). Estos modelos o sistemas de procesamiento de la información consisten en un
gran número de elementos simples llamados nodos o neuronas que están organizados en capas. Cada
neurona está conectada con otras neuronas mediante enlaces de comunicación, cada uno de los cuales
tiene asociado un peso. En los pesos se encuentra el conocimiento que tiene la RNA acerca de un
determinado problema (Palmer et al. 2000)
La RNA generalmente consta de una capa de entrada, una o más capas ocultas y una capa de
salida. La primera etapa para desarrollar un RNA consiste es definir un grupo de entrenamiento, que
corresponde a grupos de píxeles asociado a cada patrón o clase a identificar. Estos grupos de píxeles se
obtienen directamente de un grupo de imágenes seleccionadas para este fin. La segunda etapa consiste
en entrenar la RNA con el grupo de entrenamiento obtenido. Durante este proceso de aprendizaje
de la RNA, los pesos son modificados de forma iterativa de acuerdo con los valores del grupo de
entrenamiento. En esta etapa se deben definir los valores de la tasa de aprendizaje y el momento,
variables importantes para el entrenamiento de la RNA, ya que controlan el tamaño del cambio de
los pesos en cada iteración (Palmer et al. 2000). La tasa de aprendizaje es la responsable de acelerar
el proceso de aprendizaje o velocidad de respuesta de la RNA. A mayor tasa de aprendizaje, mayor
es la modificación de los pesos en cada iteración y por ende el aprendizaje será más rápido, pero
por otro lado puede ocasionar oscilaciones. Si el valor es demasiado bajo, el sistema tomará mucho
tiempo para converger a la solución. El momento se utiliza para controlar las posibles oscilaciones
o inestabilidades que se generan en la variación de los pesos y reduce la posibilidad que el sistema
converja hacia un mínimo local. La expresión matemática de la regla de modificación de los pesos se
indica a continuación (Rumelhart et al. 1986):
(1)
Donde Wji es el peso entre la neurona i y la neurona j, n es el número de la iteración, ε
corresponde a la tasa de aprendizaje, δpj es el error de la neurona j para el patrón p, xpi es la salida de
la neurona i para el patrón p y η corresponde al momento.
Entre los diversos tipos de RNA, la configuración retro propagación (back-propagation (BP)
ANN) ha sido la más popular, ya que son muy eficaces en el reconocimiento de patrones y con una
implementación bastante simple (Schmoldt et al. 2000). Este tipo de RNA ha sido utilizado en el
presente estudio.
El procedimiento de análisis de las imágenes se inició con la generación del grupo de
entrenamiento, obtenido a partir de una muestra de píxeles de las clases o patrones a identificar, en
este caso, CN, madera libre de defectos (MLD) y nudos. Este grupo de entrenamiento fue utilizado
como información de entrada por el sistema de redes neuronales, el cual posteriormente realiza el
proceso de clasificación. Valores de 0.15 y 0.5 fueron utilizados para la tasa de aprendizaje y momento,
respectivamente, con un total de 500 iteraciones. Otros valores de tasa de aprendizaje y momentun
fueron evaluados, sin embargo los utilizados presentaron mejor desempeño. Finalmente, se llevó a
cabo la clasificación de las imágenes TC, en donde se tenía una capa de entrada y una capa de salida
con tres nodos de salida correspondiente a las tres características de las trozas de Pinus radiata que
se deseaban identificar. Luego de la clasificación, los mapas temáticos fueron filtrados utilizando un
filtro de mediana de 7x7 con el objetivo de eliminar el ruido generado por el proceso de clasificación.
Finalmente, para evaluar cuantitativamente este procedimiento de clasificación de imágenes
TC, valores de precisión de detección del CN, MLD y nudos, fueron obtenidos a partir de la matriz
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de confusión determinada para cada imagen TC evaluada (Rojas y Ortiz 2009). En este método,
la precisión fue determinada mediante la comparación entre los píxeles de la imagen que fueron
correctamente clasificados y los mismos píxeles en la imagen sin clasificar (imagen de referencia ó
imagen real). El resultado de esta comparación es expresado en la forma de una matriz de confusión
o de error. En esta matriz, los datos de referencia (representados por las columnas de la matriz) son
comparados con los datos clasificados (representados por las filas de la matriz). La diagonal principal
indica el grado de acuerdo entre ambos grupos de datos. La precisión (expresada en porcentaje)
para cada clase fue determinada a partir de la matriz de confusión, dividiendo el número de píxeles
correctamente clasificados por el número total de píxeles en los datos de referencia. La precisión
global (incluye todas las clases) para una imagen clasificada fue también calculada dividiendo en total
de píxeles correctamente clasificados (suma de la diagonal principal) por el numero total de píxeles en
la matriz de confusión (Story y Congalton 1986). Un valor de 100 para los valores de precisión global
indica un acuerdo perfecto entre los resultados de la clasificación y los datos de referencia. Al mismo
tiempo, los errores de omisión y comisión pueden ser también determinados a partir de la matriz de
confusión. Los errores de omisión corresponden a píxeles que pertenecen a una clase de interés y que
han sido clasificados erróneamente en otra clase, mientras que los errores de comisión corresponden
a píxeles de otras clases y que el clasificador ha asignado a una clase de interés.
El estadístico Kappa fue también determinado como complemento a los valores de precisión
obtenidos de la matriz de confusión. Éste índice estadístico mide el grado de ajuste, exclusivamente
para la predicción de la clasificación y utiliza para el análisis los resultados de la matriz de confusión
(Jensen 1996). El estadístico K es calculado como sigue :
(2)
donde r es el numero de filas en la matriz, xii es el numero de píxeles en la fila i y columna i, xi+ y x+i
corresponden al total marginal para la fila y y columna i, respectivamente, y N es el numero total de
píxeles.
Para este estudio, se utilizaron los 270 mapas temáticos del proceso de clasificación. La
matriz de confusión asociada a cada mapa temático fue obtenida a partir de una muestra de 70 píxeles
seleccionados aleatoriamente (Richards y Jia 1999) de cada mapa temático. Este análisis también
fue realizado para cada mapa temático filtrado, utilizando exactamente los mismos 70 píxeles para
cada uno de los mapas temáticos. El procedimiento de clasificación RNA y la evaluación cuantitativa
fueron realizados utilizando el programa computacional PCI versión 10.0 (PCI 2005).
RESULTADOS Y DISCUSION
Escaneo de trozas
Un ejemplo típico del resultado del proceso de escaneo y que corresponde a una imagen TC
de una sección transversal de una troza podada de pino radiata, se presenta en la fig. 1a. Por su parte,
la fig. 1b muestra la fotografía digital de la sección transversal asociada a la imagen TC presentada
en la fig. 1a. En general, en la fig. 1a se observan claramente las características y defectos internos de
la troza tales como anillos de crecimiento, CN, MLD y nudos. Se puede observar también la zona de
madera de primavera y de verano que forma parte de los anillos de crecimiento, las cuales presentan
mayores anchos en la zona del CN que en MLD. La madera libre de defectos presenta un mayor nivel
de gris (escala de gris) que el interior del CN. Los nudos al interior del CN, se observan claramente
debido a su forma y mayor nivel de gris.
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(a)
(b)
Figura 1. (a) Imagen TC original de una sección transversal de una troza de pino radiata podada. (b)
Imagen real digital correspondiente a la imagen TC original.
Al comparar ambas figuras, se observa que la imagen TC (Fig.1a ) es una copia fiel de la
fotografía digital (Fig. 1b). Esto ratifica lo reportado en la literatura para otras especies de madera, en
el sentido que la Tomografía Computarizada presenta un gran potencial para la evaluación interna de
trozas y en particular para la especie pino radiata, objeto de evaluación en el presente estudio.
Clasificación de imágenes TC utilizando Redes Neuronales Artificiales
Los resultados obtenidos del proceso de clasificación y post clasificación de imágenes TC
utilizando RNA se presentan en las figuras 2a y 2b, respectivamente. La fig. 2a, muestra el mapa
temático (imagen TC clasificada) obtenido de la clasificación con RNA, considerando tres clases a
identificar, esto es, CN, MLD y nudos. La fig. 2b muestra el resultado de la aplicación de un filtro
de mediana 7x7 al mapa temático presentado en la fig. 2a. En general, en la fig. 2a se observa que el
CN (color azul) puede ser identificado y separado de MLD (color amarillo). Sin embargo, se observa
una superposición del nivel de gris asociado a los nudos presentes en el CN y con la zona MLD. Esta
situación se debe principalmente a la diferencia en la densidad de la madera de primavera y verano
presente en los anillos de crecimiento (Kininmonth y Whitehouse 1991) y también a la variación
del contenido de humedad en el interior de los nudos. Algo similar ocurre en la periféria de la troza,
donde también se observa una superposición del nivel de gris, que puede ser atribuido a la pérdida
de humedad debido a la ausencia de corteza. En general, la variación del contenido de humedad en
el interior de las trozas en algunos casos dificulta la identificación, situación que ha sido reportada
anteriormente por otros autores. Según (Oja 2000) el nivel de gris en todas las áreas de la sección
transversal de la troza se ve afectada por la variación del contenido de humedad. En este sentido, Rojas
et al. (2005, 2007) indicaron que la densidad verde es la propiedad física que mejor se correlaciona
con el nivel de gris de las imágenes TC.
En general, el problema de la variación del nivel de gris al interior de los anillos de
crecimiento debido a la diferencia en la densidad de la madera de primavera y de verano, es eliminado
totalmente después de aplicar un filtro de mediana de 7x7 a los mapas temáticos (Fig. 2b). En esta
figura, se observa también que el ruido generado en la periferia de la troza, asociado principalmente
a una disminución del contenido de humedad, es eliminado en forma muy leve. Por otra parte en el
interior del cilindro nudoso no se observan mayores cambios, al igual que en la zona interna de los
nudos, por lo que persiste la superposición de nivel de gris.
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Figura 2. (a) Mapa temático obtenido de la clasificación utilizando RNA.
(b) Mapa temático filtrado utilizando un filtro de mediana de 7x7.
Evaluación de la precisión
Los resultados del análisis cualitativo de los mapas temáticos indicaron que el CN puede
ser identificado utilizando el clasificador basado en RNA. Sin embargo, esto no es suficiente para
obtener conclusiones más robustas. Por esta razón, a partir de la determinación de la matriz de
confusión asociada a cada mapa temático es posible obtener valores de precisión de la capacidad de
identificación del CN, MLD y nudos.
La tabla 1 muestra la matriz de confusión obtenida a partir de la evaluación de 270 mapas
temáticos. En esta matriz se presentan los valores de precisión individual y también los valores de
precisión global. Los datos contenidos en esta matriz corresponden a número de píxeles. Estos datos
pueden ser utilizados también para calcular los errores de omisión y comisión ocurridos durante el
proceso de clasificación de las imágenes TC. Sin embargo en este estudio solo interesan los errores
de omisión. A partir de esta tabla se tiene que la precisión de detección de CN, MLD y nudos fue
de 92.7%, 84.8% y 52.8%, respectivamente, mientras que para la precisión global se obtuvo un
valor de 85.0 %. En general, estos valores individuales de precisión pueden ser explicados por el
error de omisión, el cual permite identificar las clases a las cuales los píxeles fueron asignados. Por
ejemplo, el error de omisión para el CN fue de 7.3 % (151/2078), esto significa que 151 píxeles
de CN fueron erróneamente clasificados en las clases nudos (146 píxeles) y MLD (5 píxeles). En
otras palabras, existe una leve superposición del nivel de gris particularmente entre CN y nudos,
equivalente a un 7 %. En esta dirección, para MLD se obtuvo un error de omisión de 15.2 %, del cual
el 13.3% (1.607/12.086) está asociado a la clase nudos y el 1.9% (226/12.086) al CN. En este caso, la
superposición del nivel de gris está principalmente asociada a la clase nudos. De la misma manera, un
error de omisión de 47.2 % fue obtenido para la clase nudos, el cual está asociado casi en su totalidad
a la clase MLD (46.6 %).
Tabla 1. Matriz de confusión obtenida para los mapas temáticos.
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Los resultados obtenidos para los valores de precisión individual y global, luego de aplicar
un filtro de mediana de 7 x7 a los mapas temáticos, se presentan en la tabla 2. De acuerdo a esta
tabla, valores de precisión de 96.3 %, 93.2 % y 49.1 % fueron obtenidos para el CN, MLD y nudos,
respectivamente. La precisión global fue de 92.3 %. Si comparamos estos valores de precisión con los
valores presentados en la tabla 1, se observa que la precisión de detección mejora después de filtrar
los mapas temáticos, excepto para la clase nudos la cual disminuyó en un 3.7 % aprox. También se
observa que la tendencia en los resultados se mantiene, en el sentido que CN y nudos presentan el
mayor y menor valor de precisión, respectivamente. Por otra parte, y como se esperaba, los errores de
omisión para CN y MLD disminuyeron, debido al aumento de los valores de precisión. En este caso,
y tal como se observó para los mapas temáticos sin clasificar, los principales errores de omisión están
asociados con la clase nudos.
Tabla 2. Matriz de confusión obtenida para los mapas temáticos filtrados.
Esta situación está de acuerdo con los resultados del análisis cualitativo, en el sentido
que existe un cierto nivel de superposición del nivel de gris entre estas clases. Aunque la literatura
revisada no reporta estudios sobre la identificación del CN en otras especies de coníferas, los
resultados cuantitativos obtenidos de este estudio para MLD y precisión global son comparables con
los reportados por otros investigadores. Schmoldt et al. 2000, para las tres especies de latifoliadas
evaluadas, reportaron valores de precisión de 94.7 % para MLD y valores entre 90.0 % y 97.0 % para
la precisión global. Para pino silvestre Nordmark (2002), reportó un precisión global entre 93 y 95
%. Wei et al. 2008b, para picea negra, reportaron valores de MLD y precisión global de 80.7 % y 92.4
%, respectivamente.
Finalmente, los valores obtenidos para el índice Kappa, a partir de las matrices de confusión
de los mapas temáticos y mapas temáticos filtrados, fueron de 0.607 y 0.764, respectivamente. Estos
valores indican, de acuerdo a la escala establecida por Landis y Koch (1977), la existencia de un
fuerte grado de conformidad entre los datos de referencia y los resultados del proceso de clasificación
usando RNA.
CONCLUSIONES
Este estudio investigó la posibilidad de identificar y separar el cilindro nudoso de la madera
sin defectos y nudos en imágenes TC de pino radiata usando un algoritmo de clasificación basado
en redes neuronales. El análisis cualitativo de los mapas temáticos, indicó que el cilindro nudoso
puede ser identificado y separado de la madera libre de defectos y de los nudos, debido a la diferencia
observada entre los niveles de grises asociados a estas características. Sin embargo, se observó una
leve superposición entre el nivel de gris asociado a MLD y nudos, lo cual puede estar asociado a
la variación de la densidad y contenido de humedad en el interior de la troza. Los resultados de la
evaluación cuantitativa indicaron que el cilindro nudoso puede ser identificado con una precisión
de 92.7%. Sin embargo para la precisión global se obtuvo un valor de 85,0%. Después de filtrar los
mapas temáticos los valores aumentaron a 96.3 % y 92.3 % para el cilindro nudoso y precisión global.
Los valores de índice Kappa obtenidos para los mapas temáticos (0.607) y mapas temáticos filtrados
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(0.764) indicaron que existe un fuerte grado de conformidad entre los datos de referencia y los
resultados obtenidos del proceso de clasificación usando redes neuronales. Finalmente, los resultados
obtenidos de este estudio sugieren que el algoritmo basado en redes neuronales artificiales presenta
un muy buen desempeño en la identificación y segmentación del cilindro nudoso en imágenes TC de
trozas de pino radiata.
AGRADECIMIENTOS
Los autores agradecen al personal del laboratorio de imagenología del Hospital del trabajador
por su asistencia durante la etapa de escaneo de las trozas, a la Sra. Linette Salvo, Departamento de
Ingeniería en Maderas de la Universidad del Bío-Bío, por el apoyo logístico durante el desarrollo de la
parte experimental. Esta investigación fue financiada por el Fondo Nacional de Desarrollo Científico
y Tecnológico de Chile, a través del proyecto de iniciación FONDECYT N° 11060390.
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Virtual reality in neurologic rehabilitation of spatial disorientation
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Journal of neuroengineering and rehabilitation
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Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 JNER
JOURNAL OF NEUROENGINEERING
AND REHABILITATION Open Access Abstract Background: Topographical disorientation (TD) is a severe and persistent impairment of spatial orientation and
navigation in familiar as well as new environments and a common consequence of brain damage. Virtual reality
(VR) provides a new tool for the assessment and rehabilitation of TD. In VR training programs different degrees of
active motor control over navigation may be implemented (i.e. more passive spatial navigation vs. more active). Increasing demands of active motor control may overload those visuo-spatial resources necessary for learning
spatial orientation and navigation. In the present study we used a VR-based verbally-guided passive navigation
training program to improve general spatial abilities in neurologic patients with spatial disorientation. Methods: Eleven neurologic patients with focal brain lesions, which showed deficits in spatial orientation, as well as
11 neurologic healthy controls performed a route finding training in a virtual environment. Participants learned and
recalled different routes for navigation in a virtual city over five training sessions. Before and after VR training,
general spatial abilities were assessed with standardized neuropsychological tests. Results: Route finding ability in the VR task increased over the five training sessions. Moreover, both groups
improved different aspects of spatial abilities after VR training in comparison to the spatial performance before VR
training. Conclusions: Verbally-guided passive navigation training in VR enhances general spatial cognition in neurologic
patients with spatial disorientation as well as in healthy controls and can therefore be useful in the rehabilitation of
spatial deficits associated with TD. Keywords: Topographical disorientation, Brain damage, Way-finding training, Virtual rehabilitation,
Visuo-spatial memory, Visual navigation and in an ecologically valid way [4-13]. In compari-
son to more traditional assessment methods such as
paper-and-pencil measures, VR offers the tools for
simulating realistic spatial navigation under controlled
experimental conditions. © 2013 Kober et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Virtual reality in neurologic rehabilitation of
spatial disorientation Silvia Erika Kober1*, Guilherme Wood1, Daniela Hofer1, Walter Kreuzig2, Manfred Kiefer2 and Christa Neuper1,3 * Correspondence: silvia.kober@uni-graz.at
1Department of Psychology, University of Graz, Universitaetsplatz 2/III, Graz
8010, Austria
Full list of author information is available at the end of the article Introduction In the
present study, patients were instructed to correct their
route by means of verbal feedback from the experi-
menter whenever they took a wrong route during navi-
gating in virtual scenarios. Thereby one can avoid
patients “getting completely lost” in virtual environments
and may accelerate the learning process. The fourth as-
pect of the present route finding training was to design a
standardized virtual environment which is unfamiliar to
all participants but at the same time is realistic enough to
provide a rich learning environment for participants. The
training of participants in unfamiliar environments
controls for the effects of previous experience and
habits on navigation performance [25]. Furthermore, it
increases the comparability of training outcomes across
participants and participants’ groups and also improves
the accuracy of performance assessment. version of the same hospital unit [16]. Following the VR
training, the patient was able to successfully perform
routes in the real unit. Hence, the patient easily trans-
ferred the learned performance from the virtual to the
real world [17]. Astonishingly, the same patient failed to
learn the routes in the real unit. Rose et al. (2001)
extended the findings by Brooks et al. (1999) and trained
four more patients with amnesia on route learning in
VR. The virtual training was as successful as the real
world training [18]. Moreover, Wilson et al. (1996) suc-
cessfully trained physically disabled children in a virtual
building with the goal of finding the fire extinguishers
and fire door locations in the real building [19]. Caglio
et al. (2012) used a 3D video game as navigational
training program, which led to improvements in spatial
memory in a brain damaged patient [15]. Brooks et al. (1999) attributed the superiority of VR
training over real world learning to three different
reasons: First, the routes can be performed more often
in the virtual than in the real world, since the routes can
be performed faster in VR. Second, in VR the patients
are not restricted by any physical disabilities. Different
degrees of active motor control over navigation may be
implemented depending on individual motor coordin-
ation abilities. Navigation can be self-paced by means of
a joystick [16], a keyboard [20], or verbal instructions
[21], or passive [22], when participants exert no control
over navigation. Third, in VR there are no unexpected
distractions that could interrupt the patients during
learning as in the real world. Introduction Impaired spatial orientation is a common consequence
of brain damage that greatly reduces the quality of life
and autonomy in daily living of neurologic patients. To
date, no standard rehabilitation of spatial abilities after
brain damage is in use [1]. One frequent form of spatial
disorientation is the topographical disorientation (TD)
[2,3]. Beside the use of VR as an assessment tool for spatial
deficits, there are also few studies using VR for training
spatial abilities in patients with orientation problems
[5,14,15]. Virtual training environments offer the possi-
bility to train specific spatial deficits associated with TD,
such as egocentric or allocentric (e.g. landmark agnosia)
orientation problems [2,7-9]. One of the first studies
using VR in rehabilitation of navigational skills was a
single-case study by Brooks et al. (1999). A patient with
amnesia who showed memory and orientation deficits
was trained in route finding around a real and a virtual Virtual reality (VR) technique offers the opportun-
ity
to
create
complex
individualized
and
natural
simulated
environments,
in
which
specific
spatial
deficits, such as egocentric disorientation or the abil-
ity to recognize landmarks, can be assessed precisely Full list of author information is available at the end of the article Page 2 of 13 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 requires memory and mental flexibility resources [24],
which are also necessary for route learning. Further-
more, positive effects of navigation training have been
reported for passive navigation as well [5,22,23]. This
is especially important because not every patient is
able to move a joystick or use a keyboard to control
navigation. For this reason we decided to implement a
verbally-guided passive navigation training, in which
navigation was controlled by verbal commands given
by participants to the experimenter. A pilot study in
healthy elderly participants indicated that this approach
is reasonable. The third aspect refers to the learning
mode employed in training programs. Lloyd et al. (2009) could show that patients with brain injury
benefit more from errorless learning in a virtual route
learning task compared to trial and error learning [21]. For instance, Brooks et al. (1999) corrected the patient
who underwent a spatial training in their study, when-
ever the patient took a wrong turn as well [16]. Introduction The most common etiology of the neurologic
disorders was stroke. A detailed description of the
patient group is shown in Table 1. Patients were
assigned to the study based on the diagnostics of
spatial orientation disorders made by their attending
doctors [6,9]. The neurologic healthy control group
(5 men, 6 women) was recruited from the Ortho-
pedic Unit of the same clinic (matching criteria: sex
and age). Mean age of the patient group was 66.09
(SE = 3.30) years and of the matched control group
66.18 (SE = 2.97) years. Participants who scored less
than 17 points on the Mini-Mental State Examin-
ation [8] and patients with visuo-spatial hemineglect,
severe language impairments, major psychiatric ill-
ness and depression were excluded from the study. All participants included in the study had normal or
corrected-to-normal vision. The study conforms with
the code of ethics of the World Medical Association
(WMA, Declaration of Helsinki) [26]. Neurology
Unit
of
the
Privatclinic
Lassnitzhoehe,
Austria. Ten patients had lesions in the right hemi-
sphere, one patient had lesions in the left hemi-
sphere. The most common etiology of the neurologic
disorders was stroke. A detailed description of the
patient group is shown in Table 1. Patients were
assigned to the study based on the diagnostics of
spatial orientation disorders made by their attending
doctors [6,9]. The neurologic healthy control group
(5 men, 6 women) was recruited from the Ortho-
pedic Unit of the same clinic (matching criteria: sex
and age). Mean age of the patient group was 66.09
(SE = 3.30) years and of the matched control group
66.18 (SE = 2.97) years. Participants who scored less
than 17 points on the Mini-Mental State Examin-
ation [8] and patients with visuo-spatial hemineglect,
severe language impairments, major psychiatric ill-
ness and depression were excluded from the study. All participants included in the study had normal or
corrected-to-normal vision. The study conforms with
the code of ethics of the World Medical Association
(WMA, Declaration of Helsinki) [26]. such as improving a participant’s general spatial abil-
ities and skills. The aim of VR-based spatial rehabilita-
tion should be to train improved spatial performance
of a variety of types in different spaces [14]. Introduction In the present study, we have designed a VR-based
verbally-guided
passive
navigation
training program,
which should be suitable for neurologic patients with
spatial disorientation: The first aspect of our training
program is to achieve positive results in a short period
of time. Brooks et al. (1999) and Rose et al. (2001) found
positive training effects after three and two weeks of
daily training, respectively [16,18]. For the sake of econ-
omy, the route finding training was reduced to five
training sessions of 20 min each in the present study. Hence, we assumed that five training sessions in VR are
sufficient to improve spatial orientation in neurologic
patients. The second aspect refers to the degree of
motor control necessary for performing training. Rose,
Brooks, Attree, et al. (1999) compared the influence of
active (active navigation through the VR, controlled
movement
through
the VR)
and
passive
(passively
watching, did not control movement through the VR)
navigation through a virtual building on the develop-
ment of spatial knowledge with vascular brain injury
patients and control participants. They demonstrated a
superiority of active over passive navigation [23]. How-
ever, for many patients active navigation may lead to
cognitive overload since controlling motor performance A final question of the present study is whether the
obtained
training
outcomes
can
be
generalized
to
broader aspects of spatial cognition. Are there only
learning effects, which are specific to trained routes
and training materials or can one observe more gen-
eral cognitive changes in trained patients? Is it possible
to track these changes using well established psycho-
metrically
validated
neuropsychological
instruments? Results reported by Brooks et al. (1999), Rose et al. (2001), Wilson et al. (1996), and Lloyd et al. (2009)
already present high ecological validity and demon-
strate the utility of VR-based training programs for im-
proving spatial navigation. However, these studies did
not examine how more general aspects of spatial cog-
nition may have been changed by spatial training. In
this context, Durlach et al. (2000) mentioned that prior
VR studies only trained specific spatial behaviors in
specific virtual or real spaces, and that there is a lack
of VR studies that trained spatial behavior in general, Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Page 3 of 13 Neurology
Unit
of
the
Privatclinic
Lassnitzhoehe,
Austria. Ten patients had lesions in the right hemi-
sphere, one patient had lesions in the left hemi-
sphere. Apparatus and materials In order to assess participants’ general spatial abilities,
standardized
neuropsychological
tests
were
used. Participants were asked to complete four spatial tests be-
fore and after the five VR training sessions: the Benton
Test, the LPS 50+, the LVT, and the CBTT. In pre-and
post-test measures parallel forms of the Benton Test and
the LPS 50+ were used. All tests were conducted on one
day in the pre- and post-measurement, respectively. The
overall duration of the pre- and post-measurement was
about 45 min each (including written informed consent,
assessment of demographic and basic stroke-related
data, instructions, Benton Test, LPS 50+, LVT, and
CBTT). The Benton Test, which is also called Benton Visual
Retention Test, assesses visual perception and visual
memory [27]. It is also used in clinical diagnosis of brain
damage. The participant is shown for ten seconds 15
standardized cards with geometric forms, one at a time. Then, the participant is asked to recognize the previ-
ously shown card under four different cards (multiple-
choice form). Two parallel forms of the multiple-choice
form of the Benton test are available. The highest score
one can reach is 15 points. Participants older than 55
years get an extra point. A score of 13 points is
associated with a normal visual perception and visual
memory performance. Scores lower than 12 points are
indicators of impaired visual perception and memory
[27]. For the multiple-choice form, a moderate internal
consistency is reported, with a split-half reliability of
about 0.76. The Benton Test assessing visual-spatial Introduction Therefore,
we addressed the question, whether spatial training in
a specific virtual space can enhance general spatial
abilities assessed with standardized neuropsychological
tests in neurologic patients with spatial disorientation
as well as in a healthy matched control group. In summary, the present study pursues two main
goals. The first is to demonstrate the proof-of-principle
of the applied VR-based verbally-guided passive naviga-
tion training program in neurologic patients with spatial
orientation problems and healthy participants. We ex-
pect that both patients and controls will benefit from
VR-based verbally-guided passive navigation training,
because it combines training parameters (verbally-guided
passive navigation, errorless learning mode, standardized
unfamiliar virtual environment, duration of training)
that led to performance benefits in both populations. The second aim is to address the question of how large
is the transference of improvements in VR route training
to more general aspects of spatial cognition and how
consistent are these transference effects in and across
populations. Prior
studies
showed
that
navigation
training in a virtual environment can improve visual-
spatial learning in neurologic patients [15]. Exploration
of a new and complex environment, such as the virtual
city in the present VR navigation training program,
recruits spatial memory resources (e.g. short-term spatial
memory, spatial learning rates). When the participants
need to remember a route, the demands on spatial
memory resources are particularly high. Moreover, when
the participants need to navigate on a route from a start
point to an endpoint and vice-versa, participants have to
execute a series of mental transformations on the repre-
sentation of the route. This transformation process dur-
ing the VR navigation training is related to the ability to
imagine spatial objects and to mentally transform them. Finally, the more realistic the VR model, the higher the
demands on visual orientation performance necessary to
filtrate useful input from visual noise from the back-
ground. For these reasons, we expect that positive
training effects achieved with VR-based verbally-guided
passive navigation training will potentially induce posi-
tive changes in measures of spatial memory, mental
transformations and visual orientation performance. Participants Eleven neurologic patients (5 men, 6 women) that
showed severe impairments in spatial orientation per-
formance in their ordinary environments (e.g. hospital
area, home) were recruited among the inpatients of the Page 4 of 13 Page 4 of 13 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Table 1 Patient description Table 1 Patient description
Patient code
Age (years) Sex
Diagnosis
Affected
hemisphere
Lesion location
TSO§ Further information
1
73
male
stroke
right
arteria cerebri media
5
2
79
female stroke
right
arteria cerebri media
6
Moderate memory and attention deficits
(assessed by the SKT – Syndromkurztest
zur Erfassung von Gedächtnis- und
Konzentrationsstörungen)
5
75
male
stroke
right
fronto-parietal
9
Subdural hematoma, marginal symptoms
of dementia
6
80
female stroke
right
arteria cerebri media,
basal ganglia
5
Left-sided hemiparesis
8
58
female aneurysm and
subsequent infarcts
right
arteria cerebri posterior
170
Subarachnoid hemorrhage: HUNT and
HESS II, Quadrantanopia
10
59
male
stroke
right
arteria cerebri media,
thalamus, basal ganglia
14
Left-sided hemiparesis
11
57
male
stroke
right
basal ganglia
14
Left-sided hemiparesis
12
72
male
stroke
right
arteria cerebri media
5
14
68
female cerebral haemorrhage right
arteria cerebri media
6
Diplopia, headache
17
61
female aneurysm and
subsequent infarcts
right
arteria communicans
anterior, parietal infarct
30
Organic brain syndrome, moderate memory
and attention deficits (assessed by the SKT)
18
45
female traumatic brain injury
left
hippocampus, pons
12
Attention deficits (assessed by Cognitrone),
memory deficits (assessed by Wechsler
Memory Scale)
§TSO: Time since onset (weeks). §TSO: Time since onset (weeks). initial practice phase combines the instruction and
eight practice items. In the subsequent test phase, the
participant is presented with an array of lines and
must find the end of a specified line as quickly as
possible within a given time. For the present study,
the screening form of the LVT was used containing
18 items. For statistical analyses, the total score of the
LVT and the median time of correct answers (sec)
were used. The LVT is an internally consistent meas-
ure,
with
a
Cronbach’s
alpha
coefficient
of
0.92
[30,31]. We used the LVT to assess possible changes in the
visual orientation performance for simple structures in
complex environments due to the VR training. §TSO: Time since onset (weeks). Participants Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Page 5 of 13 impairments, not all patients suffering from brain dam-
age were able to complete the computer based tests LVT
and CBTT. Therefore, only seven patients with spatial
orientation disorders completed the LVT, and eight
patients with spatial deficits completed the CBTT. training program participants gave oral commands such
as “straight ahead”, “turn left”, “turn right” and “stop” to
the experimenter, who was in charge of operating the
joystick. Therefore, participants controlled navigation in-
directly by means of verbal commands and not actively
by means of motor responses. The virtual environment was a simulation of a district
of the real world town of Graz, Austria (see Figure 1). The virtual environment was generated by the Institute
of Computer Graphics and Knowledge Visualization
(CGV) of the Graz University of Technology (www.cgv. tugraz.at). The virtual 3D model of the district of Graz
was generated by using aerial and first-person view pho-
tos
of
the
real
world
unit
with
the
framework
instantreality
(www.instantreality.org). None
of
the
participants had been in the corresponding real world
district of Graz before. The virtual city was presented on
a 2x2 m projection screen via a conventional projector
in a monoscopic view. During the VR training, different
routes were presented, which the participants had to
learn and recall correctly. Each route contained three
decision
points
(left/right
turns
or
straight-ahead
choices). Navigation speed and direction in VR were
controlled by joystick. In a pilot study, the ability of eld-
erly participants to control the joystick by themselves
was assessed. Pilot testing showed that it was too diffi-
cult for elderly to operate the navigation joystick and
simultaneously to concentrate on the routes. Further-
more, due to motor impairments, some patients were
physically unable to efficiently control the joystick. For
these reasons, in the present study a verbally-guided pas-
sive navigation training program was adopted. In this VR training program: The VR training was a route
finding training. Participants were shown one route,
completely directed by the experimenter in the learning
phase. The experimenter pointed out each junction of
the route and stated the action to be taken before exe-
cuting
it. Hence,
the
experimenter
gave
verbal
instructions such as: “We are approaching a crossroad
now. Participants memory was used to assess possible improvements in
visual-spatial learning due to the VR navigation training. The
Achievement
Measure
System
50+
(Leistungspruefsystem 50+, LPS 50+) is a German
standardized intelligence test developed for older people
between 50 and 90 years. It is based on different subtests,
which are designed to measure Thurstone’s Primary
Mental Abilities [28]. The LPS 50+ includes seven subtests
assessing verbal knowledge, non-verbal reasoning, verbal
fluency, spatial imagination, flexibility of closure and verbal
closure. For the present study, only one subtest assessing
spatial imagination was used. In this subtest, the partici-
pant is shown different geometric objects. The participant
is asked to count the number of surface areas of these
objects. For this test two parallel versions are available. The
spatial imagination subscale shows good values in reliabil-
ity, with a split-half reliability of 0.96 and a test-retest reli-
ability of 0.94 [29]. The LPS 50+ was used to assess
possible improvements in the ability to imagine spatial
objects and to mentally transform them due to the VR
navigation training. The Corsi Block-Tapping Test (CBTT) [32] is a
standardized computer based subtest of the Vienna Test
System [31]. It assesses the so called “immediate block
span”, which is associated with visual short-term mem-
ory capacity and implicit visuo-spatial learning. The par-
ticipant views nine irregularly positioned blocks on a
screen and a pointer taps on a number of these blocks
in turn. Afterwards, the participant is required to tap
with a special pencil on the same blocks in the same
order. The number of blocks increases by one after three
items. When the participant makes an error in three
successive items the test stops. The CBTT shows good
values in reliability between 0.81 and 0.89 [31]. The
CBTT assessing visual short-term memory was used to
assess possible improvements in visual-spatial learning
due to the VR navigation training. Due to motor The
Visual
Pursuit
Test
(Linienverfolgungstest,
LVT) [30] is a standardized computer based subtest of
the Vienna Test System [31]. It measures the visual
orientation performance for simple structures in a
complex environment. The participant is required to
work in a focused way, to ignore distractions, while
being placed under time pressure. Hence, this test is
also suited to assess selective visual attention. The Page 5 of 13 Kober et al. Participants We have to turn left here.” In the subsequent re-
trieval phase, participants had to call out the correct
directions to the experimenter at each junction. If the
participant
took
a
wrong
turn,
the
experimenter
informed the participant and returned to the correct
route in order to assure errorless learning [21]. In the
present study, learning and retrieval of one route were
repeated until the participant made no mistake in the re-
trieval phase. One training session took approximately
20 min in which maximal three different routes could be
learned. In each of the five training sessions different
routes were presented. The performance in the VR
training task was quantified by calculating a weighted
total score (see Appendix I for further details), including
the number of mistakes per route and the number of
correctly learned routes per training session. Higher
total scores are associated with better performance in
the VR retrieval task. Figure 1 Sample views of the virtual environment used for the VR-based spatial navigation training program, which was a district of
the real world town of Graz, Austria. Figure 1 Sample views of the virtual environment used for the VR-based spatial navigation training program, which was a district of
the real world town of Graz, Austria. Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Page 6 of 13 were observed but a non-significant interaction time ×
group (F(1, 20) = 3.24; MSE = 34.57; p = .087). Similar
results were observed in the Benton Test: Significant
main effects of time (F(1, 20) = 31.03; MSE = 76.46; p <
.001) and group (F(1, 20) = 1522; MSE = 7127; p < .001)
were observed but a non-significant interaction time ×
group (F(1, 20) = 1.32; MSE = 3.27; p = .263). In con-
trast, in the total score of the LVT no main-effect of
time (F(1,20) = 2.81; MSE = 1.84; p = .11) or interaction
time × group (F(1,20) <1 n.s.) were observed, but only
a main effect of group (F(1,20) = 178; MSE = 49178;
p < .001). In the median time score, significant main
effects of time (F(1,20) = 9.88; MSE = 4.81; p = .005)
and group (F(1,20) = 35.74; MSE = 997; p < .001)
were observed as well as the interaction time × group
(F(1,20) = 5.41; MSE = 2.64; p = .031). Training data g
The
training
data
were
assessed
using
repeated-
measures ANOVA models in which time (training
sessions 1, 2, 3, 4, and 5) was defined as a 5-levels
within-subjects factor and group (patients × matched
controls) as a between subjects factor. The training data
revealed a significant main effect of time (F(4, 80) =
3.08; MSE = 398; p = .02), which was complemented by
a linear contrast (F(1,20) = 8.12; MSE = 1561; p = .01)
and a main-effect of group (F(1, 20) = 321; MSE =
206549; p < .001). Together these results indicate that
matched controls showed superior performance in VR
training than patients regardless of the time point. Moreover, both controls and patients benefited from VR
training, as the performance of both increased with
training in a linear fashion. Participants Finally, considering
the scores of the CBTT, only a main effect of group
was observed (F(1,20) = 159; MSE = 918; p < .001). Training data To assess improvements in VR route finding ability,
weighted total scores obtained in the first and the fifth
training sessions were compared for the patient and con-
trol groups separately (Figure 2). Because of the multiple
comparisons
problem
we
reduced
the
number
of
calculated t-tests by comparing only the first and the Figure 2 Means and standard errors of the route finding
performance (weighted total score including the number of
mistakes per route and the number of correctly learned routes
per training session) in the VR way-finding training for the five
training sessions, separately for each group. Procedure The five VR training sessions and the pre- and post-
assessment of general spatial abilities using standardized
neuropsychological tests took place in the rehabilitation
clinic. In the first session, participants gave written
informed consent. Demographic and basic stroke-related
data were also collected. Afterwards, general spatial abil-
ities were assessed using the Benton Test, LPS 50+, LVT,
and CBTT. In the sessions 2–6, the five VR training
sessions were conducted. In each 20-min training ses-
sion, participants had to learn and recall up to maximal
three different routes. Before and after each training ses-
sion, participants filled out the Simulator Sickness Ques-
tionnaire
(SSQ)
[33]. The
SSQ
was
developed
to
determine whether users of virtual environments experi-
ence cybersickness symptoms, which can confound the
data. The
analysis
of
the
SSQ
revealed
that
the
participants showed no sickness symptoms during the
five VR training sessions. Additionally, patients and
controls showed no differences in their SSQ-ratings (all
p > 0.05). In the seventh session, general spatial abilities
were assessed again by using the Benton Test, LPS 50+,
LVT, and CBTT. In pre-and post-test measures parallel
forms of the Benton Test and the LPS 50+ were used to
avoid learning effects. The main effect of group indicates that the controls
performed better than the patients at all occasions. Therefore, the subsequent t-tests were calculated for the
patient and control group separately. Importantly, the
main effect of time indicates that both, controls and
patients improved their performance in measures of
general spatial abilities after VR training. Finally, the sig-
nificant interaction time × group observed in the median
time scores of the LVT indicates that patients benefited
more from VR training than matched controls. Pre-post-assessment The performance in general spatial abilities was assessed
in the pre- and post-tests using repeated-measures
ANOVA models in which time (pre-test × post-test) was
defined as a within-subjects factor and group (patients ×
matched
controls)
as
a
between
subjects
factor. Regarding performance in the LPS 50+, significant main
effects of time (F(1, 20) = 12.64; MSE = 134.75; p = .002)
and group (F(1, 20) = 1160; MSE = 124339; p < .001) Figure 2 Means and standard errors of the route finding Figure 2 Means and standard errors of the route finding
performance (weighted total score including the number of
mistakes per route and the number of correctly learned routes
per training session) in the VR way-finding training for the five
training sessions, separately for each group. Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Page 7 of 13 Table 3 VR training outcome in patients and controls
Group
Increase
Constant
Decrease
N
Patients
5
4
2
11
45%
36%
18%
100%
Controls
7
2
2
11
64%
18%
18%
100%
Number and percentage of participants showing either an increased, constant
or decreased VR navigation performance in the fifth compared to the first VR
training session. Table 3 VR training outcome in patients and controls fifth
session. Performance
in the
five VR training
sessions is illustrated in Figure 2 and presented separ-
ately for each group. In the control group, the route
finding ability in the VR task improved significantly from
the first to the fifth training session (p < 0.01) (see
Figure 2, Table 2). In the patient group, only a small
trend towards an improvement in route finding perform-
ance was observed over the five training sessions. Means
and standard errors of the behavioural data and the
results of the statistical analyses (t-tests) are summarized
in Table 2. Number and percentage of participants showing either an increased, constant
or decreased VR navigation performance in the fifth compared to the first VR
training session. Number and percentage of participants showing either an increased, constant
or decreased VR navigation performance in the fifth compared to the first VR
training session. compared to the pre-test, which is superior to a critical
difference. Pre-post-assessment
l
h To complement the investigation on VR training related
changes in general spatial abilities, performance in
neuropsychological tests was compared between pre-
and post-test separately for the patient and control
group using t-tests (Figure 3). The patient group showed
a significant higher LPS 50+ score in the post-test
compared to the pre-test (Figure 3, Table 2). Addition-
ally, after VR training the patient group answered faster
in the LVT than before VR training (Figure 3). In both
groups, the performance in the Benton Test was higher
in the post-test than in the pre-test (Figure 3). Pre-post-assessment This critical difference describes the differ-
ence in performance which, on the one side, cannot be
attributed to random performance fluctuations and, on
the other side, occurs rarely in the population. The crit-
ical difference is calculated for psychometrically well
constructed instruments and useful only for tests with
moderate or high reliability. In the present study, critical
differences were calculated for the tests LPS 50+, Benton
Test, LVT total score. No critical difference could be
calculated for the LVT median time and CBTT because
of poor or non-existent norms. As shown in Table 4,
patients reached critical differences eight times, while
controls reached critical differences five times. A critical
difference is considered significant when the difference
between
pre-
and
post-test
shown
by
the
single
participants is larger than the critical difference which
can be detected by each test (error probability α < 5%)
and only occurs in the population with a probability
lower than α < 10%. The number of participants showing increased, con-
stant or decreased performance in the VR training be-
tween the first and last training sessions was determined
separately in the two groups. A significant chi-square Χ2
(2) = 23.38, p < 0.01 revealed that more control
participants than patients benefited from route finding
training (Table 3). Nevertheless, a substantial proportion
of the patient group (45%) still showed improvement in
route finding after training (Table 3). Single-case analyses To investigate whether the VR navigation task is a valid
assessment method of spatial skills we examined if there
is a relationship between route finding ability in the VR
task and general spatial abilities assessed before and after
VR training [13,35]. For all participants, the weighted Single-case analyses based on the approach defined by
Huber [34] were conducted separately for each individ-
ual. This analysis identifies those individuals presenting
a positive difference in performance in post-test when Table 2 Means and standard errors of the behavioural data and the results of the statistical analyses (t-tests)
Patient group
Control group
Pre-test
Post-test
Pre-test
Post-test
Mean (SE)
t-value (df)
Mean (SE)
t-value (df)
LPS 50+ score [T-score]
44.73 (1.91)
50.00 (2.04)
−3.32** (10)
58.09 (2.71)
59.82 (2.51)
−1.49 (10)
Benton Test score [Raw-score]
10.18 (0.88)
13.36 (0.43)
−3.79** (10)
12.64 (0.45)
14.73 (0.36)
−4.80** (10)
LVT total score [T-score]
28.43 (1.43)
31.29 (2.77)
−0.93 (6)
46.36 (2.18)
49.36 (1.94)
−1.65 (10)
LVT median time [s]
9.17 (2.08)
7.36 (1.63)
3.28* (6)
4.35 (0.16)
4.18 (0.16)
1.27 (10)
CBTT [Raw-score]
4.38 (0.38)
4.63 (0.26)
−1.00 (7)
5.55 (0.21)
6.18 (0.46)
−1.41 (10)
VR route finding performance [weighted total score] in first (pre-test)
and fifth (post-test) VR training session
31.54 (5.44)
35.30 (5.02)
−0.73 (10)
45.36 (1.76)
62.39 (4.03)
−3.59** (10)
Significant results are marked with asterisks (*p < 0.05, **p < 0.01). ns and standard errors of the behavioural data and the results of the statistical analyses (t-tests) Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Page 8 of 13 Figure 3 Bar graphs show means and standard errors of the behavioral data separately for the patient and control group and the
results of the statistical analyses (t-tests: pre- vs. post-test). Significant results are marked with asterisks (*p < 0.05, **p < 0.01). Figure 3 Bar graphs show means and standard errors of the behavioral data separately for the patient and control group and the
results of the statistical analyses (t-tests: pre- vs. post-test). Significant results are marked with asterisks (*p < 0.05, **p < 0.01). Figure 3 Bar graphs show means and standard errors of the behavioral data separately for the patient and con
results of the statistical analyses (t-tests: pre- vs. post-test). Single-case analyses Significant results are marked with asterisks (*p < 0.05, * was correlated separately with the results of the neuro-
psychological tests (Benton Test, LPS 50+, total score
and median time of correct answers of LVT, CBTT)
assessed during the post-test (Table 5). As in the pre-test,
higher scores in general spatial ability tests were asso-
ciated with an enhanced VR performance (except for the
correlation between VR performance and the LPS 50+
score, which showed no association). Statistical com-
parisons between correlations revealed no changes in the total score of VR performance in the first training ses-
sion was correlated separately with the results of all
neuropsychological tests (Benton Test, LPS 50+, total
score and median time of correct answers of LVT,
CBTT) assessed during the pre-test (Table 5). An
increased performance in the VR route finding task was
associated with an enhanced performance in all neuro-
psychological tests. Additionally, the weighted total score
of the VR performance in the last VR training session Table 4 Results of the single-case analyses
UPN
Group (1=patient)
LPS 50+
Benton Test
LVT – total score
LVT – median time *
CBTT *
1
1
+§
+
+§
+
=
2
1
+
+§
5
1
+
+
=
=
+
6
1
+
+§
8
1
+
+
=
+
+
10
1
+
+
11
1
+§
-
-
12
1
+
+§
=
+
=
14
1
-
+
-
+
=
17
1
+§
+
=
+
+
18
1
+§
+
+
+
=
3
2
+
+
+
+
=
4
2
=
=
+
-
=
7
2
+
+
+
+
+
9
2
+
+
+§
+
=
13
2
-
=
+
+
=
15
2
=
+
-
+
+
16
2
+
+
+§
+
=
19
2
+§
+
-
=
=
20
2
-
+§
+§
+
-
21
2
+
=
-
-
+
22
2
+
+
+
+
+
§ Individuals showing a significant improvement in performance in single-case analyses (Huber, 1973). * Because of the lack of normed values for these tasks, the
single-case analysis could not be performed. In these analyses the difference in performance (pre- vs. post-test) obtained by each individual is compared to critical values obtained from published norms. § Individuals showing a significant improvement in performance in single-case analyses (Huber, 1973). * Because of the lack of normed values for these tasks, the
single-case analysis could not be performed.
In these analyses the difference in performance (pre- vs. post-test) obtained by each individual is compared to critical values obtained from published norms. (+)
Performance was higher in post- compared to pre-test; (−) Performance was lower in post- compared to pre-test; (=) Performance was in pre- and post-test
the same. gnificant improvement in performance in single-case analyses (Huber, 1973). * Because of the lack of normed values for these tasks, th
not be performed.
i
f
(
t t
t)
bt i
d b
h i di id
l i
d t
iti
l
l
bt i
d f
bli h d
(+ § Individuals showing a significant improvement in performance in single-case analyses (Huber, 1973). * Because of the lack of norme
single-case analysis could not be performed. not be performed.
erence in performance (pre- vs. post-test) obtained by each individual is compared to critical values obtained from published norms. (+
n post- compared to pre-test; (−) Performance was lower in post- compared to pre-test; (=) Performance was in pre- and post-test Single-case analyses Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Page 9 of 13 Table 5 Pearson’s correlations between the weighted total score of the VR performance in the first and last training
sessions and results of the neuropsychological tests (Benton Test, LPS 50+, total score and median time of correct
answers of LVT, CBTT) during the pre- and post-tests averaged over participants Table 5 Pearson’s correlations between the weighted total score of the VR performance in the first and last training
sessions and results of the neuropsychological tests (Benton Test, LPS 50+, total score and median time of correct
answers of LVT, CBTT) during the pre- and post-tests averaged over participants
Benton Test
(N=22)
LPS 50+
(N=22)
LVT – total score
(N=18)
LVT – median time
(N=18)
CBTT
(N=19)
Total score of VR performance in 1st training session
0.56**
0.43*
0.55*
−0.78**
0.71**
Total score of VR performance in 5th training session
0.51*
0.26 n.s. 0.56*
−0.57*
0.49*
Comparison of pre- and post-test correlations with respective
training sessions (z-test)
0.22 n.s. 0.60 n.s. −0.04 n.s. −1.09 n.s. 0.99 n.s. n.s. non-significant, *p < 0.05, **p < 0.01. The performance in the virtual route finding task
increased in a linear fashion over the five VR training
sessions
in
both,
healthy
controls
and
neurologic
patients, as indicated by the results of the ANOVA ana-
lyses. As expected, healthy elderly with no acquired
brain injuries showed a better performance in recalling
different routes in VR correctly than the patient group
in all training sessions. Hence, even healthy elderly with
no spatial deficits can benefit from spatial training in VR
and learn to improve their navigational performance due
to the used VR way-finding training paradigm. The per-
formance of the patient group also slightly increased
from the first to the last session, from an average score
of 31.54 points in the first session to 35.30 points in the
last session. Furthermore, the patients with spatial dis-
orientation became gradually more confident and also
reported to have fun during the spatial training across
five VR sessions. Therefore, the initial fear of the new
technology and the associated technology gap, which
was most prominent in the patient group, disappeared
after a few training sessions. Morganti (2004) reported
such a technology gap in older participants, too. Gener-
ally, the elderly are not familiar with VR interfaces and
particular
devices
[36]. Single-case analyses (+)
Performance was higher in post- compared to pre-test; (−) Performance was lower in post- compared to pre-test; (=) Performance was in pre- and post-test
the same. Table 4 Results of the single-case analyses
UPN
Group (1=patient)
LPS 50+
Benton Test
LVT – total score
LVT – median time *
CBTT *
1
1
+§
+
+§
+
=
2
1
+
+§
5
1
+
+
=
=
+
6
1
+
+§
8
1
+
+
=
+
+
10
1
+
+
11
1
+§
-
-
12
1
+
+§
=
+
=
14
1
-
+
-
+
=
17
1
+§
+
=
+
+
18
1
+§
+
+
+
=
3
2
+
+
+
+
=
4
2
=
=
+
-
=
7
2
+
+
+
+
+
9
2
+
+
+§
+
=
13
2
-
=
+
+
=
15
2
=
+
-
+
+
16
2
+
+
+§
+
=
19
2
+§
+
-
=
=
20
2
-
+§
+§
+
-
21
2
+
=
-
-
+
22
2
+
+
+
+
+
§ Individuals showing a significant improvement in performance in single-case analyses (Huber, 1973). * Because of the lack of normed values for these tasks, the
single-case analysis could not be performed. In these analyses the difference in performance (pre- vs. post-test) obtained by each individual is compared to critical values obtained from published norms. (+)
Performance was higher in post- compared to pre-test; (−) Performance was lower in post- compared to pre-test; (=) Performance was in pre- and post-test
the same. Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
Page 9 of 13
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http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Discussion
h In the present study we used a VR-based route finding
training to improve general spatial abilities in neurologic
patients with spatial disorientation and healthy controls. The route finding ability in the VR task increased
linearly over the five VR training sessions in both,
patients and controls. After the VR training, patients as
well as controls improved their general spatial abilities
in comparison to the spatial performance before the VR
training, assessed with standardized neuropsychological
tests. Additionally, performance in the VR route finding
task was positively correlated with performance in the
standardized neuropsychological tests. In the following
paragraphs, these results are discussed in more detail. Single-case analyses But
Morganti
(2004)
also
mentioned that older participants show a clear enthusi-
asm in embracing such type of rehabilitation when they
are “forced” to use VR devices. In this context, it seems
reasonable to assume that older participants who suffer
from a brain injury, such as the patient group in the
current study, show an even more pronounced technol-
ogy gap than healthy old people. Hence, older people
with brain damage probably need more VR training
sessions to familiarize themselves with the VR technol-
ogy than the healthy controls. Brooks et al. (1999) and
Rose et al. (2001) carried out much more than five VR
training sessions to increase spatial performance in
amnestic patients [16,18]. Additionally, 45% of the
neurologic patients could show an increased route
finding performance in the VR task after only five VR
training sessions. This result leads to the conclusion that
positive training effects can be observed in neurologic
patients as well as healthy participants after five short
VR training sessions in route finding strength of the association between neuropsychological
tests and training performance as measured in the pre-
and post-tests (Table 5). Proof-of-principle of the applied VR-based verbally-
guided passive navigation training program In the present study, we designed a VR-based verbally-
guided passive navigation training program. Our results
provide evidence that this training program is suitable
for neurologic patients with spatial disorientation as well
as neurologic healthy controls. The neurologic patient
group would not have been able to actively control the
navigation joystick adequately because some patients
had motor impairments. The errorless learning mode
assured that participants could concentrate on the cor-
rect routes in VR and that the spatial learning process
was not disturbed by taking any unplanned detours. The
standardized virtual environment was unfamiliar to all
participants as well. In summary, the verbally-guided
passive navigation mode, the errorless-learning strategy,
and the use of an unfamiliar and realistic standardized
virtual environment for the VR route finding training
were suitable for both, neurologic patients with spatial
orientation problems and healthy controls. This short
VR training led to an improvement in spatial navigation
performance in VR as well as to a performance improve-
ment in general spatial abilities. Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Page 10 of 13 However, the comparability of the present study with
the results of prior VR training studies such as the study
of Brooks et al. (1999) or Rose et al. (2001) is restricted
due to different VR systems used. For instance, in the
study of Brooks et al. (1999) the 3D non-immersive vir-
tual environment was run on a conventional computer
screen. Hence, the virtual environment was presented in
a monoscopic view on a small screen. In the present
study, the VR was presented in a monoscopic view too,
but on a large projection wall (2×2 m). There is evidence
that technological factors such as screen size can influ-
ence VR experiences, e.g. the level of immersion [37]. It
is a matter of further research to investigate the effects
of technological VR factors on the outcome of VR-based
rehabilitation programs. Furthermore, the trained neuro-
logic patient sample in the present study is not compar-
able with the amnestic patients in the studies of Brooks
et al. (1999) and Rose et al. (2001). This restricts the
comparability of the studies too. impaired visual perception and memory [27]. After VR
training, patients with spatial disorientation reached an
average score of 13.36 points, which is associated with
normal visual perception and visual memory perform-
ance. Proof-of-principle of the applied VR-based verbally-
guided passive navigation training program Hence, after navigation training in VR the visual
perception and visual memory of patients with spatial
deficits improved from a neurologic impaired level to a
normal level. The healthy control group showed a nor-
mal visual perception and visual memory performance
(12.64 points in the Benton Test) already before VR
training. Nevertheless, they could increase their per-
formance up to 14.73 points after VR training. The max-
imum score of the Benton Test is 15 points. Hence, the
control group showed a nearly perfect performance in
the Benton Test after five VR training sessions. This re-
sult is in line with the findings of Caglio et al. (2012)
who demonstrated that spatial navigation training in VR
can improve visual-spatial memory learning in a patient
with traumatic brain injury through the exploration of a
new and complex virtual environment. In the present
study, the improvement in quality of spatial memory can
be directly related to the need to memorize different
routes in a complex VR environment. Generalization of VR training to general spatial cognition
The second main research question addressed the trans-
ference of improvements in VR route training to more
general aspects of spatial cognition and the homogeneity
of these transference effects across participants. Neuro-
logic patients with spatial orientation deficits as well as
neurologic healthy controls were evaluated for different
aspects of spatial cognition before and after performing
a VR-based spatial navigation training program. p
In the subtest spatial imagination of the Achievement
Measure System 50+ (LPS 50+), the ANOVA revealed a
significant main effect of time, which indicates that
controls as well as patients improved their performance
in the LPS 50+ after VR training. However, the subse-
quent t-test showed that only the patient group showed
a significant improved performance after five VR training
sessions compared
to the
pre-test. Before
the VR
training sessions, the patient group showed a mean LPS
50+ score (T-score) of 44.73, which was more than a half
standard deviation below the average of the normal dis-
tribution (Mean = 50.00; SD = 10.00) [38]. After VR
training, neurologic patients could improve their per-
formance in the LPS 50+ with a resulting mean T-score
of 50.00. Hence, the ability to imagine spatial objects
and to mentally transform them increased in patients
with spatial disorientation after VR training. Proof-of-principle of the applied VR-based verbally-
guided passive navigation training program This indicates
that the spatial navigation task in the virtual environ-
ment is a useful tool to assess spatial abilities and that it
is largely valid. These correlation results are in accord-
ance with the findings of Kalová et al. (2005) and
Cushman et al. (2008) who could show that VR is a valid
assessment method of spatial skills [10-12]. Morganti
et al. (2007) also compared spatial performance in VR
with performances in standardized neuropsychological
tests, such as the Digit and Corsi’s span test, the Pro-
gressive Raven’s Matrixes test, the Trial Making test, the
Rey’s complex figure test, or the Benton’s line orientation
test. Therefore, they tested patients with brain damage
and healthy matched control participants. Brain damaged
patients performed worse than healthy controls in all
tasks. Hence, the patients’ performance in the VR task
was congruent with their neuropsychological evaluation
[13]. Moffat et al. (2001) also found a positive relation-
ship between spatial navigation performance in a virtual
maze and the performance in standardized neuropsycho-
logical tests, such as the Benton Visual Retention Test, in
healthy elderly [45]. In
the
computer-based
Corsi
Block-Tapping Test
(CBTT) [32] neither the healthy control group nor the
patient group showed a significant improvement in per-
formance between the pre- and post-test. Both groups
showed slightly higher scores in the post- than in the
pre-test. Nevertheless,
these
marginal
performance
improvements did not reach significance. The CBTT
primarily assesses visual short-term memory capacity
[32], which might not have improved due to the spatial
way-finding training in VR [15]. Due
to
motor
impairments,
not
all
neurologic
patients with spatial deficits were able to complete
the computer-based tests LVT and CBTT. Hence,
these computer based spatial tests are not suitable
for all patients with brain damage. In summary, these results allow to generalize the
obtained VR training outcomes to more general aspects
of spatial cognition. The spatial way-finding training in a
specific
virtual environment could
enhance
general
spatial abilities assessed with specific neuropsychological
tests in neurologic patients with spatial orientation
problems as well as in healthy controls. Hence, the VR-
based verbally guided spatial navigation training used in
the present study is associated with improvements in
some general aspects of spatial cognition. In the present study, all neurologic patients had spatial
orientation problems, although they showed various
brain lesion sites. Proof-of-principle of the applied VR-based verbally-
guided passive navigation training program The healthy control group already answered
very fast during the pre-test (4.35 s), therefore, a de-
crease in reaction time was hardly possible. No changes
in overall performance (score) of the LVT could be
found between the pre- and post-test, probably because
participants had to find the right answer while being
placed under time pressure. If the participants did not
give an answer in a predefined time window, the answer
did not count any more even when it was correct. Hence, many correct answers were not counted because
the participants answered too slowly. In contrast, for the
median time of correct answers the reaction times of all
correct answers were taken into account, even if they
were not given in the predefined time window. There-
fore, the median time of correct answers might be a
more appropriate measure when testing patients with
brain damage, who generally need more time to process
different tasks, than the absolute score of the LVT. In
summary, more general aspects of visual selective atten-
tion measured by the LVT responded positively to VR
training. One natural reason for that is the need to rap-
idly update visually presented information in both LVT
and VR tasks. an improved spatial orientation performance in the real
world after VR training, such as a more autonomous navi-
gation through the real hospital area, their own home
town, or the real world district of Graz, which was used as
VR training environment in the present study. Several
prior studies investigated the transfer of spatial knowledge
from a virtual to a corresponding real environment and
provided evidence that VR based spatial learning transfers
to improved performance in the real world [16,19,39-44]. Furthermore, a high priority for future work is to
examine changes in general spatial abilities in a control
group, defined by a neurologic patient group with spatial
orientation problems that undergoes no VR navigation
training. With such a control group one could prove if
improvements in general aspects of spatial abilities are
only caused by the VR training or if the effects are
caused by general time on task effects independent of
the intermediate spatial training. Additionally, follow-up
measurements would be necessary to examine possible
long-term training effects. In both groups, performance in the VR route finding
task was positively correlated with performance in
standardized neuropsychological tests. Proof-of-principle of the applied VR-based verbally-
guided passive navigation training program The LPS 50+
scores of neurologic healthy elderly were above the aver-
age of the normal distribution in the pre- (58.09) and
post-test (59.82). The most prominent ability common
to both LPS 50+ and VR training is mental rotation and
representation. The ability to construct a mental map of
the route might have been recruited in VR training,
since participants had to compute their routes forwards
as well as backwards, and generalized to the perform-
ance in the LPS 50+. The patient group showed an increased performance
in three out of four neuropsychological tests in the post-
compared to the pre-test, whereas the matched control
group showed a performance improvement in only one
test. Patients showed a significant higher LPS 50+ score,
a higher score in the Benton Test and faster reaction
times in the LVT after VR training compared to the pre-
test. Controls showed a performance improvement in
the Benton Test. Hence, these results indicate that
spatial training in VR can increase general spatial abilities
and that VR training seems to be most beneficial for
neurologic patients with spatial deficits. The results of the
single-case analysis support this finding. Altogether, neuro-
logic patients reached critical differences eight times (seven
out of eleven patients), which means that the performance
in the standardized neuropsychological tests was eight
times significantly increased in the post- compared to the
pre-test. Whereas the controls showed five times signifi-
cant performance improvements when comparing post-
and pre-test (four out of eleven controls). Both groups showed an increased performance in the
Benton Test after VR training compared to the pre-test. The Benton Test assesses visual perception and visual
memory [27]. Patients showed a Benton score of 10.18
points before VR training, which is associated with In the Visual Pursuit Test (LVT) [30], which is one of
the two computer based spatial tests, patients with
spatial deficits answered faster after VR training than be-
fore. The significant interaction time x group observed
in the median time score of the LVT indicates that Page 11 of 13 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Page 11 of 13 patients benefited more from VR training than matched
controls. Hence, in the patient group the reaction time
during
visual
orientation
performance
for
simple
structures in a complex environment decreased after VR
training. Appendix I performance neither in the VR task nor in standardized
neuropsychological tests such as the Corsi Block-Tapping
Test, the Benton’s line orientation test for line orientation
judgement, or the Trial Making test to assess divided atten-
tion [47]. Compared to a healthy matched control group,
patients with brain damage showed an impaired VR task
performance, such as in the current study [47]. Hence, dif-
ferent spatial deficits associated with different brain lesions
might contribute to influence the performance in VR
spatial tasks. Furthermore, when referring to the results of
the single-case analysis, no systematical differences be-
tween performance improvements in standardized neuro-
psychological tests and lesion location or time since onset
can be seen (see Table 1 and Table 4). Nevertheless, we
cannot exclude that patients with different brain lesions
who show distinct spatial impairments demonstrate differ-
ent learning effects or the absence thereof in the present
VR navigation training. Further studies are needed to in-
vestigate the influence of brain lesion site on VR navigation
performance in more detail. Calculation of weighted total score: Per training session
maximal four routes could be learned. If a previously
learned route was recalled correctly without any error in
the first run, the participant got 24 points for this route
and the learning phase of the next route started. Hence, if
all four routes were performed without any errors, the par-
ticipant could reach maximum 96 points per training ses-
sion. If no route was performed correctly, the participant
got 0 points. Each route contained three junctions where
the participants had to make a decision on the direction. The participants had to recall this route from the starting
to the endpoint and backwards after the learning phase. Hence, per run maximum 6 errors could be made. If a par-
ticipant made an error in the first run (×1 = number of
errors in first run), but completed the second run error-
free, the following formula was used to calculate the points
for the actual route: ((24-×1*4)*0.25 + 18). If a participant
made an error in the second run too (×2 = number of
errors in second run), but completed the third run error-
free, the following formula was used to calculate the points
for the actual route: ((24-×1*4)*0.25 + (18-×2*3)*0.15 + 12). Proof-of-principle of the applied VR-based verbally-
guided passive navigation training program Studies examining neuroanatomical
correlates of TD showed that TD is associated with
lesions in both hemispheres of the brain. Hence, TD can
occur after brain lesions at different sites [46]. Carelli
et al. (2011) examined spatial abilities of patients with
brain damage in VR, too. These patients also showed
differences in lesion sites [47]. Carelli et al. (2011) could
not find any relationship between lesion site and spatial However, based on our results we cannot conclude that
neurologic patients with spatial orientation problems show Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 Page 12 of 13 Appendix I If a participant also made an error in the third run (×3 =
number of errors in third run), but completed the fourth
run error-free, the following formula was used to calculate
the points for the actual route: ((24-×1*4)*0.25 + (18-×2*3)
*0.15 + (12-×3)*0.1 + 6). If a participant made an
error in the fourth run (×4 = number of errors in
fourth run), the following formula was used to calcu-
late the points for the actual route: ((24-×1*4)*0.25 +
(18-×2*3)*0.15 + (12-×3)*0.1 + 6 - ×4). Maximum 4
runs per route were possible. The resulting weighted
total score was the sum of the reached points of all
learned routes per training session. Authors’ contributions
SEK
i
d
f th
t d SEK conceived of the study and made substantial contributions to
conception and design of the study, performed the statistical analysis, made
the analysis and interpretation of data, and drafted the manuscript. GW has
been involved in the interpretation of data, drafting the manuscript and
revising it critically for important intellectual content. DH participated in the
design of the study, performed statistical analysis and collected the data. WK
& MK participated in the selection and medical care of the neurologic
patients, coordination, data collection and interpretation. CN participated in
the design of the study and interpretation of data, revised the manuscript
critically for important intellectual content. All authors read and approved
the final manuscript. To date, rehabilitation of spatial orientation ability
after brain damage is generally a part of common ther-
apy sessions at the rehabilitation hospital and there is no
explicit training of navigation skills or general spatial
abilities
in
use
[1]. Therefore,
spatial
way-finding
training in VR might provide a new and ecologically
valid rehabilitation method of spatial deficits. Our results
indicate that VR is potentially useful in the rehabilitation
of spatial deficits associated with TD. Conclusion In summary, the current study provides evidence that
our VR-based verbally-guided passive navigation training
program can enhance general aspects of spatial abilities
in neurologic patients with spatial orientation problems
as well as in healthy controls. Patients with spatial
deficits and matched controls showed an improved per-
formance
in
standardized
neuropsychological
tests
assessing general spatial abilities after only five VR
training sessions compared to the pre-test. Prior VR-
based spatial training studies focused on improvements in
specific spatial performances in particular environments
that also were used as training environments. For instance,
Lloyd et al. (2009) trained and tested participants’ route
finding ability in the same virtual town. General spatial
abilities of the patients participating in their study were not
assessed [21]. Wilson et al. (1996) and Brooks et al. (1999)
performed a spatial training in a virtual environment and
tested the acquired spatial knowledge in the corresponding
real world unit. Possible changes in general spatial abilities
due to VR training were not examined either [16,19]. Hence, this is the first study in which the influence of a
specific spatial training in VR on general spatial abilities
was investigated. Competing interests
The authors declare that they have no competing interests. Competing interests
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virtual environment navigation task. Neurobiol Aging 2001, 22:787–796. 18. Rose FD, Attree EA, Brooks BM, Andrews TK: Learning and memory in
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Published: 8 February 2013 Received: 1 March 2012 Accepted: 5 February 2013
Published: 8 February 2013 25. Marchette SA, Bakker A, Shelton AL: Cognitive mappers to creatures of
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31:15264–15268. Author details
1D
f 23. Rose FD, Brooks BM, Attree EA, Parslow DM, Leadbetter AG, McNeil JE,
Jayawardena S, Greenwood R, Potter J: A preliminary investigation into the
use of virtual environments in memory retraining after vascular brain injury:
indications for future strategy? Disabil Rehabil 1999, 21:548–554. 1Department of Psychology, University of Graz, Universitaetsplatz 2/III, Graz
8010, Austria. 2Privatclinic Lassnitzhoehe, Lassnitzhoehe, Austria. 3Laboratory
of Brain-Computer Interfaces, Institute for Knowledge Discovery, Graz
University of Technology, Graz, Austria. indications for future strategy? Disabil Rehabil 1999, 21:548–554. 24. Cirstea CM, Ptito A, Levin MF: Feedback and cognition in arm motor skill
reacquisition after stroke. Stroke 2006, 37:1237–1242. Acknowledgment g
This work was partially supported by the Neuro Center Styria (NCS) in Graz,
Austria and the European Community Seventh Framework Programme (FP7/
2007 2013), Grant Agreement nr. 258169. The authors are grateful to M. Pröll,
M. Lancelle, V. Settgast and D. Fellner of the Institute of Computer Graphics
and Knowledge Visualization (Graz University of Technology) for technical
assistance and E. Friedrich for proofreading the manuscript. Page 13 of 13 Page 13 of 13 Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 doi:10.1186/1743-0003-10-17
Cite this article as: Kober et al.: Virtual reality in neurologic rehabilitation
of spatial disorientation. Journal of NeuroEngineering and Rehabilitation
2013 10:17. Kober et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:17
http://www.jneuroengrehab.com/content/10/1/17 References Lloyd J, Riley G, Powell T: Errorless learning of novel routes through a
virtual town in people with acquired brain injury. Neuropsychol Rehabil
2009, 19:98–109. doi:10.1186/1743-0003-10-17
Cite this article as: Kober et al.: Virtual reality in neurologic rehabilitation
of spatial disorientation. Journal of NeuroEngineering and Rehabilitation
2013 10:17. 22. Brooks BM, Attree EA, Rose FD, Clifford BR, Leadbetter AG: The specificity of
memory enhancement during interaction with a virtual environment. Memory. Memory 1999, 7:65–78.
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Monolayer surface chemistry enables 2-colour single molecule localisation microscopy of adhesive ligands and adhesion proteins
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Lu, X. et al (2018) Monolayer surface chemistry enables 2-colour single molecule localisation microscopy of adhesive ligands and adhesion
proteins, Nature Communications, volume 9. Article no. 3320, https://doi.org/10.1038/s41467-018-05837-7 Statement:
Monolayer surface chemistry enables 2-colour single molecule localisation microscopy of adhesive ligands and adhesio
ommunications, volume 9. Article no. 3320, https://doi.org/10.1038/s41467-018-05837-7 Publisher Rights Statement:
Lu, X. et al (2018) Monolayer surface chemistry enables 2-colour single molecule localisation microscopy of adhesive
proteins, Nature Communications, volume 9. Article no. 3320, https://doi.org/10.1038/s41467-018-05837-7 Monolayer surface chemistry enables 2-colour
single molecule localisation microscopy of
adhesive ligands and adhesion proteins. Document Version
Publisher's PDF, also known as Version of record Citation for published version (Harvard):
Lu, X, Nicovich, PR, Zhao, M, Nieves, DJ, Mollazade, M, Vivekchand, SRC, Gaus, K & Gooding, JJ 2018,
'Monolayer surface chemistry enables 2-colour single molecule localisation microscopy of adhesive ligands and
adhesion proteins.', Nature Communications, vol. 9, 3320. https://doi.org/10.1038/s41467-018-05837-7 Link to publication on Research at Birmingham portal Citation for published version (Harvard):
Lu, X, Nicovich, PR, Zhao, M, Nieves, DJ, Mollazade, M, Vivekchand, SRC, Gaus, K & Gooding, JJ 2018,
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the work immediately and investigate. Download date: 24. Oct. 2024 ARTICLE Monolayer surface chemistry enables 2-colour
single molecule localisation microscopy of adhesive
ligands and adhesion proteins Xun Lu1, Philip R. Nicovich2,3, Manchen Zhao1, Daniel J. Nieves2, Mahdie Mollazade2, S.R.C. Vivekchand1,
Katharina Gaus2 & J. Justin Gooding1 Xun Lu1, Philip R. Nicovich2,3, Manchen Zhao1, Daniel J. Nieves2, Mahdie Mollazade2, S.R.C. Vivekchand1,
Katharina Gaus2 & J. Justin Gooding1 Nanofabricated and nanopatterned surfaces have revealed the sensitivity of cell adhesion to
nanoscale variations in the spacing of adhesive ligands such as the tripeptide arginine-
glycine-aspartic acid (RGD). To date, surface characterisation and cell adhesion are often
examined in two separate experiments so that the localisation of ligands and adhesion
proteins cannot be combined in the same image. Here we developed self-assembled
monolayer chemistry for indium tin oxide (ITO) surfaces for single molecule localisation
microscopy (SMLM). Cell adhesion and spreading were sensitive to average RGD spacing. At
low average RGD spacing, a threshold exists of 0.8 RGD peptides per µm2 that tether cells to
the substratum but this does not enable formation of focal adhesions. These findings suggest
that cells can sense and engage single adhesive ligands but ligand clustering is required for
cell spreading. Thus, our data reveal subtle differences in adhesion biology that may be
obscured in ensemble measurements. 1 School of Chemistry, Australian Centre for NanoMedicine and the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of
New South Wales, Sydney, NSW 2052, Australia. 2 EMBL Australia Node in Single Molecule Science, School of Medical Sciences and the ARC Centre of
Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, NSW 2052, Australia. 3Present address: Allen Institute for Brain Science,
Seattle, WA 98109, USA. Correspondence and requests for materials should be addressed to K.G. (email: k.gaus@unsw.edu.au)
or to J.J.G. (email: justin.gooding@unsw.edu.au) 1 NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 ligand densities, or even individual ligands, as this would require
the simultaneous detection of the location of the rare ligands and
the organisation of cellular proteins on the nanometre scale. S S
ophisticated nanofabrication tools such as photolithography
and electron beam lithography have allowed researchers to
mimic and modulate the chemistry and topography of
adhesive ligands found in the extracellular matrix in vivo on
substrata for ex vivo studies1–3. For example, colloid lithography
and
block
copolymer
micelle
nanolithography
can
create
repeating patterns on two-dimensional surfaces with the aid of
nanoparticles that self-assemble into monolayers or within
diblock copolymers4,5. Monolayer surface chemistry enables 2-colour
single molecule localisation microscopy of adhesive
ligands and adhesion proteins Such nanofabricated surfaces have pro-
vided novel insights into how cell adhesion6,7, differentiation8–10,
proliferation11,12, signalling13 and migration14,15 are influenced
by environmental parameters such as ligand spacing16. For
example, in pioneering work, Spatz and colleagues showed that
the formation of stable focal adhesions requires interligand spa-
cing of RGD-containing peptides of <70 nm and that cell polar-
isation and migration is sensitive to even nanometre variation in
ligand spacing17–20. It is currently thought that rather than ligand
availability per se, it is the nanoscale clustering of adhesive ligands
that is the minimal requirement for cell attachment to the sub-
strate and focal adhesion maturation21,22. By pairing nano-
patterned surfaces with molecular tension probes, Liu et al. recently demonstrated that sensing of ligand spacing by cells is
dependent on the forces generated by the actomyosin cytoskele-
ton and transmitted to integrin receptors, suggesting that clus-
tering
of
ligands
and
adhesion
proteins
drives
stable
attachment23. The characterisation of interfaces between substratum and cells
has been an enduring challenge in surface science2,3. Methods
such as X-ray photoelectron spectroscopy (XPS) and reflecto-
metry can give exquisite detail on the coupling yields in forming
these interfaces, the density of components in the monolayer and
the even the thickness of the layers with sub-nanometre preci-
sion42. However, as powerful as these methods are, they all
provide average information over a larger surface area without
providing information of the location of individual ligands. Scanning probe microscopy methods in contrast can provide such
localised information43 but only prior to the incubation with cells. In the absence of alternative surface characterisation technolo-
gies42, we were motivated to develop SMLM-compatible surfaces
on which ligand spacing could be varied and directly measured
after cells were seeded onto the surface. Here we report a method that gives precise presentation of
adhesive ligands to cells, using self-assembled monolayer chem-
istry on indium tin oxide (ITO) surfaces44,45, and is compatible
with SMLM imaging of ligands and adhesion proteins. The sur-
face chemistry allowed us to vary average RGD spacing over a
wide range. We performed both ensemble measurements such as
average number of RGD peptides per area and average number of
cells per area, as well as single cell measurements such as the
number of RGD beneath individual cells. Further we measured
the RGD density in and out of focal adhesions in the same cells. Monolayer surface chemistry enables 2-colour
single molecule localisation microscopy of adhesive
ligands and adhesion proteins We found that while adhesion of NIH-3T3 cells requires a
minimal average density of RGD peptides, once cells adhered and
spread, focal adhesions form independently of the position of
RGD peptides on the surface, meaning the location of adhesion
structures in spread cells does not correlate with variations in
local RGD density beneath the cell. This suggests that under these
conditions, ligand availability is not limited in the cellular
response and formation of mature focal adhesion is likely to be a
cell-intrinsic process. In contrast, on surfaces with very low
average RGD densities, we found evidence that NIH-3T3 cells can
sense and engage individual RGD peptides that allows cells to
tether to the substratum but do not spread or form focal adhe-
sions. The described approach is not just a means to simulta-
neously characterise cell adhesion and ligand distribution with
SMLM, but also illustrates that SMLM can be used to characterise
biointerfaces at the molecular level46. Such biointerfaces are
pivotal in sensors, biomaterials and model surfaces for cell
biology42. Single molecule localisation microscopy (SMLM) technolo-
gies24, such as (fluorescent) photoactivated localisation micro-
scopy ((f)PALM)25,26, (direct) stochastic optical reconstruction
microscopy ((d)STORM)27,28, point accumulation for imaging
nanoscale topography (PAINT)29, and ground-state depletion
followed by individual molecule return (GSDIM)30, have enabled
the precise mapping of protein clusters on the cell surface. This is
because SMLM generates images from the molecular coordinates
of individual fluorescence events that are temporally segregated
during data acquisition24. The distribution and clustering can
then be quantified with point-pattern algorithms31 such as Ripley
K-function32, pair correlation analysis33 and DBSCAN (density-
based spatial clustering application with noise)34. SMLM imaging
and cluster analysis approaches have thus enabled detailed
mapping of cluster morphologies and function in a range of cell
types24,35. An important insight of SMLM is that mature focal
adhesions are not homogeneous structures but consists of elon-
gated substructures36,37 with single adhesion proteins diffusing in
and out of mature adhesions38,39. In contrast, nascent adhesions
are discreet entities of ~100 nm in diameter containing ~50
activated integrins40. These studies highlight the power of SMLM
for adhesion biology and the diversity of adhesive studies in
adherent cells. To what extent ligand clustering determines the
nanoscale architecture of adhesive structures in cells is currently
not known. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 voltammetry and XPS from which an average coupling yield of
peptides per phosphohexadecanoic acid was estimated to be
12.2% (Supplementary Fig. 2). Since nonfunctional versions are
not available for all ligand−receptor pairs, this multistep sur-
face chemistry can be easily adapted to adjust the number of
surface bound ligands by diluting the number of hydroxyl-
terminated coupling points with methoxyl-terminated hexa
(ethylene oxide) molecules, as we have done previously13. It
should be noted that we observed that fluorescently labelled
RGD aggregates on the surface when it was prepared in the
absence of the nonfunctional RGE. Fluor647-labelled bovine serum albumin (BSA-A647) and unla-
belled BSA were adsorbed on glass and ITO surfaces, which
resulted in the same number of photons emitted from the
fluorophores on each surface and gave a localisation precision of
~20 nm on both surfaces for a range of BSA-A647 densities
(Fig. 1a, b). Similarly, paxillin fused to the photo-activatable
fluorescent protein mEos2 (paxillin-mEos2) expressed in NIH-
3T3 cells and phalloidin conjugated to A647 yielded similar
photon numbers and localisation precisions in cells on ITO
versus glass surfaces (Fig. 1c, d). These results confirmed that ITO
surfaces can be used for SMLM. Next, we used SMLM to estimate the relative number of RGD-
A647 peptides on surfaces with various ratios of RGD-A647 to
GRGE peptides (Fig. 2b). This resulted in surfaces with a wide
range of RGD densities from 0.01 to 600 molecules/µm2, which—
assuming a spatially random distribution—correspond to an
average RGD-to-RGD spacing of 5 µm to 20.3 nm. We next
seeded NIH-3T3 cells onto the functionalised ITO surfaces for 2
h, followed by fixation and fluorescence imaging. As reported
previously13, the average RGD spacing controls both the average
number of cell adhering to these surfaces and cell spreading
(Supplementary Fig. 3). It is notable that in ensemble measure-
ments,
the
surfaces
with
no
RGDs
effectively
prevented
nonspecific adhesion of cells (Supplementary Fig. 3a), while low
densities of RGDs, i.e. 1:103 and 1:106 RGD:RGE surfaces, also
impaired cell spreading (Supplementary Fig. 3b). Thus, the
functionalised ITO surfaces recapitulated the sensitivity of NIH-
3T3 cells to RGD spacing that was previously reported for other
cell types and surfaces13,17,18,20. Self-assembled monolayer chemistry for ITO surfaces. We
developed the self-assembled monolayer chemistry for ITO
where the biointerface is formed in multiple steps as shown in
Fig. 2a. The base monolayer was 16-phosphohexadecanoic acid
to which hydroxyl-terminated 1-aminohexa(ethylene oxide)
was attached. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 The close packing of the monolayer, and hence
compatability with building well-defined interfaces, was verified
electrochemically (Supplementary Fig. 1) by showing that redox
species in solution could not access the underlying ITO surface
and hence redox peaks were absent in the cyclic voltammetry. To this layer, the peptides GRGDC, conjugated to Alexa Fluor
647 (RGD-A647), and nonlabelled RGE-containing peptides
were coupled. The cell adhesive ligand density on the surface
can be varied by coupling different ratios of adhesive ligands
(RGD) to nonfunctional ligands to the surface (RGE). Impor-
tantly, although they have different cell adhesive properties,
RGD and RGE peptides otherwise have very similar surface
properties and hence changing the ratio of the two peptides
does not alter the surface other than in terms of cell adhesion48. The
surfaces
were
characterised
extensively
with
cyclic Focal adhesions on RGD-modified ITO surfaces. Next we
imaged adhesive structures of paxillin-mEos2 in NIH-3T3 cells The
surfaces
were
characterised
extensively
with
cyclic
imaged adhesive structures of paxillin mEos2 in NIH 3T3 cells
1000
25
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
*
20
15
Localisation precision (nm)
10
5
0
30
Paxillin-
mEos2
Phalloidin-
A647
Paxillin-
mEos2
Phalloidin-
A647
20
Localisation precision (nm)
Number of photons/molecule
10
0
800
600
400
200
0
800
c
600
400
200
0
1:640
1:320
1:40
1:640
1:320
1:40
BSA-A647: BSA ratio
BSA-A647: BSA ratio
1:10
1:10
Number of photons/event
d
b
a
Fig. 1 dSTORM data quality is similar on ITO and glass surfaces. a, b BSA and BSA labelled with Alexa Fluor 647 (BSA-A647) were adsorbed onto ITO
(black bars) and glass (grey bars) surfaces at the indicated ratios and imaged with dSTORM under identical conditions. Fluorescent events were identified
and grouped as described in the Methods to extract the number of photons (a) and the localisation precision (b) as a function of labelled BSA. c, d NIH-
3T3-cells-expressing paxillin fused to mEos2 were plated onto ITO (black bars) and glass (grey bars) surfaces and stained with phalloidin conjugated to
Alexa Fluor 647. PALM and dSTORM images were recorded under identical imaging conditions for both surfaces and the number of photons per molecule
(c) and localisation precision (d) calculated. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 Bars and error bars in a−d indicate average and standard deviation, respectively, of n = 5 (a, b) and n = 4 (c,
d) independent experiments; ns not significant; *P ≤0.05 (two-tailed t test assuming equal variance)
NATURE COMMUNICATIONS | (2018)9 3320 | DOI 10 1038/ 41467 018 05837 7 |
t
/
t
i
ti
3 1000
25
ns
ns
ns
ns
ns
ns
ns
*
20
15
Localisation precision (nm)
10
5
0
800
600
400
200
0
1:640
1:320
1:40
1:640
1:320
1:40
BSA-A647: BSA ratio
BSA-A647: BSA ratio
1:10
1:10
Number of photons/event
b
a 25
ns
ns
ns
*
20
15
Localisation precision (nm)
10
5
0
1:640
1:320
1:40
BSA-A647: BSA ratio
1:10
b 1000
ns
ns
ns
ns
800
600
400
200
0
1:640
1:320
1:40
BSA-A647: BSA ratio
1:10
Number of photons/event
a ns
ns
Paxillin-
mEos2
Phalloidin-
A647
Number of photons/molecule
800
c
600
400
200
0 ns
ns
30
Paxillin-
mEos2
Phalloidin-
A647
20
Localisation precision (nm)
10
0
d d c Fig. 1 dSTORM data quality is similar on ITO and glass surfaces. a, b BSA and BSA labelled with Alexa Fluor 647 (BSA-A647) were adsorbed onto ITO
(black bars) and glass (grey bars) surfaces at the indicated ratios and imaged with dSTORM under identical conditions. Fluorescent events were identified
and grouped as described in the Methods to extract the number of photons (a) and the localisation precision (b) as a function of labelled BSA. c, d NIH-
3T3-cells-expressing paxillin fused to mEos2 were plated onto ITO (black bars) and glass (grey bars) surfaces and stained with phalloidin conjugated to
Alexa Fluor 647. PALM and dSTORM images were recorded under identical imaging conditions for both surfaces and the number of photons per molecule
(c) and localisation precision (d) calculated. Bars and error bars in a−d indicate average and standard deviation, respectively, of n = 5 (a, b) and n = 4 (c,
d) independent experiments; ns not significant; *P ≤0.05 (two-tailed t test assuming equal variance) Fig. 1 dSTORM data quality is similar on ITO and glass surfaces. a, b BSA and BSA labelled with Alexa Fluor 647 (BSA-A647) were adsorbed onto ITO
(black bars) and glass (grey bars) surfaces at the indicated ratios and imaged with dSTORM under identical conditions. Results Indium tin oxide (ITO) surfaces are suitable for SMLM. Self-
assembled monolayers have been shown to be exceedingly suc-
cessful in providing molecular level control over ligand pre-
sentation in biointerfaces as they are often stable in biological
media, have antifouling properties (i.e. prevent nonspecific
adsorption) and offer precise control over the number of coupling
points to which ligands are attached2. To date, the dominant self-
assembled monolayer systems are alkanethiols on gold, alkenes
and alkynes on silicon and organosilanes on glass42. The chal-
lenge is that neither gold and silicon are compatible with SMLM
while the organosilane systems for modifying glass surfaces are
prone to forming multilayers and can be unstable in biological
media. This means that unambiguous presentation of ligands on
glass is not achieved42. We have addressed this conundrum by
developing organophosphonate self-assembled monolayer chem-
istry for ITO surfaces44,45,47. However, we first evaluated that the
optical properties of ITO surfaces were suitable for SMLM. Alexa It would be a logical extension to combine nanofabricated
substrates with SMLM imaging and cluster analysis. However,
many types of nanofabricated substrates are incompatible with
SMLM, as they do not have the optical requirements for single
molecule fluorescence41. Indeed, in the field of adhesion biology,
surface characterisation and cell measurements are often con-
ducted in separate parallel experiments2. This means that the
average surface parameters such as average ligand spacing are
used to interpret the biological responses and inversely, cell
behaviours are averaged over large surface areas. This puts
additional constraints on the nanofabrication methods in terms of
reproducibility between samples and across surface areas, and
potentially masked heterogeneity in cellular responses22. The
separation of surface characterisation and cell experiments has
also made it challenging to measure the cellular sensitivity to low NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 We noticed that the average density of RGD peptides
has a significant impact on paxillin clustering with lower average
ligand density resulting in fewer paxillin molecules in clusters,
smaller clusters and lower paxillin densities in adhesive structures
(1:0 RGD:RGE versus 1:103 RGD:RGE surfaces, Fig. 3b−d). These data highlight the details and diversity of adhesive struc-
tures SMLM imaging and analysis can reveal. on functionalised ITO surfaces with SMLM (Fig. 3). Since it was
previously reported that mature adhesions are not homogenous
clusters,
but
consist
of
substructures
within
a
mature
adhesion37,39, we first identified adhesive structures in total
internal reflection fluorescence (TIRF) images and then quanti-
fied the
properties of paxillin-mEos2 clusters within these
adhesive structures after segmentation by DBSCAN34,35. This
afforded us the opportunity to compare the cluster morphology of
paxillin-mEos2 in adhesive structures induced by RGD-A647
peptides compared to RGD peptides without the fluorophore. Given that the number of paxillin-mEos2 in clusters (Fig. 3b),
cluster area (Fig. 3c) and the density of paxillin-mEos2 molecules
(Fig. 3d) in adhesive structures were similar for RGD-A647- and
RGD-functionalised ITO surfaces, we concluded that the A647
fluorophore on RGD peptides did not interfere with adhesion
formation. We noticed that the average density of RGD peptides
has a significant impact on paxillin clustering with lower average
ligand density resulting in fewer paxillin molecules in clusters,
smaller clusters and lower paxillin densities in adhesive structures
(1:0 RGD:RGE versus 1:103 RGD:RGE surfaces, Fig. 3b−d). These data highlight the details and diversity of adhesive struc-
tures SMLM imaging and analysis can reveal. Finally, we plated paxillin-mEos2-expressing cells on RGD-
A647-modified surfaces and obtained SMLM images of both the
individual RGD peptides and the adhesion protein paxillin-
mEos2 with the two-colour SMLM (Fig. 4a). Cells on surfaces
with RGD:RGE ratios of 1:0, 1:10 and 1:100 formed visible focal
adhesions. On 1:103 and 1:106 RGD:RGE surfaces, a few cells
adhered, but did not spread; those cells had many smaller, less
pronounced adhesion structures compared to surfaces with
higher RGD densities. On the 0:1 RGD:RGE surfaces, onto
which only a few RGD-A647 peptides adsorbed nonspecifically,
hardly any cells adhered, and those that did were round with no
visible adhesive structures (Fig. 4a, Supplementary Fig. 3b). Although the monolayer chemistry limits nonspecific adsorp-
tion, and affords control over average RGD density, individual
RGD-A647 peptides were not completely randomly distributed. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 a
ITO (as received)
Cleaning
OH
O
P
O
O
O
P
O
O
O
O
O
OH
OH
6
6
OH OH OH
Self assembly
EDC/NHS
2
1
3 a
ITO (as received)
Cleaning
OH
O
P
O
O
O
P
O
O
P
O
O
O
O
N
H
N
H
N
H
N
H
N
H
H
N
H
N
N
H
O
O
O
O
O
O
O
O
C
C
GRGE
GRGDC - Alexa Fluor 647
6
6
6
6
O
O
O
O
O
O
O
O
O
O
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
O
O
O
O
O
O
O
O
O
O
N
H
N
C
N
C
6
6
6
6
6
H
6
6
6
N
OH
OH
6
6
6
6
OH OH OH
Self assembly
EDC/NHS
DSC/DMAP
Incubation with GRGDC-
Alexa Fluor 647 and GRGE
Incubation with EO6
4
5
6
2
1
3 b
a
ITO (as received)
Cleaning
OH
O
P
O
O
O
P
O
O
P
O
O
O
O
N
H
N
H
N
H
N
H
N
H
H
N
H
N
N
H
O
O
O
O
O
O
O
O
C
C
GRGE
GRGDC - Alexa Fluor 647
6
6
6
6
O
O
O
O
O
O
O
O
O
O
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
P
O
O
O
O
O
O
O
O
O
O
O
O
O
N
H
N
C
N
C
6
6
6
6
6
H
6
6
6
N
OH
OH
6
6
6
6
OH OH OH
Self assembly
EDC/NHS
DSC/DMAP
Incubation with GRGDC-
Alexa Fluor 647 and GRGE
Incubation with EO6
4
5
6
103
102
101
100
10–1
101
102
103
Mean spacing (nm)
104
10–2
10–3
1:0
1:10
1:102
RGD:RGE ratio
Number of RGD/µm2
1:103
1:106
0:1
2
1
3
Fig. 2 Multistep surface chemistry to functionalise ITO surfaces to control average RGD spacing. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 a Schematic of the formation of the interface where the
surface density of RGD peptides attached to the otherwise cell-inert surface (facilitated by hexa(ethylene oxide) molecules, EO6) was controlled by
controlling the ratio of the molar ratio of GRGDC-Alexa Fluor 647 (RGD) and unlabelled, nonfunctional GRGE (RGE) peptides. b RGD peptides imaged by
dSTORM showing the number of RGD per μm2 on functionalised ITO surfaces. RGD density was determined from SMLM data of n = 4 independent
experiments. Mean spacing was calculated from RGD densities, assuming a spatially random distribution a Incubation with GRGDC-
Alexa Fluor 647 and GRGE Self assembly EDC/NHS
3 DSC/DMAP
5 b
103
102
101
100
10–1
101
102
103
Mean spacing (nm)
104
10–2
10–3
1:0
1:10
1:102
RGD:RGE ratio
Number of RGD/µm2
1:103
1:106
0:1 b Fig. 2 Multistep surface chemistry to functionalise ITO surfaces to control average RGD spacing. a Schematic of the formation of the interface where the
surface density of RGD peptides attached to the otherwise cell-inert surface (facilitated by hexa(ethylene oxide) molecules, EO6) was controlled by
controlling the ratio of the molar ratio of GRGDC-Alexa Fluor 647 (RGD) and unlabelled, nonfunctional GRGE (RGE) peptides. b RGD peptides imaged by
dSTORM showing the number of RGD per μm2 on functionalised ITO surfaces. RGD density was determined from SMLM data of n = 4 independent
experiments. Mean spacing was calculated from RGD densities, assuming a spatially random distribution on functionalised ITO surfaces with SMLM (Fig. 3). Since it was
previously reported that mature adhesions are not homogenous
clusters,
but
consist
of
substructures
within
a
mature
adhesion37,39, we first identified adhesive structures in total
internal reflection fluorescence (TIRF) images and then quanti-
fied the
properties of paxillin-mEos2 clusters within these
adhesive structures after segmentation by DBSCAN34,35. This
afforded us the opportunity to compare the cluster morphology of
paxillin-mEos2 in adhesive structures induced by RGD-A647
peptides compared to RGD peptides without the fluorophore. Given that the number of paxillin-mEos2 in clusters (Fig. 3b),
cluster area (Fig. 3c) and the density of paxillin-mEos2 molecules
(Fig. 3d) in adhesive structures were similar for RGD-A647- and
RGD-functionalised ITO surfaces, we concluded that the A647
fluorophore on RGD peptides did not interfere with adhesion
formation. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 Fluorescent events were identified
and grouped as described in the Methods to extract the number of photons (a) and the localisation precision (b) as a function of labelled BSA. c, d NIH-
3T3-cells-expressing paxillin fused to mEos2 were plated onto ITO (black bars) and glass (grey bars) surfaces and stained with phalloidin conjugated to
Alexa Fluor 647. PALM and dSTORM images were recorded under identical imaging conditions for both surfaces and the number of photons per molecule
(c) and localisation precision (d) calculated. Bars and error bars in a−d indicate average and standard deviation, respectively, of n = 5 (a, b) and n = 4 (c,
d) independent experiments; ns not significant; *P ≤0.05 (two-tailed t test assuming equal variance) 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 +A647
–A647
150
b
a
c
d
ns
ns
ns
***
ns
100
30,000
20,000
10,000
2000
1500
1000
500
0
0
No. molecules/cluster
Cluster area (nm2)
Density (molecules/µm2)
50
0
1:0
(+A647)
1:0
(–A647)
RGD:RGE ratio
RGD:RGE ratio
RGD:RGE ratio
1:103
(+A647)
1:103
(–A647)
1:0
(+A647)
1:0
(–A647)
1:103
(+A647)
1:103
(–A647)
1:0
(+A647)
0.1
0.5
1.0
0.1
0.5
1.0
Normalized molecular density
Normalized molecular density
1:0
(–A647)
1:103
(+A647)
1:103
(–A647)
***
ns
ns
****
Fig. 3 Paxillin-mEos2 clusters on RGD-A647- and RGD-functionalised ITO surfaces. a PALM images of paxillin-mEos2 in NIH-3T3 cells adhering onto ITO
surfaces that were functionalised with RGD-A647 (left) and unlabelled RGD (right). Top: PALM images of paxillin-mEos2; scale bar = 5 µm. Middle:
zoomed regions (black boxes in top row) of an individual adhesive structure with the region for analysis traced (blue line); scale bar = 1 µm. Bottom:
paxillin-mEos2 clusters inside identified adhesive structures; colours indicated molecular densities. Images are representative images of n = 3−5
independent experiments. b Number of paxillin-mEos2 in clusters, c cluster area, and d density of paxillin-mEos2 molecules inside adhesive structures i
NIH-3T3 cells on ITO surfaces with 1:0 and 1:103 of RGD-A647:RGE or RGD:RGE. Paxillin-mEos2 clustering inside adhesive structures was dependent o
the RGD density on the surface, but not affected by A647 fluorophore labelled on RGD peptides; ns not significant (P > 0.05), ***P < 0.001 and ****P <
0.0001 (two-way ANOVA with Tukey post-testing). Bars and error bars are mean and standard deviation, respectively, n = 3−5 independent experiment
(including 16−27 independent regions of interest)
|
/ 150
b
ns
***
ns
100
No. molecules/cluster
50
0
1:0
(+A647)
1:0
(–A647)
RGD RGE
i
1:103
(+A647)
1:103
(–A647) –A647 b a +A647 c
ns
ns
30,000
20,000
10,000
0
Cluster area (nm2)
RGD:RGE ratio
1:0
(+A647)
1:0
(–A647)
1:103
(+A647)
1:103
(–A647)
*** c d
2000
1500
1000
500
0
Density (molecules/µm2)
RGD:RGE ratio
1:0
(+A647)
1:0
(–A647)
1:103
(+A647)
1:103
(–A647)
ns
ns
**** 0.1
0.5
1.0
Normalized molecular density 0.1
0.5
1.0
Normalized molecular density 1.0 1.0 Fig. 3 Paxillin-mEos2 clusters on RGD-A647- and RGD-functionalised ITO surfaces. a PALM images of paxillin-mEos2 in NIH-3T3 cells adhering onto ITO
surfaces that were functionalised with RGD-A647 (left) and unlabelled RGD (right). Top: PALM images of paxillin-mEos2; scale bar = 5 µm. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 A
Ripley K-function analysis revealed spatial heterogeneities that
peaked on the 60–160 nm spatial scale (Supplementary Fig. 4). These local heterogeneities in RGD ligands could be caused by
irregular protrusions in the underlying ITO coating that exhibit a
similar size range44. It was noticeable that the nonhomogeneity of
the RGD distributions (reflected in the L(r)-r values, Supplemen-
tary Fig. 4) was greater at low average RGD densities. This raises NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications 4 4 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 Middle:
zoomed regions (black boxes in top row) of an individual adhesive structure with the region for analysis traced (blue line); scale bar = 1 µm. Bottom:
paxillin-mEos2 clusters inside identified adhesive structures; colours indicated molecular densities. Images are representative images of n = 3−5
independent experiments. b Number of paxillin-mEos2 in clusters, c cluster area, and d density of paxillin-mEos2 molecules inside adhesive structures in
NIH-3T3 cells on ITO surfaces with 1:0 and 1:103 of RGD-A647:RGE or RGD:RGE. Paxillin-mEos2 clustering inside adhesive structures was dependent on
the RGD density on the surface, but not affected by A647 fluorophore labelled on RGD peptides; ns not significant (P > 0.05), ***P < 0.001 and ****P <
0.0001 (two-way ANOVA with Tukey post-testing). Bars and error bars are mean and standard deviation, respectively, n = 3−5 independent experiments
(including 16−27 independent regions of interest) of the average surface density (Fig. 4b). It was particularly
noticeable on 1:106 RGD:RGE surfaces and 0:1 RGD:RGE
surfaces that those average RGD densities were much lower than
0.8 RGD peptides per µm2. Interestingly, the 1:103 RGD:RGE
surfaces—surfaces that supported cell adhesion, but limited cell
spreading (Supplementary Fig. 3)—had an average RGD density
of 0.8 RGD peptides per µm2. Thus, it appears that cells had
detached from areas with less than 0.8 RGD peptides per µm2. However, the nonrandom distributions of RGD peptides meant the question whether NIH-3T3 cells adhered and formed
adhesive structures predominantly at sites where local RGD
density was higher, a question that can only be addressed with
simultaneous imaging of RGD ligands and adhesion molecules. We thus compared (i) the density of RGD beneath cells versus the
surface average and (ii) RGD and paxillin densities inside and
outside of adhesive structures. We first noticed that NIH-3T3 cells only adhered on areas of
the ITO surfaces that had >0.8 RGD peptides per µm2 irrespective NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications 5 Discussion
l A few drops of the solution (50 μL) were
then pipetted onto clean glass or ITO-coated glass coverslips; the incubation lasted
for 20 min in dark condition at room temperature. The incubated substrates were
then gently rinsed with PBS (3 × 15 mL) and checked immediately under dSTORM. Preparation and modification of ITO and glass substrates. Glass and ITO
substrates were first cleaned in an ultrasonicator with dichloromethane and then
with methanol for 10 min each, followed by treatment with 0.5 M K2CO3 in a 3:1 Fig. 4 Local RGD density thresholds for adhesion formation and cell tethering to substratum. a dSTORM images of RGD ligands on ITO surfaces and
paxillin in NIH-3T3 cells. NIH-3T3-cells-expressing paxillin fused to mEos2 were plated onto ITO surfaces that were functionalised with RGD-A647 and
RGE peptides at the indicated ratios. Top: TIRF images of paxillin-mEos2; scale bar = 10 µm. Middle and bottom: Merged PALM images of paxillin-mEos2
(green) and dSTORM images of RGD-A647 peptides (red); scale bar = 10 µm. Zoomed regions of individual adhesions are shown in the second and third
row; scale bar = 1 µm. Images are representative images of n = 3−4 independent experiments. b RGD density averaged over the entire surface (black bars)
and averaged over the area occupied by cells (grey bars) obtained from dSTORM images of RGD-A647 on ITO surfaces with various ratios of RGD-A647:
RGE. Bars and error bars are mean and standard deviation, respectively of n = 5 independent experiments; ns not significant; *P ≤0.05, **P < 0.01, ***P <
0.001 and ****P < 0.0001 (paired t test). c Density of paxillin-mEos2 in adhesive structures (black bars) and outside adhesive structures (grey bars) in
NIH-3T3 cells on ITO surfaces with various densities of RGD-A647:RGE. Paxillin-mEos2 densities in versus out of adhesive structures were significantly
different on all surfaces (****P < 0.0001, unpaired t test) while paxillin-mEos2 inside adhesive structures was dependent on the RGD-A647:RGE ratio,
except where indicated as ns, not significant (one-way ANOVA with Tukey post-testing). d Density of RGD-A647 in adhesive paxillin-mEos2 structures
(black bars) and outside adhesive structures (grey bars) in NIH-3T3 cells on ITO surfaces with various densities of RGD-A647:RGE. Discussion
l In conclusion, we describe a monolayer chemistry to functionalise
ITO surfaces that enables the characterisation of ligand dis-
tribution and localisation of adhesion protein in a single experi-
ment via the two-colour SMLM imaging. This affords the
opportunity to probe both the local cellular environment and the
protein distributions within cells in response to engineered sur-
faces. By examining surfaces with low average RGD densities, our
single molecule imaging experiments revealed that NIH-3T3 cells
can tether to the substratum in areas of ~0.8 RGD peptides per
µm2. This suggests that cells can sense and engage individual
adhesive ligands. Interestingly, it was recently reported that
during the initial phase of the adhesion process, the force per
single integrin of ~3 pN is independent of ligand spacing23. A few
such low-force integrin−ligand interactions may be sufficient to
tether cells to the substratum, but not for cell spreading or the
formation of nascent and mature focal adhesions. Indeed, we
observed that that 4−7 RGD peptides per µm2 was needed for cell
spreading and adhesion formation. Such higher local density may
facilitate integrin clustering, so that the ligand−integrin interac-
tions can withstand forces of ~ 6 pN (ref. 23). The generation and
maintenance of such high-force ligand−integrin interactions
require that ligands are spaced less than 58 nm apart23. In our
experiments, the location of mature focal adhesion within a NIH- Synthesis and surface adsorption of BSA-Alexa Fluor 647. A solution of Alexa
Fluor 647 C2 maleimide (10 mM, 10 μL) was added to a solution of BSA (2.5 mg/mL,
625 μL) in PBS. The reaction vessel was sealed and placed in the dark at 4 °C. After 24
h the reaction solution was pipetted into Zeba spin desalting column (7K MYCO,
Thermo Fisher Scientific) to remove the excess Alexa Fluor 647 C2 maleimide. The
degree of labelling of BSA molecules with Alexa Fluor 647 was 90% as determined by
UV-Vis spectroscopy. According to the reaction mechanism, each labelled BSA
molecule is labelled only by one Alexa Fluor 647 molecule, as the BSA has only one
free cysteine on its surface. A solution of BSA-Alexa Fluor 647 was diluted to 1000 nM in PBS and mixed
with different amount of 1000 nM BSA (unlabelled) to make 0.156, 0.313, 0.625,
1.25, 2.5% BSA-Alexa Fluor 647 samples. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 that same cells could still be tethered to surfaces with lower
average ligand spacing. This illustrates that local RGD density
rather than average RGD density is the biological relevant
parameter for cell interactions with the substratum. structures by manually identifying adhesive structures based on
the TIRF images of paxillin-mEos2, as done above. As expected,
paxillin density was vastly higher in adhesive structures as
compared with regions outside adhesive structures (Fig. 4c). On
surfaces with high average RGD densities (i.e. 1:0, 1:10 and 1:100
RGD:RGE
surfaces),
the
RGD
cluster
sizes
were
similar
(Supplementary Fig. 5a-b) suggesting that at these average RGD p
We also examined the density of paxillin molecules (Fig. 4c),
the size of paxillin clusters and the number of paxillin molecules
in clusters (Supplementary Fig. 5a-b) in and out of adhesive 103
*
*
**
**
****
**** ****
****
****
****
****
***
102
101
10–1
10–2
500
400
300
200
100
0
10–3
Number of RGD/µm2
Density (molecules/µm2)
Density (molecules/µm2)
1:0
ns
ns
1:10
1:102
RGD:RGE ratio
RGD:RGE ratio
RGD:RGE ratio
RGD:RGE ratio
2000
1500
1000
500
0
1:103
NS
1:106
0:1
1:0
a
1:10
1:102
1:103
1:106
0:1
1:0
1:10
1:102 1:103 1:106
1:0
1:10
1:102
100
b
c
d
NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications RGD:RGE ratio
1:0
a
1:10
1:102
1:103
1:106
0:1 a 103
*
*
**
**
****
102
101
10–1
10–2
10–3
Number of RGD/µm2
1:0
1:10
1:102
RGD:RGE ratio
1:103
NS
1:106
0:1
100
b b **** ****
****
****
****
****
***
500
400
300
200
100
0
Density (molecules/µm2)
Density (molecules/µm2)
ns
ns
RGD:RGE ratio
RGD:RGE ratio
2000
1500
1000
500
0
1:0
1:10
1:102 1:103 1:106
1:0
1:10
1:102
c
d
NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications d c NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 3T3 cell did not correlate with the positioning of the RGD pep-
tides beneath the spread cells suggesting that once cells adhered
and spread, a cell-intrinsic process such as actin-generated forces
takes over to form mature adhesion structures with a complex
molecular suborganisation37. Methods
Ch
i
l Chemicals and materials. Indium tin oxide-coated glass (ITO) coverslips (0.17
mm thickness) were purchased from SPI, USA (6480-AB, 15−30 Ω cm, 0.17 mm
thickness) and glass coverslips were purchased from ProSciTech, (Sydney, Aus-
tralia). All chemicals, unless noted otherwise, were of analytical grade and used as
received. Aqueous solutions were prepared with Milli-Q water of 18.2 MΩ cm
resistivity. Potassium ferricyanide (K3Fe(CN)6) used as redox active species in
electrochemical experiments was obtained from Sigma-Aldrich (Sydney, Australia). N,N’-disuccinimidyl carbonate (DSC), dimethylaminopyridine (DMAP),
phosphate-buffered saline (PBS), cysteamine, 4-(2-hydroxyethyl)-1-piper-
azineethanesulfonic acid (HEPES), glucose, glycerol, horseradish peroxidase
(HRP), N-hydroxysuccinimide (NHS) and 16-phosphonohexadecanoic acid
(PHDA) were obtained from Sigma-Aldrich (Sydney, Australia). The antifouling
unit 1-aminohexa(ethylene oxide) was obtained from Biomatrik (China). Dichloromethane, tetrahydrofuran (THF), methanol, acetonitrile, ethyl acetate,
potassium carbonate, potassium chloride, sodium hydroxide and sodium dihy-
drogen phosphate were obtained from Ajax (UK). 1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide (EDC) was obtained from Alfa Aesar (UK). Paraformaldehyde
(PFA, 16%), BSA and Alexa Fluor 647 C2 maleimide were obtained from Life
Technologies (USA). Alexa Fluor 647-labelled GRGDC peptide and unlabelled
GRGD peptide were purchased from Cambridge Research Biochemicals (UK). GRGE peptide was purchased from Genscript (USA). All solvents used were
analytical grade unless further indicated. Milli-Q water was used for all aqueous
solutions, buffer preparations, rinsing and washing steps. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 SMLM imaging of both protein
clustering in adherent cells on model surfaces and the distribution
and location of ligands beneath the cells could thus reveal insights
into adhesion biology that may have been obscured when surface
characterisation and cell adhesion are examined separately. densities, NIH-3T3 cells formed mature focal adhesions. Surpris-
ingly, RGD densities on these surfaces were not significantly
different in and out of adhesive structures (Fig. 4d), indicating
that cells did not place adhesive structures onto individual RGD
peptides. This strongly suggests that local RGD density, but not
the positioning of RGD peptides beneath the adherent cells, was
the determining factor in focal adhesion formation. g
On surfaces with low average RGD densities and on which cell
spreading was limited (i.e. 1:103, 1:106 and 0:1 RGD:RGE
surfaces), few paxillin clusters formed and clusters were smaller
(Supplementary Fig. 5a-b). Here too RGD densities in and out of
adhesive structures were similar. A colocalisation analysis31,49
revealed that the percentage of paxillin molecules that colocalised
with RGD peptides was not significantly different in and out of
adhesive structures (Supplementary Fig. 5c), confirming that the
position of adhesive structures within cells did not correlate with
the position of RGD peptides beneath the cell. This strongly
suggests that once cells had adhered and spread on the surface,
the formation of adhesive structure was no longer guided by the
position of the adhesive ligands on the surface and may have
occurred via a cell-intrinsic process such as the clustering of
integrin and other adhesion proteins. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 Here, 500 μL of
PBS were first loaded into the Chamlide microscope chamber with 15 μL of
colloidal spherical gold nanoparticles (From BBI solutions, 250 nm diameter,
~5000 nanoparticles) solution and the location of the transfected cells was
identified under 488 nm. Subsequently, this solution was replaced with 500 μL of
dSTORM buffer and a dSTORM image of Alexa Fluor 647 was taken under 488
and 647 nm lasers. After dSTORM imaging, a PALM image of mEos2 was then
taken under 405 and 561 nm lasers at the same area. y
ITO coverslips functionalised with an antifouling layer were incubated in a
solution of DSC (100 mM) and DMAP (100 mM) in dry acetonitrile in sealed vials
(purged with argon) for 20 h. Samples were then removed from solution and
immediately rinsed with dry acetonitrile (3 × 10 mL), ethyl acetate (3 × 10 mL),
dichloromethane (3 × 10 mL) and dried with nitrogen. A few drops (100 µL) of
GRGE and GRGDC-Alexa Fluor 647 or unlabelled GRGD (RGD-A647:RGE 1:0,
1:10, 1:102, 1:103, 1:106, 0:1; RGD:RGE 1:0, 1:103) solution (5 µg/mL in PBS) were
placed on the conductive side and left for 15 min. Samples were then rinsed with
PBS and stored in PBS in dark at 4 °C before use. The captured data were analysed using PALM processing algorithms (Zeiss
ZEN 2012) as described previously35,50 as well as here. After Gaussian filtering,
fluorescent blinking events were identified as I −M > 6S, where I is the event
intensity, M the mean image intensity and S the standard deviation of the image
intensity. The peak mark size was set at nine pixels for Alexa Fluor 647 and six
pixels for mEos2, which allowed for the detection of dye molecules on the surface
and minimised counts arising from background noises. Each event corresponding
to a point-spread function was fitted to a two-dimensional Gaussian distribution to
calculate its centre, accounting for the possibility of overlapping peaks. The
localisation precision was calculated based on the definition of Mortensen et al.51. Events within a radius of 100 nm were grouped together if they appeared in the
same area, last for no more than five frames (on-time) and the gap between
blinking events was no more than 50 frames (off-gap). Image analysis. SMLM data were analysed using custom software written in
MATLAB (MathWorks). Image analysis. SMLM data were analysed using custom software written in
MATLAB (MathWorks). Ripley K-function32 was used to determine the extent of clustering of a
population of molecules compared to a random distribution at the same density. In
brief, the Ripley K-function calculates the number of neighbour molecules for each
molecule within a given radius r corrected by the total density and then the average
is calculated for each radius and all molecules. It therefore provides ensemble
information on the whole region of interest. A density-based spatial clustering application with noise analysis (DBSCAN)
was used for cluster identification and segmentation34. DBSCAN identifies clusters
in large datasets of points in a propagative fashion52 based on the search radius (r
= 20 nm) and the minimum number of neighbours (ε = 3). The DBSCAN routine
is implemented in MATLAB and subsequently coded in C++ and compiled in an
MEX file (Matlab executable file) to improve the speed of processing as we are
working with large data files. Gene transfection. LipofectamineTM LTX with PLUSTM reagent was purchased
from Life TechnologiesTM. NIH-3T3 cells were trypsinised and counted before
transfection. Then 5 × 105 cells were plated in a six-well plate and cultured in a
standard incubator until 80% confluent. For each well in the six-well plate, a
mixture of 0.5 µg of DNA and 0.5 µL of the PLUSTM Reagent in 100 µL of Opti-
MEM® reduced serum medium was added. The resulting solution was mixed gently
and incubated for 5–15 min at room temperature. For each well of cells, 5 µL of
LipofectamineTM LTX was pipetted into the solution, mixed gently and incubated
for 25 min at room temperature to form DNA-LipofectamineTM LTX complexes. Then the growth medium was removed from cells and replaced with 3 mL of
complete growth medium. One hundred microlitres of the DNA- LipofectamineTM
LTX complexes was added directly to each well and mixed gently by rocking the
plate back and forth. Finally, the cells were incubated at 37 °C in a CO2 incubator
for 24 h post-transfection before assaying for transgene expression. Two-colour SMLM data were also analysed with a degree-of-colocalisation
(DoC) analysis. As previously described31,35,49, the local density of each channel
and each molecule is calculated at increasing radius size (10–500 nm), providing
the density gradient around that molecule for each channel. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 SMLM imaging (dSTORM/PALM). SMLM was performed on a LSM 7 EYLRA
system equipped with 405, 488, 561 and 642 nm lasers and a Plan-Apochromat
×100/1.46 Oil DIC M27 objective lens. Laser power was adjusted and a TIRF angle
between 64° and 67° was used for acquisition. Pretreated glass and ITO coverslips
were loaded into an 18 mm × 18 mm square Chamlide microscope chamber (Live
Cell Instrument, CM-B-30), and were submerged in 500 μL of oxygen scavenging
buffer. In a typical dSTORM experiment, the illumination was focused on an area
of ~ 25 μm × 25 μm under continual radiation of 488 and 647 nm laser. 10,000
−100,000 frames (or until blinking events ceased) were captured with an exposure
time of 30 ms and a camera gain of 100. For a typical PALM experiment, the
surface was under continual radiation of 405 and 561 nm laser. A total of 20,000
frames were captured with an exposure time of 30 ms and a camera EM gain of
100. methanol:Milli-Q water mixture for 45 min under sonication. The substrates were
then rinsed with copious amounts of Milli-Q water. methanol:Milli-Q water mixture for 45 min under sonication. The substrates were
then rinsed with copious amounts of Milli-Q water. ITO surfaces were modified using the procedure outlined previously44 and
described here. Clean ITO surfaces were immersed in a PHDA solution (1 mM) in
THF for 24 h. Then the substrates were then placed in an annealing desiccator
under vacuum and annealed at 200 °C for 48 h to promote stable covalent bonding
formation. After annealing, the substrates were then rinsed with copious amount of
THF to remove possible multilayers and weak bond molecules. Subsequently, the
substrates were thoroughly rinsed with Milli-Q water. The PHDA-modified ITO
substrates were immediately immersed in a mixed solution of EDC and NHS
solution (5 mM) in water for 1 h to activate the carboxyl groups. The EDC/NHS-
activated ITO surfaces were rinsed with Milli-Q water (5 × 10 mL) and immediately
incubated in the antifouling unit 1-aminohexa(ethylene oxide) solution (200 mM)
in dry acetonitrile for 24 h. For imaging cell adhesions and RGD modified on ITO surface, the two-colour
imaging (dSTORM/PALM) was applied on the same imaging area. Discussion
l RGD-A647 densities in
versus out of adhesive structures were not statistically significant for all surfaces (unpaired t test, not shown) while RGD-A647 densities on 1:0 RGD:RGE
surfaces was significantly higher in and out of adhesive structures compared to other presented (***P < 0.001, ****P < 0.0001, one-way ANOVA with
Tukey post-testing). In b−d, data are average and standard deviation from n = 3−4 independent experiments NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications 7 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 To correct for potential
sample drift, we used a built-in drift correction function from the Zen software that
uses a method based on the triangulation of localisations and a piecewise-linear
drift model. Finally, tables containing the x−y particle coordinates of each spot
detected in the acquisition were generated and used subsequently for DBSCAN and
Ripley K-function analysis. Surface characterisation. Electrochemical measurements were performed with a
BAS-100B electrochemical analyser (Bioanalytical System Inc., Lafayette, IL) and a
conventional three-electrode system, comprising an ITO working electrode, a
platinum wire as the auxiliary electrode, and an Ag/AgCl 3.0 M NaCl electrode
(CH Instrument, USA) as reference. XPS measurements were taken using an
ESCALAB 220iXL spectrometer with Al Kα monochromatic source (1486.6 eV),
hemispherical analyser, and multichannel detector. Spectra were analysed using
AVANTAGE 4.54 software. The fitting of the spectra was performed by a non-
linear least-squares procedure using simple Lorentzian−Gaussian line shapes; prior
to peak fitting, a background subtraction was performed using the Shirley method. Cell culture and staining. NIH-3T3 were maintained in Dulbecco’s modified
Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) FBS, 100 U/mL
penicillin, and 100 μg/mL streptomycin, and grown at 37 °C in a 5% CO2 standard
incubator. Cells were at >90% confluency when trypsinised and incubated on the
modified ITO substrates. The substrates were put in a six-well plate and 5 × 105
cells were placed in each well and incubated for 2 h at 37 °C in a 5% CO2 humi-
dified incubator. Then the medium was removed and the substrates were rinsed
with PBS three times. Then 3 mL (for six-well plate) of 4% PFA was placed on each
substrate, and the well plate was kept in dark at room temperature for 15 min and
then rinsed three times with PBS. 0.1% Triton X100 in PBS was placed on each
substrate at room temperature for 5 min and then the substrates were rinsed with
PBS three times. For focal adhesion staining, the substrates were incubated in 1%
BSA in PBS for 1 h and rinsed with PBS three times. A 1:200 Phalloidin-Alexa
647 solution in PBS was prepared and added on the substrates and incubated for
30 min in dark at room temperature. Then the substrates were rinsed with PBS for
three times and imaged under dSTORM. Image analysis. SMLM data were analysed using custom software written in
MATLAB (MathWorks). The two density
gradients are tested for correlation with the Spearman criteria, which score
monotonic dependence and corrected with nearest neighbour distance to account
for long distance interactions. As a result, each molecule is assigned a DoC value
ranging from −1 to 1, with −1 characterising anticolocalisation (or segregation), 0
corresponding to single species and 1 defining high colocalisation. A paxillin
molecule was regarded as colocalised with an RGD peptide if its DoC value was
≥0.4. The GUI enabled drawing of multiple subregions within each image and
returned the output of the DBSCAN and DoC analysis for each region individually. Cell outlines and regions representing adhesive structures were manually
segmented using TIRF images. Regions representing nonadhesive structures were
inside in the cell outline and not belonging to the adhesion structures. Preparation of oxygen scavenging buffer for dSTORM imaging. Oxygen
scavenging buffer was prepared in the following way: (1) A base buffer stock
containing PBS (1×), HEPES (25 mM), glucose (25 mM) and glycerol (5%) was
adjusted to pH 8.0 and filtered through a 0.22 µm filter (Millipore, 47 mm
regenerated cellulose); (2) frozen stock solutions of glucose oxidase (GOx, 10 mg/
mL in 50 mM phosphate buffer pH 5.1) and HRP (10 mg/mL in 100 mM phos-
phate buffer pH 6.0) were added to the base buffer solution for a final con-
centration of 0.05 mg/mL GOx and 0.025 mg/mL HRP; (3) cysteamine
hydrochloride solution (1 M) was prepared just prior to the experiment and added
to the base buffer containing GO and HRP for a final cysteamine concentration of
10 mM. Statistical analysis. All statistical analyses were performed with Prism software
(Graphpad Software). Unless stated otherwise, all data are presented as mean ±
standard deviation from at least three independent experiments. Unpaired two-
tailed Student’s t tests were performed for testing of statistical significance between
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analysis of variance (ANOVA) followed by Tukey’s test. In statistical analysis, P >
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(2007). Author contributions X.L. performed experiments and analysed data. P.R.N. established and performed ana-
lysis and helped writing the manuscript. M.Z. and D.J.N. performed experiments and
analysed data, M.M. performed image analysis. S.R.C.V. performed and provided gui-
dance with experiments. K.G. and J.J.G. designed the project, interpreted the data and
wrote the manuscript. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05837-7 Science and Technology (CE140100036), the Australian Research Council for an Aus-
tralian Laureate Fellowship (FL150100060) and a National Health and Medical Research
Council of Australia programme grant (1091261). S.R.C.V acknowledges the University
of New South Wales for a UNSW Vice-Chancellor’s Research Fellowship. M.Z. acknowledges the support received through an “Australian Government Research
Training Program Scholarship”. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. acknowledges the support received through an “Australian Government Research
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regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. Acknowledgements We acknowledge technical assistance by the BioMedical Imaging Facility, University of
New South Wales. K.G. and P.R.N. acknowledge funding from the ARC Centre of
Excellence in Advanced Molecular Imaging (CE140100011 to K.G.), Australian Research
Council (LP140100967 to K.G. and P.R.N. and DP130100269 to K.G.) and National
Health and Medical Research Council of Australia (1059278 and 1037320 to K.G.). J.J.G. acknowledges funding from the ARC Centre of Excellence in Convergent Bio-Nano We acknowledge technical assistance by the BioMedical Imaging Facility, University of
New South Wales. K.G. and P.R.N. acknowledge funding from the ARC Centre of
Excellence in Advanced Molecular Imaging (CE140100011 to K.G.), Australian Research
Council (LP140100967 to K.G. and P.R.N. and DP130100269 to K.G.) and National
Health and Medical Research Council of Australia (1059278 and 1037320 to K.G.). J.J.G. acknowledges funding from the ARC Centre of Excellence in Convergent Bio-Nano y
25. Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging
by fluorescence photoactivation localization microscopy. Biophys. J. 91,
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resolution. Science 313, 1642–1645 (2006). 9 NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications 9 Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
018-05837-7. © The Author(s) 2018 © The Author(s) 2018 Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ reprintsandpermissions/ 10 NATURE COMMUNICATIONS | (2018) 9:3320 | DOI: 10.1038/s41467-018-05837-7 | www.nature.com/naturecommunications
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English
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Effect of nanostructured carbon coatings on the electrochemical performance of Li<sub>1.4</sub>Ni<sub>0.5</sub>Mn<sub>0.5</sub>O<sub>2+</sub><i><sub>x</sub></i>-based cathode materials
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Beilstein journal of nanotechnology
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Abstract Nanocomposites of Li1.4Ni0.5Mn0.5O2+x and amorphous carbon were obtained by the pyrolysis of linear and cross-linked
poly(vinyl alcohol) (PVA) in presence of Li1.4Ni0.5Mn0.5O2+x. In the case of linear PVA, the formation of nanostructured carbon
coatings on Li1.4Ni0.5Mn0.5O2+x particles is observed, while for cross-linked PVA islands of mesoporous carbon are located on the
boundaries of Li1.4Ni0.5Mn0.5O2+x particles. The presence of the carbon framework leads to a decrease of the polarization upon
cycling and of the charge transfer resistance and to an increase in the apparent Li+ diffusion coefficient from 10−16 cm2·s−1 (pure
Li1.4Ni0.5Mn0.5O2+x) to 10−13 cm2·s−1. The nanosized carbon coatings also reduce the deep electrochemical degradation of
Li1.4Ni0.5Mn0.5O2+x during electrochemical cycling. The nanocomposite obtained by the pyrolysis of linear PVA demonstrates
higher values of the apparent lithium diffusion coefficient, a higher specific capacity and lower values of charge transfer resistance,
which can be related to the more uniform carbon coatings and to the significant content of sp2-hybridized carbon detected by XPS
and by Raman spectroscopy. Effect of nanostructured carbon coatings on the
electrochemical performance of Li1.4Ni0.5Mn0.5O2+x-based
cathode materials Full Research Paper
Open Access
Address:
1Department of Chemistry, M. V. Lomonosov Moscow State
University, 119991 Moscow, Russia and 2Department of Materials
Sciences, M. V. Lomonosov Moscow State University, 119991
Moscow, Russia
Email:
Oleg A. Shlyakhtin* - oleg@inorg.chem.msu.ru
* Corresponding author
Keywords:
carbon coatings; electrode materials; Li-ion batteries;
nanocomposites; nanostructures
Beilstein J. Nanotechnol. 2016, 7, 1960–1970. doi:10.3762/bjnano.7.187
Received: 16 July 2016
Accepted: 17 November 2016
Published: 09 December 2016
This article is part of the Thematic Series "Physics, chemistry and biology
of functional nanostructures III". Guest Editor: A. S. Sidorenko
© 2016 Kurilenko et al.; licensee Beilstein-Institut. License and terms: see end of document. Results and Discussion esu ts a d
scuss o
TEM micrographs of pure Li1.4Ni0.5Mn0.5O2+x (LNM) and
LNM/C composites obtained by the pyrolysis of both linear and
cross-linked PVA are shown in Figure 1. In the case of LNM/C
composites obtained from linear PVA, 5–7 nm thick carbon
coatings can be clearly observed on the particle surface as well
as amorphous carbon bottlenecks between particles forming a
consolidated 3D network (Figure 1B). Concerning the LNM/C
composites obtained from cross-linked PVA, the continuous
carbonaceous coatings on oxide particles are absent. The car-
bon is localized as separate islands on the boundaries of
Li1.4Ni0.5Mn0.5O2+x particles (Figure 1C). The carbon obtained
from cross-linked PVA has a sponge-like microstructure with
nanometer-sized mesopores (Figure 1D). During the assembly of the lithium-ion cells, the cathode mate-
rials are always mechanically mixed with carbon black in order
to enhance the electronic conductivity. However, this method of
carbon introduction provides the contact only between the
external surfaces of cathode materials aggregates, but it does
not improve the coherence and hence the electrical conductivity
inside the aggregates. Both kinds of problems can be solved by the fabrication of con-
ducting carbon coatings on the surface of Li(Ni,Mn,Co)O2+x
particles [19-22]. Such coatings can be obtained by the impreg-
nation of oxide powders with the solution or the melt of organic
compounds accompanied by subsequent heat treatment in the
absence of oxygen or at limited oxygen access [23-25]. These
carbonaceous coatings improve the electrokinetic properties of
cathode materials, which results in enhanced capacities at high
discharge rates [26]. The variation in the electrochemical prop-
erties is governed by the increase in the electronic conductivity
of the obtained coatings, which depends on both the organic
precursor composition and the preparation conditions. Thus, the
investigation of the influence of physicochemical properties
of carbon coatings on the electrochemical properties of
Li(Ni,Mn,Co)O2+x/C composites proves to be an important
task. The choice of an organic precursor is an important chal-
lenge. In the case of Li(Ni,Mn)O2, it is rather complicated due
to the high oxidizing ability of this material, which leads to its
intensive interaction with both the products of the pyrolysis of
organic compounds [27] and carbon [28]. As it has been shown Thermogravimetry (TG) curves of the linear and cross-linked
PVA demonstrate the intense pyrolysis of the linear PVA at
240–310 °C (Figure 2A) and the continuous decomposition
process of the cross-linked PVA at 240–600 °C (Figure 2B). Introduction reasonable combination of their electrochemical properties
(C = 160–180 mAh·g−1 at C/10; U = 2.5–4.6 V) remains attrac-
tive until now [2-4]. Most of the studies deal with Li(Ni,Mn)O2 LiNi0.5Mn0.5O2-based electrode materials [1] were proposed as
a less expensive alternative to LiCoO2 for high energy density
Li-ion batteries containing less toxic elements than cobalt. The 1960
1960 Beilstein J. Nanotechnol. 2016, 7, 1960–1970. before, the rate of this interaction could be significantly reduced
by using some polymer precursors [28]. They allow for a solid
conducting film consisting of pyrolysis products to be obtained
at moderate temperatures (300–350 °С). Poly(vinyl alcohol)
(PVA) could be one of such precursors. It eliminates water at
temperatures higher than 230 °С, forming carbon residues with
a system of conjugated double bonds [29,30]. with equimolar amounts of nickel and manganese. The influ-
ence of the Ni/Mn ratio on the properties of these materials is
discussed in [5]. One of the obstacles to the practical applica-
tion of LiNi0.5Mn0.5O2 is related to the internal ion exchange
(ion mixing) of Li+ and Ni2+ due to the very similar ionic radii. This process could be partially suppressed by the introduction
of extra lithium into LiNi0.5Mn0.5O2. Extra lithium ions are
usually located in the (Ni,Mn) sublattice with the formation of
Li[LixNi0.5−(x/2)Mn0.5−(x/2)]O2−δ [6,7]. The practical applica-
tion of these materials is limited by the insufficient electronic
conductivity of Li1+x(Ni,Mn)O2 materials [8] and their ability
to catalyze the organic electrolyte decomposition at high poten-
tials and currents [9,10]. In the present paper, the electrochemical properties of
Li1.4Ni0.5Mn0.5O2+x/C composites are discussed, the prepara-
tion and characterization of which have been described before
in detail [28,31]. The melts of linear or cross-linked PVA
served as carbon source. We investigated the influence of com-
position and micro/nanomorphology of the carbonaceous coat-
ings obtained by the pyrolysis of various kinds of PVA on the
kinetics of lithium intercalation–deintercalation and the electro-
chemical properties of Li1.4Ni0.5Mn0.5O2+x/C composites. In the present paper, the electrochemical properties of
Li1.4Ni0.5Mn0.5O2+x/C composites are discussed, the prepara-
tion and characterization of which have been described before
in detail [28,31]. The melts of linear or cross-linked PVA
served as carbon source. We investigated the influence of com-
position and micro/nanomorphology of the carbonaceous coat-
ings obtained by the pyrolysis of various kinds of PVA on the
kinetics of lithium intercalation–deintercalation and the electro-
chemical properties of Li1.4Ni0.5Mn0.5O2+x/C composites. Introduction The most common way of overcoming this problem is the mod-
ification of cathode materials by introducing additives and by
depositing coatings that would suppress the interaction of elec-
trolyte and the surface of particles. Various kinds of materials
have been tested for surface modification, namely other cathode
materials (LiMnPO4 [11], LiMn2O4 [12], LiCoO2 [13], LiNiO2
[14]) or simple binary compounds such as CaF2 [15], TiO2 [16],
ZnO [17] and Al2O3 [18]. Results and Discussion The latter shows a significantly higher thermal stability of the
cross-linked polymer. The analysis of differential scanning cal-
orimetry (DSC) curves allows one to identify a small “endo”-
effect at 230 °C before the decomposition of the linear PVA
(Figure 2A) that could be associated with its melting [32]. The
corresponding effect is absent in the DSC curve of the cross-
linked polymer (Figure 2B). The reasons for the different localization of carbon in the sam-
ples can be associated with the observed features of the pyroly-
sis of precursors. Melting of linear PVA causes a wetting of
LNM grains with the polymer melt followed by a relatively
uniform pyrolysis of the thin polymer films on the surface of
oxide crystallites. In the case of cross-linked PVA, the polymer 1961 Beilstein J. Nanotechnol. 2016, 7, 1960–1970. Figure 1: TEM micrographs: A) pure LNM; B) LNM + carbon (linear PVA); C) LNM + carbon (cross-linked PVA); D) carbon, obtained by the
isothermal heat treatment of cross-linked PVA at 350 °С for 15 min in argon. 196
Figure 1: TEM micrographs: A) pure LNM; B) LNM + carbon (linear PVA); C) LNM + carbon (cross-linked PVA); D) carbon, obtained by the
isothermal heat treatment of cross-linked PVA at 350 °С for 15 min in argon. Figure 2: TG-DSC curves of the linear (A) and cross-linked (B) PVA in argon. Figure 1: TEM micrographs: A) pure LNM; B) LNM + carbon (linear PVA); C) LNM + carbon (cross-linked PVA); D) carbon, obtained by the
isothermal heat treatment of cross-linked PVA at 350 °С for 15 min in argon. Figure 2: TG-DSC curves of the linear (A) and cross-linked (B) PVA in argon. Figure 2: TG-DSC curves of the linear (A) and cross-linked (B) PVA in argon. 1962 Beilstein J. Nanotechnol. 2016, 7, 1960–1970. it takes place over a wider potential range. It means that the
carbon nanocoatings enable the polarization decrease upon
lithium intercalation [23]. After galvanostatic cycling at
20–100 mA·g−1, CVA curves were recorded at the 15th cycle
(Figure 4В). In the case of pure Li1.4Ni0.5Mn0.5O2+x, the poten-
tial range of lithium intercalation expands and in the anodic
region, the main lithium deintercalation peak shifts towards
higher potentials (from 3.78 V at the 1st cycle to 3.85 V at the
15th cycle). This fact confirms a poorer reversibility of lithium
insertion/extraction into the pure Li1.4Ni0.5Mn0.5O2+x. pyrolysis tentatively occurs without preliminary melting. Results and Discussion The
pyrolysis of PVA particles allocated in the voids between LNM
crystallites results in forming mesoporous particulates of amor-
phous carbon in the interparticular space. Another difference between the pyrolysis products of linear and
cross-linked PVA concerns their different chemical composi-
tion. According to the C 1s XPS spectra of carbon-coated LNM
(Figure 3, Table 1), the pyrolysis products of linear PVA
contain larger amounts of C–C and C=C bonds compared to the
pyrolysis products of cross-linked PVA. This difference could
be associated to the easier dehydration of linear PVA at 350 °C
resulting in the formation of C=C bonds: –(CH2–CH(OH))n– →
–(CH=CH)n– + nH2O compared to the complicated thermal de-
composition of –(СН2–CH–O–CH–CH2)n– chains of cross-
linked PVA. The electrokinetic properties of LNM/C nanocomposite ob-
tained by the pyrolysis of linear PVA are significantly better
than the ones of the corresponding composite obtained by the
pyrolysis of cross-linked PVA. In particular, after galvanostatic
cycling at 20–100 mA·g−1, the cathodic peak at ca. 3.6 V is
more pronounced and the range of lithium intercalation poten-
tials is narrower (Figure 4). Apparently, this phenomenon could
be ascribed to a more uniform carbon distribution and to the
higher content of conductive sp2-hybridized carbon in the coat-
ings obtained by pyrolysis of linear PVA compared to the pyrol-
ysis of cross-linked PVA [28]. Another reason for the superior
electrokinetic properties of the LNM/C nanocomposite ob-
tained from linear PVA could be associated with the absence of
the reversible structural transition in the partially delithiated The electrochemical properties of as-obtained nanocomposites
were studied by cyclic voltammetry (CVA) to estimate the in-
fluence of carbon coatings on the lithium intercalation–deinter-
calation in Li1.4Ni0.5Mn0.5O2+x (Figure 4). Concerning the
LNM/C composites, the cathodic peaks are substantially
narrower than the ones for pure Li1.4Ni0.5Mn0.5O2+x. In the
case of LNM/C composites, the lithium intercalation is almost
completely finished at 3.64 V, but for pure Li1.4Ni0.5Mn0.5O2+x Figure 3: XPS spectra of C 1s of the LNM/C composite samples derived from the linear (A) and cross-linked (B) PVA. Figure 3: XPS spectra of C 1s of the LNM/C composite samples derived from the linear (A) and cross-linked (B) PVA. Table 1: Fractions of C 1s XPS spectra components of the LNM/C composite samples derived from the linear and cross-linked PVA. Results and Discussion sample
fraction of C 1s components, %
285.0 eV
286.3 eV
287.2 eV
288.3 eV
290.1 eV
291.7 eV
С=С (С–С)
C–O
C=O
O–C=O
–CO32−
—
LNM + linear PVA
50.7
9.7
8.2
7.8
19.8
3.8
LNM + cross-linked PVA
35.9
15.5
9.2
5.1
28.1
6.2 Beilstein J. Nanotechnol. 2016, 7, 1960–1970. Figure 4: CVA curves of pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocomposites at the 1st cycle (A) and after galvanostatic cycling (B) at discharge
currents of 20–100 mA·g−1 (the 15th cycle). Potential scan rate is 50 µV·s−1. Figure 4: CVA curves of pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocomposites at the 1st cycle (A) and after galvanostatic cycling (B) at d
currents of 20–100 mA·g−1 (the 15th cycle). Potential scan rate is 50 µV·s−1. action of Li1.4Ni0.5Mn0.5O2+x particles with the organic elec-
trolyte. Li1.4Ni0.5Mn0.5O2+x that could be indicated by the absence of
anodic peak at 4.3 V [33] clearly visible for the other two sam-
ples. The appearance of this transition, which causes additional
internal stress during cycling in the more polarized cathode ma-
terials, could be promoted by a less homogeneous Li distribu-
tion inside the Li1.4Ni0.5Mn0.5O2+x lattice during the electro-
chemically driven intercalation–deintercalation processes. At
potentials higher than 4.5 V, the current increase could result
from the partial oxidation of the electrolyte upon cycling. In order to clarify the influence of carbon nanocoating, the
apparent Li+ diffusion coefficients (D) were estimated both for
pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocomposites. These
diffusion coefficients are calculated from EI data using the
following equation [36]: Electrochemical impedance (EI) measurements were performed
to investigate the details of the lithium insertion-extraction pro-
cesses in LNM/C nanocomposite cathodes (Figure 5A–D). All
the plots are mainly composed of a small intercept at high
frequencies, a semicircle at high to medium frequencies and a
linear part in the low-frequency region. The real and complex
EI parts are normalized to the real surface area of each elec-
trode calculated according to the procedure described earlier
[31]. The impedance spectra are fitted using the equivalent
circuit model (Figure 5E) [34,35]. The small intercept is almost
the same for the electrodes and corresponds to the solution
resistance of the cell (Re). Rf and CPEf stand for the Li+ migra-
tion resistance and the capacity of the surface layer, respective-
ly. Rct and CPEct stand for the related charge-transfer resis-
tance and the double-layer capacitance, respectively. Results and Discussion for the one obtained from cross-linked PVA at 3.75 and 3.72 V,
respectively. This difference can be explained by the different
morphologies and compositions of carbon-based nanocoatings,
in particular, by the higher ratio of sp2- to sp3-hybridized car-
bon in the LNM/C composite obtained from linear PVA [28]. This results in a higher electronic conductivity and higher D
values. The different amounts of residual intermediates of py-
rolysis also play a role. For all the investigated materials, the charge–discharge curves
for the intermediate cycles obtained at different rates are shown
in Figure 7A. In contrast to pure Li1.4Ni0.5Mn0.5O2+x, both
LNM/C nanocomposites exhibit is a well-pronounced plateau at
3.75 V in the discharge curves, especially at a current density of
20 mA·g−1. Apparently, this fact confirms the polarization de-
crease for the LNM/C composites discussed earlier during the
analysis of CVA results. Figure 6: Apparent lithium diffusion coefficients as functions of the dis-
charge potential for pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocompos-
ites obtained from linear and cross-linked PVA. Figure 5: Nyquist plots of pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocomposites at the charge potential of 3.9 V: A) 1st cycle, B) after cycling at dis-
charge currents of 20–100 mA·g−1 (15th cycle); at the discharge potential of 3.7 V: С) 1st cycle, D) after cycling at discharge currents of
20–100 mA·g−1 (15th cycle). E) Equivalent circuit used for fitting the EI spectra. i0.5Mn0.5O2+x and LNM/C nanocomposites at the charge potential of 3.9 V: A) 1st cycle, B) after cycling at dis-
5th cycle); at the discharge potential of 3.7 V: С) 1st cycle, D) after cycling at discharge currents of
valent circuit used for fitting the EI spectra. inked PVA at 3.75 and 3.72 V,
be explained by the different
of carbon-based nanocoatings,
of sp2- to sp3-hybridized car-
btained from linear PVA [28]. nic conductivity and higher D
f residual intermediates of py-
, the charge–discharge curves
ned at different rates are shown
re Li1.4Ni0.5Mn0.5O2+x, both
s a well-pronounced plateau at
specially at a current density of
confirms the polarization de-
es discussed earlier during the
Figure 6: Apparent lithium diffusion coefficients as functions of the dis-
charge potential for pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocompos-
ites obtained from linear and cross-linked PVA. Results and Discussion Zw repre-
sents the diffusion-controlled Warburg impedance in the low-
frequency region [34,35]. where n = 1, σ is the Warburg parameter (Ω·s−0.5), F is the
Faraday constant (96500 C·mol−1), s is the real surface of the
electrode (cm2), Vm is the molar volume of the electrode materi-
al (cm3·mol−1) and dE/dx is the derivative of the potential
versus lithium content. where n = 1, σ is the Warburg parameter (Ω·s−0.5), F is the
Faraday constant (96500 C·mol−1), s is the real surface of the
electrode (cm2), Vm is the molar volume of the electrode materi-
al (cm3·mol−1) and dE/dx is the derivative of the potential
versus lithium content. While discharging, the values of the apparent diffusion coeffi-
cient of lithium ions for the pure Li1.4Ni0.5Mn0.5O2+x are in the
range from 10−17 to 10−15 cm2·s−1 with a minimal value of
6.8 × 10−17 cm2·s−1 at 3.67 V (Figure 6). Concerning the
LNM/C nanocomposites, the variations of D with the applied
potential are substantially different. The D values are higher
practically over the whole potential range, and at potentials
higher than 3.75 V they exceed the ones for pure
Li1.4Ni0.5Mn0.5O2+x by three orders of magnitude. Most likely,
the 3D carbon framework decreases the polarization and the
charge-transfer resistance, which promotes the faster Li trans-
port through the electrode–electrolyte interface. The variations
of D of both LNM/C nanocomposites are rather similar, which
points to resemblances in the Li+ intercalation processes. The
minimal values of D are 6.4 × 10−16 cm2·s−1 for the LNM/C
composite obtained from linear PVA and 7.3 × 10−17 cm2·s−1 Concerning the 1st cycle, the values of Rct are almost identical
for pure Li1.4Ni0.5Mn0.5O2+x and LMN/C nanocomposites
(Figure 5А,С). However, after cycling at 20–100 mA·g−1, for
pure Li1.4Ni0.5Mn0.5O2+x Rct substantially increases in compar-
ison with LNM/C composites (Figure 5B,D). One can suppose
that the carbon additives lead to an enhancement of the elec-
tronic conductivity of Li1.4Ni0.5Mn0.5O2+x and hinder the inter- 1964 Beilstein J. Nanotechnol. 2016, 7, 1960–1970. Figure 5: Nyquist plots of pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocomposites at the charge potential of 3.9 V: A) 1st cycle, B) after cycling at dis-
charge currents of 20–100 mA·g−1 (15th cycle); at the discharge potential of 3.7 V: С) 1st cycle, D) after cycling at discharge currents of
20–100 mA·g−1 (15th cycle). E) Equivalent circuit used for fitting the EI spectra. Results and Discussion Figure 5: Nyquist plots of pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocomposites at the charge potential of 3.9 V: A) 1st cycle, B) after cycling at dis-
charge currents of 20–100 mA·g−1 (15th cycle); at the discharge potential of 3.7 V: С) 1st cycle, D) after cycling at discharge currents of
20–100 mA·g−1 (15th cycle). E) Equivalent circuit used for fitting the EI spectra. Figure 6: Apparent lithium diffusion coefficients as functions of the dis-
charge potential for pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocompos-
ites obtained from linear and cross-linked PVA. for the one obtained from cross-linked PVA at 3.75 and 3.72 V,
respectively. This difference can be explained by the different
morphologies and compositions of carbon-based nanocoatings,
in particular, by the higher ratio of sp2- to sp3-hybridized car-
bon in the LNM/C composite obtained from linear PVA [28]. This results in a higher electronic conductivity and higher D
values. The different amounts of residual intermediates of py-
rolysis also play a role. For all the investigated materials, the charge–discharge curves
for the intermediate cycles obtained at different rates are shown
in Figure 7A. In contrast to pure Li1.4Ni0.5Mn0.5O2+x, both
LNM/C nanocomposites exhibit is a well-pronounced plateau at
3.75 V in the discharge curves, especially at a current density of
20 mA·g−1. Apparently, this fact confirms the polarization de-
crease for the LNM/C composites discussed earlier during the
analysis of CVA results. Figure 6: Apparent lithium diffusion coefficients as functions of the dis-
charge potential for pure Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocompos-
ites obtained from linear and cross-linked PVA. 1965 Beilstein J. Nanotechnol. 2016, 7, 1960–1970. Figure 7: Discharge curves (А) and specific capacities (В) of pure
Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocomposites at discharge currents
of 20–100 mA·g−1. Figure 8 displays the XRD patterns of electrode materials after
electrochemical cycling at high discharge currents. It can
be seen that the splitting of (018)/(110) reflections is much
higher in carbon-coated materials compared to the pure
Li1.4Ni0.5Mn0.5O2+x (Figure 8, inset). Figure 8: XRD patterns of pure and carbon-coated Li1.4Ni0.5Mn0.5O2+x
after electrochemical cycling at 20–100 mAh·g−1. The asterisks denote
reflections of Li2CO3 (PC PDF 22-1141). Figure 8: XRD patterns of pure and carbon-coated Li1.4Ni0.5Mn0.5O2+x
after electrochemical cycling at 20–100 mAh·g−1. The asterisks denote
reflections of Li2CO3 (PC PDF 22-1141). Results and Discussion According to the selected area electron diffraction (SAED)
patterns, the preservation of the hexagonal structure of
Li1.4Ni0.5Mn0.5O2+x is especially visible in the carbon-coated
material obtained from linear PVA (Figure 9B). At high potentials during cycling, the contact of
Li1.4Ni0.5Mn0.5O2+x with electrolyte results in an inter-
action leading to the formation of a Li-deficient spinel
Lix(Ni0.5Mn0.5)2O4, and, later, cubic (Ni0.5Mn0.5)O [37]. Other
authors observed a slow transformation of the hexagonal phase
into a cubic phase in the course of electrochemical cycling of
LiMeO2 [38,39]. This process was also accompanied by a
systematic decrease in (018)/(110) splitting. The observed pro-
cesses of the deep crystallochemical degradation of pure
Li1.4Ni0.5Mn0.5O2+x (Figure 8) lead to kinetic obstacles to the
solid-state diffusion of Li+ in Li1.4Ni0.5Mn0.5O2+x crystallites
and, hence, to a substantial decrease in its electrochemical
capacity values. Figure 7: Discharge curves (А) and specific capacities (В) of pure
Li1.4Ni0.5Mn0.5O2+x and LNM/C nanocomposites at discharge currents
of 20–100 mA·g−1. The discharge capacity variations upon cycling are represented
in Figure 7B. In the case of pure Li1.4Ni0.5Mn0.5O2+x, the
capacity rapidly drops at the 4th cycle. For the LNM/C nano-
composites, the initial capacity values are lower but the stability
upon cycling is significantly better. For the LNM/C nanocom-
posite obtained from linear PVA, the capacity values are usually
higher than for the one obtained from cross-linked PVA. They attain a value of 165 mAh·g−1 at a current density of
20 mA·g−1. This fact correlates quite well with the higher elec-
tronic conductivity of this material and the higher values of the
Li+ diffusion coefficient. The electrode–electrolyte interface in composite electrode mate-
rials is reduced by the thin carbon nanocoating that protects a
part of Li1.4Ni0.5Mn0.5O2+x surface from electrochemical deg-
radation. SAED patterns show the presence of Li2CO3 in all
cycled samples, although the intensity of the (002) reflections of
lithium carbonate is rather weak (Figure 9B,D,F). However,
X-ray diffraction patterns demonstrate the presence of a 1966 Beilstein J. Nanotechnol. 2016, 7, 1960–1970. gure 9: TEM micrographs and SAED patterns of the cycled electrode materials based on Li1.4Ni0.5Mn0.5O2+x (A,B) and LNM/C nanocomposites o
ned by the pyrolysis of linear (C,D) and cross-linked (E,F) PVA. The cycling conditions are shown in Figure 7B. Figure 9: TEM micrographs and SAED patterns of the cycled electrode materials based on Li1.4Ni0.5Mn0.5O2+x (A,B) and LNM/C nanocomposites ob-
tained by the pyrolysis of linear (C,D) and cross-linked (E,F) PVA. Experimental The mixed hydroxide (Ni0.5Mn0.5)(ОН)n·xH2O was precipitat-
ed from a solution containing Ni and Mn acetates (С ≈ 1.5 М) at
a given molar ratio by adding 1 М NaOH aqueous solution at
room temperature. The precipitates were filtered, repeatedly
washed, then placed into a freeze-dryer chamber (Labconco
Freezone 7948030 (USA)) and subjected to freeze-drying (P =
0.1–0.3 mbar, T = −40 °C to +30 °C, 72 h). The obtained mixed
hydroxide powders were annealed at 500 °С for 2 h, then mixed
with LiOH·H2O (≥99%, Fluka) (molar ratio Li/(Ni + Mn) = 1.4)
and pressed into pellets. The pellets were subsequently annealed
at 500 and 900 °С for 4 h in air at each temperature. Hence, it was demonstrated that even a partial modification of
the synthesis technique (additional stage of thermal cross-
linking of PVA) leads to LNM/C composites with different
properties of the carbon coatings, including a different micro-/
nanomorphology and a different ratio of sp2- to sp3-hybridized
carbon. The last feature is also confirmed by the different inte-
gral intensities of D- and G-peaks in the Raman spectra of the
pyrolysis products of linear and cross-linked PVA [28,40]. The
variation of sp2- and sp3-hybridized carbon content has to do
with the features of the composition and pyrolysis of organic
polymer precursors. During the pyrolysis of linear PVA at
200–230 °C, its dehydration is accompanied by the formation of
C=C chains with conjugated double-bond systems. In the case
of cross-linked PVA, the number of free OH-groups is substan-
tially lower due to the formation of chemical bonds between
neighboring linear PVA chains. Hence in the course of the
dehydration of this polymer, the formation of C=C double
bonds is much less probable. As-prepared Li1.4Ni0.5Mn0.5O2+x powders were mixed with
15 wt % of linear or cross-linked PVA (Mr = (5–200) × 103,
Grade 16/1, Reachim). The cross-linked PVA was obtained by
thermal processing of linear PVA according to the procedure
described in [26]. The mixtures of Li1.4Ni0.5Mn0.5O2+x and
PVA were heated argon up to 350 °С with subsequent dwelling
for 15 min, then they were cooled down to room temperature at
a rate of 5 °C·min−1 in argon [25]. Experimental Due to the different char-
acter of the pyrolysis processes of linear and cross-linked PVA,
the amount of PVA was optimized in order to ensure the
comparable carbon content in the final composites (4 and
5.5 wt % for the linear and cross-linked PVA, respectively
[28]). Results and Discussion The cycling conditions are shown in Figure 7B. 1967 Beilstein J. Nanotechnol. 2016, 7, 1960–1970. detectable amount of lithium carbonate only in the cycling
products of composite electrodes (Figure 8). more homogeneous carbon distribution and a higher content of
sp2-hybridized carbon were obtained. The synergistic effect of
these features of ultrathin carbon coatings results in higher
values of the lithium diffusion coefficient and a lower charge-
transfer resistance of the whole nanocomposite and, hence, to
the enhancement of specific capacity of as-obtained cathode
material at moderate discharge rates. Taking into account the lack of other traces of
Li1.4Ni0.5Mn0.5O2+x degradation in the C-coated samples, the
formation of Li2CO3 in nanocomposites could be attributed to
an interaction of the electrolyte rather with the carbon coating
than with the bare Li1.4Ni0.5Mn0.5O2+x. The better electrochem-
ical performance of nanocomposite electrodes during cycling
indicates that the formation of Li2CO3 particles and other
related products has a negligible effect on the electrochemical
performance of the carbon-coated electrode materials. Conclusion Soc. 2001, 148, A680–A686. ,
;
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g,
;
,
;
,
;
,
Lee, J.-Y. J. Electrochem. Soc. 2001, 148, A680–A686. The galvanostatic curves and cyclic voltammograms (CVA)
were registered using 100N Metrohm Autolab equipment. The
cells were cycled in a potential range of 2–4.6 V at a current
density of 20–100 mA·g−1 at room temperature. The potential
scan rate was 50 µV·s−1. 14.Cho, J.; Kim, T.-J.; Kim, Y. J.; Park, B. Electrochem. Solid-State Lett. 14.Cho, J.; Kim, T.-J.; Kim, Y. J.; Park, B. Electrochem. Solid-State Lett. 2001, 4, A159–A161. doi:10.1149/1.1398556
15.Shi, S. J.; Tu, J. P.; Mai, Y. J.; Zhang, Y. Q.; Tang, Y. Y.; Wang, X. L. Electrochim. Acta 2012, 83, 105–112. doi:10.1016/j.electacta.2012.08.029 15.Shi, S. J.; Tu, J. P.; Mai, Y. J.; Zhang, Y. Q.; Tang, Y. Y.; Wang, X. L. Electrochim. Acta 2012, 83, 105–112. doi:10.1016/j.electacta.2012.08.029 16.Wu, F.; Wang, M.; Su, Y.; Chen, S.; Xu, B. J. Power Sources 2009,
191, 628–632. doi:10.1016/j.jpowsour.2009.02.063 Electrochemical impedance (EI) measurements were performed
on a “Solartron 1255B” using the cells assembled as described
above. The amplitude of the AC signal was 5 mV over a fre-
quency range from 1 MHz to 10 mHz. The Corrware 2 and
CorrView 2 software (Scribner Associates) was employed for
the electrochemical experiments and the obtained hodographs
were treated using “ZView-Impedance Software”. 17.Guo, R.; Shi, P.; Cheng, X.; Sun, L. Electrochim. Acta 2009, 54, 17.Guo, R.; Shi, P.; Cheng, X.; Sun, L. Electrochim. Acta 2009, 54, 5796–5803. doi:10.1016/j.electacta.2009.05.034 18.Lee, J.-T.; Wang, F.-M.; Cheng, C.-S.; Li,
Electrochim. Acta 2010, 55, 4002–4006. doi:10.1016/j.electacta.2010.02.043 19.Kim, H.-S.; Kong, M.; Kim, K.; Kim, I.-J.; Gu, H.-B. J. Power Sources
2007 171 917 921 d i 10 1016/j j
2007 06 028 19.Kim, H.-S.; Kong, M.; Kim, K.; Kim, I.-J.; Gu, H.-B. J. Power Sources
2007, 171, 917–921. doi:10.1016/j.jpowsour.2007.06.028 20.Nayak, P. K.; Grinblat, J.; Levi, M.; Aurbach, D. Electrochim. Acta 20.Nayak, P. K.; Grinblat, J.; Levi, M.; Aurbach, D. Electrochim. Acta 2014, 137, 546–556. doi:10.1016/j.electacta.2014.06.055 2014, 137, 546–556. doi:10.1016/j.electacta.2014.06.055 Acknowledgements 21.Liu, Y.; Lu, J.; Liu, S.; Chen, L.; Chen, X. Powder Technol. 2013, 239,
461–466. doi:10.1016/j.powtec.2013.02.039 21.Liu, Y.; Lu, J.; Liu, S.; Chen, L.; Chen, X. Powder Technol. 2013, 239,
461–466. doi:10.1016/j.powtec.2013.02.039 O.A. Shlyakhtin, K.A. Kurilenko and O.A. Brylev are grateful
to the Russian Foundation for Basic Research (grant 16-08-
00900) for the kind support of their studies. The activity of D.I. Petukhov is supported by the Russian Foundation for Basic
Research (individual grant 16-33-60195). Authors are also
thankful to the M.V. Lomonosov Moscow State University
Program of Development for the partial support of instrumental
studies. 22.Hashem, A. M.; Abdel Ghany, A. E.; Nikolowski, K.; Ehrenberg, H. 22.Hashem, A. M.; Abdel Ghany, A. E.; Nikolowski, K.; Ehrenberg, H
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doi:10.1016/j.jallcom.2008.05.102 doi:10.1016/j.jallcom.2008.05.102 24.Kim, H.-S.; Kim, K.; Moon, S.-I.; Kim, I.-J.; Gu, H.-B. 24.Kim, H.-S.; Kim, K.; Moon, S.-I.; Kim, I.-J.; Gu, H.-B. J. Solid State Electrochem. 2008, 12, 867–872. doi:10.1007/s10008-008-0552-0 J. Solid State Electrochem. 2008, 12, 867–872. doi:10.1007/s10008-008-0552-0 25.Cushing, B. L.; Goodenough, J. B. Solid State Sci. 2002, 4,
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both the kinetics of lithium insertion–extraction and the electro-
chemical properties of LNM/C composites. The presence of
carbon results in a decrease of the cell polarization and the
charge transfer resistance. This causes an increase of the specif-
ic capacity in comparison with pure Li1.4Ni0.5Mn0.5O2+x. Analyzing the CVA, EI and galvanostatic cycling data for
LNM/C composites obtained using linear and cross-linked
PVA, it can be seen that the presence of a carbon nanocoating
and/or a mesoporous carbon framework does not alter
the mechanism of lithium insertion–extraction into the
Li1.4Ni0.5Mn0.5O2 structure but improves its kinetic and elec-
trochemical properties. It is shown that even the nanosized layer
of carbon complicates the transformation of hexagonal
Li1.4Ni0.5Mn0.5O2+x into the polymorphic cubic structure
during cycling through the limitation of electrochemical degra-
dation of Li1.4Ni0.5Mn0.5O2+x at the electrode–electrolyte inter-
face. In the case of linear PVA, LNM/C nanocomposites with a The samples were characterized by XRD using a D/MAX 2500
diffractometer (Rigaku) in the reflection mode with Cu Kα radi-
ation and a curved-graphite [2] monochromator placed in the re-
flected beam (2θ range 10–90°, step 0.02°, acquisition time 3 s
per step).The analysis of diffraction patterns was performed by
using WinXPow software and PDF-2 powder diffraction data-
base. The thermal analysis of the linear and cross-linked PVA
was performed in argon by heating to 700 °C at a rate of
10 °C·min−1 (STA 209 PC Luxx thermal analyzer (Netzsch)). The morphology of the composites and their electron diffrac-
tion patterns were analyzed by transmission electron microsco-
py (Libra 200 MC, Carl Zeiss) at an accelerating voltage of
200 kV and a magnification of 30,000–300,000×. 1968 Beilstein J. Nanotechnol. 2016, 7, 1960–1970. 3. Lu, Y.-X.; Jiang, Y.; Yang, Z.; Han, J.-T.; Huang, Y.-H.; Ma, J. J. Alloys Compd. 2013, 559, 203–208. doi:10.1016/j.jallcom.2013.01.065 The X-ray photoelectron spectra were acquired with a Kratos
Ultra DLD spectrometer using a monochromatic Al Kα X-ray
source that possesses an analysis area of 300 μm × 700 μm. The
spectra were recorded in a constant analyzer pass energy mode
of 5 eV resulting in a resolution better than 0.3 eV. J. Alloys Compd. 2013, 559, 203–208. doi:10.1016/j.jallcom.2013.01.065 4. Gao, T.; Yang, G.; Yang, B.; Dai, Y. J. Alloys Compd. 2016, 680,
694–700. doi:10.1016/j.jallcom.2016.04.187 4. Gao, T.; Yang, G.; Yang, B.; Dai, Y. J. Alloys Compd. 2016, 680 4. Conclusion Gao, T.; Yang, G.; Yang, B.; Dai, Y. J. Alloys
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material (85 wt %), acetylene black (10 wt %, Timcal) and
polyvinylidene fluoride (5 wt %, solved in N-methyl-2-pyrroli-
done) was coated on a stainless steel net (0.05 mm thick). After
pressing, the electrodes were dried in vacuum (0.5–1.0 mbar) at
120 °C for 8 h. The active electrode, counter electrode (Li) and
reference electrode (Li) were placed in a hermetic Teflon cell
using a porous polypropylene separator. 1 M LiPF6 solution in
ethylene carbonate (EC)–dimethyl carbonate (DMC)–diethyl
carbonate (DEC) (1:1:1 by volume) was used as an electrolyte. According to Fischer titration data, the water content in the
electrolyte did not exceed 25 ppm. In the course of cell
assembly, the water content in the glove box did not exceed
0.5 ppm. All the potential values in this manuscript are referred
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2003, 35, 342–348. doi:10.1023/B:FICH.0000012188.90919.d9 30.Gilman, J. W.; VanderHart, D. L.; Kashiwagi, T. ACS Symp. Ser. 1994, 30.Gilman, J. W.; VanderHart, D. L.; Kashiwagi, T. ACS Symp. Ser. 1994,
599, 161–185. doi:10.1021/bk-1995-0599.ch011 30.Gilman, J. W.; VanderHart, D. L.; Kashiwagi, T. AC
599, 161–185. doi:10.1021/bk-1995-0599.ch011 599, 161–185. doi:10.1021/bk-1995-0599.ch011 31.Kurilenko, K. A.; Shlyakhtin, O. A.; Brylev, O. A.; Drozhzhin, O. A. Electrochim. Acta 2015, 152, 255–264. 31.Kurilenko, K. A.; Shlyakhtin, O. A.; Brylev, O. A.; Drozhzhin, O. A. Electrochim. Acta 2015, 152, 255–264. doi:10.1016/j.electacta.2014.11.045 doi:10.1016/j.electacta.2014.11.045 32.Tubbs, R. K. J. Polym. Sci., Part A: Gen. Pap. 1965, 3, 4181–4189. doi:10.1002/pol.1965.100031213 32.Tubbs, R. K. J. Polym. Sci., Part A: Gen. Pap. 1965, 3, 4181–4189. License and Terms 1970
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O poder das ideias
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Conversas & Controvérsias
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Recebido em: 10 ago. 2020.
Aprovado em: 20 nov. 2020.
Publicado em: 08 jun. 2021. Felipe Rocha de
Carvalho1 Resumo: O objetivo principal deste artigo é discutir as principais contribuições
da Teoria Construtivista enquanto uma abordagem teórica alternativa às esco
las clássicas de Relações Internacionais. Primeiramente, é realizada uma breve
contextualização histórica e acadêmica do surgimento da teoria, ocorrido em um
contexto de mudança da ordem bipolar no Sistema Internacional. Em seguida,
é feita uma revisão teórica sobre as definições construtivistas de alguns dos
principais conceitos das Relações Internacionais, tais como anarquia, identidade
e interesses. Finalmente, argumenta-se que o Construtivismo consiste em uma
opção viável, não apenas para a análise de acontecimentos no final da Guerra
Fria, mas também acerca de fenômenos contemporâneos, como a crise do
modelo clássico de democracia. Carvalho
orcid.org/0000-0002-0679-6092
feliperochacarvalho@gmail.com Palavras-chave: Construtivismo. Relações Internacionais. Anarquia. Identidade. Abstract: The main objective of this article is to discuss the main contributions
of the Constructivist Theory as an alternative theoretical approach to classical
schools of thought in International Relations. First, a brief historical and academic
contextualization of the emergence of the theory is carried out, which was given
in a context of change in the bipolar order of the International System. Then, a
theoretical review is made regarding the constructivist definitions of some of the
main concepts of International Relations, such as anarchy, identity and interests. Finally, it is argued that Constructivism is a viable option not only for analyzing
events at the end of the Cold War, but also about contemporary phenomena
such as the crisis of the classic model of democracy. Artigo está licenciado sob forma de uma licença
Creative Commons Atribuição 4.0 Internacional. Keywords: Constructivism. International Relations. Anarchy. Identity. lhttp://dx.doi.org/10.15448/2178-5694.2020.2.39115 lhttp://dx.doi.org/10.15448/2178-5694.2020.2.39115 CONVERSAS &
CONTROVÉRSIAS Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020
e-ISSN: 2178-5694 O poder das ideias: a teoria construtivista como um
paradigma alternativo nas Relações Internacionais The power of ideas: the constructivist theory as an alternative paradigm in International
Relations 1 Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brasil. Introdução Durante grande parte do século XX, era evidente a prevalência das
teorias clássicas nas produções acadêmicas de Relações Internacionais,
em especial as correntes das escolas Realista e Liberal. Um dos pontos
centrais de tais teorias é compreender de que maneira a ação dos Estados
é influenciada pela estrutura na qual este se encontra. No entanto, Wendt
(1995) afirma que as teorias tradicionais falham ao reduzir os efeitos das
estruturas simplesmente ao comportamento estatal, ignorando o papel
na formação da identidade e dos interesses de um país. Dessa forma, os teóricos construtivistas “[...] possuem um interesse
normativo em promover mudança social, mas o buscam por meio da
tentativa de explicar como estruturas sociais aparentemente naturais, 1 Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brasil. 2/10
Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020 | e-39115 2/10 Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020 | e-39115 1992). Sob esse ponto de vista, as dinâmicas da
anarquia e do equilíbrio de poder poderiam ser
melhor analisadas se for dado um enfoque aos
pensamentos e ideias. como autoajuda ou a Guerra Fria, são efeitos de
práticas [...]” (Wendt 1995, 74, tradução nossa). Assim, as estruturas sociais não estariam relacio
nadas às capacidades materiais, como defendido
por outras teorias, mas, sim, às práticas. É dada
uma maior ênfase ao papel das ideias, visto que
elementos como poder e interesse não estão
dissociados do conhecimento que permite a
aplicação desse papel (Barros 2006). Cabe destacar que os teóricos construtivis
tas de Relações Internacionais também foram
influenciados por autores de outras áreas do
conhecimento, dentre as quais está a Sociologia. Anthony Giddens, em seu livro The Constitution
of Society: Outline of the Theory of Structuration
(1984), estabeleceu o conceito de estruturação
como uma ferramenta de análise das relações
entre estruturas e agentes. Segundo o sociólogo,
as estruturas não são as únicas determinantes das
ações dos agentes. Ao invés disso, tais relações
estão permeadas pela compreensão intersub
jetiva e pelos significados atribuídos às próprias
relações (Giddens 1984). Sendo assim, não é
negado que exista uma influência da estrutura
sobre os agentes, mas é argumentado que essas
mesmas estruturas podem ser alteradas por meio
de novas ações por partes dos agentes, as quais
são geradas devido à reflexão sobre elas. Introdução O presente artigo busca analisar os principais
conceitos da Teoria Construtivista de Relações
Internacionais, que são comuns às obras de al
guns de seus principais autores: Alexander Wendt,
Emanuel Adler e Nicholas Onuf. Para tanto, é rea
lizada uma contextualização no que diz respeito
ao surgimento enquanto teoria social, bem como
à posição desta em relação às demais teorias de
Relações Internacionais. Em seguida, é realizada
uma revisão teórica, e posteriormente a discussão
de alguns elementos centrais em contraposição
à visão neorrealista de Relações Internacionais,
nomeadamente as noções de anarquia, identidade,
interesses e instituições. Por fim, são elencadas
algumas considerações sobre as possíveis apli
cações do Construtivismo nos dias de hoje. Nesse contexto, Adler (1999) salienta que os
agentes descritos por Giddens não se tratam de
passivos estruturais, mas constroem suas práti
cas e estruturas. Além disso, ações sob regras
instituídas também estão condicionadas aos
interesses, que, por sua vez, estão relacionados
às suas identidades, direitos e obrigações. Um
aspecto acrescentado ao pensamento de Gid
dens pelos construtivistas foi a estruturação das
próprias ideias. “Em primeiro lugar, as ideias [...]
são o meio e o propulsor da ação social; definem
os limites do que é cognitivamente possível ou
impossível para os indivíduos” (Adler 1999, 210). Ainda, o conhecimento serve como base para
as práticas, resultando das mesmas interações
entre os indivíduos e pautado em ideias e inter
pretações das circunstâncias. O surgimento do Construtivismo no
contexto do fim da ordem bipolar Durante a década de 1980 — em um cenário
de desintegração da União Soviética e, por con
seguinte, do modelo de Sistema Internacional
vigente na Guerra Fria —, teve início um debate
teórico acerca da aplicabilidade das teorias ne
orrealistas de Relações Internacionais, tendo em
vista a incerteza acerca das futuras possibilidades
de distribuição de poder (Jackson e Sorensen
2006). De acordo com a visão neorrealista, outros
Estados perceberiam a necessidade de esta
belecer um equilíbrio de poder com a grande
potência, os Estados Unidos, uma vez que esse
país seria o único meio para garantir a segurança. A inocorrência desse fato serviu de argumento
para que uma nova corrente de teóricos — os
construtivistas — afirmassem que as teorias clás
sicas possuíam um foco exacerbado no aspecto
materialista das relações internacionais (Wendt O Construtivismo é definido por Adler (1999,
205) como “[...] a perspectiva segundo a qual o
modo pelo qual o mundo material forma a, e é
formado pela, ação e interação humana depen
de de interpretações normativas e epistêmicas
dinâmicas do mundo material”. Sendo assim, Felipe Rocha de Carvalho
O poder das ideias: a teoria construtivista como um paradigma alternativo nas Relações Internacionais Felipe Rocha de Carvalho 3/10 de práticas de discurso, estão centradas no ar
gumento de que as estruturas de discurso são as
únicas constituintes da subjetividade individual. tendo em vista o caráter essencialmente social
das relações internacionais, é possível afirmar
que o acordo humano é necessário para que os
fatos sociais em questão passem a existir. Ainda,
a ênfase dos estudos não é colocada, primeira
mente, em metodologia, mas, sim, em aspectos
ontológico e epistemológico. Portanto, apesar do respeito dos pós-estrutu
ralistas e pós-modernos ao paradoxo agente/
estrutura como uma oposição na qual nunca é
possível a escolha de uma proposição frente a
outra, eles são capazes apenas de descrever
histórias de práticas discursivas; no entanto,
a história e entendida em sua pluralidade in
trínseca, como um vasto texto de incontáveis
textos (Adler 1999, 219-220). [...] faz-se crucial esclarecer [...] que o centro
do debate sobre o construtivismo não é sobre
ciência versus interpretação literária ou “rela
tos”, mas sobre a própria natureza da ciência
social e, portanto, da disciplina de relações
internacionais. O surgimento do Construtivismo no
contexto do fim da ordem bipolar Em outras palavras, a questão
contrapõe uma concepção naturalista de ciên
cia, quase inteiramente baseada em filosofias
da ciência concorrentes e teorias que a física há
muito abandonou, a uma concepção de ciência
social que é – social (Adler 1999, 202-203). Além disso, a base epistemológica do Construti
vismo não possui como objetivos centrais a eman
cipação ou a descoberta de estruturas de poder
que constrangem os Estados não centrais, mas,
sim, prover análises mais aprimoradas da sociedade. Enquanto os pós-modernos e pós-estruturalistas não
concentram suas pesquisas em casos particulares,
os construtivistas buscam analisar, detalhadamente,
de que forma as regras formam as identidades e
interesses dos agentes internacionais em casos
específicos (Adler 1999; Wendt 1992). É válido ressaltar que o Construtivismo não
consiste em uma teoria da política em sentido
estrito, se comparado ao Realismo ou ao Libe
ralismo. Em oposição, é uma teoria de caráter
social que serve de base para que sejam reali
zadas análises de política internacional, por meio
dos instrumentos e interpretações acerca de
fenômenos, tanto internos quanto externos aos
Estados. Tal fator permite que seja dado destaque
a alguns pontos, por vezes negligenciados pelas
demais teorias, o que repercute diretamente no
desenvolvimento de pesquisas empíricas. Estática ou mutável? A anarquia sob a
visão construtivista Segundo o pensamento neorrealista, a estru
tura política pode ser definida em três pontos:
princípios ordenadores, princípios de diferen
ciação e distribuição de capacidades. A anar
quia está incluída na primeira categoria e seria
a responsável pela geração de uma “balança
de ameaças”, que moldaria a ação dos Esta
dos (Waltz 1979). Embora as relações de poder
sejam uma premissa de análise primariamente
realista, é possível afirmar que o Neorrealismo
de Waltz se diferencia por meio da afirmação
de que os efeitos dessas relações são formados
quase exclusivamente por força material bruta
(Wendt 1999). Nesse contexto, o oposto dessa
conceituação seria o argumento de que o poder
é constituído, essencialmente, por ideias. Wendt (1992) havia estabelecido uma matriz,
opondo materialismo e individualismo, na qual o
Construtivismo estava situado na mesma catego
ria do Pós-Modernismo e do Pós-Estruturalismo. Para Adler, entretanto, a Teoria Construtivista
está no meio dessa matriz, visto que também
pretende entender como o mundo material, em
interação com o subjetivo e o intersubjetivo, in
fluencia na construção social da realidade. Assim,
ademais de explicar como as estruturas moldam
a identidade e os interesses dos agentes, a teoria
objetiva compreender o contexto social no qual
os agentes desenvolvem essas estruturas. Assim, para os neorrealistas, a anarquia do Siste
ma Internacional é uma característica permanente,
tendo em vista que as unidades desse sistema
— os Estados — possuem igual soberania. Sendo
assim, os Estados constituem a autoridade máxima Outro aspecto a ser abordado é a crítica reali
zada por Adler (1999) em relação às abordagens
pós-estruturalistas e pós-modernas. Tais pers
pectivas, ainda que considerem a importância da
ação do indivíduo enquanto produtor e reprodutor 4/10 Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020 | e-39115 Para Wendt (1999), a anarquia em si não possui
um sentido fixo, mas adquire um significado en
quanto função na estrutura em que for colocada. Adler (1999) assevera que a teoria adota uma
espécie de causalidade social, que enxerga as
motivações como causas, o que implica fazer o
que é requerido em determinadas circunstâncias. do Sistema Internacional, não existindo nenhuma
instância superior a estes. Para Waltz (1979, 113), A política internacional é o reino do poder,
da luta e da acomodação. O reino interna
cional é preeminentemente político. Estática ou mutável? A anarquia sob a
visão construtivista O reino
nacional é descrito como sendo hierárquico,
vertical, centralizado, heterogêneo, direcio
nado e planejado; o reino internacional, como
sendo anárquico, horizontal, descentralizado,
homogêneo, não-direcionado e mutualmente
adaptável. Quanto mais centralizada a ordem,
mais perto do topo se encontra o local de
decisão. Internacionalmente, as decisões são
feitas no nível mais baixo, visto que não existe
outro. [...] Ajustes são feitos internacionalmente,
mas são feitos sem um ajustador formal ou
autoritário” (tradução nossa). q
q
Dessa forma, o Construtivismo afirma que
existe a possibilidade de que, mesmo em um
contexto de anarquia, a natureza das interações
entre os Estados pode variar consideravelmente. Wendt (1999) utiliza o exemplo das relações entre
Estados Unidos e União Soviética e, em seguida,
entre Estados Unidos e Rússia, afirmando ser
inegável a distinção dentre elas, mesmo que
ambas ocorressem em um Sistema Internacional
de ordem anárquica. Nesse contexto, o autor
estabelece três cenários, nos quais o mesmo
sistema anárquico pode ser um ambiente para
relações de cunho distinto: a cultura hobbesiana,
a cultura lockeana e a cultura kantiana. Cada um
destes panoramas possui uma representação
distinta dos conceitos de “ego” e “outro” no que
diz respeito ao uso da violência. Wendt (1999) interpreta as afirmações de Waltz
como argumentos para sustentar a ideia de que
a estrutura do Sistema Internacional afeta dire
tamente os Estados, ainda que não desejem ou
não estejam conscientes dessa influência. Uma
vez que as intenções particulares de cada Esta
do não são centrais na análise neorrealista, são
elencados dois pressupostos principais acerca
do comportamento estatal. O primeiro deles é o
de que a segurança consiste na principal meta
dos governos, visto que é a única maneira de
assegurar a sobrevivência estatal. O segundo
está relacionado à característica primariamente
egoísta dos Estados, a qual acarreta na imprevi
sibilidade de suas ações (Waltz 1979). Wendt (1999) afirma que a anarquia hobbesiana,
ainda que não possua um elo necessário com o
Neorrealismo, é um caso importante de estudo
para o Construtivismo. Isso se deve ao fato de
que o alto nível de violência — característica
primordial desse cenário — dificulta a formação
de ideias compartilhadas; “[...] Se elas se foram,
ainda é difícil observar por quê os Estados teriam
participação nelas, o que é indicado pela propo
sição construtivista de que ideias internalizadas
constituem identidades e interesses” (Wendt 1999,
259, tradução nossa). Estática ou mutável? A anarquia sob a
visão construtivista Assim, a premissa da qual se parte é
a de que não haverá uma tentativa de conquista
ou dominação, o que é aliado ao reconhecimento
do direito à propriedade. O fato de a soberania estatal ser reconhecida
por parte dos outros Estados, contribui para o pen
samento de que a soberania não se trata de uma
propriedade de Estados isolados, mas, sim, de uma
instituição compartilhada por vários deles. Na visão
construtivista, há uma expectativa mútua de que os
Estados não realizarão tentativas de suprimir a liber
dade dos demais (Adler 1999). Por meio do sistema
westfaliano de Estados, essa expectativa adquiriu
a forma de direito internacional, fazendo parte de
uma estrutura maior de política internacional. O pensamento construtivista compreende
identidade como os entendimentos e expec
tativas de um agente sobre si próprio, que são
adquiridos por meio da participação nos signi
ficados coletivos mencionados anteriormente. Wendt (1992) argumenta que as identidades são,
por definição, elementos relacionais, estando
sempre presentes em um cenário específico e
socialmente construído. Dessa maneira, trata-se
de um significado intrinsecamente social do ator
e que é baseado nas teorias que os atores pos
suem de si mesmos e dos demais, o que forja a
estrutura do mundo social (Barnett 2008). O último tipo de anarquia discutido por Wendt
(1999) é a kantiana. Definida como uma visão de
caráter idealista, a cultura kantiana pressupõe
um cenário no qual as práticas políticas estejam
embasadas na não-violência e na cooperação. Por
meio disso, não haveria a possibilidade de retor
no para os outros cenários. Cabe ressaltar que,
apesar de ser denominado kantiano — devido ao
trabalho de Kant em A Paz Perpétua —, esse tipo
de cultura não requer, necessariamente, um sis
tema formado apenas por Estados republicanos. Em oposição aos conceitos de hostilidade e
rivalidade anteriormente apresentados, a cultura
kantiana introduz uma estrutura de amizade nas
relações entre os agentes. Wendt (1999) lembra
que tal conceito, se comparado aos demais,
possui menos teorização no campo das Relações
Internacionais. Isso se deve, em grande medida,
ao fato de que relações hostis representam pro
blemas consideravelmente maiores à política
internacional. Por conta disso, o posicionamento
da noção de amizade em um ambiente anárquico
é visto, por teóricos realistas, como utópico. Estática ou mutável? A anarquia sob a
visão construtivista Entretanto, embora concor
de com a ideia de que a cultura hobbesiana seja
permeada por um ambiente de “guerra de todos
contra todos”, o autor afirma que esse estado não
provém da anarquia ou da natureza humana. A visão de Wendt (1992) sobre a anarquia, por
outro lado, afirma que a definição neorrealista
não é capaz de prever as dinâmicas da própria
anarquia, tendo em vista o fato de não abarcar
fatores intersubjetivos que também influenciam
a segurança dos Estados, tais como a estrutura
de identidade e interesses no sistema. Logo: Analisar a construção social da política in
ternacional é analisar como os processos de
interação produzem e reproduzem as estrutu
ras sociais – cooperativas ou conflitivas – que
moldam as identidades e interesses dos atores
e a significância de seus contextos materiais
(Wendt 1995, 81, tradução nossa). No que diz respeito à cultura lockeana, cabe
destacar que o conceito de hostilidade é subs
tituído pelo de rivalidade. De modo semelhante
ao que ocorre em relação ao inimigo, a represen
tação do rival inclui a diferenciação entre “ego”
e “outro” em termos de utilização da violência. Nesse sentido, é válido destacar o princípio
construtivista de que as pessoas possuem deter
minadas ações em relação aos objetos baseadas
no significado que tais objetos têm para elas. Felipe Rocha de Carvalho
O poder das ideias: a teoria construtivista como um paradigma alternativo nas Relações Internacionais Felipe Rocha de Carvalho 5/10 nuance não é considerada pelas análises das
teorias de Relações Internacionais, o que faz com
que outros aspectos assumam papeis centrais
na explicação das dinâmicas entre os Estados. O
Neorrealismo, por exemplo, ao afirmar que a au
toajuda é a única forma de configuração possível
e que os atores não participantes desse modelo
podem ser excluídos do sistema, não considera
os processos de aprendizagem complexa e as
definições de identidade e interesses pelas quais
os Estados podem passar. Os teóricos neoliberais,
por sua vez, embora admitam que possa existir
um processo de aprendizagem por parte dos
atores, não desenvolvem explicações acerca de
como acontecem as transformações das estrutu
ras que, em suma, são ocasionadas por mudanças
nas identidades e interesses (Wendt 1992). Contudo, o rival consiste em uma figura com
um grau menor de ameaça para o agente, haja
vista a expectativa de reconhecimento mútuo da
soberania. Estática ou mutável? A anarquia sob a
visão construtivista Onuf (1998) ressalta que, embora agentes não
necessitem de certo nível de autoconsciência para
que lhes seja atribuída uma identidade, na maior
parte dos casos, possuem consciência de suas
identidades, tanto singular quanto coletiva. Quando
há um grande número de indivíduos operando de
forma coletiva, há a formação de um grupo. Caso
também existam agentes operando por eles, bem
como uma medida considerável de identidade,
esse grupo de pessoas constitui um país. Por vários séculos, os agentes tiveram o inte
resse consistente em discutir países como se
estes fossem independentes uns dos outros
e de qualquer outra construção social. Isto se
torna mais claro ao se definir soberania como
independência absoluta e ao descrever países
como Estados soberanos. Como construtivis
tas, entretanto, devemos sempre manter em As dinâmicas da identidade estatal O conceito de identidade também é essencial
para a compreensão da percepção construtivis
ta acerca das interações estatais. Em geral, tal 6/10
Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020 | e-39115 Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020 | e-39115 6/10
Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020 | e-39115 mente que independência total é uma ficção
útil e que soberania é uma questão de medida
(Onuf 1998, s.p., tradução nossa). essa base é o próprio corpo; no caso dos Estados,
um conjunto de corpos e território. Além disso, a principal distinção da identidade
pessoal de agentes conscientes é a noção do
“ego” enquanto um espaço separado de pensa
mento e ação. Wendt (1999) assevera que, mes
mo que sejam entidades diferentes em termos
biológicos, os indivíduos não são agentes caso
não possuam consciência, memória e senso
de “ego”. No caso dos Estados, tal argumento é
ainda mais incisivo, uma vez que não possuem
sequer corpos se os membros não possuem uma
forma de identidade coletiva. “O Estado é um
‘ego grupal’ capaz de cognição ao nível grupal. Estas ideias de ‘ego’ possuem uma qualidade
‘autogenética’, e assim identidades pessoais e
corporativas são, por constituição, exógenas ao
‘outro’” (Wendt 1999, 225, tradução nossa). Nesse sentido, segundo Berger (1966), con
juntos de identidades estão presentes em todas
as sociedades e fazem parte do conhecimento
objetivo de seus integrantes. Assim, a medida que
tais indivíduos se socializam, essas identidades
são internalizadas. Para o autor, as identidades
constituem partes essenciais das consciências
individuais. O processo de socialização gera uma
simetria entre realidade objetiva e realidade
subjetiva, bem como entre identidade objetiva e
identidade subjetiva. Ao conhecer as característi
cas, o agente pode, então, escolher as alternativas
de ação, de forma espontânea. Para Wendt (1992), os argumentos de Berger
(1966) são de essencial importância para com
preender a deficiência das teorias de cunho
racionalista em tratar o Sistema Internacional
anárquico como uma entidade separada dos
processos de formação e internalização das
identidades estatais. A identidade pessoal ou corporativa pode dar
origem a outras formas de identidade. A identi
dade de tipo, por exemplo, faz referência a um
grupo de indivíduos — ou outros agentes — que
compartilham uma ou diversas características,
tais como padrões de comportamentos, valores
e semelhanças históricas (Wendt 1999). As dinâmicas da identidade estatal No cenário
internacional atual, por exemplo, o fato de dois
Estados possuírem sistemas parlamentaristas não
pressupõe que essa seja uma característica signi
ficativa em termos de identidade compartilhada. O último tipo de identidade trazido por Wendt
(1999) é a identidade coletiva. Em suma, essa
identidade engloba a relação entre “ego” e “ou
tro” em seu último estágio: a identificação. Esse
estágio pode ser definido como um processo no
qual as distinções entre os agentes se tornam
mínimas e são, eventualmente, ultrapassadas. Assim, “ego” passa a ser caracterizado como
“outro”. Em geral, tal identificação se restringe a
assuntos específicos, não deixando de se tratar
de um cenário no qual os limites dos agentes se
expandem para incluir os demais. No que concerne à identidade de função, a
relação com o aspecto cultural e com os de
mais agentes é mais aprofundada. Ao passo
que a identidade de tipo prevê características
intrínsecas aos agentes, a identidade de função
existe apenas quando há interação com o “outro”. Sendo assim, um Estado não pode assumir uma
identidade de função por si próprio. É neces
sário lembrar, porém, que estruturas sociais já
existentes podem facilitar a institucionalização
de funções das quais a identidade de função
depende para existir (Wendt 1999). Nesse sentido, a identidade coletiva vai além
das identidades de tipo e função. Enquanto a
identidade de função requer o desempenho de
funções distintas por parte dos agentes, a identi
dade coletiva possui o objetivo de mesclá-los em
uma identidade única. A identidade coletiva, de
modo semelhante à identidade de tipo, envolve
características compartilhadas, mas nem todas
as identidades podem ser caracterizadas como
coletivas pelo fato de não possuírem o elemento
da identificação (Wendt 1999). Ademais, Wendt (1999) questiona o fato de o
conceito de função não ser utilizado por parte
das teorias estruturais de Relações Internacio
nais, haja vista a consideração de uma estrutura
social nas relações entre agentes. Isso pode ser
atribuído à difícil aplicação da ideia de identidade
de função trazida pela baixa institucionalização
do Sistema Internacional. Entretanto, pode haver
uma tendência da Literatura atual em desconsi
derar as identidades de função de instituições,
tendo como exemplo a igualdade de soberania
entre os Estados. As dinâmicas da identidade estatal Por conta
disso, um mesmo agente pode possuir várias
identidades de tipo. Todavia, cabe ressaltar que,
para que seja considerada enquanto identidade
de tipo, uma característica compartilhada precisa
ter um significado ou conteúdo social. Em um
contexto histórico, tais características podem
variar, também ocasionando variações quanto ao
comportamento dos “outros” em relação a elas. Ao negar ou menosprezar a autoria coletiva de
suas identidades e interesses por parte dos
Estados, em outras palavras, a aliança realis
ta-racionalista nega ou menospreza o fato de
que políticas de poder competitivas ajudam a
criar o mesmo “problema de ordem” que de
veria ser resolvido por eles — que o Realismo
é uma profecia autorrealizável. Longe de ser
dado de forma exógena, o conhecimento inter
subjetivo que constitui identidades e interesses
competitivos é construído todos os dias por
processos de “formação da vontade social”. É o que os Estados têm feito de si mesmos
(Wendt 1992, 410, tradução nossa). Em um ambiente de interação entre Estados,
as identidades de tipo podem ser facilmente
atribuídas a de regime ou formas estatais, tais
como Estados capitalistas, socialistas e monár
quicos. Wendt (1999, 226, tradução nossa) afirma
que, “ao contrário de identidades de função e
coletivas, [...] as características que baseiam as
identidades de tipo são em sua base intrínsecas
aos agentes”. Desse modo, mesmo que Estados
tenham assumido determinadas características
somente após uma interação com outro Estado,
eles não dependem do outro para garantir sua
existência. Assim, também é válido ressaltar que No que diz respeito às formas de identidade
existentes, levando em consideração o caráter
variante das relações internas e externas dos
agentes, Wendt (1999) as divide em quatro cate
gorias: pessoal ou corporativa, de tipo, de função
e coletiva. O primeiro modelo, a identidade pes
soal, é formado por estruturas auto-organizadas
que fazem dos agentes unidades distintas. Sendo
assim, os Estados, enquanto agentes com pro
priedades específicas, estão diretamente rela
cionados a este tipo de identidade, que sempre
possui uma base material. No caso dos indivíduos, Felipe Rocha de Carvalho
O poder das ideias: a teoria construtivista como um paradigma alternativo nas Relações Internacionais
7/10 Felipe Rocha de Carvalho
O poder das ideias: a teoria construtivista como um paradigma alternativo nas Relações Internacionais um agente e o “outro” também é determinante
na definição da identidade de função. nem todas as características compartilhadas se
transformam em identidades de tipo. As dinâmicas da identidade estatal Ao contrário do que afirmam os
neorrealistas, teóricos construtivistas defendem
a noção de que a soberania não é uma carac
terística estatal inerente, mas, sim, uma identi
dade de função por causa da necessidade de
reconhecimento por parte dos demais Estados. Os interesses dos agentes No tocante ao conceito de interesses, é neces
sário destacar a relação direta com o conceito de
identidade. Embora os quatro tipos de identidade
expostos no tópico anterior não se detenham
somente aos interesses dos agentes, não é pos
sível dissociar o caráter de um agente de seus
objetivos. Sendo assim, os interesses constituem
as motivações, por meio das quais pode ser expli
cado o comportamento do agente (Gecas 1982). Além disso, as identidades constituem as bases
dos interesses, visto que são estabelecidos no
processo de definição das situações (Wendt 1992). Ainda sobre identidade de função, é comum
o argumento de que esse cenário pressupõe
a existência de integração e cooperação en
tre Estados. Essa afirmação possui um caráter
essencialmente materialista, que é defendido
pelas teorias estruturais de cunho racionalista. No entanto, o Construtivismo afirma que as ideias
compartilhadas podem ter caráter tanto coopera
tivo quanto conflitivo, o que remete aos possíveis
posicionamentos do “outro” enquanto inimigo,
rival ou amigo. O grau de interdependência entre Onuf (1998) utiliza o argumento de que os
agentes tendem a utilizar os meios disponíveis
para atingir suas metas. Esses meios, por sua vez,
incluem elementos materiais disponíveis no mun
do, embora não estejam limitados a eles. Sendo
assim, levando em consideração o caráter social
no qual os agentes estão inseridos, tais elementos 8/10 Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020 | e-39115 Pelo fato de a constante ameaça de uma in
terpretação errônea de identidade levar à perda
dela, um dos principais dilemas enfrentados pelos
agentes é o de compatibilizar seus interesses
objetivos e subjetivos. Ainda, não há uma distin
ção tão rígida entre os “desejos” e crenças de um
agente, sendo que aqueles são uma categoria
destes. Desse modo, podem ser transformados em recursos a serem
utilizados pelos próprios. Se um agente é capaz
de justificar seus motivos para utilizar os recursos
disponíveis, podem ser observados os interesses
de tal. O autor também aponta que um agente
não necessita saber quais são seus interesses para
agir sobre eles, visto que esse conhecimento pode
ser adquirido por meio da observação de outros
em relação a seus próprios interesses. Os interesses dos agentes Algumas crenças constituem quem somos
(identidades e suas necessidades adjacentes),
outras os objetivos que acreditamos que nos
ajudarão a atender a tais necessidades (inte
resses subjetivos ou desejos), e ainda outras
crenças que relacionam tais objetivos ao am
biente externo (a interpretação racionalista de
“Crença”). Nenhuma destas determina qualquer
uma das outras diretamente, mesmo que elas
não estejam tampouco dissociadas (Wendt
1999, 233, tradução nossa). Em referência aos tipos de interesses que os
agentes possuem, Wendt (1999) elenca duas
possibilidades: interesses objetivos e interesses
subjetivos. Os interesses objetivos consistem nas
necessidades que precisam ser atendidos, no intui
to de reproduzir determinada identidade. Esse fator
se aplica aos quatro tipos de identidades — pessoal
ou corporativa, de tipo, de função e coletiva. Ademais, a partir do momento em que são in
ternalizadas as identidades, os agentes adquirem
também duas disposições: compreender suas
necessidades e agir em relação a essa com
preensão. Em outras palavras, a internalização
das identidades requer, necessariamente, que
o agente atue com o objetivo de garantir sua
reprodução. No entanto, Wendt (1999) lembra
que o fato de um agente buscar compreender
identidade e necessidades não é garantia de
que serão interpretadas de forma correta. Assim,
tanto os indivíduos quanto os Estados podem
se equivocar e, por conta disso, agir de maneira
contrária ao que seria ideal. Em relação às escolhas realizadas pelos agen
tes, Onuf (1998) assevera que regras facilitam o
processo de escolha. Nesse contexto, parte do
agente a decisão de seguir determinada regra
ou de desrespeitá-la. Na maior parte dos casos,
as possíveis consequências de não seguir uma
regra são facilmente calculadas. Contudo, devido
ao fato de consequências imprevistas também
serem possíveis, as mesmas regras proporcionam
a possibilidade de realizar escolhas racionais,
fornecendo uma segurança de que a decisão
tomada seja a melhor possível. Ademais, o autor
também afirma que os agentes podem agir de
acordo com o estabelecido por uma regra de
maneira inconsciente. Entretanto, em geral, qual
quer agente possui a capacidade — caso tenha
informação suficiente — de formular o conteúdo
de uma regra sob o mesmo formato. Os interesses subjetivos, por sua vez, estão
ligados às crenças dos agentes acerca de como
devem agir para suprir as necessidades da iden
tidade, o que está intimamente relacionado às
motivações para o comportamento dos agentes. Em termos filosóficos, tais interesses podem ser
conceituados como os “desejos” dos agentes. As instituições na Teoria Construtivista
de Relações Internacionais tadas” (Onuf 1998, s.p., tradução nossa). A balança
de poder pode ser citada como um modelo de
instituição que possui tais características. As
esferas de influência, por sua vez, são exemplos
de instituições formadas por regras informais. No que tange ao conceito de instituições, é
preciso vislumbrá-los como uma estrutura de
identidades e interesses. Como ressalta Wendt
(1992), apesar de possuírem regras e normas, tais
conjuntos só possuem força motivacional devido
à socialização dos agentes e à participação no
conhecimento coletivo. Sendo assim, as institui
ções não são exógenas às ideias acerca de como
o mundo opera; não obstante, exercem coerção
sobre os indivíduos sob a forma de fatores sociais,
ainda que os atores o saibam coletivamente. Segundo Adler (1999), as instituições também
auxiliam na formação da realidade social. Cabe salientar que as instituições também pos
suem um caráter de conhecimento compartilhado
entre os agentes, forma sob a qual possuem uma
existência que transcende os indivíduos que as
assimilam (Wendt 1992). Assim, os fatores sociais
trazidos pelas instituições podem assumir um
viés coercivo, não deixando de ser uma função
conhecida pelos de forma coletiva. Além disso, o
fato de algumas instituições serem constituídas
por regras menos formais pode dificultar sua iden
tificação. Devido a isso, os agentes conseguem
observar mais facilmente instituições como os
tratados, os quais são amplamente conhecidos
como derivados do direito internacional — que,
em si, também é uma instituição (Onuf 1998). As identidades e cognições coletivas não exis
tem separadamente, são “mutualmente constitu
tivas”. Sob essa visão, a institucionalização é um
processo de internalização de novas identidades
e interesses, não algo que ocorre fora e que afeta
apenas o comportamento; a socialização é um
processo cognitivo, e não apenas comportamen
tal. Concebidas dessa maneira, as instituições
podem ser cooperativas ou conflitivas, um ponto
por vezes perdido no estudo sobre regimes in
ternacionais, que tende a igualar instituições e
cooperação (Wendt 1992, 399, tradução nossa). Por fim, é válido ressaltar que os teóricos cons
trutivistas também enxergam o próprio contex
to, no qual uma instituição assume o papel de
agente como uma instituição. A sociedade, por
exemplo, se trata de uma instituição complexa,
na qual estão inseridas uma série de outras. Do
mesmo modo, Estados são sociedades, nas quais
o desenvolvimento das instituições é conside
ravelmente elevado no intuito de estabelecer
vínculos com outros Estados. Os interesses dos agentes Porém, tal afirmação não implica em uma prio
rização dessas preferências em detrimento da
estratégia. Isso se alia ao fato de que o compor
tamento de um indivíduo ou de um Estado não
está condicionado somente às suas pretensões,
mas também ao grau de possibilidade de sucesso
da ação em questão (Wendt 1999). Logo, pode-se perceber que existe uma li
gação direta entre definição de interesses, for
mulação de estratégias para o suprimento das
necessidades do agente e manutenção da iden
tidade. Além disso, fatores, como a informação
disponível, influenciam na compreensão dos
agentes no tocante às suas ações, uma vez que
tais atos podem também contribuir para o alcance
dos objetivos de outros. Felipe Rocha de Carvalho
O poder das ideias: a teoria construtivista como um paradigma alternativo nas Relações Internacionais
9/10 Felipe Rocha de Carvalho
O poder das ideias: a teoria construtivista como um paradigma alternativo nas Relações Internacionais
9 Felipe Rocha de Carvalho
O poder das ideias: a teoria construtivista como um paradigma alternativo nas Relações Internacionais Felipe Rocha de Carvalho 9/10 As instituições na Teoria Construtivista
de Relações Internacionais De acordo com Onuf
(1998), a sociedade internacional é um ambiente
no qual os Estados desempenham o papel de
agentes simplesmente pelo fato de possuírem
relações entre si. Além dos Estados, existem
também inúmeras outras instituições, com maior
ou menor grau de complexidade. Tradicionalmente, a junção de regras e prá
ticas — nesse caso, também de identidades e
interesses — é chamada de regime. Uma maneira
pela qual os regimes internacionais podem ser
diferenciados é pelo tamanho. Também podem
ser identificadas regras como funcionam ou são
aplicadas de formas distintas. Ainda, a medida
que regimes distintos possuem regras que ofe
reçam suportes para outras regras, também é um
importante meio de diferenciação (Onuf 1998). Considerações Finais Nesse sentido, as instituições podem variar
nos mesmos pontos. As regras de cada um po
dem variar não apenas em aspecto formal, mas
também em quantidade e disposição. “Algumas
instituições simples consistem de um número
pequeno de regras cujo conteúdo faz delas
uma família, mesmo que as regras pareçam dar
pouco suporte umas as outros e buscar suporte
de outras instituições com as quais estão conec No contexto das Relações Internacionais, o
Construtivismo consiste em uma teoria que, sob a
afirmação de que as dinâmicas internacionais são
processos socialmente construídos, busca oferecer
uma interpretação alternativa para tais dinâmicas. Argumenta-se que os Estados, por serem consti
tuídos por indivíduos, são agentes que possuem
características semelhantes a estes, nomeadamente Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020 | e-39115 Conversas & Controvérsias, Porto Alegre, v. 7, n. 2, p. 1-10, jul.-dez. 2020 | e-39115 identidades e interesses, que, por sua vez, moldam
o comportamento em relação aos demais. Jackson, Robert; Sorensen, Georg. 2006. Introduction
to International Relations Theories and Approaches. Oxford: Oxford University Press. Ao contrário das visões tradicionais defendidas por
grande parte das teorias de Relações Internacionais,
o Construtivismo defende a ideia de que a anarquia
não deriva do constrangimento imposto aos Estados
pelo Sistema Internacional ou da igualdade destes
enquanto entes soberanos, mas, sim, dos significa
dos atribuídos pelos Estados a seus pares. Assim,
como um meio-termo entre abordagens positivistas
e pós-positivistas, as relações internacionais consis
tiriam primariamente em fatos sociais, os quais são
fatos apenas por acordo humano. Levitsky, Steven; Ziblatt, Daniel. 2018. Como as demo
cracias morrem. Rio de Janeiro: Zahar. Nye, Joseph S. 1987. “Nuclear learning and U.S.-Soviet
security regimes”. International Organization 41, 3: 371-
402. https://doi.org/10.1017/S0020818300027521 Onuf, Nicholas. 1998. “Constructivism: A User’s Manu
al”. In International Relations in a Constructed World. Kubálková, Vendulka; Onuf, Nicholas; Kowert, Paul. Armonk: M. E. Sharpe. Waltz, Kenneth N. 1979. Theory of International Politics. Reading: Addison-Wesley Publishing Company. Wendt, Alexander. 1992. “Anarchy is what States Make of
It”. International Organization. Cambridge 46, 2: 391-425. Considerações Finais https://doi.org/10.1017/S0020818300027764 No contexto atual de crise dos modelos tradi
cionais de democracia (Levitsky & Ziblatt 2018), o
Construtivismo pode auxiliar na compreensão acerca
de como mudanças nas identidades e interesses
estatais — ocasionadas, por vezes, pela ascensão
ao poder de indivíduos com diferentes sistemas de
crenças — podem gerar alterações, não somente
em atuação em âmbito doméstico, mas também
em postura nas relações com outros Estados. Or
ganizações internacionais, por sua vez, também
constituem ambientes propícios para a observação
de tais modificações e afetam a visão de países
sobre temas como direitos humanos, preservação
ambiental e combate à pobreza, por exemplo. Wendt, Alexander. 1995. “Constructing International
Politics”. International Security 20, 01: 71-81. https://doi. org/10.2307/2539217 Wendt, Alexander. 1999. Social Theory of International
Politics. Cambridge: Cambridge University Press. Wendt, Alexander. 1999. Social Theory of International
Politics. Cambridge: Cambridge University Press. Felipe Rocha de Carvalho Doutorando em Ciências Sociais pela Pontifícia Uni
versidade Católica do Rio Grande do Sul (PUCRS), em
Porto Alegre, RS, Brasil. Bolsista Capes. Os textos deste artigo foram revisados por Zeppe
lini Publishers e submetidos para validação do(s)
autor(es) antes da publicação. Referências Adler, Emanuel. 1999. “O Construtivismo no Estudo das
Relações Internacionais”. Lua Nova – Revista de Cultura
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64451999000200011 Barnett, Michael. 2008. “Social Constructivism”. In The
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Contexto Internacional 28, 01: 259-267. https://doi. org/10.1590/S0102-85292006000100005 Berger, Peter. 1966. “Identity as a Problem in the Socio
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105-115. https://doi.org/10.1017/S0003975600001351 Gecas, Viktor. 1982. “The Self-Concept”. Annual Review
of Sociology 8: 1-33. https://doi.org/10.1146/annurev. so.08.080182.000245
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English
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Organic materials in planetary and protoplanetary systems: nature or nurture?
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Astronomy & astrophysics
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cc-by
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To cite this version: C. M. Dalle Ore, M. Fulchignoni, D. P. Cruikshank, M. A. Barucci, R. Brunetto, et al.. Organic
materials in planetary and protoplanetary systems: nature or nurture?. Astronomy and Astrophysics
- A&A, 2011, 533, 10.1051/0004-6361/201116599. insu-03623459 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: insu-03623459
https://insu.hal.science/insu-03623459v1
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abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Astronomy
&
Astrophysics A&A 533, A98 (2011)
DOI: 10.1051/0004-6361/201116599
c⃝ESO 2011 A&A 533, A98 (2011)
DOI: 10.1051/0004-6361/201116599
c⃝ESO 2011 ABSTRACT Aims. The objective of this work is to summarize the discussion of a workshop aimed at investigating the properties, origins, and
evolution of the materials that are responsible for the red coloration of the small objects in the outer parts of the solar system. Because
of limitations or inconsistencies in the observations and, until recently, the limited availability of laboratory data, there are still many
questions on the subject. Our goal is to approach two of the main questions in a systematic way: – Is coloring an original signature of materials that are presolar in origin (“nature”) or stems from post-formational c
or weathering (“nurture”)? g (
)
chemical signature of the material that causes spectra to be sloped towards the red in the visible? – What is the chemical signature of the material that causes spectra to be sloped towards the red in the visible? We examine evidence available both from the laboratory and from observations sampling different parts of the solar system and cir-
cumstellar regions (disks). What is the chemical signature of the material that causes spectra to be sloped towards the red in the visible? We examine evidence available both from the laboratory and from observations sampling different parts of the solar system and cir-
cumstellar regions (disks). dial vs. evolutionary origin for the material that reddens the small objects in the outer parts of our, as well as in other, planetary systems. We proceed by first summarizing laboratory results followed by observational data collected at various distances from the Sun. Results. While laboratory experiments show clear evidence of irradiation effects, particularly from ion bombardment, the first obsta-
cle often resides in the ability to unequivocally identify the organic material in the observations. The lack of extended spectral data of
good quality and resolution is at the base of this problem. Furthermore, that both mechanisms, weathering and presolar, act on the icy
materials in a spectroscopically indistinguishable way makes our goal of defining the impact of each mechanism challenging. Conclusions. Through a review of some of the workshop presentations and discussions, encompassing laboratory experiments as well
as observational data, we infer that both “nature” and “nurture” are instrumental in the coloration of small objects in the outer parts y
g
j
p
p
y y
We proceed by first summarizing laboratory results followed by observational data collected at various distances from the Sun. 13 University of Hawaii, Institute for Astronomy, USA
e-mail: owen@ifa.hawaii.edu 13 University of Hawaii, Institute for Astronomy, USA e-mail: owen@ifa.hawaii.edu 14 Space Telescope Science Institute, Baltimore, MD 21218, USA 14 Space Telescope Science Institute, Baltimore, MD 21218, USA 14 Space Telescope Science Institute, Baltimore, MD 21218, USA
15 p
p
15 Department of Physics and Astronomy, Johns Hopkins University, Baltimore, MD 21218, USA
e-mail: Pascucci@stsci.edu 15 Department of Physics and Astronomy, Johns Hopkins University, Baltimore, MD 21218, USA
e-mail: Pascucci@stsci.edu 16 NASA Lunar Science Institute, Mail Stop 17-1, Moffett Field, CA 94035, USA nar Science Institute, Mail Stop 17-1, Moffett Field, CA 940 NASA Lunar Science Institute, Mail Stop 17-1, Moffett Fi 17 Nasa Postdoctoral Program, NASA Ames Research Center, Moffett Field, CA 94035, USA
e-mail: noemi.pinilla-alonso@nasa.gov 17 Nasa Postdoctoral Program, NASA Ames Research Center, Moffett Field, CA 94035, USA
e-mail: noemi.pinilla-alonso@nasa.gov 18 INAF – Osservatorio Astrofisico di Catania, via S. Sofia 78, 95123 Catania, Italy
e-mail: Gianni@oact.inaf.it 18 INAF – Osservatorio Astrofisico di Catania, via S. Sofia 78, 95123 Catania, Italy
e-mail: Gianni@oact.inaf.it Received 27 January 2011 / Accepted 6 June 2011 Article published by EDP Sciences Organic materials in planetary and protoplanetary
systems: nature or nurture? C. M. Dalle Ore1,2,3, M. Fulchignoni4, D. P. Cruikshank3, M. A. Barucci4, R. Brunetto5, H. Campins6
C. de Bergh3, J. H. Debes7, E. Dotto8, J. P. Emery9, W. M. Grundy10, A. P. Jones5, V. Mennella11,
F. R. Orthous-Daunay12, T. Owen13, I. Pascucci14,15, Y. J. Pendleton16, N. Pinilla-Alonso17,
E. Quirico12, and G. Strazzulla18 1 Centre International d’Ateliers Scientifiques de l’Observatoire de Paris, Paris, France
2 SETI Institute, 189 Bernardo Ave., Mountain View, CA 94043, USA q
2 SETI Institute, 189 Bernardo Ave., Mountain View, CA 94043, USA 2 SETI Institute, 189 Bernardo Ave., Mountain View, CA 94043, USA e-mail: Cristina.M.DalleOre@nasa.gov 3 NASA Ames Research Center, Mail Stop 245-6, Moffett Field, CA 94035, USA
e-mail: Dale.P.Cruikshank@nasa.gov 3 NASA Ames Research Center, Mail Stop 245-6, Moffett Field, CA 94035, US
e-mail: Dale.P.Cruikshank@nasa.gov 4 LESIA, Observatoire de Paris, 92195 Meudon Principal Cedex, France
e-mail: [marcello.fulchignoni,Antonella.Barucci, Catherine 4 LESIA, Observatoire de Paris, 92195 Meudon Principal Cedex, France e-mail: [marcello.fulchignoni,Antonella.Barucci, Catherine.deBergh]@obspm.fr
5 5 Institute d’Astrophysique Spatiale (IAS), Université Paris 11 and CNRS, 91405 Orsay, France 5 Institute d’Astrophysique Spatiale (IAS), Université Paris 11 and CNRS, 91405 Orsay, France 6 Physics Department, University of Central Florida, Orlando, FL 32816, USA
7 6 Physics Department, University of Central Florida, Orlando, FL 32816, USA
7 7 NASA Postdoctoral Program Fellow, Goddard Space Flight Center, USA 7 NASA Postdoctoral Program Fellow, Goddard Space Flight Center, USA 8 INAF – Osservatorio Astronomico di Roma, via Frascati 33, 00040 Monteporzio Catone (Roma), Italy
e-mail: Dotto@mporzio.astro.it 9 Earth and Planetary Sciences Dept, University of Tennessee, Knoxville, TN 37919, USA
e-mail: JEmery2@utk.edu 9 Earth and Planetary Sciences Dept, University of Tennessee, Knoxville, TN 37919, USA
il JE
2@
k
d y
10 Lowell Observatory, 1400 W.Mars Hill Rd., FlagstaffAZ 86001, USA y
10 Lowell Observatory, 1400 W.Mars
e-mail: W.Grundy@lowell.edu 10 Lowell Observatory, 1400 W.Mars Hill Rd., FlagstaffAZ 86001, USA
e-mail: W.Grundy@lowell.edu
11 owell Observatory, 1400 W.Mars Hill Rd., FlagstaffAZ 86 e-mail: W.Grundy@lowell.edu
11 11 INAF Osservatorio Astronomico di Capodimonte, via Moiariello, 16, 80131 Napoli, Italy y
12 UJF-Grenoble 1/CNRS-INSU, Institut de Planétologie et d’Astrophysique de Grenoble (IPAG) UMR 5274,
France 13 University of Hawaii, Institute for Astronomy, USA
e-mail: owen@ifa.hawaii.edu 13 University of Hawaii, Institute for Astronomy, USA
e-mail: owen@ifa.hawaii.edu 1. Introduction Similarly, the regions of Saturn’s rings that exhibit
red color may acquire their reflectance and coloration properties
from the presence of iron or iron oxides (Cuzzi et al. 2009). With the exception of the new interpretation by Clark et al. (2008), so far, the coloration of solid bodies in the outer solar
system and in debris disks of other planetary systems has been
attributed to “organic” materials that can be either native to icy
bodies or the products of the irradiation of volatile ices, or a
combination of the two. We call the scenario of a surface be-
ing reddened by irradiation “nurture”. We note, however, that
both Cruikshank et al. (2001) and Emery & Brown (2004) found
that scattering models with mafic silicates can match the spectral
reflectance of the red colored jovian Trojan asteroids for wave-
lengths shorter than 2.5 μm, as discussed further below. An alter-
native view to the “nurture” scenario is offered by Grundy (2009)
where the “nurture” idea is matched with a “nature” counter-
part, amply supported in recent literature (Grundy & Stansberry
2003; Grundy 2009, and references therein). The “nature” coun-
terpart described in detail below consists of the case in which
the reddening is caused by sub-micron particles of organic mate-
rial of presolar origin trapped in ice from which comes the label
“nature”. According to Grundy (2009) the degree of reddening
varies with the concentration of the organics with respect to ice. We summarize the idea and offer laboratory and observational
evidence to support the two views in an effort to unravel the
puzzle that has impacted our understanding of outer solar sys-
tem and planetary system chemistry. We also assess whether the
evidence weighs in favor of either scenario to provide a guideline
for future work and investigations. Solid organic complexes (tholins) produced synthetically by
a corona or a spark discharge in a gaseous mixture of N2 and CH4
(and other molecules in some experiments) are strongly colored,
and are effective when used in scattering models to reproduce the
color of Titan’s atmospheric aerosols (e.g., Imanaka et al. 2004;
Bernard et al. 2006). Similarly, tholins are an effective color-
ing agent in models of solid surfaces (e.g., Hoffman et al. 1993;
Dalle Ore et al. 2009; Owen et al. 2001; and Cruikshank et al. 2005). New views of the red coloration of solid bodies in the solar
system (Fig. 1. Introduction The colors of solar system bodies are often the main (or sole)
source of information available for determining the composi-
tion of the surface materials, especially in the absence of spectra
showing diagnostic absorption or emission features. These com-
bined with information on the absolute reflectance (e.g., geomet-
ric albedo), the color of reflected sunlight measured over a range
of wavelengths in the visible and near-infrared (0.3−2.5 μm)
spectral region, are usually enough to discriminate between
mineral- and ice-covered surfaces. In general terms, surfaces
with a high albedo and neutral color are usually ice-covered
(with H2O being most common), while medium high albedo
and neutral color may indicate pulverized minerals. Low albedo
and a moderately red color are suggestive of mafic minerals
(e.g., olivine and pyroxene), while low albedo and very red color
are usually taken to indicate a surface rich in complex, macro-
molecular carbonaceous material similar to the kind found in
primitive carbonaceous meteorites. This latter material is often
referred to by the shorthand terms, kerogen-like or “organics”,
but it is necessary to keep in mind the rather longer but more
descriptive name given in the previous sentence when using
this simplification. Furthermore, we use the term “reddening”
when referring to either the amount of organics or the effects of
“weathering”, as explained in the text. The term “weathering” is
used with reference to the gradual changes to the surface of the
bodies caused by irradiation. Fig. 1. Measured spectral reflectance of several solar system small bod-
ies. Key: Pholus = Centaur object, Iapetus = Saturn Satellite, Hektor,
Odysseus & Euphrosyne = Trojan asteroids, Himalia = Jupiter Satellite,
Borrelly = Jupiter-family comet. Figure from J. P. Emery. The idea of complex organic materials constituting the col-
oring agent for low-albedo asteroids appears to have originated
with Gradie and Veverka (1980), who suggested that “... very
opaque, very red, polymer-type organic compounds, which are
structurally similar to aromatic-type kerogen”, mixed with mont-
morillonite (clay), magnetite (iron oxide), and carbon black,
could match the colors of Trojan asteroids in the spectral range
0.3−1.1 μm. The organic material they used in their laboratory
simulations was derived from coal tar by removing the soluble
components and leaving the insoluble complex solid material
called kerogen. may be material that is intrinsically red, such as metallic iron or
iron oxides. ABSTRACT planets and satellites: composition – comets: general – circumstellar matter – Kuiper belt: general – dust, extinction Key words. planets and satellites: composition – comets: general – circumstellar matter – Kuiper belt: general – dust Fig. 1. Measured spectral reflectance of several solar system small bod-
ies. Key: Pholus = Centaur object, Iapetus = Saturn Satellite, Hektor,
Odysseus & Euphrosyne = Trojan asteroids, Himalia = Jupiter Satellite,
Borrelly = Jupiter-family comet. Figure from J. P. Emery. ABSTRACT p
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Results. While laboratory experiments show clear evidence of irradiation effects, particularly from ion bombardment, the first obsta-
cle often resides in the ability to unequivocally identify the organic material in the observations. The lack of extended spectral data of
good quality and resolution is at the base of this problem. Furthermore, that both mechanisms, weathering and presolar, act on the icy
materials in a spectroscopically indistinguishable way makes our goal of defining the impact of each mechanism challenging. y
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good quality and resolution is at the base of this problem. Furthermore, that both mechanisms, weathering and presolar, act on the icy
materials in a spectroscopically indistinguishable way makes our goal of defining the impact of each mechanism challenging. Conclusions. Through a review of some of the workshop presentations and discussions, encompassing laboratory experiments as well
as observational data, we infer that both “nature” and “nurture” are instrumental in the coloration of small objects in the outer parts p
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Conclusions. Through a review of some of the workshop presentations and discussions, encompassing laboratory experiments as well
as observational data, we infer that both “nature” and “nurture” are instrumental in the coloration of small objects in the outer parts Article published by EDP Sciences A98, page 1 of 14 A&A 533, A98 (2011) of the solar system. While in the case of some observations it is clear that the organic reddening material originated before the solar nebula
(i.e. presolar grains found in meteorites), for many other cases pointers are not as clear and indicate a concurrence of both processes. of the solar system. While in the case of some observations it is clear that the organic reddening material origina
(i.e. presolar grains found in meteorites), for many other cases pointers are not as clear and indicate a concurrence of b hile in the case of some observations it is clear that the organic reddening material originated before the solar ne
nd in meteorites), for many other cases pointers are not as clear and indicate a concurrence of both processes. (i.e. presolar grains found in meteorites), for many other cases pointers are not as clear and indicate a concurrence of both processes. Key words. planets and satellites: composition – comets: general – circumstellar matter – Kuiper belt: general – dust, extinction Key words. 3.1. Laboratory studies of refractory carbonaceous materials Carbonaceous and organic materials in the solar system and in
the interstellar medium (ISM) of our own, as well as external
galaxies, display a wide range of composition and structure, il-
lustrating the rich chemistry that occurs in space. Experimental
studies play a fundamental role in understanding this diver-
sity: in the laboratory it is possible to analyze collected extrater-
restrial carbon-rich materials [meteorites (see the spectrum of
Orgueil in Fig. 4), micro-meteorites, IDPs, and cometary grains
collected by the Stardust mission], and to synthesize, charac-
terize, and process laboratory analogues to gain insight into the
formation, origin, and evolution of the natural samples. Among these processes UV irradiation and cosmic ion irra-
diation play an important role in the life-cycle of interstellar dust
(Mennella et al. 2003), in the production of complex organics in
the presolar nebula (Muñoz-Caro et al. 2006) as well as on the
surface of TNOs and other minor bodies (Brunetto et al. 2006),
and in the alteration of pre-existing carbon materials (Moroz
et al. 2004; Baratta et al. 2008). Many studies point out that the
early Sun was much more active than today, passing through an
active T Tauri phase before reaching the main sequence (e.g.,
Feigelson & Montmerle 1999); such strong particle and photon
radiation could have had major influences on processing mate-
rial during the planetary accretion phase, and more specifically
on the carbon chemistry. Although there is abundant evidence that icy surfaces that
include carbon-bearing species are rapidly reddened and dark-
ened as a result of radiation damage, there are also several lines
of evidence to suggest that the surface colors of TNOs are not
due to thin, modern radiolytic veneers, but in fact reflect their
bulk compositions. Specifically, the color clusters reported by
Barucci et al. (2005) are hard to explain if the surfaces of TNOs
are progressively altered by radiation damage, but occasionally
refreshed by impact excavation of pristine material. The colors
of primary and secondary bodies among small binary TNOs
are also very closely correlated (Benecchi et al. 2009). This
pattern is inconsistent with time-dependent surface coloration
from radiation damage, since impact erosion rates depend on
object size. 1. Introduction 1) are emerging with improvements in computa-
tional methods for scattering models and new laboratory data,
and with a large and homogeneous observational data set for
the spectral reflectances of Saturn’s satellites and rings. Clark
et al. (2008) have shown that a brightness peak in the blue spec-
tral region seen in most of Saturn’s satellites can be explained
as a Rayleigh scattering phenomenon caused by submicrometer
particles that form a thin coating on the ice or other surface ma-
terials. For neutral-colored surfaces, these small particles may be
composed of carbon, while for red-colored surfaces, the particles A98, page 2 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems 2. Sub-micron organic particles and their possible
influence on the colors of primitive bodies:
the “nature” hypothesis The hollow rims of red organic globules such as seen in the
Tagish Lake meteorite are also worth mentioning in this context. These shells are thought to have been produced by photolysis of
the surfaces of small icy grains prior to their accretion into larger
bodies (e.g., Nakamura-Messenger et al. 2006). Subsequently,
the remaining ice sublimated away, leaving the less volatile or-
ganic shell. These particles tell us that at least some icy grains
were already well-irradiated before they accreted into planetes-
imals, so we may not require the modern-day radiation envi-
ronment to explain the existence of reddish organic materials in
primitive bodies (although ongoing radiolysis and photolysis of
icy surfaces surely does also occur). Micron and submicron sized organic and carbonaceous parti-
cles occur in primitive meteorites, interplanetary dust particles
(IDPs), and comet dust. Some could have formed in the proto-
planetary nebula, via the irradiation of icy particles. Others could
have survived from the cloud of gas and dust from which the
Sun formed. In regions where the volatiles condensed to form
ices in the outer parts of the proto-planetary nebula, these par-
ticles could have served as condensation nuclei, acquiring icy
mantles, perhaps with distinct regional compositions very dif-
ferent from the mix of ices prevalent in the presolar cloud. Small carbonaceous particles and ices were likely the main
solid ingredients from which planetesimals accreted in the outer
proto-planetary nebula although comet samples retrieved by the
Stardust mission also contain silicates and sulfides as significant
components (Sandford et al. 2006; Sandford 2008; Clemett et al. 2010). 3. Evidence from laboratory work Both the “nature” and the “nurture” scenarios are likely to have
played an important role in the primordial nebula before plan-
etesimals formed. We describe some of the processes that affect
carbon-rich materials and are thought to be predominant: they
are mostly “nurture” mechanisms. Of the large number of differ-
ent forms of carbon we consider those that are most relevant and
abundant in space. At visible wavelengths, the addition of colorless ice to oth-
erwise dark organic material can dramatically increase the red-
ness as well as the reflectance, if they are mixed at spatial scales
of the order of the wavelength or less (Grundy & Stansberry
2003; Grundy 2009). The behavior of such mixtures suggests
a resolution to the dilemma posed by the inconsistent behav-
ior of the colors and albedos of trans-neptunian objects (TNOs),
Centaurs, and ecliptic comet (EC) nuclei (e.g., Jewitt 2002;
Lamy & Toth 2009). In fact, although these objects are thought
to be genetically linked, much redder colors and higher albedos
are found among TNOs and Centaurs than among the EC nu-
clei (Doressoundiram et al. 2008), culminating in consistently
red colors and high albedos among dynamically cold classical
belt TNOs. Cold classical TNOs, with their near circular orbits,
presumably formed close to where they currently are. They may
or may not be direct progenitors of Centaurs and thence ECs
(Volk & Malhotra 2008; Horner & Lykawka 2010) but, because
of their distance from the Sun, their surface compositions could
offer relatively little-altered samples of the solids accreted in the
outermost parts of the nebula. Radiative transfer mixing models
can produce a wide variety of albedos and colors. By includ-
ing sublimation loss of ice, they can also reproduce the observed
color and albedo evolution as TNOs progress from the far fringes
of the solar system through the giant planet-crossing Centaur
zone to become ECs. 3.1. Laboratory studies of refractory carbonaceous materials TNOs could be more resilient to radiation damage
than previously thought, if their surface ices do not include low
molecular weight organic molecules, as could happen if they
mostly consist of already-highly-processed carbonaceous parti-
cles plus relatively clean H2O ice distilled by sublimation and
re-condensation in the nebula. Several laboratories have concentrated their efforts on the
production, characterization, and alteration of refractory car-
bonaceous materials of astrophysical interest, and in particular
on the extensively studied amorphous carbons, with different
degrees of hydrogenation. Different types of amorphous car-
bon can be produced by several techniques, such as laser py-
rolysis, laser ablation or arc discharge (e.g., Scott & Duley
1996; Mennella et al. 1999), and ion and photon irradiation of
hydrocarbon-rich ices (e.g., Strazzulla & Baratta 1991; Dartois
et al. 2005). Recent studies show that the IR spectra of these
materials can be used to interpret observations of the dust in
the diffuse ISM of galaxies, where an important fraction of the
available carbon is locked in the dust (Pendleton & Allamandola
2002; Dartois et al. 2007; Dartois & Muñoz-Caro 2007). Carbon
soot is another interesting material (Jäger et al. 2006), due to A98, page 3 of 14 A&A 533, A98 (2011) Fig. 2. Results of irradiation of CH4 ice (Brunetto et al. 2006):
eV/16 amu units refer to the energy (eV) adsorbed per 16 atomic mass
unit. The dotted line shows the spectrum of the residue at a higher tem-
perature (250 K). its possible connection with polycyclic aromatic hydrocarbons
(PAHs), and to the fact that its IR spectral features may help to
interpret the nature of some of the ubiquitously observed aro-
matic IR bands (Pino et al. 2008). Aromatic compounds are
stable molecules made of six-fold rings of carbon and hydro-
gen atoms (hydrocarbon). The most basic aromatic compound is
the benzene ring (C6H6): PAHs are complex molecules made of
fused aromatic (benzene) rings and in a terrestrial setting are a
product of combustion. Visible-IR spectra of soot and amorphous carbons dif-
fer in their chemical (e.g., hydrogen content, sp2/sp3 ratio,
i.e. graphite-like to diamond-like bonding character), optical
(e.g., variation of the optical gap), and structural (e.g., bond dis-
order, crystallites size) properties. These properties can vary ac-
cording to different formation processes (“nature” scenario) but
are also altered by photon and ion irradiation processes (“nur-
ture” scenario) similar to that which is observed for irradiated
organic residues. 3.3. Laboratory evidence conclusion The observed reddening material could be carbonaceous in na-
ture or could be the result of sublimation and irradiation of
carbon-rich compounds (nurture). The characteristics of car-
bonaceous materials themselves are related to different forma-
tion processes, but can also be transformed by irradiation: their
colors do not seem to correlate directly with their mode of
formation, highlighting the difficulty in defining the “nature”
and “nurture” scenarios. Important constraints should be sought 3.2. Ion irradiation and the colors of C-rich materials Several experiments have been performed to investigate the ef-
fects of ion irradiation on carbon-rich materials (e.g. Thompson
et al. 1987). Three different kinds of materials chosen to best rep-
resent those thought to be present on the surfaces of atmosphere-
less outer solar system objects (i.e. ices, initially red solids, and
polymer-like initially transparent solids), have been irradiated. They are: frozen (16 K) methane (CH4) (Brunetto et al. 2006),
asphaltite (a natural bitumen) (Moroz et al. 2004), and a film of
polystyrene deposited on a silicate (olivine) (Kanuchova et al. 2010). 4.1. Interstellar medium (ISM) Fundamental to the evolution of the biogenic molecules, to the
process of planetary system formation, and perhaps to the origin
of life, is the connection between the organic material found in
the interstellar medium and that which was incorporated in the
most primitive solar system bodies. Understanding the connec-
tion between interstellar ices and organics can elucidate the in-
ventory of materials available as the most primitive solar system
bodies formed. Star formation and the subsequent evolution of planetary
systems occur in dense molecular clouds, which are comprised,
in part, of interstellar dust grains gathered from the diffuse inter-
stellar medium (DISM). Carbonaceous materials are a primary
component of interstellar dust, forming in the outflow of car-
bon stars and the DISM. Over time, the low density DISM is
swept into dense molecular clouds, the principal formation sites
and repositories of most interstellar molecules. Organic com-
pounds created in these clouds represent the first step towards
the complex materials that might be responsible of the reddening
in the outer solar system. In this section we examine their rela-
tionship to ices, an essential ingredient in the “nature” scenario. 3.1. Laboratory studies of refractory carbonaceous materials The colors of amorphous carbon vary non-
uniquely as a function of the above properties, suggesting that
colors might be a poor parameter to constrain the “nature” and
“nurture” scenarios in carbonaceous materials. Fig. 2. Results of irradiation of CH4 ice (Brunetto et al. 2006):
eV/16 amu units refer to the energy (eV) adsorbed per 16 atomic mass
unit. The dotted line shows the spectrum of the residue at a higher tem-
perature (250 K). in the laboratory analysis of collected extraterrestrial primitive
carbons (e.g., cometary grains) to further understand the weight
of one scenario over the other. On the other hand, the study of
carbon-rich irradiated materials shows that weathering plays an
important role in modifying the residue of H2O ice sublimation. It becomes apparent that a clear distinction between the “nature”
and “nurture” scenario is very difficult when analyzing the ma-
terials that we think are responsible for most of the reddening
in the solar system. Therefore, when examining the solar sys-
tem evidence, we will make the simplifying assumption that the
weathering process began with low-order, volatile-rich organics
that are presumed to be presolar materials. We then try to in-
fer whether “nature” or “nurture” prevails at the later stages of
evolution of the materials that constitute the building blocks of
planetary systems. 4. Observational evidence outside the solar system
4.1. Interstellar medium (ISM) The irradiation of frozen CH4 shows a strong reddening
and darkening of the spectra, due to the formation of an or-
ganic (C-rich) refractory residue. The color is preserved after
the sublimation of the volatile species (methane and other com-
pounds formed by irradiation) as shown by the spectrum of the
residue at higher temperatures (250 K, Fig. 2). Further irradia-
tion of such a solid polymer-like residue, as well as of natural red
solids (e.g., asphaltite), produces a darkening and a flattening of
the spectra. Recently, samples of silicates (olivine) covered with
polystyrene layers have been considered. Polystyrene is consid-
ered a model for an originally transparent polymer-like material. Before irradiation, it is a transparent medium that, as ion flu-
ence increases, exhibits spectral reddening and darkening. The
reddening increases and reaches a maximum at a given fluence. Further irradiation produces further darkening, but is accompa-
nied by a flattening of the spectrum. This process is what we
refer through this paper as weathering and is considered one of
the main “nurture” mechanisms. 4.1.1. The nature of interstellar organics
and their relationship to ices Infrared observational studies provide details of the solid-state
features in dust grains. A series of absorption bands have been A98, page 4 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems observed in the 3.4 μm wavelength region towards bright in-
frared objects that are seen through high column densities of
dust along multiple sightlines throughout the diffuse interstellar
medium (DISM) (Pendleton et al. 1994). the physical and chemical properties of a-C:H solids are sur-
prisingly complex. Their properties can vary in response to
the external conditions (e.g., temperature; photon, ion or elec-
tron irradiation). For example, a-C:H can undergo an equivalent
process known as “photo-darkening”, “graphitisation” or “aro-
matisation” when heated or exposed to UV- or ion-irradiation. This transformation leads to a closing of the optical gap and a
concomitant change in the optical properties at near-IR to
visible-UV wavelengths. Comparisons of organic residues, produced under a vari-
ety of laboratory conditions, to the diffuse interstellar medium
observations have shown that aliphatic chain-like hydrocarbon
grains are responsible for the spectral absorption features ob-
served near 3.4 μm (∼2940 cm−1). The hydrocarbons in the
DISM appear to carry the -CH2- and -CH3 functional groups
in the abundance ratio CH2/CH3 ∼2.5, and the amount of car-
bon tied up in this component is greater than 4% of the cosmic
carbon available (Sandford et al. 1991), highlighting the impor-
tance of these materials. The Murchison carbonaceous meteorite
has been shown a good analog of the interstellar observations, re-
vealing a striking similarity between some of the hydrocarbons
in the meteorite and the DISM (Pendleton & Allamandola 2002). Dartois et al. (2004) indeed show that the thermal anneal-
ing of hydrogenated amorphous carbon materials is accompa-
nied by an increase in the aromatic component, i.e. an “arom-
atization”. This type of evolution for interstellar hydrocarbons
was proposed previously (Duley et al. 1989; Jones 1990; Jones
et al. 1990) and it was suggested to be the result of the photon-
driven processing of hydrogen-rich a-C:H mantles accreted onto
silicate grains in the ISM. In this model the progressive loss
of hydrogen, and the associated “aromatisation” of the struc-
ture was assumed to lead to materials with a lower hydrogen
content, a smaller band gap, and an increased aromatic content. 4.1.1. The nature of interstellar organics
and their relationship to ices The end point of this evolutionary process is expected to be a
low-density, aromatic-rich material that can disaggregate into its
constituent aromatic molecular components (PAH-like species)
with associated sp3 and sp2 carbon and hydrogen atom bridging
structures (e.g., Duley 2000; Petrie et al. 2003; Pety et al. 2005;
Jones 2005) as a result of UV photo-processing or fragmentation
in grain-grain collisions (Jones 2005; Jones et al. 1996). Ground- and space-based telescopic observations trace in-
terstellar organics from the diffuse to dense interstellar clouds,
revealing that organic material in the DISM is predominantly
hydrocarbon in nature, possessing little N or O, and with the C
distributed between aromatic ring-clusters and aliphatic forms. These data provide the first step towards identifying the nature of
the reddening material in the DISM and giving us a possible link
with solar system materials. The transition between the diffuse
and dense cloud regions provides an interesting place to study
the onset of interstellar ice chemistry. The hardy, refractory com-
ponent of the DISM is expected to survive incorporation into the
dense cloud environment, but to date there is no observational
evidence that the DISM 3.4-μm band is present in dense molec-
ular clouds. The deep ice band at 3.0 μm detected toward many
deeply embedded protostellar objects and through the quiescent
dust through which background field stars are observed (Smith
et al. 1993) often has a broad wing on the long-wavelength side
of the band that is assumed to arise from some type of simple hy-
drocarbon (Sellgren et al. 1994). However, the wavelength peak
of this feature and its overall structure differ appreciably from
the symmetric and asymmetric aliphatic hydrocarbon bands of
the DISM (Chiar & Pendleton 2008). Mennella (2008a, 2010)
proposed a scenario in which the different conditions in dense
clouds with respect to diffuse regions (e.g. ice coating of the
dust grains, H atom temperature) may inhibit the formation of
CH2 and CH3 groups, while the formation of the 3.47 μm band
can proceed (see also Sect. 4.3). Thus, it seems that a viable source of interstellar, circum-
stellar or solar system “PAHs” could be provided by the natural
evolution of interstellar hydrogenated amorphous carbon grains. 4.1.1. The nature of interstellar organics
and their relationship to ices However, in this case any “released” aromatic rich, “molecular”
carriers are not the pristine and classic PAHs generally discussed
in the PAH literature, but are probably more complex and con-
tain carbon in other than purely aromatic form. 4.2. Dense clouds In order to study the ice chemistry of dense clouds from the ear-
liest stages, one needs to look at the transition zone between the
diffuse and dense clouds where extinction is high enough that the
grains are shielded from the harsh interstellar radiation field of
the DISM allowing for ice formation. The spectroscopy of back-
ground stars seen through quiescent clouds with no star forma-
tion activity reveals that chemistry occurs before star formation
begins. In fact, star formation might well begin with dust already
coated with a fairly complex mixture of ices (Chiar et al. 2011). NH3-ice is an important mantle constituent in all dense cloud en-
vironments, and therefore an important reservoir of elemental N
in these regions. 4.3. Evolution of organics from diffuse interstellar clouds
to the solar system The results of experiments aimed at simulating the evolution of
the aliphatic carbon component in space have recently shown
that an evolutionary link between the organics observed in the
two environments is possible (Mennella 2008b, 2010). In fact,
the same carbon grain population may absorb at 3.4 μm in dif-
fuse regions of the ISM, and at 3.47 μm in dense regions. The
carbon materials absorbing at 3.47 μm in dense cloud/protostars,
included in a comet during the formation process in the cold
outer edge of the solar nebula, may thermally evolve to de-
velop the CH2 and CH3 groups, as testified by laboratory simu-
lations. Therefore, a unifying interpretation of how the aliphatic
component appears in the main phases of its evolution in space
is possible. As the inner part of the disk clears out, the observed infrared
spectra dramatically change. No water molecules and no organ-
ics are detected, just atomic ionic species and the very high J
transition of OH, pointing to dissociation of water by the now
stronger UV field that is no longer shielded by the dust (Najita
et al. 2010). The outer disk does not seem to be affected by these
changes: the molecular complexity observed at this stage is the
same as that of optically thick gas-rich disks. Studies of the interaction of hydrogen atoms at 80 K with
carbon grains covered with a water ice layer at 12 K confirm
that exposure of the samples to H atoms induces the activation of
the band at 3.47 μm with no evidence for the formation of aro-
matic and aliphatic C-H bonds in the CH2 and CH3 functional
groups. A penetration depth of 100 nm has been estimated for
H atoms in the porous water ice covering carbon grains. Sample
warm up to room temperature causes the activation of the IR fea-
tures representing the vibrations of the CH2 and CH3 aliphatic
functional groups. The presence of the 3.47 μm band carrier is
compatible with the evolutionary time scale limit imposed by
fast cycling of materials between dense and diffuse regions of
the interstellar medium. In diffuse regions the formation of the
CH2 and CH3 aliphatic bands, inhibited in dense regions, takes
place, masking the 3.47 μm band. 4.1.2. The evolution of hydrocarbonaceous dust in the ISM Through laboratory simulations, Dartois et al. (2004) have
shown that hydrogenated amorphous carbon (denoted a-C:H)
can explain not only the 3.4-μm, but also the 6.85- and 7.25-μm
interstellar absorption bands if the a-C:H is hydrogen-rich
(>50 atomic percent H). Given that recent studies of carbon
dust and PAH evolution in the ISM indicate that these materials
are rather susceptible to destructive processing in SN-generated
shock waves, carbonaceous solids must be rather rapidly re-
formed there (Serra Díaz-Cano & Jones 2008; Micelotta et al. 2010a,b, 2011; Jones & Nuth III 2011). Therefore it appears
that a large fraction of the carbon dust in the ISM is likely to
be in the form of an amorphous, re-accreted, a-C:H-like carbon,
rather than in the form of crystalline graphite (pure carbon) that
would be very difficult to condense in the low-density ISM. Thus, graphite is probably not an abundant component of inter-
stellar carbon dust (e.g. Serra Díaz-Cano & Jones 2008). For a
material containing the atoms of only two elements (C and H) g
Regarding the dust component of interstellar grains, the pos-
sibility of the presence of PAH-like chemicals (aromatic hydro-
carbons) in the building blocks of the solar system has been es-
tablished. The next step is to understand if and how such basic
primitive components could have survived the primordial neb-
ula environment to become part of the current outer solar system
bodies. Aliphatic hydrocarbonsare observed in the diffuse ISM, they
are present in IDPs, cometary grains and meteorites, while they
are absent in dense cloud environments. There are two possible
interpretations: 1. There is no evolutionary link between the aliphatic com-
pounds observed in the diffuse regions of the ISM and
those present in solar system materials. The observation of 1. There is no evolutionary link between the aliphatic com-
pounds observed in the diffuse regions of the ISM and
those present in solar system materials. The observation of A98, page 5 of 14 A&A 533, A98 (2011) organics in the two environments is a mere coincidence. In
diffuse interstellar clouds, the C-H bonds of the CH2 and
CH3 groups form as a result of processing of carbon particles
by H atoms (at thermal energies), UV photons and cosmic
ions and they are destroyed when carbon grains enter into
dense clouds (e.g., Mennella et al. 2002, 2003). 4.1.2. The evolution of hydrocarbonaceous dust in the ISM Other pro-
cesses, e.g., photoprocessing of C bearing ices, during solar
system formation and evolution, form the aliphatic materials
observed in solar system materials. solar system. The chemical transformation of complex organic
molecules during accretion and disk evolution and the level
of complexity that they achieve as the protoplanetary disk en-
ters the planet-formation stage are current research topics being
pushed forward by recent dramatic advances in infrared and mil-
limeter astronomy. Recent observations point to chemical evolution during the
first 30 Myr of the disk lifetime. Due to the very high tem-
peratures necessary for star formation, all materials, unless far
enough from the protostar, are in gaseous form. During the con-
densation, planet accretion and formation phases they return
to be seen in solid form. Optically thick gas-rich disks around
young solar analogs are found to have rich infrared spectra indi-
cating the presence of water, OH, and organic molecules such as
C2H2 and HCN in the warm (∼700 K) disk atmosphere within
a few AU from the star (Carr & Najita 2008; Pontoppidan et al. 2010). The large abundances of these molecules suggest that
they form not only from evaporation of inward migrating ices
but also from additional chemistry in the gas phase. Atomic and
ionic species such as ionized Ne are also frequently detected
(Pascucci et al. 2007; Güdel et al. 2010) and trace the very hot
disk surface (several thousands K gas) likely photoevaporating
from the star-disk system (Pascucci & Sterzik 2009). CN, HCN,
H2CO and some deuterated species are also detected at large
disk radii (beyond 100 AU) via interferometric millimeter ob-
servations (Öberg et al. 2010). 2. An evolutionary link between the aliphatic observed in dif-
fuse ISM and solar system materials exists. In this case,
a fundamental constraint to be considered is the absence of
the aliphatic component in dense clouds: going from diffuse
ISM to the formation site of comets/parent bodies one has to
pass through the dense cloud phase where the solar system
formed. 4.3. Evolution of organics from diffuse interstellar clouds
to the solar system The activation of the CH2 and
CH3 aliphatic vibrational modes at the end of H processing after
sample warm up represents the first experimental evidence sup-
porting an evolutionary connection between the interstellar car-
bon grain population, which is responsible for the 3.4 μm band
(diffuse regions) and contributes to the absorption at 3.47 μm
(dense regions), and the organics observed in interplanetary dust
particles and cometary Stardust grains. Finally, as the disk clears out most of its primordial dust
(and likely gas), for systems older than 10 Myr, infrared and
millimeter spectra are strikingly lacking a number of molecules,
atomic and ionic species (Pascucci et al. 2006) such as H2 IR gas
lines, forbidden atomic lines or CO mm lines, that are instead
observed in younger disks. This fact is attributed to a decrease
of gas in the disk. According to Pascucci et al. (2006) there is
less than 0.1 Jupiter mass of gas in disks that are ∼10 Myr old or
older, implying that giant planet formation should occur in less
than 10 Myr, in agreement with current giant planet formation
theories. Along with time evolution, we also have evidence for the
effect of different UV fields from the different types of molecu-
lar species detected toward stars of different luminosities. While
stars that are a few times more massive than the Sun with
stronger UV lack almost any molecular detections in their spec-
tra (Fedele et al. 2011; Pontoppidan et al. 2010), disks around
very low-mass stars present a rich chemistry (Pascucci et al. 2009). What remains to be seen is how much of the chemistry in
the disk mid-plane is impacted by the different radiation fields
and what are the effects on the composition of forming planets. A98, page 6 of 14 4.4. The chemistry of protoplanetary disks and the effect
of stellar UV irradiation Porous aggregates of carbon, water ice, and silicates can also
fit the scattered light and IR spectral energy distribution (SED)
of HR 4796A, providing an alternate explanation (Köhler et al. 2008). However, there are problems with this interpretation, in-
cluding whether water ice should survive in the UV environment
of an A star (Grigorieva et al. 2007). 0
50
100
150
200
Scattering Angle
100
1000
Normalized Surface Brightness
Porous Ice mixtures
Tholins Furthermore, neither pure tholins nor porous aggregates ad-
equately fit the observed phase function of HR 4796A’s ring in
the visible (Debes et al., in prep.). Analysis of the HR 4796A
ring surface brightness in the visible as a function of scattering
angle for the north-east and south-west lobes of the disk show a
remarkably flat inferred phase function for the constituent dust. Figure 3 shows that the data are poorly fit by either a Henyey-
Greenstein phase function (as assumed by Köhler et al. 2008)
or Mie scattering phase functions of pure micron-sized tholin
grains (as assumed by Debes et al. 2008). Further study is re-
quired to determine the composition of the dust in the HR 4796A
disk and how representative it is compared to other debris disk
systems. More information on the “nature” versus “nurture”
problem at the late stages of planet formation may come as more
debris disks are observed in scattered light in the visible and
near-IR. Fig. 3. (Top left) Scattering efficiency Fdisk/F⋆as a function of wave-
length for the HR 4796A debris disk. Horizontal error bars represent the
width of the filter for each image of the disk, and the solid line is the best
fitting mixture of Titan tholins and astronomical silicates (amin = 1 μm
(grain size), 20% Silicates, 80% Tholins). (Top right) Contours of the
68.5% and 98% confidence levels for the models of HR 4796A’s scat-
tered light as a function of minimum grainsize and Tholin fraction. The X represents the best fitting model with a χ2
ν = 0.98. (Bottom)
Relative scattered light surface brightness at visible wavelengths for the
HR 4796A debris disk as a function of inferred scattering angle for dust
grains in the disk. Grey squares represent the south-west lobe of the
ring corrected for the brightness asymmetry present in the ansae of the
disk (Schneider et al. 2009) while black squares represent the north-east
lobe. 5. Observational evidence in the solar system Having laid the foundations of the investigation by studying the
building blocks of the solar system, we proceed to examine the
observational evidence at increasing distance from the Sun. We
first search for evidence of organic signatures and try to quan-
tify the organic matter content in the interplanetary material as
well as meteoritic samples analyzed in the laboratory. We follow
up on this with a systematic search for signs of organics in the
spectra and photometric colors of small bodies (i.e. asteroids,
satellites, TNOs, comets) from the asteroids in the Main Belt
to the region beyond Neptune. Lastly, we analyze the behavior
of colors in the outer solar system looking for correlations with
the quantity of ice. This scheme is based on the fact that, ac-
cording to our current knowledge of the solar nebula formation
and evolution, the quantity of H2O ice increases with decreasing
temperature and increasing distance from the Sun. chemical routes to its formation have so far not been clearly
elucidated. The D and 15N enrichments found in these mate-
rials are consistent with synthesis in a cold medium through
ion-molecule reactions, but the context where these enrichments
took place (molecular cloud, proto-planetary disk) is unknown
(e.g., Alexander et al. 2007). The origin of the soluble organic
matter (SOM) – i.e. molecules released with organic solvents or
water – has been attributed to the effects of hydrothermal pro-
cesses in the parent body (Alexander et al. 1998; Yabuta et al. 2005), in which SOM could be the hydrolysis product of IOM. An alternative view considers SOM as a component of the ices
that were accreted along with refractory phases, and therefore
with no connection with IOM (Remusat et al. 2005). Among and within chondrites, the IOM presents a wide
range of isotopic and elemental compositions, as well as large
chemical and structural variations (Alexander et al. 2007;
Quirico et al. 2003). These variations are controlled by hetero-
geneities in the precursors accreted by the parent bodies and
by the effects of post-accretion processes (Cody & Alexander
2005; Quirico et al. 2009). Post-accretion processes include
low-temperature hydrothermalism (Cody & Alexander 2005;
Yabuta et al. 2005; Orthous-Daunay et al. 2010a), long-duration
radiogenic thermal metamorphism (Bonal et al. 2006a, 2007; 4.4. The chemistry of protoplanetary disks and the effect
of stellar UV irradiation Once the gas disperses in a protoplanetary disk, gas phase chem-
istry ceases and rocky bodies and dust are all that remain in
a young planetary system. Often the collision or evaporation
of planetesimals form second generation dusty debris disks. In
these systems, the dust provides a unique remote laboratory
to study the composition and thermal processing in the disk Interstellar clouds evolve into proto-planetary disks and then
protostars. Observations of proto-planetary disks can be used
to monitor the chemical variations during the early phases of
planetary evolution and to infer the chemistry in the primordial A98, page 6 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems 0.0
0.5
1.0
1.5
2.0
2.5
Wavelength (μm)
3
4
5
6
7
8
9
Fdisk/F* (x10−4)
30
40
50
60
70
80
90
100
Fraction of Tholin Dust (%)
0
1
2
3
4
5
Grain Size (μm)
0
50
100
150
200
Scattering Angle
100
1000
Normalized Surface Brightness
Porous Ice mixtures
Tholins
Fig. 3. (Top left) Scattering efficiency Fdisk/F⋆as a function of wave-
length for the HR 4796A debris disk. Horizontal error bars represent the
width of the filter for each image of the disk, and the solid line is the best
fitting mixture of Titan tholins and astronomical silicates (amin = 1 μm
(grain size), 20% Silicates, 80% Tholins). (Top right) Contours of the
68.5% and 98% confidence levels for the models of HR 4796A’s scat-
tered light as a function of minimum grainsize and Tholin fraction. The X represents the best fitting model with a χ2
ν = 0.98. (Bottom)
Relative scattered light surface brightness at visible wavelengths for the
HR 4796A debris disk as a function of inferred scattering angle for dust
grains in the disk. Grey squares represent the south-west lobe of the
ring corrected for the brightness asymmetry present in the ansae of the
disk (Schneider et al. 2009) while black squares represent the north-east
lobe. Overplotted are phase functions of a) a porous mixture of ices,
silicates, and carbonaceous material assuming a Henyey-Greenstein ap-
proximation to the phase function and b) Tholins assuming a Mie scat-
tering model phase function. 4.4. The chemistry of protoplanetary disks and the effect
of stellar UV irradiation Overplotted are phase functions of a) a porous mixture of ices,
silicates, and carbonaceous material assuming a Henyey-Greenstein ap-
proximation to the phase function and b) Tholins assuming a Mie scat-
tering model phase function. 4.4. The chemistry of protoplanetary disks and the effect
of stellar UV irradiation 0.0
0.5
1.0
1.5
2.0
2.5
Wavelength (μm)
3
4
5
6
7
8
9
Fdisk/F* (x10−4)
30
40
50
60
70
80
90
100
Fraction of Tholin Dust (%)
0
1
2
3
4
5
Grain Size (μm) (see Apai et al. 2010, for a recent review). Both scattered light
and thermal emission from these disks can provide composi-
tional information, on the processing of organic compounds. Unfortunately, many debris disks are cold and have few spec-
tral features in the mid-IR. If they are spatially resolved in scat-
tered light, they are often imaged at one or two wavelengths. Therefore, because of the infancy of such observations, more
questions than answers remain. The most detailed scattered light
analysis of a disk comes from observations of the HR 4796A
debris disk observed in seven wavelength bands that span the
visible to the near-IR. From the analysis of Debes et al. (2008),
a mixture of silicates and tholins were found to be the best-
fitting explanation for the red character of the disk, assuming
a purely Mie scattering model of solid spheres (See Fig. 3). Porous aggregates of carbon, water ice, and silicates can also
fit the scattered light and IR spectral energy distribution (SED)
of HR 4796A, providing an alternate explanation (Köhler et al. 2008). However, there are problems with this interpretation, in-
cluding whether water ice should survive in the UV environment
of an A star (Grigorieva et al. 2007). (see Apai et al. 2010, for a recent review). Both scattered light
and thermal emission from these disks can provide composi-
tional information, on the processing of organic compounds. Unfortunately, many debris disks are cold and have few spec-
tral features in the mid-IR. If they are spatially resolved in scat-
tered light, they are often imaged at one or two wavelengths. Therefore, because of the infancy of such observations, more
questions than answers remain. The most detailed scattered light
analysis of a disk comes from observations of the HR 4796A
debris disk observed in seven wavelength bands that span the
visible to the near-IR. From the analysis of Debes et al. (2008),
a mixture of silicates and tholins were found to be the best-
fitting explanation for the red character of the disk, assuming
a purely Mie scattering model of solid spheres (See Fig. 3). 5.1. Structural and chemical characterization of organic
matter in meteoritic chondrites and interplanetary dust In order to identify the source of reddening in meteoritic chon-
drites and interplanetary dust particles (IDPs) we separate the
organic matter into its main components. In order to identify the source of reddening in meteoritic chon-
drites and interplanetary dust particles (IDPs) we separate the
organic matter into its main components. Insoluble organic matter (IOM) is the predominant carbon
material in chondrite meteorites, stratospheric IDPs and antarc-
tic micrometeorites (AMMs). The given environment and the A98, page 7 of 14 A&A 533, A98 (2011) 2.8
3.0
3.2
3.4
3.6
3.8
4.0
0.04
0.05
0.06
0.07
0.08
24 Themis
65 Cybele
PAHs
Orgueil 2.8
3.0
3.2
3.4
3.6
3.8
4.0
0.04
0.05
0.06
0.07
0.08
24 Themis
65 Cybele
PAHs
Orgueil
Fig. 4. Reflectance spectra of 65 Cybele and 24 Themis from Licandro
et al. (2011) show similarities in the band between 2.85 and 3.5 μm. The model shown for 24 Themis (Fig. 1, Rivkin & Emery 2010) and
that for 65 Cybele, include water ice, but not organics. A spectrum of
the insoluble organic material Orgueil (Orthous-Daunay et al. 2010b)
and one showing an average of six polycyclic aromatic hydrocarbons
(Colangeli et al. 1992) are included for comparison. Busemann et al. 2007), and short-duration thermal metamor-
phism (Yabuta et al. 2010). IOM in interplanetary dust particules
(IDPs) and antarctic micrometeorites (AMMs) has a fairly, but
not strictly, similar polyaromatic structure to that of chondrites
(Quirico et al. 2005; Bonal et al. 2006b; Dobrica et al. 2009). Thermal effects occurring during the slowing down of the par-
ticles in Earth’s atmosphere could partly account for these dif-
ferences. The fact that IOM is ubiquitous in IDPs, AMMs and
chondrites precludes its formation in a parent body through hy-
drothermalism (Flynn et al. 2003). This latter process, in com-
parison with thermal metamorphism, has generally weak effects
on the composition of IOM. p
IOM
chemical
heterogeneity is
reflected
in
the
de-
tailed structure of the 3.4-μm band (aliphatic hydrocarbons)
(Orthous-Daunay et al. 2010b). Therefore, tracking of the
3.4-μm band is a potential observational test to infer the pres-
ence or absence of IOM in astrophysical contexts. For exam-
ple in the DISM, infrared spectra point to the presence of an
amorphous hydrogenated carbon material, and lack of IOM as
observed in chondrites (Dartois et al. 2005). 5.2. The nature of asteroids 24 Themis and 65 Cybele 5.2. The nature of asteroids 24 Themis and 65 Cybele While a number of volatile organic molecules have been
identified in the gas phase in cometary comae, of particular
interest for the present study is the more refractory solid car-
bonaceous component of comets. Fomenkova (1999) deduced,
from mass spectrometer observations obtained from the comet
P/Halley flyby mission, that 60% of the carbon on Halley is
in the form of complex organic material. This material affects
the albedo and color of a comet’s nucleus when spatially iso-
lated or mixed with ices. It is of additional interest because this
richly organic molecular material can be dispersed in both the
inner and outer solar system as a consequence of the dynami-
cal properties of comets. Refractory components of comet dust
(organic solids plus minerals) have been collected by aircraft in
the stratosphere (a component of interplanetary dust particles,
as described above), and by the Stardust spacecraft during its
encounter with 81P/Wild 2 (Sandford et al. 2006; Sandford
2008; Clemett et al. 2010). Rotationally-resolved infrared (2−4 μm) spectra of 24 Themis
and 65 Cybele indicate that ice and organics are present on
the surfaces of these asteroids (Campins et al. 2010; Rivkin &
Emery 2010; Licandro et al. 2011) as indicated by the absorp-
tion bands in the range 2.85 to 3.5 μm (Fig. 4). A spectrum of
the IOM Orgueil (Orthous-Daunay et al. 2010b), highlighting
the position of the 3.4 μm, and one showing the average of six
polycyclic aromatic hydrocarbons (Colangeli et al. 1992), are
also shown for comparison. These are the first detections of wa-
ter and organics on asteroid surfaces. Spectral models for these objects include a mixture of sili-
cates, amorphous carbon and water ice and are shown in Fig. 4 as
solid lines. Complex organics are not included in the model: their
absence is highlighted by the discrepancy between model and
observations. The presence of any surface ice on these asteroids
is surprising because of the instability of exposed water ice at
their heliocentric distances (∼3.3 AU). The presence of H2O ice and absence of hydrated silicates
indicate that 24 Themis and 65 Cybele have not undergone the
widespread aqueous alteration or thermal metamorphism rec-
ognized in most carbonaceous meteorites. This argues against
parent-body processes (such as those described above for SOM
in meteorites) as a source of these asteroidal organics. an indication of presolar material (“nature”) or irradiation prod-
ucts (“nurture”). an indication of presolar material (“nature”) or irradiation prod-
ucts (“nurture”). g
p
Finally, we should note that some grains in IDPs and organic
meteorites appear to have intact presolar organics that are highly
enriched in deuterium, suggesting that at least some cold molec-
ular cloud particles experienced relatively non-violent condi-
tions on arrival in the nebula preserving their original signature,
which would be consistent with the “nature” idea. It is especially
striking how the highly enriched grains are found adjacent to
less enriched grains in meteorites (Duprat et al. 2010; Busemann
et al. 2006; Messenger 2002), implying that particles with a wide
variety of histories have coalesced, which would probably sup-
port a scenario of mixing between “nature” and “nurture”. The
importance of sample return from various primitive bodies, per-
haps not so far represented in the meteorite collections, therefore
becomes evident. 5.3. Organic molecules in cometary components Comets are highly relevant to the broad issue of the nature and
distribution of complex organic solid material in the solar sys-
tem. Recent investigations have shown a great compositional
variety among comets. The prevalence of solid opaque materi-
als on their surfaces, either as a consolidated crust or as a mantle
of loose particles, may distinguish short-period comets from dy-
namically new comets. The surfaces of comets 9P/Tempel and
103P/Hartley were imaged in 2005 and 2010, respectively, by
the Deep Impact spacecraft as part of its original mission and
its extended EPOXI mission (see Fig. 5). Both comets appear
to have surfaces that are a combination of crusted regions and
regions covered with fine particles, having low albedos and red-
dish colors characteristic of a combination of mafic silicates and
carbonaceous materials. 5.1. Structural and chemical characterization of organic
matter in meteoritic chondrites and interplanetary dust The 3.4-μm band
could also help connect chondrite chemical classes to asteroids,
still an appreciable challenge, considering the currently achiev-
able signal precision (Licandro et al. 2011). Fig. 4. Reflectance spectra of 65 Cybele and 24 Themis from Licandro
et al. (2011) show similarities in the band between 2.85 and 3.5 μm. The model shown for 24 Themis (Fig. 1, Rivkin & Emery 2010) and
that for 65 Cybele, include water ice, but not organics. A spectrum of
the insoluble organic material Orgueil (Orthous-Daunay et al. 2010b)
and one showing an average of six polycyclic aromatic hydrocarbons
(Colangeli et al. 1992) are included for comparison. Fig. 4. Reflectance spectra of 65 Cybele and 24 Themis from Licandro
et al. (2011) show similarities in the band between 2.85 and 3.5 μm. The model shown for 24 Themis (Fig. 1, Rivkin & Emery 2010) and
that for 65 Cybele, include water ice, but not organics. A spectrum of
the insoluble organic material Orgueil (Orthous-Daunay et al. 2010b)
and one showing an average of six polycyclic aromatic hydrocarbons
(Colangeli et al. 1992) are included for comparison. A98, page 8 of 14 5.4. The surface composition of Jupiter Trojans A frac-
tion of cometary PAHs containing N atoms in the form of aro-
matic nitriles represent a significant difference from meteorite
organics (Clemett et al. 2010). Both O and N atoms are found in
a variety of bonding states, while some particles contain abun-
dant C atoms that are not in aromatic structures (Sandford 2008). Those particles lacking “aromatization” are instead more likely
to support the “nature” situation. Initial compositional interpretations ascribed their low albe-
dos and red, featureless visible spectral slopes at least partly to
the presence of organics (Gradie & Veverka 1980). Comparisons
with comets and other outer solar system bodies are not uncom-
mon (e.g., Jewitt 2002), given the similarly dark, spectrally red
surfaces, but many low-albedo Main Belt asteroids also exhibit
many of the same spectral qualities. Detecting and characteriz-
ing the possible organic component of JTs would move us a step
closer to understanding the origin and evolutionary processes
important for small bodies throughout the solar system. p
g
y
Statistical analyses of the featureless reflectance spectra have
provided some insights. Szabó et al. (2007) reported a corre-
lation between visible colors (from Sloan Digital Sky Survey
[SDSS] measurements) and orbital inclination for JTs that man-
ifested itself as an apparent bimodal distribution of spectral
slopes. Roig et al. (2008) confirm the SDSS bimodal spec-
tral slope distribution and find a similar distribution in previ-
ously published visible spectra. Emery et al. (2011) identify
a much stronger bimodality in near-infrared spectral slopes of
JTs. This bimodality is present in both the leading and trailing
swarms and among the background (non-family member) JTs,
and therefore appears to be a signature of the primordial pop-
ulation rather than an artifact of collisional families or capture
mechanisms. Spectral similarities between the redder group and
some Centaurs and the less-red group and some outer Main Belt
asteroids may indicate multiple source regions for JTs. Organic molecules such as CH4, C2H2, C2H6 and CH3OH
have been identified in the gas phase of comets and in solid
phases from IDPs and Stardust samples. It is therefore reason-
able to infer that many short-period comet nuclei, the only ones
we have been able to observe so far, have their low albedos and
red colors, at least in part, due to the presence of a wide range of
complex molecules. 5.4. The surface composition of Jupiter Trojans Jupiter Trojans (JTs) are one of the most intriguing populations
of small bodies in the solar system. Currently residing in sta-
ble orbits in the Lagrangian points of Jupiter, JTs are believed
to have formed at relatively large heliocentric distances, where
the solar nebula was rich in frozen volatiles and organics (Dotto
et al. 2008; Marzari et al. 2002). Jupiter Trojans (JTs) are one of the most intriguing populations
of small bodies in the solar system. Currently residing in sta-
ble orbits in the Lagrangian points of Jupiter, JTs are believed
to have formed at relatively large heliocentric distances, where
the solar nebula was rich in frozen volatiles and organics (Dotto
et al. 2008; Marzari et al. 2002). The origin of JTs and how they were trapped in librating or-
bits around the Jupiter Lagrangian points are still a matter of de-
bate. Several mechanisms have been so far proposed, including
capture of fragments of Jovian satellites, trapping of planetesi-
mals orbiting nearby a proto-Jupiter, mass growth of the planet
or gas drag capture of short-period comets (Marzari et al. 2002). Morbidelli et al. (2005) suggested that JTs, like Kuiper belt ob-
jects (KBOs) and the scattered disk, originated in the planetes-
imal disk which drove the planetary migration. Therefore, the
analysis of the physical properties, in particular the composition
of JTs, can help us place important constraints on their origin
and early evolution. Fig. 5. The surfaces of comets 9P/Tempel and 103P/Hartley imaged,
in 2005 and 2010, respectively, by the Deep Impact spacecraft in its
original mission and its extended EPOXI mission. P/Tempel is 7.6 km
across and Hartley is 2.2 km in its long dimension. The figure shows
them to the same scale. Image credit: NASA/JPL-Caltech/UMD. exactly. The Stardust mission particles show extreme hetero-
geneity (Sandford et al. 2006); some show evidence of a high
degree of heating expected from formation in the inner solar sys-
tem, while others could not have survived such temperatures and
must have originated further out. These data corroborate what
was previously known from cometary IR spectra showing the
existence of crystalline silicates in comets in contrast to their
apparent scarcity in the ISM (Ehrenfreund et al. 2004, and ref-
erences therein). This is also an argument in favor of mixing
of “high-temperature” and “low-temperature” materials in the
comet-forming region. 5.2. The nature of asteroids 24 Themis and 65 Cybele The
presence of C-H bonds and possibly aromatic groups could be The organic molecules of cometary origin in the Stardust
samples are similar, in many respects, to those found in the
matrix material of some carbonaceous chondrite meteorites and
IDPs. A detailed analysis of the X-ray absorption spectra of
the Stardust particles by Wirick et al. (2009) has shown that
these spectra are closer to the spectra of IDPs than those
of carbonaceous chondrites, but do not match either category A98, page 8 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems Fig. 5. The surfaces of comets 9P/Tempel and 103P/Hartley imaged,
in 2005 and 2010, respectively, by the Deep Impact spacecraft in its
original mission and its extended EPOXI mission. P/Tempel is 7.6 km
across and Hartley is 2.2 km in its long dimension. The figure shows
them to the same scale. Image credit: NASA/JPL-Caltech/UMD. comet nuclei may include complex molecules analogous to the
soluble fraction in the meteorites (e.g., amino and hydroxylic
acids, and nitrogen heterocycles). Therefore, comets are plausi-
bly a mixture of native and irradiation products, with their initial
composition differing and being a function of their place of for-
mation (Ehrenfreund et al. 2004, and references therein), and
advocating for both the “nature” and “nurture” scenarios. 5.4. The surface composition of Jupiter Trojans The “high temperature” group supports
the idea of a “nurture” scenario, while the “low-temperature”
one could be presolar in origin and promote the “nature” alter-
native. Aliphatic compounds appear to have withstood temper-
atures of ∼550 C, probably because of protection by minerals
such as sulfides. Specific identifications include PAHs, with a
predominance of single (benzene) and double ring (naphthalene)
molecules, but also including much larger PAHs (extending up
to ∼800 a.m.u.), as well as their alkylation series resulting from
the successive addition of CH2- subunits. However, it is possible
that PAHs formed as a result of the high temperature exposure,
which would vouch for “nurture” rather than “nature”. A frac-
tion of cometary PAHs containing N atoms in the form of aro-
matic nitriles represent a significant difference from meteorite
organics (Clemett et al. 2010). Both O and N atoms are found in
a variety of bonding states, while some particles contain abun-
dant C atoms that are not in aromatic structures (Sandford 2008). Those particles lacking “aromatization” are instead more likely
to support the “nature” situation. exactly. The Stardust mission particles show extreme hetero-
geneity (Sandford et al. 2006); some show evidence of a high
degree of heating expected from formation in the inner solar sys-
tem, while others could not have survived such temperatures and
must have originated further out. These data corroborate what
was previously known from cometary IR spectra showing the
existence of crystalline silicates in comets in contrast to their
apparent scarcity in the ISM (Ehrenfreund et al. 2004, and ref-
erences therein). This is also an argument in favor of mixing
of “high-temperature” and “low-temperature” materials in the
comet-forming region. The “high temperature” group supports
the idea of a “nurture” scenario, while the “low-temperature”
one could be presolar in origin and promote the “nature” alter-
native. Aliphatic compounds appear to have withstood temper-
atures of ∼550 C, probably because of protection by minerals
such as sulfides. Specific identifications include PAHs, with a
predominance of single (benzene) and double ring (naphthalene)
molecules, but also including much larger PAHs (extending up
to ∼800 a.m.u.), as well as their alkylation series resulting from
the successive addition of CH2- subunits. However, it is possible
that PAHs formed as a result of the high temperature exposure,
which would vouch for “nurture” rather than “nature”. 5.4. The surface composition of Jupiter Trojans Such molecular complexes can occur both
in recognizable structures and in more complex aggregates sim-
ilar to kerogen-like materials in the insoluble organic matter
in carbonaceous meteorites. Similarly, the volatile fraction in Of particular interest in the present context are direct
searches for organic materials. Strong fundamental vibrational
bands of C-H, O-H, and N-H bonds in complex organics occur at
wavelengths in the 2.7 to 3.6 μm region, and overtones and com-
binations of these, though significantly weaker, occur at shorter
wavelengths in the reflectance spectra of organics. Several au-
thors (e.g. Cruikshank et al. 2001; Emery & Brown 2003; Dotto
et al. 2006; Fornasier et al. 2007; de Luise et al. 2010; Emery
et al. 2011; Yang & Jewitt 2011) have published visible and near-
infrared reflectance spectra of JTs. Despite the expectation that A98, page 9 of 14 A&A 533, A98 (2011) JTs formed with abundant volatiles and organics, as for KBOs,
they do not appear to exhibit any of the spectral features of these
materials on their surfaces. However, as there is more than an or-
der of magnitude difference in the solar flux on Trojans, at 5 AU,
compared to the KBOs, the difference in spectral properties of
the uppermost surfaces of Trojans and KBOs does not necessar-
ily preclude a similar origin. A thermal model of the evolution of Iapetus’ surface (Spencer &
Denk 2010), in which H2O ice is mobilized by the higher tem-
peratures in the dark hemisphere and re-condensed elsewhere,
convincingly reproduces the albedo pattern seen today. An absorption band at 3.28 μm, attributed to polycyclic aro-
matic hydrocarbons, has been identified in the low-albedo mate-
rial on Iapetus (Cruikshank et al. 2008). The data for that study
came from the Visible- Infrared Mapping Spectrometer (VIMS)
aboard the Cassini spacecraft that has been operating in orbit
around Saturn since late 2004. The detection of PAHs is the first
clear identification of a significant chemical component. Current
studies are attempting to discern which of the two low-albedo
components presents the PAH signature. The characterization of
the dark components of Iapetus is important as it sheds light on
the origin of the materials and their evolution, as well as the “na-
ture” versus “nurture” question regarding the satellites of Saturn. Furthermore, the featureless visible spectra seem to rule
out a significant quantity of hydrated silicates, crystalline an-
hydrous silicates, organic materials and ices. 5.6. The surface composition of trans-neptunian objects
and Centaurs The TNOs or KBOs are small icy bodies (the diameters of
known objects range between about 50 and 3000 km) that orbit
the Sun beyond Neptune. More than 1200 of them have been dis-
covered so far. A number of spectroscopic and dynamical stud-
ies (Barucci et al. 2008a, and references therein) have shown
that TNOs are remnants of the formation of the solar system
that have been subjected to minimal chemical evolution since
their formation. Their upper surface layers have however been
altered by various space weathering processes (solar and cosmic
ion irradiation, collisions) and, for the largest of them, surface-
atmosphere interaction and, possibly, cryovolcanism. A study of
their surface compositions can therefore provide clues on solar
nebula composition and solar system evolution as well as the
origin of their reddening material. The detection of organic features in the 3.3 to 3.5 μm region
on two outer Main Belt asteroids, 24 Themis and 65 Cybele,
as noted in Sect. 5.2, is instructive. Only one of the JT spectra
has high enough S/N in this wavelength range to have detected a
Themis- or Cybele-like absorption feature. It therefore remains
possible that, though not yet detected, organics may be a minor
component on the surfaces of JTs. It is likely though, considering
their relative proximity to the Sun, that their organic material, if
present, could be partially the product of the irradiation of the
original surface components. Based on the amount of processing
that these surface have undergone the “nurture” scenario would
likely be the prevalent one. The dynamical evolution of TNOs is believed to have been
cataclysmic. A currently popular dynamical model (the Nice
model, Levison & Morbidelli 2003) describes a series of events
in the young solar system that caused the migration of the outer
giant planets Neptune and Uranus, and the scattering of the plan-
etesimals left over from their formation. TNOs are distinguished
(Elliot et al. 2005; Gladman et al. 2008) into several different
classes that describe their current locations and orbital character-
istics: the classical objects, the resonant objects (such as Pluto,
which is in 2:3 orbital resonance with Neptune), the scattered
disk objects and the detached objects (like Sedna). In addition,
Centaurs (such as Pholus or Chiron) have orbits between those
of Jupiter and Neptune. 5.6. The surface composition of trans-neptunian objects
and Centaurs They may be escapees from the Kuiper
belt (former TNOs), they are therefore studied in parallel with
TNOs, but being closer to us, they are usually brighter. 5.4. The surface composition of Jupiter Trojans Amorphous sili-
cates have been suggested to model the red slopes of JT spectra
(Cruikshank et al. 2001; Emery & Brown 2004), but feature-
less spectra provide at best a weak constraint on inferred com-
positions. However, thermal emission spectra of several Trojans
show strong spectral emissivity peaks near 10 and 20 μm, in-
dicating the presence of fine-grained, largely amorphous, anhy-
drous silicates (Emery et al. 2006; Mueller et al. 2010). An absorption feature at ∼2.3 μm found in the spectra of the
Centaur Pholus and the TNO 2002 VE95 has been attributed to
organic materials (Cruikshank et al. 1998; Barucci et al. 2006),
and specifically to frozen CH3OH or (less likely) hexamethylte-
tramine. However, near-infrared spectra of ∼100 JTs have been
published by various authors, and none of them show this fea-
ture. Spectroscopic searches for the strong fundamental absorp-
tions at longer wavelengths (2.8 to 4.0 μm) have been published
for eight JTs. No absorption features have been found in these
spectra, which strongly limits the type and abundance of organic
material on the surfaces of JTs (Cruikshank et al. 2001; Emery
& Brown 2004). C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems Quaoar best fitting model (solid thin line) includes about
30% H2O, 20% N2, and 13% CH4 with traces of C2H6 ice mixed in with
organic materials (Dalle Ore et al. 2009). Hydrocarbon ices features are
labeled in the figure. Quaoar is an RR object. The observations were
scaled to Stansberry et al. (2008) visual geometric albedo. Fig. 6. Quaoar best fitting model (solid thin line) includes about
30% H2O, 20% N2, and 13% CH4 with traces of C2H6 ice mixed in with
organic materials (Dalle Ore et al. 2009). Hydrocarbon ices features are
labeled in the figure. Quaoar is an RR object. The observations were
scaled to Stansberry et al. (2008) visual geometric albedo. Table 1. Selected objects’ compositions. Table 1. Selected objects’ compositions. Object
Dyn
Taxon
a∗
%
%
%
class
reddening darkening
ices
5145 Pholusa
C
RR
20.3
34
61
15
8405 Asbolusb
C
BR
18.1
32
59
9
1999TC36c
R
RR
39.7
70
22
8
50000 Quaoard
C
RR
43.6
27
0
73
90377 Sednae
D
RR
518.6
76
0
24
90482 Orcusf
R
BB
39.2
1
58
40
Notes. (∗) Semi-major axis measurements (a) are from JPL Small-Body
Database Browser at http://ssd.jpl.nasa.gov/sbdb.cgi
References. (a) Cruikshank et al. (1998); (b) Poulet et al. (2002); (c) Dotto
et al. (2003); (d) Dalle Ore et al. (2009); (e) Barucci et al. (2010);
(f) Delsanti et al. (2010). p
y
μ
When spectroscopic data are not available, colors have been
obtained for a relatively large sample of objects (around 200). TNOs as well as Centaurs have very diverse colors, from neutral
to very red. A taxonomic scheme based on multivariate statis-
tics of the photometric color was proposed by Barucci et al. (2005): four classes were identified indicating different composi-
tion and/or evolutional history, with increasingly red colors from
the BB (blue objects) to the RR class (reddest objects). The rela-
tionship between taxonomical and dynamical classifications has
been investigated in detail (Perna et al. 2010) yielding somewhat
inconclusive results. No direct correlation between color and dy-
namical parameters (in particular semi-major axis) was found. However, recent work by Romanishin et al. C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems (2010) shows some
evidence for a color gradient with distance from the Sun, if the
Inner Classical KBOs (ICKBOs) are considered to be an exten-
sion of the Cold Classical population: the Cold Classical objects
are predominately red, and the Inner Classical belt objects are a
mixture of neutral and red. function of distance. However, the measured semi-major axes
describe the current position of these objects, after the migration
of Neptune had scattered them: it therefore comes as no surprise
that their compositions do not relate to their locations. Out to the Kuiper belt, we expect that current weathering
from solar irradiation would affect the surface compositions as
a function of distance from the Sun, with the closest objects
being most processed. This picture becomes more complicated
at distances beyond 50 AU where cosmic rays become pre-
dominant (Cooper et al. 2006). As seen in Sect. 5.4 close to
the Sun weathering happens on very short timescales, imply-
ing that some weathering effects should be seen. In fact, we
do see the effects of weathering on the IKBOs, as mentioned
above. However, for the TNOs, based on the complete lack of
correlation between their composition, and their current semi-
major axis, particularly for those that are within the 50 AU ra-
dius, two possible explanations become apparent. The first is
that not a lot of weathering has happened since Neptune mi-
gration repositioned objects to their current locations. The sec-
ond that the collision rate is comparable to the weathering rate,
keeping the surfaces fresh (Melita et al. 2009). In both cases
the direct implication is that the weathering that we observe, at
least in some of the objects (i.e., the presence of a population
of objects with highly processed, dark materials, such as Orcus),
must have preceded the migration and must have happened on a
short timescale early on. When looking at the TNOs, a limited number of high resolu-
tion TNO spectra have been modeled using a variety of com-
positions that include several ices, carbonaceous, and organic
materials varying in proportion from object to object. Figure 6
shows the best fitting model of Quaoar, an RR object, orbit-
ing at about 44 AU. C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems 1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
0.2
0.4
0.6
0.8
1.0
Geometric Albedo
Wavelength (μm)
(50000) Quaoar
Dalle Ore et al. (2009)
N2
C2H6
CH4
Fig. 6. Quaoar best fitting model (solid thin line) includes about
30% H2O, 20% N2, and 13% CH4 with traces of C2H6 ice mixed in with
organic materials (Dalle Ore et al. 2009). Hydrocarbon ices features are
labeled in the figure. Quaoar is an RR object. The observations were
scaled to Stansberry et al. (2008) visual geometric albedo. 1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
0.2
0.4
0.6
0.8
1.0
Geometric Albedo
(50000) Quaoar
Dalle Ore et al. (2009)
N2
C2H6
CH4 simple distribution was apparently altered by giant planet migra-
tion and planetesimal scattering like that described in the Nice
model, yielding the much more complex picture that we observe
today. Another complication in the unraveling of the chemical his-
tory of solar system bodies lies with the processing due to so-
lar and cosmic radiation that has weathered their surfaces, more
effectively for those closer to the Sun. The best candidates for
our study are probably the smaller TNOs, which are possibly far
enough from the Sun to be sufficiently pristine to be useful in our
investigation. Larger ones are not as reliable as internal melting
and differentiation occurred in their history. Information about TNO and Centaur surface compositions
or physical properties comes from albedo measurements, color
measurements, polarimetric observations and spectroscopy. The
measured albedos are low (between 0.04 and 0.20), except for
a few objects that have albedos of about 0.6 (Pluto, Makemake,
Haumea) or even higher (∼0.86 for Eris, Stansberry et al. 2008
and 2000 TX300; a member of the Haumea family, Elliot et al. 2010). Their generally low albedos, their distances, and their
sizes make them very faint and therefore difficult to observe. Even with the largest telescopes available (8 to 10 m class),
only the brightest TNOs can be studied spectroscopically to
obtain compositional information. Furthermore, the available
TNO spectra extend only to about 2.5 μm (Barucci et al. 2008b),
a limiting factor when trying to identify surface components
whose fundamental spectral features often fall beyond 2.5 μm. Wavelength (μm) Wavelength (μm) Fig. 6. 5.5. Polycyclic aromatic and aliphatic hydrocarbons
on Saturn’s satellite Iapetus Iapetus, Saturn’s third largest satellite, is an icy moon show-
ing spectral signatures of H2O ice over more than half its sur-
face. The low-albedo material on Iapetus’ surface is concen-
trated primarily on the leading hemisphere in the sense of the
satellite’s locked synchronous orbital motion around Saturn, but
telescopic studies undertaken since the discovery of the albedo
dichotomy by G. D. Cassini in 1671 have failed to convincingly
establish the composition of the dark material. New studies,
based on data from the Cassini spacecraft demonstrate that there
are two low-albedo surface components, one very red in color
and the other more nearly neutral (Denk et al. 2010), one native
to Iapetus, the other exogenous in nature. The native material is
probably ubiquitously present to some degree and was originally
evenly distributed on the surface. Both low-albedo components
contribute to the dark coloration of the leading hemisphere of
Iapetus and are the cause of a temperature enhancement detected
in this area of the satellite surface. They are ultimately respon-
sible for the material segregation on the surface of the satellite. The chemical history of TNOs is unfortunately not as clearly
understood. The concept of “snow lines” in the original neb-
ula, i.e. the distance from the Sun at which materials can con-
dense, provides us with some idea of what should have been the
distribution of materials before the neptunian migration. In fact,
we can envision that planetesimals, originally formed close to
Jupiter, have little more than rock, metal, and water ice as part of
their composition. On the other hand, planetesimals originating
outward of Saturn would consist of more volatile ices as well as
water ice and rock and metal depending on their distance from
the Sun and size (Schaller & Brown 2007). As a result, we ex-
pect that the original material distribution would have followed
a composition pattern from simple to more complex in travel-
ing from the inner to the outer solar system. Unfortunately this A98, page 10 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems Acknowledgements. This paper is based on the conclusions of the work-
shop “Organic material in planetary system: “nature” or “nurture”?” held
on May 27−29, 2010 at the Centre International d’Ateliers Scientifiques de
l’Observatoire de Paris with the participation of all the authors. C.M.D.O.
aknowledges support from the Centre International d’Ateliers Scientifiques de
l’Observatoire de Paris. N.P.A. acknowledges support from the NASA postdoc-
toral program at Ames Research Center administered by Oak Ridge Associated
Universities through a contract with NASA. C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems Its composition is listed among a few rep-
resentative cases in Table 1, where the published quantities of
the different components have been grouped together into three
categories: reddening components (mostly tholins), darkening
components (mostly amorphous carbon) and ices (hydrocar-
bons, H2O, CO2, CO, CH3OH ices). From a compilation of
objects with spectra available in the literature (examples shown
in Table 1) it appears that composition and semi-major axis (a)
also do not correlate: ice abundance does not map with distance
and likewise objects with a high abundance of processed dark
materials do not orbit closest to the Sun. Grundy’s idea (i.e. the “nature” scenario) of the reddening
material being primarily primordial in origin, would imply that
the color of the objects vary with the quantity of ice and as a A98, page 11 of 14 A98, page 11 of 14 A&A 533, A98 (2011) Table 2. “Nature” vs. “nurture” summary. Evidence
“Nature”
“Nurture”
? Laboratory studies of refractory carbonaceous materials
X
X
–
Ion irradiation and colors of C-rich materials
–
X
–
The nature of interstellar organics
X
–
–
Hydrocarbonaceous dust in the ISM
X
–
–
Dense clouds
X
–
–
Evolution of organics from diffuse interstellar clouds to the solar system
X
–
–
Chemistry of protoplanetary disks
–
–
X
Evidence for organics in dusty disks
–
–
X
Refractory organic matter in chondrites and interplanetary dust
X
X
–
Asteroids 24 Themis & 65 Cybele
–
–
X
Comets
X
X
–
The surface composition of Jupiter Trojans
–
–
X
PAHs and aliphatic hydrocarbons on Iapetus
X
X
–
The surface composition of trans-Neptunian objects and centaurs
X
X
– Some of the key factors outlined above are within reach with
the current state-of-the-art telescopes, and observing instruments
(e.g. d above), others are far from being solvable. We can therefore conclude, for the TNO case, that the
“nature” reddening mechanism, whose traces are apparent in
those objects with a lot of ices and red coloration, as well as
the “weathering” component visible in the processed materials
(“nurture”) could be interpreted, for some of the objects, as to
date from a time previous to the neptunian migration. 6. Conclusions We present an examination of some recent evidence, encom-
passing a number of laboratory experiments as well as obser-
vational data, in an effort to characterize the reddening material
common to planetary systems. The available information, dis-
cussed at length among the authors, leads us to infer that the red
coloration of a variety of objects in the solar system could be
due mainly to one of two mechanisms, or more likely to a com-
bination of them: weathering and primordial composition, i.e. “nurture” and “nature”. Nevertheless, materials such as PAHs, indicative of the pres-
ence of organics, are difficult to interpret, as their origin could
be due to “nurture” as well as “nature”. The spectroscopic ef-
fects of the “nature” and “nurture” mechanisms on the icy ma-
terial are practically indistinguishable: while intrinsically differ-
ent, macroscopically, they both reduce the ratio of H to C atoms. Further, with the current state of the art instruments we cannot
yet detect the signature features of the organic material, whether
primordial or resulting from irradiation, in most of the available
observations. A few exceptions include Cassini VIMS data for
the Saturn satellites, Triton, Pluto, 24 Themis and 65 Cybele
data. Laboratory work has given, and is still in the process of giv-
ing, us a few tools to aid us in discriminating between the differ-
ent choices. In this paper we explore in the observations avail-
able in the literature the evidence for one process or the other,
and view them in the frame of the knowledge acquired through
the laboratory studies. Finally, a model of the dynamical evolution of the outer solar
system has to be kept in the picture as the current location of the
objects probably does not reflect their original position, therefore
complicating the history of the weathering contributions. Table 2 summarizes the current status of the “nature” vs. “nurture” debate. In summary, the question at the basis of the “nature” vs. “nur-
ture” debate, i.e. whether ice indeed magnifies the effect of the
reddening material, is still to be fully addressed. While some as-
pects of the problem have been answered or will be in the near
future, such as the determination of the amount of ice present in
red objects, others, such as the ability to discriminate between
reddening agents, are still far from being solvable. C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems At that
time both mechanisms might have played a role, but their rela-
tive importance is too difficult to trace as their effect on the spec-
trum of the material is so macroscopically similar as to make it
impossible, at this time, to discern the importance of one vs. the other. The identification of the material dwells primarily on the
ability to obtain spectroscopic data past the 2.5 μm current limit
for a number of well chosen objects representing the main tax-
onomic groups of TNOs, Centaurs, asteroids and Trojans in the
hope that the behavior in the near-IR will shed light on their
compositions, help map the reddening material(s) throughout
the outer solar system, and distinguish minerals from complex
organics. This information along with dynamical data might
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https://openalex.org/W2972084011
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https://www.frontiersin.org/articles/10.3389/fpsyg.2019.02135/pdf
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English
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Surface Stickiness Perception by Auditory, Tactile, and Visual Cues
|
Frontiers in psychology
| 2,019
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cc-by
| 6,327
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Edited by: Edited by:
Joachim Lange,
Heinrich Heine University Düsseldorf,
Germany Reviewed by:
Julian Keil,
Kiel University, Germany
Wanjoo Park,
New York University Abu Dhabi,
United Arab Emirates
*Correspondence:
Junsuk Kim
junsuk.kim@skku.edu Reviewed by:
Julian Keil,
Kiel University, Germany
Wanjoo Park,
New York University Abu Dhabi,
United Arab Emirates New York University Abu Dhabi,
United Arab Emirates Surface Stickiness Perception by
Auditory, Tactile, and Visual Cues Hyungeol Lee1, Eunsil Lee2, Jiye Jung3 and Junsuk Kim4,5* 1 Department of Philosophy, Sogang University, Seoul, South Korea, 2 Department of Psychology, Duksung Women’s
University, Seoul, South Korea, 3 Department of Psychology, Chung-Ang University, Seoul, South Korea, 4 Center
for Neuroscience Imaging Research, Institute for Basic Science, Suwon, South Korea, 5 Department of Biomedical
Engineering, Sungkyunkwan University, Suwon, South Korea This study aimed to explore the psychophysical bases of multisensory surface stickiness
perception by investigating how sensitively humans perceive different levels of stickiness
intensity conveyed by auditory, tactile, and visual cues. First, we sorted five different
sticky stimuli by perceived intensity in ascending order for each modality separately and
evaluated the discrimination sensitivities of each participant using a fitted psychometric
curve. Results showed that perceptual intensity orders were not identical to physical
intensity order and that the sequential order of perceived intensities for different
modalities was inconsistent. Moreover, estimated perceptual sensitivities to surface
stickiness indicated that auditory cues result in better discrimination sensitivity than
tactile and visual cues. Second, we calculated the relative perceptual distances
of stickiness intensities using multidimensional scaling. A follow-up statistical test
demonstrated that the perceptual mapping of vision and touch are similar but that
auditory perception is different. These results suggest that the discriminability of
stickiness intensity is best served by auditory cues and that texture information
processing in the auditory domain is distinctive from that of other modalities. INTRODUCTION *Correspondence:
Junsuk Kim To interact effectively with surrounding objects, humans need to acquire surface texture
information from objects using different sensory modalities. For example, when perceiving surface
characteristics of a certain object, we could obtain texture information (1) by looking at the surface,
(2) by touching it with our hands, or (3) by hearing sounds generated by interacting with it. Previous research has shown that information from the visual channel, relative to other modalities,
is weighed most strongly for object perception (Heller, 1982) and this also applies to surface
texture perception (Tiest and Kappers, 2007). On the other hand, it has been demonstrated that
tactile information is essential in perceiving the characteristics of surface texture (Lederman and
Klatzky, 2009) and auditory cues play important roles in texture discrimination tasks (Lederman
and Abbott, 1981; Lederman et al., 2002; Drewing et al., 2004). Based on these previous studies,
there appears to be no fixed sensory dominance for texture perception and it seems that sensory
dominance is largely dependent on specific aspects of surface texture, e.g., particle size of rough
surfaces (Lederman and Klatzky, 2004). Specialty section:
This article was submitted to
Perception Science,
a section of the journal
Frontiers in Psychology Received: 14 May 2019
Accepted: 03 September 2019
Published: 18 September 2019 Received: 14 May 2019
Accepted: 03 September 2019
Published: 18 September 2019 Keywords: texture perception, surface stickiness, auditory cues, tactile cues, visual cues BRIEF RESEARCH REPORT
published: 18 September 2019
doi: 10.3389/fpsyg.2019.02135 Stimuli Five different kinds of repositionable tape (9415PC, 665, 9183,
9495, and 9071; 3M Center, St. Paul, MN, United States) were
prepared for the experiment. To measure physical stickiness
strength of these tapes, we employed a “probe tack test”
that measures the peak value of adhesive force indicating the
instantaneous adhesion property. Using a probe in the shape
of a stainless steel ball (1-inch diameter), we measured the
adhesive force that occurs when a probe is peeled offat peeling
rate 2 mm/sec. Previous studies have considered this test as
a qualitative approach to evaluate tactile sensations of human
(Mith et al., 2008; Cakmak et al., 2011). The physical stickiness
intensities of tape 9415PC, 665, 9183, 9495, and 9071 were
estimated as 22.9, 124.5, 330.0, 419.2, and 558.7 gf (gram-force),
respectively. According to these values, we labeled five different
physical stickiness intensities as level 1 to level 5: 9415PC (level
1), 665 (level 2), 9183 (level 3), 9495 (level 4), and 9071 (level 5). With these tapes, we created auditory, tactile, and visual stimuli. Since this is the first study of the stickiness dimension
using various sensory modalities, we formulated a hypothesis
based on previous findings of multisensory roughness perception
(Lederman and Klatzky, 2004; Klatzky and Lederman, 2010). If the perceptual characteristics of surface roughness hold for
stickiness stimuli, we can assume that sensitivity in stickiness
perception would be similar for vision and touch and less for
the auditory modality (Lederman and Klatzky, 2004). However,
there is an obvious difference in visual perception for rough
compared to that for sticky surfaces. Roughness intensity can be
perceived visually by looking at the surface texture (e.g., particle
size or inter-particle distance), whereas it is difficult to measure
stickiness intensity by observing the surface texture. Moreover,
in our daily life, we are far more familiar with tactile stickiness
than with visual and auditory cues for stickiness. Therefore, we
hypothesized that the sense of touch would show the highest
sensitivity in surface stickiness perception. • Auditory stimuli: Sound clips of tactile explorations (i.e., a
sound when touching and detaching with an index finger)
were recorded with a mobile condenser microphone. Each
clip lasted 3 s and was sub-divided into a touching and then
a detaching period of about 1.5 s each. • Tactile stimuli: Tapes were prepared in a size of 5 × 1.9 cm
and attached to an acrylic plate sized 5 × 8 cm. Stimuli The plastic
plate was used to enable the experimenter to present the
stimuli easily and without direct contact. All tactile stimuli
were used only once, then replaced by new tape. • Visual stimuli: Video clips of tactile explorations were
recorded with a resolution of 1920 × 1080 at 60 frames per
second (Figure 1). Each clip displayed a right index finger
touching one of the five tapes and lifting off. The recording
video camera was positioned at a distance of 20 cm from the
stimulus surface and 5 cm above the tabletop. Each video
clip lasted 3 s. In this psychophysical study, we investigated how differently
stickiness perception is mediated by auditory, tactile, and visual
cues. To be specific, we examined (1) in which modality
people are the most sensitive in terms of stickiness intensity
discrimination using the just noticeable difference (JND), (2)
the relative similarity of stickiness intensities across modalities
using multidimensional scaling (MDS), and (3) the relationship
between physical and perceived stickiness intensities. Note that we did not match the absolute perceptual intensities
across sensory modalities. We were aware of that stimulus
intensities can be perceived in a different scale depending on the
modalities, but the primary consideration was that our stimuli
should be expressed its unique characteristics as fully as possible. Citation: Moreover, in
these studies, the authors have used sticky stimuli such as liquid
glue, prunes, molasses, and jelly (Zigler, 1923), or silicone-
based sticky objects into which fast catalysts were mixed in
several proportions (Yeon et al., 2017). All these sticky stimuli
are difficult to quantify in terms of the physical intensities of
stickiness. To make up for these shortcomings and improve
the reproducibility of the research, we used adhesive tapes
which are relatively easy to obtain and have stickiness intensity
of several levels. (IRB# 2018-05-001) and the study was conducted in accordance
with the Declaration of Helsinki. All participants were informed
about the experimental procedure and gave written informed
consent prior to their participation. Citation: Lee H, Lee E, Jung J and Kim J
(2019) Surface Stickiness Perception
by Auditory, Tactile, and Visual Cues. Front. Psychol. 10:2135. doi: 10.3389/fpsyg.2019.02135 It
is
well-known
that
the
perception
of
surface
texture
is
multidimensional,
e.g.,
roughness/smoothness,
hardness/softness,
stickiness/slipperiness,
and
warmth/coolness September 2019 | Volume 10 | Article 2135 1 Frontiers in Psychology | www.frontiersin.org Lee et al. Surface Stickiness Perception (Hollins et al., 1993; Bensmaia and Hollins, 2005; Bensmaia,
2016). Compared to other dimensions, stickiness is one of the
least investigated properties of surface perception (Bensmaia,
2016). Stickiness can be defined as a mechanical sensation
related to the friction between skin and surface, or stretch of
skin (Bensmaia, 2016). More specifically, it can be sub-divided
into non-slipperiness arising from horizontal movement and
stickiness stimulated by vertical movement. Note that the current
study focuses on stickiness perception evoked by vertical pull-
offmovements. There are several studies on stickiness perception
and its neural mechanism (Zigler, 1923; Kim et al., 2017; Yeon
et al., 2017), but they examined solely stickiness from the tactile
sense, and not from visual and auditory senses. Moreover, in
these studies, the authors have used sticky stimuli such as liquid
glue, prunes, molasses, and jelly (Zigler, 1923), or silicone-
based sticky objects into which fast catalysts were mixed in
several proportions (Yeon et al., 2017). All these sticky stimuli
are difficult to quantify in terms of the physical intensities of
stickiness. To make up for these shortcomings and improve
the reproducibility of the research, we used adhesive tapes
which are relatively easy to obtain and have stickiness intensity
of several levels. (Hollins et al., 1993; Bensmaia and Hollins, 2005; Bensmaia,
2016). Compared to other dimensions, stickiness is one of the
least investigated properties of surface perception (Bensmaia,
2016). Stickiness can be defined as a mechanical sensation
related to the friction between skin and surface, or stretch of
skin (Bensmaia, 2016). More specifically, it can be sub-divided
into non-slipperiness arising from horizontal movement and
stickiness stimulated by vertical movement. Note that the current
study focuses on stickiness perception evoked by vertical pull-
offmovements. There are several studies on stickiness perception
and its neural mechanism (Zigler, 1923; Kim et al., 2017; Yeon
et al., 2017), but they examined solely stickiness from the tactile
sense, and not from visual and auditory senses. MATERIALS AND METHODS Twenty-five
paired
stimuli
(counterbalanced
pairwise
combinations of five distinct intensities, plus five pairs of
the same intensity; 5P2 + 5 = 25) were used for each modality
condition and these pairs were presented once in each block. Participants performed 5 blocks for each modality, thus there
were a total of 375 trials (3 modalities × 5 blocks × 25 pairs). A single trial presented two stimulus pairs one after another
for 3 s and there was a pause of 1.5 s between them (Figure 1). Participants and Ethics Approval Participants and Ethics Approval Twenty-seven volunteers (10 females, 24.52 ± 2.79 years old,
age range: 20–34 years) participated in this experiment. All
participants were all right-handed and had no deficits in auditory,
tactile, and visual processing. Experimental procedures were
approved by the Ethical Committee of Sungkyunkwan University September 2019 | Volume 10 | Article 2135 Frontiers in Psychology | www.frontiersin.org 2 Lee et al. Surface Stickiness Perception FIGURE 1 | Structure and time course of the experimental design. Participants completed five blocks per modality condition. Following the instruction on the screen,
participants perceived stickiness intensity levels through the sensory cues, i.e., auditory, tactile, and visual cues. In each trial, two stimuli were presented one after
another for 3 s each and then participants were asked to respond to questions (1) “Does the second stimulus feel stickier than the first one?” and (2) “How much
stickier was the second stimulus compared to the first one?”. FIGURE 1 | Structure and time course of the experimental design. Participants completed five blocks per modality condition. Following the instruction on the screen,
participants perceived stickiness intensity levels through the sensory cues, i.e., auditory, tactile, and visual cues. In each trial, two stimuli were presented one after
another for 3 s each and then participants were asked to respond to questions (1) “Does the second stimulus feel stickier than the first one?” and (2) “How much
stickier was the second stimulus compared to the first one?”. also could respond 0 if the pair of stimuli were felt to be
of the same intensity. The larger the response value, the
greater the difference in perceived intensity between the
stimulus pair. Participants were instructed to perceive stickiness intensity from
the sensory cues (auditory, tactile, and visual information) and
to conduct two different tasks, i.e., 2-Alternative Forced Choice
(2-AFC) and magnitude estimation of perceived dissimilarity. The 2-AFC and the magnitude estimation are the common
psychophysical methods for measuring the subjective experience
(Lederman and Klatzky, 2009). In tactile conditions, participants
performed tasks following the instruction “Touch” and “Detach”
displayed on a monitor. To give more detail, a tactile stimulus
was given to the participants prior to the stimulation periods. When “Touch” was on the monitor, participants put their
index finger on the tape and lifted offas soon as they saw
“Detach” on the monitor. Participants and Ethics Approval Participants were instructed to perceive stickiness intensity from
the sensory cues (auditory, tactile, and visual information) and
to conduct two different tasks, i.e., 2-Alternative Forced Choice
(2-AFC) and magnitude estimation of perceived dissimilarity. The 2-AFC and the magnitude estimation are the common
psychophysical methods for measuring the subjective experience
(Lederman and Klatzky, 2009). In tactile conditions, participants
performed tasks following the instruction “Touch” and “Detach”
displayed on a monitor. To give more detail, a tactile stimulus
was given to the participants prior to the stimulation periods. When “Touch” was on the monitor, participants put their
index finger on the tape and lifted offas soon as they saw
“Detach” on the monitor. A short break was provided between the blocks and the
entire experiment took approximately 60 min. The presentation
order of stimuli as well as modality was randomized to remove
ordering effects. Note that we did not notify participants of
the number of intensity levels throughout the entire experiment
(Supplementary Material). Frontiers in Psychology | www.frontiersin.org 2-Alternative Forced Choice • 2-AFC: Participants were asked to respond to the question
“Does the second stimulus feel stickier than the first one?”
If the second stimulus was felt to be stickier, participants
pressed a “Yes” button on the keyboard, otherwise they
pressed a “No” button. We first computed the sequential order of perceived intensity
for the five sticky stimuli to find a relationship between stimulus
intensity and sensation. In addition, to fit a psychometric curve
to the behavioral responses, we calculated “Yes” answer rates for
each of the stimulus pairs. • Magnitude estimation: Participants were asked to respond
to the question “How much stickier was the second
stimulus compared to the first one?” The response range
was from −10 to 10. Participants reported a negative value
if the second stimulus was felt to be less sticky and a positive
value if the second stimulus was felt to be more sticky. They • Ordering perceived intensity: To test whether the order
of perceived intensity is consistent with the physical
intensity order, we compared the average number of
“Yes” responses for each stimulus pair. Our experiment
asked the participants to answer whether the secondly • Ordering perceived intensity: To test whether the order
of perceived intensity is consistent with the physical
intensity order, we compared the average number of
“Yes” responses for each stimulus pair. Our experiment
asked the participants to answer whether the secondly September 2019 | Volume 10 | Article 2135 Frontiers in Psychology | www.frontiersin.org 3 Lee et al. Surface Stickiness Perception presented stimulus was felt stickier than the first one. To
minimize these sequential effects of stimulus presentation,
we presented each pair of stimuli in both ascending and
descending order, e.g., level 3 - 4 and level 4 - 3. For
example, if the average number of “Yes” across participants
was 4.13 for a stimulus pair of levels 3 - 4 and 2.58 for a
pair of level 4 - 3, we considered that the perceived intensity
of level 4 was greater than the perceived intensity of level
3. Through this process of comparing average number of
“Yes” answer for every stimulus pair, the ascending order of
perceived intensity of stimuli was obtained. a better discriminability. Specifically, the discriminability of
each participant was estimated as a difference of perceived
intensity values between the 25th and 75th percentiles. This
difference is known as the just noticeable difference, i.e., JND. Magnitude Estimation To
compute
a
dissimilarity
matrix
for
each
modality,
we
normalized
participants’
responses
in
the
magnitude
estimation tasks so that the mean value became 0 and the
standard
deviation
became
1. The
normalized
responses
were averaged across participants to obtain a group-level
dissimilarity matrix. An entry at the j-th row and the k-th
column of the dissimilarity matrix was the average perceived
difference in a normalized scale when the j-th intensity was
presented followed by the k-th intensity. Then, an MDS
was applied to this dissimilarity matrix to yield the spatial
organization of perceptual responses for the five different
stickiness intensities. Psychometric curves were fitted to the “Yes” answer rates. In particular, to set the values of the x-axis, we assumed that
the intensity difference between perceptually adjacent stimuli
has a quantity of 1 regardless of the actual physical intensity
difference. Hence, the x-axis was set to indicate the perceived
intensity difference ranging from −4 to 4, and the y-axis
indicated the “Yes” answer rates. A psychometric function
was derived using the Palamedes toolbox for Matlab, which
implements a maximum-likelihood method (Wichmann and
Hill, 2001; Prins and Kingdom, 2018). It showed a cumulative
probability that the stickiness intensity of the second stimulus
was perceived to be stickier than the first stimulus, as a function
of its relative stickiness. Therefore, a steeper slope indicated To
further
examine
the
correspondence
of
perceived
dissimilarities between sensory modalities, we employed the
Mantel test to calculate a correlation between the matrices
(Saarimaki et al., 2016). The significance was tested from
the probability distribution obtained from 10,000 repeated FIGURE 2 | Sequential order of physical and perceived intensities for the 5 sticky tapes. The left side: Based on participants’ responses to the 2-AFC task, the 5
stickiness intensities were sorted in ascending order for each modality condition. The right side: The number of “Yes” responses of 27 participants are depicted
over the physical stickiness values. Each dot indicates a participant. The solid lines indicate the average number of “Yes” responses of each stimulus pair for both
ascending and descending order. FIGURE 2 | Sequential order of physical and perceived intensities for the 5 sticky tapes. The left side: Based on participants’ responses to the 2-AFC task, the 5
stickiness intensities were sorted in ascending order for each modality condition. The right side: The number of “Yes” responses of 27 participants are depicted
over the physical stickiness values. 2-Alternative Forced Choice To determine the reliability of the JND, we estimated each
participant’s goodness of fit. We fitted a logistic function to
the psychometric curve and the individual deviances were
evaluated to measure goodness of fit (Fruend et al., 2011). A 95%
confidence interval was calculated based on simulations from the
bootstrapping procedure (n = 1000). If the observed deviance was
outside the 95% confidence limit, we considered the participant’s
data as an outlier. • “Yes” answer rates: These rates were derived by the ratio of
the number of “Yes” responses to the number of stimulus
pairs presented. For example, if the “Yes” answer rate was
0.3, it meant that the second stimulus was perceived to
be stickier three times when a stimulus pair is presented
ten times. Magnitude Estimation Each dot indicates a participant. The solid lines indicate the average number of “Yes” responses of each stimulus pair for both
ascending and descending order. September 2019 | Volume 10 | Article 2135 4 Frontiers in Psychology | www.frontiersin.org Lee et al. Surface Stickiness Perception permutations. For each permutation, we permuted the values of
the dissimilarity matrix and determined the expected distribution
of the statistics under the null hypothesis. The probability of
the observed correlation arising by chance was then yielded by
observing where the statistic calculated from our data fell in the
permuted distribution. the order for auditory perception was shown to be 1-2-3-
5-4, and for tactile and the visual perception was shown
to be 1-2-5-3-4. The JNDs, the point of subject equality (PSE), and the
goodness of fit values (i.e., deviance) of all participants across
modalities are summarized in Table 1. Among the data of the
27 participants, data from participants 15 and 18 were not
well-fitted and we therefore excluded them from the analyses. The mean and the standard error values in the table were
calculated after excluding the outlier data. We carried out a one-
way repeated measures ANOVA on JND values and found a
significant difference in the values for each modality (F2,75 = 20.1,
p < 0.01). Tukey’s post hoc test revealed that JND values for the
auditory condition were significantly smaller than those for the
visual and tactile conditions (p < 0.01). No significant difference
was found between visual and tactile conditions. 2-Alternative Forced Choice Figure 2 shows the sequential order of perceived intensity
for 5 sticky tapes and the number of “Yes” responses of
each participant (Weissgerber et al., 2015). It is noticeable
that physical and perceived intensities did not coincide. More interestingly, the perceptual intensity orders of the
three sensory modalities were not identical, i.e., the order
of perceived intensity levels for auditory perception differs
from those for visual and tactile perception. Based on the
ascending order of physical intensity levels (i.e., 1-2-3-4-5), ∗Data from participants 15 and 18 were considered as outliers and excluded from the analyses. 5 and 18 were considered as outliers and excluded from the analyses. Frontiers in Psychology | www.frontiersin.org Magnitude Estimation Distance matrices for each modality condition were computed based on the perceived difference of stickiness
intensities. The values in each cell indicate the perceptual distances of each stimulus pair. Based on these values, relative positions of the five stickiness intensities
were computed and depicted in a 2-dimensional MDS map. FIGURE 3 | Results of the magnitude estimation task. Distance matrices for each modality condition were computed based on the perceived difference of stickiness
intensities. The values in each cell indicate the perceptual distances of each stimulus pair. Based on these values, relative positions of the five stickiness intensities
were computed and depicted in a 2-dimensional MDS map. map (Figure 3). To statistically estimate how similarly the
spatial configurations were mapped across sensory conditions,
we employed the Mantel test to calculate the correlation between
modalities. We observed a significant correlation between visual
and tactile modalities (r = 0.98, p < 0.01). However, no other
condition-pairs showed any significant correlation (auditory-
tactile: r = 0.50, p = 0.16; auditory-visual: r = 0.49, p = 0.14). magnitude estimation tasks. A follow-up Mantel test revealed that
the perceptual mapping for vision and touch were statistically
similar, but that auditory perception was different. These results
thus suggest that the discriminability of stickiness intensity
is best served by auditory cues and that texture information
processing in the auditory domain is distinctive from that of
other modalities. Our results clearly showed a discrepancy between perceptual
and physical stickiness intensities. On the one hand, this may
be attributed to the material difference of contacting areas. Participants perceived stickiness intensities by touching with
their own fingers or watching/hearing touch by another’s finger. However, for the measurement of physical stickiness, the probe
tack test measured the intensity when lifting a steel ball offthe
surface. Namely, the difference between the physical properties
of steel and skin could have brought about dissimilar interactions
with the sticky surface, resulting in this discrepancy. On the
other hand, the non-linear nature of human intensity perception
could cause confusion in the sequential ordering. We observed
that the orders of perceived and physical intensity were identical
for relatively low physical stickiness intensities (levels 1 and 2)
across the modalities, but that this was different for relatively
high intensities (levels 3, 4, and 5). Magnitude Estimation We computed dissimilarity matrices using perceived intensity
differences between stimulus pairs and the corresponding MDS TABLE 1 | A summary of the behavioral responses (2-AFC task). Auditory
Tactile
Visual
Participant
JND
PSE
Deviance
JND
PSE
Deviance
JND
PSE
Deviance
1
0.55
0.43
0.21
2.15
0.74
11.06
2.59
−0.03
6.40
2
1.39
0.12
6.81
2.65
0.21
3.54
3.04
−0.22
6.66
3
1.43
−0.26
10.04
2.11
0.35
3.64
2.86
−0.69
3.72
4
0.97
0.71
5.86
2.08
0.81
12.26
1.46
0.12
3.42
5
1.68
0.84
7.47
1.35
1.39
2.50
2.37
1.63
6.12
6
0.84
0.46
6.03
1.77
0.76
5.91
2.44
−0.14
4.89
7
1.42
−0.31
2.51
1.94
−0.45
3.60
2.48
−0.72
6.09
8
1.40
0.36
4.69
2.23
−0.08
6.28
1.84
−0.33
9.50
9
1.23
−0.21
10.38
2.09
0.13
3.14
2.27
−0.53
13.64
10
1.43
1.12
13.19
3.58
1.01
10.03
2.15
−1.20
5.92
11
1.50
0.27
12.21
2.30
0.03
9.13
2.57
−0.26
7.80
12
2.78
1.34
6.17
2.67
0.03
9.43
2.27
0.30
4.26
13
1.04
0.29
1.71
1.67
0.17
1.93
2.20
0.52
6.36
14
1.37
0.21
6.19
2.67
−0.03
4.93
1.54
0.07
0.91
15∗
1.39
−0.12
16.66∗
3.08
0.67
20.53∗
2.16
0.03
6.30
16
0.85
0.79
0.71
2.45
−0.43
7.73
3.12
−0.29
3.28
17
1.66
0.68
3.41
2.09
−0.03
8.61
1.68
−0.03
5.11
18∗
1.84
0.33
16.27∗
2.48
0.32
5.46
2.59
0.15
6.20
19
1.66
0.68
3.41
2.22
−0.05
4.45
2.17
0.41
4.03
20
1.19
0.30
3.22
2.12
−0.25
6.76
2.00
0.24
5.62
21
1.22
0.64
3.92
2.53
−0.43
2.72
2.23
−0.14
2.71
22
1.09
0.07
1.69
1.88
−0.13
2.29
3.45
−0.37
11.71
23
0.81
0.37
5.40
1.87
0.44
10.02
2.51
0.67
15.00
24
1.38
0.17
1.83
1.64
−0.27
2.37
3.13
0.55
1.99
25
2.88
0.21
4.78
2.12
0.79
8.03
1.99
0.94
8.16
26
0.84
0.33
0.73
0.82
0.74
5.35
1.78
0.49
2.68
27
2.59
−0.03
7.79
1.74
−0.18
5.19
2.24
−0.41
5.28
Mean
1.41
0.38
5.22
2.11
0.21
6.04
2.33
0.02
6.05
Stdandard
Error
0.59
0.40
3.54
0.52
0.50
3.10
0.51
0.60
3.45
∗Data from participants 15 and 18 were considered as outliers and excluded from the analyses. TABLE 1 | A summary of the behavioral responses (2-AFC task). September 2019 | Volume 10 | Article 2135 Frontiers in Psychology | www.frontiersin.org 5 Lee et al. Surface Stickiness Perception FIGURE 3 | Results of the magnitude estimation task. Magnitude Estimation In line with the Fechner’s
law describing that the magnitude of a subjective sensation
increases in proportion with the logarithm of stimulus intensity
(Weber, 1996), this observation can be interpreted as participants Frontiers in Psychology | www.frontiersin.org September 2019 | Volume 10 | Article 2135 DISCUSSION In this study, we investigated how humans perceive intensity
information regarding surface stickiness when it is conveyed
via auditory, tactile, and visual cues. Results of the 2-AFC
tasks indicated that perceptual stickiness intensities were distinct
from physical stickiness intensities. The sequential orders of the
perceived intensity for three different modalities were also not
identical, suggesting a distinct perceptual process for surface
stickiness across sensory modalities. Moreover, we derived JNDs
from the psychometric functions for evaluating each participant’s
perceptual sensitivity to tactile stickiness. The results indicate that
auditory cues resulted in better discriminative sensitivity than
tactile and visual cues. Intriguingly, this result contradicts our
hypothesis that tactile cues support better perceptual sensitivity. Furthermore, to explore spatial configurations of five levels of
stickiness intensity, MDS was applied on the responses in the September 2019 | Volume 10 | Article 2135 Frontiers in Psychology | www.frontiersin.org 6 Surface Stickiness Perception Lee et al. requiring a greater perceptual difference to discriminate as
physical intensity increased. Further studies will be needed to
identify various causes that affect surface stickiness perception. is approximately 60 dB. Since we focused to capture the clear
sound of moment of detachment, we did not notice the loudness
of the sound. Third, the sensitivity of tactile stimuli may be
varying depending on the size of the tape or the area of the
finger. Prior to the experiment, participants practiced finger
postures following instruction of the experimenter to standardize
their finger movements across participants as well as across
trials. However, we cannot completely rule out the unexpected
confounding due to the stimuli and movements. y
p
p
Another key finding of the current study is that participants
were most sensitive in perceiving and distinguishing stickiness
intensity using auditory sensation. In line with our findings,
previous studies have reported that auditory sense plays
important roles in texture perception (Bresciani et al., 2005;
Avanzini and Crosato, 2006). For instance, Avanzini and
colleagues presented sound clips of a contact between a rigid
probe and objects with different levels of stiffness and reported
that participants’ perceived stiffness was correlated with the
auditory information (Avanzini and Crosato, 2006). Moreover,
Giordano investigated auditory perception of hardness and
demonstrated that contact time is a crucial factor for auditory
perception, suggesting that tactile discriminability is strongly
dependent on a certain feature of auditory stimuli (Giordano,
2005). ETHICS STATEMENT Experimental
procedures
were
approved
by
the
Ethical
Committee
of
Sungkyunkwan
University
(IRB#
2018-05-
001) and the study was conducted in accordance with the
Declaration of Helsinki. All participants were informed about
the experimental procedure and gave informed consent prior to
their participation. FUNDING This
work
was
supported
by
the
Institute
for
Basic
Science (IBS-R015-Y2). Although we tried to minimize potential influences due to
our stimuli throughout the experiment, there is still a chance
of unexpected confounding. First, we checked the sound onsets
carefully and found that there was a subtle difference: The largest
timing difference between onsets was 0.003 s. It is unlikely
that participants could recognize the timing difference, but we
cannot completely rule out its potential influences. Second,
the sound of touching a sticky surface was rather loud (e.g.,
71.9 dB) considering that the loudness of a normal conversation AUTHOR CONTRIBUTIONS HL, EL, and JJ conducted the experiments, analyzed the results,
and wrote the original draft of the manuscript. JK conceived of
the project and supervised the project. Frontiers in Psychology | www.frontiersin.org DATA AVAILABILITY All datasets generated for this study are included in the
manuscript and/or Supplementary Files. DISCUSSION Following this, in our experimental paradigm, which
feature of the auditory stimuli was beneficial to discriminative
performance? There are at least two possible candidates. First, perceived stickiness intensity may be determined by the
loudness of stimuli. We examined the physical characteristics
of the auditory stimuli and observed a clear interrelationship
between amplitude (i.e., loudness) and intensity perception. The amplitudes of auditory stimuli were 57.5, 61.1, 64.3, 71.9,
and 64.5 (dB) for levels 1 to 5, respectively. Interestingly, the
ascending order of the amplitude values was in accordance
with the order of perceived intensities, i.e., 1-2-3-5-4. Hence,
it seems that participants perceived intensity according to the
loudness of the stimuli. In the case of visual perception, there
were far more factors to consider so as to distinguish the
intensities, e.g., velocity of vertical movement of the finger and
stretching of skin at the moment of lifting off, etc. In the
case of tactile perception, factors such as the pressure applied
to the sticky surface, or subtle changes in the stickiness as
time passes by, might have affected intensity discrimination. Therefore, it is likely that more complex perceptual processes
were required for vision and tactile sensation than for auditory
sensation. Second, the distinctive perceptual relationship between
the five intensities in the auditory domain might lead to a
higher discriminability. We investigated the relative positioning
of stimulus intensities using MDS and the follow-up Mantel
test revealed that the spatial configuration in the auditory
domain was different from that of the others. This suggests
that the human auditory system has different perceptual
processes to that of visual and tactile system, and that auditory
information processing could be more efficient for stickiness
intensity perception. In this study, we investigated how cues in different sensory
modalities (auditory, tactile, and visual cues) have an influence
on the discriminability of stickiness intensity. Our results showed
that the physical and perceived intensity of surface stickiness
is different and auditory cues were the most beneficial for
stickiness perception. More work will be needed to examine
various aspects of surface stickiness perception, but we have
provided fundamental evidence on stickiness perception by
different sensory modalities. As future work, we will study
which sensory modality dominates in stickiness perception using
multimodal and incongruent conditions, etc. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online
at:
https://www.frontiersin.org/articles/10.3389/fpsyg.2019. 02135/full#supplementary-material DATASET S1 | Raw behavioral data for each of the 27 participants. September 2019 | Volume 10 | Article 2135 7 Surface Stickiness Perception Lee et al. REFERENCES Lederman, S. J., and Klatzky, R. L. (2009). Haptic perception: a tutorial. Atten. Percept. Psychophys. 71,
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5615-6_12 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Lederman, S. J., and Abbott, S. G. (1981). Texture perception: studies of
intersensory organization using a discrepancy paradigm, and visual versus
tactual psychophysics. J. Exp. Psychol. Hum. Percept. Perform. 7, 902–915. doi:
10.1037/0096-1523.7.4.902 Copyright © 2019 Lee, Lee, Jung and Kim. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms. Copyright © 2019 Lee, Lee, Jung and Kim. September 2019 | Volume 10 | Article 2135 Frontiers in Psychology | www.frontiersin.org REFERENCES This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms. Lederman, S. J., and Klatzky, R. L. (2004). “Multisensory texture perception,” in
Handbook of multisensory processes, eds E. Calvert, C. Spence and B. Stein
(Cambridge, MA: MIT Press), 107–122. September 2019 | Volume 10 | Article 2135 Frontiers in Psychology | www.frontiersin.org 8
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Molecular approaches for manipulating astrocytic signaling in vivo
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Molecular approaches for
manipulating astrocytic signaling
in vivo
Alison X. Xie 1, Jeremy Petravicz 2 and Ken D. McCarthy 1*
1 Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA,
2 Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of
Technology, Cambridge, MA, USA Astrocytes are the predominant glial type in the central nervous system and play
important roles in assisting neuronal function and network activity. Astrocytes exhibit
complex signaling systems that are essential for their normal function and the
homeostasis of the neural network. Altered signaling in astrocytes is closely associated
with neurological and psychiatric diseases, suggesting tremendous therapeutic potential
of these cells. To further understand astrocyte function in health and disease, it is
important to study astrocytic signaling in vivo. In this review, we discuss molecular
tools that enable the selective manipulation of astrocytic signaling, including the tools
to selectively activate and inactivate astrocyte signaling in vivo. Lastly, we highlight a few
tools in development that present strong potential for advancing our understanding of
the role of astrocytes in physiology, behavior, and pathology. Edited by:
Tycho M. Hoogland,
Netherlands Institute for
Neuroscience, Netherlands Edited by:
Tycho M. Hoogland,
Netherlands Institute for
Neuroscience, Netherlands Keywords: astrocyte, in vivo, GPCR signaling, DREADD, IP3R2 KO REVIEW
published: 21 April 2015
doi: 10.3389/fncel.2015.00144 Keywords: astrocyte, in vivo, GPCR signaling, DREADD, IP3R2 KO Introduction Reviewed by:
Eliana Scemes,
Albert Einstein College of Medicine,
USA Reviewed by:
Eliana Scemes,
Albert Einstein College of Medicine,
USA
Erlend Arnulf Nagelhus,
University of Oslo, Norway
*Correspondence:
Ken D. McCarthy,
Department of Pharmacology, School
of Medicine, UNC-Chapel Hill, 120
Mason Farm Road, Chapel Hill,
NC 27599-7365, USA
ken_mccarthy@med.unc.edu Reviewed by:
Eliana Scemes,
Albert Einstein College of Medicine,
USA Albert Einstein College of Medicine,
USA G protein coupled receptors (GPCRs) are the primary molecules through which non-excitable
cells transduce information from external cues to biological responses. There are four major
families of GPCRs that are distinguished by their composition, ability to activate intracellular
signaling cascades, and the functional consequences associated with their activation. The impor-
tance of GPCRs is underscored by the fact that these molecules are the most common tar-
geted class of proteins of therapeutic agents. Astrocytes express each of the major classes of
GPCRs (Porter and McCarthy, 1997) clearly demonstrating that these cells are dynamically cou-
pled to the activity of their surrounding cellular and chemical milieu. It is likely that astro-
cyte GPCRs are activated by neurotransmitters released from neurons synaptically as well as
through volume transmission. It is also likely that neighboring non-neuronal cells including
microglia, vascular endothelial cells, astrocytes, and other resident CNS cells release molecules
that activate astrocyte GPCRs and modulate astrocyte activity. In certain situations, low lev-
els of ambient neurotransmitters might tonically activate astrocyte GPCRs. However, it is likely
that in most cases astrocyte GPCRs are spatially restricted to discrete signaling domains that
are activated with different temporal characteristics dependent on the source of the signal and
biological response being affected. Layered onto this signaling complexity is the morpholog-
ical complexity of astrocytes; the fine processes of an individual astrocyte within the CA1
region of the hippocampus can associate with ∼100,000 synapses (Bushong et al., 2002). This
being the case, different regions of an individual astrocyte are likely responding simultaneously Erlend Arnulf Nagelhus,
University of Oslo, Norway University of Oslo, Norway
*Correspondence:
Ken D. McCarthy,
Department of Pharmacology, School
of Medicine, UNC-Chapel Hill, 120
Mason Farm Road, Chapel Hill,
NC 27599-7365, USA
ken_mccarthy@med.unc.edu Received: 05 February 2015
Accepted: 27 March 2015
Published: 21 April 2015 Citation: Xie AX, Petravicz J and McCarthy KD
(2015) Molecular approaches for
manipulating astrocytic signaling
in vivo. Front. Cell. Neurosci. 9:144. doi: 10.3389/fncel.2015.00144 April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 1 Manipulating astrocytic signaling in vivo Xie et al. to local signals (from neurons or other cell types) with different
functional outcomes. Collectively, this information underscores
how difficult it is to replicate in vitro or in situ the complicated
pattern of GPCR activation that normally occurs in vivo. variant ChR2 (C128S) or an optogenetic proton pump (ArchT). Activation of these optogenetic tools lead to, respectively, acidi-
fication or alkalization of astrocytes which modulated glutamate
release and ischemic damage in vivo. The remainder of studies
using ChR2 or its variants have been performed using culture or
slice preparations, but provide valuable insights into the mecha-
nisms of their ability to activate astrocytes signaling (Figueiredo
et al., 2014). A second variant, CatCh (Ca2+ translocating ChR,
which has improved Ca2+ permeability) (Kleinlogel et al., 2011)
when expressed in cultured astrocytes, was found to also increase
Ca2+ but with varying reliability compared to ChR2 (H134R) (Li
et al., 2012; Figueiredo et al., 2014). A non-ChR2 light activated
channel, the light-gated Ca2+-permeable ionotropic GluR6 gluta-
mate receptor (LiGluR), has also been used in cultured astrocytes,
with again varying results (Li et al., 2012). By far, the emphasis in astrocyte GPCR signaling activity has
been on the regulation of Ca2+. This is not surprising given
that Ca2+ fluxes play a very important role in regulating bio-
logical processes and Ca2+ is the only signaling molecule that
we can readily monitor selectively in astrocytes in complex tis-
sue such as brain. Consequently, we know a lot about astrocyte
Ca2+ responses following the activation of Gq-GPCRs and the
consequences of increasing astrocyte Ca2+ by a number of differ-
ent approaches. Two important points need to be kept in mind
when considering findings in this area. First, most investigations
linking increases in astrocyte Ca2+ with functional responses use
pharmacological methods to increase astrocyte Ca2+ and con-
sequently findings may not reflect physiological responses. Citation: Sec-
ond, while the field has focused on the role of astrocyte Ca2+ in
functional responses, the activation of Gq-coupled GPCRs leads
to the modulation of a broad set of signaling cascades beyond
changes in Ca2+; the variety of effector proteins in Gαq “interac-
tome” may affect astrocyte Ca2+ responses as well as play impor-
tant roles in physiological responses to Gq-GPCR activation
(Figure 1; Sanchez-Fernandez et al., 2014). The advantages of using optogenetics to activate astrocytes
are the ability to temporally and spatially control the extent of
activation. However, there are a number of important caveats
involved in the use of optogenetics to activate astrocyte signal-
ing cascades. First, it is unresolved as to the source of Ca2+
when using these optogenetic actuators in astrocytes. Extracel-
lular Ca2+ entry through ChR2 variants seems the most logical
mechanism, but a recent comparative study found that intracel-
lular stores are the primary source (Figueiredo et al., 2014). These
finding are at odds with studies from other laboratories (Li et al.,
2012), and previous research from the same authors (Gourine
et al., 2010). Current evidence indicates that both external and
internal Ca2+ sources are involved in the ChR2 induced Ca2+
signal. Second, ChR2 and its variants are non-selective cation
channels, allowing the entry of Na+ K+, H+, and Ca2+ upon
activation (Nagel et al., 2003). ChR2 expressed in the cultured
astroglia cell line GL261 display significant influx of Na+ and
Ca2+ across the cell membrane based upon fluorescent indicator
imaging (Ono et al., 2014). Additionally, significant alterations
in intracellular pH were also observed in the same study, which
has been corroborated in the recent study by Beppu et al. (2014). Interestingly, the ChR2 (H134R) used in the two current in vivo
studies of astrocyte function was engineered to increase its ability
to depolarize cultured HEK293 and neuronal cells over wild type
ChR2 (Nagel et al., 2005; Beppu et al., 2014). Astrocytes process
intracellular changes in Na+, Ca2+, and protons in different ways
that may affect several different astrocyte functions. For example,
increases in intracellular sodium and membrane depolarization,
which is known to occur with ChR2-stimulation (Gourine et al.,
2010), could alter glutamate reuptake efficacy (Djukic et al., 2007;
Unichenko et al., 2012; Verkhratsky et al., 2013). Citation: Alterations to
intracellular pH in astrocytes are important in the regulation of
ischemic damage in vivo (Benesova et al., 2009; Beppu et al.,
2014) and gap junction connectivity (Duffy et al., 2004). It is not
unreasonable to hypothesize that ChR2 activation leads to mem-
brane depolarization of astrocytes and interferes with a number
of transport systems, most importantly glutamate, which may
lead to effects on neuronal activity. In addition, to use optoge-
netics in vivo it is necessary to use a viral vector to deliver the
optogenetic construct and may require the insertion of a light
probe to activate the optogenetic channel in deeper brain regions. It seems very likely that these insults will lead to pathological Frontiers in Cellular Neuroscience | www.frontiersin.org Temporal Control of Astrocyte Activation In Vivo
Using Optogenetics These signaling molecules
include: Epithelial sodium channel (ENaC) (Miller and Loewy, 2013),
PLA2 (Ha et al., 2014), Myristoylated alanine-rich C-kinase substrate
(MARCKS) (Vitkovic et al., 2005), Wiskott–Aldrich syndrome protein
(WASP) (Murk et al., 2013), profilin (Molotkov et al., 2013), vinculin, talin,
and paxillin (Kalman and Szabo, 2001), ERM protein family (Persson
et al., 2010), α1-syntrophin (Masaki et al., 2010), ankyrin (Lee et al.,
2012), adipocyte protein 2 (AP2) (Rossello et al., 2012), and clathirin
(Pascual-Lucas et al., 2014). Danussi et al., 2013), Rock (Holtje et al., 2005; Lau et al., 2011), and
mDia1 (Shinohara et al., 2012); (4) PLC regulation of PtdIns(3,4,5)P3
(PIP3)/protein kinase B (PKB or Akt) pathway (DiNuzzo et al., 2013;
Kong et al., 2013), PtdIns(4,5)P2(PIP2)/diacylglycerol (DAG)/protein kinase
C (PKC) pathway, and PIP2/Ins(1,4,5)P3(IP3) pathway. Moreover, PKC
activation in astrocytes (Wang et al., 2002) engages PLD (Servitja et al.,
2003), c-src tyrosine kinase (CSK) (Jo et al., 2014), glycogen synthase
kinase (GSK) (Sanchez et al., 2003), and cAMP signaling. Many of
these signaling pathways are known to trigger cellular responses that
are important for astrocyte function including gene transcription and cell
migration. Box 1. Selected PIP2-induced signaling pathways in which
key molecules are expressed by astrocytes. These signaling molecules
include: Epithelial sodium channel (ENaC) (Miller and Loewy, 2013),
PLA2 (Ha et al., 2014), Myristoylated alanine-rich C-kinase substrate
(MARCKS) (Vitkovic et al., 2005), Wiskott–Aldrich syndrome protein
(WASP) (Murk et al., 2013), profilin (Molotkov et al., 2013), vinculin, talin,
and paxillin (Kalman and Szabo, 2001), ERM protein family (Persson
et al., 2010), α1-syntrophin (Masaki et al., 2010), ankyrin (Lee et al.,
2012), adipocyte protein 2 (AP2) (Rossello et al., 2012), and clathirin
(Pascual-Lucas et al., 2014). FIGURE 1 | A fabric of signaling cascades can be activated by
astrocyte Gq-GPCRs. Upon Gq-GPCR activation, Gαq subunits are
known to interact with G protein-coupled receptor kinases (GRKs),
β-arrestins, Rho family of guanine exchange factors (RhoGEFs) and
phospholipase C (PLC) in astrocytes or astroglia. In addition, Gβγ
subunits are able to regulate ion channel properties, as well as to
interact with signaling molecules including cdc42, PAK-PIXα,
phospholipase A (PLA), PLC, and Ras/MAPK/ERK1/2. Many of the key
signaling molecules regulated by Gβγ are expressed in astrocytes or
astroglia. Temporal Control of Astrocyte Activation In Vivo
Using Optogenetics Therefore, Gq-GPCR activation in astrocytes is likely to
activate an entire fabric of downstream signaling pathways that may
include (1) GRKs-mediated glutamate transporter (GLAST) localization
(Nijboer et al., 2013), gene transcription (Atkinson et al., 2009),
metabotropic glutamate receptor 5 (mGluR5) expression (Sorensen and
Conn, 2003), and astrocytoma growth (Woerner et al., 2012); (2)
β-arrestin-mediated gene silencing (McLennan et al., 2008; Miyatake
et al., 2009; Zhu and Reiser, 2014); (3) RhoGEFs-mediated activation of
Kinectin (Santama et al., 2004), citron kinase (Ackman et al., 2007),
phospholipase D (PLD) (Burkhardt et al., 2014), diacylglycerol kinase
(DGKa) (Kefas et al., 2013), Rhophilin2 (Vedrenne and Hauri, 2006; Temporal Control of Astrocyte Activation In Vivo
Using Optogenetics Optogenetics is an extremely powerful tool for activating and
inactivating neuronal circuits in vivo with high temporal resolu-
tion. Optogenetic regulation of neuronal activity generally occurs
through the flux of ions that either depolarize or hyperpolar-
ize neurons with high temporal and spatial resolution (Schoe-
nenberger et al., 2011; Lin, 2012). More recently, a number of
investigators have used this technology to activate astrocyte sig-
naling in situ and in vivo (Gourine et al., 2010; Perea et al., 2014;
Yamashita et al., 2014). It is important to understand that while
optogenetic activation of astrocytes can lead to changes in ion
fluxes across plasma and intracellular compartment membranes,
these fluxes do not remotely reflect the changes in signaling that
occur following the activation of astrocyte GPCRs, the primary
mode for activating astrocyte signaling (Agulhon et al., 2012;
Sanchez-Fernandez et al., 2014). Multiple variants of channelrhodopsin (ChR2) have been
expressed in astrocytes, primarily to elicit Ca2+ responses
(Table 1). In vivo, activation of the ChR2 variant ChR2 (H134R)
(Nagel et al., 2005) in astrocytes in the ventral surface of the
medulla oblongata was shown to lead to increases in intracellu-
lar Ca2+ and alteration in respiratory activity of rats (Gourine
et al., 2010). This same variant of ChR2 was employed to illus-
trate the ability of astrocyte Ca2+ elevations to alter the firing
and orientation responses of mouse primary visual cortex exci-
tatory and inhibitory neurons in vivo (Perea et al., 2014). Most
recently, Beppu et al. (2014) created mice expressing the ChR April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 2 Manipulating astrocytic signaling in vivo Xie et al. Xie et al. Danussi et al., 2013), Rock (Holtje et al., 2005; Lau et al., 2011), and
mDia1 (Shinohara et al., 2012); (4) PLC regulation of PtdIns(3,4,5)P3
(PIP3)/protein kinase B (PKB or Akt) pathway (DiNuzzo et al., 2013;
Kong et al., 2013), PtdIns(4,5)P2(PIP2)/diacylglycerol (DAG)/protein kinase
C (PKC) pathway, and PIP2/Ins(1,4,5)P3(IP3) pathway. Moreover, PKC
activation in astrocytes (Wang et al., 2002) engages PLD (Servitja et al.,
2003), c-src tyrosine kinase (CSK) (Jo et al., 2014), glycogen synthase
kinase (GSK) (Sanchez et al., 2003), and cAMP signaling. Many of
these signaling pathways are known to trigger cellular responses that
are important for astrocyte function including gene transcription and cell
migration. Box 1. Selected PIP2-induced signaling pathways in which
key molecules are expressed by astrocytes. Selective Activation of Astrocyte Gq-GPCR
Signaling with MrgA-1 changes that alter astrocyte responses. Overall, while optogenetic
tools are great tools to activate or silence neurons, using opto-
genetics to increase astrocyte Ca2+ bypasses the majority of sig-
naling pathways that are activated by GPCRs, as well as result
in aberrant ion fluxes that likely never occur under physiological
conditions (Fiacco et al., 2007; Agulhon et al., 2012; Wang et al.,
2013). The first experimental system that enabled the specific activa-
tion of endogenous astrocyte signaling cascades in complex tissue
was the Mas-related gene A1 (MrgA-1) transgenic mouse line
(Fiacco et al., 2007) (Table 1). MrgA-1 is a Gq-GPCR that is
normally expressed in a subsets of nociceptive sensory neurons April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 3 Manipulating astrocytic signaling in vivo Xie et al. Xie et al. TABLE 1 | Tools for selectively activation of astrocyte signaling in vivo. Class
Tools
Molecule
Application method
Comments
Selected references
Optogenetics
Photo-activated cation
channel, ChR2 variant
ChR2
(H134R)
Lenti-viral vectors
Localized, high temporal control of glial
activation, yet bring in non-physiological
sources of Ca2+ and significantly alter
intracellular pH; Non-selective to all cations;
Require viral injections and light-source
Gourine et al., 2010;
Figueiredo et al., 2014;
Perea et al., 2014
ChR2
(L132C);
CatCh
AAV vector
Improved Ca2+ permeability compared to
ChR2 (H134R) with varying reliability; Tested
in cultured astroglia and no in vivo data
Figueiredo et al., 2014;
Li et al., 2012
ChR2
(C128S)
tetO-ChR2(C128S)-EYFP
β-actin locus knockin mice
(crossed to Mlc1-tTA mice)
Does not require virus injection but requires
light activation. Highly sensitive to light
activation. Selective Activation of Astrocyte Gq-GPCR
Signaling with MrgA-1 Provide high temporal control of
glial acidification
Tanaka et al., 2012,
2010; Beppu et al.,
2014
Photo-activated outward
proton pump
Archaerhodopsin
(ArchT)
tetO-ArchT-EGFP β-actin
locus knockin mice (crossed
to Mlc1-tTA mice)
Does not require virus injection but requires
light activation; High temporal control; Only
available tool for glial alkalization
Tanaka et al., 2010;
Tsunematsu et al., 2013;
Beppu et al., 2014
Photo-activated
Ca2+-permeable iGluR6
LiGluR
Lipofectamine 2000
transfection
Varying reliability; Tested in cultured astroglia
and no in vivo data
Li et al., 2012
Pharmacogenetics
Endogenous Gq GPCR
expressed outside of the
brain
MrgA-1
tetO-MrgA-1 transgenic
mouse line (crossing to
GFAP-tTA*)
Stable expression in brain astrocytes;
Peptide ligands do not cross BBB, therefore
requires brain infusion, injection or
implantation for in vivo activation;
Low temporal control; Potential
over-activation
Fiacco et al., 2007;
Agulhon et al., 2010;
Cao et al., 2013
Engineered Gq GPCR
Gq-DREADD
(hM3Dq)
GFAP-hM3Dq transgenic
mouse line**; AAV-GFAP***
Stable expression in brain astrocytes; ligand
crosses BBB and bio-inert; Long-lasting
physiological and behavioral phenotypes;
Also expressed in PNS GFAP+ glial cells and
certain non-neural cells;
Low temporal control; Potential
over-activation
Armbruster et al., 2007;
Agulhon et al., 2013;
Bonder and McCarthy,
2014
Engineered Gi GPCR
Gi-DREADD
(hM4Di)
Transgenic mouse line
Under characterization
Armbruster et al., 2007
Engineered Gs GPCR
Gs-DREADD
Transgenic mouse line;
AAV-GFAP***
Under characterization
Guettier et al., 2009
*Mouse line available in the Jackson Laboratory, stock No. 005964; **For various DREADD constructs and transgenic mouse lines currently available, see http://pdspit3.mml. unc.edu/projects/dreadd/wiki/WikiStart; ***AAV-GFAP minimal promotor available via UNC Vector core. TABLE 1 | Tools for selectively activation of astrocyte signaling in vivo. *Mouse line available in the Jackson Laboratory, stock No. 005964; **For various DREADD constructs and transgenic mouse lines cu
unc.edu/projects/dreadd/wiki/WikiStart; ***AAV-GFAP minimal promotor available via UNC Vector core. (Dong et al., 2001) but not in the brain. The ligand used to acti-
vate MrgA-1, the peptide FMRF, does not activate endogenous
brain Gq-GPCRs (Fiacco et al., 2007). To achieve cell speci-
ficity in the brain, MrgA-1 expression was controlled by using
a tetracycline-controlled inducible expression system (tetO sys-
tem), which requires a second transgene, the transcription trans-
activator (tTA). By crossing tetO-MrgA-1 transgenic mouse to
GFAP-tTA transgenic mouse, GFAP+ glia become the only cells
in the brain that express both tTA and tetO-MrgA-1, therefore the
only CNS cells to express MrgA-1 receptor (Fiacco et al., 2007). Pharmacogenetic Activation of Astrocytic
Signaling In Vivo Using DREADDs g
g
g
In 2007, a new family of engineered GPCRs, Designer Recep-
tor Exclusively Activated by Designer Drugs (DREADD) were
developed (Armbruster et al., 2007) and have become the best
option for activating GPCR signaling in specific cell popula-
tions in vivo (Rogan and Roth, 2011) (Table 1). A significant
advantage of DREADDs compared to MrgA-1 is that the ligand
of DREADDs, clozapine N-oxide (CNO), crosses BBB (Bender
et al., 1994), therefore enabling non-invasive manipulation of
receptor activity via peripheral injections (e.g., intraperitoneal or
intravenous injections) and even via drinking water (Jain et al.,
2013). The M3 muscarinic cholinergic receptor (M3AChR) was
engineered through directed molecular evolution (Dong et al.,
2010) that led to a striking decrease the affinity of this receptor
for its native agonist (acetylcholine) as well as to a large increase
in affinity for CNO. In addition, DREADDs do not exhibit consti-
tutive activity and CNO is pharmacologically inert in the absence
of DREADDs (Armbruster et al., 2007; Nichols and Roth, 2009;
Dong et al., 2010). Consequently, mice expressing DREADDs do
not exhibit a phenotype in the absence of CNO and CNO does
not lead to a phenotype in wild type mice (Alexander et al., 2009;
Guettier et al., 2009; Agulhon et al., 2013). Since their devel-
opment, DREADDs have been extensively used to chronically
and acutely activate (Gq-DREADDs) and silence (Gi-DREADDs)
specific subsets of neurons in vivo (Wess et al., 2013). There are caveats associated with using pharmacogenetic sys-
tems to study the role of astrocyte Gq-GPCR signaling in vivo. First, engineered GPCRs are driven by an exogenous promoter
system and consequently the levels of expression are likely to be
different than that of endogenous astrocytic GPCRs. While this
does not appear to lead to markedly different Ca2+ responses
compared to endogenous receptors, it has not been confirmed
that the engineered GPCR signaling cascades are regulated in
a similar manner. Second, it is impossible to mimic the tem-
poral and spatial characteristics of in vivo GPCR activation, a
caveat associated with all pharmacological stimulation. Follow-
ing an i.p. injection of CNO, most of the physiological pheno-
types were observed in 5 min and peak in 30 ∼45 min (Agulhon
et al., 2013). Selective Activation of Astrocyte Gq-GPCR
Signaling with MrgA-1 As a native GPCR, MrgA-1 activation triggers the entire fabric
of signaling cascades normally activated by endogenous astro-
cyte Gq-GPCRs; an important component that is absent when
selectively increasing specific signaling molecules such as IP3
and Ca2+. With respect to Ca2+, MrgA-1 activation leads to a
similar spatial and temporal response as endogenous Gq-GPCRs
in hippocampal astrocytes (Fiacco et al., 2007). MrgA-1 transgenic mice were used to prepare brain slices to
test the gliotransmission hypothesis at hippocampal CA3-CA1
synapses (Fiacco et al., 2007; Agulhon et al., 2010; Wang et al.,
2012, 2013; Devaraju et al., 2013). Initial reports showed that
bath application of FMRFa induced widespread Ca2+ elevations
in stratum radiatum astrocytes from MrgA-1 mice, while CA1
neuronal Ca2+ activity, excitatory synaptic transmission and
short- or long-term excitatory synaptic plasticity in CA3-CA1
synapses were not affected (Fiacco et al., 2007; Agulhon et al.,
2010). Later Wang et al. reported that although MrgA-1 medi-
ated astrocyte activation did not change neuronal excitability and
miniature excitatory synaptic currents (mEPSCs) in neurons near
the surface of hippocampal slices, both bath and microinjection
of FMRFa led to a transient hyperpolarization and decreased
mEPSC frequency in neurons below 80 µm depth in the slices
(Wang et al., 2012, 2013). These studies suggested that selective
activation of Gq-GPCR signaling in astrocytes increased activity Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org April 2015 | Volume 9 | Article 144 4 Manipulating astrocytic signaling in vivo Xie et al. Xie et al. immunostaining studies demonstrated that the expression of
Gq-DREADD was restricted to GFAP+ glia in the CNS and
PNS (Agulhon et al., 2013). Bath application of CNO in situ
or i.p. injection of CNO in vivo led to Ca2+ increases in brain
astrocytes, without affecting Ca2+ in nearby neurons; CNO
induced Ca2+ increases occurred throughout astrocytes includ-
ing their fine processes within the neuropil. The development
of this model enabled, for the first time, examination of the
behavioral and physiological consequences of specifically acti-
vating Gq-GPCR signaling in GFAP+ glia. CNO administra-
tion to GFAP-Gq-DREADD transgenic mice revealed robust
and unexpected behavioral and physiological phenotypes that
were absent in litter mate controls; phenotypic changes include
robust increases in heart rate and blood pressure, saliva for-
mation, a decrease in body temperature, and increased seda-
tion in the presence of a GABA receptor agonist (Agulhon
et al., 2013). Selective Activation of Astrocyte Gq-GPCR
Signaling with MrgA-1 These findings suggest that GFAP+ glia have the
potential for modulating a number of important physiological
processes. of the Na+/K+ ATPase, resulting in a reduction of extracellular
K+ which consequently hyperpolarized neurons and suppressed
excitatory transmission (Wang et al., 2012). The Gq-GPCR acti-
vated change in [K+] was hard to detect in the superficial layer of
the slices, where the constant bath perfusion buffered the effects
(Wang et al., 2012). The K+ removal hypothesis was supported
by an independent study from Devaraju et al. who found that
both Schaffer Collaterals stimulation and selective stimulation
of astrocytic MrgA-1s potentiated inward K+ current and glu-
tamate uptake in hippocampal astrocytes (Devaraju et al., 2013). These data suggest that astrocytic Gq-GPCR activation may regu-
late neuronal excitability and modulate neuronal network activity
indirectly rather than inducing the release of gliotransmitters. The MrgA-1 mouse line is rarely used for studying astro-
cyte function in vivo because the peptide agonists do not effec-
tively cross blood brain barrier. Recently, Cao et al. used MrgA-1
mouse line to study the role of astrocytic activation in behavior
by implanting infusion cannula or osmotic pumps into the brain
of MrgA-1 mouse (Cao et al., 2013). Brain infusion of the pep-
tide agonist of MrgA-1 mice induced antidepressant-like effect
in forced swimming test and reversed depression-like behavior
in MrgA-1 mice suggesting that astrocytic Gq-GPCR signaling
is capable of modulating depressive-like behaviors (Cao et al.,
2013). In addition to GFAP-Gq-DREADD mice, transgenic mouse
lines expressing Gs- and Gi-DREADD specifically in GFAP+ glia
were developed and are currently under characterization in the
McCarthy laboratory. GFAP-DREADD transgenic mice offer the
best system to non-invasively and simultaneously activate widely
distributed astrocyte populations. Other systems for manipu-
lating astrocytic activity in vivo, including MrgA-1 transgenic
mice and optogenetics, requires direct application of ligand/light
to brain tissue and thus have spatial limitations with regard to
cells being activated at a given time. Region-specific expression
DREADD can be achieved via viral delivery (Bull et al., 2014). Once the expression pattern is established, one can activate a
subset of astrocytes acutely or chronically in dose-dependent
manner, and behavior and physiological outcome can be mea-
sured from free-moving, awake mice. For the first time in astro-
cyte research, we can now test the contribution of astrocytes
GPCR signaling in physiology and behavior, as well as verify the
previously known astrocytic function in intact animals. Frontiers in Cellular Neuroscience | www.frontiersin.org Selective Astrocyte Gene Rescue in Mice with
Global Gene Deletion Another challenge of using viral injection to express con-
structs in astrocytes is cell-specificity. AAVs show tropism to all
cell types in the CNS (Aschauer et al., 2013; Gholizadeh et al.,
2013; Petrosyan et al., 2014; Yang et al., 2014). Even with an
astrocytic-specific promoter, it is important to carefully verify
astrocyte specific transduction using the most sensitive meth-
ods available. A low level of GPCR expression in non-astrocyte
cell types could lead to significant downstream signaling and
confound the interpretation of findings. The optogenetic and pharmacogenetic approaches for acti-
vating astrocyte in vivo share a common pitfall of poten-
tial over-activation. Recently, a conditional endogenous gene
repair approach was used to isolate the role of astrocyte-
specific endogenous signaling in vivo in a mouse model of Rett’s
syndrome (RTT). RTT is an X-chromosome-linked autism spectrum disor-
der due to the loss of function of the transcriptional regula-
tor methyl-CpG-binding protein 2 (MeCP2) in the brain (Amir
et al., 1999; Guy et al., 2007). Because MeCP2 is expressed in
all CNS cell types (Ballas et al., 2009), a conditional knock-in
mice, MeCP2loxp mice, was developed to study cell-specific dis-
ease mechanisms. In this model, the endogenous Mecp2 gene
is silenced by insertion of a loxP-Stop cassette, but can be acti-
vated when combined with Cre- or Cre-ER system (Guy et al.,
2007). When MeCP2loxp mice were crossed to hGFAP-CreERT2
mice (Hirrlinger et al., 2006), the expression of MeCP2 was selec-
tively restored in GFAP+ astrocytes when mice were treated
with tamoxifen (Lioy et al., 2011). The specific re-expression of
MeCP2 in astrocytes significantly improved RTT phenotype pos-
sibly by restoring normal dendritic morphology and levels of the
excitatory glutamate transporter VGLUT1 (Lioy et al., 2011). This
model illustrates the potential of using conditional astrocyte-
specific rescue model to isolate the function of astrocyte signaling
in vivo and in disease. One final consideration to keep in mind is that it is cur-
rently impossible to transduce functionally distinct populations
of astrocytes in a manner analogous to transducing a subpopu-
lation of functionally distinct neurons; this will only be solved
as new subpopulation specific astrocyte transcriptional units are
identified. Pharmacogenetic Activation of Astrocytic
Signaling In Vivo Using DREADDs Increased temporal and spatial resolution can be
obtained by either microinjecting CNO into the region of interest
or uncaging CNO with laser pulse activation; the latter approach
has temporal and spatial resolution similar to optogenetic activa-
tion; caged CNO has recently been prepared (Brian Roth, per-
sonal communication). Overall, DREADD technology enables
activation of the entire fabric of endogenous signaling cascades
in specific cell types that are generally stimulated by GPCRs; this Gq-DREADD was introduced into astrocyte research stud-
ies with the development of GFAP-Gq-DREADD mice for
specifically activating GFAP+ glial Gq-GPCR signaling in vivo
(Agulhon et al., 2013). Gq-DREADD expression was regu-
lated by the 2.2 Kb human GFAP promotor fragment; a
hemagglutinin (HA) tag was added to the N-terminus of the
Gq-DREADD for highly specific antibody staining. Extensive April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 5 Manipulating astrocytic signaling in vivo Xie et al. is a striking advantage over most other methods used to activate
glial signaling in situ or in vivo. studies and GFAP expression levels (Xie et al., 2010; Shigetomi
et al., 2013a). However, it is likely that more subtle undetected
changes in astrocytes occur that could influence their functional
interactions with surrounding cells. Specific GPCR Knockout Models To date, most investigators have used optogenetic, pharmaco-
genetic or pharmacological tools to determine the consequences
of activating astrocyte signaling cascades (Agulhon et al., 2008). None of these approaches recapitulate the complex regulation of
signaling cascades occurring in vivo. Findings from these stud-
ies (see review by Agulhon et al., 2008) provide insight into the
potential outcome(s) of astrocyte signaling. However, one has
to keep in mind that these highly artificial types of stimulation
may lead to outcomes that rarely, or perhaps never, occur in
physiology. To determine the functional significance of astrocytic
signaling, a more powerful approach is to demonstrate that the
loss-of-function of a particular pathway affects physiological pro-
cesses such as synaptic transmission or behavior. Here we review
current knockout (KO) and conditional KO (cKO) models for
deleting specific astrocytic signaling pathways (Table 2). Spatial Control of Astrocytic Signaling via Viral
Delivery—Advantages and Disadvantages At this time, there are no astrocyte transcriptional units that can
be used to target specific populations of astrocytes in mature
brain. Consequently, a large number of investigators have used
viral vectors to perturb signaling in subpopulations of astrocytes
using both adeno-associated viruses (AAV) and lentiviral vectors
(Figueiredo et al., 2014). Several AAV serotypes show tropism
toward astrocytes, including AAV 2/5 and AAV 8 (Koerber et al.,
2009; Aschauer et al., 2013; Petrosyan et al., 2014). In combi-
nation with an astrocytic selective promoter (Lee et al., 2008;
Pfrieger and Slezak, 2012), these AAVs are expected to express
the gene of interest selectively in astrocytes in specific brain
regions. Frontiers in Cellular Neuroscience | www.frontiersin.org GFAP-CB1R-KO One of the first GPCRs targeted for a cKO in astrocytes was
the cannabinoid type-1 receptor (CB1R), a GPCR predominately
coupled to Gi signaling (Han et al., 2012). GFAP-CB1R cKO
mouse line was accomplished by crossing floxed CB1R mouse
line with an inducible Cre system driven by the GFAP promoter
(GFAP-CreERT2) (Hirrlinger et al., 2006). Following tamoxifen
administration, CB1R expression was reduced in GFAP+ astro-
cytes in the brain, providing the first model to study the con-
tribution of astrocytes in cannabinoid induced working memory
impairment in vivo. Han et al. found that conditional knockout
of astrocytic CB1R abolished cannabinoid-induced impairment
of spatial working memory and in vivo long-term depression at
hippocampal CA3-CA1 synapses. In contrast, a cKO of CB1R in
glutamatergic or GABAergic neurons did not rescue cannabinoid
induced deficit (Han et al., 2012). This study revealed significant
impact of a single astrocyte GPCR signaling system on synaptic The disadvantages of using viral vectors to express genes in
astrocytes should not be overlooked. The most obvious concerns
are tissue damage and reactive gliosis induced by viral injection. Reactive astrocytes display more robust, frequent and widely-
spread intracellular Ca2+ activity, and intercellular coupling and
Ca2+ waves are exaggerated among reactive astrocytes (Agulhon
et al., 2012). A recent study showed that AAV 2/5 vector can be
used to induce astrogliosis and disrupt the glutamate-glutamine
cycle in astrocytes, which led to glutamate-reversible hyperac-
tivity of nearby neurons (Ortinski et al., 2010). The primary
methods for assessing whether or not astrocytes are pathologi-
cally transformed by viral infection are through morphological April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 6 Manipulating astrocytic signaling in vivo Xie et al. TABLE 2 | Current molecular tools for perturbing astrocyte signaling in vivo. GFAP-CB1R-KO Class
Mouse line
Confirmed expression
profile
In vivo phenotype
Comments
Selected
references
GPCR KO
Floxed CB1R mouse
line (crossed to
GFAP-CreERT2)
79% reduction in the
number of CB1R+ CA1
astrocytes by
immunohistochemistry
Cannabinoid-induced
impairment of spatial working
memory and in vivo long-term
depression at hippocampal
CA3-CA1 synapses were
abolished
For GFAP-CreERT2 please see
Hirrlinger et al., 2006
Marsicano et al.,
2003
Floxed DrD2 mouse
line (crossed to
hGFAP-Cre)
No marked excision
detected in striatal tissue by
qPCR analysis
Mild increase in pro-inflammatory
mediators in the striatum was
detected by qPCR; Neuronal
development, neuronal numbers
and astrogliosis in advanced age
appear normal in the substantia
nigra (SNC) and the ventral
tegmental area (VTA)
Compared to global DrD2 KO,
the pro-inflammatory
phenotypes of astrocytes in the
SNC and in the striatum are very
mild. Further characterization in
other brain regions needed
Shao et al., 2013
(created by
Shanghai Research
Center for Model
Organisms)
Perturbing Ca2+ signaling
Global IP3R2 KO
Global knockout
Loss of astrocyte GPCR
mediated Ca2+ increase;
conflicting evidence for
alterations in synaptic
transmission; minor behavioral
alterations; no evidence for
altered vascular control;
alterations in astrocyte
processes mobility in response
to LTP-inducing sensory stimuli
There are two sources for the
global IP3R2 KO: Ju Chen lab at
UCSD (used by McCarthy lab)
and Katsuhiko Mikoshiba lab at
RIKEN Brain Science Institute. GFAP-CB1R-KO There has been no direct
comparison of these two lines for
differences
Petravicz et al.,
2008; Agulhon et al.,
2010; Navarrete
et al., 2012; Bonder
and McCarthy, 2014
Floxed IP3R2
(crossed to hGFAP-Cre)
Conditional knockout, with
80–95% reduction in the
number of IP3R2+
astrocytes in brain by
immuno-histochemistry and
Gq GPCR agonist-evoked
Ca2+ imaging
Loss of astrocyte GPCR
mediated Ca2+ increases; no
reported behavioral phenotypes;
reduced cholinergic modulation
of visual responses
For hGFAP-Cre, please see
Casper and McCarthy, 2006
Chen et al., 2012;
Petravicz et al.,
2014
AAV2/5-
gfaABC1D-p130PH
Reduced ATP-evoked Ca2+
signaling in vitro and in vivo
No effect on spontaneous Ca2+
increase
Xie et al., 2010
tetO-GST-“IP3 sponge”
transgenic mice
(crossed to GLT-1-tTA
mice)
80–90% S100β+ cells are
lacZ+ in many brain regions
including CA1; Each
astrocyte on average
express 3800 “IP3 sponge”
molecules
Reduced spontaneous and
GPCR evoked Ca2+ responses;
reduced synaptic coverage by
astrocyte processes; impaired
glutamate uptake; impaired
spatial reference memory and
contextual fear responses
While alterations to the listed
behaviors and impaired
glutamate uptake were
observed, there we no significant
alterations to any synaptic
measures such as LTP and LTD
Tanaka et al., 2013
Blocking vesicular release
tetO-dnSNARE
(crossed to GFAP-tTA)
Global astrocyte expression;
recently discovered cortical
neuronal expression
Changes in sleep-wake cycle,
sleep loss related cognitive
functions, cortical slow waves,
depressive-like behavior, LTP
induction threshold in
hippocampus, neuronal survival
after stroke, development of
temporal lobe epilepsy, and
baseline mechanical nociception
Recent in-depth analysis of
dnSNARE expression in large
population of neurons suggest
re-evaluation of thought-to-be
astrocyte-driven in vivo
phenotypes
Pascual et al., 2005;
Nam et al., 2012;
Fujita et al., 2014
TRE-loxP-STOP-loxP-
TeNT11-GFP (crossed
to GFAP-tTA and
GFAP-CreERt2 lines)
No direct data on the
percentage of astrocytes
expressing TeNT in different
brain regions from the
transgenic animals
Unaltered sleep patterns, only
the EEG power in low-gamma
range during wakefulness was
reduced; Unaltered basal
synaptic transmission as well as
normal short- and long-term
plasticity in hippocampus
Characterization of TeNT
expression profile is needed at
least in the region-of-interest in
future studies. The expression is
expected to be variable among
users due to differences in the
induction protocols
Lee et al., 2014 TABLE 2 | Current molecular tools for perturbing astrocyte signaling in vivo. Compared to global DrD2 KO,
the pro-inflammatory
phenotypes of astrocytes in the
SNC and in the striatum are very
mild. Petravicz et al.,
2008; Agulhon et al.,
2010; Navarrete
et al., 2012; Bonder
and McCarthy, 2014 GFAP-CB1R-KO Further characterization in
other brain regions needed Reduced spontaneous and
GPCR evoked Ca2+ responses;
reduced synaptic coverage by
astrocyte processes; impaired
glutamate uptake; impaired
spatial reference memory and
contextual fear responses Changes in sleep-wake cycle,
sleep loss related cognitive
functions, cortical slow waves,
depressive-like behavior, LTP
induction threshold in
hippocampus, neuronal survival
after stroke, development of
temporal lobe epilepsy, and
baseline mechanical nociception Characterization of TeNT
expression profile is needed at
least in the region-of-interest in
future studies. The expression is
expected to be variable among
users due to differences in the
induction protocols Unaltered sleep patterns, only
the EEG power in low-gamma
range during wakefulness was
reduced; Unaltered basal
synaptic transmission as well as
normal short- and long-term
plasticity in hippocampus April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 7 Manipulating astrocytic signaling in vivo Xie et al. Xie et al. GFAP-CB1R-KO TABLE 2 | Continued
Class
Mouse line
Confirmed expression
profile
In vivo phenotype
Comments
Selected
references
Glutamate transporter KO
Global Glt-1 KO
Global knockout
Reduced body weight, increased
morbidity, excitotoxic neuronal
death
Tanaka et al., 1997
Heterozygous global
knockout
Behavioral alterations in
sensorimotor function, locomotor
activity, anxiety, contextual and
cue-based fear conditioning
Kiryk et al., 2008
Floxed Glt-1
(crossed to
GLAST-CreERT2)
Conditional knockout
Excessive repetitive and injurious
self-grooming, bodily tics,
increased excitatory transmission
at corticostriatal synapses
Aida et al., 2015
Global GLAST KO
Global knockout
Altered glutamate/glucose
uptake and sensory evoked
neuronal oscillations in olfactory
bulbs; impaired vestibular activity
in aged mice; reduced sociability,
reduced acoustic startle
response; impaired visual
discrimination; hyperactive
locomotor activity
Karlsson et al.,
2008, 2009; Martin
et al., 2012;
Schraven et al.,
2012
NT synthesis enzyme KO
Nitrous oxide synthase
2 KO
Global knockout
Increased NO production in vivo;
stress and anxiety-related
behavior alterations
Buskila et al., 2007;
Abu-Ghanem et al.,
2008
Serine-racemase cKO
Conditional knockout;
Analyses of astrocyte
specific D-serine KO mice
show only a modest
reduction in serine
racemase activity
No alteration to hippocampal
LTP in situ; No in vivo phenotype
to date
Benneyworth et al.,
2012
Ion channel KO
Kir4.1 cKO
Conditional knockout;
Complete loss of Kir4.1
expression from S100β+
and GLAST+ astrocytes as
well as CNP+
oligodendrocytes
throughout brain and spinal
cord
Increased morbidity, ataxia,
stress-induced seizures, brain
anatomical changes,
white-matter vacuolization;
reduced potassium buffering
in vivo
Disorganized and fragmented
processes of cerebellar Bergman
glia, spinal cord white matter
astrocytes, and motor neurons
Djukic et al., 2007;
Chever et al., 2010
Connexin KOs
Cx30 and Cx43 KO
and cKO
Multiple global and
conditional knockout
models
Alterations to spatial working
memory, motor coordination,
sensorimotor adaptation, anxiety,
and pain perception
There exist multiple global,
conditional and combinatorial
knockouts (for example global
Cx30 KO with astrocyte specific
Cx43 knockout)
For comprehensive
reviews of connexin
genetic models,
please see reviews
by Giaume and
Theis (2010) and
Pannasch and
Rouach (2013) Disorganized and fragmented
processes of cerebellar Bergman
glia, spinal cord white matter
astrocytes, and motor neurons
Djukic et al., 2007;
Chever et al., 2010 Alterations to spatial working
memory, motor coordination,
sensorimotor adaptation, anxiety,
and pain perception modulation and behavior, as well as presented an elegant in vivo
system for temporal control of astrocytic signaling. is expressed in astrocytes in vivo (Khan et al., 2001). GFAP-CB1R-KO During
aging, DRD2 expression is downregulated in the brain (Kaasi-
nen et al., 2000), suggesting potential involvement of DRD2 in
aging related neuroinflammation. In this study DRD2-deficient
astrocytes were found to produce more proinflammatory media-
tors compared to wild-type astrocytes. Further, this effect appears
to be mediated through a decrease in αB-crystallin (CRYAB) sig-
naling, a small heat-shock protein known to negatively regulate There exist multiple global,
conditional and combinatorial
knockouts (for example global
Cx30 KO with astrocyte specific
Cx43 knockout) GFAP-DRD2-cKO In 2013, an astrocyte dopamine D2 receptor (DRD2) condi-
tional knockout mouse was generated to study the role of astro-
cytic GPCR signaling in aging-related neuroinflammation (Shao
et al., 2013). DRD2 couples to Gi (Missale et al., 1998) and April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 8 Manipulating astrocytic signaling in vivo Xie et al. kidneys (Fujino et al., 1995); this leads to potential confounding
issues in the use of this model in vivo when assessing the role
of astrocyte GPCR-dependent Ca2+ fluxes in behavior. Addition-
ally, concerns over compensation due to the role of intracellular
Ca2+ signaling during development have been raised regarding
this model; however to date no evidence for altered development
leading to compensation has been reported. pro-inflammatory mediator production and to display neuro-
protective effect (Ousman et al., 2007). Interestingly, DRD2-
deficient astrocytes also show robust GFAP upregulation, and
a reactive morphology in the substantia nigra and the striatum
of aged mice (Shao et al., 2013), suggesting the possible link
between astrocytic GPCR signaling and age-related impairments
in cognitive and motor function. g
In an attempt to address some of these issues, a conditional
IP3R2 knockout mouse model was developed by our laboratory. This model was generated by crossing the original floxed IP3R2
mouse developed by Dr. Ju Chen at UCSF (Li et al., 2005) to a
GFAP-Cre recombinase mouse (Stehlik et al., 2006) to restrict the
deletion of IP3R2 to GFAP+ cells in the CNS. This model recom-
bines the floxed IP3R2 allele at a high rate (>80–85% GFAP+
cells lack IP3R2), significantly reducing the number of astrocytes
responding to Gq-GPCR activation or neuronal activity in mul-
tiple brain regions (Petravicz et al., 2008; Chen et al., 2012). The
use of the GFAP-Cre system spatially and temporally restricts the
IP3R2 deletion to GFAP+ glia, thereby making it more appropri-
ate for in vivo analysis such as behavioral characterization and
reduces potential developmental compensation. Our lab recently
published a behavioral analysis of the IP3R2 cKO mice and found
no significant alteration to behavior (Petravicz et al., 2014). Most
importantly, no alterations to learning and memory as assessed
by the Morris Water Maze test were observed in these mice,
despite previous literature proposing an important role for astro-
cyte IP3R-mediated Ca2+ signaling in hippocampal LTP. GFAP-DRD2-cKO It is
unlikely that developmental compensation occurs such that alter-
native ions substitute for Ca2+ in physiological processes or that
global rewiring of neuronal circuits occurs to compensate for the
loss of astrocyte Ca2+ fluxes. Nevertheless, to completely rule
out developmental compensation, it will be necessary to prepare
inducible IP3R2 cKO mice using mice expressing floxed IP3R2
and an astrocyte specific inducible Cre system. IP3R2 Germline and Conditional IP3R2 Knockout Astrocytic Gq-GPCR/PLC/IP3 signaling is the most intensely
studied pathway in the proposed modulation of neuronal activity
and cerebral blood flow by astrocytes and has been the subject
of numerous reviews (Haydon and Carmignoto, 2006; Agulhon
et al., 2008, 2012; Fiacco et al., 2009; Halassa and Haydon, 2010;
Hamilton and Attwell, 2010). The activation of this pathway
results in the release of Ca2+ from IP3 receptor (IP3R) regu-
lated intracellular stores in the endoplasmic reticulum. Astro-
cytes express only one of three subtypes of this receptor, IP3R
type 2 (IP3R2) (Sharp et al., 1999; Holtzclaw et al., 2002; Foskett
et al., 2007; Hertle and Yeckel, 2007). The first functional report
to confirm this was published by Petravicz et al. (2008), in which
the germline IP3R2 knockout mouse (generated by the Ju Chen
lab; Li et al., 2005) was found to lack somatic increases in intra-
cellular Ca2+ upon activation of multiple subtypes of Gq-GPCRs
known to exist on astrocytes. Further experiments using this
model have confirmed that astrocytes lack Gq-GCPR elicited,
IP3R-dependent Ca2+ signals in their processes and soma (Agul-
hon et al., 2010; Di Castro et al., 2011; Panatier et al., 2011;
Takata et al., 2011; Navarrete et al., 2012; Tamamushi et al., 2012;
Nizar et al., 2013). The IP3R2 KO mouse is fertile, displays no
overt alterations in brain development, and displays no obvious
behavioral alterations (in contrast to the IP3R1 KO, the primary
neuronal IP3R Matsumoto and Nagata, 1999). Due to these fea-
tures, this mouse model has become one the most utilized mouse
models in astrocyte research. Use of this model has led to novel
findings (Panatier et al., 2011; Navarrete et al., 2012; Haustein
et al., 2014; Perez-Alvarez et al., 2014), several of which have been
contradictory with previously held theories concerning astrocyte-
neuron communication and vascular control (Fiacco et al., 2007;
Petravicz et al., 2008; Agulhon et al., 2010; Nizar et al., 2013;
Takata et al., 2013; Bonder and McCarthy, 2014). Recently, evi-
dence of residual Ca2+ signaling of a non-IP3R origin has been
published using genetically encoded Ca2+ indicators (GECIs) in
the IP3R2 KO model (Haustein et al., 2014; Kanemaru et al.,
2014), further illustrating the model’s usefulness for discovery of
novel signaling events in astrocytes; importantly, there is no evi-
dence suggesting that this residual Ca2+ signaling is regulated by
neuronal activity. Frontiers in Cellular Neuroscience | www.frontiersin.org Blocking Vesicular Release from Astrocytes with
dnSNARE or Tetanus Toxin Ca2+ dependent release of neurotransmitters from astrocytes,
termed “gliotransmission,” is one of the most important con-
cepts presented in glial biology over the past several decades
(Araque et al., 2014). While several mechanisms have been sug-
gested to underlie gliotransmission, most studies support a pro-
cess dependent on a vesicular release system (i.e., a SNARE
dependent process) similar to that found in neurons (Zorec et al.,
2012; Sahlender et al., 2014). To test the significance of SNARE-
mediated gliotransmission to synaptic function, a mouse line that
expresses a dominant negative form of SNARE (dnSNARE) in
astrocytes was developed in our laboratory in 2005 (Pascual et al.,
2005). This line was prepared by coinjecting three independent
constructs (tetO-lacZ, tetO-dnSNARE, and tetO-eGFP) into fer-
tilized zygotes. In dnSNARE transgenic mice, the expression of
the cytosolic portion of the SNARE domain of synaptobrevin 2,
lacZ, and eGFP are controlled by tetracycline regulatory system. When crossed with GFAP-tTA mice, the expression of dnSNARE,
lacZ, and eGFP are independently controlled by doxycycline. In
the absence of doxycycline, dnSNARE is expressed and inter-
feres with SNARE-dependent vesicular release. A limitation of
this model is that since dnSNARE was not directly tagged, there
is no way in situ to verify that it is not expressed in cells other
than astrocytes. Nevertheless, the dnSNARE mice have been used
in a large number of studies to demonstrate a role for gliotrans-
mission in synaptic transmission, synaptic plasticity, as well as
behavior. However, a recent paper performed an in-depth anal-
ysis of dnSNARE mice and found that that the expression of
dnSNARE was also expressed by a large population of neurons
(Fujita et al., 2014). Given the critical role of the SNARE complex
in neurotransmitter release, these findings bring into question the
validity of this model and the findings obtained using this system. Studies using dnSNARE mice suggest that SNARE-mediated
astrocytic release of ATP and subsequent adenosine receptor acti-
vation regulates neuronal excitability and synaptic plasticity in
many brain regions as well as modulate certain behaviors includ-
ing sleep (Nam et al., 2012). The dnSNARE mice exhibit a weak Memory deficit was also detected in the mice expressing TeNT
in astrocytes using novel object recognition test (Lee et al., 2014). Fast local field potential oscillations in the gamma frequency
are closely correlated with many cognitive functions, including
learning, memory storage and retrieval and attention (Basar-
Eroglu et al., 1996). IP3 Sponges These stud-
ies suggest that astrocytes release ATP in vesicular manner and
that this plays an important role in sleep patterns and cortical
oscillations. The expression of a fragment of the IP3 binding domain
of IP3R1, an “IP3 sponge” (Iwasaki et al., 2002) can also be
used to suppress IP3 induced Ca2+ release in astrocytes in vivo
(Tanaka et al., 2013). These investigators found that suppres-
sion of astrocyte Ca2+ responses affected several behavioral
responses, but no underlying evidence for alterations to synaptic
transmission was found. Surprisingly, astrocytes expressing the
“IP3 sponge” exhibited process retraction surrounding synapses,
which was attributed to underlie the behavioral phenotypes. Recently, evidence that astrocyte processes retract in response
to LTP-inducing stimuli in vivo was reported, and that this fea-
ture was lacking in IP3R2 KO mice (Perez-Alvarez et al., 2014). These findings appear to contradict those reported by Tanaka
et al. (2013). Further comparison between these two methods of
blocking astrocyte Ca2+ increases will be required to clarify this
contradiction. Tenanus toxins (TeNTs) are known to interfere with synap-
tic vesicular release as well as other processes dependent on
vesicular protein trafficking (Galli et al., 1994). Recently, a trans-
genic model system was developed using TeNT to block vesicular
release in astrocytes in vivo (Lee et al., 2014). This transgenic
model took advantage of both the tetracycline inducible regu-
latory system and the Cre-dependent inducible system to block
vesicular release from astrocytes. A transgenic line was prepared
that contained the tetracycline response element (TRE) followed
by a floxed stop cassette and a cassette that when expressed led
to the expression of eGFP tagged TeNT (TRE-loxP-STOP-loxP-
TeNT11-GFP). In this system, the expression of TeNT required
Cre expression to remove the floxed stop cassette and tetracycline
transactivator (tTA) to activate the TRE. TRE-loxP-STOP-loxP-
TeNT11-GFP mice were crossed with GFAP-tTA and GFAP-
CreERt2 lines to create a triple transgenic mouse line. In the triple
transgenic mice TeNT can be expressed in astrocytes following
tamoxifen treatment; the expression of TeNT is suppressed in the
presence of doxycycline. In this model, astrocyte expression of
TeNT requires that two different transgenes driven by the GFAP
promoter be expressed in the same astrocyte markedly increasing
the probability of astrocyte specific expression. Further, as TeNT
was directly tagged with eGFP, it is possible to identify all cells
expressing TeNT. IP3 Sponges Activation of Phospholipase C beta (PLCβ) and sequential release
of IP3 are the key steps in Gq-GPCR mediated intracellular Ca2+
elevations. Traditional approaches to abolish Gq-GPCR medi-
ated Ca2+ elevation in astrocytes include chelating intracellular
Ca2+ with BAPTA, or preventing IP3-mediated release of Ca2+
from ER using IP3R2 KO mice. Recently, the Pleckstrin Homol-
ogy domain of PLC-like protein (p130PH), which binds cytosolic
IP3 molecules, was used to suppress astrocytic Ca2+ signaling
in vitro and ATP-induced astrocytic Ca2+ responses in vivo (Xie
et al., 2010). In this study, p130PH was selectively expressed in
cortical astrocytes in vivo using rAAV2/5 vector in combination
with a specific astrocyte promoter, gfaABC(1)D (Lee et al., 2008;
Xie et al., 2010). p130PH transduced astrocytes in the somatosen-
sory cortex exhibited reduced amplitude and frequency of Ca2+
activity in response to direct ATP application on cortex com-
pared to non-transduced astrocytes, whereas the characteristics
of spontaneous Ca2+ activity in p130PH-transduced astrocytes
remained unchanged (Xie et al., 2010). Therefore, p130PH serves
as a more selective tool to suppress Gq-GPCR induced Ca2+ ele-
vations without chelating Ca2+ activity in astrocytes completely. This system serves as a nice addition to IP3R2KO and IP3R2 cKO Due to its restricted expression pattern in the CNS, a germline
knockout of IP3R2 provides a clean and reliable model to block
the release of intracellular Ca2+ in astrocytes elicited by Gq-
GPCR activity in vivo. However, this model suffers in that it lacks
tissue specificity, as do all germline knockout models. IP3R2 is
expressed in multiple tissues outside the CNS including the heart
(Li et al., 2005), pancreas (Orabi et al., 2012), lungs, liver, and Frontiers in Cellular Neuroscience | www.frontiersin.org April 2015 | Volume 9 | Article 144 9 Manipulating astrocytic signaling in vivo Xie et al. Xie et al. mouse lines to distinguish astrocyte functions regulated by ER
released Ca2+ vs. channel mediated Ca2+ in vivo. sleep phenotype under basal conditions, as well as an atten-
uated “rebound” response to sleep deprivation (Halassa et al.,
2009). Cortical slow oscillations, a rhythm characterizing non-
rapid eye movement (non-REM) sleep was also found impaired
in dnSNARE mice (Fellin et al., 2009). Further, the hippocam-
pal dependent memory deficits produced by sleep deprivation
were rescued in dnSNARE mice (Florian et al., 2011). IP3 Sponges In contrast to the impaired sleep pattern in
dnSNARE mice, astrocytic TeNT-expressing mice showed unal-
tered sleep patterns compared to triple transgenic mice without
tamoxifen or double transgenic mice with tamoxifin (Lee et al.,
2014). Studies using this mouse line indicate that basal synaptic
transmission as well as normal short- and long-term plasticity in
hippocampus in situ is not altered by the expression of TeNT (Lee
et al., 2014); these findings bring into question the concept that
astrocytes release gliotransmitters that modulate synaptic trans-
mission and plasticity via a vesicular dependent process. Interest-
ingly, the EEG power in low-gamma range during wakefulness
was reduced, whereas the EEG power during but not non-REM
sleep remain unchanged (Lee et al., 2014). These observations
in sleep regulation from astrocytic TeNT-expressing mice do not
match with those from dnSNARE mice. Frontiers in Cellular Neuroscience | www.frontiersin.org Astrocytic Serine-Racemase Conditional Knockout Astrocytic Serine-Racemase Conditional Knockout
D-serine has long been considered one of the three primary
gliotransmitters along with ATP/adenosine and glutamate, with
putative astrocyte derived D-serine reported to modulate neu-
ronal NMDA receptors (Panatier et al., 2006; Billard, 2008; Oliet
and Mothet, 2009; Martineau, 2013; Shigetomi et al., 2013b; Sild
and Van Horn, 2013). The enzyme responsible for D-serine pro-
duction (serine racemase, SR) was initially found to be primarily
expressed by astrocytes with some modest expression in neu-
rons (Schell et al., 1995; Mothet et al., 2005). However, more
recent studies have called this expression pattern into question
(Miya et al., 2008; Ding et al., 2011; Ehmsen et al., 2013). The
most recent study of SR localization in mice and human brains
finds that nearly all immunostaining for SR is found in neurons
and not astrocytes (Balu et al., 2014). Recently, cell type specific
knockouts of SR were generated to examine the relative contri-
butions of astrocytes and neurons in the forebrain of mice (Ben-
neyworth et al., 2012). The astrocyte specific knockout of SR led
to a modest (∼15%) reduction in SR expression, while the neu-
ronal knockout in forebrain neurons reduced SR by much larger
amounts (∼65%). Further, the neuronal specific SR knockout dis-
played alterations to LTP at hippocampal synapses that were not
found in the astrocyte SR knockout. These findings raise new
questions in how astrocytes may be regulating D-serine availabil-
ity in the brain, which the astrocyte specific SR knockout will be
crucial to resolving. Blocking Vesicular Release from Astrocytes with
dnSNARE or Tetanus Toxin Astrocytic TeNT-expressing mice did not
show deficit in other behavior tests that involves simpler form of
memory processing or are less dependent on cortical processing. The reduction of gamma oscillation power and significant deficit y
g
g
y
Studies using dnSNARE mice suggest that SNARE-mediated
astrocytic release of ATP and subsequent adenosine receptor acti-
vation regulates neuronal excitability and synaptic plasticity in
many brain regions as well as modulate certain behaviors includ-
ing sleep (Nam et al., 2012). The dnSNARE mice exhibit a weak Frontiers in Cellular Neuroscience | www.frontiersin.org April 2015 | Volume 9 | Article 144 10 Manipulating astrocytic signaling in vivo Xie et al. in novel object recognition suggest that the fast neural circuit
oscillations are regulated by astrocytes (Lee et al., 2014). net increase in overall NO concentrations in the brains of mutant
mice. Interesting, the increase in NO originated from astrocytes
through an alternate mechanism without alterations in the rela-
tive levels of NOS isoforms (Buskila et al., 2007). Further, these
mice display stress and anxiety-related alterations to behavior
suggesting a role for astrocyte-derived NO in the modulation of
neural circuits (Abu-Ghanem et al., 2008). Currently, the role of
astrocyte-derived NO in modulation of neuronal circuit activity
remains an understudied area of glial research. Transporter Knockout Mouse Models Astrocytes are responsible for 80–90% of glutamate reuptake in
the brain (Tzingounis and Wadiche, 2007), and two of the five
glutamate transporters (GluTs) are primarily expressed in astro-
cytes: Glt-1/EAAT2 and GLAST/EAAT1 (Danbolt, 2001; Huang
and Bergles, 2004). There are currently germline knockout mouse
models for both Glt-1 (Tanaka et al., 1997) and GLAST (Harada
et al., 1998); however their use in vivo has been limited. The Glt-
1 germline knockout mouse model suffers from reduced body
weight, increased morbidity, and progressive neuronal death due
to excitotoxocity (Tanaka et al., 1997). This limits the ability to
conduct in vivo experiments, which typically require older mice,
to examine the role of astrocytic glutamate reuptake via Glt-1. Recently, the heterozygous Glt-1 (Glt-1 Het) knockout model
has become an attractive model for studying the role of Glt-
1 in vivo. The Glt-1 Het model does not suffer from the more
obvious adverse effects of the full Glt-1 knockout, and displays
several interesting behavioral phenotypes and has allowed for the
study of Glt-1 in several brain pathologies (Kiryk et al., 2008). The GLAST knockout mice are viable, enabling in vivo studies. GLAST is primarily expressed in the cerebellum and olfactory
bulb (Regan et al., 2007), and this mouse model has led to inter-
esting findings concerning the physiological (Martin et al., 2012;
Schraven et al., 2012) and pathological (Karlsson et al., 2008,
2009) functions of GLAST. Recently, a Glt-1 floxed mouse model
has been developed (Aida et al., 2015). Induced knockout of Glt-
1 in adult animals resulted in development of repetitive behav-
iors and alterations to excitatory transmission due to reduced
glutamate uptake. The floxed Glt-1 model when combined with
inducible Cre systems will open up new avenues of research into
the role of Glt-1 in vivo that were not possible due to the lethality
of the germline Glt-1 KO. Current Limitations in Genetically Targeting
Astrocytes y
At this time, there is not a single gene delivery system that can be
used to exclusively express transgenes or recombine naïve genes
in astrocytes. The common astrocyte marker proteins (GFAP,
S100b, glutamine synthetase, aquaporin 4, connexin43, GLAST,
GLT1, ALDH1L1) are either expressed in alternate subsets of
mature CNS cells (Dunham et al., 1992; Zhuo et al., 2001; Su
et al., 2004; Hachem et al., 2005; Regan et al., 2007; Donato et al.,
2013), in progenitor CNS cells that give rise to multiple CNS cell
types (Hartfuss et al., 2001; Casper and McCarthy, 2006), or are
expressed outside the CNS (Jessen et al., 1990; Rinholm et al.,
2007; Darlot et al., 2008; Meabon et al., 2012; Rutkovskiy et al.,
2012; Jesus et al., 2014; Kato et al., 2014). The GFAP transcrip-
tional regulatory unit (TRU) is probably the best characterized
and most frequently used TRU for regulating gene expression in
astrocytes and serves as a good example to illustrate the difficul-
ties in targeting genes to astrocytes. The GFAP TRU is active in
progenitor cells that give rise to astrocytes, neurons, and oligo-
dendrocytes (Casper and McCarthy, 2006). Even in the mature
CNS, certain populations of neurons express GFAP (Zhuo et al.,
2001; Su et al., 2004; Regan et al., 2007). Further, in the periph-
ery, non-myelinating peripheral glia (Jessen et al., 1990) as well
as certain populations of non-neural cells (e.g., stellate cells in
the liver) are GFAP+ (Lim et al., 2008) and will express trans-
genes driven by the GFAP TRU. To avoid transgene expression
in progenitor cells, many laboratories have developed inducible
gene regulatory systems (Casper and McCarthy, 2006; Hirrlinger
et al., 2006; Mori et al., 2006). This approach circumvents gene
expression in progenitors during development but does not affect
the expression of transgenes in peripheral glia, adult stem cells or In summary, genetic manipulation of astrocytes is a very
powerful tool for assessing the role of these cells in physiology,
disease, and behavior. However, just as one has to verify the speci-
ficity of pharmacological reagents, great care must be used to
insure the specificity of genetic manipulations. Connexins Astrocytes in the brain exist not only as single units occupy-
ing a discrete domain, but also as a network of cells connected
by connexin (Cx) gap junctions. Astrocytes express two major
connexin proteins (Cx43 and Cx30) with germline and condi-
tional knockout mouse models existing (Dermietzel et al., 2000;
Teubner et al., 2003; Wiencken-Barger et al., 2007). These mouse
models display a variety of alterations to behavior, synaptic
transmission, metabolic support, and ion homeostasis (Giaume
and Theis, 2010; Pannasch and Rouach, 2013). However, there
are a large number of open questions concerning the role of
gap junction communication and astrocyte networks in vivo. Currently, the only in vivo experiments in these models have
involved behavioral studies, indicating a significant impact on
neuronal circuit function (for an excellent review please see
Pannasch and Rouach, 2013). Further exploration of neuronal
activity in vivo utilizing these models, as well as generation of
inducible knockout systems, represents novel avenues of research
for understanding astrocyte network function. The above discussion assumes that the astrocyte TRU is acting
with the fidelity of the endogenous TRU. Unfortunately, this is
often not the case and is largely dependent on the genomic con-
struct used to prepare the TRU. Most typically, investigators use
a fragment of the TRU to drive transgene expression. As trans-
genes generally integrate somewhat randomly at active sites in the
genome, the activity of surrounding genomic regulatory units can
markedly affect transgene expression levels as well as the cells the
transgene is expressed. This problem is markedly reduced using a
BAC approach where very large genomic segments containing the
TRU and inserted transgene are used to prepare transgenic lines. One final difficulty is that there are no astrocyte TRU systems that
can be used to target subpopulations of astrocytes. Currently, the
only way to genetically-manipulate subpopulations of astrocytes
is to transduce these cells using viral vectors. Unfortunately, this
generally restricts the size of the TRU used to target astrocytes
and requires viral injection that may lead to subtle or striking
changes in astrocyte function. Kir4.1 In the central nervous system nitric oxide serves a number of
roles, and has been shown to act at glutamatergic synapses to
enhance glutamate release (Garthwaite, 2008). While neurons in
many brain regions are known to produce and release NO via
nitric oxide synthase (nNOS), this does not account fully for the
activity of NO in several brain regions where excitatory neurons
lack nNOS expression. Astrocytes are known to express all three
isoforms of NOS, and are the sole expressers of an inducible
form of NOS (iNOS or NOS2) that is activated in response to
physiological stress in a Ca2+ dependent manner (Murphy, 2000;
Buskila et al., 2005; Amitai, 2010). Astrocyte-derived NO has
been shown to enhance LTP of presynaptic afferents in the spinal
cord, as well as enhance synaptic transmission in the neocortex
in acute slice preparations (Ikeda and Murase, 2004; Buskila and
Amitai, 2010). In vivo evidence for astrocytic-derived NO being
a modulator of neuronal transmission has primarily come from
the use of a NOS2 knockout mouse, in which it was discovered
that deletion of the calmodulin-binding domain of NOS2 led to a One of the major roles in the CNS for astrocytes is the buffer-
ing of potassium ions in response to neuronal activity. Astrocytes
express a variety of potassium channels, but among them Kir4.1
plays a predominant role in their K+ buffering capacity (Takumi
et al., 1995; Higashi et al., 2001; Djukic et al., 2007). A germline
full Kir4.1 knockout model has provided insight into the role
of potassium buffering in response to hyperammonemic condi-
tions (Stephan et al., 2012) and the channel’s role in regulating
astrocyte membrane potential during development (Seifert et al.,
2009). An astrocyte conditional knockout model for Kir4.1 was
generated by the McCarthy lab to provide a cleaner animal model
alternative to the germline KO (Djukic et al., 2007). The condi-
tional knockout astrocytes display reduced glutamate clearance
and decreased resting membrane voltage, while neuronal plastic-
ity was enhanced implicating Kir4.1 as an important mediator
of extracellular potassium regulation. These finding have been
confirmed in an in vivo study utilizing the Kir4.1 cKO, with the Frontiers in Cellular Neuroscience | www.frontiersin.org April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 11 Manipulating astrocytic signaling in vivo Xie et al. Kir4.1 cKO mice found to have reduced capacity to regulate extracel-
lular potassium levels compared to controls (Chever et al., 2010). Usage of this model in awake mice to examine the effect of altered
potassium homeostasis may provide unique insights into how
astrocytes regulate neuronal networks. small populations of neurons that normally express GFAP. How
much of a problem this presents depends on the question being
asked. When studying a transgene in the mature CNS that can
be assumed not to affect developmental processes nor lead to a
peripheral phenotype it is reasonable to use many of the available
astrocyte TRU to drive transgenes to astrocytes. For example, the
GFAP TRU can be used to express eGFP, GCaMP, or DREADD
receptors that must be activated by an exogenous ligand without
preventative concern about developmental expression. However,
when using the GFAP TRU to drive bioactive molecules such as
DREADD receptors or inducible Cre recombinase, it is impor-
tant to remain cognizant that in addition to astrocytes, peripheral
GFAP+ cells, adult stem cells, and certain neurons will also be
affected. Alternatively, when expressing molecules that innately
affect biological processes (e.g., a dominant negative mutation,
constitutively active signaling molecule or Cre recombinase), it
is very important to consider the consequences of expression
during development. Conditional Gαq/Gα11 KO GPCR KO mouse lines for all the known Gα subunits have been
developed to analyze the physiological function of GPCR signal-
ing in vivo (Offermanns, 1999), many of which show deficiencies
in CNS-related physiology (Offermanns, 2001). Although these
KO mouse lines are not astrocyte specific, inducible and con-
ditional Gαq/Gα11 KO mice are available (Wettschureck et al.,
2001) and can be combined with astrocytic specific Cre mouse
line to selectively knock out Gq-GPCR signaling in astrocytes. Recently, the concept of using opsin based-pigments to
develop optogenetic tools for modulating GPCR signaling was
suggested (Koyanagi and Terakita, 2014). One of the candi-
dates is melanopsin (OPN4), a Gq-coupled opsin that is orig-
inally found in a subtype of retinal ganglion cells (Hatori and
Panda, 2010; Sexton et al., 2012). Several groups have used ectopic
expression of OPN4 to control intracellular Ca2+ dynamics in
neurons (Koizumi et al., 2013). In 2013, Karunarathne and col-
leagues used non-rhodopsin opsins to activate native Gq, Gi/o,
and Gs signaling in localized regions of a single cell, and were
able to gain spatial-temporal control over immune cell migra-
tion (Karunarathne et al., 2013b) as well as neurite initiation and
extension (Karunarathne et al., 2013a). These studies suggest high
potential of opsins as optogenetic GPCRs for in vivo astrocyte
research. y
q
g
g
y
The conditional Gαq/Gα11 KO system was developed to
study the role of Gαq/Gα11 signaling in specific tissues with-
out developmental problems exhibited in the constitutive KO
mouse line for both genes. This mouse line was developed using
the Cre/LoxP system where the Gαq gene (gnaq) is condition-
ally inactivated in Gα11 KO (gna11–/–) mice, which do not
have obvious behavior defect (Stanislaus et al., 1998). There-
fore, mice homozygous for gnaqflox gene appear normal until
Cre recombinase is introduced. The conditional Gαq/Gα11 KO
mouse was first used with MLC2a-Cre to obtain cardiomyocyte-
specific Gαq/Gα11 deficiency, resulting in a nearly complete
recombination of gnaqflox in cardiomyocytes (Wettschureck
et al., 2001). Studying G-Protein Independent Signaling Using
Biased DREADDs One important aspect of GPCR signaling in astrocytes that has
rarely been explored is the role of G-protein independent sig-
naling. Endogenous GPCR activation not only initiates signaling
via heterotrimeric G proteins, but also recruits proteins of the
arrestin family, which act as scaffolding proteins and promote
G protein-independent signaling (Pierce et al., 2002; Rajagopal
et al., 2010; Shukla et al., 2011). Research has shown that arrestin
3 (β-arrestin 2) is expressed in astrocytes ex vivo (Bruchas et al.,
2006; McLennan et al., 2008), and it is involved in kappa opioid
receptor (KOR)-induced proliferation (McLennan et al., 2008;
Miyatake et al., 2009), reduction of chemical-induced apoptosis
(Zhu and Reiser, 2014), CXCR7 mediated inflammatory response
(Odemis et al., 2012; Lipfert et al., 2013) and beta 2-adrenergic
receptor (β2AR)-mediated glycogenolysis (Dong et al., 2001; Du
et al., 2010) in astrocytes. As a scaffolding protein, β-arrestins also
mediate internalization and ubiquitylation for many ion chan-
nels and transporters expressed in astrocytes (Shukla et al., 2011). As the list of signaling pathways that β-arrestins can regulate in
astrocytes grows, it is important to dissect the relative contri-
bution of G-protein independent signaling pathway to known
functions of astrocytic GPCR signaling. In 2006, the Gαq/Gα11 cKO mice was crossed to mice
that express Cre under the control of the promoter of the
Ca2+/calmodulin-dependent protein kinase IIα gene (Camkcre4
mice) to generate forebrain specific and neuronal Gαq/Gα11
double KO mice (Wettschureck et al., 2004, 2006; Broicher et al.,
2008), resulting in impaired endocannabinoid levels, increased
seizure susceptibility (Wettschureck et al., 2006), and lack of
maternal behavior in females (Wettschureck et al., 2004). The
Gαq/Gα11 cKO line was also used to disrupt glial Gq/11 signal-
ing in combination with Nestin-Cre mouse (Wettschureck et al.,
2005), resulting in loss of Gq/11 in the neural stem cells that gives
rise to both neurons and astrocytes. Although the Gq/11 signal-
ing deficiency did not cause gross morphological changes in the
developing nervous system, pups with insufficient Gq/11 signal-
ing suffers from hypothalamic growth hormone deficiency and
somatotroph hypoplasia, dwarfism, and anorexia (Wettschureck
et al., 2005). Given the availability of astrocytic-specific Cre mice,
it will be possible to isolate the contribution of astrocytic Gq/11
signaling in vivo. Temporal Control of GPCR Signaling Using
Optogenetic GPCRs Optogenetically activated GPCR signaling is a reasonable alter-
native to ChR2 stimulation in astrocytes. There are two sys-
tems based on adrenergic receptors: Opto-alpha-1 (Gq linked)
and opto-beta-1 (Gs linked) (Airan et al., 2009). Activation of
these systems use similar experimental methods as optogenet-
ics, but have the advantage of activating endogenous signaling
cascades that exist in astrocytes. To date, these systems have
not been tested for astrocytes in vivo, only in astroglia in cul-
ture (Figueiredo et al., 2014). While the Gq-linked opto-alpha-1 April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 12 Manipulating astrocytic signaling in vivo Xie et al. messages, but can promote CNO-dependent and arrestin-
dependent signaling in biological systems (Nakajima and Wess,
2012). This novel GPCR represents an excellent tool to study
the relative contribution of G protein-dependent and indepen-
dent pathways in the known function of astrocytes, as well
as reveal the physiological roles of astrocytic-arrestin signaling
in vivo. predictably elicited Ca2+ increases via release from intracellu-
lar stores, the Gs-linked opto-beta-1 was found to also trigger
Ca2+ increases in a cyclic AMP-dependent manner. In culture
systems, it has been shown that the Gβγ subunit of Gs-coupled
GPCRs is capable of directly gating IP3Rs (Zeng et al., 2003). Recently, experiments in HEK293 cells discovered that IP3R2
complexes with Gαs and type 6 adenyl cyclase (AC6), and facili-
tates crosstalk between the two signaling pathways (Tovey et al.,
2008, 2010). Further study into the mechanism behind increases
in Ca2+ in astrocytes may lead to the identification of novel
pathways regulating Ca2+ signaling. Frontiers in Cellular Neuroscience | www.frontiersin.org Summary and Future Directions Recently, a modified Gq-DREADD that has strong biases
toward arrestin-signaling was developed (Nakajima and Wess,
2012). This receptor was generated by introducing a point muta-
tion within the highly conserved DRY motif [Rq(R165L)] of
Gq-DREADD, which results in lack of ability to activate het-
erotrimetic G proteins. Therefore, CNO-induced Rq(R165L)
activation has no effect on the levels of conventional second There is no doubt that genetic tools will play an important role
in understanding the role of astrocytes in physiology, behavior,
and neurological disorders. Studies cited above provide strong
evidence that astrocytes are doing much more than simply
insulating synapses and providing nutrients to neurons. It is not April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 13 Manipulating astrocytic signaling in vivo Xie et al. surprising that findings using genetic tools may conflict with pre-
vious findings using pharmacological methods to perturb astro-
cyte function. It is also not surprising that different genetic
approaches (e.g., dnSNARE and tetanus toxin expression) may
yield different results. These conflicts require additional studies
using multiple tools (pharmacological and genetic) to refine our
understanding of astrocyte function. Currently, there are several
important limitations in the genetic tools available for perturb-
ing astrocyte function in vivo. First, we are extremely limited
in the transcriptional units (promoters) available for expressing
molecules in astrocytes. Currently, all available transcriptional
units drive transgene expression in cells other than astrocytes. Further, during development, most astrocyte promoters drive
gene expression in progenitor cells that give rise to neurons,
oligodendrocytes, and astrocytes (Casper and McCarthy, 2006). Consequently, it is necessary to use inducible systems linked to
astrocyte promoters to avoid transgene expression in progenitors
during development. Second, when expressing transgenes that
drive function, it is important to remember that the transgene
may be overexpressed and targeted to cellular compartments not
normally found. While this can be partially overcome using an inducible gene regulatory system, it is likely that this will remain
a caveat until we know a great deal more about the cell machin-
ery in astrocytes that target molecules to specific cellular com-
partments. Third, we currently lack promoters that can be used
to drive transgene expression in subtypes of astrocytes. Subtype
specific astrocyte promoters will enable important advances with
respect to the heterogeneity of astrocytes as well as their different
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early stages of development, genetic models have already pro-
vided important insight into the role of astrocytes in physiol-
ogy, behavior, and neurological diseases. It seems very likely
that future advances in this field will depend largely on
genetic approaches currently available as well as those under
development. Acknowledgments We thank Dr. T. Kendall Harden for his generous help with the
preparation of Figure 1. The work in McCarthy laboratory is
supported by NINDS RO1-NS20212-26 and RO1-MH099564. References Neuropsychopharmacology
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distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, pro-
vided the original author(s) or licensor are credited and that the original publi-
cation in this journal is cited, in accordance with accepted academic practice. No
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10.1016/S0960-9822(03)00330-0 April 2015 | Volume 9 | Article 144 Frontiers in Cellular Neuroscience | www.frontiersin.org 20
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Dental arch spatial changes after premature loss of first primary molars: a systematic review and meta-analysis of split-mouth studies
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RESEARCH Open Access Abstract Keywords Space loss, Premature tooth loss, Primary first molar, Spatial changes, Space maintainer *Correspondence:
Xiurong Qin
qinxiurong0531@163.com
1Department of Orthodontics, East Hospital District, Jinan Stomatological
Hospital, Jinan, Shandong Province, China
2Department of Stomatology, Harbin Institute of Technology Hospital,
Harbin, Heilongjiang Province, China
3Department of Endodontics, Yantai Stomatological Hospital Affiliated to
Binzhou Medical College, Yantai, Shandong Province, China
4Department of Pediatric Dentistry, Jinan Stomatological Hospital, Jinan,
Shandong Province, China *Correspondence:
Xiurong Qin
qinxiurong0531@163.com
1Department of Orthodontics, East Hospital District, Jinan Stomatological
Hospital, Jinan, Shandong Province, China
2Department of Stomatology, Harbin Institute of Technology Hospital,
Harbin, Heilongjiang Province, China
3Department of Endodontics, Yantai Stomatological Hospital Affiliated to
Binzhou Medical College, Yantai, Shandong Province, China
4Department of Pediatric Dentistry, Jinan Stomatological Hospital, Jinan,
Shandong Province, China © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included
in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data. BMC Oral Health BMC Oral Health Zhao et al. BMC Oral Health (2023) 23:430
https://doi.org/10.1186/s12903-023-03111-x Dental arch spatial changes after premature
loss of first primary molars: a systematic
review and meta-analysis of split-mouth
studies Jingzi Zhao1, Hua Jin2, Xiaoning Li3 and Xiurong Qin4* Jingzi Zhao1, Hua Jin2, Xiaoning Li3 and Xiurong Qin4* Abstract
Background This study aimed to evaluate spatial changes in dental arches resulting from premature loss of first
primary molars and assess the necessity of a space maintainer. Methods We searched the electronic databases PubMed, Cochrane Library, ClinicalTrials, and EMBASE. Split-mouth
studies involving unilateral premature loss of a primary first molar were included. Quality assessment of selected
studies made use of the ROBINS-I tool. Mean space differences were calculated for the D + E and D spaces, arch width,
arch length, arch perimeter. Results Of the 329 studies considered, 11 split-mouth studies were selected, including 246 cases in the maxilla and
217 in the mandible from 477 individuals aged 5–10 years. Over the medium-term follow-up period (6–24 months),
space loss was 0.65 mm in the maxillary D + E (MD 0.65, 95% CI 0.15–1.16, P = 0.01), 1.24 mm in the mandibular D + E
(MD 1.24, 95% CI 0.60–1.89, P < 0.01), and 1.47 mm in the mandibular D (MD 1.47, 95% CI 0.66–2.28, P < 0.01). There
was no significant change in arch width, length, or arch perimeter between the initial and follow-up examinations
(P > 0.05). Conclusions After premature loss of first primary molars, space can be lost, but the amount of loss would not affect
arch width, length, or arch perimeter over the 6–24 months follow-up period. Keywords Space loss, Premature tooth loss, Primary first molar, Spatial changes, Space maintainer Abstract Background This study aimed to evaluate spatial changes in dental arches resulting from premature loss of first
primary molars and assess the necessity of a space maintainer. Methods We searched the electronic databases PubMed, Cochrane Library, ClinicalTrials, and EMBASE. Split-mouth
studies involving unilateral premature loss of a primary first molar were included. Quality assessment of selected
studies made use of the ROBINS-I tool. Mean space differences were calculated for the D + E and D spaces, arch width,
arch length, arch perimeter. Results Of the 329 studies considered, 11 split-mouth studies were selected, including 246 cases in the maxilla and
217 in the mandible from 477 individuals aged 5–10 years. Over the medium-term follow-up period (6–24 months),
space loss was 0.65 mm in the maxillary D + E (MD 0.65, 95% CI 0.15–1.16, P = 0.01), 1.24 mm in the mandibular D + E
(MD 1.24, 95% CI 0.60–1.89, P < 0.01), and 1.47 mm in the mandibular D (MD 1.47, 95% CI 0.66–2.28, P < 0.01). There
was no significant change in arch width, length, or arch perimeter between the initial and follow-up examinations
(P > 0.05). Results Of the 329 studies considered, 11 split-mouth studies were selected, including 246 cases in the maxilla and
217 in the mandible from 477 individuals aged 5–10 years. Over the medium-term follow-up period (6–24 months),
space loss was 0.65 mm in the maxillary D + E (MD 0.65, 95% CI 0.15–1.16, P = 0.01), 1.24 mm in the mandibular D + E
(MD 1.24, 95% CI 0.60–1.89, P < 0.01), and 1.47 mm in the mandibular D (MD 1.47, 95% CI 0.66–2.28, P < 0.01). There
was no significant change in arch width, length, or arch perimeter between the initial and follow-up examinations
(P > 0.05). Conclusions After premature loss of first primary molars, space can be lost, but the amount of loss would not affect
arch width, length, or arch perimeter over the 6–24 months follow-up period. Zhao et al. BMC Oral Health (2023) 23:430 Page 2 of 14 Zhao et al. BMC Oral Health (2023) 23:430 Zhao et al. BMC Oral Health Research question and study protocolh This study was registered in the PROSPERO database
(Registration number: CRD 42,022,372,202) and con
ducted in accordance with the Preferred Reporting Items
for Systematic Reviews and Meta-Analysis (PRISMA)
2020 guidelines [27]. The research question of this review
was based on the PECOS framework: Population (P):
children with unilateral first primary molar premature
loss in primary dentition and mixed dentition; Exposure
(E): unilateral premature loss of a primary first molar;
Comparison (C): between side with premature loss of
first primary molar and the control side with no loss of
molars; Outcome (O): the dental arch spatial changes;
and Study Design (S): split-mouth studies were retrieved
for analysis. There are differences between the maxilla and man
dible in terms of the space changes that occur follow
ing premature loss of primary first molars. Some studies
have reported that space loss in the mandible is greater
than that in the maxilla [13], while others have claimed
the opposite [14]. Studies have shown more than one
cause of space loss after premature loss of the primary
first molar. Lin’s research showed that distal movement
of the primary canine could occur after premature loss
of the primary first molar on the maxilla and mandible
[8], and mesial movement of permanent molars or tilt
ing of the primary second molar did not occur [15]. Love
and Adams [16] found that space loss caused by mesial
movement of posterior teeth was higher than that caused
by distal movement of anterior teeth, especially in the
maxilla. In addition, some studies showed that space loss
occurred in the maxilla due to mesial movement of the
molars and mandible resulting from distal movement
of the anterior teeth [17–19]. One study reported that
premature loss of the primary mandibular first molar
mainly results in distal drifting of the primary mandib
ular canine, while in the maxilla, mesial drifting of the
primary second molar into the extraction space is more
common [13]. Eligibility criteria
l d Data were included if dental arch spatial changes after
unilateral premature loss of first primary molars were
investigated in human split-mouth studies. Reviews,
abstracts, case reports and series, comments, letters to
the editor, conference proceedings, in vitro investiga
tions, and animal studies were excluded. Introductionh mixed dentition. Although there is little debate about the
need for a space maintainer after premature loss of the
primary second molar, there is disagreement about the
need after premature loss of the primary first molar [15]. Some scholars believe that premature loss of the primary
first molar will lead to loss of space, so the use of space
maintainers becomes necessary [4, 7, 15]. However, oth
ers believe that arch length does not change after prema
ture loss of the primary first molar, so the use of a space
maintainer is unnecessary [13, 22, 23]. Some with views
between these two extremes suggest that use of a space
maintainer following premature loss of a primary first
molar should be based on the dentist’s experience and
the patient’s orofacial condition [24–26]. The purpose of
this study was to evaluate dental arch spatial changes fol
lowing premature loss of first primary molars and assess
the need for a space maintainer. The concept of premature loss of primary teeth has been
defined as exfoliation on the arch more than 12 months
prior to the normal period of permanent tooth eruption
[1, 2], which exceeds normal variability of the exfoliation
sequences of temporary teeth. Premature loss of primary
molars is usually caused by early extraction due to caries
and/or failed pulp therapy, which may cause migration
of adjacent teeth, space loss, crowding and impaction or
dislocation of the permanent teeth, leading to the need
for complex orthodontic treatment [1, 3–6]. In 1887,
Davenport [7] described the concept of space loss caused
by premature loss of primary teeth, and this problem has
been studied since then. In 1998, Lin and Chang were the
first to quantify the amount of space loss caused by pre
mature loss of primary molars [8]. Since then, research
on space changes following premature loss of primary
teeth has included cross-sectional and longitudinal stud
ies. At present, there are extensive clinical studies in the
literature describing space changes resulting from pre
mature loss of primary molars, including but not limited
to the direction of the space change, the amount of space
loss, and the need for space maintenance [9–12].hf Literature search strategy and selection of papersh BMC Oral Health Table 1 Search strategy
Database
Key Words
Results
PubMed
((((primary first molar ) OR (primary maxillary first molar ) OR (primary mandibular first molar))) AND (((tooth migration) OR
(tooth drift)OR (mesial movement) OR (distal movement) OR (space loss) OR (arch changes)))) AND (((premature tooth loss )
OR (premature loss) OR (tooth loss) OR (tooth extraction) OR (tooth exfoliation)))
Last update posted on or before 09/15/2022
100
Web of Science ((((primary first molar ) OR (primary maxillary first molar ) OR (primary mandibular first molar))) AND (((tooth migration) OR
(tooth drift)OR (mesial movement) OR (distal movement) OR (space loss) OR (arch changes)))) AND (((premature tooth loss )
OR (premature loss) OR (tooth loss) OR (tooth extraction) OR (tooth exfoliation)))
Last update posted on or before 09/15/2022
63
Cochrane
Libray
((((primary first molar ) OR (primary maxillary first molar ) OR (primary mandibular first molar))) AND (((tooth migration) OR
(tooth drift)OR (mesial movement) OR (distal movement) OR (space loss) OR (arch changes)))) AND (((premature tooth loss )
OR (premature loss) OR (tooth loss) OR (tooth extraction) OR (tooth exfoliation))) in Title Abstract Keywords
Last update posted on or before 09/15/2022
77
EMBASE
(‘primary first molar’ OR (primary AND first AND (‘molar’/exp OR molar)) OR ‘primary maxillary first molar’ OR (primary AND
(‘maxillary’/exp OR maxillary) AND first AND (‘molar’/exp OR molar)) OR ‘primary mandibular first molar’ OR (primary AND
mandibular AND first AND (‘molar’/exp OR molar))) AND (premature AND tooth AND loss OR (premature AND loss) OR
(tooth AND loss) OR (tooth AND extraction) OR (tooth AND exfoliation)) AND (space AND loss OR (tooth AND migration )
OR (tooth AND drift ) OR (mesial AND movement) OR (distal AND movement) OR (arch AND changes))
Last update posted on or before 09/15/2022
87
Fig. 1 PRISMA flow diagram summarizing the study selection process 100 Fig. 1 PRISMA flow diagram summarizing the study selection process Fig. 1 PRISMA flow diagram summarizing the study selection process changes,” and “premature tooth loss OR premature loss
OR tooth loss OR tooth extraction OR tooth exfoliation.”
The search terms were used alone or in combination
using the Boolean operators OR and AND (Table 1). those authors, none responded to our inquiries. Research
publications from the same authors or institutions were
scrutinized to eliminate any data redundancy. In the case
of redundancy, only results from the most recent publica
tions were included. Literature search strategy and selection of papersh Literature search strategy and selection of papers
The PubMed, Cochrane, ClinicalTrials, and EMBASE
databases were searched for studies through September
15, 2022. No restrictions were placed on the language
and date of publication. Manual searching of references
in the relevant published articles was also performed. Three corresponding authors were contacted to either
acquire unpublished study results of published trial pro
tocols relevant to our study or clarify information in
the original manuscripts. Despite our following up with Space maintenance is considered to play an impor
tant role in maintaining the integrity of the dental arch
after premature loss of primary teeth [20]. Choonara [21]
reported that many orthodontic cases involving crowd
ing and insufficient space in permanent dentition could
have been prevented or mitigated if the dentist had main
tained sufficient space during the initial treatment of Page 3 of 14 Zhao et al. BMC Oral Health (2023) 23:430 (2023) 23:430 Zhao et al. Literature search strategy and selection of papersh g
The searches were imported into the EndNote (v. 20)
library. Duplications were identified and removed. Titles
and abstracts of each retrieved record were screened to
exclude any papers not fulfilling inclusion criteria. Two
independent reviewers (JZ Zhao and H Jin) evaluated The search terms included “primary first molar OR
primary maxillary first molar OR primary mandibular
first molar,” “tooth migration OR tooth drift OR mesial
movement OR distal movement OR space loss OR arch Page 4 of 14 Page 4 of 14 Zhao et al. BMC Oral Health (2023) 23:430 Zhao et al. BMC Oral Health Author
Study
design
Age(years)
Sam
ple
size
Arch(n)
Data
collection
Follow-up
Period
Evaluation
indicators
D + E Space loss(mm)
Heidari et
al. 2022
[37]
cross-
sectional
Split-mouth
8–10
(9.08 ± 0.58)
47
Maxilla(25)
Mandible(22)
Plaster casts
11.85 ± 5.81 m(6-
24 m)
midline, molar and
canine relation
ship, facial growth
pattern, Canine’s
inclination, space
loss, crowding
Maxilla: 0.54
Mandible: 0.58
Mosharra
fian et al. 2021 [13]
cross-
sectional
Split-mouth
6–8
(7.30 ± 0.68)
50
Maxilla(25)
Mandible(25)
Plaster casts
13.54 ± 6.28 m
(6-24 m)
midline, molar and
canine relation
ship, facial growth
pattern, space loss,
crowding
Maxilla: 1.32
Mandible: 1.40
Kobylńska
et al. 2019
[36]
longitudi
nal study
Split-mouth
5–7
(6.64 ± 1.01)
44*
Maxilla(16)
Mandible(14)
Plaster casts
1,3,6,12 m
midline, inter-arch
tooth alignment,
Angle’s class,
vertical bite, lat
eral teeth contact,
radiological assess
ment, space loss
Maxilla: 1.156
Mandible: 1.000
Lin et al. 2017 [9]
longitudi
nal study
Split-mouth
5–7
(6.0 ± 0.42)
9
Maxilla(9)
Plaster casts
81 m
arch width, arch
length, intercanine
width, intercanine
length, and arch
perimeter, space
loss
Not mentioned
Alexander
et al. 2015
[34]
longitudi
nal study
Split-mouth
7.7–8.2
226
Maxilla(111)
Mandible(115)
Direct intraoral
measurement
9 m
facial growth pat
tern, space loss
Maxilla:(Leptoprosopic/
End-On:1.75 ± 0.31;
Leptoprosopic/Class
I:0.89 ± 0.16; Mesopro
i /E
i /
Table 2 General characteristics of the studies included in the systematic review and meta-analysis Table 2 General characteristics of the studies included in the systematic review and meta-analysis racteristics of the studies included in the systematic review and meta-analysis sopic/Class I:0.08 ± 0.04)
Lin et al. 2011 [15]
longitudi
nal study
Split-mouth
6–9
(6.0 ± 0.74)
13
Maxilla(13)
Plaster casts
12 m
arch width, arch
length, intercanine
width, intercanine
length, arch perim
eter, space loss
Maxilla: 0.82. Macena
et al. Literature search strategy and selection of papersh 2011
[33]
longitudi
nal study
Split-mouth
6–9
20
Maxilla (12)
Mandible (8)
Plaster casts
3、6、10 m
arch length, arch
hemi- perimeter,
space loss
Maxilla: 0.2&
Mandible: 1.0&
Park et al. 2009 [32]
cross-
sectional
Split-mouth
5–10
13
Maxilla(13)
Digitized
plaster casts
12 m (8-23 m)
space loss, arch
width, arch length,
arch perimeter,
the inclination and
angulation
Maxilla: 0.57 ± 0.83
Kim et al. 2008 [35]
cross-
sectional
Split-mouth
6–10
6
Maxilla(3)
Mandible(3)
Digitized
plaster casts
Maxilla: 15.3 m
(6-23 m)
Mandible:
13.6 m (9-20 m)
space loss, arch
width, arch length,
arch perimeter,
the inclination and
angulation
Maxilla: 0.43
Mandible: 1.78 Page 5 of 14 Zhao et al. BMC Oral Health (2023) 23:430 Zhao et al. BMC Oral Health Author
Study
design
Age(years)
Sam
ple
size
Arch(n)
Data
collection
Follow-up
Period
Evaluation
indicators
D + E Space loss(mm)
Lin et al. 2007 [31]
longitudi
nal study
Split-mouth
4–7
(5.9 ± 0.74)
19
Maxilla(19)
Plaster casts
6 m
arch width, arch
length, intercanine
width, intercanine
length, and arch
perimeter, space
loss
Maxilla: 1.08
Padam et
al. 2006
[30]
longitudi
nal study
Split-mouth
6–9
40**
Mandible(30)
Plaster casts
2、4、6、8 m
arch length, arch
perimeter, arch
width, space loss
Mandible: 1.83&
*: 17 individuals were excluded from the studies because of malocclusion (n = 4), lack of second premolar buds on examination (n = 1), the presence of mesiodens
(n = 2) and further tooth extractions (n = 10)
**:10 individuals were excluded because of no further follow up
& : D space loss
Table 2 (continued) **:10 individuals were excluded because of no further follow up and (3) Post-intervention—biases due to deviations from
intended interventions, biases due to missing data, biases
in measurement of outcomes, and biases in selection of
the reported results. Studies were categorized four levels
as critical, serious, moderate, and low respectively. the studies extracted from the searches. When there was
uncertainty on the eligibility of an article, the study was
adjudicated based on discussion and consensus between
the two reviewers and a third reviewer (XR Qin). Statistical analysis Relevant data from the included papers were indepen
dently extracted in duplicate by two authors (JZ Zhao
and H Jin). Data extracted from the selected studies
included study information (author, year, and study
design), patient information (age, sample size, follow-up
time, tooth, research methods, and indicators), diagnos
tic information (facial type, molar occlusal relationship,
canine occlusal relationship, crowding, and midline), and
follow-up evaluations (D + E and D space: D and E are the
first and second primary molar respectively, while D + E
space is the space occupied by the first and second pri
mary molars; arch width, length, and perimeter). Because
the follow-up times were different in the longitudinal
studies, they were divided into three periods, short-term
(≤ 6 m), medium-term (6–24 m), and long-term (> 24 m),
in order to evaluate results for different follow-up times. Duplication of sample data in meta-analyses was avoided
by selecting for analysis only the final data in a longitudi
nal study. SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA) was
used to apply the Kappa test for assessment of article
identification, screening, data extraction, and quality to
evaluate agreement among reviewers. g
g
Meta-analysis was performed when warranted by the
quality and quantity of data. Review manager 5 software
(Revman5.4) was used to analyze the combined effect,
heterogeneity, and publication bias. To test for statisti
cal significance in the differences in spaces between the
extraction and control sides, and space losses between
baseline and final examination values, mean differences
(MD) were calculated for D + E and D spaces and space
losses, and for arch widths, lengths, and perimeters. Het
erogeneity was assessed using I2 statistics and Cochrane’s
Q test, with I2 > 50% or P < 0.10 on Cochrane’s Q test indi
cating substantial heterogeneity [28]. P-values < 0.05 were
considered statistically significant. Publication bias was
evaluated by visual inspection of funnel plot [29]. Stata
software (Stata 15.1) was used to analyze the results of
Begg’s and Egger’s tests and perform sensitivity analyses. Quality assessment Quality assessment was carried out independently by two
authors (JZ Zhao and H Jin), and any disagreements were
resolved by consensus. The ROBINS-I (Risk Of Bias In
Non-randomized Studies – of Interventions)(http://cre
ativecommons.org/licenses/by-nc-nd/4.0/) tool was used
to assess the quality of the studies. The ROBINS-I tool
evaluates the methodological quality of individual stud
ies based on seven domains grouped into three phases:
(1) Pre-intervention—biases due to confounding and
biases in selection of participants into the study; (2) At
intervention—biases in classification of interventions; Quality assessment and kappa’s test or nonunilateral premature loss of a primary first molar
(n = 1) [1, 4, 14, 26, 38, 39] (Online Resource 1). Accord
ing to the quality and quantity of data, seven articles were
selected for meta-analysis [15, 30, 32, 34–36]. Details on
the selection process of research articles are presented in
a flow diagram (Fig. 1). or nonunilateral premature loss of a primary first molar
(n = 1) [1, 4, 14, 26, 38, 39] (Online Resource 1). Accord
ing to the quality and quantity of data, seven articles were
selected for meta-analysis [15, 30, 32, 34–36]. Details on
the selection process of research articles are presented in
a flow diagram (Fig. 1). Quality assessment of the included studies is shown in
Table 3. Overall, nine studies were considered to be mod
erate risk of bias [9, 15, 30–33, 35–37], and two were low
risk of bias [13, 34]. The Kappa coefficients of the reviewers involved in
article identification and screening, data extraction,
and quality assessment were 0.895, 0.892, and 1.000,
respectively(Online Resource 2). All were greater than
0.800, indicating strong agreement among reviewers [40]. A total of 477 individuals aged 5–10 years were
included, comprising 246 cases of premature loss of first
primary molars from the maxilla and 217 cases from
the mandible. Individuals were excluded from the stud
ies for a variety of reasons (n = 27), including malocclu
sion (n = 4), lack of second premolar buds on examination
(n = 1), the presence of mesiodens (n = 2), further tooth
extractions (n = 10), and no further follow up (n = 10)
(Table 2). Fig. 2 Forest plot of space differences (D/D + E) between the extraction and control sides
(A) D + E space differences in the maxilla. (B) D space differences in the mandible. (C) D + E space differences in the mandible. Space was significantly
reduced compared with the control side (P < 0.05 each) Systematic searchh The primary search identified 329 published papers, of
which 16 satisfied the initial inclusion criteria. Reading
of complete texts resulted in the inclusion of 11 stud
ies, 9 longitudinal [9, 15, 30–36] and 2 cross-sectional
[13, 37]. Among them, there were five studies on Cauca
sians [13, 30, 33, 34, 37], five on Mongolians [9, 15, 31,
32, 35], and one on a Central European [36]. Five studies
were excluded because they were non split-mouth (n = 5) Zhao et al. BMC Oral Health (2023) 23:430 Page 6 of 14 Zhao et al. BMC Oral Health (2023) 23:430 Zhao et al. BMC Oral Health Quality assessment and kappa’s test Characteristics of the clinical protocol (A) D + E space changes in the maxilla. (B) D space changes in the mandible. (C) D + E space changes in the mandible. Space was significantly reduced
compared with the initial baseline values (P < 0.05 each) Fig. 3 Forest plot of space changes (D/D + E) on the extraction side between the initial baseline values and those at the final follow-up examination. (A) D + E space changes in the maxilla. (B) D space changes in the mandible. (C) D + E space changes in the mandible. Space was significantly reduced
compared with the initial baseline values (P < 0.05 each) Fig. 3 Forest plot of space changes (D/D + E) on the extraction side between the initial baseline values and those at the final follow-up examination. (A) D + E space changes in the maxilla. (B) D space changes in the mandible. (C) D + E space changes in the mandible. Space was significantly reduced
compared with the initial baseline values (P < 0.05 each) Fig. 3 Forest plot of space changes (D/D + E) on the extraction side between the initial baseline values and those at the final follow-up examination. (A) D + E space changes in the maxilla. (B) D space changes in the mandible. (C) D + E space changes in the mandible. Space was significantly reduced
compared with the initial baseline values (P < 0.05 each) Table 3 Quality assessment according to ROBINS-I tool of the included observational studies
Study
Pre-intervention
At intervention
Post-intervention
Overall
risk of bias
Due to
confounding
Selection of
participants
into the study
Classifica
tion of
interventions
Deviations
from intended
interventions
Miss
ing
data
Measure
ment of
outcomes
Selection
of the
reported
result
Low/
Moderate/
Serious/
Critical
Heidari et al. 2022 [37]
Low
Low
Low
Low
Low
Moderate
Low
Moderate
Mosharrafian et al. 2021
[13]
Low
Low
Low
Low
Low
Moderate
Low
Low
Kobylńska et al. 2019 [36]
Moderate
Low
Low
Low
Low
Moderate
Low
Moderate
Lin et al. 2017 [9]
Moderate
Low
Low
Low
Low
Moderate
Low
Moderate
Alexander et al. 2015 [34]
Low
Low
Low
Low
Low
Moderate
Low
Low
Lin et al. 2011 [15]
Moderate
Low
Low
Low
Low
Moderate
Low
Moderate
Macena et al. 2011 [33]
Moderate
Low
Low
Low
Low
Moderate
Low
Moderate
Park et al. 2009 [32]
Moderate
Low
Low
Low
Low
Low
Low
Moderate
Lin et al. Characteristics of the clinical protocol In general, the children included in these studies were
expected to need the unilateral premature extraction of
a primary first molar because of caries and/or failed pulp
therapy, with an intact contralateral primary first molar
available for use as the control. The research data were
obtained in three ways: plaster cast [9, 13, 15, 30, 31, 33,
36, 37], digital plaster cast [32, 35], and direct intraoral Fig. 2 Forest plot of space differences (D/D + E) between the extraction and control sides
(A) D + E space differences in the maxilla. (B) D space differences in the mandible. (C) D + E space differences in the mandible. Space was significant
educed compared with the control side (P < 0.05 each) Fig. 2 Forest plot of space differences (D/D + E) between the extraction and control sides
(A) D + E space differences in the maxilla. (B) D space differences in the mandible. (C) D + E space differences in the mandible. Space was significantly
reduced compared with the control side (P < 0.05 each) Fig. 2 Forest plot of space differences (D/D + E) between the extraction and control sides
(A) D + E space differences in the maxilla. (B) D space differences in the mandible. (C) D + E space differences in the mandible. Space was significantly
reduced compared with the control side (P < 0.05 each) Fig. 2 Forest plot of space differences (D/D + E) between the extraction and control sides
(A) D + E space differences in the maxilla. (B) D space differences in the mandible. (C) D + E space differences in the mandible. Space was significantly
reduced compared with the control side (P < 0.05 each) Zhao et al. BMC Oral Health (2023) 23:430 Page 7 of 14 Page 7 of 14 Zhao et al. BMC Oral Health Fig. 3 Forest plot of space changes (D/D + E) on the extraction side between the initial baseline values and those at the final follow-up examination. (A) D + E space changes in the maxilla. (B) D space changes in the mandible. (C) D + E space changes in the mandible. Space was significantly reduced
compared with the initial baseline values (P < 0.05 each) Fig. 3 Forest plot of space changes (D/D + E) on the extraction side between the initial baseline values and those at the final follow-up examination. Characteristics of the clinical protocol 2007 [31]
Moderate
Low
Low
Low
Low
Moderate
Low
Moderate
Padam et al. 2006 [30]
Moderate
Low
Low
Low
Low
Moderate
Low
Moderate
Kim et al. 2008 [35]
Moderate
Low
Low
Low
Low
Moderate
Low
Moderate Table 3 Quality assessment according to ROBINS-I tool of the included observational studies Zhao et al. BMC Oral Health (2023) 23:430 Page 8 of 14 Page 8 of 14 Page 8 of 14 Zhao et al. BMC Oral Health measurement [34]. The initial study used plaster casts
made from alginate impressions just before extraction
of the primary first molar [30, 32] or 2–14 days after
extraction [9, 15, 31, 36]. Study outcomes were measured
before or after tooth loss and at specified time points
during the follow-up period. The outcomes were spa
tial changes between the intervention and control sides
as represented by primary molar D + E or D space, and
arch width, length, and perimeter. These quantities were
measured in all the selected studies except for two that
included space loss, midline/molar/canine relationships,
facial growth patterns, canine inclination, and crowding
[13, 37]. The follow-up time varied from 2 to 81 months
[9], with one at 81 months being the extreme, while all
others were less than 24 months. measurement [34]. The initial study used plaster casts
made from alginate impressions just before extraction
of the primary first molar [30, 32] or 2–14 days after
extraction [9, 15, 31, 36]. Study outcomes were measured
before or after tooth loss and at specified time points
during the follow-up period. The outcomes were spa
tial changes between the intervention and control sides
as represented by primary molar D + E or D space, and
arch width, length, and perimeter. These quantities were
measured in all the selected studies except for two that
included space loss, midline/molar/canine relationships,
facial growth patterns, canine inclination, and crowding
[13, 37]. The follow-up time varied from 2 to 81 months
[9], with one at 81 months being the extreme, while all
others were less than 24 months. Five studies assessed dental arch changes [15, 30, 33,
35, 36]. However, no significant differences were found
in arch width, arch length, or arch perimeter between
the initial examination and the medium-term follow-up
examination ( P > 0.05) (Fig. 4A–F). Factors influencing space change after premature losshf Factors influencing space change after premature loss
There were two studies [13, 37] of multiple factors affect
ing space change after the first primary molar loss,
including age (years); tooth extraction time (months);
molar relationships on the control side; facial patterns;
and canine movements, crowding, and relationships on
the control side, midline and jaw. Factors such as age,
facial pattern, duration of tooth loss, molar relation
ships on the control side, and canine-to-lateral distance
influenced space loss following premature loss of the first
primary molars [13, 37]. However, other factors such as
crowding, midline deviation, and canine relationships on
the control side did not significantly affect space loss [37]. Over the medium-term follow-up period (6–24
months) [15, 30, 32–36], the D and D + E spaces on the
extraction side were significantly smaller than those on
the control side in both the maxilla and mandible (D + E
space difference in the maxilla: MD 0.48, 95% CI 0.18–
0.78, P < 0.01; D space difference in the mandible: MD
1.22, 95% CI 0.19–2.25, P = 0.02; D + E space difference
in the mandible: MD 1.07, 95% CI 0.66–1.48, P = 0.02)
(Fig. 2A–C). The space loss in the maxillary D + E was
0.65 mm (MD 0.65, 95% CI 0.15–1.16, P = 0.01) (Fig. 3A). The space loss in the mandibular D + E was 1.24 mm (MD
1.24, 95% CI 0.60–1.89, P < 0.01), and that in the man
dibular D was 1.47 mm (MD 1.47, 95% CI 0.66–2.28,
P < 0.01) (Fig. 3B–C). Characteristics of the clinical protocol In addition, there
were three articles from one institution evaluating both
intercanine width and length [9, 15, 31] in which the
authors found that both were significantly larger at the 6,
12, and 81 month follow-ups. Sensitivity analysis showed that the combined effect
did not change after excluding any of the studies, sug
gesting that the results were reliable, except for the meta-
analysis of D space and loss (Fig. 5). Only two studies
were included, and the results could not be obtained by
removing the study. Therefore, further research is needed
to verify these results. Funnel plots, Begg’s tests, and Egg
er’s tests showed that there was no publication bias in the
included studies (Fig. 6; Table 4, Online Resource 3 and
4). Short-term (≤ 6 m) space changes Four articles described short-term follow up (≤ 6 m) [30,
31, 33, 36], including 1–4 and 6 months. However, there
were only one or two research articles for each follow-up
time, which could not be combined for meta-analysis. However, in these studies, space loss was detected at
an early stage. Padma and Retnakumari [30] found the
greatest space loss to occur in the 4 months immediately
following premature extraction. Long-term (> 24 m) space changes Lin et al. [9] evaluated unilateral premature loss of the
primary maxillary first molar in nine children (6.0 ± 0.42
years old), involving a follow-up period of 81 months,
which was the longest followup period in all the included
studies. The arch width, arch length, and intercanine
width and length significantly increased over that period. Medium-term (6–24 m) space changes (meta-analysis)i Medium term (6 24 m) space changes (meta analysis)
Only two of the included studies assessed first primary
molar D space in the maxilla [33, 36], and meta-analysis
was not feasible because of incomplete standard devia
tion data in Kobylińska’s study [36]. In Alexander’s study,
the population was divided into four groups according to
occlusal relationship and facial type: leptoprosopic with
end-on molar occlusions, mesoprosopic/euryprosopic
with end-on molar occlusions, leptoprosopic with Class
I molar occlusions, and mesoprosopic/euryprosopic with
Class I molar occlusions. Each group was included in the
meta-analysis as an independent sample. The follow-up
period was 6–24 months in the studies included in this
meta-analysis. Discussionh The present systematic review and meta-analysis identi
fied studies that evaluated space changes following pre
mature loss of the primary first molar. Because systematic
reviews are based on rigorous inclusion, exclusion, and
methodological criteria, few articles addressing this topic
are available, with most lacking a self-controlled group [1,
4]. Additionally, in some articles, there is more than one Zhao et al. BMC Oral Health (2023) 23:430 Zhao et al. BMC Oral Health Page 9 of 14 ig. 4 Forest plot of dental arch changes after premature loss of the first primary molar (A) Arch width of the maxilla. (B) Arch length of the maxilla. (C)
rch perimeter of the maxilla. (D) Arch width of the mandible. (E) Arch length of the mandible. (F) Arch perimeter of the mandible. No statistically signifi
ant differences were observed between the initial baseline values and those of the final follow up examinations (P>0 05 each) Fig. 4 Forest plot of dental arch changes after premature loss of the first primary molar (A) Arch width of the maxilla. (B) Arch length of the maxilla. (C)
Arch perimeter of the maxilla. (D) Arch width of the mandible. (E) Arch length of the mandible. (F) Arch perimeter of the mandible. No statistically signifi
cant differences were observed between the initial baseline values and those of the final follow-up examinations (P > 0.05 each) Fig. 4 Forest plot of dental arch changes after premature loss of the first primary molar (A) Arch width of the maxilla. (B) Arch length of the maxilla. (C)
Arch perimeter of the maxilla. (D) Arch width of the mandible. (E) Arch length of the mandible. (F) Arch perimeter of the mandible. No statistically signifi
cant differences were observed between the initial baseline values and those of the final follow-up examinations (P > 0.05 each) Zhao et al. BMC Oral Health (2023) 23:430 Zhao et al. BMC Oral Health Page 10 of 14 Fig. 5 Sensitivity analyses of the included articles
(A) D + E space changes in the maxilla on the extraction side. (B) D space changes in the mandible on the extraction side. (C) D + E space changes in
the mandible on the extraction side. (D) D + E space differences in the maxilla between the extraction and control sides. (E) D space differences in the
mandible between the extraction and control sides. Discussionh (F) D + E space differences in the mandible between the extraction and control sides. (G) Arch width
of the maxilla. (H) Arch length of the maxilla. (I) Arch perimeter of the maxilla. (J) Arch width of the mandible. (K) Arch length of the mandible. (L) Arch
perimeter of the mandible. Sensitivity analysis showed that, except for the meta-analysis of D space and D loss, the combined effects did not change after
excluding any single study, suggesting that the results were generally reliable Fig. 5 Sensitivity analyses of the included articles
(A) D + E space changes in the maxilla on the extraction side. (B) D space changes in the mandible on the extraction side. (C) D + E space changes in
the mandible on the extraction side. (D) D + E space differences in the maxilla between the extraction and control sides. (E) D space differences in the
mandible between the extraction and control sides. (F) D + E space differences in the mandible between the extraction and control sides. (G) Arch width
of the maxilla. (H) Arch length of the maxilla. (I) Arch perimeter of the maxilla. (J) Arch width of the mandible. (K) Arch length of the mandible. (L) Arch
perimeter of the mandible. Sensitivity analysis showed that, except for the meta-analysis of D space and D loss, the combined effects did not change after
excluding any single study, suggesting that the results were generally reliable Fig. 5 Sensitivity analyses of the included articles
(A) D + E space changes in the maxilla on the extraction side. (B) D space changes in the mandible on the extraction side. (C) D + E space changes in
the mandible on the extraction side. (D) D + E space differences in the maxilla between the extraction and control sides. (E) D space differences in the
mandible between the extraction and control sides. (F) D + E space differences in the mandible between the extraction and control sides. (G) Arch width
of the maxilla. (H) Arch length of the maxilla. (I) Arch perimeter of the maxilla. (J) Arch width of the mandible. (K) Arch length of the mandible. (L) Arch
perimeter of the mandible. Sensitivity analysis showed that, except for the meta-analysis of D space and D loss, the combined effects did not change after
excluding any single study, suggesting that the results were generally reliable Fig. Discussionh 5 Sensitivity analyses of the included articles missing molar in a single jaw [4, 26, 41, 42], which may be
another reason for relatively limited research in this area. followed up for less than 24 months, which may reduce
the validity of the outcomes. missing molar in a single jaw [4, 26, 41, 42], which may be
another reason for relatively limited research in this area. Nine studies were considered to be moderate risk
of bias [9, 15, 30–33, 35–37], and two were low risk of
bias [13, 34]. In two articles [30, 36], 27 individuals were
excluded because of malocclusion, lack of second pre
molar buds on examination, the presence of mesiodens,
additional tooth extractions, or no further follow up,
which may not be serious sources of bias for the study
outcomes. However, except for one study [9] that was
followed up for 81 months, all included studies were Over the medium-term follow-up period (6–24
months), the D or D + E space on the extraction side was
significantly smaller than on the control side in both
the maxilla and mandible (D + E space difference in the
maxilla: MD 0.48, 95% CI 0.18–0.78, P < 0.01; D space
difference in the mandible: MD 1.22, 95% CI 0.19–2.25,
P = 0.02; D + E space difference in the mandible: MD
1.07, 95% CI 0.66–1.48, P = 0.02), which means that if the
tooth loss lasts for more than 6 months, the space will Nine studies were considered to be moderate risk
of bias [9, 15, 30–33, 35–37], and two were low risk of
bias [13, 34]. In two articles [30, 36], 27 individuals were
excluded because of malocclusion, lack of second pre
molar buds on examination, the presence of mesiodens,
additional tooth extractions, or no further follow up,
which may not be serious sources of bias for the study
outcomes. However, except for one study [9] that was
followed up for 81 months, all included studies were (2023) 23:430 Page 11 of 14 Zhao et al. BMC Oral Health Fig. 6 Funnel plot of the included articles
(A) D + E space changes in the maxilla on the extraction side. (B) D space changes in the mandible on the extraction side. (C) D + E space changes in the
mandible on the extraction side. (D) D + E space differences in the maxilla between the extraction and control sides. Discussionh (E) D space differences in the man
dible between extraction and control sides. (F) D + E space differences in the mandible between the extraction and control sides. (G) Arch width of the
maxilla. (H) Arch length of the maxilla. (I) Arch perimeter of the maxilla. (J) Arch width of the mandible. (K) Arch length of the mandible. (L) Arch perimeter
of the mandible. The funnel plots indicated that there was no obvious heterogeneity among the included studies Fig. 6 Funnel plot of the included articles Fig. 6 Funnel plot of the included articles
(A) D + E space changes in the maxilla on the extraction side. (B) D space changes in the mandible on the extraction side. (C) D + E space changes in the
mandible on the extraction side. (D) D + E space differences in the maxilla between the extraction and control sides. (E) D space differences in the man
dible between extraction and control sides. (F) D + E space differences in the mandible between the extraction and control sides. (G) Arch width of the
maxilla. (H) Arch length of the maxilla. (I) Arch perimeter of the maxilla. (J) Arch width of the mandible. (K) Arch length of the mandible. (L) Arch perimeter
of the mandible. The funnel plots indicated that there was no obvious heterogeneity among the included studies g
p
(A) D + E space changes in the maxilla on the extraction side. (B) D space changes in the mandible on the extraction side. (C) D + E space changes in the
mandible on the extraction side. (D) D + E space differences in the maxilla between the extraction and control sides. (E) D space differences in the man
dible between extraction and control sides. (F) D + E space differences in the mandible between the extraction and control sides. (G) Arch width of the
maxilla. (H) Arch length of the maxilla. (I) Arch perimeter of the maxilla. (J) Arch width of the mandible. (K) Arch length of the mandible. (L) Arch perimeter
of the mandible. The funnel plots indicated that there was no obvious heterogeneity among the included studies be significantly reduced. The space loss in the maxillary
D + E was 0.65 mm (MD 0.65, 95% CI 0.15–1.16, P = 0.01),
similar to that in Heidari’s study (0.54 mm) [37] but dif
ferent from that in Tunison’s study (≤ 1 mm) [43]. Discussionh BMC Oral Health Table 4 Begg’s and Egger’s tests of D and D + E space changes
and dental arch changes
Begg’s test
Egger’s test
Z
P
t
P
D + E space of Maxilla on extraction
side
0.30
0.764
-0.21
0.845
D space of Mandible on extraction
side
0.00
1.000
-*
-*
D + E space of Mandible on extraction
side
0.24
0.806
-0.19
0.861
D + E space changes of Maxilla be
tween extraction and control side
0.62
0.536
-0.80
0.457
D space changes of Mandible be
tween extraction and control side
0.00
1.000
-*
-*
D + E space changes of Mandible
between extraction and control side
0.38
0.707
-0.25
0.816
Arch width of Maxilla
0.00
1.000
-1.68
0.341
Arch width of Mandible
0.00
1.000
1.80
0.322
Arch length of Maxilla
0.34
0.734
0.86
0.430
Arch length of Mandible
1.02
0.308
3.00
0.096
Arch perimeter of Maxilla
0.00
1.000
1.23
0.434
Arch perimeter of Mandible
0.00
1.000
0.02
0.985
* There were only two articles included, so the Egger’s test was not performed Table 4 Begg’s and Egger’s tests of D and D + E space changes
and dental arch changes 15, 31], which may provide enough space for eruption of
the successor permanent teeth, partially compensating
for earlier space loss. p
In these split-mouth longitudinal studies, space loss
was detected at an early follow-up time (≤ 6 mm) [30,
31, 33, 36]. Padma and Retnakumari [30] observed the
greatest space loss in the first 4 months after premature
extraction, but space loss subsequently increased gradu
ally and became stable over 6–24 months [30, 33, 34, 36]. Lin and Chang found more than 1 mm space loss in the
maxilla at 6 and 12 months, however, and at 81 months
follow up, 88.9% of the subjects did not show crowded
permanent successors or canine block-out at the extrac
tion site, which suggested that space maintainers were
not needed for children aged about 6 years when the
permanent first molars were about to erupt or had just
erupted. In Heidari’s study [37], space loss resulting
from extraction of the first primary molars in late mixed
dentition at 8–10 years old was neither statistically nor
clinically significant. In addition, some other factors
influenced space change after premature loss of the first
primary molar. Discussionh The
space loss in the mandibular D + E was 1.24 mm (MD
1.24, 95% CI 0.60–1.89, P < 0.01), and that in the man
dibular D was 1.47 mm (MD 1.47, 95% CI 0.66–2.28,
P < 0.01), which was similar to the values reported by
Andreeva et al. (1.12–1.50 mm) [26, 43, 44]. be significantly reduced. The space loss in the maxillary
D + E was 0.65 mm (MD 0.65, 95% CI 0.15–1.16, P = 0.01),
similar to that in Heidari’s study (0.54 mm) [37] but dif
ferent from that in Tunison’s study (≤ 1 mm) [43]. The
space loss in the mandibular D + E was 1.24 mm (MD
1.24, 95% CI 0.60–1.89, P < 0.01), and that in the man
dibular D was 1.47 mm (MD 1.47, 95% CI 0.66–2.28,
P < 0.01), which was similar to the values reported by
Andreeva et al. (1.12–1.50 mm) [26, 43, 44]. space was due to distalization of the primary canine and
mesialization of the second primary molar on both the
maxillary and mandibular arches. In the maxillary arch,
the space changes consisted mainly of distal drift of the
primary canine, but there was no observation of mesial
movement of permanent molars or tilting of the primary
molars [15, 31]. In the mandibular arch, both mesial
migration of posterior teeth and distal movement of
anterior teeth were observed, but the distal movement of
the primary canine toward the extraction space was most
likely responsible for the early space change [30]. In Park
and Kim’s studies [32, 35], inclination and angulation of In all the included studies, seven articles mentioned the
movement of adjacent teeth on the extraction side [15,
30–32, 34–36]. Kobylńska et al. [36] found that the loss of Page 12 of 14 Zhao et al. BMC Oral Health (2023) 23:430 Zhao et al. Discussionh In these studies using multifactor linear
regression [13, 37], it was found that factors such as age,
facial pattern, duration of tooth loss, and molar relation
ships influenced space loss following premature loss of
the first primary molars. However, two articles are insuf
ficient to confirm this conclusion, and further multifacto
rial research is needed. adjacent teeth were measured by 3D scanning and super
imposing the initial and final dental casts, but there were
no consistent findings concerning inclination and angula
tion changes on the extraction side in their studies. According to the seven articles included, the factors
influencing the movement of the adjacent teeth may be
related to the dental arch, the eruptive status of the first
permanent molar, and the occlusal relationship of the
molar and facial types [15, 30–32, 34–36]. Alexander et
al. [34] analyzed the pattern of space loss in people with
different facial types and molar occlusal relationships and
observed different movements of adjacent teeth. In peo
ple with leptoprosopic facial forms, maxillary space loss
occurred by mesial migration of distal segments in all
subjects; in the mandibular arch, space loss in more than
80% of subjects occurred by mesial migration of distal
segments and distal tipping of the canine, and less than
20% occurred only by mesial migration. In people with
end-on molar occlusions and mesoprosopic/eurypro
sopic facial forms, space loss occurred by mesial migra
tion of distal segments and distal tipping of the canine
in 88.37% of subjects, and by mesial migration in only
11.63% of subjects in the mandibular extraction group. No significant differences were found in arch width, arch
length, or arch perimeter between the initial baseline val
ues and those after the medium-term follow-up period
(6–24 months) (P > 0.05) [15, 30, 33, 35, 36], which may
suggest that premature loss of a first primary molar will
not affect the development of the dental arch. In addition,
the authors found an increase in length and width of the
intercanine arch at the 6, 12, and 81 month follow-ups [9, The present study had several limitations. First, the
meta-analysis of D space and loss thereof included only
two articles, which cannot be improved by removing one
article to improve the reliability of the results. Abbreviations
ROBINS-I
Risk Of Bias In Non-randomized Studies – of Interventions
MD
Mean differences. Discussionh Second,
follow-up times differed among the included studies,
with most having been less than 24 months, so it may be
necessary to extend the follow-up times until eruption of
the successor permanent teeth occurs. Conclusions Over the medium-term follow-up period (6–24 months),
space loss was 0.65 mm for the maxillary D + E, 1.24 mm
for the mandibular D + E, and 1.47 mm for the mandibu
lar D. After premature loss of first primary molars, space
can be lost, but the amount of loss would not affect arch
width, length, or arch perimeter over the 6–24 months
follow-up period Factors such as age, time since tooth
extraction, facial pattern, and molar relationships also
influenced the space change after the premature loss of
the first primary molar. It is advisable to precisely assess
these related factors to decide whether to place a space
maintainer for a prematurely lost primary first molar. The online version contains supplementary material available at https://doi.
org/10.1186/s12903-023-03111-x. Premature loss of primary teeth: part V, treatment plan
ning with due respect to the significance of drifting patterns. ASDC J Dent
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dental arches of man, with a consideration of the changes which occur as a
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dental arches of man, with a consideration of the changes which occur as a
result of their artificial derangement by filling or by the extraction of teeth. Dent Cosmos. 1877;29:413–39. 8. Lin YT, Chang LC. Space changes after premature loss of the mandibular
primary first molar: a longitudinal study. J Clin Pediatr Dent. 1998;22:311–6. 9
Lin YJ Lin YT Long term space changes after premature loss of a primary 8. Lin YT, Chang LC. Space changes after premature loss of the mandibular
primary first molar: a longitudinal study. J Clin Pediatr Dent. 1998;22:311–6. 8. Lin YT, Chang LC. Space changes after premature loss of the mandibular
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yi
g
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in the mandible on the extraction side. (D) D + E space differences in the
maxilla between the extraction and control sides. (E) D space differences
in the mandible between the extraction and control sides. (F) D + E space
differences in the mandible between the extraction and control sides. (G)
Arch width of the maxilla. (H) Arch length of the maxilla. (I) Arch perimeter
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dible. (L) Arch perimeter of the mandible. Begg’s test indicated that there
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Effect of Valve- and Patient- Related Factors on the Effective and Geometric Orifice Areas: An In Vitro Study with the CoreValve
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Computer methods in biomechanics and biomedical engineering
| 2,017
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cc-by
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Effect of Valve- and Patient- Related Factors on the
Effective and Geometric Orifice Areas: An In Vitro
Study with the CoreValve
V. Stanova, A. S. Zense, R. Rieu, L. Kadem, P. Pibarot V. Stanova, A. S. Zense, R. Rieu, L. Kadem, P. Pibarot To cite this version: V. Stanova, A. S. Zense, R. Rieu, L. Kadem, P. Pibarot. Effect of Valve- and Patient-
Related Factors on the Effective and Geometric Orifice Areas:
An In Vitro Study with the
CoreValve. Computer Methods in Biomechanics and Biomedical Engineering, 2017, 20 (1), pp.195-196. 10.1080/10255842.2017.1382929. hal-01691478 HAL Id: hal-01691478
https://hal.science/hal-01691478v1
Submitted on 10 Apr 2018 L’archive ouverte pluridisciplinaire HAL, est
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http://www.tandfonline.com/action/journalInformation?journalCode=gcmb20 Effect of Valve- and Patient- Related Factors on the
Effective and Geometric Orifice Areas: An In Vitro
Study with the CoreValve V. Stanová, AS. Zense, R. Rieu, L. Kadem & P. Pibarot Effect of Valve- and Patient- Related Factors on the Effective and Geometric
Orifice Areas: An In Vitro Study with the CoreValve V. Stanová , AS. Zense
, R. Rieu , L. Kadem and P. Pibarot
aAix Marseille Univ, CNRS, ISM, Inst Movement Sci, GIBoc, Marseille, France; bQuebec Heart and Lung Institute, Laval Univ, Quebec, Canada;
cDepartment of Mechanical and Industrial Engineering, Concordia Univ, Montreal, QC, Canada
KEYWORDS Aortic hemodynamics; aortic flow; effective orifice area; geometric orifice area 1. Introduction Vivid 7). Determination of GOA was proceeded using pro-
gram inspired by Chang and Vese (2001). The program was
modified by adding the initialization mask that allows to
focalize on one precise region in order to determine the
valve GOA. This program was optimized for our applica-
tion by adding the area calculation based on a personalized
Matlab application after image calibration (Figure 1). Effective orifice area (EOA) is a standard parameter for
clinical assessment of the severity of valve stenosis, but it
is also used to compare the hydrodynamic performance of
various prosthetic heart valves. However, the EOA meas-
ured by Doppler echocardiography provides an estimation
of the cross-sectional area of the flow jet at the vena con-
tracta. On the other hand, geometric orifice area (GOA)
provides an estimation of the area of the valve orifice. Both
parameters may be influenced by prosthetic valve-related
factors (model and size) as well as patient-related factors
(aortic annulus size, level of transvalvular flow).h As the limiting factor in terms of hemodynamic perfor-
mance is the most restrictive area (Figure 1, 120 iterations:
green line), GOA was not measured at the prosthesis free
leaflet edge (Figure 1, 120 iterations: red line) as in case of
mechanical or surgical bioprostheses, but in the inner area
of the valve. Generally, the difference between measured l
The objective of this study was to examine, in a pulse
duplicator, the impact of flow and different sizes of
CoreValve (CV) on valve effective orifice area and geomet-
ric orifice area. Figure 1. GOA determination using region-based active contour
segmentation. The green contour represents the GOA as
determined at the base of the leaflets. The red contour represents
the GOA as determined at the tip of valve leaflets. © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
CONTACT V. Stanová
viktoria.stanova@univ-amu.fr V. Stanová, AS. Zense, R. Rieu, L. Kadem & P. Pibarot To cite this article: V. Stanová, AS. Zense, R. Rieu, L. Kadem & P. Pibarot (2017) Effect of Valve-
and Patient- Related Factors on the Effective and Geometric Orifice Areas: An In Vitro Study
with the CoreValve, Computer Methods in Biomechanics and Biomedical Engineering, 20:sup1,
195-196, DOI: 10.1080/10255842.2017.1382929 To link to this article: https://doi.org/10.1080/10255842.2017.1382929 © 2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group
Published online: 27 Oct 2017. Submit your article to this journal
Article views: 82
View related articles
View Crossmark data © 2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group
Published online: 27 Oct 2017. Submit your article to this journal
Article views: 82
View related articles
View Crossmark data Computer Methods in Biomechanics and Biomedical Engineering, 2017
VOL. 20, NO. S1, S195–S196 Computer Methods in Biomechanics and Biomedical Engineering, 2017
VOL. 20, NO. S1, S195–S196
https://doi.org/10.1080/10255842.2017.1382929 https://doi.org/10.1080/10255842.2017.1382929 OPEN ACCESS 2. Methods Three different sizes of percutaneous aortic valves,
CoreValve (Medtronic, Inc.), were tested on a pulse
duplicator under the following configurations: CV 23
mm implanted in aortic annuli (AA) ranging from 17 to
20 mm; 2) CV 26 mm implanted in AA ranging from 20
to 23 mm; 3) CV 29 mm implanted in AA ranging from
23 to 26 mm. For each configuration tested, the heart rate
was set to 70 bpm, mean aortic pressure to 100 mmHg
while the stroke volume (SV) was varied (30, 50, 70, 90
ml). A high-speed camera (Photron, 1000 images/second)
was used to acquire images during systole (290 images)
and diastole (570 images). In this abstract, we will focus
mainly on one size of CV (23 mm). Figure 1. GOA determination using region-based active contour
segmentation. The green contour represents the GOA as
determined at the base of the leaflets. The red contour represents
the GOA as determined at the tip of valve leaflets. Doppler EOA was estimated using the standard elec-
tro-magnetic flowmeter (Carolina Medical Inc. CME 500
Series) by Doppler velocity-time integral (General Electric CONTACT V. Stanová y Informa UK Limited, trading as Taylor & Francis Group.
buted under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
ny medium, provided the original work is properly cited. CONTACT V. Stanová S196
V. STANOVÁ ET AL. S196
V. STA V. STANOVÁ ET AL. S196 Table 1. EOA (cm2), GOA (cm2) and contraction coefficient for CV 23 mm as a function of AA diameter and SV. Aortic valve area (cm²) for CV 23 mm in aortic annuli
17 mm
18 mm
19 mm
20 mm
SV (ml)
EOA
GOA
Cc
EOA
GOA
Cc
EOA
GOA
Cc
EOA
GOA
Cc
30
0.82
1.17
0.69
0.84
1.32
0.64
0.90
1.41
0.64
1.03
1,49
0.69
50
0.95
1.25
0.75
0.99
1.35
0.73
1.12
1.55
0.72
1.24
1,67
0.74
70
0.99
1.30
0.76
1.04
1.36
0.76
1.22
1.57
0.77
1.26
1,69
0.75
90
1,06
1.31
0.81
1.11
1.39
0.80
1.29
1.61
0.80
1.31
1,71
0.77 Table 1. EOA (cm2), GOA (cm2) and contraction coefficient for CV 23 mm as a function of AA diameter and SV. mm as a function of AA diameter and SV. ²) for CV 23 mm in aortic annuli
19 mm
20 mm
c
EOA
GOA
Cc
EOA
GOA
Cc
64
0.90
1.41
0.64
1.03
1,49
0.69
73
1.12
1.55
0.72
1.24
1,67
0.74
76
1.22
1.57
0.77
1.26
1,69
0.75
80
1.29
1.61
0.80
1.31
1,71
0.77
Figure 2. EOA for CV 23 mm as a function of stroke volume (2A),
aortic annulus diameter (2B) and Cc vs. stroke volume (2C). GOA and GOA on the tip of leaflets was 33%. The ratio of
EOA to GOA was termed the contraction coefficient (Cc)
and is usually 0.6–0.9 depending on the left ventricular
outflow tract geometry and aortic valve morphology. l
g
y
p
gy
A Student t-tests was used to test for significant differences
(P value < 0.05 was considered statistically significant). EOA = - 0.186 + 0.674 × GOA + 0.03 × SV (r = 0.97) EOA = - 0.186 + 0.674 × GOA + 0.03 × SV (r = 0.97) The EOA and hence the Cc (Figure 2C) are both directly
related to the stroke volume (Cc = 0.74 ± 0.17, p < 0.001). At low flow states, the EOA, and hence the Cc display
lower values. 3. Results and discussion For CV 23 mm, the EOA increased significantly with aor-
tic annulus diameter (0.96 ± 0.10 to 1.21 ± 0.12 cm2, p
< 0.001) and SV (range from 0.82 ± 0.01 to 1.31 ± 0.04
cm2, p < 0.001). GOA also increased significantly with AA
diameter (1.25 ± 0.14 to 1.64 ± 0.10 cm2, p < 0.001) and
SV (range: 1.17 to 1.71 cm2, p = 0.001) for given size and
flow conditions (Table 1, Figure 2A, 2B). Figure 2. EOA for CV 23 mm as a function of stroke volume (2A),
aortic annulus diameter (2B) and Cc vs. stroke volume (2C). l
g
EOA (0.82 ± 0.10 cm2) and GOA (1.17 cm2) smallest
values were obtained in CV 23 mm implanted in aortic
annulus of 17 mm with a SV of 30 ml (Table 1) and the
largest values (1.31 ± 0.04 cm2 and 1.71 cm2 for EOA and
GOA respectively) in the CV 23 mm in 20 mm aortic
annulus with a stroke volume of 90 ml. 4. Conclusions The main findings of this study are: firstly, both the EOA
and the GOA decrease with the flow (28% and 11% respec-
tively). Hence, the valve opening is incomplete at low flow
states. Secondly, the EOA/GOA ratio decreases with the
flow (18%) thus indicating that the flow contraction is
more pronounced at low flow states. Hence, compared
to the GOA, the EOA decreases to a larger extent with
decreasing flow. For a given flow, the EOA and the GOA
of the CV are essentially determined by the AA size. Furthermore, due to the valvular and vascular determi-
nants of transvalvular gradients, EOA is not constant for a
specific GOA. Finally, similar results have been observed
with other valves: CV23, CV26 and CV 29 mm. A multiple linear analysis showed that the relationship
between EOA and GOA, and therefore the value of Cc,
was dependent on the flow (p < 0.001, r² = 0.80), but not
on AA diameter (p = 0.42). The following relations to pre-
dict the contraction coefficient and effective orifice area,
in the CV23, were found: Cc = 0.566 + 0.002 × SV (r = 0.80) Chan TF, Vese LA. 2001. Active Contours without Edges. IEEE
Transactions on Image Processing. 10(2):266–2.
Garcia D, Kadem L. 2006. What Do You Mean by Aortic Valve
Area: Geometric Orifice Area, Effective Orifice Area, or
Gorlin Area? Journal of Heart Valve Disease. 15(5):601.
Schoephoerster R, Yearwood TL, Chandran KB. 1989.
Prediction of Stenotic Valve Orifice Area: An in Vitro Study
on a Bioprosthesis. Catheterization and Cardiovascular
Diagnosis 18, no.1 References Chan TF, Vese LA. 2001. Active Contours without Edges. IEEE
Transactions on Image Processing. 10(2):266–2. Garcia D, Kadem L. 2006. What Do You Mean by Aortic Valve
Area: Geometric Orifice Area, Effective Orifice Area, or
Gorlin Area? Journal of Heart Valve Disease. 15(5):601. Schoephoerster R, Yearwood TL, Chandran KB. 1989. Prediction of Stenotic Valve Orifice Area: An in Vitro Study
on a Bioprosthesis. Catheterization and Cardiovascular
Diagnosis 18, no.1 Chan TF, Vese LA. 2001. Active Contours without Edges. IEEE
Transactions on Image Processing. 10(2):266–2. The overall mean difference for CV 23 between the
GOA and the EOA was 0.37 cm2, representing 30% (mean
differences of: 0.45, 0.38, 0.35, 0.31 cm2 for SV of 30, 50,
70, 90 ml respectively). Thus, the difference between the
EOA and the GOA was even higher at lower stroke vol-
umes. Similar results have been observed with two other
valves tested: CV 26 (26%) and CV 29 (27%).
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Nursing workload in mental health
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Rev Rene
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DOI: 10.15253/2175-6783.2016000500014
www.revistarene.ufc.br DOI: 10.15253/2175-6783.2016000500014
www.revistarene.ufc.br Original Article DOI: 10.15253/2175-6783.2016000500014
www.revistarene.ufc.br 1Universidade Estadual do Oeste do Paraná. Cascavel, PR, Brazil.
2Centro Universitário de Maringá. Maringá, PR, Brazil. Received: Maio 30th 2016; Accepted: July 5th 2016. Corresponding author: Sidnei Roberto Alves Corresponding author: Sidnei Roberto Alves
Avenida Tancredo Neves, 3224 - Santa Cruz. CEP:85806-470. Cascavel, PR, Brazil. E-mail: sidneiunioeste@yahoo.com.br Corresponding author: Sidnei Roberto Alves Methods Research with descriptive-analytic design
carried out in two psychiatric services (one public and
one private) located in the southern Brazil. At the same time, there was the expansion of
the Unified Health System, with the incorporation
of integral concepts, health care, decentralization,
regionalization and democratization to the daily
life of health services(2). However, the overwork in
mental health services affects health and the welfare
of workers, as well as the quality of care provided to
patients. Thus, personal wear in the labor exercise of
health professionals in psychiatric services has been
the focus of recent research(3-4). Both services (public and private) are located
in the same municipality of the northern Paraná,
Brazil. In the public service, called Municipal Hospital
of Maringá, there are 90 beds, 26 intended for mental
service and having 22 nursing professionals. In
this service, there are patients of the Unified Health
System assisted, classified as low complexity and it
began operations in 2003. The private psychiatric
service, called at the Psychiatric Hospital of Maringá,
had 240 beds for 61 nursing members. It was founded
in 1962, with about 20 beds, being rapidly expanded
due to the great demand of patients and families in the
region and the state that was repressed. Nevertheless, it is appropriate to note that
diseases related to professional activity are a source
of suffering and losses in the labor market. According
to the International Labor Organization, 2.3 million
deaths occur annually worldwide, and they have some
connection with the activity of the worker. According
to the report Prevention of occupational illnesses,
about 2 million deaths in the world are a result of the
development of professional illnesses and 321,000 are
the result of accidents - about one death per accident
for every six deaths by disease(5). The study included all members of the nursing
staff of both institutions, being in labor activity in their
respective institutions at the time of data collection. Thus, subjects in medical, leave, vacation certificate
or who refused to participate in the study were
excluded. From this, the sample of this research was
70 participants. The data were collected regarding
age, marital status, gender, bonding time with the
institution, number of employments, the level of
education, professional training to characterize the
study population. Moreover, the new model of mental health care
and its application are directly related to the skills
and commitment of its employees. Introduction public service, work overload favored the psychic
and physical wear of professionals. Such wear is
directly reflected in the professional job satisfaction(7). Therefore, the aim of this study was to compare the
workload among nursing mental health professionals
who work in public service and the private service. The mental health care services have undergone
several changes, and the cause of these changes
was the movement of the Psychiatric Reform, which
promoted several changes in treatment to psychiatric
patients in Brazil, being no longer centered on the
biomedical model but in the individual´s disease. Meanwhile, the practice of psychiatric nursing is also
suffering innovations over the years(1). Sobrecarga de trabalho da Enfermagem em saúde mental Sidnei Roberto Alves1, Reginaldo Passoni dos Santos1, Régio Marcio Toesca Gimenes2, Mirian Ueda Yamaguchi2 Sidnei Roberto Alves1, Reginaldo Passoni dos Santos1, Régio Marcio Toesca Gimenes2, Mirian Ue Objective: to compare the workload in nursing mental health professionals who work in the public service and
the private service. Methods: this is a quantitative research conducted with 70 nursing professionals in two
services. For data collection, the evaluation scale of the work impact on mental health services was applied. Results: in the overall analysis, there was no statistically significant difference in the average score for workload
among professionals of both institutions. However, all subscale averages had statistical differences, and the
subscale “emotional repercussions at work” showed the highest significance. Conclusion: the nursing staff from
the private service had higher workload average. Descriptors: Mental Health; Nursing; Working Conditions. Objetivo: comparar a sobrecarga de trabalho entre profissionais de enfermagem em saúde mental que atuam em
serviço público e aqueles do serviço privado. Métodos: pesquisa quantitativa, realizada com 70 profissionais de
enfermagem em dois serviços. Para coleta de dados, aplicou-se a escala de avaliação do impacto do trabalho em
serviços de saúde mental. Resultados: na análise global, não se observou diferença estatística significativa na
média de pontos para sobrecarga de trabalho entre os profissionais das duas instituições. Entretanto, todas as
médias das subescalas apresentaram diferenças estatísticas, sendo que a subescala “repercussões emocionais
no trabalho” apresentou maior significância. Conclusão: a equipe de enfermagem do serviço privado apresentou
maiores médias de sobrecarga. Descritores: Saúde Mental; Enfermagem; Condições de Trabalho. Corresponding author: Sidnei Roberto Alves
Avenida Tancredo Neves, 3224 - Santa Cruz. CEP:85806-470. Cascavel, PR, Brazil. E-mail: sidneiunioeste@yahoo.com.br Rev Rene. 2016 Sept-Oct; 17(5):684-90. Received: Maio 30th 2016; Accepted: July 5th 2016. 684 Nursing workload in mental health Results in Brazil, already used in other similar studies(8-9). This
scale is self-administered and quantitative containing
18 items and three open questions. In this study, the
results obtained from the answers to the quantitative
items were shown, which address the perception of
professionals about work overload and the gradual
responses of Likert-type with five points were present:
1 – “in any way” ; 2 – “not much”; 3 – “more or less”;
4 – “very much” and 5 – “extremely”. From the answers
given by the professionals in the 18 quantitative items,
they were grouped into three factors (subscales):
effects on physical and mental health (whose internal
consistency during the validation process was high
and submitted alpha value (α) equal to 0.78); the
effects on the functioning of the team (value of
α=0.77); emotional impact of the work (with α=0.70). There were 21 of the 70 participants (30.0%)
belonging to the Municipal Hospital of Maringá and
49 (70.0%) to the Psychiatric Hospital of Maringá. When comparing the profile of the professionals, it
was found that regardless of the institution, women,
with an average age above 30 years old, constituted
the majority and who played mid-level/nursing
technician activities (Table 1). Table 1 - Profile of nursing professionals according to
the psychiatric institution they worked
Variables
Municipal
Hospital of
Maringá(n=21)
Psychiatric
Hospital of
Maringá (n=49)
n(%)
n(%)
Gender
Female
17(80.9)
33(67.3)
Male
4(19.1)
16(32.6)
Age (average)
40.4
37.1
Education level
Mid-level/technical level
11(52.0)
39(80.0)
Graduation
2(10.0)
5(10.0)
Post-Graduation
8(38.0)
5(10.0)
Graduation
Nursing Technician
6(28.5)
36(73.5)
Nurse
4(19.2)
4(8.2)
Operating nurse
-
3(6.1)
Average time of service (years)
6.67
5.47
Another work link
Yes
8(38.1)
21(43.9)
No
13(61.9)
28(57.1) Table 1 - Profile of nursing professionals according to
the psychiatric institution they worked Table 1 - Profile of nursing professionals according to
the psychiatric institution they worked Data collection took place between April and
May 2013, with a table in Excel spreadsheet. The
variables for the profile of the participants had an
analysis on the distribution of absolute and relative
frequencies. Comparative analysis of the workload
among the participants took the average of points
obtained in the quantitative items of the Labor
Impact Scale Evaluation in Mental Health Services. In this sense, the normality assumptions were
checked by Shapiro-Wilk test, and homogeneity by
F test (analysis of variance). Methods Thus, the indicator
used to check overloading of workers could help to
assess the mental health services, but also to promote
proposals for the implementation to improve the
quality of working life(6). The Impact Evaluation for Mental Health Work
Services was used to assess the workload, which refers
to a range built by the mental health division of the
World Health Organization and subsequently validated Following this, it is important to stress that
in a survey of the nursing staff in mental health Rev Rene. 2016 Sept-Oct; 17(5):684-90. 685 Alves SR, Santos RP, Gimenes RMT, Yamaguchi MU Nursing workload in mental health Nursing workload in mental health In the comparative analysis, there was
no significant statistical difference between the
professionals of both institutions, concerning the
global burden (p=0.110).However, the comparison
in each subscale points out that the three factors
assessed in the private hospital nursing staff presented
significantly greater average scores (Table 2). critical analysis of the conditions and the nursing
work process in mental health services, where the
professionals may be predisposed to the levels of
work overload. There was a predominance of female partici
pation among nursing human resources of both ins
titutions. The inclusion of women in the formal labor
market has been significant in many areas of the labor
sector, due to increased education and the achieve
ment of economic autonomy, among others. The heal
th professions have absorbed part of the contingent of
this working class(11). Table 2 - Mental average score (standard deviation)
of the factors related to the workload of professionals
according to the mental health service. Scale
Municipal
Hospital of
Maringá
Psychiatric
Hospital of
Maringá
p*
Global overload
1.48 (0.15)
1.72 (0.17)
0.110
Subscale1
Effects of the team in the physi
cal and mental health
1.30 (0.17)
1.43 (0.25)
0.029
Subscale2
Impact on the team working
1.57 (0.33)
1.81 (0.57)
0.026
Subscale3
Emotional impact of working
1.58 (0.37)
1.93 (0.52)
0.036
*T-test for paired sample Scale
Municipal
Hospital of
Maringá
Psychiatric
Hospital of
Maringá
p*
Global overload
1.48 (0.15)
1.72 (0.17)
0.110
Subscale1
Effects of the team in the physi
cal and mental health
1.30 (0.17)
1.43 (0.25)
0.029
Subscale2
Impact on the team working
1.57 (0.33)
1.81 (0.57)
0.026
Subscale3
Emotional impact of working
1.58 (0.37)
1.93 (0.52)
0.036
*T-test for paired sample In this same perspective, the profile of nursing
professionals in mental health observed in this study
is consistent with the profile of the working class as
a whole. Recent study data conducted by the Federal
Nursing Council in partnership with Oswaldo Cruz
Foundation (Fiocruz) showed that the Brazilian
nursing is predominantly female(12). Also on the profile of the participants, there is
the need to have other employment showed higher
among workers of a private institution. This fact leads
to inferring that there is the low rate of payment among
the factors associated with this feature. Nursing workload in mental health In addition
to receiving lower salaries, the private health care
worker often has no career plan and other benefits
offered by the public sector, which motivates them to
seek other work to supplement the family income(13). Results Thus, considering that
the normal assumptions about homogeneity were
accepted, the average of the variables was compared
by applying the t-test for paired samples. Descriptive
and inferential analysis were performed using the
“R” software(10), giving the differences statistically
significant presenting p-value <0.05. The study complied with the formal require
ments contained in the national and international
regulatory standards for research involving human
beings. Rev Rene. 2016 Sept-Oct; 17(5):684-90. Discussion The limitation of this study is linked to the
expansion of a multidisciplinary team of mental
health and with the highest number of participants. Moreover, the development of research in only two
psychiatric services prevented the largest selection of
the sample. Another factor regarding this instrument
was used to measure the workload of the nursing
staff working in psychiatric services, restricting the
analysis of the results. Further studies are needed to
make more effective this assessment, with questions
related to factors related to other situations, such as
the causes of absenteeism at work, sickness, among
others. Comparing the workload, there was no
significant difference in the evaluation of the overall
overload between the groups. However, the nursing
staff of private mental health service showed averages
higher overloads in all aspects when considering the
individual assessment of each subscale. In this sense, the results showed that in the
subscale1 (effects of the team in the physical and
mental health) participants from both the public and
the private service had the lowest average scores
points (1.30±0.17 and 1.43±0.25, respectively). Nevertheless, the literature indicates that the work of The results of this study can contribute to the Rev Rene. 2016 Sept-Oct; 17(5):684-90. 687 Alves SR, Santos RP, Gimenes RMT, Yamaguchi MU conditions of work and reduced autonomy, combined
with physical overload and lack of labor cooperation
between team members, are factors that affect the
psyche of professionals in mental health services and
often, they are reasons for suffering and emotional
lability among workers of this area(1,3-4,15). On the
other hand, the workload directly influences the
assistance, undermining the quality of service,
compromising the safety of the patient. In the same
direction, it is emphasized that working conditions
have repercussions on the quality of working life on
the nursing team(16). the professional with the mental patients can cause
interference with physical complaints and work-
related stress in mental health. Thus, it is necessary
that the nurse managers are attentive to the signs and
symptoms presented by the team that often cannot
verbally report such events, but manifest them in the
form of absenteeism(3). However, this is different from
a study conducted in two pediatric hospitals in the
United States that show the factors that generate work
overloads, such as stress at work, burnout, emotional
exhaustion, job dissatisfaction and burnout by work
overload of these professionals(14). Conclusion 5. Organização Internacional do Trabalho. A preven
ção das doenças profissionais [Internet]. 2013
[citado 2013 out 10]. Disponível em: http://www. ilo.org/public/portugue/region/eurpro/lisbon/
pdf/safeday2013_relatorio.pdf The nursing professionals of service they
worked showed similar characteristics of their profile,
and similar to the Brazilian nursing profile. 6. Camilo CA, Bandeira M, Leal RMAC, Scalon JD. Avaliação da satisfação e sobrecarga em um ser
viço de saúde mental. Cad Saúde Coletiva. 2012;
20(1):82-92. About the workload in the overall evaluation,
there were no statistical differences in the average
overload between the groups. However, the nursing
staff in the private service had higher overload average
in the analysis of each subscale. 7. Souza IAS, Pereira MO, Oliveira MAF, Pinho PH,
Gonçalves RMDA. Work process and its impact
on mental health nursing professionals. Acta Paul
Enferm. 2015; 28(5):447-53. Discussion Nursing workload in mental health Collaborations 8. Leal RMAC, Bandeira MB, Azevedo KRN. Avaliação
da qualidade de um serviço de saúde mental na
perspectiva do trabalhador: satisfação, sobrecarga
e condições de trabalho dos profissionais. Psicol
Teor Prat. 2012; 14(1):15-25. Alves SR and Yamaguchi MU contributed to the
design, data collection, analysis, data interpretation
and writing of the article. Santos RP and Gimenes
RMT contributed to the design and final approval of
the version to be published. 9. Alves AP, Guidetti GECB, Diniz MA, Ferreira LA,
Zuffi FB. Avaliação do impacto do trabalho em
profissionais de saúde mental de uma instituição
psiquiátrica. Rev Min Enferm. 2013; 17(2):424-8. Discussion It is necessary to be aware of the fact that all
these aspects related to work wear of the nursing
team are reflected in job satisfaction with the service
in which they operate and in health care quality
dispensed to patients. Thus, it is very important and
emerging to conduct a situational, strategic planning
in the context of psychiatric institutions to identify
possibilities, minimizing the excessive workload that
the professionals are exposed(7). In another study
conducted with 400 nurses in Iran, it was described
that the workload is influenced by several factors,
including environmental conditions, structural work
process(17). The subscale on the impact of overload on the
operation of the team showed the most significant
difference between the teams (p=0.026). On this, there
is the professional´s fear regarding the possibility of
being attacked, the general state of physical health and
sick leave as factors associated with overload and they
can cause stress to the entire team and disagreements
between its members, affecting labor structure and
dynamics of the service as a whole(15). It
was
found
that
in
private
service,
professionals have a lower average time (in years) of
service, and more employments. This may explain the
high in the statistical difference observed in subscale2
since there can be no close relationship of proximity
between the couple among these professionals. This
closeness between the staff in the workplace promotes
unity and cooperation of the members and therefore
confirms that there may be a joint elaboration of
strategies for coping with difficulties arising from
overload(4). In this regard, improvements in the labor
process, the increase in the number of human
resources in nursing and greater appreciation of them,
music education in the workplace, team training for
the implementation of an interdisciplinary approach
activities and multidisciplinary cooperation, aiming at
comprehensive care are some of the strategies listed
in the literature(3-4,6-7,18). When analyzing the results, although the
aspects related to “emotional repercussions of work”
(subscale3), had the highest average overload among
workers of both institutions (1.58±0.37 for the
professionals in the public service and 1.93±0.52 for
the private service staff). Therefore, it is expected that this research
will provide discussions and actions to reduce the
workload for professionals working in mental health
services, and new studies are suggested that allow
comparison with results found in this investigation of
the studied topic. Therefore,
it
is
noted
that
inadequate Rev Rene. 2016 Sept-Oct; 17(5):684-90. References 10. R Development Core Team. R: a language and
environment for statistical computing. Vienna: R
Foundation for Statistical Computing [Internet]. 2013 [cited 2016 Mar 13]. Available from https://
cran.r-project.org/doc/manuals/r-release/
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view/6295/pdf_7391 11. Nunes EFPA, Santini SML, Carvalho BG, Junior LC. Força de trabalho em saúde na atenção básica em
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KH, Brown R L, et al. A human factors framework
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Efeitos da adubação nitrogenada e potássica na produção e na qualidade de frutos de laranjeira-'Valência'
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Revista brasileira de fruticultura
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______________________________________________________________
1 (Trabalho 061/2001). Recebido: 12/03/2001. Aceito para publicação: 08/02/2002.
2 Engo Agro Mestre em Produção Vegetal (FCAV-UNESP. Câmpus de Jaboticabal). Rua Prudente de Moraes 45, Apto 51, Caixa Postal 29. 14730-
000. Monte Azul Paulista, SP.
3 Professor Titular do Departamento de Solos e Adubos da FCAV-UNESP, Câmpus de Jaboticabal. Via de acesso Prof. Paulo Donato Castellane S/
N. 14884-900. Jaboticabal, SP. 282 282 COMUNICAÇÃO CIENTÍFICA EFFECTS OF NITROGEN AND POTASSIUM FERTILIZATION ON PRODUCTION
AND FRUIT QUALITY OF CITRUS ‘ VALÊNCIA’ ABSTRACT – Nitrogen and potassium fertilization criteria for citrus orchards was not clearly stablished yet, based on regional
experimentation. This experiment was carried out in order to compare the effects of combined levels of N and K fertilization on citrus
orchard of ‘Valência’, cultivaded in Adolfo (Northen of São Paulo State – Brazil). Three levels of nitrogen, 94, 188 and 376 kg.ha-1 as
ammonium nitrate and three levels of potassium, 38, 75 and 150 kg ha-1 of K2O as potassium chloride, were combined in factorial design
3 x 3 with 3 replications, during three harvest (1997 to 1999). Fruit productions, fruit tecnological analysis and foliar diagnosis were
evaluated yearly. Data showed that the trees kept the productivity with the mininum levels of N and K2O for three years and no
response in productivity was observed in this period. Only in the second year, when yield was decreased by climatic factors, it was
observed effects of combined levels of N and K2O on juice acidity and soluble solids contents. EFEITOS DA ADUBAÇÃO NITROGENADA E POTÁSSICA NA PRODUÇÃO E
NA QUALIDADE DE FRUTOS DE LARANJEIRA-‘VALÊNCIA’1
MARCELO CARMINATI DE ALMEIDA2, JOSÉ GERALDO BAUMGARTNER3 RESUMO - Os critérios para recomendação de adubação nitrogenada e potássica em pomares cítricos carecem de experimentação
regional. O objetivo deste experimento foi avaliar os efeitos de doses combinadas de N e de K2O para a laranjeira-Valência cultivada em
solo representativo do município de Adolfo, Norte do Estado de São Paulo, de citricultura desenvolvida. Em delineamento tipo fatorial
3 x 3, com 3 repetições, foram combinadas as doses de 94; 188 e 376 kg ha-1 de N, como nitrato de amônio e 38; 75 e 150 kg ha-1 de K2O,
como cloreto de potássio, durante três safras (1997 a 1999). Anualmente, foram feitas avaliações de diagnose foliar para nitrogênio e
potássio, de produção e de análise de suco para acidez titulável e teor total de sólidos solúveis. Os dados obtidos mostraram que os
pomares mantiveram a produtividade com as doses mínimas de N e de K2O durante os três anos, não havendo resposta em produção
às doses crescentes desses nutrientes. Os teores foliares de N e de K mantiveram-se dentro das faixas consideradas adequadas, nos
tratamentos com doses mínimas de N e de K2O, nas três safras. Apenas no segundo ano, em que a produção foi diminuída por fatores
climáticos, foram observados efeitos significativos de combinações de doses de N e de K2O sobre a acidez e o teor de sólidos solúveis
do suco. Termos para indexação: citros, nitrogênio, potássio, adubação. Rev. Bras. Frutic., Jaboticabal - SP, v. 24, n. 1, p. 282-284, abril 2002 Termos para indexação: citros, nitrogênio, potássio, adubação. Index terms: citrus, nitrogen, potassium, fertilization. Te o re s to tais na m até ria s e c a (g kg
-1)
D o s e s (kg ha
-1)
1 9 9 7
1 9 9 8
1 9 9 9
N itro gê nio (N )
N
K
N
K
N
K
9 4
2 7 ,0
1 3 ,4
2 4 ,8 b
1 0 ,2
2 5 ,1 b
1 2 ,5
1 8 8
2 7 ,2
1 2 ,8
2 5 ,1 ab
9 ,1
2 8 ,5 a
1 1 ,6
3 7 6
2 7 ,3
1 3 ,7
2 6 ,6 a
1 0 ,2
2 8 ,3 a
1 0 ,5
P o tás s io (K 2O )
3 8
2 6 ,8
1 3 ,4 a
2 5 ,6
1 0 ,1
2 7 ,7
1 0 ,7
7 5
2 7 ,3
1 4 ,2 a
2 5 ,8
1 0 ,0
2 7 ,7
1 1 ,6
1 5 0
2 7 ,3
1 2 ,4 b
2 5 ,2
9 ,5
2 6 ,5
1 2 ,3
F (do s e s de N )
0 ,1 3
3 ,2 9
4 ,6 9 *
3 ,1 9
5 ,9 3 *
0 ,7 8
F (do s e s de K )
0 ,7 3
1 2 ,3 8 * *
0 ,4 3
0 ,9 1
0 ,8 4
0 ,5 4
F (N x K )
1 ,2 2
1 ,9 2
1 ,1 1
0 ,9 6
0 ,3 7
0 ,8 8 TABELA 3 – Teores totais de N e K nas terceiras e quartas folhas de ramos frutíferos. -1 TABELA 3 – Teores totais de N e K nas terceiras e quartas folhas de ramos frutíferos. Rev. Bras. Frutic., Jaboticabal - SP, v. 24, n. 1, p. 282-284, abril 2002
TABELA 4 – Acidez e teor de sólidos solúveis do suco de frutos de laranjeira-‘Valência’. D o s e s de N
(kg ha
- 1 )
D o s e s de K 2 O
kg ha
- 1
A c ide z ( % ac . c ítric o )
A go s to / 9 8
S o l. Index terms: citrus, nitrogen, potassium, fertilization. c ítric o )
A
/ 9 8
S o l. s o lúve is (
0 B rix)
S
b
/ 9 8 TABELA 1 – Análise prévia de fertilidade de amostras coletadas nas faixas adubadas. TABELA 2 – Produção de frutos (kg/planta) de laranjeira-Valência. 2 0 - 4 0
5 ,1
5
1 ,7
2 4
1 3
1 8
6 8
P ro duç ão (kg/planta)
D o se s (kg ha
-1)
N itro gê nio (N )
1 9 9 7
1 9 9 8
1 9 9 9
M é dias
9 4
2 0 5
9 4
2 1 6
1 7 2
1 8 8
2 0 8
1 0 4
2 2 5
1 7 9
3 7 6
2 0 9
9 7
2 1 1
1 7 2
P o tássio (K 2O )
3 8
1 9 9
1 0 3
2 2 3
1 7 5
7 5
2 1 3
9 7
2 2 0
1 7 7
1 5 0
2 0 9
9 5
2 1 0
1 7 1
F (do se s de N )
0 ,0 4
0 ,6 0
0 ,7 2
-
F do se s de (K )
0 ,3 9
0 ,3 8
0 ,6 0
-
F (N x K )
0 ,0 6
1 ,9 2
0 ,4 6
-
C V (% )
1 5 ,8 1
1 9 ,8 8
1 2 ,2 1
- TABELA 2 – Produção de frutos (kg/planta) de laranjeira-Valência. TABELA 3 – Teores totais de N e K nas terceiras e quartas folhas de ramos frutíferos. Letras diferentes nas colunas significam médias diferentes, pelo teste de Tukey, a 5%. Index terms: citrus, nitrogen, potassium, fertilization. Index terms: citrus, nitrogen, potassium, fertilization. Index terms: citrus, nitrogen, potassium, fertilization. Vermelho-Amarelo eutrófico, textura arenosa a média, em relevo
suavemente ondulado, foi coletado em amostras compostas, nas
faixas adubadas e nas profundidades de 0-20cm e 20-40cm que
foram submetidas à análise de fertilidade, segundo os métodos
descritos por Raij & Quaggio, 1983. Os resultados analíticos estão
na Tabela 1. Com base na análise de solo, estimativa de safra e
análises foliares anteriores, foram estabelecidas as doses de N
(kg ha-1): N1 = 94; N2 = 188 e N3 = 376 e de K2O (kg ha-1): K1 = 38;
K2 = 75 e K3 = 150. As parcelas foram compostas por 3 linhas de
6 plantas, considerando-se como área útil as quatro plantas
centrais. O pomar foi plantado no espaçamento de 8 x 4 m de
forma que cada parcela tinha área de 576m2. O delineamento
experimental adotado foi tipo fatorial 3 x 3, em blocos ao acaso,
com 3 repetições. Os adubos utilizados foram o nitrato de amônio
(34% de N) e o cloreto de potássio (58% de K2O), granulados. A O nitrogênio é o problema central na recomendação de
adubos na citricultura, pois é complexa a avaliação da ciclagem
desse elemento no ambiente do pomar. A análise de fertilidade
do solo não permite estimativa de disponibilidade de nitrogênio,
e a análise do teor total deste nas folhas também tem sido
questionada como critério diagnóstico. As plantas, de um modo
geral, exigem potássio na medida da capacidade de metabolização
do nitrogênio. Na citricultura, a relação N/K nos tecidos foliares
afeta a produção e a qualidade dos frutos como já observaram
diferentes pesquisadores (Reese & Koo, 1975; DuPlessis & Koen,
1988). O objetivo deste experimento foi avaliar efeitos de doses
combinadas de nitrogênio e potássio sobre a produção e a
qualidade dos frutos de laranjeira-Valência, cultivada no município
de Adolfo, Norte do Estado de São Paulo. O solo, Podzólico Rev. Bras. Frutic., Jaboticabal - SP, v. 24, n. 1, p. 282-284, abril 2002 Frutic., Jaboticabal - SP, v. 24, n. 1, p. 282-284, abril 200 M. C. ALMEIDA & J. G. BAUMGARTNER 283 TABELA 1 – Análise prévia de fertilidade de amostras coletadas nas faixas adubadas. TABELA 2 – Produção de frutos (kg/planta) de laranjeira-Valência. TABELA 3 – Teores totais de N e K nas terceiras e quartas folhas de ramos frutíferos. Index terms: citrus, nitrogen, potassium, fertilization. Letras diferentes nas colunas significam médias diferentes, pelo teste de Tukey, a 5%. TABELA 4 – Acidez e teor de sólidos solúveis do suco de frutos de laranjeira-‘Valência’. Index terms: citrus, nitrogen, potassium, fertilization. K
+
C a
2 +
M g
2 +
H + A l
P r o f u n d i d a d e s
c m
p H
C a C l 2
P
m g d m
- 3
m m o l c d m
- 3
V %
0 - 2 0
5 ,6
2 0
4 ,0
2 9
8
1 7
7 0
2 0 - 4 0
5 ,1
5
1 ,7
2 4
1 3
1 8
6 8
P ro duç ão (kg/planta)
D o se s (kg ha
-1)
N itro gê nio (N )
1 9 9 7
1 9 9 8
1 9 9 9
M é dias
9 4
2 0 5
9 4
2 1 6
1 7 2
1 8 8
2 0 8
1 0 4
2 2 5
1 7 9
3 7 6
2 0 9
9 7
2 1 1
1 7 2
P o tássio (K 2O )
3 8
1 9 9
1 0 3
2 2 3
1 7 5
7 5
2 1 3
9 7
2 2 0
1 7 7
1 5 0
2 0 9
9 5
2 1 0
1 7 1
F (do se s de N )
0 ,0 4
0 ,6 0
0 ,7 2
-
F do se s de (K )
0 ,3 9
0 ,3 8
0 ,6 0
-
F (N x K )
0 ,0 6
1 ,9 2
0 ,4 6
-
C V (% )
1 5 ,8 1
1 9 ,8 8
1 2 ,2 1
-
Te o re s to tais na m até ria s e c a (g kg
-1)
D o s e s (kg ha
-1)
1 9 9 7
1 9 9 8
1 9 9 9
N itro gê nio (N )
N
K
N
K
N
K
9 4
2 7 ,0
1 3 ,4
2 4 ,8 b
1 0 ,2
2 5 ,1 b
1 2 ,5
1 8 8
2 7 ,2
1 2 ,8
2 5 ,1 ab
9 ,1
2 8 ,5 a
1 1 ,6
3 7 6
2 7 ,3
1 3 ,7
2 6 ,6 a
1 0 ,2
2 8 ,3 a
1 0 ,5
P o tás s io (K 2O )
3 8
2 6 ,8
1 3 ,4 a
2 5 ,6
1 0 ,1
2 7 ,7
1 0 ,7
7 5
2 7 ,3
1 4 ,2 a
2 5 ,8
1 0 ,0
2 7 ,7
1 1 ,6
1 5 0
2 7 ,3
1 2 ,4 b
2 5 ,2
9 ,5
2 6 ,5
1 2 ,3
F (do s e s de N )
0 ,1 3
3 ,2 9
4 ,6 9 *
3 ,1 9
5 ,9 3 *
0 ,7 8
F (do s e s de K )
0 ,7 3
1 2 ,3 8 * *
0 ,4 3
0 ,9 1
0 ,8 4
0 ,5 4
F (N x K )
1 ,2 2
1 ,9 2
1 ,1 1
0 ,9 6
0 ,3 7
0 ,8 8
D o s e s de N
(k
h
- 1 )
D o s e s de K 2 O
k
h
- 1
A c ide z ( % ac . Index terms: citrus, nitrogen, potassium, fertilization. s o lúve is (
0 B rix)
S e te m br o / 9 8
9 4
3 8
1 ,0 3 b
1 0 ,3 9 a
9 4
7 5
0 ,9 9 b
1 0 ,0 6 b
9 4
1 5 0
1 ,1 5 a
1 0 ,4 5 a
1 8 8
3 8
1 ,1 3 a
1 0 ,5 1 a
1 8 8
7 5
1 ,0 7 ab
1 0 ,3 1 ab
1 8 8
1 5 0
1 ,0 6 b
1 0 ,7 1 a
3 7 6
3 8
1 ,0 6 b
1 0 ,4 0 a
3 7 6
7 5
1 ,1 7 a
1 0 ,8 3 a
3 7 6
1 5 0
1 ,1 2 ab
1 0 ,2 7 ab
F ( N x K )
3 ,0 5 *
3 ,4 0 *
dm s (Tuke y 5 % )
0 ,0 8
0 ,3 2
C V ( % )
6 ,4 5
2 ,5 9 TABELA 4 – Acidez e teor de sólidos solúveis do suco de frutos de laranjeira-‘Valência’. EFEITOS DA ADUBAÇÃO NITROGENADA E POTÁSSICA NA PRODUÇÃO E NA QUALIDADE DOS FRUTOS 284 de doses de N e K sobre as características de acidez e teor total
de sólidos solúveis. Houve interação significativa entre doses
de N e K para ambas as variáveis. Dessa forma, a maior dose de
K (150 kg ha-1 de K2O), comparada às outras, causou aumento da
acidez do suco apenas dentro da menor dose de nitrogênio (94
kgha-1 de N), ao passo que a dose intermediária de K (75 kg ha-1
de K2O) causou diminuição no teor de sólidos solúveis, apenas
dentro da maior dose de nitrogênio. Esses efeitos foram presentes
só nas primeiras amostragens de frutos, isto é, sobre a acidez
nas amostras de frutos coletados na mês de agosto e sobre o
teor total de sólidos solúveis, na amostragem de setembro. O
efeito de doses de K sobre a acidificação do suco está coerente
com o que já foi observado por diferentes pesquisadores (Reese
& Koo, 1975). Pode-se observar ainda que, fixando a dose
intermediária de potássio (K2 = 75 kg ha-1 de K2O), à medida que
aumentou a dose de N, aumentou também o teor de sólidos
solúveis do suco. Index terms: citrus, nitrogen, potassium, fertilization. Já no segundo ano, a produção
foi prejudicada por condições climáticas desfavoráveis e pode-
se inferir que a colheita obtida pouco exigiu quanto à
disponibilidade de nutrientes. A não-resposta à adubação NK
foi inesperada na terceira safra, pois pode-se considerar as doses
menores (N1 e K1), como uma subadubação, visto que as doses
centrais N2 e K2 foram baseadas nas recomendações do Grupo
Paulista (1994) e, portanto, as doses N3 e K3 podem ser
consideradas excessivas. Entretanto, as plantas mantiveram a
mesma produtividade, quer recebendo 94 e 38 kg ha-1 de N e de
K2O, quer 376 e 150 kg ha-1 de N e de K2O, respectivamente, por
três anos consecutivos. Este contraste confirma a capacidade
de as plantas cítricas bem conduzidas manterem a produtividade
às custas de suas próprias reservas e das do solo por período de
tempo relativamente longo. Os dados da Tabela 3 mostram que
os teores foliares de nitrogênio e potássio foram pouco afetados
pelos tratamentos de adubação, ocorrendo pequenas oscilações
dentro das faixas consideradas adequadas (N = 23 - 27 g kg-1 e K
= 10 - 15 g kg-1).Pode-se verificar, ainda, que o ano agrícola teve
maior influência nos teores foliares de N e de K que os níveis de
adubação. Todavia, os teores foliares de N aumentaram
significativamente em função das doses crescentes do nutriente,
apenas nos anos de 1998 e 1999. A variação obtida sobre os
teores foliares de K, em função de doses do nutriente, foi estranha,
pois constatou-se diminuição no teor foliar com aumento da dose,
apenas no primeiro ano. Isso, entretanto, é passível de ocorrer,
por dois fatores não excludentes: diluição do nutriente em copa
de maior vegetação e concentração salina na faixa adubada,
diminuindo a absorção via radicelas. Das análises tecnológicas dos frutos, constataram-se
efeitos esparsos dos tratamentos sobre a acidez (gramas de ácido Index terms: citrus, nitrogen, potassium, fertilization. Diferentes autores têm considerado como
inconsistentes os efeitos de doses de nitrogênio sobre o teor de
sólidos do suco (Reese & Koo, 1975; Embleton et al., 1973). adubação foi feita em faixas laterais, dos dois lados das plantas,
em três parcelas, na época de chuvas nos anos de 1996 a 1999. Nos meses de março de cada ano, cerca de 40 dias após a última
parcela de adubação, foram coletadas amostras de folhas para
fins de diagnose foliar. As amostras foram preparadas e analisadas
para teores totais de nitrogênio e de potássio conforme métodos
descritos por Bataglia et al., 1983. Nos meses de agosto, setembro,
outubro e novembro de cada ano, foram coletadas amostras de
48 frutos por parcela, determinando-se, nos mesmos, a acidez
titulável e o teor de sólidos solúveis (0Brix) segundo métodos
descritos por Reed et al., 1986. Em novembro de cada ano, foi
feita a colheita total de frutos maduros, determinando-se o peso
dos mesmos por parcela (Tabela 2). Esses dados mostram que
não houve efeitos significativos das doses combinadas de
nitrogênio e potássio na produção da laranjeira-Valência durante
três safras consecutivas. As produções nos anos de 1997 e 1999
foram normais, de acordo com os padrões da laranjeira-“Valência”
indicados por Figueiredo (1991). No ano de 1998, a produção
caiu pela metade devido à irregularidade de chuvas, afetando o
florescimento e o pegamento dos frutos. Os dados do primeiro
ano de não-resposta à adubação NK são considerados previsíveis
em função do histórico do pomar. Já no segundo ano, a produção
foi prejudicada por condições climáticas desfavoráveis e pode-
se inferir que a colheita obtida pouco exigiu quanto à
disponibilidade de nutrientes. A não-resposta à adubação NK
foi inesperada na terceira safra, pois pode-se considerar as doses
menores (N1 e K1), como uma subadubação, visto que as doses
centrais N2 e K2 foram baseadas nas recomendações do Grupo
Paulista (1994) e, portanto, as doses N3 e K3 podem ser
consideradas excessivas. Entretanto, as plantas mantiveram a
mesma produtividade, quer recebendo 94 e 38 kg ha-1 de N e de
K2O, quer 376 e 150 kg ha-1 de N e de K2O, respectivamente, por
três anos consecutivos. Este contraste confirma a capacidade
de as plantas cítricas bem conduzidas manterem a produtividade
às custas de suas próprias reservas e das do solo por período de
tempo relativamente longo. Rev. Bras. Frutic., Jaboticabal - SP, v. 24, n. 1, p. 282-284, abril 2002 Index terms: citrus, nitrogen, potassium, fertilization. Os dados da Tabela 3 mostram que
os teores foliares de nitrogênio e potássio foram pouco afetados
pelos tratamentos de adubação, ocorrendo pequenas oscilações
dentro das faixas consideradas adequadas (N = 23 - 27 g kg-1 e K
= 10 - 15 g kg-1).Pode-se verificar, ainda, que o ano agrícola teve
maior influência nos teores foliares de N e de K que os níveis de
adubação. Todavia, os teores foliares de N aumentaram
significativamente em função das doses crescentes do nutriente,
apenas nos anos de 1998 e 1999. A variação obtida sobre os
teores foliares de K, em função de doses do nutriente, foi estranha,
pois constatou-se diminuição no teor foliar com aumento da dose,
apenas no primeiro ano. Isso, entretanto, é passível de ocorrer,
por dois fatores não excludentes: diluição do nutriente em copa
de maior vegetação e concentração salina na faixa adubada,
diminuindo a absorção via radicelas. Das análises tecnológicas dos frutos, constataram-se adubação foi feita em faixas laterais, dos dois lados das plantas,
em três parcelas, na época de chuvas nos anos de 1996 a 1999. Nos meses de março de cada ano, cerca de 40 dias após a última
parcela de adubação, foram coletadas amostras de folhas para
fins de diagnose foliar. As amostras foram preparadas e analisadas
para teores totais de nitrogênio e de potássio conforme métodos
descritos por Bataglia et al., 1983. Nos meses de agosto, setembro,
outubro e novembro de cada ano, foram coletadas amostras de
48 frutos por parcela, determinando-se, nos mesmos, a acidez
titulável e o teor de sólidos solúveis (0Brix) segundo métodos
descritos por Reed et al., 1986. Em novembro de cada ano, foi
feita a colheita total de frutos maduros, determinando-se o peso
dos mesmos por parcela (Tabela 2). Esses dados mostram que
não houve efeitos significativos das doses combinadas de
nitrogênio e potássio na produção da laranjeira-Valência durante
três safras consecutivas. As produções nos anos de 1997 e 1999
foram normais, de acordo com os padrões da laranjeira-“Valência”
indicados por Figueiredo (1991). No ano de 1998, a produção
caiu pela metade devido à irregularidade de chuvas, afetando o
florescimento e o pegamento dos frutos. Os dados do primeiro
ano de não-resposta à adubação NK são considerados previsíveis
em função do histórico do pomar. REFERÊNCIAS BIBLIOGRÁFICAS BATAGLIA, O. C; FURLANI, A. M. C; TEIXEIRA, J. P. F;
FURLANI, P. R.; GALLO, J. R. Métodos de análises químicas de
plantas. Campinas: Instituto Agronômico, 1983. 48p. (Boletim
Técnico, 78). DuPLESSIS, S. F; KOEN, T. J. The effect of N and K fertilization
on yield and fruit size of ‘Valência’: In: INTERNATIONAL
CITRUS CONGRESS, 6., 1988, Israel. Proceedings. P. 663-672. EMBLETON, T. W; WINSTON, W. J; LABANAUSKAS, C. K;
REUTHER, W. Leaf analysis as a diagnostic tool and guide to
fertilization. In: REUTHER, W, BATCHELOR, L. D, WEBBER, H. J (Ed). The citrus industry. Berkeley: University of Califórnia,
Division of Agricultural Sciences, 1973, v. 3, p. 183-210. FIGUEIREDO, J. O. Variedades-copa de valor comercial. In:
RODRIGUEZ, O; VIEGAS, F; POMPEU JR, J; AMARO, A. S. (ed). Citricultura Brasileira 2. ed. rev. ampl. Campinas: Fundação
Cargill 1991. v 1, 492p. GRUPO PAULISTA DE ADUBAÇÃO E CALAGEM PARA
CITROS. Recomendações de adubação e calagem para citros no
Estado de São Paulo. 3. ed. rev. atual. Cordeirópolis: Laranja,
1994. 27p. Edição especial. RAIJ, B. van; QUAGGIO, J. A. Métodos de análises de solo para
fins de fertilidade. Campinas: Instituto Agronômico, 1983. 31 p. (Boletim Técnico, 81). Das análises tecnológicas dos frutos, constataram-se
efeitos esparsos dos tratamentos sobre a acidez (gramas de ácido
cítrico / 100 mL de suco) e teor de sólidos solúveis (0Brix). Os
efeitos significativos, identificados pelo teste F, foram
desdobrados e as médias comparadas pelo teste de Tukey a 5%
(Tabela 4). Os dados mostram que apenas na safra de menor
produção, em razão de fatores climáticos (1998), ocorreram efeitos REED, J. B; HENDRIX, J. R, C. M; HENDRIX, D. L. Quality control
manual for citrus processing plantas. Flórida: Intercit, 1986. 250p. REESE, R. L; KOO, R. G. J. Effects of N and K fertilization on
internal fruit quality of three major Flórida oranges cultivars. J. Am. Soc. Hort. Sci. nº 100, v 2, p.425-428, 1975. Rev. Bras. Frutic., Jaboticabal - SP, v. 24, n. 1, p. 282-284, abril 2002
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Food Preferences and Foraging Strategies of Wood-Feeding Termites in a West African Savanna
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To cite this version: A. N’dri, J. Gignoux, S. Konaté. Food Preferences and Foraging Strategies of Wood-Feeding Termites
in a West African Savanna. Current Science, 2018, 114 (01), pp.186. 10.18520/cs/v114/i01/186-192. hal-02388766 Food Preferences and Foraging Strategies of
Wood-Feeding Termites in a West African Savanna
A. N’dri, J. Gignoux, S. Konaté To cite this version:
A. N’dri, J. Gignoux, S. Konaté. Food Preferences and Foraging Strategies of Wood-Feeding Termites
in a West African Savanna. Current Science, 2018, 114 (01), pp.186. 10.18520/cs/v114/i01/186-192. hal-02388766 Food Preferences and Foraging Strategies of
Wood-Feeding Termites in a West African Savanna
A. N’dri, J. Gignoux, S. Konaté
To cite this version:
A. N’dri, J. Gignoux, S. Konaté. Food Preferences and Foraging Strategies of Wood-Feeding Termites
in a West African Savanna. Current Science, 2018, 114 (01), pp.186. 10.18520/cs/v114/i01/186-192.
hal-02388766 Food Preferences and Foraging Strategies of
Wood-Feeding Termites in a West African Savanna
A. N’dri, J. Gignoux, S. Konaté Distributed under a Creative Commons Attribution 4.0 International License Food preferences and foraging strategies of
wood-feeding termites in a West African
savanna A. B. N’Dri1,*, J. Gignoux2 and S. Konaté1
1UFR des Sciences de la Nature, Station d’Ecologie de Lamto/CRE, Pôle de Recherche Environnement et Dév
Université Nangui Abrogoua, 02 BP 801 Abidjan 02, Côte d'Ivoire
2IEES-Paris (CNRS, IRD, UPMC, INRA, UPEC), UPMC, 4 place Jussieu, 75252 Paris cedex 05, France A. B. N’Dri1,*, J. Gignoux2 and S. Konaté1
1UFR des Sciences de la Nature, Station d’Ecologie de Lamto/CRE, Pôle de Recherche Environnement et Développement Durable,
Université Nangui Abrogoua, 02 BP 801 Abidjan 02, Côte d'Ivoire
2IEES-Paris (CNRS, IRD, UPMC, INRA, UPEC), UPMC, 4 place Jussieu, 75252 Paris cedex 05, France termites towards feeding on and colonizing the trunks of
particular tree species. Termite food preferences might, in
the end, be responsible for the whole savanna physio-
gnomy through their impacts on tree population dynam-
ics, as suggested by Gould et al.11. The feeding preferences of termites in a Guinean
savanna (Lamto, Côte d’Ivoire) were studied in the
field to better understand the incidence of termite
damages observed on adult trees, which, on interaction
with fire, affect the dynamics of the tree community. Two separate multiple-choice test experiments were
conducted on the six dominant tree species, aiming at
determining preferences for the species and for wood
conditions naturally encountered and likely to affect
termite consumption behaviour. The preferences of
termites were determined by their occurrence on
standard-sized wood pieces and their behaviour during
the first attack. Two fungus-growing termites, Ancis-
trotermes and Microtermes were frequently encountered
on wood pieces, whereas wood feeders were absent. The two genera have different preferences, and they do
not compete for the same food. Wood density and
water content did not explain termite preference. Holes in the bark seem to facilitate termite entry into
pieces of wood, whereas the action of fire on wood ap-
parently makes it less attractive to termites. In the
field, the decision by termites to forage on a given tree
species seems to be more driven by habitat and acces-
sibility differences than real food quality differences. We tested the feeding preferences of termites through
field experiments in order to understand their impact on
the adult tree populations. Termites interact with fire to
cause external openings on trees9. This has population-
level consequences, trees with external openings reach
smaller sizes both in height and basal diameter9. It
also affects adult mortality of some dominant tree spe-
cies10. HAL Id: hal-02388766
https://cnrs.hal.science/hal-02388766v1
Submitted on 7 Jul 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License RESEARCH ARTICLES Food preferences and foraging strategies of
wood-feeding termites in a West African
savanna Although a plausible scenario of termite–fire inter-
action has been proposed, the precise mechanisms of
interaction are unknown. In particular, we do not know
which are the termite genera responsible for the impacts
observed on trees; which tree species are more suitable
for termites; how are trees entered, and in particular,
whether holes caused by fire on trees allow termites to
enter them. Our experiments address these questions. Ex-
periment 1 was a multiple-choice test designed to test rel-
ative preferences among tree species, whereas experiment
2 was a multiple-choice test designed to test relative pre-
ferences among five different wood conditions. In order
to better understand the tree–termite interactions, we
identified (1) termite genera responsible for tree damage,
(2) tree species more suitable to termites and (3) condi-
tion of the wood preferred by termites. Keywords: Food preference, foraging strategies, tree
species, wood-feeding termites. ABOUT 2600 species belonging to 281 genera of termites
(Isoptera) have been described1. Termites are abundant in
tropical ecosystems. They affect the decay of plants
debris in soil, playing an important role in soil nutrients
cycling and fertility2. Many species are known to move
organic matter from the surface to deeper layers of the
soil, improving and maintaining its porosity and aera-
tion3,4. Their negative impact on crops and houses is rela-
tively well understood5–8. Recently, they have been
proven to affect the mechanical resistance of living trees
in fire-prone savanna ecosystems9, and the population
dynamics of trees through their mortality rate10. It is
therefore of key importance to understand what drives *For correspondence. (e-mail: ndri.brigitte@yahoo.fr) Study site The experiments were conducted at the Lamto Research
Station in Côte d’Ivoire (613N, 502W). The major tree
species are shrubby and usually <10 m, high, and more
than 90% of the woody layer is composed of four species:
Bridelia ferruginea Benth. (Phyllantaceae), Crossopteryx
febrifuga (Afzel. ex G. Don) Benth. (Rubiaceae), Cus-
sonia barteri A. Rich. (Araliaceae), and Piliostigma thon-
ningii (Schum.) Milne-Redhead (Caesalpiniaceae)12. Shoots of woody species such as Annona senengalensis CURRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018 186 RESEARCH ARTICLES Pers. (Annonaceae) grow in the grass layer, whereas in
the upper stratum we find Terminalia schimperiana
Hochst. (Combretaceae). holes left by fire and broken branches on the living trees
as a test of the ‘access point’ hypothesis30,31, as observed
in N’Dri et al.9. According to N’Dri et al.9, termites are
better able to consume parts of trees impacted by fire than
fresh ones and use them to get into the trees. These ob-
servations were tested using burned (B) and charred (C)
pieces of wood in order to simulate the impact of fire on
living trees. To standardize their water content, the dry,
burned and charred wood blocks were oven-dried at
100C for 72 h. Burned and charred treatment was ob-
tained by placing blocks in dry grass (cropped in the field
and drying) fires, until char appeared on the block surface
for ‘C’ and less than 2 min, for ‘B’, according to the de-
scription of savanna fire behaviour in the literature32–34. Dry wood (D) was added to test whether dead wood was
preferred over fresh (F) wood by termites, as suggested
by Josens15 and Grassé35. All the wood blocks (FH, B, C,
D, F) were tagged and placed on each of the two plots. (
)
With biomass reaching 3.4–19.6 g m–2 (fresh weight),
termites are one of the major animal components of the
Lamto ecosystem13,14. The main trophic groups are the
fungus-growing (from 1.15 to 5.93 g m–2), humivorous
(from 2.14 to 11.65 g m–2), and xylophageous and grass-
foraging (from 0.17 to 1.98 g m–2) termites14–16. Their
activities result in the formation of biogenic structures
which are frequently encountered in all savanna types17:
above-ground termitaria; below-ground nests; comb
chambers; sheetings and galleries. The latter two are built
by workers, generally using soil-impregnated saliva
covering the food supply or termite pathway. Termite sampling methods on experimental plots To estimate the overall termite diversity of an experimental
habitat, the whole experimental plot was searched for ter-
mites. A modified standardized method of the protocol for
rapid assessment of termite diversity by Jones and Eggle-
ton22 was used9. Sampled termites were identified to the ge-
nus level using standard determination keys23–26 and species
descriptions27. Following Josens15, Deligne28 and Sands29,
they were classified into fungus-growers, soil-feeders,
wood-feeders and grass-feeders. Wood-feeders and fungus-
growers were expected to be actively feeding on wood. Wood blocks were visited daily for termite impacts, to
determine the attack order of different tree species. A
block was considered impacted by termites when it was
covered by biogenic structures (sheetings or/and tunnel-
galleries). Biogenic structure type (gallery or sheeting)
was recorded to determine the use of wood blocks by
termites, tunnel-galleries being built for prospection and
sheetings for feeding36. When termite attacks were found
on all wood blocks, observations were done every second
week from April 2009 to January 2010. F and FH wood
blocks were changed at each sample. Termites and their
biogenic structures were systematically searched in order
to determine the termite genera. When termites were
found, representative (~10) individuals of the soldier
caste and/or worker were collected and preserved in 75%
alcohol and later identified to the genus level. Study site Also,
termite mounds which are very large, complex biogenic
structures grouping below-ground nests of different
termite species, are frequently encountered in all savanna
types13,18. All Lamto savannas are subject to annual
fires19,20. Termites, which are known to consume only
dead material21, have recently been observed to use the
burned parts of trees to enter the trunk9. Experiments
were conducted in two burned shrubby savanna habitats
(the most frequent savanna type in Lamto), replicated
twice in each area. Experimental plots were selected as
far as possible from termite mounds, nests and trees,
which could bias the sampling because they favour the
presence of one species of termite. Our first experiment focused on termite preference for
wood of a particular tree species (species preference test,
SPT). Wood blocks of the six species in the same condi-
tion were grouped at one field location, directly on the
soil and 10 cm apart (distance chosen to give the same
probability for a wood block to be consumed by foraging
termites) from each other. Five groups of wood blocks,
one for each wood condition, were randomly placed 35 m
apart from each other, which corresponds to the approxi-
mate range of termite prospection (S.K., pers. commun.). The second experiment focused on termite preference for
a particular wood condition (condition preference test,
CPT). It was setup like the first experiment, but reversing
the role of species and wood conditions. Each of these
experiments was replicated twice in each shrubby
savanna (four replicates overall). Tree species and experimental procedures A total of six dominant tree species of the Lamto reserve
were used as potential food for termites: A. senegalensis;
B. ferruginea; C. febrifuga; Cussonia arborea; P. thon-
ningii and T. schimperiana. Five wood blocks (~10 cm in length) were cut from
living (around 6 cm dia) branches of each of the six spe-
cies and additional treatments were applied to the blocks
to mimic the possible conditions of wood debris that
could be encountered by termites in the field. The fresh
wood with holes (FH) was obtained by making holes with
nails through the bark of wood blocks to simulate the Measurement of wood species density According to Behr et al.37, termites preferentially con-
sume lower density wood. To test this, we estimated the CURRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018 187 RESEARCH ARTICLES wood blocks significantly differed with termite genus
( 2 = 5530.69, df = 7, P < 0.001). wood blocks significantly differed with termite genus
( 2 = 5530.69, df = 7, P < 0.001). wood blocks significantly differed with termite genus
( 2 = 5530.69, df = 7, P < 0.001). wood density of five of the dominant tree species of the
Lamto reserve (B. ferruginea, C. febrifuga, C. arborea,
P. thonningii and T. shimperiana) in two different condi-
tions (fresh and dry). One wood block was cut from liv-
ing branches of five different trees for each species, then
each wood block was weighed and its volume determined
by immersion in water in a 1 litre beaker graduated to the
nearest cubic centimetre, density being then obtained as
the ratio of sample mass over its volume. The average
humidity content of each wood species was determined
simultaneously by weighing the samples after oven-
drying at 100C during 72 h. The impact of average water
content of each tree species on the feeding preference of
termite genera was then tested. Ancistrotermes and Microtermes feeding The occurrence of termites on different tree species
changed according to their genera (ANOVA, F1,10 =
45.61; P < 0.001; Figure 2). Occurrence of the Ancistrotermes on wood blocks varied
according to tree species (ANOVA with logit transfor-
mation, F5,60 = 4.91; P = 0.001). No experiment-type Figure 1. Occurrence of different termite genera on wood blocks. Adai, Adaiphrotermes; Ami, Amitermes; Anc, Ancistrotermes; Microc,
Microcerotermes; Micro, Microtermes; Odonto, Odontotermes; Pseu,
Pseudacanthotermes and Triner, Trinervitermes. Statistical analyses All analyses were performed using the R software
(http://www.r-project.org/). The distribution of different
termite genera on wood blocks, and of sheetings and gal-
leries on wood blocks during the first termite attack were
compared using 2 tests. Linear models (LM) with logit
transformation were used to analyse the effect of tree
species, wood condition and experiment design on: (1)
the feeding preference of two termite genera (i.e. Ancis-
trotermes and Microtermes) and (2) the occurrence of
biogenic structures (galleries and sheetings) on tree spe-
cies and wood condition during the first attack. Linear
models were also used for analysing the wood humidity
and density according to wood species and condition. Post-hoc Tukey’s HSD test was used to identify (1) the
more preferred tree species and wood condition by ter-
mites and (2) the wood block conditions and species pre-
senting the highest density and humidity. Generalized
linear models (GLM) were used for analysing the occur-
rence of biogenic structures during the first attack. Figure 1. Occurrence of different termite genera on wood blocks. Adai, Adaiphrotermes; Ami, Amitermes; Anc, Ancistrotermes; Microc,
Microcerotermes; Micro, Microtermes; Odonto, Odontotermes; Pseu,
Pseudacanthotermes and Triner, Trinervitermes. Figure 1. Occurrence of different termite genera on wood blocks. Adai, Adaiphrotermes; Ami, Amitermes; Anc, Ancistrotermes; Microc,
Microcerotermes; Micro, Microtermes; Odonto, Odontotermes; Pseu,
Pseudacanthotermes and Triner, Trinervitermes. CURRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018
Figure 2. Absence of competition between Ancistrotermes and
Microtermes for tree species preferences. ANN, Annona senegalensis;
BRI, Bridelia ferruginea; CRO, Crossopteryx febrifuga; CUS, Cus-
sonia arborea; PIL, Piliostigma thonningii and TER, Terminalia
schimperiana. Termite diversity in the study site At the experiment sites, 10 termite genera distributed into
four functional groups were collected. Five fungus-
growers (Ancistrotermes, Macrotermes, Microtermes,
Pseudacanthotermes and Odontotermes), two soil-feeders
(Adaiphrotermes and Amitermes), two wood-feeders
(Microcerotermes and Fulleritermes) and one grass-
feeder (Trinervitermes) were found. Results Termite diversity in the study site Wood density and humidity per species The interaction between wood condition and experi-
ment type was statistically significant (ANOVA with
logit transformation, F4,180 = 4.35; P = 0.002). When
individuals of Microtermes had to choose between several
wood conditions (CPT), fresh wood with holes (FH) was
the most preferred and the least preferred was charred
wood (C; ANOVA with logit transformation, F4,15 = 3.86;
P = 0.024, post hoc Tukey’s HSD test, 95% confidence
interval; Figure 4). Wood block density varied with tree species and condi-
tion (ANOVA, F4,40 = 14.53; P < 0.001 and F3,40 =
118.28; P < 0.001). Highest densities were observed in
dry woods of C. febrifuga, B. ferruginea P. thonningii
and T. schimperiana, whereas C. arborea presented the
lowest value. Intermediate density was observed in fresh
woods of T. schimperiana (Figure 6). Wood humidity
averaged ~30%, but varied according to species (ANOVA,
F4,20 = 3.12; P = 0.04). C. arborea presented the highest
water content, whereas B. ferruginea presented the lowest
value (Figure 7). Foraging behaviour of termites according to tree
species and wood condition The occurrence of galleries and sheetings during the first
attack of termites changed according to experiment type
(ANOVA
with
logit
transformation,
F1,117 = 5.24;
P = 0.02). The occurrence of this biogenic structure
changed significantly according to tree species in SPT
(binomial GLM model with logit link; deviance = 12.95,
df = 5, 2 = 136.53, P = 0.023, Figure 5). In addition, Termite genera on the wood baits Ancistrotermes and Microtermes were the only genera
frequently found on wood blocks for statistical analyses
to be performed (Figure 1). The occurrence of termites on 188 CURRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018 RESEARCH ARTICLES effect was observed on their occurrence (ANOVA with
logit transformation, F1,60 = 1.40; P = 0.24). The most
preferred wood species was T. schimperiana and the least
preferred was B. ferruginea (Figure 3). more sheetings (67%) were built than galleries (33%)
during the first attack of termites in SPT ( 2 = 13, df = 1,
P 0.001). effect was observed on their occurrence (ANOVA with
logit transformation, F1,60 = 1.40; P = 0.24). The most
preferred wood species was T. schimperiana and the least
preferred was B. ferruginea (Figure 3). more sheetings (67%) were built than galleries (33%)
during the first attack of termites in SPT ( 2 = 13, df = 1,
P 0.001). CPT did not reveal any termite preference for a specific
wood condition during the first attack. No significant
difference was noted on the occurrence of sheetings and
galleries (binomial GLM model with logit link; devi-
ance = 3.055, df = 4, 2 = 159.38, P = 0.55; Figure 5). Termites built nearly as many galleries (46%) as sheetings
(54%) during their first attack ( 2 = 0.68, df = 1, P = 0.41). The occurrence of Microtermes also varied with tree
species (ANOVA with logit transformation, F5,180 = 8.61;
P < 0.001); the most preferred species was P. thonningii
and the least preferred were C. febrifuga and C. arborea
(Figure 3). Experiment type did not influence Microter-
mes consumption (ANOVA with logit transformation,
F1,180 = 0.007; P = 0.93). Discussion Among the eight termite genera collected in this work,
only Ancistrotermes and Microtermes were significantly
abundant. As wood-feeders were frequently found in
living trees9, we expected to collect more of them on wood CURRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018
189
Figure 3. Tree species preferences of Ancistrotermes and Microter-
mes. Letters indicate groups of non-significantly different values at the
95% level according to per-termite genus Tukey’s HSD post-hoc tests. Species codes as in Figure 2. Figure 4. Microtermes preferences in the wood condition preference
test. Letters indicate groups of non-significantly different values at the
95% level according to Tukey’s HSD post-hoc tests. B, Burned; C,
Charred; D, Dry; F, Fresh; FH, Fresh with holes. Figure 4. Microtermes preferences in the wood condition preference
test. Letters indicate groups of non-significantly different values at the
95% level according to Tukey’s HSD post-hoc tests. B, Burned; C, Figure 4. Microtermes preferences in the wood condition preference
test. Letters indicate groups of non-significantly different values at the
95% level according to Tukey’s HSD post-hoc tests. B, Burned; C,
Charred; D, Dry; F, Fresh; FH, Fresh with holes. Figure 3. Tree species preferences of Ancistrotermes and Microter-
mes. Letters indicate groups of non-significantly different values at the
95% level according to per-termite genus Tukey’s HSD post-hoc tests. Species codes as in Figure 2. URRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018 189 RESEARCH ARTICLES blocks, but found fungus-growing termites instead, as
observed by Basu38 in Lamto on food preference of fun-
gus-growing termites. Fungus-growers (Termitidae :
Macrotermitinae) need plant material for the establish-
ment of their fungus combs, known as the substratum of
their symbiotic fungi39. In addition, termites of these two
genera have been classified as pests in many studies in
human-managed ecosystems40–43 and naturals ones44. Together with the fact that Ancistrotermes and Microter-
mes are known as the most common fungus-growing
termites15,38,44 in Lamto, their habit of digging galleries
into living trees was also revealed in natural ecosys-
tems9,15,45,46. species, suggesting that the termites are able to choose
their food. Indeed, food preference of termites has been
reported in several studies38,49–51. Among the many factors which could explain this
preference of termites, we tested a possible effect of
wood density and wood average humidity. Inta et al.49
studied the material properties on termite food choice. Discussion According to Behr et al.37, termites prefer lower density
wood because wood density affects the fragmentation
process by termites52. According to our density measure-
ments, wood densities were remarkably similar among
species, except for C. arborea which had low density and
high water content. The uniformity in density prevents
any explanation of preferences based on this factor alone. The absence of wood-feeders on wood samples could
be due to the preference for higher quantity of wood
found on a whole tree: wood-feeders often build arboreal
nests on living trees9,47 and would therefore preferably
exploit the trees they live on rather than risk exploration
in the open to search for smaller wood quantities. For
wood-feeders, wood is not only a food, but also a habitat. Figure 6. Density according to wood species and condition. Species
codes as in Figure 2. Letters indicate groups of non-significantly dif-
ferent values at the 95% level according to per-wood density Tukey’s
HSD post-hoc tests. As suggested by Stewart and Zalucki48, aggregation or
occurrence on different wood blocks was used as a meas-
ure of preference. The ability of termites to be attracted
by one wood species or condition during their first attack
was also used to measure preference. The occurrence of termites on wood samples changed
according to tree species. They also built directly more
feeding sheetings than prospection galleries on wood dur-
ing their first attack when presented with different wood CURRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018
190
Figure 5. Biogenic structures build by termites on wood during the
first attack on (a) SPT and (b) CPT. Species and wood conditions codes
as in Figures 2 and 4. Figure 6. Density according to wood species and condition. Species
codes as in Figure 2. Letters indicate groups of non-significantly dif-
ferent values at the 95% level according to per-wood density Tukey’s
HSD post-hoc tests. Figure 7. Water content of different wood species. Species codes as
in Figure 2. Letters indicate groups of non-significantly different values
at the 95% level according to per-wood humidity Tukey’s HSD post-
hoc tests. Figure 6. Density according to wood species and condition. Species
codes as in Figure 2. Letters indicate groups of non-significantly dif-
ferent values at the 95% level according to per-wood density Tukey’s
HSD post-hoc tests. CURRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018
Figure 7. Water content of different wood species. Discussion C., Status and prospects of termite
problems and control in Brazil. Sociobiology, 1990, 17, 45–56. 8. Han, S. H., Dégâts causés par les termites sur les bâtiments dans la
région de Dakar au sénégal. Actes Coll. Insect. Soc., 2000, 13, 61–
64. Fresh wood with holes was preferred by termites than
the other wood conditions, probably due to the fact that
holes constitute easy entry points into the wood9. Charred
wood blocks were the least preferred, suggesting that the
positive effect of fire on termite trunk-digging activity is
more related to the creation of entry points and exposure
of dead wood by fire than by an improvement in food
quality. 9. N’Dri, A. B., Gignoux, J., Konaté, S., Dembélé, A. and Aïdara,
D., Origin of trunk damage in West African savanna trees: the
interaction of fire and termites. J. Trop. Ecol., 2011, 27,
269–278. 10. N’Dri, A. B., Gignoux, J., Konaté, S. and Aïdara, D., The dynam-
ics of hollowing in annually burnt savanna trees and its effect on
adult tree mortality. Plant Ecol., 2014, 215, 27–37. 11. Gould, M. S., Lowe, A. J. and Clarke, G. P., The frequency of
termite (Isoptera) Damage to tree species in Namakutwa forest,
Tanzania. Sociobiology, 1993, 23, 189–198. Food preferences at the wood block level did not match
observations on adult trees9. These authors9 observed that
C. febrifuga was a preferred target for termites, most
individuals of this species being entirely piped, while in
the present study T. schimperiana and P. thonningii were
the two most preferred species. However, N’Dri et al.10
have observed that P. thonningii (more preferred in this
field experiment) suffered a higher mortality as a result of
termite and fire impact. 12. Menaut, J. C. and César, J., Structure and primary productivity of
Lamto savannas (Ivory Coast). Ecology, 1979, 60, 1197–1210. 13. Abbadie, L., Lepage, M. and Le Roux, X., Soil fauna at the forest–
savanna boundary: role of termite mounds in nutrient cycling. In
Nature and Dynamics of Forest-Savanna Boundaries (eds Furley,
P. A., Proctor, J. and Ratter, J. A.), Chapman and Hall, London,
UK, 1992, pp. 473–484. 14. Konaté, S., Le Roux, X., Verdier, D. and Lepage, M., Effect of
underground fungus-growing termites on carbon dioxide emission
from soils at the chamber- and landscape-scales in an African
savanna. Funct. Ecol., 2003, 17, 305–314. Discussion Our results show that (1) wood-feeders are not inter-
ested in small pieces of wood, while fungus-growers are;
(2) the two different genera studied have different prefer-
ences, but they do not seem to compete for the same
food; (3) neither wood density nor water content is corre-
lated with preferences; (4) holes in the bark seem to fa-
cilitate termite entry into pieces of wood; (5) the action of
fire on wood apparently makes it less attractive to ter-
mites. These results tend to show that, in the field, the
decision of termites to forage on a given tree species is
driven by habitat and accessibility differences (points (1)
and (4)) than real food quality differences (point (3)). From points (4) and (5) above, it is clear that fire im-
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Figure 7. Water content of different wood species. Species codes as
in Figure 2. Letters indicate groups of non-significantly different values
at the 95% level according to per-wood humidity Tukey’s HSD post-
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on the manuscript. This work was supported by RIPIECSA-Côte
d’Ivoire Project (Recherche Interdisciplianaire et Participative sur les
Interactions entre les Ecosystèmes, le Climat et les Sociétés en Afrique
de l’Ouest). A.B.N’.D. thanks the BDI fellowship programme of CNRS
(Centre National de la Recherche Scientifique) for scholarship. RESEARCH ARTICLES tropical moist savanna (Lamto, Central Côte d’Ivoire). J. Trop. Ecol., 2010, 26, 323–334. 25. Bouillon, A. and Mathot, G., Quel est ce termite africain? Sup-
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plément N° 1. Université de Lovanium, Kinshassa, 1966. 191 CURRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018 RESEARCH ARTICLES Received 6 June 2016; accepted 12 July 2017 RESEARCH ARTICLES We
thank François N’Guessan, Honoré Kounan, Hubert Kouadio Kouakou,
Alexis N'DRI, Bruno Brou, Drissa Coulibaly, Issa Quattara for support,
and technical staff of Lamto Reserve, for assistance during field
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from the Darfur region of the Sudan with comments on their pest
status. J. Arid Environ., 1995, 30, 197–206. 42. Rajagopal, D., Economically important termite species in India. Sociobiology, 2002, 40, 33–46. 43. Rouland-Lefèvre, C. and Mora, P., Control of Ancistrotermes gui-
neensis Silvestri (Termitidae: Macrotermitinae), a pest of sugar-
cane in Chad. Int. J. Pest Manage., 2002, 48, 81–86. 44. Dosso, K., Konaté, S., Aidara, D. and Linsenmair, K. E., Termite
diversity and abundance across fire-induced habitat variability in a doi: 10.18520/cs/v114/i01/186-192 CURRENT SCIENCE, VOL. 114, NO. 1, 10 JANUARY 2018 192
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De novo assembly of highly polymorphic metagenomic data using in situ generated reference sequences and a novel BLAST-based assembly pipeline
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© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: youylin@ntu.edu.tw; hurngyi@ntu.edu.tw
1Department of Life Science, National Taiwan University, Taipei 106, Taiwan
4Graduate Institute of Clinical Medicine, National Taiwan University, Taipei
100, Taiwan
Full list of author information is available at the end of the article De novo assembly of highly polymorphic
metagenomic data using in situ generated
reference sequences and a novel BLAST-
based assembly pipeline De novo assembly of highly polymorphic
metagenomic data using in situ generated
reference sequences and a novel BLAST-
based assembly pipeline You-Yu Lin1,4*
, Chia-Hung Hsieh2, Jiun-Hong Chen1, Xuemei Lu3, Jia-Horng Kao4, Pei-Jer Chen4,
Ding-Shinn Chen4,5 and Hurng-Yi Wang4,6,7* Abstract Background: The accuracy of metagenomic assembly is usually compromised by high levels of polymorphism due
to divergent reads from the same genomic region recognized as different loci when sequenced and assembled
together. A viral quasispecies is a group of abundant and diversified genetically related viruses found in a single
carrier. Current mainstream assembly methods, such as Velvet and SOAPdenovo, were not originally intended for
the assembly of such metagenomics data, and therefore demands for new methods to provide accurate and
informative assembly results for metagenomic data. Results: In this study, we present a hybrid method for assembling highly polymorphic data combining the partial
de novo-reference assembly (PDR) strategy and the BLAST-based assembly pipeline (BBAP). The PDR strategy
generates in situ reference sequences through de novo assembly of a randomly extracted partial data set which is
subsequently used for the reference assembly for the full data set. BBAP employs a greedy algorithm to assemble
polymorphic reads. We used 12 hepatitis B virus quasispecies NGS data sets from a previous study to assess and
compare the performance of both PDR and BBAP. Analyses suggest the high polymorphism of a full metagenomic
data set leads to fragmentized de novo assembly results, whereas the biased or limited representation of external
reference sequences included fewer reads into the assembly with lower assembly accuracy and variation sensitivity. In comparison, the PDR generated in situ reference sequence incorporated more reads into the final PDR assembly
of the full metagenomics data set along with greater accuracy and higher variation sensitivity. BBAP assembly
results also suggest higher assembly efficiency and accuracy compared to other assembly methods. Additionally,
BBAP assembly recovered HBV structural variants that were not observed amongst assembly results of other
methods. Together, PDR/BBAP assembly results were significantly better than other compared methods. Conclusions: Both PDR and BBAP independently increased the assembly efficiency and accuracy of highly
polymorphic data, and assembly performances were further improved when used together. BBAP also provides
nucleotide frequency information. Together, PDR and BBAP provide powerful tools for metagenomic data studies. Keywords: Next generation sequencing, Metagenomics, Hepatitis B virus, Sequence assembly, Asse generation sequencing, Metagenomics, Hepatitis B virus, Sequence assembly, Assembly pipeline ,
Full list of author information is available at the end of the article © The Author(s). Lin et al. BMC Bioinformatics (2017) 18:223
DOI 10.1186/s12859-017-1630-z Lin et al. BMC Bioinformatics (2017) 18:223
DOI 10.1186/s12859-017-1630-z Background development of de novo assembly algorithms for meta-
genomes, such as MetaVelvet [22] and Genovo [23]. g
Next-generation sequencing (NGS) has become the
mainstream method for obtaining high quantities of gen-
omic data during the past decade, and the increased ac-
cessibility of massive datasets has driven up the need for
compatible analytic algorithms and software [1]. There
are several key components for an assembly algorithm,
including the capacity to handle massive data sets, the
accuracy and efficiency of the assembly, the nature of
the data set itself, and the intended use of the assembly
results. The former two are dependent of the hardware
and algorithms implemented, whereas the latter two in-
fluences the optimization strategy and the type of infor-
mation to be extracted during assembly. For example,
metagenomic studies commonly aim to understand the
composition and relative abundances of the data set as
well as the intra-species or inter-population heterogen-
eity, therefore the assembly depth and length as well as
accuracy are prioritized for such data sets [2]. In this study, we propose a partial de novo-reference
assembly strategy, PDR, which is a de novo-reference hy-
brid assembly strategy that utilizes the completeness of de
novo assembly while complementing its low-efficiency
with reference assembly. PDR generates an in situ refer-
ence sequence by de novo assembly of a smaller yet less di-
verse partial data set followed by the reference assembly of
the full data set. Results show that the PDR assembly re-
sults are more complete and accurate than direct de novo
or reference assembly of highly polymorphic metagenomic
data sets. We also present a novel BLAST-based assembly
pipeline, BBAP, capable of both de novo and reference as-
sembly specifically designed for assembly of metagenomic
data sets. The assembly efficiency and accuracy of both
PDR and BBAP were examined using actual NGS data sets
as well as in silico generated simulated NGS data sets and
compared with the assembly results of other assembly
methods. A viral quasispecies is a group of highly genetically re-
lated viruses found in a single carrier and can be both
abundant (viral titer ≈106-109 ge/ml) and greatly diversified
(nucleotide diversity ≈10−2-10−3) within patient carriers
[3–5]. Two main NGS platforms, 454/Roche pyrose-
quencing [6] and Illumina Genome Analyzer [7], have
been commonly used for recent quasispecies-related
studies. Abstract 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lin et al. BMC Bioinformatics (2017) 18:223 Page 2 of 10 Page 2 of 10 Background Pyrosequencing has longer sequence reads and
typically does not require data set assembly [8–10], al-
though some studies still performed de novo assembly
[11] or reference sequence assembly [12, 13]. Illumina
sequencing generates much larger data sets compared
to pyrosequencing, but its shorter read length limits the
efficiency for de novo assembly [2]. Therefore, Illumina se-
quenced viral quasispecies data sets are usually assembled
using reference sequences as templates [14–17] while de
novo assembly is applicable but not commonly used [18]. Results To examine the performance of BBAP and the proposed
hybrid assembly strategy, we acquired 12 NGS data sets
of HBV viral quasispecies from 7 HBV patient samples
[24]. The 12 data sets used for assembly consisted of an
average of 21,494,295 101-bp raw reads (RRs), 14,388,844
high quality reads (HQRs, quality score ≧20 for all bases;
i.e., sequencing error rate = 1%), and 60,228 HRURs (high
redundancy unique representative reads; unique represen-
tative reads with redundancy ≧5, Table 1 and Additional
file 1: Table S1). The optimized parameters for BBAP as-
sembly are listed in Additional file 1: Table S2. The same
parameters were used for all BBAP assemblies in this
study unless mentioned otherwise. y
pp
y
The high throughput Illumina platform, compared to
the pyrosequencing platform, is capable of detecting
greater amounts of genetic variation within viral quasis-
pecies [15]. However, a major challenge for Illumina
quasispecies NGS studies is the sequence assembly of
the data sets. Sequence assembly using a reference ap-
proach is not only subject to bias of the chosen refer-
ence sequence, but also assembles less reads and thus
less genetic variation information in the assembly [15]. De novo assembly should be able to provide the most
complete and accurate genetic information of NGS data,
but can be hindered by regions with high levels of diver-
sity. The commonly used de novo assembly algorithms,
such as Velvet [19], SOAPdenovo [20], CLC Genomics
Workbench (CLC, CLC bio, Aarhus, Denmark), and
Euler-SR [21], were not originally intended for the as-
sembly of metagenomics data with high diversity and
coverage depth. Recent progress have been made in the BBAP de novo assembly of full and partial data sets BMC Bioinformatics (2017) 18:223 Page 3 of 10 Page 3 of 10 Table 1 Average assembly statistics of all 12 data sets using
BBAP with multiple approaches
PDa
FDb
SRc
PDRd
RRs
214,942
21,494,295
21,494,295
21,494,295
HQRs
143,912
14,388,844
14,388,844
14,388,844
URs
27,150
860,144
860,144
860,144
HRURs
6264
60,228
60,228
60,228
RiHRURs
116,555
13,388,423
13,388,423
13,388,423
Contigs assemblede
2.1
46.0
1.0
3.9
Max contig length
3119
1473
3,207
3148
Average contig length
2319
321
3207
1268
% of Mapped HRURs
95.9%
70.3%
67.4%
69.9%
% of Mapped RiHRURs
80.4%
68.7%
82.7%
84.5%
The full data sets were used in the BBAP assembly with FD, SR, and PDR
approaches, whereas partial data sets consisting of 1% of randomly selected
RRs were used in the BBAP PD assembly approach
aPartial data set de novo assembly
bFull data set de novo assembly
cSanger reference assembly
dPartial data set reference assembly of the full data set
eOnly minimum assembled contig length > 150 bp was shown
RRs raw reads, HQRs high quality reads (quality score threshold = 20, i.e.,
sequencing error rate = 1%), URs unique representative reads, HRURs high
redundancy unique representative reads (unique representative reads with
redundancy threshold = 5), RiHRURs reads included in high redundancy unique
representative reads Table 1 Average assembly statistics of all 12 data sets using
BBAP with multiple approaches BBAP as different haplotypes and subsequently assembled
into separate clusters. The proportion of polymorphic sites
in overlapping contig regions of D2_1 FD assembly was 10
times higher than that in non-overlapping regions (0.238
vs. 0.022; p < 10−10). A similar trend was also found in
D2_1 PD assembly (Additional file 1: Table S4). The
shorter FD assembled contigs (<300 bp) had a significantly
higher proportion of polymorphic sites than the longer FD
assembled contigs (Additional file 2: Figure S2, Student’s
t-test, p < 0.05). HRURs that were included or excluded in
the partial data sets (for PD assembly) had average re-
dundancies of 1,808X (n = 75,173) and 38X (n = 647,561),
respectively, within the full data set. Additionally, the re-
dundancies of the included HRURs in the full and partial
data sets were highly correlated (R2 = 0.9997). This sug-
gests the random selection partial data sets was unbiased
and effectively excluded HRURs of low redundancies,
resulting in lower polymorphism levels and, in turn, less
fragmented assembly results. BBAP de novo assembly of full and partial data sets BBAP de novo assembly of full and partial data sets
The de novo assembly of the full data sets (FD) resulted
in an average of 46.0 contigs (minimum length of 150 bp)
for each library with an average contig length of 321 bp,
suggesting that the assembly results were fragmentized
(Table 1 and Additional file 1: Table S3). For de novo
assembly of partial data sets (PD) of each data set, five
partial data sets were initially randomly generated and
assembled independently. Because the PD assembly re-
sults of the partial data sets from each library were
highly similar (data not shown), a single partial data set
and its assembly results were used for representation of
the sample in further analyses. The PD assembly yielded
fewer number of contigs and longer average maximum
contig lengths, indicating the PD assembly results were
not as fragmentized as FD assembly. Furthermore, PD
assembly required fewer contigs than the FD assembly
to span the full genome to recover the full length HBV Lin et al. BBAP reference assembly with different reference
sequences MetaVel-
vet assembly results, which are based on initial Velvet
assembly results, did not show any improvement and
were completely identical to Velvet assembly results for
both full and partial data set. SOAPdenovo generated 8
assembled contigs with maximum and average lengths
of 934 bp and 340 bp, respectively, and covered 14% of
the HBV genome (Fig. 1c). Genovo assembly for the
D2_1 data set resulted in a total of 60 contigs with max-
imum and average contig lengths of 1352 bp and 395 bp,
respectively, but only 44% of the HBV genome were recov-
ered (Fig. 1d, Additional file 2: Figure S1). D2_1 data set. Similar to BBAP FD, the full data set as-
semblies by Velvet, MetaVelvet, SOAPdenovo, and Gen-
ovo resulted in fragmented contigs. De novo assembly of
full data set with Velvet resulted in 13 contigs with max-
imum and average lengths of 1102 bp and 303 bp, re-
spectively (Table 2), and recovered only 19% of the HBV
genome (Fig. 1b, Additional file 2: Figure S1). MetaVel-
vet assembly results, which are based on initial Velvet
assembly results, did not show any improvement and
were completely identical to Velvet assembly results for
both full and partial data set. SOAPdenovo generated 8
assembled contigs with maximum and average lengths
of 934 bp and 340 bp, respectively, and covered 14% of
the HBV genome (Fig. 1c). Genovo assembly for the
D2_1 data set resulted in a total of 60 contigs with max-
imum and average contig lengths of 1352 bp and 395 bp,
respectively, but only 44% of the HBV genome were recov-
ered (Fig. 1d, Additional file 2: Figure S1). polymorphism in the consensus sequences (Additional
file 1: Table S5), but the PDR assembly additionally identi-
fied HBV structural variants (Additional file 1: Table S6,
Additional file 2: Figure S3-S5 and Additional file 3: SA). g
PDR alignment accuracy was also higher than SR. SR
assembly of D2_1 resulted in a single contig with 50,587
HRURs, but only 50,211 of the SR assembled HRURs
were mapped to the two main PDR assembled contigs
(M1 and M2; Additional file 2: Figure S6, 50,396 HRURs)
covering the full HBV genome and have identical se-
quences as the SR contig. BBAP reference assembly with different reference
sequences Not only did the remaining 376
HRURs all mapped to one of the nine PDR assembled
variant contigs, but the SR alignment qualities of those
376 HRURs was less optimal than the 50,211 HRURs,
shown by the significantly greater BLAST e-value and
lower BLAST alignment score (Wilcoxon rank-sum test,
p < 0.001), both supporting the higher alignment accuracy
of PDR assembly. Overall, results of SR assembly and PDR
assembly were similar in recovering sequence variation,
but the latter included more HRURs and RiHRURs with
increased accuracy due to the additional mapping options
of the shorter HBV variant contigs provided by the de
novo assembly of the partial data set, whereas the lower
assembly accuracy of the former resulted in low quality
alignments and slightly more polymorphic sites. We proposed that the high polymorphic nature of
virus quasispecies may have hindered the efficiency of
sequence assembly, and a randomly extracted yet less
polymorphic partial data set may provide a better start
for initial assembly as shown in FD vs. PD assemblies. Assembly results of different methods all show that the
assembly of the partial data set not only generated lon-
ger contigs, but also recovered more than 90% of the full
HBV genome, demonstrating that exclusion of low re-
dundant HRURs by random selection of partial data ef-
fectively reduced level of polymorphism which, in turn,
improved the assembly results as judged by contig length
and coverage (Table 2, Additional file 2: Figure S1). We were able to measure the polymorphism level of
BBAP assembly results (Additional file 2: Figure S6) by
calculating the nucleotide frequencies for each position
(Additional file 1: Table S7, Additional file 2: Figure S7
and Additional file 3: SB). Furthermore, the nucleotide
frequencies derived from BBAP PDR assembly were vali-
dated by pyrosequencing (Additional file 1: Table S8),
demonstrating the assembly results of BBAP are reliable. We also noticed that BBAP had better performance in
recovering structural variants than the other methods
tested. While some of BBAP assembled HBV variants
were validated by PCR sequencing (Fig. 2), both Velvet/
MetaVelvet and SOAPdenovo did not identify any contigs
with HBV structural variation. Although Genovo assembled
34 structural containing contigs, their accuracies were BBAP reference assembly with different reference
sequences To fully represent the full data set, the PD assembled
contigs were used as references for the reference assem-
bly of the full data set (PDR). For comparison purposes,
a Sanger sequence from each patient sample was chosen
as the reference sequence for the reference assembly of
the full data set (SR). SR assembly resulted in single con-
tigs with average lengths of 3207 bp, whereas PDR assem-
bly produced an average of 3.9 contigs with maximum and
average lengths of 3148 bp and 1268 bp, respectively
(Table 1 and Additional file 1: Table S3). Both PDR and
SR recovered full HBV genomes and similar levels of genome (Fig. 1a, Additional file 2: Figure S1). PD as-
sembly also yielded a higher proportion of mapped
HRURs (95.9% vs 70.3%) and RiHRURs (reads included
in high redundancy unique representative reads, 80.4%
vs. 68.7%) than FD, further demonstrating its better as-
sembly efficiency. Fragmentation is possibly due to high polymorphic
reads from the same genomic regions recognized by Fig. 1 Assembly results of full and partial D2_1 data set by a BBAP, b Velvet, c SOAPdenovo, and d Genovo. The contigs were aligned to the
Sanger reference sequence. MetaVelvet assembly results for both full and partial D2_1 data set were identical to those of Velvet and thus not shown vet, c SOAPdenovo, and d Genovo. The contigs were aligned to the
rtial D2_1 data set were identical to those of Velvet and thus not shown Fig. 1 Assembly results of full and partial D2_1 data set by a BBAP, b Velvet, c SOAPdenovo, and d Genovo. The contigs were aligned to the
Sanger reference sequence. MetaVelvet assembly results for both full and partial D2_1 data set were identical to those of Velvet and thus not shown Lin et al. BMC Bioinformatics (2017) 18:223 Page 4 of 10 Page 4 of 10 D2_1 data set. Similar to BBAP FD, the full data set as-
semblies by Velvet, MetaVelvet, SOAPdenovo, and Gen-
ovo resulted in fragmented contigs. De novo assembly of
full data set with Velvet resulted in 13 contigs with max-
imum and average lengths of 1102 bp and 303 bp, re-
spectively (Table 2), and recovered only 19% of the HBV
genome (Fig. 1b, Additional file 2: Figure S1). BBAP assembly results compared with other assembly
methods We next compared the efficiency and accuracy of BBAP
to different assembly methods using both full and partial Table 2 Comparison of D2_1 assembly results with different methods and different data set sizes
Max length
Average length
Number of contigs
% of HBV genome
recovered
Contigs that
reference HBV Table 2 Comparison of D2_1 assembly results with different methods and different data set sizes
Max length
Average length
Number of contigs
% of HBV genome
recovered
Contigs that map to
reference HBV genome
Contigs with HBV structural
variants
BBAP/FD
998
263
52
100%
16
30
Velvet/Full
1102
303
13
19%
4
0
MetaVelvet/Full
1102
303
13
19%
4
0
SOAPdenovo/Full
934
340
8
14%
3
0
Genovo/Full
1352
395
60
44%
4
34
BBAP/PD
2924
692
6
100%
3
3
Velvet/Partial
2576
973
3
89%
3
0
MetaVelvet/Partial
2576
973
3
89%
3
0
SOAPdenovo/Partial
1723
390
10
95%
10
0
Genovo/Partial
2427
481
12
91%
4
7 son of D2_1 assembly results with different methods and different data set sizes Lin et al. BMC Bioinformatics (2017) 18:223 Page 5 of 10 Fig. 2 Schematic summary of corresponding HBV genome (NC_003977) regions for assembled contigs identified as HBV variants. Arrows indicate
5’ to 3’ direction. Only reads containing the sequences spanning the junction regions were assembled separately into variant contigs; reads
spanning non-junction regions of the variant contigs (dotted lines) were assembled into the main HBV contig. The L1 sequence, which is similar
to T5, resulted from HBV variant validation with PCR using specialized primers followed by Sanger sequencing. Positions are in correspondence with
NC_003977, with dotted lines representing the remaining portion of the circular HBV genome, and the boxed section indicating the encapsidation
signal (or episilon, ε) Fig. 2 Schematic summary of corresponding HBV genome (NC_003977) regions for assembled contigs identified as HBV variants. Arrows indicate
5’ to 3’ direction. Only reads containing the sequences spanning the junction regions were assembled separately into variant contigs; reads
spanning non-junction regions of the variant contigs (dotted lines) were assembled into the main HBV contig. The L1 sequence, which is similar
to T5, resulted from HBV variant validation with PCR using specialized primers followed by Sanger sequencing. BBAP assembly results compared with other assembly
methods Positions are in correspondence with
NC_003977, with dotted lines representing the remaining portion of the circular HBV genome, and the boxed section indicating the encapsidation
signal (or episilon, ε) questionable as most of them with non-retraceable
junction regions (Additional file 2: Figure S8 and Additional
file 3: SC). with low error rates and/or small data set sizes were as-
sembled with near perfect coverage and accuracy, whereas
both large data sets and high error rates were poorly as-
sembled. As the degree and amount of polymorphism are
proportional to the error rate and data set size, respect-
ively, results suggest Velvet is inefficient in assembling
highly polymorphic data sets. Unlike the assembly results
for D2_1 data sets, MetaVelvet in silico data set assembly
results, compared to Velvet results, were improved with
higher coverage and less fragmentation (Additional file 1:
Table S11). MetaVelvet has wider parameter handling
range than Velvet, but was still unable to assemble highly
polymorphic data sets with high error rates and large data
set sizes. Similar to that of Velvet and MetaVelvet, SOAP-
denovo could not efficiently assemble data sets of high
polymorphism (large data set size and high error rate). In
addition, SOAPdenovo also performed poorly when as-
sembling data sets of low polymorphism (low error rate
and small data set size). Only data sets of medium sizes
and error rates were efficiently assembled by SOAPdenovo
(Additional file 1: Table S12). Genovo assembly of smaller
data set sizes (55X, 557X, and 5,579X), regardless of the
error rate, were highly consistent, with only a single nu-
cleotide assembly error among all 45 assembly results
(Additional file 1: Table S13). The assembly result for the
largest data sets (55,799X) were slightly fragmentized
across all error rates and on average 4 assembly errors
were identified among high error rate (0.01) data sets. Results of in silico data set assembly For a more general assessment and comparison of BBAP
performance, in silico NGS data sets were generated
from the NCBI HBV complete genome and assembled
separately using BBAP FD, Velvet, MetaVelvet, SOAPde-
novo, and Genovo. Data set sizes were set to 1,726,462
(55,799X), 172,646 (5,579X), 17,264 (557X), and 1726
(55X) HQRs in combination with error rates of 10−2, 10−3,
and 10−4/site. Due to computing time considerations, the
maximum simulated data set size of 55,799X was approxi-
mately 10% of the D2_1 data set size. Five independent
data sets were generated for each parameter combination. BBAP assembly results were highly consistent regardless
of the data set parameter values. All but one of the 60 as-
sembly results had both perfect coverage and accuracy;
the lone standout assembly result had perfect coverage
but a 0.9996 (3214/3215) accuracy (Table 3 and Additional
file 1: Table S9). The single “inaccurate” nucleotide was
not an assembly error, but rather a degenerate nucleotide
(Y) representing the reference nucleotide (T, 2/3 or 0.67)
and the in silico generated erroneous nucleotide (C, 1/3 or
0.33). The corresponding in silico data set was generated
with the highest error rate (0.01) and smallest data set size
(55X), which is the most likely parameter value combin-
ation for erroneous nucleotides to exceed the minimum
nucleotide frequency threshold (0.2). Discussion In turn, the following ref-
erence assembly not only assembles more reads due to
the accurate representation of the reference sequences,
but also has increased assembly accuracy than both
straight-up de novo and reference assemblies (Table 1). More importantly, the improved quality of assembly
resulting from this hybrid PDR approach was not lim-
ited to BBAP, as better assembly results using partial
data sets were also demonstrated by Velvet, MetaVelvet,
SOAPdenovo, and Genovo (Table 2). We also introduce a new partial de novo-reference
(PDR) assembly strategy, which in situ generates refer-
ence sequences by de novo assembly of a randomly ex-
tracted partial data set to be subsequently used for the
reference assembly of the full data set. Current assembly
approaches typically assemble the full data set straight-
forward with either de novo or reference assembly
methods, each with their respective advantages and dis-
advantages. Reference assembly is a much more direct
process than de novo assembly which reduces alignment
ambiguities and low coverage issues. However, the qual-
ity of reference assembly is reliant on the representation
level of the reference sequence, as the assembly result will
be biased towards the reference sequence and sequence
variations not represented by the reference sequence will
not be captured. De novo assembly, which is independent
of reference sequences, possesses the potential to generate
a more complete assembly result including majority con-
sensus sequences and minor variant sequences, but can be
hindered by coverage gaps that lack sequencing informa-
tion and polymorphic regions with high levels of diversity
as shown in Tables 1 and 2. The assembly efficiency of metagenomics data sets is
also dependent on the algorithms each assembly method
employs. Velvet, MetaVelvet, and SOAPdenovo all as-
semble NGS data sets through the construction of de
Bruijn graphs and Eulerian paths. De Bruijn graphs con-
tain overlapping sequence information represented by
branching nodes and stemming vertices, and is extremely
sensitive and results quickly deteriorate even with the
slightest amount of polymorphism [21]. The assembly al-
gorithm of Velvet and SOAPdenovo both manipulate the
constructed de Bruijn graph with error removal and sim-
plification to generate optimal assembly results, which ef-
fectively excludes the essential polymorphism information
vital to metagenomics data sets during assembly. In con-
trast, MetaVelvet decomposes the de Bruijn graphs into
individual subgraphs and assembles each subgraphs into
separate contigs. Discussion Velvet assembly of the in silico data sets produced
mixed results (Additional file 1: Table S10). Data sets We developed BBAP, an assembly pipeline designed for
the accurate and efficient assembly of highly polymorphic Lin et al. BMC Bioinformatics (2017) 18:223 Page 6 of 10 Table 3 Assembled results of in silico generated data sets from the reference HBV genome by different methodsa
Data
set size
Method
BBAP FD
Velvet
SOAPdenovo
Genovo
Error rate
10−4
10−3
10−2
10−4
10−3
10−2
10−4
10−3
10−2
10−4
10−3
10−2
55X
Coverage
1
1
1
1
1
1
0
0
1
1
1
1
Accuracy
1
1
0.99
1
1
1
0
0
1
1
1
1
# of contigs
1
1
2
1
1
1
0
0
1
1
1
1
557X
Coverage
1
1
1
1
0.99
1
0
1
0.27
1
1
1
Accuracy
1
1
1
1
1
0.99
0
0.99
0.99
1
1
1
# of contigs
1
1
2
1
1
9
0
1
5
1
1
1
5,579X
Coverage
1
1
1
0.99
0.96
0.03
1
0.01
0.43
1
1
1
Accuracy
1
1
1
1
1
0.59
1
0.20
0.99
1
1
0.99
# of contigs
1
1
1
1
6
1
1
0
11
1
1
1
55,799X
Coverage
1
1
1
0.98
0
0.11
0.02
0
0.04
1
1
1
Accuracy
1
1
1
1
0
0.97
0.40
0
0.80
1
1
0.99
# of contigs
1
1
1
3
0
2
0
0
1
3
2
5
aResults represent averages of the assembly results of 5 replicate data sets. Bold areas indicate average assembly results with <80% coverage metagenomic NGS data sets. BBAP implements a unique
BLAST-based greedy algorithm to assemble data set reads
and provides multiple intuitive parameters, depending on
the nature of the data set, the sequencing platform, and
information demands, to adjust the threshold for read
alignment, variant retention, and error removal during as-
sembly. BBAP assembly results of both real and simulated
NGS data sets were of higher quality than assembly results
of other methods compared. results that are more complete and highly representa-
tive of both majority sequence as well as minor variant
sequences in the full data set. BLAST-based assembly pipeline y p p
The BLAST-based assembly pipeline, BBAP, is divided
into four major steps: quality control (QC), blast and
cluster (BC), alignment and consensus determination (AC),
and contig assembly (CA) (Fig. 3a). BBAP assembles high
quality sequences into contigs according to BLAST results. Alignment files of the assembled contigs are generated
as a result. The contigs are further assembled into
extended-contigs and resulting in contig sequences, a
log file, and a statistical analysis of the assembly. All
steps, with the sole exception of BLAST, used in-house
developed perl scripts. The QC step excludes sequences with low quality
scores, trims sequences from both ends, removes redun-
dant identical sequences, and filters unique representa-
tive sequences with low redundancy. First, raw reads
(RRs) that include any called base with a quality score
less than the given threshold is omitted. The remaining
high quality reads (HQRs) are trimmed from both ends
for the given length to remove barcodes, artificial sequences
such as linker, adapters or vectors, and error-prone regions
that are more frequently found in the terminal regions for
some sequencing platforms. Identical HQRs are com-
pressed and represented by a single unique representative
read (UR) while retaining the redundancy count informa-
tion. Unique representative reads with redundancy counts
greater than or equal to the given threshold, high redun-
dancy unique representative reads (HRURs), are retained
for further assembly. Viral pathogens are responsible for the majority of
pandemic and epidemic diseases listed by the World
Health Organization. Recent studies have utilized the ad-
vantages of NGS data sets of the viral quasispecies genome
to construct genome-wide diversity profiles for studying the
virus-host interactions during infection and, treatment and
vaccination [8, 10, 11, 15, 17]. Resistance associated variants
and novel variants of the viral quasispecies usually are
rare and not detectable by conventional or low depth
sequencing, therefore detection of minor variants is
clinically important for customizing patient manage-
ment and treatment strategies [10, 16]. Our results
show that BBAP and PDR not only provided an accurate
assembly sequence but also generates a high resolution di-
versity profile of the data set. Additionally, we were able to
detect and recover novel variants that were otherwise un-
detectable to alternative assembly methods. y
For de novo assembly, the BLAST and cluster step
(BC) is initiated with the reciprocal BLAST of the
HRURs fasta file. Discussion On the other hand, BBAP adopts a
greedy assembly approach by incorporating and clustering
sequence reads through BLAST results, and Genovo im-
plements a Bayesian-based probabilistic model and takes
into account the potential presence of multiple genomes
in the data set. Therefore, it was reasonably expected for
BBAP, MetaVelvet, and Genovo to have better assembly The partial de novo-reference assembly strategy utilizes
the advantages of both traditional approaches to contem-
plate each other. De novo assembly of a randomly extracted
yet less polymorphic partial data set provides assembly Lin et al. BMC Bioinformatics (2017) 18:223 Page 7 of 10 results than Velvet and SOAPdenovo when assembling
metagenomics data sets, and this was consistent with our
results that support BBAP, MetaVelvet, and Genovo are
better equipped to assemble metagenomics data sets than
Velvet or SOAPdenovo. results than Velvet and SOAPdenovo when assembling
metagenomics data sets, and this was consistent with our
results that support BBAP, MetaVelvet, and Genovo are
better equipped to assemble metagenomics data sets than
Velvet or SOAPdenovo. de novo-reference assembly strategy will provide a power-
ful tool for future metagenomic and viral quasispecies
studies. BLAST-based assembly pipeline The BLAST parameter of repeat mask-
ing was set to include repetitive regions into the results
(-F “”). BLAST results with gaps or e-value, identity, or
BLAST length not meeting the given thresholds were
excluded from further assembly. During clustering, if
two reads are BLASTed to one another and are both un-
assigned, then they are assigned to a same new cluster. If only one read has been assigned a cluster, then the un-
assigned read is added to the cluster of the assigned
read. If both have been separately assigned to different
clusters, then the two clusters are merged into one sin-
gle cluster. Finally, clusters with number of assigned
reads less than the given threshold sequence number are
excluded from further assembly. Methods We compared the average assembly times for in silico
and NGS data sets on our server (E5310 1.6GHz x4 x2,
12GB RAM) between all methods to further assess the
performance of both BBAP and PDR. For smaller in
silico data sets (data set size ≦5,579X or 17.44 Mb)
BBAP assembly time was slightly longer than Velvet,
MetaVelvet, and SOAPdenovo, but still within a couple
minutes (Additional file 1: Table S14). BBAP assembly
time for the largest in silico data sets tested (data set
size = 55,799X or 174 Mb) were similar to the assembly
time by the other methods except Genovo, which re-
quired considerably much more assembly time than
BBAP or the other methods for all in silico data sets. The average BBAP PDR assembly time (624 s) for the 12
NGS data sets was drastically faster than the average
BBAP FD assembly time (14,347 s). Overall, results sug-
gest not only do both BBAP and PDR individually increase
assembly efficiency and accuracy compared to their re-
spective counterparts, but the combination of BBAP and
PDR together further improves the overall assembly qual-
ity of metagenomic data sets. Conclusions Assembly of a highly polymorphic NGS data set is a
complicated process as it involves multiple steps (such
as quality control, read assembly and error removal) and
is dependent of several prerequisite factors (data set type,
sequencing platform, intended use of results, etc.). In
addition, a functional understanding of the algorithms and
sufficient parameters are important for the optimization
of assembly results. We believe both BBAP and the partial The BC step of reference assembly is similar to that of de
novo assembly but with some minor differences. Instead of
reciprocal BLAST, the HRURs fasta file is BLASTed to the
reference sequences. If a read has identical e-values for Lin et al. BMC Bioinformatics (2017) 18:223 Page 8 of 10 Fig. 3 Flow chart summary of BBAP. a The pipeline is divided into four major steps: quality control (QC), blast and cluster (BC), alignment and
consensus determination (AC), and contig assembly (CA). b BBAP can perform de novo assembly, reference assembly, or partial de novo-reference
assembly, which includes the initial de novo assembly of a randomly extracted partial data set followed by the reference assembly of the full data
set using the assembly results from the initial assembly as reference sequences e is divided into four major steps: quality control (QC) blast and cluster (BC) alignment and Fig. 3 Flow chart summary of BBAP. a The pipeline is divided into four major steps: quality control (QC), blast and cluster (BC), alignment and
consensus determination (AC), and contig assembly (CA). b BBAP can perform de novo assembly, reference assembly, or partial de novo-reference
assembly, which includes the initial de novo assembly of a randomly extracted partial data set followed by the reference assembly of the full data
set using the assembly results from the initial assembly as reference sequences multiple reference sequences, the read will be assigned to
the reference sequence with the longest sequence length. BBAP can assemble data sets with or without a refer-
ence sequence by reference assembly or de novo assem-
bly, respectively. We also introduce a third assembly
strategy, the partial de novo-reference assembly approach
(Fig. 3b). A randomly extracted partial data set is first de
novo assembled, and then the resulting contig sequences
are used as reference sequences to assemble the entire
data set through reference assembly. Conclusions The alignment and consensus determination step (AC)
calculates the alignment position for each read of a clus-
ter based on its BLAST results. Only top BLAST results
with identity and BLAST length greater than the given
thresholds were used for alignment. Consensus se-
quences were calculated for each base according to the
alignment results. Nucleotides with frequencies greater
than or equal to the given threshold are retained for
polymorphic sites. Next generation sequencing data set assembly and
statistical analyses NGS data sets were downloaded from a previous study
[24], which consisted of 12 libraries derived from 7 pa-
tients chronically infected with HBV within a single fam-
ily (Additional file 1: Table S15). The full data set was
separately assembled with BBAP through full data set de
novo (FD) assembly, Sanger reference (SR) assembly, and
partial de novo-reference (PDR) assembly. A single Sanger
sequence from each patient sample was chosen and used as
the reference sequence for the SR assembly of the corre-
sponding full data set. For the PDR assembly, partial data
sets were constructed independently by randomly choosing
1% of the RRs from the full data set and assembled de novo,
and the results of the partial data set de novo (PD) assembly
were used as reference sequences for the reference assem-
bly of the full data set. Partial data sets of different ratios
were assembled and 1% partial data sets generated the most
optimal assembly results (Additional file 1: Table S16 and
Additional file 3: SD). Assembly results of different BBAP
methods were then compared to each other. Contigs with identical terminal sequences longer than
the given threshold are merged together into extended-
contigs. Identical terminal sequences were identified
by self-BLAST of contigs. This step is optional and
dependent on the nature of the data set. Overall, BBAP uses BLAST results (reciprocal BLAST
for de novo assembly, and data set to reference sequence
BLAST for reference assembly) to cluster reads into contig
groups to increase computation efficiency of following
steps. The reads in each contig group are then positioned/
aligned according to their respective BLAST results into
contigs. The grouped reads are then extended into contigs
according to positioning/alignment information provided
from the BLAST results in a greedy strategy manner. Ex-
tension of contigs and prevention of assembly artifacts
(such as artificial chimeras) are directly dictated by the
BLAST identify and length threshold parameters, and in-
directly effected by quality control parameters, including
the QC-score threshold and the redundancy threshold. Lin et al. BMC Bioinformatics (2017) 18:223 Page 9 of 10 Variant contigs were identified by BLAST against the
NCBI HBV complete genome sequence (NC_003977),
the Sanger reference sequence, and the NCBI nr/nt data-
base. Funding g
This work was supported by the Ministry of Science and Technology
[103-2621-B-002 -003 and 104-2621-B-002 -006] and National Taiwan
University [101R7836, 102R7836, and 103R7836]. Both funding bodies listed
above (MOST and NTU) did not play any role in the study or conclusions of this
study. Availability of data and materials The datasets generated during and/or analysed during the current study are
available in the NCBI SRA repository (accession number SRP051091). BBAP is
implemented in perl and is publicly available at http://homepage.ntu.edu.tw/
~youylin/BBAP.html. In silico data set assembly y
We also compared the performance of different assem-
bly methods by using simulated data sets. In silico data
sets were generated by randomly generating 101 bp
reads from the reference NCBI HBV complete genome,
NC_003977. To mimic observed polymorphism from
virus diversity or sequencing error of NGS, different
error rates, 10−2, 10−3, and 10−4/site, were applied to the
simulated reads. Data set sizes were set to 1,726,462
(55,799X), 172,646 (5,579X), 17,264 (557X), and 1726
(55X) HQRs. Five independent data sets were generated
for each parameter combination, error rate and dataset
size. Data sets were assembled using BBAP FD assembly,
Velvet, MetaVelvet, SOAPdenovo, and Genovo. All in
silico data sets, except for data sets of high error rate
(0.01) coupled with small data set sizes (55X and 557X),
used the same BBAP parameter values for NGS de novo
assembly. For the high error rate-low coverage depth
data sets, the redundancy threshold was reduced from 5
to 1 to compensate for its low redundancy. For Velvet,
MetaVelvet, and SOAPdenovo assembly, the k-mer size
was optimally set to 57, 57, and 63, respectively. For
Genovo assembly, different numbers of iterations were
used for data sets of different coverage depths because
of the extreme long run time for larger data sets; the
number of iterations for data sets with coverage depths
of 55,799X, 5,579X, 557X and 55X was 10, 2000, 10,000,
and 10,000, respectively. Additional file 3: A. Variant sequences and human genome sequences. B. Diversity profile of D2_1 HBV quasispecies. C. Structure variation by
Genovo. D. Determining optimal size of partial data set. (DOC 67 kb) Next generation sequencing data set assembly and
statistical analyses Table S13: Results of
Genovo assembled in silico NCBI HBV complete genome (NC_003977) data
sets (n = 5). Table S14: Assembly time required for in silico data sets by BBAP, Velvet, MetaVelvet, SOAPdenovo, and Genovo. Table S15: Summary
of study subjects and samples. Table S16: Summary of assembly results for
D2_1 partial data sets of different size ratio. (DOC 461 kb) The full data set and partial data sets of one library,
D2_1 (Additional file 1: Table S15), were also assembled
using all methods. Statistical analyses and comparisons
between assembly methods were performed with perl
scripts. Additional file 2: Figure S1. Comparison of HBV recover ratio by BBAP,
Velvet, SOAPdenovo, and Genovo assembly of full and partial D2_1 data
sets. Figure S2: Correlation between assembled scaffold length and
scaffold degeneracy for all 12 data sets. Figure S3: (a) Nucleotide
sequence of the R1 scaffold. (b) Schematic alignment of the R1 scaffold,
HBV X gene and HBV precore/core gene. Figure S4: Schematic diagram
of the T1 scaffold and its corresponding HBV genome regions. Figure S5:
Schematic diagram of the T6 scaffold and its corresponding HBV genome
and Sanger reference sequence regions. Figure S6: Alignment of Sanger
(SR) and partial D2_1 data set assembled scaffolds reference assembled
(PDR) scaffolds to the Sanger reference sequence. Figure S7: Diversity
profile of D2_1 HBV quasispecies according to assembly results of partial
data set reference assembly of the full data set. Figure S8: Schematic
diagram of two Genovo assembled scaffolds with identified HBV structural
variants and its corresponding HBV genome regions. (PDF 489 kb) Authors’ contributions Conception and design of the study: JHC, JHK, PJC, DSC, HYW. Performed
experiments: YYL, XL. Data analysis: YYL, HYW. Manuscript preparation: YYL,
CHH, JHC, HYW. All authors read and approved the final manuscript. Additional files Additional file 1: Table S1. Statistics of next generation sequencing
data set of HBV genome from patient serum. Table S2: Parameters used
for de novo, reference, and partial de novo reference BBAP assembly. Table S3: Assembly results of individual data sets using BBAP with
multiple approaches. Table S4: Comparison of polymorphism between
non-overlapping and overlapping regions of D2_1 assembled contigs
alignment. Table S5: Comparison of polymorphism levels between
assembly results of BBAP PDR and SR assemblies. Table S6: Summary
of assembled contigs from the PDR assembly of D2_1 NGS data set. Table S7: Top ten non-synonymous frequency positions of the HBV
quasispecies. Table S8: Nucleotide frequencies derived from BBAP Acknowledgement We would like to acknowledge Chia-Hua Chen for discussions and suggestions
during this study. We would like to acknowledge Chia-Hua Chen for discussions and suggestions
during this study. Abbreviations
AC Ali AC: Alignment and consensus determination; BBAP: BLAST-based assembly
pipeline; BC: Blast and cluster; CA: Contig assembly; CLC: CLC Genomics
Workbench; FD: De novo assembly of the full data set; HQRs: High quality
reads; HRURs: High redundancy unique representative reads; NGS: Next
generation sequencing; PD: De novo assembly of the partial data set;
PDR: Partial de novo-reference assembly; QC: Quality control; RiHRURs: Reads
included in HRURs; RRs: Raw reads; SR: Reference assembly of the full data
set with Sanger generated reference sequences; URs: Unique representative
reads Next generation sequencing data set assembly and
statistical analyses To verify that the identified variants were not arti-
facts of incorrect assembly by BBAP, sequences of at
least 20 bp and spanning the junction regions of the
structural variations were searched for in both the RRs
and HQRs fasta files. PDR assembly and pyrosequencing. Table S9: Results of BBAP de novo
assembled in silico NCBI HBV complete genome (NC_003977) data sets
(n = 5). Table S10: Results of Velvet assembled in silico NCBI HBV
complete genome (NC_003977) data sets (n = 5). Table S11: Results of
MetaVelvet assembled in silico NCBI HBV complete genome (NC_003977)
data sets (n = 5). Table S12: Results of SOAPdenovo assembled in silico NCBI
HBV complete genome (NC_003977) data sets (n = 5). Table S13: Results of
Genovo assembled in silico NCBI HBV complete genome (NC_003977) data
sets (n = 5). Table S14: Assembly time required for in silico data sets by
BBAP, Velvet, MetaVelvet, SOAPdenovo, and Genovo. Table S15: Summary
of study subjects and samples. Table S16: Summary of assembly results for
D2_1 partial data sets of different size ratio. (DOC 461 kb) PDR assembly and pyrosequencing. Table S9: Results of BBAP de novo
assembled in silico NCBI HBV complete genome (NC_003977) data sets
(n = 5). Table S10: Results of Velvet assembled in silico NCBI HBV
complete genome (NC_003977) data sets (n = 5). Table S11: Results of
MetaVelvet assembled in silico NCBI HBV complete genome (NC_003977)
data sets (n = 5). Table S12: Results of SOAPdenovo assembled in silico NCBI
HBV complete genome (NC_003977) data sets (n = 5). Table S13: Results of
Genovo assembled in silico NCBI HBV complete genome (NC_003977) data
sets (n = 5). Table S14: Assembly time required for in silico data sets by
BBAP, Velvet, MetaVelvet, SOAPdenovo, and Genovo. Table S15: Summary
of study subjects and samples. Table S16: Summary of assembly results for
D2_1 partial data sets of different size ratio. (DOC 461 kb) (n
5). Table S10: Results of Velvet assembled in silico NCBI HBV
complete genome (NC_003977) data sets (n = 5). Table S11: Results of
MetaVelvet assembled in silico NCBI HBV complete genome (NC_003977)
data sets (n = 5). Table S12: Results of SOAPdenovo assembled in silico NCBI HBV complete genome (NC_003977) data sets (n = 5). Table S13: Results of
Genovo assembled in silico NCBI HBV complete genome (NC_003977) data HBV complete genome (NC_003977) data sets (n = 5). Competing interests g
The authors declare that they have no competing interests. g
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1 1Department of Life Science, National Taiwan University, Taipei 106, Taiwan. 2Department of Forestry and Nature Conservation, Chinese Culture
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Individualized Medicine, Beijing Institute of Genomics, the Chinese Academy
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National Taiwan University, Taipei 100, Taiwan. 5Genomics Research Center,
Academia Sinica, Taipei 115, Taiwan. 6Institute of Ecology and Evolutionary
Biology, National Taiwan University, Taipei 106, Taiwan. 7Research Center for
Developmental Biology and Regenerative Medicine, National Taiwan
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Precision measurement of the B<sup>0</sup><sub>s</sub>–$\overline{\rm B}{}^0_{\rm s}$ oscillation frequency with the decay B<sup>0</sup><sub>s</sub>→ D<sup>−</sup><sub>s</sub><i>π</i><sup>+</sup>
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Aaij, R. et al. (2013) Precision measurement of the B0s–B0s oscillation
frequency with the decay B0s → D−s π. New Journal of Physics, 15 . Art. 053021. ISSN 1367-2630 Aaij, R. et al. (2013) Precision measurement of the B0s–B0s oscillation
frequency with the decay B0s → D−s π. New Journal of Physics, 15 . Art. 053021. ISSN 1367-2630 Copyright © 2013 CERN, for the benefit of the LHCb collaboration Precision measurement of the B0
s–B0
s oscillation
frequency with the decay B0
s →D−
s π + The LHCb Collaboration
New Journal of Physics 15 (2013) 053021 (15pp)
Received 18 April 2013
Published 14 May 2013
Online at http://www.njp.org/
doi:10.1088/1367-2630/15/5/053021
E-mail: wandernoth@physi.uni-heidelberg.de Abstract. A key ingredient to searches for physics beyond the Standard Model
in B0
s mixing phenomena is the measurement of the B0
s– B
0
s oscillation frequency,
which is equivalent to the mass difference 1ms of the B0
s mass eigenstates. Using
the world’s largest B0
s meson sample accumulated in a dataset, corresponding
to an integrated luminosity of 1.0 fb−1, collected by the LHCb experiment at
the CERN LHC in 2011, a measurement of 1ms is presented. A total of about
34 000 B0
s →D−
s π + signal decays are reconstructed, with an average decay time
resolution of 44 fs. The oscillation frequency is measured to be 1ms = 17.768 ±
0.023 (stat) ± 0.006 (syst) ps−1, which is the most precise measurement to date. New Journal of Physics 15 (2013) 053021
1367-2630/13/053021+15$33.00 http://eprints.gla.ac.uk/82340/ Deposited on: 19 July 2013 Enlighten – Research publications by members of the University of Glasgow
http://eprints.gla.ac.uk Contents 1. Introduction
2
2. The LHCb experiment
3
3. Signal selection and analysis strategy
4
4. Invariant mass description
5
5. Decay time description
6
6. Flavour tagging
7
7. Measurement of 1ms
8
8. Systematic uncertainties
9
9. Conclusion
10
Acknowledgments
10
The LHCb Collaboration
10
References
14 1. Introduction
2. The LHCb experiment
3. Signal selection and analysis strategy
4. Invariant mass description
5. Decay time description
6. Flavour tagging
7. Measurement of 1ms
8. Systematic uncertainties
9. Conclusion
Acknowledgments
The LHCb Collaboration
References 1. Introduction
2. The LHCb experiment
3. Signal selection and analysis strategy
4. Invariant mass description
5. Decay time description
6. Flavour tagging
7. Measurement of 1ms
8. Systematic uncertainties
9. Conclusion
Acknowledgments
The LHCb Collaboration
References New Journal of Physics 15 (2013) 053021
1367-2630/13/053021+15$33.00 © CERN 2013 for the benefit of the LHCb Collaboration, published under the terms of the Creative Commons Attribution 3.0
licence by IOP Publishing Ltd and Deutsche Physikalische Gesellschaft. Any further distribution of this work must maintain
attribution to the author(s) and the published article’s title, journal citation and DOI. 2 1. Introduction The Standard Model (SM) of particle physics, despite its great success in describing
experimental data, is considered an effective theory valid only at low energies, below the TeV
scale. At higher energies, new physics phenomena are predicted to emerge. For analyses looking
for physics beyond the SM (BSM), there are two conceptually different approaches: direct and
indirect searches. Direct searches are performed at the highest available energies and aim at
producing and detecting new heavy particles. Indirect searches focus on precision measurements
of quantum-loop-induced processes. Accurate theoretical predictions are available for the heavy
quark sector in the SM. It is therefore an excellent place to search for new phenomena [1, 2],
since any deviation from these predictions can be attributed to contributions from BSM. In the SM, transitions between quark families (flavours) are possible via the charged current
weak interaction. Flavour changing neutral currents (FCNC) are forbidden at lowest order,
but are allowed in higher order processes. Since new particles can contribute to these loop
diagrams, such processes are highly sensitive to contributions from BSM. An example FCNC
transition is neutral meson mixing, where neutral mesons can transform into their antiparticles. Particle–antiparticle oscillations have been observed in the K0–K
0 system [3], the B0–B
0 system
[4], the B0
s–B
0
s system [5, 6] and the D0–D
0 system [7–10]. The frequency of B0
s– B
0
s oscillations
is the highest. On average, a B0
s meson changes its flavour nine times between production and
decay. This poses a challenge to the detector for the measurement of the decay time. Another key
ingredient of this measurement is the determination of the flavour of the B0
s meson at production,
which relies heavily on good particle identification and the separation of tracks from the primary
interaction point. The observed particle and antiparticle states B0
s and B
0
s are linear combinations of the mass
eigenstates BH and BL with masses mH and mL and decay widths 0H and 0L, respectively [11]. The B0
s oscillation frequency is equivalent to the mass difference 1ms = mH −mL. The
parameter 1ms is an essential ingredient for all studies of time-dependent matter–antimatter New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 3 asymmetries involving B0
s mesons, such as the B0
s mixing phase φs in the decay B0
s →
J/ψφ [12]. 2. The LHCb experiment The LHCb experiment is designed for precision measurements in the beauty and charm hadron
systems. At a centre-of-mass energy of √s = 7 TeV, about 3 × 1011 bb pairs were produced in
2011. The LHCb detector [14] is a single-arm forward spectrometer covering the pseudorapidity
range from two to five. The excellent decay time resolution necessary to resolve the fast B0
s– B
0
s
oscillation is provided by a silicon-strip vertex detector surrounding the pp interaction region. At nominal position, the sensitive region of the vertex detector is only 8 mm away from the
beam. An impact parameter (IP) resolution of 20 µm for tracks with high transverse momentum
(pT) is achieved. Charged particle momenta are measured with the LHCb tracking system consisting of the
aforementioned vertex detector, a large-area silicon-strip detector located upstream of a dipole
magnet with a bending power of about 4 T m, and three stations of silicon-strip detectors and
straw drift tubes placed downstream. The combined tracking system has momentum resolution
1p/p that varies from 0.4% at 5 GeV/c to 0.6% at 100 GeV/c. Since this analysis is performed with decays involving only hadrons in the final state,
excellent particle identification is crucial to suppress background. Charged hadrons are
identified using two ring-imaging Cherenkov detectors [15]. Photon, electron and hadron
candidates are identified by a calorimeter system consisting of scintillating-pad and preshower
detectors, an electromagnetic calorimeter and a hadronic calorimeter. Muons are identified by a
system composed of alternating layers of iron and multiwire proportional chambers. The first stage of the trigger [16] is implemented in hardware, based on information
from the calorimeter and muon systems, and selects events that contain candidates with large
transverse energy and transverse momentum. This is followed by a software stage that applies
a full event reconstruction. The software trigger used in this analysis requires a two-, three-
or four-track secondary vertex with a significant displacement from the primary interaction, a
large sum of pT of the tracks, and at least one track with pT > 1.7 GeV/c. In addition, an IP χ2
with respect to the primary interaction greater than 16 and a track fit χ 2 per degree of freedom
<2 is required. The IP χ2 is defined as the difference between the χ2 of the primary vertex
reconstructed with and without the considered track. 1. Introduction It was first observed by the Collider Detector at Fermilab (CDF) [6]. The Large
Hadron Collider beauty experiment (LHCb) published a measurement of this frequency using
a dataset, corresponding to an integrated luminosity of 37 pb−1, taken in 2010 [13]. This
analysis complements the previous result and is obtained in a similar way, using a data sample,
corresponding to an integrated luminosity of 1.0 fb−1, collected by LHCb in 2011. 3. Signal selection and analysis strategy The analysis uses B0
s candidates reconstructed in the flavour-specific decay mode1 B0
s →D−
s π +
in five D−
s decay modes, namely D−
s →φ(K+K−)π −, D−
s →K∗0(K+π −)K−, D−
s →K+K−π−
nonresonant, D−
s →K−π+π−and D−
s →π −π +π −. To avoid double counting, events that
contain a candidate passing the selection criteria of one mode are not considered for the
subsequent modes, using the order listed above. All reconstructed decays are flavour-specific
final states; thus the flavour of the B0
s candidate at the time of its decay is given by the charges of
the final state particles. A combination of tagging algorithms is used to identify the B0
s flavour at
production. The algorithms provide for each candidate a tagging decision as well as an estimate
of the probability that this decision is wrong (mistag probability). These algorithms have been
optimized using large event samples of flavour-specific decays [24, 25]. To be able to study the effect of selection criteria that influence the decay time spectrum,
we restrict the analysis to those events in which the signal candidate passed the requirements of
the software trigger algorithm used in this analysis. Specific features, such as the masses of the
intermediate φ and K∗0 resonances or the Dalitz structure of the D−
s →π −π +π −decay mode,
are exploited for the five decay modes. The most powerful quantity to separate signal from
background common to all decay modes is the output of a boosted decision tree (BDT) [26]. The BDT exploits the long B0
s lifetime by using as input the IP χ 2 of the daughter tracks, the
angle of the reconstructed B0
s momentum relative to the line between the reconstructed primary
vertex, and the B0
s vertex and the radial flight distance in the transverse plane of both the B0
s and
the D−
s mesons. Additional requirements are applied on the sum of the pT of the B0
s candidate’s
decay products as well as on particle identification variables, and on track and vertex quality. The reconstructed D−
s mass is required to be consistent with the known value [27]. After this
selection, a total of about 47 800 candidates remain in the B0
s →D−
s π + invariant mass window
of 5.32–5.98 GeV/c2. 2. The LHCb experiment A multivariate algorithm is used for the
identification of the secondary vertices. For the simulation, pp collisions are generated using Pythia 6.4 [17] with a specific LHCb
configuration [18]. Decays of hadronic particles are described by EvtGen [19], in which final
state radiation is generated using Photos [20]. The interaction of the generated particles with
the detector and its response are implemented using the Geant4 toolkit [21, 22], as described
in [23]. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 4 1 Unless explicitly stated, inclusion of charge-conjugated modes is implied. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 3. Signal selection and analysis strategy 0 An unbinned likelihood method is employed to simultaneously fit the B0
s invariant mass
and decay time distributions of the five decay modes. The probability density functions (PDFs)
for signal and background in each of the five modes can be written as P = Pm(m) Pt(t, q|σt, η) Pσt(σt) Pη(η),
(1) (1) where m is the reconstructed invariant mass of the B0
s candidate, t is its reconstructed decay
time and σt is an event-by-event estimate of the decay time resolution. The tagging decision q
can be 0 if no tag is found, −1 for events with different flavour at production and decay (mixed)
or +1 for events with the same flavour at production and decay (unmixed). The predicted event-
by-event mistag probability η can take values between 0 and 0.5. The functions Pm and Pt
describe the invariant mass and the decay time probability distributions, respectively. Pt is a
conditional probability depending on σt and η. The functions Pσt and Pη are required to ensure
the proper relative normalization of Pt for signal and background [28]. The functions Pσt and
Pη are determined from data, using the measured distribution in the upper B0
s invariant mass
sideband for the background PDF and the sideband subtracted distribution in the invariant mass
signal region for the signal PDF. This measurement has been performed ‘blinded’, meaning that during the analysis process
the fitted value of 1ms was shifted by an unknown value, which was removed after the analysis
procedure had been finalized. 1 Unless explicitly stated, inclusion of charge-conjugated modes is implied. 1 Unless explicitly stated, inclusion of charge-conjugated modes is implied. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 5 5 ]
2
c
) invariant mass [MeV/
+
π
−
s
(D
5350
5400
5450
5500
5550
)
2
c
candidates / (15 MeV/
0
2000
4000
data
fit
+
π
−
s
D
→
0
s
B
+
K
−
s
D
→
0
s
B
misid bkg. comb bkg. a)
LHCb
−
π
φ
→
−
s
D
]
2
c
) invariant mass [MeV/
+
π
−
s
(D
5350
5400
5450
5500
5550
)
2
c
candidates / (15 MeV/
0
2000
data
fit
+
π
−
s
D
→
0
s
B
+
K
−
s
D
→
0
s
B
misid. bkg. comb. bkg. 3. Signal selection and analysis strategy −
π
+
π
−
K
→
−
s
D
LHCb
d) ]
2
c
) invariant mass [MeV/
+
π
−
s
(D
5350
5400
5450
5500
5550
)
2
c
candidates / (15 MeV/
0
1000
data
fit
+
π
−
s
D
→
0
s
B
+
K
−
s
D
→
0
s
B
comb. bkg. −
π
−
π
+
π
→
−
s
D
LHCb
e) ]
2
c
) invariant mass [MeV/
+
π
−
s
(D
5400
5450
5500
555 Figure 1. Invariant mass distributions for B0
s →D−
s π+ candidates with the D−
s
meson decaying as (a) D−
s →φ(K+K−)π −, (b) D−
s →K∗0(K+π −)K−, (c) D−
s →
K+K−π−nonresonant, (d) D−
s →K−π +π −and (e) D−
s →π −π+π−. The fits
and the various background components are described in the text. Misidentified
backgrounds refer to background from B0 and 30
b decays with one misidentified
daughter particle. 3. Signal selection and analysis strategy −
K*K
→
−
s
D
LHCb
b)
]
2
c
) invariant mass [MeV/
+
π
−
s
(D
5350
5400
5450
5500
5550
)
2
c
candidates / (15 MeV/
0
1000
2000
data
fit
+
π
−
s
D
→
0
s
B
+
K
−
s
D
→
0
s
B
misid. bkg. comb. bkg. −
π
−
K
+
K
→
−
s
D
LHCb
c)
]
2
c
) invariant mass [MeV/
+
π
−
s
(D
5350
5400
5450
5500
5550
)
2
c
candidates / (15 MeV/
0
500
data
fit
+
π
−
s
D
→
0
s
B
+
K
−
s
D
→
0
s
B
misid. bkg. comb. bkg. −
π
+
π
−
K
→
−
s
D
LHCb
d)
]
2
c
) invariant mass [MeV/
+
π
−
s
(D
5350
5400
5450
5500
5550
)
2
c
candidates / (15 MeV/
0
1000
data
fit
+
π
−
s
D
→
0
s
B
+
K
−
s
D
→
0
s
B
comb. bkg. −
π
−
π
+
π
→
−
s
D
LHCb
e)
Figure 1. Invariant mass distributions for B0
s →D−
s π+ candidates with the D−
s
meson decaying as (a) D−
s →φ(K+K−)π −, (b) D−
s →K∗0(K+π −)K−, (c) D−
s →
K+K−π−nonresonant, (d) D−
s →K−π +π −and (e) D−
s →π −π+π−. The fits
and the various background components are described in the text. Misidentified
backgrounds refer to background from B0 and 30
b decays with one misidentified
daughter particle. ]
2
c
) invariant mass [MeV/
+
π
−
s
(D
5350
5400
5450
5500
5550
)
2
c
candidates / (15 MeV/
0
1000
2000
data
fit
+
π
−
s
D
→
0
s
B
+
K
−
s
D
→
0
s
B
misid. bkg. comb. bkg. −
π
−
K
+
K
→
−
s
D
LHCb
c)
]
2
c
) invariant mass [MeV/
+
π
−
s
(D
5350
5400
5450
5500
5550
)
2
c
candidates / (15 MeV/
0
500
data
fit
+
π
−
s
D
→
0
s
B
+
K
−
s
D
→
0
s
B
misid. bkg. comb. bkg. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 4. Invariant mass description The invariant mass of each B0
s candidate is determined in a vertex fit constraining the D−
s
invariant mass to its known value [27]. The invariant mass spectra for the five decay modes
after all the selection criteria are applied are shown in figure 1. The fit to the five distributions
takes into account contributions from signal, combinatorial background and b-hadron decay
backgrounds. The signal components are described by the sum of two Crystal Ball (CB)
functions [29], which are constrained to have the same peak parameter. The parameters of the
CB function describing the tails are fixed to values obtained from simulation, whereas the mean
and the two widths are allowed to vary. These are constrained to be the same for all five decay
modes. It has been checked on data that the mass resolution is compatible among all modes. The b-hadron decay background includes B0 and 30
b decays with one misidentified
daughter particle. Their mass shapes are derived from simulated samples. The yields for the New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 6 6 Table 1. Number of candidates and B0
s signal fractions in the mass range
5.32–5.98 GeV/c2. Table 1. Number of candidates and B0
s signal fractions in the mass
5.32–5.98 GeV/c2. Decay mode
(D−
s π+) candidates
fB0s →D−s π+
fB0s →D∓s K±
D−
s →φ(K+K−)π−
14 691
0.834 ± 0.008
D−
s →K∗0(K+π−)K−
10 866
0.857 ± 0.009
D−
s →K+K−π−nonresonant
11 262
0.595 ± 0.009
D−
s →K−π+π−
4288
0.437 ± 0.014
D−
s →π−π+π−
6674
0.599 ± 0.008
0.019 ± 0.010
Total
47 781
0.714 ± 0.004
0.019 ± 0.010 different b-hadron decay backgrounds are allowed to vary individually for each of the five
decay modes. Another component originates from B0
s →D∓
s K± decays, in which the kaon is
misidentified as a pion. This contribution is treated as a signal in the decay time analysis. different b-hadron decay backgrounds are allowed to vary individually for each of the five
decay modes. Another component originates from B0
s →D∓
s K± decays, in which the kaon is
misidentified as a pion. This contribution is treated as a signal in the decay time analysis. 4. Invariant mass description The requirement that the invariant mass be larger than 5.32 GeV/c2 rejects background
candidates from B0
s decays with additional particles in the decay not reconstructed, such as
B0
s →D∗−
s π+(D∗−
s
→D−
s π 0 or D−
s γ ). The fitted number of signal candidates does not change
with respect to a fit in a larger mass window. The high mass sideband region 5.55–5.98 GeV/c2
provides a sample of mainly combinatorial background candidates. The mass distribution is
described by an exponential function, whose parameters are allowed to vary individually for the
five decay modes. By including this region in the fit, we are able to determine the decay time
distribution as well as the tagging behaviour of the combinatorial background. The number of used candidates along with the signal fractions extracted from the two-
dimensional fit in mass and decay time are reported in table 1. One complication arises from
the fact that the shape of the invariant mass distribution of the B0
s →D∓
s K± events is very
similar to that of the B0 background. Therefore, the fraction of B0
s →D∓
s K± candidates has
been determined in a fit to the D−
s →π −π+π−mode only, in which no B0 background is present. Subsequently this value is used for all the other modes. 5. Decay time description The decay time of a particle is measured as New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) The decay time of a particle is measured as t = Lm
p , (2) where L is the distance between the production vertex and the decay vertex of the particle,
m its reconstructed invariant mass and p its reconstructed momentum. We use the decay time
calculated without the D−
s mass constraint to avoid a systematic dependence of the B0
s decay time
on the reconstructed invariant mass. The theoretical distribution of the decay time, t, ignoring
the oscillation and any detector resolution, is Pt ∝0s e−0st cosh
10s
2 t
θ(t),
(3) Pt ∝0s e−0st cosh
10s
2 t
θ(t), (3) New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 7 where 0s is the B0
s decay width and 10s the decay width difference between the light and
heavy mass eigenstates2. The value for 10s is fixed to the latest value measured by LHCb [12]
10s = 0.106 ± 0.011 ± 0.007 ps−1. It is varied within its uncertainties to assess the systematic
effect on the measurement of 1ms. The Heaviside step function θ(t) restricts the PDF to positive
decay times. To account for detector resolution effects, the decay time PDF is convolved with a Gaussian
distribution. The width σt is taken from an event-by-event estimate returned by the fitting
algorithm that reconstructs the B0
s decay vertex. Due to tracking detector resolution effects, σt
needs to be calibrated. A data-driven method, combining prompt D−
s mesons from the primary
interaction with random π + mesons, forms fake B0
s candidates. The decay time distribution of
these candidates, each divided by its event-by-event σt, is fitted with a Gaussian function. The
width provides a scale factor Sσt = 1.37, by which each σt is multiplied, such that it represents
the correct resolution. By inspecting different regions of phase space of the fake B0
s candidates,
the uncertainty range on this number is found to be 1.25 < Sσt < 1.45. The variation is taken
into account as part of the 1ms systematic studies. The resulting average decay time resolution
is Sσt × ⟨σt⟩= 44 fs. Some of the selection criteria influence the shape of the decay time distribution, e.g. the
requirement of a large IP for B0
s daughter tracks. Thus, a decay time acceptance function Et(t)
has to be taken into account. The decay time of a particle is measured as Its parametrization is determined from simulated data and the
parameter describing its shape is allowed to vary in the fit to the data, while 0s is fixed to the
nominal value [27]. Taking into account resolution and decay time acceptance, the PDF given
in equation (3) is modified to Pt(t|σt) ∝
0s e−0s t cosh
10s
2 t
θ(t)
⊗G(t; 0, Sσtσt)Et(t)
(4) (4) with G(t; 0, Sσtσt) being the resolution function determined by the method mentioned above. The decay time PDFs for the B0 and 30
b backgrounds are identical to the signal PDF, except for
10 being zero, and 0s being replaced by their respective decay widths [27]. The shape of the
decay time distribution of the combinatorial background is determined with high mass sideband
data. It is parametrized by the sum of two exponential functions multiplied by a second-order
polynomial distribution. The exponential and polynomial parameters are allowed to vary in the
fit and are constrained to be the same for the five decay modes. 2 10s and 1ms are measured in units with ¯h = 1 throughout this paper. 6. Flavour tagging To determine the flavour of the B0
s meson at production, both opposite-side (OST) and same-
side (SST) tagging algorithms are used. The OST exploits the fact that b quarks at the LHC
are predominantly produced in quark–antiquark pairs. By partially reconstructing the second
b hadron in the event, conclusions on the flavour at production of the signal B0
s candidate can
be drawn. The OST has been optimized on large samples of B+ →J/ψ K +, B →µ+D∗−X and
B0 →D−π + decays [24]. The SST takes advantage of the fact that the net strangeness of the pp collision is zero. Therefore, the s quark needed for the hadronization of the B0
s meson must have been produced in
association with an s quark, which in about 50% of the cases hadronizes to form a charged kaon. 2 10s and 1ms are measured in units with ¯h = 1 throughout this paper. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 8 By identifying this kaon, the flavour at production of the signal B0
s candidate is determined. The
optimization of the SST was performed on a data sample of B0
s →D−
s π+ decays, which has a
large overlap with the sample used in this analysis [25]. However, since the oscillation frequency
is not correlated with the parameters describing tagging performance, this does not bias the 1ms
measurement. The decisions given by both tagging algorithms have a probability ω to be incorrect. Each
tagging algorithm provides an estimate for the mistag probability η; which is the output of
a neural network combining various event properties. The true mistag probability ω can be
parametrized as a linear function of the estimate η [24, 25]: ω = p0 + p1 × (η −⟨η⟩)
(5) ω = p0 + p1 × (η −⟨η⟩)
(5) (5) ω = p0 + p1 × (η −⟨η⟩) with ⟨η⟩being the mean of the distribution of η. This parametrization is chosen to minimize the
correlations between p0 and p1. The calibration is performed separately for the OST and SST. The sets of calibration parameters (p0, p1)OST and (p0, p1)SST are allowed to vary in the fit. The figure of merit of these tagging algorithms is called the effective tagging efficiency εeff. It
gives the factor by which the statistical power of the sample is reduced due to imperfect tagging
decisions. 6. Flavour tagging In this analysis, εeff is found to be (2.6 ± 0.4)% for the OST and (1.2 ± 0.3)% for the
SST. Uncertainties are statistical only. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 7. Measurement of 1ms Adding the information of the flavour tagging algorithms, the decay time PDF for tagged signal
candidates is modified to Adding the information of the flavour tagging algorithms, the decay time PDF for tagged signal
candidates is modified to Pt(t|σt) ∝
0s e−0s t 1
2
cosh
10s
2 t
+ q [1 −2ω(ηOST, ηSST)] cos(1mst)
θ(t)
⊗G(t, Sσtσt) Et(t) ϵ,
( (6) ⊗G(t, Sσtσt) Et(t) ϵ,
(6) ⊗G(t, Sσtσt) Et(t) ϵ, where ϵ gives the fraction of candidates with a tagging decision. Signal candidates without a
tagging decision are still described by equation (4) multiplied by an additional factor (1 −ϵ) to
ensure relative normalization. The information provided by the opposite-side and same-side taggers for the signal is
combined to a single tagging decision q and a single mistag probability ω(ηOST, ηSST) using their
respective calibration parameters p0OST/SST and p1OST/SST. The individual background components
show different tagging characteristics for candidates tagged by the OST or SST. The b hadron
backgrounds show the same opposite-side tagging behaviour (q and ω) as the signal, while
the combinatorial background shows random tagging behaviour. For same-side tagged events,
we assume random tagging behaviour for all background components. We introduce tagging
asymmetry parameters to allow for different numbers of candidates being tagged as mixed
or unmixed, and other parameters to describe the tagging efficiencies for these backgrounds. As expected, the fitted values of these asymmetry parameters are consistent with zero within
uncertainties. All tagging parameters, as well as the value for 1ms, are constrained to be the same for
the five decay modes. The result is 1ms = 17.768 ± 0.023 ps−1 (statistical uncertainty only). The likelihood profile was examined and found to have a Gaussian shape up to nine standard
deviations. The decay time distributions for candidates tagged as mixed or unmixed are shown
in figure 2, together with the decay time projections of the PDF distributions resulting from the
fit. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) decay time [ps]
0
1
2
3
4
candidates / (0.1 ps)
0
200
400
Tagged mixed
Tagged unmixed
Fit mixed
Fit unmixed
LHCb
Figure 2. Decay time distribution for the sum of the five decay modes for
candidates tagged as mixed (different flavour at decay and production; red,
continuous line) or unmixed (same flavour at decay and production; blue, dotted
line). 7. Measurement of 1ms The data and the fit projections are plotted in a signal window around the
reconstructed B0
s mass of 5.32–5.55 GeV/c2. decay time [ps]
0
1
2
3
4
candidates / (0.1 ps)
0
200
400
Tagged mixed
Tagged unmixed
Fit mixed
Fit unmixed
LHCb 9 Figure 2. Decay time distribution for the sum of the five decay modes for
candidates tagged as mixed (different flavour at decay and production; red,
continuous line) or unmixed (same flavour at decay and production; blue, dotted
line). The data and the fit projections are plotted in a signal window around the
reconstructed B0
s mass of 5.32–5.55 GeV/c2. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 8. Systematic uncertainties The total systematic
uncertainty is calculated as the quadratic sum of the individual contributions. 8. Systematic uncertainties With respect to the first measurement of 1ms at LHCb [13], all sources of systematic
uncertainties have been reevaluated. With respect to the first measurement of 1ms at LHCb [13], all sources of systematic
uncertainties have been reevaluated. The dominant source is related to the knowledge of the absolute value of the decay time. This has two main contributions. First, the imperfect knowledge of the longitudinal (z) scale
of the detector contributes to the systematic uncertainty. It is obtained by comparing the track-
based alignment and survey data and evaluating the track distribution in the vertex detector. This results in 0.02% uncertainty on the decay time scale and thus an absolute uncertainty of
±0.004 ps−1 on 1ms. The second contribution to the uncertainty of the decay time scale comes from the
knowledge of the overall momentum scale. This has been evaluated by an independent study
using mass measurements of well-known resonances. Deviations from the reference values [27]
are measured to be within 0.15%. However, since both the measured invariant mass and
momentum enter the calculation of the decay time, this effect cancels to some extent. The
resulting systematic uncertainty on the decay time scale is evaluated from simulation to be
0.02%. This again translates to an absolute uncertainty of ±0.004 ps−1 on 1ms. The next largest systematic uncertainty is due to a possible bias of the measured decay time
given by the track reconstruction and the selection procedure. This is estimated from simulated
data to be less than about 0.2 fs, and results in ±0.001 ps−1 systematic uncertainty on 1ms. Various other sources contributing to the systematic uncertainty have been studied such
as the decay time acceptance, decay time resolution, variations of the value of 10s, different
signal models for the invariant mass and the decay time resolution, variations of the signal
fraction and the fraction of B0
s →D∓
s K± candidates. They are all found to be negligible. The
sources of systematic uncertainty on the measurement of 1ms are summarized in table 2. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 10 10 Table 2. Systematic uncertainties on the 1ms measurement. The total systematic
uncertainty is calculated as the quadratic sum of the individual contributions. Source
Uncertainty (ps−1)
z-scale
0.004
Momentum scale
0.004
Decay time bias
0.001
Total systematic uncertainty
0.006 Table 2. Systematic uncertainties on the 1ms measurement. Acknowledgments We express our gratitude to our colleagues in the CERN accelerator departments for the
excellent performance of the LHC. We thank the technical and administrative staff at the
LHCb institutes. We acknowledge support from CERN and from the national agencies: CAPES,
CNPq, FAPERJ and FINEP (Brazil); NSFC (China); CNRS/IN2P3 and Region Auvergne
(France); BMBF, DFG, HGF and MPG (Germany); SFI (Ireland); INFN (Italy); FOM and
NWO (The Netherlands); SCSR (Poland); ANCS/IFA (Romania); MinES, Rosatom, RFBR and
NRC ‘Kurchatov Institute’ (Russia); MinECo, XuntaGal and GENCAT (Spain); SNSF and SER
(Switzerland); NAS Ukraine (Ukraine); STFC (UK); and NSF (USA). We also acknowledge the
support received from the ERC under FP7. The Tier1 computing centres are supported by IN2P3
(France), KIT and BMBF (Germany), INFN (Italy), NWO and SURF (The Netherlands), PIC
(Spain) and GridPP (UK). We are thankful for the computing resources put at our disposal by
Yandex LLC (Russia), as well as to the communities behind the multiple open source software
packages that we depend on. 9. Conclusion A measurement of the B0
s– B
0
s oscillation frequency 1ms is performed using B0
s →D−
s π +
decays in five different D−
s decay channels. Using a data sample corresponding to an integrated
luminosity of 1.0 fb−1 collected by LHCb in 2011, the oscillation frequency is found to be 1ms = 17.768 ± 0.023 (stat) ± 0.006 (syst) ps−1, in good agreement with the first result reported by the LHCb experiment [13] and the current
world average, 17.69 ± 0.08 ps−1 [27]. This is the most precise measurement of 1ms to date,
and will be a crucial ingredient in future searches for BSM physics in B0
s oscillations. New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 11
A Berezhnoy31, R Bernet39, M-O Bettler46, M van Beuzekom40, A Bien11, S Bifani44, T Bird53,
A Bizzeti17,67, P M Bjørnstad53, T Blake37, F Blanc38, J Blouw11, S Blusk57, V Bocci24,
A Bondar33, N Bondar29, W Bonivento15, S Borghi53, A Borgia57, T J V Bowcock51,
E Bowen39, C Bozzi16, T Brambach9, J van den Brand41, J Bressieux38, D Brett53,
M Britsch10, T Britton57, N H Brook45, H Brown51, I Burducea28, A Bursche39, G Busetto21,76,
J Buytaert37, S Cadeddu15, O Callot7, M Calvi20,69, M Calvo Gomez35,73, A Camboni35,
P Campana18,37, D Campora Perez37, A Carbone14,62, G Carboni23,70, R Cardinale19,68,
A Cardini15, H Carranza-Mejia49, L Carson52, K Carvalho Akiba2, G Casse51, M Cattaneo37,
Ch Cauet9, M Charles54, Ph Charpentier37, P Chen3,38, N Chiapolini39, M Chrzaszcz25,
K Ciba37, X Cid Vidal37, G Ciezarek52, P E L Clarke49, M Clemencic37, H V Cliff46,
J Closier37, C Coca28, V Coco40, J Cogan6, E Cogneras5, P Collins37, A Comerma-Montells35,
A Contu15,37, A Cook45, M Coombes45, S Coquereau8, G Corti37, B Couturier37, G A Cowan49,
D C Craik47, S Cunliffe52, R Currie49, C D’Ambrosio37, P David8, P N Y David40,
I De Bonis4, K De Bruyn40, S De Capua53, M De Cian39, J M De Miranda1, L De Paula2,
W De Silva56, P De Simone18, D Decamp4, M Deckenhoff9, L Del Buono8, D Derkach14,
O Deschamps5, F Dettori41, A Di Canto11, H Dijkstra37, M Dogaru28, S Donleavy51,
F Dordei11, A Dosil Su´arez36, D Dossett47, A Dovbnya42, F Dupertuis38, R Dzhelyadin34,
A Dziurda25, A Dzyuba29, S Easo48,37, U Egede52, V Egorychev30, S Eidelman33, D van Eijk40,
S Eisenhardt49, U Eitschberger9, R Ekelhof9, L Eklund50,37, I El Rifai5, Ch Elsasser39,
D Elsby44, A Falabella14,64, C F¨arber11, G Fardell49, C Farinelli40, S Farry12, V Fave38,
D Ferguson49, V Fernandez Albor36, F Ferreira Rodrigues1, M Ferro-Luzzi37, S Filippov32,
M Fiore16, C Fitzpatrick37, M Fontana10, F Fontanelli19,68, R Forty37, O Francisco2, M Frank37,
C Frei37, M Frosini17,65, S Furcas20, E Furfaro23,70, A Gallas Torreira36, D Galli14,62,
M Gandelman2, P Gandini57, Y Gao3, J Garofoli57, P Garosi53, J Garra Tico46, L Garrido35,
C Gaspar37, R Gauld54, E Gersabeck11, M Gersabeck53, T Gershon47,37, Ph Ghez4, V Gibson46,
V V Gligorov37, C G¨obel58, D Golubkov30, A Golutvin52,30,37, A Gomes2, H Gordon54,
M Grabalosa G´andara5, R Graciani Diaz35, L A Granado Cardoso37, E Graug´es35, G Graziani17,
A Grecu28, E Greening54, S Gregson46, O Gr¨unberg59, B Gui57, E Gushchin32, Yu Guz34,37,
T Gys37, C Hadjivasiliou57, G Haefeli38, C Haen37, S C Haines46, S Hall52, T Hampson45,
S Hansmann-Menzemer11, N Harnew54, S T Harnew45, J Harrison53, T Hartmann59, J He37,
V Heijne40, K Hennessy51, P Henrard5, J A Hernando Morata36, E van Herwijnen37, E Hicks51,
D Hill54, M Hoballah5, C Hombach53, P Hopchev4, W Hulsbergen40, P Hunt54, T Huse51,
N Hussain54, D Hutchcroft51, D Hynds50, V Iakovenko43, M Idzik26, P Ilten12, R Jacobsson37,
A Jaeger11, E Jans40, P Jaton38, F Jing3, M John54, D Johnson54, C R Jones46, B Jost37,
M Kaballo9, S Kandybei42, M Karacson37, T M Karbach37, I R Kenyon44, U Kerzel37, T Ketel41,
A Keune38, B Khanji20, O Kochebina7, I Komarov38, R F Koopman41, P Koppenburg40,
M Korolev31, A Kozlinskiy40, L Kravchuk32, K Kreplin11, M Kreps47, G Krocker11,
P Krokovny33, F Kruse9, M Kucharczyk20,25,69, V Kudryavtsev33, T Kvaratskheliya30,37,
V N La Thi38, D Lacarrere37, G Lafferty53, A Lai15, D Lambert49, R W Lambert41,
E Lanciotti37, G Lanfranchi18, C Langenbruch37, T Latham47, C Lazzeroni44, R Le Gac6,
J van Leerdam40, J-P Lees4, R Lef`evre5, A Leflat31, J Lefran¸cois7, S Leo22, O Leroy6, T Lesiak25,
B Leverington11, Y Li3, L Li Gioi5, M Liles51, R Lindner37, C Linn11, B Liu3, G Liu37, S Lohn37,
I Longstaff50, J H Lopes2, E Lopez Asamar35, N Lopez-March38, H Lu3, D Lucchesi21,76,
J Luisier38, H Luo49, F Machefert7, I V Machikhiliyan4,30, F Maciuc28, O Maev29,37, S Malde54,
G Manca15,63, G Mancinelli6, U Marconi14, R M¨arki38, J Marks11, G Martellotti24, A Martens8,
L Martin54, A Mart´ın S´anchez7, M Martinelli40, D Martinez Santos41, D Martins Tostes2, New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) The LHCb Collaboration R Aaij40, C Abellan Beteta35,73, B Adeva36, M Adinolfi45, C Adrover6, A Affolder51,
Z
Ajaltouni5,
J
Albrecht9,
F
Alessio37,
M
Alexander50,
S
Ali40,
G
Alkhazov29,
P Alvarez Cartelle36, A A Alves Jr24,37, S Amato2, S Amerio21, Y Amhis7, L Anderlini17,65,
J Anderson39, R Andreassen56, R B Appleby53, O Aquines Gutierrez10, F Archilli18,
A Artamonov34, M Artuso57, E Aslanides6, G Auriemma24,72, S Bachmann11, J J Back47,
C Baesso58, V Balagura30, W Baldini16, R J Barlow53, C Barschel37, S Barsuk7, W Barter46,
Th Bauer40, A Bay38, J Beddow50, F Bedeschi22, I Bediaga1, S Belogurov30, K Belous34,
I Belyaev30, E Ben-Haim8, M Benayoun8, G Bencivenni18, S Benson49, J Benton45,
New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 11 A Berezhnoy31, R Bernet39, M-O Bettler46, M van Beuzekom40, A Bien11, S Bifani44, T Bird53,
A Bizzeti17,67, P M Bjørnstad53, T Blake37, F Blanc38, J Blouw11, S Blusk57, V Bocci24,
A Bondar33, N Bondar29, W Bonivento15, S Borghi53, A Borgia57, T J V Bowcock51,
E Bowen39, C Bozzi16, T Brambach9, J van den Brand41, J Bressieux38, D Brett53,
M Britsch10, T Britton57, N H Brook45, H Brown51, I Burducea28, A Bursche39, G Busetto21,76,
J Buytaert37, S Cadeddu15, O Callot7, M Calvi20,69, M Calvo Gomez35,73, A Camboni35,
P Campana18,37, D Campora Perez37, A Carbone14,62, G Carboni23,70, R Cardinale19,68,
A Cardini15, H Carranza-Mejia49, L Carson52, K Carvalho Akiba2, G Casse51, M Cattaneo37,
Ch Cauet9, M Charles54, Ph Charpentier37, P Chen3,38, N Chiapolini39, M Chrzaszcz25,
K Ciba37, X Cid Vidal37, G Ciezarek52, P E L Clarke49, M Clemencic37, H V Cliff46,
J Closier37, C Coca28, V Coco40, J Cogan6, E Cogneras5, P Collins37, A Comerma-Montells35,
A Contu15,37, A Cook45, M Coombes45, S Coquereau8, G Corti37, B Couturier37, G A Cowan49,
D C Craik47, S Cunliffe52, R Currie49, C D’Ambrosio37, P David8, P N Y David40,
I De Bonis4, K De Bruyn40, S De Capua53, M De Cian39, J M De Miranda1, L De Paula2,
W De Silva56, P De Simone18, D Decamp4, M Deckenhoff9, L Del Buono8, D Derkach14,
O Deschamps5, F Dettori41, A Di Canto11, H Dijkstra37, M Dogaru28, S Donleavy51,
F Dordei11, A Dosil Su´arez36, D Dossett47, A Dovbnya42, F Dupertuis38, R Dzhelyadin34,
A Dziurda25, A Dzyuba29, S Easo48,37, U Egede52, V Egorychev30, S Eidelman33, D van Eijk40,
S Eisenhardt49, U Eitschberger9, R Ekelhof9, L Eklund50,37, I El Rifai5, Ch Elsasser39,
D Elsby44, A Falabella14,64, C F¨arber11, G Fardell49, C Farinelli40, S Farry12, V Fave38,
D Ferguson49, V Fernandez Albor36, F Ferreira Rodrigues1, M Ferro-Luzzi37, S Filippov32,
M Fiore16, C Fitzpatrick37, M Fontana10, F Fontanelli19,68, R Forty37, O Francisco2, M Frank37,
C Frei37, M Frosini17,65, S Furcas20, E Furfaro23,70, A Gallas Torreira36, D Galli14,62,
M Gandelman2, P Gandini57, Y Gao3, J Garofoli57, P Garosi53, J Garra Tico46, L Garrido35,
C Gaspar37, R Gauld54, E Gersabeck11, M Gersabeck53, T Gershon47,37, Ph Ghez4, V Gibson46,
V V Gligorov37, C G¨obel58, D Golubkov30, A Golutvin52,30,37, A Gomes2, H Gordon54,
M Grabalosa G´andara5, R Graciani Diaz35, L A Granado Cardoso37, E Graug´es35, G Graziani17,
A Grecu28, E Greening54, S Gregson46, O Gr¨unberg59, B Gui57, E Gushchin32, Yu Guz34,37,
T Gys37, C Hadjivasiliou57, G Haefeli38, C Haen37, S C Haines46, S Hall52, T Hampson45,
S Hansmann-Menzemer11, N Harnew54, S T Harnew45, J Harrison53, T Hartmann59, J He37,
V Heijne40, K Hennessy51, P Henrard5, J A Hernando Morata36, E van Herwijnen37, E Hicks51,
D Hill54, M Hoballah5, C Hombach53, P Hopchev4, W Hulsbergen40, P Hunt54, T Huse51,
N Hussain54, D Hutchcroft51, D Hynds50, V Iakovenko43, M Idzik26, P Ilten12, R Jacobsson37,
A Jaeger11, E Jans40, P Jaton38, F Jing3, M John54, D Johnson54, C R Jones46, B Jost37,
M Kaballo9, S Kandybei42, M Karacson37, T M Karbach37, I R Kenyon44, U Kerzel37, T Ketel41,
A Keune38, B Khanji20, O Kochebina7, I Komarov38, R F Koopman41, P Koppenburg40,
M Korolev31, A Kozlinskiy40, L Kravchuk32, K Kreplin11, M Kreps47, G Krocker11,
P Krokovny33, F Kruse9, M Kucharczyk20,25,69, V Kudryavtsev33, T Kvaratskheliya30,37,
V N La Thi38, D Lacarrere37, G Lafferty53, A Lai15, D Lambert49, R W Lambert41,
E Lanciotti37, G Lanfranchi18, C Langenbruch37, T Latham47, C Lazzeroni44, R Le Gac6,
J van Leerdam40, J-P Lees4, R Lef`evre5, A Leflat31, J Lefran¸cois7, S Leo22, O Leroy6, T Lesiak25,
B Leverington11, Y Li3, L Li Gioi5, M Liles51, R Lindner37, C Linn11, B Liu3, G Liu37, S Lohn37,
I Longstaff50, J H Lopes2, E Lopez Asamar35, N Lopez-March38, H Lu3, D Lucchesi21,76,
J Luisier38, H Luo49, F Machefert7, I V Machikhiliyan4,30, F Maciuc28, O Maev29,37, S Malde54,
G Manca15,63, G Mancinelli6, U Marconi14, R M¨arki38, J Marks11, G Martellotti24, A Martens8,
L Martin54, A Mart´ın S´anchez7, M Martinelli40, D Martinez Santos41, D Martins Tostes2, New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 12 12
A Massafferri1, R Matev37, Z Mathe37, C Matteuzzi20, E Maurice6, A Mazurov16,32,37,64,
J McCarthy44, A McNab53, R McNulty12, B Meadows56,54, F Meier9, M Meissner11,
M Merk40, D A Milanes8, M-N Minard4, J Molina Rodriguez58, S Monteil5, D Moran53,
P Morawski25, M J Morello22,78, R Mountain57, I Mous40, F Muheim49, K M¨uller39,
R Muresan28, B Muryn26, B Muster38, P Naik45, T Nakada38, R Nandakumar48, I Nasteva1,
M Needham49, N Neufeld37, A D Nguyen38, T D Nguyen38, C Nguyen-Mau38,75, M Nicol7,
V Niess5, R Niet9, N Nikitin31, T Nikodem11, A Nomerotski54, A Novoselov34, A Oblakowska-
Mucha26, V Obraztsov34, S Oggero40, S Ogilvy50, O Okhrimenko43, R Oldeman15,63,
M Orlandea28, J M Otalora Goicochea2, P Owen52, A Oyanguren
35,74,
B K Pal57,
A Palano13,61, M Palutan18, J Panman37, A Papanestis48, M Pappagallo50, C Parkes53,
C J Parkinson52, G Passaleva17, G D Patel51, M Patel52, G N Patrick48, C Patrignani19,68,
C Pavel-Nicorescu28, A Pazos Alvarez36, A Pellegrino40, G Penso24,71, M Pepe Altarelli37,
S Perazzini14,62, D L Perego20,69, E Perez Trigo36, A P´erez-Calero Yzquierdo35, P Perret5,
M Perrin-Terrin6, G Pessina20, K Petridis52, A Petrolini19,68, A Phan57, E Picatoste Olloqui35,
B Pietrzyk4, T Pilaˇr47, D Pinci24, S Playfer49, M Plo Casasus36, F Polci8, G Polok25,
A Poluektov47,33, E Polycarpo2, D Popov10, B Popovici28, C Potterat35, A Powell54,
J Prisciandaro38, V Pugatch43, A Puig Navarro38, G Punzi22,77, W Qian4, J H Rademacker45,
B Rakotomiaramanana38, M S Rangel2, I Raniuk42, N Rauschmayr37, G Raven41, S Redford54,
M M Reid47, A C dos Reis1, S Ricciardi48, A Richards52, K Rinnert51, V Rives Molina35,
D A Roa Romero5, P Robbe7, E Rodrigues53, P Rodriguez Perez36, S Roiser37, V Romanovsky34,
A Romero Vidal36, J Rouvinet38, T Ruf37, F Ruffini22, H Ruiz35, P Ruiz Valls35,74,
G Sabatino24,70, J J Saborido Silva36, N Sagidova29, P Sail50, B Saitta15,63, C Salzmann39,
B Sanmartin Sedes36, M Sannino19,68, R Santacesaria24, C Santamarina Rios36, E Santovetti23,70,
M Sapunov6, A Sarti18,71, C Satriano24,72, A Satta23, M Savrie16,64, D Savrina30,31, P Schaack52,
M Schiller41, H Schindler37, M Schlupp9, M Schmelling10, B Schmidt37, O Schneider38,
A Schopper37, M-H Schune7, R Schwemmer37, B Sciascia18, A Sciubba24, M Seco36,
A Semennikov30, K Senderowska26, I Sepp52, N Serra39, J Serrano6, P Seyfert11, M Shapkin34,
I Shapoval16,42, P Shatalov30, Y Shcheglov29, T Shears51,37, L Shekhtman33, O Shevchenko42,
V Shevchenko30, A Shires52, R Silva Coutinho47, T Skwarnicki57, N A Smith51, E Smith54,48,
M Smith53, M D Sokoloff56, F J P Soler50, F Soomro18, D Souza45, B Souza De Paula2,
B Spaan9, A Sparkes49, P Spradlin50, F Stagni37, S Stahl11, O Steinkamp39, S Stoica28, S Stone57,
B Storaci39, M Straticiuc28, U Straumann39, V K Subbiah37, S Swientek9, V Syropoulos41,
M Szczekowski27, P Szczypka38,37, T Szumlak26, S T’Jampens4, M Teklishyn7, E Teodorescu28,
F Teubert37, C Thomas54, E Thomas37, J van Tilburg11, V Tisserand4, M Tobin38, S Tolk41,
D Tonelli37, S Topp-Joergensen54, N Torr54, E Tournefier4,52, S Tourneur38, M T Tran38,
M Tresch39, A Tsaregorodtsev6, P Tsopelas40, N Tuning40, M Ubeda Garcia37, A Ukleja27,
D Urner53, U Uwer11, V Vagnoni14, G Valenti14, R Vazquez Gomez35, P Vazquez Regueiro36,
S Vecchi16, J J Velthuis45, M Veltri17,66, G Veneziano38, M Vesterinen37, B Viaud7,
D Vieira2, X Vilasis-Cardona35,73, A Vollhardt39, D Volyanskyy10, D Voong45, A Vorobyev29,
V Vorobyev33, C Voß59, H Voss10, R Waldi59, R Wallace12, S Wandernoth11,79, J Wang57,
D R Ward46, N K Watson44, A D Webber53, D Websdale52, M Whitehead47, J Wicht37,
J Wiechczynski25, D Wiedner11, L Wiggers40, G Wilkinson54, M P Williams47,48, M Williams55,
F F Wilson48, J Wishahi9, M Witek25, S A Wotton46, S Wright46, S Wu3, K Wyllie37,
Y Xie49,37, F Xing54, Z Xing57, Z Yang3, R Young49, X Yuan3, O Yushchenko34, M Zangoli14,
M Zavertyaev10,60, F Zhang3, L Zhang57, W C Zhang12, Y Zhang3, A Zhelezov11, A Zhokhov30,
L Zhong3 and A Zvyagin37 New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 12
A Massafferri1, R Matev37, Z Mathe37, C Matteuzzi20, E Maurice6, A Mazurov16,32,37,64,
J McCarthy44, A McNab53, R McNulty12, B Meadows56,54, F Meier9, M Meissner11,
M Merk40, D A Milanes8, M-N Minard4, J Molina Rodriguez58, S Monteil5, D Moran53,
P Morawski25, M J Morello22,78, R Mountain57, I Mous40, F Muheim49, K M¨uller39,
R Muresan28, B Muryn26, B Muster38, P Naik45, T Nakada38, R Nandakumar48, I Nasteva1,
M Needham49, N Neufeld37, A D Nguyen38, T D Nguyen38, C Nguyen-Mau38,75, M Nicol7,
V Niess5, R Niet9, N Nikitin31, T Nikodem11, A Nomerotski54, A Novoselov34, A Oblakowska-
Mucha26, V Obraztsov34, S Oggero40, S Ogilvy50, O Okhrimenko43, R Oldeman15,63,
M Orlandea28, J M Otalora Goicochea2, P Owen52, A Oyanguren
35,74,
B K Pal57,
A Palano13,61, M Palutan18, J Panman37, A Papanestis48, M Pappagallo50, C Parkes53,
C J Parkinson52, G Passaleva17, G D Patel51, M Patel52, G N Patrick48, C Patrignani19,68,
C Pavel-Nicorescu28, A Pazos Alvarez36, A Pellegrino40, G Penso24,71, M Pepe Altarelli37,
S Perazzini14,62, D L Perego20,69, E Perez Trigo36, A P´erez-Calero Yzquierdo35, P Perret5,
M Perrin-Terrin6, G Pessina20, K Petridis52, A Petrolini19,68, A Phan57, E Picatoste Olloqui35,
B Pietrzyk4, T Pilaˇr47, D Pinci24, S Playfer49, M Plo Casasus36, F Polci8, G Polok25,
A Poluektov47,33, E Polycarpo2, D Popov10, B Popovici28, C Potterat35, A Powell54,
J Prisciandaro38, V Pugatch43, A Puig Navarro38, G Punzi22,77, W Qian4, J H Rademacker45,
B Rakotomiaramanana38, M S Rangel2, I Raniuk42, N Rauschmayr37, G Raven41, S Redford54,
M M Reid47, A C dos Reis1, S Ricciardi48, A Richards52, K Rinnert51, V Rives Molina35,
D A Roa Romero5, P Robbe7, E Rodrigues53, P Rodriguez Perez36, S Roiser37, V Romanovsky34,
A Romero Vidal36, J Rouvinet38, T Ruf37, F Ruffini22, H Ruiz35, P Ruiz Valls35,74,
G Sabatino24,70, J J Saborido Silva36, N Sagidova29, P Sail50, B Saitta15,63, C Salzmann39,
B Sanmartin Sedes36, M Sannino19,68, R Santacesaria24, C Santamarina Rios36, E Santovetti23,70,
M Sapunov6, A Sarti18,71, C Satriano24,72, A Satta23, M Savrie16,64, D Savrina30,31, P Schaack52,
M Schiller41, H Schindler37, M Schlupp9, M Schmelling10, B Schmidt37, O Schneider38,
A Schopper37, M-H Schune7, R Schwemmer37, B Sciascia18, A Sciubba24, M Seco36,
A Semennikov30, K Senderowska26, I Sepp52, N Serra39, J Serrano6, P Seyfert11, M Shapkin34,
I Shapoval16,42, P Shatalov30, Y Shcheglov29, T Shears51,37, L Shekhtman33, O Shevchenko42,
V Shevchenko30, A Shires52, R Silva Coutinho47, T Skwarnicki57, N A Smith51, E Smith54,48,
M Smith53, M D Sokoloff56, F J P Soler50, F Soomro18, D Souza45, B Souza De Paula2,
B Spaan9, A Sparkes49, P Spradlin50, F Stagni37, S Stahl11, O Steinkamp39, S Stoica28, S Stone57,
B Storaci39, M Straticiuc28, U Straumann39, V K Subbiah37, S Swientek9, V Syropoulos41,
M Szczekowski27, P Szczypka38,37, T Szumlak26, S T’Jampens4, M Teklishyn7, E Teodorescu28,
F Teubert37, C Thomas54, E Thomas37, J van Tilburg11, V Tisserand4, M Tobin38, S Tolk41,
D Tonelli37, S Topp-Joergensen54, N Torr54, E Tournefier4,52, S Tourneur38, M T Tran38,
M Tresch39, A Tsaregorodtsev6, P Tsopelas40, N Tuning40, M Ubeda Garcia37, A Ukleja27,
D Urner53, U Uwer11, V Vagnoni14, G Valenti14, R Vazquez Gomez35, P Vazquez Regueiro36,
S Vecchi16, J J Velthuis45, M Veltri17,66, G Veneziano38, M Vesterinen37, B Viaud7,
D Vieira2, X Vilasis-Cardona35,73, A Vollhardt39, D Volyanskyy10, D Voong45, A Vorobyev29,
V Vorobyev33, C Voß59, H Voss10, R Waldi59, R Wallace12, S Wandernoth11,79, J Wang57,
D R Ward46, N K Watson44, A D Webber53, D Websdale52, M Whitehead47, J Wicht37,
J Wiechczynski25, D Wiedner11, L Wiggers40, G Wilkinson54, M P Williams47,48, M Williams55,
F F Wilson48, J Wishahi9, M Witek25, S A Wotton46, S Wright46, S Wu3, K Wyllie37,
Y Xie49,37, F Xing54, Z Xing57, Z Yang3, R Young49, X Yuan3, O Yushchenko34, M Zangoli14,
M Zavertyaev10,60, F Zhang3, L Zhang57, W C Zhang12, Y Zhang3, A Zhelezov11, A Zhokhov30,
L Zhong3 and A Zvyagin37 A Massafferri1, R Matev37, Z Mathe37, C Matteuzzi20, E Maurice6, A Mazurov16,32,37,64,
J McCarthy44, A McNab53, R McNulty12, B Meadows56,54, F Meier9, M Meissner11,
M Merk40, D A Milanes8, M-N Minard4, J Molina Rodriguez58, S Monteil5, D Moran53,
P Morawski25, M J Morello22,78, R Mountain57, I Mous40, F Muheim49, K M¨uller39,
R Muresan28, B Muryn26, B Muster38, P Naik45, T Nakada38, R Nandakumar48, I Nasteva1,
M Needham49, N Neufeld37, A D Nguyen38, T D Nguyen38, C Nguyen-Mau38,75, M Nicol7,
V Niess5, R Niet9, N Nikitin31, T Nikodem11, A Nomerotski54, A Novoselov34, A Oblakowska-
Mucha26, V Obraztsov34, S Oggero40, S Ogilvy50, O Okhrimenko43, R Oldeman15,63,
M Orlandea28, J M Otalora Goicochea2, P Owen52, A Oyanguren
35,74,
B K Pal57,
A Palano13,61, M Palutan18, J Panman37, A Papanestis48, M Pappagallo50, C Parkes53,
C J Parkinson52, G Passaleva17, G D Patel51, M Patel52, G N Patrick48, C Patrignani19,68,
C Pavel-Nicorescu28, A Pazos Alvarez36, A Pellegrino40, G Penso24,71, M Pepe Altarelli37,
S Perazzini14,62, D L Perego20,69, E Perez Trigo36, A P´erez-Calero Yzquierdo35, P Perret5,
M Perrin-Terrin6, G Pessina20, K Petridis52, A Petrolini19,68, A Phan57, E Picatoste Olloqui35,
B Pietrzyk4, T Pilaˇr47, D Pinci24, S Playfer49, M Plo Casasus36, F Polci8, G Polok25,
A Poluektov47,33, E Polycarpo2, D Popov10, B Popovici28, C Potterat35, A Powell54,
J Prisciandaro38, V Pugatch43, A Puig Navarro38, G Punzi22,77, W Qian4, J H Rademacker45,
B Rakotomiaramanana38, M S Rangel2, I Raniuk42, N Rauschmayr37, G Raven41, S Redford54,
M M Reid47, A C dos Reis1, S Ricciardi48, A Richards52, K Rinnert51, V Rives Molina35,
D A Roa Romero5, P Robbe7, E Rodrigues53, P Rodriguez Perez36, S Roiser37, V Romanovsky34,
A Romero Vidal36, J Rouvinet38, T Ruf37, F Ruffini22, H Ruiz35, P Ruiz Valls35,74,
G Sabatino24,70, J J Saborido Silva36, N Sagidova29, P Sail50, B Saitta15,63, C Salzmann39,
B Sanmartin Sedes36, M Sannino19,68, R Santacesaria24, C Santamarina Rios36, E Santovetti23,70,
M Sapunov6, A Sarti18,71, C Satriano24,72, A Satta23, M Savrie16,64, D Savrina30,31, P Schaack52,
M Schiller41, H Schindler37, M Schlupp9, M Schmelling10, B Schmidt37, O Schneider38,
A Schopper37, M-H Schune7, R Schwemmer37, B Sciascia18, A Sciubba24, M Seco36,
A Semennikov30, K Senderowska26, I Sepp52, N Serra39, J Serrano6, P Seyfert11, M Shapkin34,
I Shapoval16,42, P Shatalov30, Y Shcheglov29, T Shears51,37, L Shekhtman33, O Shevchenko42,
V Shevchenko30, A Shires52, R Silva Coutinho47, T Skwarnicki57, N A Smith51, E Smith54,48,
M Smith53, M D Sokoloff56, F J P Soler50, F Soomro18, D Souza45, B Souza De Paula2,
B Spaan9, A Sparkes49, P Spradlin50, F Stagni37, S Stahl11, O Steinkamp39, S Stoica28, S Stone57,
B Storaci39, M Straticiuc28, U Straumann39, V K Subbiah37, S Swientek9, V Syropoulos41,
M Szczekowski27, P Szczypka38,37, T Szumlak26, S T’Jampens4, M Teklishyn7, E Teodorescu28,
F Teubert37, C Thomas54, E Thomas37, J van Tilburg11, V Tisserand4, M Tobin38, S Tolk41,
D Tonelli37, S Topp-Joergensen54, N Torr54, E Tournefier4,52, S Tourneur38, M T Tran38,
M Tresch39, A Tsaregorodtsev6, P Tsopelas40, N Tuning40, M Ubeda Garcia37, A Ukleja27,
D Urner53, U Uwer11, V Vagnoni14, G Valenti14, R Vazquez Gomez35, P Vazquez Regueiro36,
S Vecchi16, J J Velthuis45, M Veltri17,66, G Veneziano38, M Vesterinen37, B Viaud7,
D Vieira2, X Vilasis-Cardona35,73, A Vollhardt39, D Volyanskyy10, D Voong45, A Vorobyev29,
V Vorobyev33, C Voß59, H Voss10, R Waldi59, R Wallace12, S Wandernoth11,79, J Wang57,
D R Ward46, N K Watson44, A D Webber53, D Websdale52, M Whitehead47, J Wicht37,
J Wiechczynski25, D Wiedner11, L Wiggers40, G Wilkinson54, M P Williams47,48, M Williams55,
F F Wilson48, J Wishahi9, M Witek25, S A Wotton46, S Wright46, S Wu3, K Wyllie37,
Y Xie49,37, F Xing54, Z Xing57, Z Yang3, R Young49, X Yuan3, O Yushchenko34, M Zangoli14,
M Zavertyaev10,60, F Zhang3, L Zhang57, W C Zhang12, Y Zhang3, A Zhelezov11, A Zhokhov30,
L Zhong3 and A Zvyagin37 New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 13 41 Nikhef National Institute for Subatomic Physics and VU University Amsterdam, Amsterdam,
The Netherlands 41 Nikhef National Institute for Subatomic Physics and VU University Amsterdam, Amsterdam,
The Netherlands 2 NSC Kharkiv Institute of Physics and Technology (NSC KIPT), Kharkiv, Ukraine SC Kharkiv Institute of Physics and Technology (NSC KIPT), Kharkiv, Ukraine y
gy (
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43 Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukraine
44 nstitute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukrain 43 Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, U 44 University of Birmingham, Birmingham, UK 5 H H Wills Physics Laboratory, University of Bristol, Bristol, UK
6 45 H H Wills Physics Laboratory, University of Bristol, Bristol, UK 6 Cavendish Laboratory, University of Cambridge, Cambridge, UK 46 Cavendish Laboratory, University of Cambridge, Cambridge, UK 7 Department of Physics, University of Warwick, Coventry, UK 47 Department of Physics, University of Warwick, Coventry, UK 48 STFC Rutherford Appleton Laboratory, Didcot, UK 9 School of Physics and Astronomy, University of Edinburgh, Edinburgh, UK 49 School of Physics and Astronomy, University of Edinburgh, Edinburgh, UK 0 School of Physics and Astronomy, University of Glasgow, Glasgow, UK 50 School of Physics and Astronomy, University of Glasgow, Glasgow, UK 1 Oliver Lodge Laboratory, University of Liverpool, Liverpool, UK 51 Oliver Lodge Laboratory, University of Liverpool, Liverpool, UK 52 Imperial College London, London, UK 53 School of Physics and Astronomy, University of Manchester, Manchester, UK 53 School of Physics and Astronomy, University of Manchester, M 54 Department of Physics, University of Oxford, Oxford, UK 54 Department of Physics, University of Oxford, Oxford, UK 55 Massachusetts Institute of Technology, Cambridge, MA, USA 55 Massachusetts Institute of Technology, Cambridge, MA, USA 56 University of Cincinnati, Cincinnati, OH, USA 56 University of Cincinnati, Cincinnati, OH, USA 57 Syracuse University, Syracuse, NY, USA 57 Syracuse University, Syracuse, NY, USA 58 Pontif´ıcia Universidade Cat´olica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil,
associated to Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 58 Pontif´ıcia Universidade Cat´olica do Rio de Janeiro (PUC-Rio), Rio de Janeiro
i
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(UFRJ) Ri d J
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il Pontifıcia Universidade Catolica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil,
associated to Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil associated to Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
5 59 Institut f¨ur Physik, Universit¨at Rostock, Rostock, Germany, associated to Physikalisches
Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 60 P N Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, R 60 P N Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia
61 Universit`a di Bari, Bari, Italy 61 Universit`a di Bari, Bari, Italy
6 61 Universit`a di Bari, Bari, Italy
62 62 Universit`a di Bologna, Bologna, Italy 62 Universit`a di Bologna, Bologna, Italy
63 63 Universit`a di Cagliari, Cagliari, Italy 63 Universit`a di Cagliari, Cagliari, Italy
64 64 Universit`a di Ferrara, Ferrara, Italy 64 Universit`a di Ferrara, Ferrara, Italy 65 Universit`a di Firenze, Firenze, Italy 65 Universit`a di Firenze, Firenze, Italy 66 Universit`a di Urbino, Urbino, Italy 66 Universit`a di Urbino, Urbino, Italy 67 Universit`a di Modena e Reggio Emilia, Modena, Italy 67 Universit`a di Modena e Reggio Emilia, Modena, Italy 68 Universit`a di Genova, Genova, Italy 68 Universit`a di Genova, Genova, Italy 69 Universit`a di Milano Bicocca, Milano, Italy 70 Universit`a di Roma Tor Vergata, Roma, Italy 71 Universit`a di Roma La Sapienza, Roma, Italy 72 Universit`a della Basilicata, Potenza, Italy 73 LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain 74 IFIC, Universitat de Valencia-CSIC, Valencia, Spain 75 Hanoi University of Science, Hanoi, Vietnam 76 Universit`a di Padova, Padova, Italy 78 Scuola Normale Superiore, Pisa, Italy 1 Centro Brasileiro de Pesquisas F´ısicas (CBPF), Rio de Janeiro, Brazil 2 Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 3 Center for High Energy Physics, Tsinghua University, Beijing, People 4 LAPP, Universit´e de Savoie, CNRS/IN2P3, Annecy-Le-Vieux, France 5 Clermont Universit´e, Universit´e Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferr 6 CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France
7
A
i
i ´
i S d C
S/
2 3 O 6 CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France 7 LAL, Universit´e Paris-Sud, CNRS/IN2P3, Orsay, France 7 LAL, Universit´e Paris-Sud, CNRS/IN2P3, Orsay, France 8 LPNHE, Universit´e Pierre et Marie Curie, Universit´e Paris Diderot, CNRS/IN2P3, Paris,
France 8 LPNHE, Universit´e Pierre et Marie Curie, Universit´e Paris Diderot, CNRS/IN2P3, Paris,
France Fakult¨at Physik, Technische Universit¨at Dortmund, Dortmund, Germany 9 Fakult¨at Physik, Technische Universit¨at Dortmund, Dortmund, Germany 0 Max-Planck-Institut f¨ur Kernphysik (MPIK), Heidelberg, Germany 10 Max-Planck-Institut f¨ur Kernphysik (MPIK), Heidelberg, Germany 1 Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 11 Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, G 2 School of Physics, University College Dublin, Dublin, Ireland 12 School of Physics, University College Dublin, Dublin, Ireland 13 Sezione INFN di Bari, Bari, Italy 14 Sezione INFN di Bologna, Bologna, Italy 14 Sezione INFN di Bologna, Bologna, Italy 15 Sezione INFN di Cagliari, Cagliari, Italy 15 Sezione INFN di Cagliari, Cagliari, Italy 16 Sezione INFN di Ferrara, Ferrara, Italy 17 Sezione INFN di Firenze, Firenze, Italy 18 Laboratori Nazionali dell’INFN di Frascati, Frascati, Italy 18 Laboratori Nazionali dell’INFN di Frascati, Frascati, Italy 19 Sezione INFN di Genova, Genova, Italy 20 Sezione INFN di Milano Bicocca, Milano, Italy 20 Sezione INFN di Milano Bicocca, Milano, Italy 21 Sezione INFN di Padova, Padova, Italy 22 Sezione INFN di Pisa, Pisa, Italy 23 Sezione INFN di Roma Tor Vergata, Roma, Italy 23 Sezione INFN di Roma Tor Vergata, Roma, Italy 24 Sezione INFN di Roma La Sapienza, Roma, Italy 24 Sezione INFN di Roma La Sapienza, Roma, Italy 25 Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Krak´ow,
Poland 25 Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Krak´ow,
Poland 26 AGH—University of Science and Technology, Faculty of Physics and Applied Computer
Science, Krak´ow, Poland 26 AGH—University of Science and Technology, Faculty of Physics and Applied Computer
Science, Krak´ow, Poland 27 National Center for Nuclear Research (NCBJ), Warsaw, Poland 27 National Center for Nuclear Research (NCBJ), Warsaw, Poland 28 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest-Magurele,
Romania 29 Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia 29 Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia 30 Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia 30 Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia 1 Institute of Nuclear Physics, Moscow State University (SINP MSU), Moscow, Russia
2 32 Institute for Nuclear Research of the Russian Academy of Sciences (INR RAN), Moscow,
Russia 33 Budker Institute of Nuclear Physics (SB RAS) and Novosibirsk State University, Novosibirsk,
Russia
34 34 Institute for High Energy Physics (IHEP), Protvino, Russia 34 Institute for High Energy Physics (IHEP), Protvino, Russia
5 35 Universitat de Barcelona, Barcelona, Spain 35 Universitat de Barcelona, Barcelona, Spain 36 Universidad de Santiago de Compostela, Santiago de Compostela, Spain 36 Universidad de Santiago de Compostela, Santiago de Compostela, Sp 38 Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausanne, Switzerland 40 Nikhef National Institute for Subatomic Physics, Amsterdam, The Netherlands New Journal of Physics 15 (2013) 053021 (http://www.njp.org/) 14 [2] Aaij R et al (LHCb Collaboration) 2013 Implications of LHCb measurements and future prospects Eur. Phys. J. C 73 2373 [3] Lande K et al 1956 Observation of long-lived neutral V, particles Phys. Rev. 103 1901
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[15] AdinolfiM et al 2012 Performance of the LHCb RICH detector at the LHC Eur. Phys. J. References [1] Adeva B et al (LHCb Collaboration) 2009 Roadmap for selected key measurements of LHCb
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Nuclear Science Symp. Conf. Record (NSS/MIC) (Piscataway, NJ: IEEE) p 1155 [19] Lange D J 2001 The EvtGen particle decay simulation package Nucl. Instrum. Methods A 462 152 [20] Golonka P and Was Z 2006 PHOTOS Monte Carlo: a, precision tool for QED corrections in Z and W decays
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T
1986
A
study
of
the
radiative
cascade
transitions
between
the
upsilon-prime
and
upsilon
resonances
PhD
Thesis
Institute
of
Nuclear
Physics
Krakow
DESY-F31-86-02
(http://inspirehep.net/record/230779/files/230779.pdf) New Journal of Physics 15 (2013) 053021 (http://www.njp.org/)
|
https://openalex.org/W1894554624
|
https://zenodo.org/records/2295188/files/article.pdf
|
English
| null |
A STUDY OF EUGENIC FORCES
|
The American journal of psychiatry
| 1,915
|
public-domain
| 14,045
|
No.2 Voi. LXXII. OCTOBER,
1q15. AMERICAN
JOURNAL
OF
INSANITY JOURNAL
OF
INSANITY
A
STUDY
OF
EUGENIC
FORCES. PARTICULARLY
OF
SOCIAL
CONDITIONS
WHICH
BRING
ABOUT
THE
SEGREGATION
OF
NEUROPATHIC
PERSONS
IN
SPECIAL
INSTITUTIONS. B
A. J. ROSANOFF,
M. D.,
Kings
Park
State
Hospital,
Kings
Park,
N. V. A
STUDY
OF
EUGENIC
FORCES. A
STUDY
OF
EUGENIC
FORCES. PARTICULARLY
OF
SOCIAL
CONDITIONS
WHICH
BRING
ABOUT
THE
SEGREGATION
OF
NEUROPATHIC
PERSONS
IN
SPECIAL
INSTITUTIONS. B
A. J. ROSANOFF,
M. D.,
Kings
Park
State
Hospital,
Kings
Park,
N. V. Whoever
is
somewhat
familiar
with
the
history
of
the
care
of
the
insane
in
this
country
or
in
other
civilized
countries
is
also
familiar
with
the
fact
that
the
number
of
the
insane
in
asylums
and
hospitals
has
been
on
the
increase
during
the
past
several
decades
at
a
more
rapid
rate
than
the
general
population. This
fact
has
led
some
to
draw
the
conclusion
that
the
incidence
of
insanity
has
increased
among
civilized
nations;
but
even
those
who
hold
this
view
generally
admit
that
at
least
a
part
of
the
increase
of
the
insane
in
institutions
has
been
due
to
the
almost
universal
improvement
in
the
kind
and
adequateness
of
facilities
for
the
care
of
such
patients;
and
from
this
it
would
seem
that
the
increase
of
insane
in
institutions
is
to
be
regarded
not
neces-
sarily
with
alarm,
but,
from
the
eugenic
standpoint,
as
a
salutary
phenomenon. It
is important
to
note
that
the
origin
and
remarkable
extension
of
the
practice
of
segregating
neuropathic
persons
in
special
insti-
tutions
has
been
wholly
independent
of
any
consciously
eugenic
movement;
which
would
indicate
that
in
modern
civilized
com-
munities,
even
in
the
absence
of
such
a movement,
powerful
social
forces
are
at
work
which
are
eugenic
in
effect. Now
a
new
force
is
being
added,
the
force
of
a
consciously
eugenic
movement. It
would
seem
that
this
movement
might
be
strengthened
through
systematization
and
economy
of
effort,
if
we
could
estimate
even
but
roughly
the
relative
effectiveness
of Is A
STUDY
OF
EUGENIC
FORCES 224 [Oct. the
various
forces
that
have
been
at
work
in
the
past. It
may
be
found
that
the
energies
of
this
movement
might
most
profitably
be
expended
in
merely
directing
these
forces
in
a more
purposeful
way
rather
than
in
promoting
experimental
legislation. To
attempt
an
analysis
of
conditions
which
have,
independently
of
any
eugenic
movement,
determined
in
the
various
states
of
the
Union
the
extent
of
the
practice
of
segregating
neuropathic
per-
sons
in
special
institutions
is
the
main
object
of
this
study. ‘These
figures,
as
well
as
others
like
them
to
be
given
farther
on,
have
been
obtained
by
dividing
the
sum
of
the
gross
values
of
all
agricultural,
mineral,
and
manufactured
products,
as
furnished
by
the
census
for
the
given
year
and
for
the
given
state
or
group
of
states,
as
the
case
may
be,
by
the
total
population. The
expression
“neuropathic
persons”
is
applied
here
to
the
insane,
epileptic,
feeble-minded,
and
inebriate,
and
more
particularly
to
those
who
are
inmates
of
public
or
private
institutions
provided
especially
for
their
care. §I.
CORRELATION
BETWEEN
WEALTH
PRODUCTION
AND
NUMBER
OF
INSTITUTION
INMATES. The
increase
in
the
number
of
neuropathic
persons
in
institu-
tions
during
the
past
several
decades
has
occurred
in
rather
close
correlation
with
the
increase
in
per
capita
wealth
production
also
observed
during
the
same
period. Thus,
according
to
the
enumerations
of
the
Tenth,
Eleventh,
Twelfth
and
Thirteenth
Censuses,
there
were
on
the
dates
of
enumeration
(1880-1910),
respectively,
86.5,
126.6,
204.4,
and
232.0
neuropathic
persons1
in
special
institutions
per
ioo,ooo
of
the
general
population. The
per
capita
wealth
production,
according
to
statistics
derived
from
the
same
sources,
was
$158.53,
$197.30,
$243.75,
and
$327.88
for
the
respective
years.’ However,
a
part
of
this
apparent
increase
in
wealth
production
is
undoubtedly
but
an
expression
of
increased
prices
of
commodi-
ties. Such
prices
are
not
apt
to
vary
so
much
at
any
one
time
in
different
parts
of
the
country
as
in
the
entire
country
in
different
decades. For
this
reason
we
computed
the
data
separately
fpr
each
of
the
four
census
years
by
groups
of
states
according
to
per
capita
wealth
production. The
resulting
statistics
are
given
in
Table
i,
and
the
showing
is
such
as
to
force
us
to
assume
the 1915] 225 A. J. ROSANOFF existence
of
a connection
of
some
sort
between
the
wealth
produc-
tion
of
communities
and
the
practice
of
segregating
neuropathic
persons
in
special
institutions. TABLE
1.-NUMBER
OF
NEUROPATHIC
PERSONS
IN
INSTITUTIONS
PER
100,000
OF
THE
GENERAL
PoPuI..&TI0N
IN
CENSUS
YF&is
IN
GROUPS
OF
STATES
DISTINGUISHED
ACCORDING
TO
PER
CAPITA
WRALTI! PRODUCTION. Census
Years. Group
i. j Group
2. Group
3. Group
. Group
. Per
capita
wealth
production
Under
$ioo
$ioo.$soo
$2oo43oo
$3oo.$4oo
Over
$400
i88o
‘890
1900
1910
40.4
57.6
95.8
.... 83.9
99.4
123.3
115.8
123.2
151.2
227.2
205.4
i6.i
192.9
300.0
257.3
192.8
295.6
301.1 TABLE
1.-NUMBER
OF
NEUROPATHIC
PERSONS
IN
INSTITUTIONS
PER
100,000
OF
THE
GENERAL
PoPuI..&TI0N
IN
CENSUS
YF&is
IN
GROUPS
OF
STATES
DISTINGUISHED
ACCORDING
TO
PER
CAPITA
WRALTI! PRODUCTION. Perhaps
it
is
hardly
necessary
to
add
that
it
is
not
all
merely
a
matter
of
dollars
and
cents;
the
per
capita
value
of
the
products
of
a
community,
being
an
indication
of
its
wealth-producing
power,
is
at
the
same
time
an
indication
of
the
diligence
and
pur-
posefulness
of
its
labors,
its
intelligence,
resourcefulness
and
enterprise
and,
indirectly,
of
the
general
standard
of
its
civiliza-
tion
and
conditions
of
living. §I.
CORRELATION
BETWEEN
WEALTH
PRODUCTION
AND
NUMBER
OF
INSTITUTION
INMATES. A
closer
examination
of
the
statistics
in
Table
I will
show
at
once
that
conditions
other
than
per
capita
wealth
production
have
also
influenced
the
practice
of
segregating
neuropathic
persons
in
institutions. While
the
figures
in
the
horizontal
columns
show
a
close
correlation
between
per
capita
wealth
production
and
relative
number
of
neuropathic
persons
in
institutions,
the
figures
in
the
vertical
colunms
show
that
in
different
decades
between
1880
and
1910
groups
of
the
same
per
capita
wealth
production
present
great
variations
in
relative
number
of
neuropathic
persons
in
institu-
tions,
the
trend
being
mainly
in
the
direction
of
progressive
increase. The
inference
is
that
during
those
decades
some
other
factor
or
factors
were
at
work
simultaneously
with
that
of
per
capita
wealth
production
and
exerting
an
influence
in
the
same
direction. [Oct. 226 A
STUDY
OF
EUGENIC
FORCES Further
evidence
is
to
be
found
in
the
statistics
of
individual
states
given
in
the
full
table
of
data
appended
at
the
end. The
states
of
Colorado,
Delaware,
IllinoiS,
Maine,
Nebraska,
New
Hampshire,
Oklahoma,
Pennsylvania,
Rhode
Island,
Tennessee,
Texas,
Utah
and
Wyoming
have
shown
during
the
entire
period
of
four
decades
a
number
of
neuropathic
persons
in
institutions
far
below
that
which
would
correspond
with
their
per
capita
wealth
production
according
to
the
correlation
statistics
in
Table
i;
the
opposite
is
true
for
the
states
of
California,
Kentucky,
Massachusetts,
New
York,
Oregon
and
Virginia;
while
at
various
times
marked
fluctuations
in
both
directions
were
shown
by
the
states
of
Arizona,
Connecticut,
Michigan,
Nevada,
North
Caro-
lina,
North
Dakota,
South
Carolina,
Washington,
West
Virginia
and
Wisconsin. Occasional
and
slighter
fluctuations
were,
fur-
thermore,
shown
by
almost
all
the
remaining
states,
only
three,
in
fact,
showing
at
all
times
practically
an
absence
of
such
fluctua-
tions,
namely,
the
states
of
Florida,
Maryland
and
Mississippi. It
would
seem,
therefore,
that
conditions,
other
than
per
capita
wealth
production,
exerting
an
influence
on
the
proportion
of
neuropathic
persons
in
institutions,
not
only
exist,
but
are
prob-
ably
numerous
and
heterogeneous,
some
effecting
an
increase,
others
a reduction. §2.
CoRRELATION
BETWEEN
PERCENTAGE
OF
URBAN
POPULATION
AND
NUMBER
OF
INSTITUTION
INMATES. The
segregation
of
neuropathic
persons
in
special
institutions
is
essentially
an
urban
practice;
and
if
to-day
it
is
found
also
in
rural
communities,
it
can
easily
be
shown
in
every
case
to
be
the
result
of
more
or
less
direct
urban
influence. The
history
of
the
care
of
the
insane
in
all
civilized
countries
as
well
as
the
history
of
the
establishment
of
individual
institutions
bears
out
this
con-
tention. The
explanation
is
obvious:
the
problem
of
insanity
in
a given
community
must
attain
a certain
magnitude
before
it
may
be
expected
to
receive
special
attention
from
the
public
authorities
or
from
the
people
themselves;
the
organization
of
elaborate
special
provisions
will
not
be
undertaken
in
order
to
facilitate
the
care
of
an
occasional
patient
here
and
there;
care
at
home,
con-
finement
in
jail,
placing
in
the
almshouse,
according
to
the
special
indications,
is
the
rule
under
such
conditions. 1915] 227 A. J. ROSANOFF Accordingly,
when
we
compare
the
conditions
in
various
groups
of
states
distinguished
according
to
percentage
of
urban
popula-
tion,
we
find
very
consistently
a
correlation
between
percentage
of
urban
‘and
relative
number
of
neuropathic
persons
in
institutions. This
is
shown
in
Table
2. TLE
2.-NUMBER
OF
NEUROPATHIC
PERSONS
IN
INSTITUTIONS
PER
100,000
OF
THE
GENERAL
POPULATION
IN
CENSUS
YEARs
IN
GROUPS
OF
STATES
DISTINGUISHED
ACCORDING
TO
PERCENTAGE
OF
URBAN
POPULATION. Census
Years. Groupi. Group2. f Groups. Group. Groups. Percentage
of
urban
population. Under
i%
15-25%
25.50%
50.75%
Over
75%
i8go
1900
1910
62.6
90.4
129.8
213.8
199.8
99.3
138.9
223.8
296.9
.8
1o4.8
124.9
234.2
250.3
373.2 TLE
2.-NUMBER
OF
NEUROPATHIC
PERSONS
IN
INSTITUTIONS
PER
100,000
OF
THE
GENERAL
POPULATION
IN
CENSUS
YEARs
IN
GROUPS
OF
STATES
DISTINGUISHED
ACCORDING
TO
PERCENTAGE
OF
URBAN
POPULATION. It
will
be
noticed,
by
reference
to
the
full
table
of
data
appended
at
the
end,
that
the
groups
of
states
distinguished
according
to
percentage
of
urban
population
differ
but
slightly
from
the
groups
distinguished
according
to
per
capita
wealth
production. (i,
Table
i.)
It
is,
in
fact,
highly
probable
that
throughout
the
land
increased
wealth
production
is
itself
in
part
but
a
result
of
the
organization
of
urban
centers,
i. e.,
centers
of
manufacture,
com-
merce,
enlightenment. Yet
it
can
be
shown
that
to
some
extent
these
two
factors
act
independently. ‘The
expression
“urban
population”
is here
used,
as
in
the
U.
S.
Census
since
1890,
to
designate
all
that
part
of
the
general
population
which
resides
in
cities,
towns,
or
other
incorporated
places
of
2500
inhabitants
or
more. §2.
CoRRELATION
BETWEEN
PERCENTAGE
OF
URBAN
POPULATION
AND
NUMBER
OF
INSTITUTION
INMATES. The
states
of
Montana,
Nevada,
North
Dakota
and
South
Dakota
are
peculiar
in
that
they
present
a high
per
capita
wealth
production
and
at
the
same
time
a
low
percentage
of
urban
popu-
lation;
thus
they
afford
an
opportunity
of
examining
separately
the
influence
of
these
two
factors
on
the
number
of
neuropathic
persons
in
institutions. In
these
four
states
as
a group
the
per
capita
wealth
production
is
$406.47,
and
the
percentage
of
urban
population
is
17.7
(Thir- [Oct. A
STUDY
OF
EUGENIC
FORCES 228 teenth
Census). The
number
of
institution
inmates
per
ioo,ooo
of
the
general
population
is
161.4. This
group
may
be
compared,
firstly,
with
another
wherein
all
states
have
a
percentage
of
urban
population
below
25
and
a
per
capita
wealth
production
under
$3oo;
the
states
thus
selected
present,
as
a group,
19.1
per
cent
urban
population
and
per
capita
wealth
production
of
$175.91. The
number
of
institution
inmates
per
ioo,ooo
of
the
general
population
is but
117.0. This
difference
is
obviously
not
due
to
any
difference
in
percentage
of
urban
pop-
ulation,
which,
in
fact,
is
even
slightly
lower
for
the
first
men-
tioned
group:
the
correlation
is
here
only
with
per
capita
wealth
production. The
second
comparison
may
be
made
with
a
third
group
of
states
wherein
both
percentage
of
urban
population
and
per
capita
wealth
production
are
high,
being,
respectively,
above
25
per
cent
and
above
$300;
the
states
thus
selected
present,
as
a group,
59.3
per
cent
urban
population
and
per
capita
wealth
production
of
$402.16. The
number
of
institution
inmates
per
ioo,ooo
of
the
general
population
is
285.3. This
difference
is
obviously
not
due
to
any
difference
in
per
capita
wealth
production,
which,
in
fact,
is
somewhat
higher
in
the
first
mentioned
group;
the
correlation
is
here
only
with
percentage
of
urban
population. ‘Jarvis,
E.
“Influence
of
Distance
from
and
Nearness
to
an
Insane
Hos-
pital
on
its
Use
by
the
People.”
American
Journal
of
Insanity,
VoL
22.
i86-i866. §.
CORRELATION
BETWEEN
AMOUNT
OF
ILLITERACY
AND
NUMBER
OF
INSTITTJTION
INMATES. Public
education
is
a
costly
thing;
therefore,
adequacy
of
pro-
vision
for
it may
be
expected
to
run
more
or
less
parallel
with
per
capita
wealth
production. Again,
public
education
can
be
more
conveniently
provided
under
urban
than
under
rural
conditions;
therefore,
other
things
being
equal,
adequacy
of
provision
for
it
may
be
expected
to
run
more
or
less
parallel
with
percentage
of
urban
population. If,
then,
the
states
are
dassified
in
groups
according
to
amount
of
illiteracy,
as
in
Table
3,
it
will
be
found,
by
reference
to
the
full
table
of
data
appended
at
the
end,
that
the
grouping
follows
closely
those
which
are
made
according
to
per
capita
wealth
production
and
percentage
of
urban
population,
excepting
that
it
is
in
reverse
order. The
figures
in
Table
3
show
clearly
that
a
reverse
correlation
exists
between
amount
of
illiteracy
and
proportion
of
neuropathic 1915] 229 A. J. ROSANOFF persons
in
institutions;
but
from
what
we
have
already
seen,
it
would
appear
that
this
correlation
is
due
to
a
large
extent
to
the
factors
of
per
capita
wealth
production
and
percentage
of
urban
population. On
the
other
hand,
it
would
seem
equally
certain, TARLE
3.-PER
CAPITA
WEALTH
PRODUCTION,
PERCENTAGE
OF
URBx
Popu-
LATION,
AND
NUMBER
OF
NEUROPATHIC
PERSONS
IN
INSTITUTIONS
PER
100,000
OF
THE
GENERAL
POPULATION
IN
GROUPS
OF
STATES
Dis-
TINGUISHED
ACCORDING
TO
AMOUNT
OF
ILLITERACY. Groupi. f
Group
a. Group
3. Amount
of
Illiteracy. Under
io%
Io.2o%
Over
a0%
Wealth
Production
Urban
Population.. ,.. Institution
Inmates
$375.88
$175.27
$171.86
54.2%
20.0%
19.0%
262.9
128.5
113.3 though
perhaps
hardly
susceptible
of
actual
demonstration,
that
illiteracy
is
itself
a
factor
exerting
at
least
an
indirect
influence. If
an
unduly
large
amount
of
illiteracy
is
in
one
generation
a
result
of
financial
poverty
and
rural
stagnation,
it becomes
for
the
next
generation
a
cause
of
such
conditions. However,
the
case
is
not
so
simple,
being
complicated
by
a
highly
important
relationship
which
exists
between
illiteracy
and
neuropathic
conditions,
a
matter
of
which
a
full
consideration
will
not
be
undertaken
here. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. Nearly
50
years
ago,
Dr. Edward
Jarvis
published
an
admirable
study
of
the
part
played
by
the
factor
of
accessibility
of
institu-
tions
in
determining
the
number
of
patients
committed
to
them.’
He
observed
that
the
number
of
patients
committed
from
any
locality
to
a
hospital
for
the
insane
depended
in
a
large
measure [Oct. A
STUDY
OF
EUGENIC
FORCES 230 on
its
distance
from
and
means
of
transportation
to
the
hospital. This
principle
was
very
well
demonstrated
between
the
years
1843
and
1865
by
admission
rates
to
the
Utica
State
Hospital
in
New
York
from
all
the
counties
of
the
state
excepting
New
York
and
Kings,
which
had
hospitals
of
their
own. Mr. Jarvis
divided
all
the
counties
into
four
districts;
the
first
district
was
Oneida
County,
in
which
the
hospital
was
situated;
the
second
district
consisted
of
the
ii
counties
in
the
first
tier
surrounding
Oneida
County,
which
were
mostly
within
6o
miles
of
Utica;
the
third
district
included
17
counties
which
were
from
6o
to
120
miles
distant;
and
the
fourth
district
included
the
most
distant
counties,
being
120
and
350
miles
from
Utica. From
these
four
districts
the
average
annual
admission
rates
in
relation
to
the
general
popu-
lation
during
the
years
mentioned
were,
respectively,
I
to
2772,
I
to
5820,
I
to
7351,
and
I
to
11,535. In
a
similar
way,
Dr. Jarvis
examined
statistics
from
insane
hospitals
in
21
other
states
and
in
Canada. The
table
in
which
he TARIx
4.-POPULATION
TO
ONE
PATIENT
ANNUALLY
SENT
TO
LUNATIC
Hos-
PITALS
(FROM
A
STUDY
BY
DL
EDWARD
JARVIS). State. Years. 1
District
i. District
a. District
3. JDistrict
. Districts. Maine. N. Hampshire. Mass
R. Island
New
York
New
Jersey.... Pa
Pa.-East
Pa.-West
Maryland. Virginia
N. Carolina.... Mississippi
.... Louisiana
Tennessee
Kentucky
Ohio
Illinois
Michigan
Missouri
Canada
Nova
Scotia... ‘There
is
apparently
something
unexplained
in
the
record
of
one
county
in
each
of
these
districts.
‘E
l di ‘There
is
apparently
something
unexplained
in
the
record
of
one
county
in
each
of
these
districts.
‘Excluding
1844.47.
‘Excluding
1861-63. these
districts.
‘Excluding
1844.47.
‘Excluding
1861-63. these
districts.
‘Excluding
1844.47.
‘Excluding
1861 63 Excluding
1844.47.
‘Excluding
1861-63. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. 1840-65
I8-65
1833-53
1849-65
1843-65
1848-66
1850-57
1857-66
1857-66
1850-64
1828-59
I856-6o
1858
1848-58
1852-59
I824_552
1838-66
1847-64
1859-65
1851-64’
1853-66
1858-64
2,835
2,440
2,229
3,094
2,772
2,253
6,o#{243}i
5,884
3,650
7,034
5,472
4,875
i,oi8’
6,653
3,923
3,198
5,060
3,306
3,162
5,910
3,184
467
5,171
3,470
3,872
5,279
5,820
3,714
10,793
10,497
xo,85
10,122
12,314
6,433
7,026
15,235
8,318
10,670
7,304
7,865
9,229
12,553
7,227
1,023
5,630
6,28o
4,953
7,351
5,905
17,686
17,414
22,382
23,009
21,579
9,707
13,890
16,645
13,164
12,964
11,712
9,317
11,089
13,989
7,744
1,768
7,890
11,535
23,748
53,629
24,433
10,982
i6,ii
21,399
20,440
24,132
28,873
11,753
14,208
15,983
12,608
3,057
...... 25,105
45,779
21,276
25,822
15,826’
27,801
15,585
58,039
26,933
14,582
‘There
is
apparently
something
unexplained
in
the
record
of
one
county
in
each
of TARIx
4.-POPULATION
TO
ONE
PATIENT
ANNUALLY
SENT
TO
LUNATIC
Hos-
PITALS
(FROM
A
STUDY
BY
DL
EDWARD
JARVIS). 1915] 231 A. J. ROSANOFF presented
a summary
of
his
findings
is here
reproduced
(Table
4). His
comments
in
this
connection
are
of
interest: In
all
these
states
the
privileges
of
the
hospitals
are
offered
equally
to
the
people
of
the
counties. The
patients
of
Oneida
and
Allegany
counties
in
New
York,
of
Mercer
and
Warren
counties
in
New
Jersey,
of
Dauphin
and
Venango
counties
in
Pennsylvania,
can
enter
on
the
same
terms,
enjoy
the
same
advantages,
and
for
the
same
price. The
only
difference
is the
burden
of
cost,
care
and
labor
of
travel
from
their
homes
to
the
place
of
healing. And
yet,
the
actual
use
of
the
hospitals
by,
and
the
practical
value
of
these
institutions
to
the
people
of
the
remote
districts
have
been
only
one-fourth
as
great
in
New
York,
about
one-third
as
great
in
New
Jersey,
and
less
than
one-third
as
great
in
Pennsylvania
as
they
have
been
in
the
districts
near
to
them. Similar
discrepancies
in
favor
of
the
central
counties
and
against
the
distant
counties
are
seen
to
have
existed
in all
the
other
states
whose
record
has
been
obtained. In
the
same
study
Dr. Jarvis
analyzed
the
effect
of
multiplying
hospitals
in
states. To
quote
him
further: This
principle
has
been
remarkably
manifested
whenever
and
wherever
a
second
hospital
has
been
opened
in
any
state
and
placed
in
a
district
remote
from
the
one
previously
in
operation. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. The
people
who
sent
a
few
patients
to
the
distant
institution
now
send
many
to
the
hospital
which
was
brought
to
their
neighborhood. The
number
of
lunatics
that
found
a place
of
healing
was
suddenly
and
permanently
increased. In
Massachusetts,
the
hospital
at
Worcester
was
the
only
state
institu-
tion
for
the
insane
in
the
commonwealth
from
1833
to
1854,
when
the
second
hospital
was
opened
in
Taunton,
Bristol
County,
for
the
south-
eastern
part
of
the
state. The
Worcester
establishment
continued
to
re-
ceive
all
the
patients
from
the
northern,
central
and
western
counties
until
1858,
when
the
third
hospital
was
opened
in
Northampton,
Hampshire
County,
for
the
western
district. In
both
of
these
districts,
there
was
a
sudden
and
large
increase
of
the
insane,
whose
friends
sought
and
used
these
new
places
of
healing
for
them. The
statistics
show
that
following
the
opening
of
the
new
institutions
the
admission
rates
from
the
counties
of
Barnstable,
Bristol,
Dukes,
Nantucket,
and
Plymouth
increased
by
42.9
per
cent;
and
from
the
counties
of
Berkshire,
Franklin,
Hampshire,
and
Hampden
by
52.7
per
cent. In
Ohio,
the
State
Hospital
at
Columbus
received
patients
from
all
the
counties
from
1838
to
when
the
Northern
Asylum
at
Newburgh,
Cuyahoga
County,
and
the
Southern
Asylum
at
Dayton,
Montgomery
County,
went
into
operation
and
received
patients
from
certain
surround-
ing
districts
which
were
defined
by
the
law. Following
the
opening
of
the
new
institutions
the
admission
rate
from
the
northern
district
increased
by
o
per
cent,
and
from
the
southern
district
by
305
per
cent! [Oct. 232 A
STUDY
OF
EUGENIC
FORCES Dr. Jarvis
further
shows
that
a
similar
effect
is produced
by
railroads
and
other
facilities
of
travel: Dr. Jarvis
further
shows
that
a
similar
effect
is produced
by
railroads
and
other
facilities
of
travel: Facilities
of
travel,
navigable
rivers,
canals,
railroads,
public
highways,
public
conveyances,
which
render
communication
easy
and
cheap,
and
inter-
course
familiar,
and
virtually
diminish
distance
from
the
hospital,
increase
the
ratio
of
patients
that
are
sent
to
it. We,
therefore,
find
that
those
counties
which
are
situated
along
the
course
of
rivers,
canals,
roads,
etc.,
leading
directly
to
the
situation
of
the
hospitals,
have
sent
more
patients
to
these
institutions
than
other
counties
of
equal
population
and
at
equal
distances,
but
not
favored
with
these
facilities
of
communication. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. Ten
counties
in
New
York
along
the
line
of
railroad,
canal,
etc.,
east
and
west
of
Utica,
with
easy
means
of
travel,
having
a
sum
of
annual
popula-
tions
equal
to
15,622,250,
sent
2151
patients
to
Utica
(I
in
7263);
while,
during
the
same
period,
ten
other
counties,
northeast
and
southwest
of
Utica,
with
no
easy
means
of
communication,
with
a sum
of
7,840,684
annual
populations,
sent
647
patients,
or
I
in
11,934
of
their
number,
to
the
state
hospital. The
experience
of
more
recent
times
throughout
the
country
affords
ample
corroboration
of
Dr. Jarvis’
observations
and
con-
cluSions,
as
may
be
seen
from
the
statistics
given
in
Table
5. The
states
in
which
the
area
in
square
miles
per
institution
is
great
have
relatively
a
small
number
of
neuropathic
persons
in
institu-
tions,
while
the
opposite
is
true
of
the
states
in
which
the
area
in
square
miles
per
institution
is
small. TABLE
5.-PER
CAPITA
WEALTH
PRODUCTION,
PERCENTAGE
OF
URBAN
POPU-
LATION,
AND
NUMBER
OF
NEUROPATHIC
PERSONS
IN
INSTITUTIONS
PER
100,000
OF
THE
GENERAL
POPULATION
IN
GROUPS
DISTINGUISHED
Ac00RD-
ING
TO
LAND
Aas
IN
SQUARE
MILEs
PER
INSTITUTION. Census
Years. Groupi. J Groupa. Group*. Group. Area
in
sq. mi. per
institution. Under
2,000
a,ooo
to
10.000
io,00o
to
40,000
Over
40,000
1890
(
I
‘900
I
1910
I
I. Wealth
Production.. Urban
Population... Institution
Inmates. Wealth
Production.. Urban
Population... Institution
Inmates. Wealth
Production.. Urban
Population.. Institution
Inmates. $358.69
78.9%
175.1
$360.43
78.0%
306.05
$418.66
68.%
323.6
$271.05
48.5%
iO#{244}.i
$310.79
53.7%
269.3
$331.83
43.9%
220.9
$139.12
24.2%
99.6
$190.46
25.7%
185.6
$254.18
28.1%
161.3
$103.67
16.2%
68.02
$136.78
i8.8%
117.1
$211.66
25.3%
119.05 TABLE
5.-PER
CAPITA
WEALTH
PRODUCTION,
PERCENTAGE
OF
URBAN
POPU-
LATION,
AND
NUMBER
OF
NEUROPATHIC
PERSONS
IN
INSTITUTIONS
PER
100,000
OF
THE
GENERAL
POPULATION
IN
GROUPS
DISTINGUISHED
Ac00RD-
ING
TO
LAND
Aas
IN
SQUARE
MILEs
PER
INSTITUTION. 1915] 233 A. J. ROSANOFF Table
5 shows
also
that
the
factor
of
accessibility
of
institutions,
like
that
of
amount
of
illiteracy,
runs
somewhat
parallel
to
those
of
per
capita
wealth
production
and
percentage
of
urban
popu-
lation. It
seems
obvious,
indeed,
that
the
erection
of
institutions
at
short
distances
from
one
another
in
a state
is but
an
expression
of
the
financial
prosperity
and
high
state
of
social
organization
of
the
community. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. It
would
seem,
further,
that
under
such
conditions
the
factor
of
accessibility
of
institutions
can
hardly
be
regarded
as
an
independent
one,
but
rather
as
merely
constituting
the
mech-
anism
by
means
of
which
the
increase
in
the
number
of
institution
inmates
is
brought
about. Such
is
apparently
the
general
import
of
the
statistics;
yet
a
detailed
examination
of
the
figures
for
individual
states,
as
given
in
the
full
table
of
data
appended
at
the
end,
would
show
that
to
some
extent
the
appearance
and
influence
of
the
factor
of
accessi-
bility
of
institutions
is
independent
of
the
other
factors. That
is
to
say,
given
certain
conditions
of
wealth
production,
percentage
of
urban
population
and
public
education,
institutions
may
or
may
not
be
erected
at
convenient
distances
from
population
districts,
depending,
perhaps,
on
the
amount
of
special
attention
that
the
people
or their
representatives
have
given
to
the
matter
of
the
care
of
the
insane,
feeble-minded,
or
other
neuropathic
persons,
such
special
attention
being,
in its turn,
determined
by
the
quality
of the
leadership
in
the
community,
as
emanating
from
its governing
authorities,
charitable
associations,
medical
and
scientific
bodies,
etc. Perhaps
the
most
striking
evidence
of
the
independent
influence
of
the
factor
of
accessibility
of
institutions
is presented
by
the
conditions
in
Kentucky,
Tennessee,
Virginia
and
West
Virginia,
in
1910,
in
contrast
with
those
in
Georgia,
Idaho,
Louisiana,
Okla-
homa
and
Texas
in
the
same
year. In
these
two
groups
of
states
the
factors
of
per
capita
wealth
production
and
percentage
of
urban
population
do
not
differ
materially,
being,
respectively,
$194.03
and
21.9
per
cent
for
the
first
group
and
$181.89
and
23.3
per
cent
for
the
second
group. The
difference,
however,
in
the
factor
of
accessibility
of
institutions
is
great:
in
the
first
group
the
average
land
area
in
square
miles
per
institution
is
6643.3,
while
in the
second
group
it is
37,092.9;
and
we
find
that,
in spite
of
the
similarity
of
conditions
as
regards
wealth
production
and [Oct. A
STUDY
OF
EUGENIC
FORCES 234 percentage
of
urban
population,
the
number
of
institution
inmates
per
Ioo,Ooo
of
the
general
population
differs
very
considerably,
being
150.9
for
the
first
group
and
113.6
for
the
second. §5. CORRELATION
BETWEEN
INTRA-MURAL
CONDITIONS
AND
NUMBER
OF
INSTITUTION
INMATES.-INSANITY
LAWS. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. Probably
no
factor
is
more
obviously
connected
with
the
pro-
gressive
increase
in
the
number
of
neuropathic
persons
in
institu-
tions
than
that
of
improved
intra-mural
conditions. In
times,
not
very
long
past,
when
the
commitment
of
a
person
to
an
insane
institution
meant
almost
invariably
his
subjection
to
mechanical
restraint,
seclusion
in
a cell,
and
measures
of
punishment
or
disci-
pline,
with
inadequate
feeding,
raiment
and
shelter,
under
condi-
tions
of
revolting
filth,
with
hardly
any
medical
care,
such
commitment
was,
naturally,
resorted
to
as
but
the
last
measure,
justified
only
by
extreme
necessity,
a measure
greatly
deplored
as
the
worst
calamity
that
could
befall
any
one. With
the
gradual
improvement
of
intra-mural
conditions
the
popular
horror
of
insane
institutions
has,
in
large
measure,
disappeared,
so
that
to-day,
in
the
states
of
the
most
advanced
development,
we
find
evidence
of
a
different
feeling,
mainly
in
the
shape
of
increasing
numbers
of
voluntary
admissions:
patients
themselves
and
their
friends
now
have
hope
of
being
benefited
by
a
sojourn
in
a
state
hospital. Obvious
as
the
influence
of
intra-mural
conditions
seems
to
be,
it
is
at
the
same
time
rather
imponderable
and
very
difficult
to
subject
to
measurement
and
correlation
with
other
phenomena. For
present
purposes
statistics
of
per
capita
cost
of
maintenance
in
institutions
have
been
selected
as
being,
possibly,
the
most
satis-
factory
index
of
intra-mural
conditions
among
all
the
available
data. It
is
freely
admitted
that
it
is
at
best
but
an
indirect
index
and
that
its use
introduces
certain
sources
of
error;
the
cost
of
commodities,
prevailing
wages,
etc.,
vary
in
different
parts
of
the
country;
the
expense
of
heating
is high
in
Northern
and
Central
states,
and
low
in
Southern
and
Coast
states;
institutions
of
large
size
can
be
conducted
at
a lower
per
capita
cost
than
those
of
small
size, other
things
being
equal;
dishonest
or
incompetent
management
of
an
institution
may
result
in
very
low
intra-mural
standards
in comparison
with
other
institutions,
better
managed, 1915] 235 A. J. ROSANOFF but
at no
higher
per
capita
cost;
finally,
the
statistics
of
different
institutions
are
perhaps
not
strictly
comparable
on
account
of
differences
in
methods
of
bookkeeping:
what
one
institution
reports
as
an
item
of
maintenance
expense
another
may
report
as
one
of
construction
or
extraordinary
repairs. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. Nevertheless,
it
is self-evident
that
definite
standards
of
intra-
mural
conditions
correspond
to
certain
minima
of
per
capita
cost
of
maintenance;
therefore,
when
a
certain
per
capita
cost
is
reported
by
a
given
institution,
we
may
know
approximately
the
best
that
can
be
expected
in
such
matters
as
quality
and
adequate-
ness
of
rations
and
raiment,
number
of
attendants
in
relation
to
that
of
patients,
amusements
and
recreation,
etc. The
necessary
statistical
data
are
given,
for
most
of
the
states,
in
the
Eleventh
and
Twelfth
Censuses;
the
Tenth
Census
gives
all
disbursements
of
institutions,
including
those
for
construction
and
extraordinary
repairs,
but
not
the
maintenance
expenditures
separately;
the
Thirteenth
Census
gives
no
statistics
of
cost. The
states
for
which
the
necessary
data
are
available
have
been
divided
into
three
groups
according
to
per
capita
cost
of
main-
tenance
of
patients
in
institutions,
and
the
number
of
neuropathic
persons
in
institutions
per
oo,ooo
of
the
general
population
has
been
calculated
for
each
group. The
resulting
figures,
given
in
Table
6,
show
plainly
that
a
correlation
exists
between
per
capita
expenditures
for
maintenance
and
number
of
institution
inmates. TABLE
6.-NUMBER
OF
NEUROPATHIC
PERSONS
IN
INSTITUTIONS
PER
100,000
OF
THE
GENERAL
POPULATION
IN
GROUPS
OF
STATES
DISTINGUISHED
ACCORDING
TO
PER
CAPITA
COST
OF
MAINTENANCE
OF
INSTITUTION
INMATES. Census
Years. Group
I. Group
Group. 3. Annual
per
capita
cost
of
maintenance
in
institutions. Under
$125
$125-$Iso
Over
$‘o
1890
1900
96.7
100.2
131.9
129.7
198.3
277.6 TABLE
6.-NUMBER
OF
NEUROPATHIC
PERSONS
IN
INSTITUTIONS
PER
100,000
OF
THE
GENERAL
POPULATION
IN
GROUPS
OF
STATES
DISTINGUISHED
ACCORDING
TO
PER
CAPITA
COST
OF
MAINTENANCE
OF
INSTITUTION
INMATES. [Oct. 236 A
STUDY
OF
EUGENIC
FORCES There
can
be
no
doubt
that
to-day
the
influence
of
the
factor
of
intra-mural
c(snditions,
as
indicated
by
per
capita
cost
of
main-
tenance,
is
as
potent
as
it
was
10,
20,
or
more
years
ago. p
y
g
To
take
a
striking
instance,
the
annual
per
capita
cost
of
patients
in
the
state
of
Rhode
Island,
according
to
the
statistics
furnished
in
the
report
of
the
superintendent
of
the
state
hospital
for
the
insane
at
Howard
for
the
year
ending
December
31,
1910,
was
$133.67. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. The
per
capita
wealth
production
in
that
state,
according
to the
Thirteenth
Census,
was
$53246;
the
percentage
of
urban
population
was
96.7;
the
area
of the
state
being
but
1067
square
miles
and
the
transportation
facilities
being
very
good,
the
hospital
may
be
judged
to
be
readily
accessible
to all
parts
of
its
district. Rhode
Island
may
be
fairly
compared
with
Connecticut,
Massa-
chusetts
and
New
York,
which,
as
a
group,
show
a
per
capita
wealth
production
of
$427.47
and
a
percentage
of
urban
popula-
tion
of
83.2. In
these
states
the
average
per
capita
cost
of
main-
tenance
in
institutions
in
1910
was
$197.17,
that
is
to
say,
by
47.5
per
cent
higher
than
in
Rhode
Island. As
a
result
we
find
that
the
number
of
institution
inmates
per
100,000
of
the
general
popu-
lation
was,
in
that
year,
237.8
for
Rhode
Island
and
396.5
for
Connecticut,
Massachusetts
and
New
York,
as
a
group. We
turn
with
interest
to
the
report
of
the
Rhode
Island
insti-
tution
in
the
hope
of
gaining
more
direct
information
about
its
intra-mural
conditions. The
following
passages
quoted
from
the
report
convey
some
idea
as
to
how
the
“economy”
is
accom-
plished: In
the
period
of
three
years
ending
December
31,
1910,
our
daily
average
number
has
increased
141
patients. At
the
beginning
of
the
three-year
period
of
which
I speak
we
were
already
beginning
to
feel
stress
from
lack
of
space
for
patients. We
have
managed
to
house
the
additional
accumulation
of
141
patients
by
filling
the
proper
day
spaces
with
beds;
we
are
fast
approaching
the
ioo
mark. Another
fact
which
must
be
borne
in
mind
is
that
some
of
our
buildings
are
wooden
structures,
fast
falling
into
irreparable
decay,
and
containing
equipment
long
since
obsolete. Physically
these
buildings
are
unsafe
and
unsanitary;
add
to
these
two
latter
facts
the
crowded
state
of
these
wards,
as
described
above,
and
we
have
a condi-
tion
created
to
which
we
may
well
look
with
apprehension. The
recommendations
are,
that
we
replace
these
dilapidated
wood
build-
ings
with
proper
modern
construction. Our
laundry
facilities
are
entirely
inadequate. We
need
a
new
laundry
building
this
year. We
can
not 1915] 237 A. J. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. ROSANOFF properly
keep
up
with
the
demands
upon
the
laundry
at
the
present
time
and
I
fail
to
see
how
we
can
open
the
new
reception
hospital
without
this
demand
for
increased
laundry
facilities
being
met. The
need
of
an
administration
building
for
the
entire
hospital
for
the
insane
is greater
than
ever. Our
small
office
space
is
crowded
and
cramped
beyond
measure. There
are
many
states
in
which
provision
for
the
insane
is
much
poorer
and
less
adequate
than
in
Rhode
Island. The
exam-
ple
of
Rhode
Island
has
been
selected
because
it affords
a particu-
larly
clear
demonstration
of
the
cacogenic
influences
of
bad
intra-mural
conditions;
for
in
that
state
the
lack
of
thoroughness
of
segregation
of
neuropathic
persons
cannot
be
ascribed
to
lack
of
means
or
to
difficulties
resulting
from
a
large,
thinly-settled
area
with
imperfect
transportation
facilities,
but
apparently
rather
to
a
deliberate
pursuit
by
the
governing
authorities
of
a
cruel
and
short-sighted
penny-wise
policy. Another
feature
of
intra-mural
conditions
which
hinders
the
segregation
of
neuropathic
persons
in
special
institutions
is
simply
lack
of
room. Overcrowding
exists
in
almost
all
institutions
in
this
country
to
a greater
or
less
extent;
it
seems
to
be
the
general
practice
of
the
legislative
bodies
of
all
states
to
ignore
the
recom-
mendations
of
hospital
superintendents,
boards
of
administra-
tion,
or
other
authorities
who
are
charged
with
the
duty
of
caring
for
the
insane
and
who
are
best
informed
as
to
the
needs,
being
in
immediate
contact
with
the
problems. But
in
some
states
the
conditions
are
extreme
and
thoroughly
disgraceful. Such
an
extreme
instance
is presented
by
the
state
of
Louisiana,
as
may
be
judged
from
a
rather
remarkable
statistical
table
pub-
lished
in
the
biennial
report
of
the
Board
of
Administrators. The
data
for
this
table
have
been
collected
by
the
superintendents
of
both
the
state
asylums
from
the
sheriffs
of
the
various
parishes
of
the
state
by
correspondence. Of
the
6o
parish
sheriffs
in
the
state,
only
36
responded
to
the
letters
of
inquiry,
so
that
the
resulting
statistics
reveal
only
in
part
the
existing
conditions. Following
is
a
summary
of
these
statistics: Number
of
persons
in
jails
awaiting
admission
139
Number
who
died
in
jails
during
the
biennial
period
while
awaiting
admission
so
Number
of
applications
for
admission
to
either
asylum... §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. 609 In
some
states
it
is
the
declared
policy,
embodied
in
the
law,
to
limit
the
provision-such
as
it
is
in
those
states-to
the
care
of 238 A
STUDY
OF
EUGENIC
FORCES [Oct. certain
variously
selected
groups
of
the
insane
and
to
require
the
carrying
out
of
cumbersome
procedures
in
each
case
to
determine
the
patient’s
eligibility
for
admission
and
his
claim
to
it. As
fur-
nishing
a
typical
instance
may
be
cited
the
law
of
the
state
of
Arkansas. Upon
a written
statement
filed
by
any
reputable
citizen
of
the
state
with
the
county
and
probate
judge
alleging
that
any
person
is
entitled
to
admis-
sion
to
the
state
hospital
for
the
insane,
the
county
or
probate
judge
must
appoint
a
time
as
soon
as
practicable
to
hear
the
testimony
of
witnesses,
and
must
cause
the
insane
person
to
be
examined
separately
by
two
repu-
table,
competent,
and
disinterested
physicians,
who
shall
severally
present
to
the
county
judge
a sworn
statement
of
the
result
of
their
examinations. If
the
judge
finds
the
person
insane
he
must
without
delay
transmit
to
the
superintendent
of
the
state
hospital
his
decision
in
writing,
with
copies
of
the
original
statement
filed
with
him
by
the
citizen,
and
the
statement
of
the
physicians
including
interrogatories
and
answers. The
superintendent
of
the
state
hospital
must
thereupon
immediately
notify
the
judge
of
his
readiness
to
admit
the
insane
person
if
there
is
room
unoccupied. If
there
is
no
room,
he
must
notify
the
judge,
and
return
to
him
the
documents
in
the
case. But
in
such
instances,
the
name
and
county
of
the
insane
person
must
be
recorded
at
the
hospital
in
the
order
in
which
the
decision
of
the
judge
was
received,
and
he
is entitled
to
precedence
over
all
who
may
apply
for
admission
later. ‘The
above
digest
of
the
insanity
law
of
the
state
of
Arkansas
is quoted
from
“Summaries
of
Laws
relating
to
the
Commitment
and
Care
of
the
Insane
in
the
United
States,”
prepared
by
John
Koren
and
published
by
The
National
Committee
for
Mental
Hygiene,
o
Union
Square,
New
York,
1912. §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. No
appeal
may
be
had
from
the
decision
of
the
superintendent
in
matters
of
admission
or
discharge
of
inmates,
except
to
the
board
of
trustees
of
the
asylum,
who
may
direct
the
superintendent
to
admit
or
discharge
any
person
under
any
circumstances
not
involving
a violation
of
the
law. When
by
reason
of
recovery
or
necessity
for
the
benefit
of
an
insane
person
of
the
“acute”
class
the
superintendent
of
the
state
hospital
thinks
proper
to
discharge
any
inmate,
he
must
notify
the
county
or
probate
judge
of
the
county
from
which
the
inmate
was
committed,
who
shall
then
direct
the
removal
of
the
inmate
without
delay
to
his
guardian
or
home. If
the
person
has
no
guardian
or
home
he
must
be
delivered
to
such
party
and
place
in
the
county
as
may
be
provided
for
his
further
custody
and
mainte-
nance,
if
he
be
not
recovered
and
capable
of
taking
care
of
himself. An
inmate
of
the
hospital
having
recovered
his
reason
may
unofficially
be
removed
from
the
asylum
by
consent
of
the
superintendent,
or
by
his
friends
without
consent
of
the
superintendent,
or
by
direction
of
the
board
of
trustees. But
notice
of
removal
must
be
sent
at
once
to
the
county
or
probate
judge
of
the
county
from
whidi
the
person
was
removed
or
com-
mitted.1 §4.
CoRRELATION
BETWEEN
ACCESSIBILITY
OF
INSTITUTIONS
AND
NUMBER
OF
INSTITUTION
INMATES. All
persons
found
to
be
insane,
for
whom
application
for
admission
to
the
state
insane
asylum
is
made,
are
classified
as
“acute,”
“chronic,”
“probably
incurable,”
or
“incurable.”
All
cases
of
less
than
one
year’s
duration
from
first
recognized
symptoms
of
insanity
are
classified
as
“acute”;
all
cases
of
one
year’s
duration
as
“chronic”;
all
cases
com-
plicated
with
epilepsy,
original
imbecility
or
feeble-mindedness,
deformities
of
skull
from
injuries,
old
age
or
paralysis
as
“probably
incurable”;
and
all
other
cases
as
“incurable,”
provided
that
no
person
of
either
classifica-
tion
whether
curable
or
not, and
whether
the
imbecility
or
insanity
be
idiotic
or
congenital
or
not,
may
be
refused
admission
as
long
as
there
is
unoccupied
room
for
patients
in
the
hospital. Insane
persons
found
at
large,
and
not
in
the
care
of
some
discreet
person,
must
be
arrested
by
any
peace
officer
and
taken
beforea
magistrate
of
the
county,
city
or
town
in
which
the
arrest
is
made,
who
shall
make
the
neces-
sary
orders
to
keep
him
in
restraint
until
he
can
be
sent
to
the
insane
hospital. If
the
insane
person
has
no
friends
to
whose
custody
the
magistrate
can
commit
him,
he
may
order
him
to
be
confined
in
the
county
or
city
jail,
giving
immediate
notice
to
the
county
judge
or
city
attorney,
whose
duty
it
is
to
take
the
proper
proceedings
for
having
the
insane
person
sent
to
the
hospital. When
there
is
no
available
room
in
the
hospital,
the
superintendent
must,
as
soon
as
practicable,
in
order
to
make
room
for
a
patient
suffering 1915] 239 A. J. ROSANOFF from
an
“acute”
form
of
insanity,
discharge
some
inmate
belonging
to
the
“incurable”
class,
if
there
is
such,
or
one
belonging
to
the
“probably
incurable”
class
if
there
is
no
one
dischargeable
from
the
fourth
class,
or
one
belonging
to
the
“chronic”
class
if
there
is
no
one
dischargeable
from
the
third
class. In
making
a
selection
for
discharge,
the
superintendent
must
give
preference
to
one
who
has
been
longest
in
the
hospital,
if
not
violent
or
dangerous
to
the
community,
or
whose
discharge
will
effect
the
least
possible
inconvenience
or
cost. §6.
SIGNIFICANCE
OF
THE
DATA
EXHIBITED
IN
THE
PRECEDING
SECTIONS. The
Financial
Aspect.-The
fact
that
the
number
of
neuro-
pathic
persons
in
institutions
is
in
correlation
with
per
capita
wealth
production
would
indicate
that
this
factor
controls,
in
a
measure,
the
kind
and
adequateness
of
the
facilities
possessed
by
states
for
the
care
of
such
persons. Yet,
it
can
be
shown
that
such
control
is,
in
the
nature
of
things,
not
inevitable,
and
owes
its
existence
merely
to
a
fallacious
financial
policy. The
benefit
that
is
gained
for
public
welfare
by
the
segregation
of
neuropathic
persons
instead
of
allowing
them
to
remain
at i6 [Oct. A
STUDY
OF
EUGENIC
FORCES 240 large
is
mainly
in
the
shape
of
greater
protection
against
vice
and
crime. This
subject
has
been
more
fully
considered
else-
where,’
and
will
not
be
further
referred
to
here,
beyond
pointing
out
that
money
spent
in
segregation
is
not
without
some
balance
on
the
credit
side
which
results
from
the
saving
through
diminu-
tion
of
the
amount
of
vice
and
crime. But
the
benefits
that
hospitals
and
asylums
afford
are
to
be
felt
not
only
by
the
present
generation,
but
also,
and
possibly
even
to
a
greater
extent,
by
generations
to
come;
it
would
seem
fair,
there-
fore,
that
a
part
of
the
large
expense
of
segregation
should
be
borne
by
them. Large
enterprises,
whether
of
governments
or
of
private
cor-
porations,
are
capitalized
with
the
aid
of
public
loans
of
long
term,
while
capital
that
is
otherwise
available
is
used
merely
for
current
expenses
of
operation. Thus
public
schools,
highways,
canals,
railroads,
telegraph
and
telephone
systems,
manufacturing
plants,
power
plants,
mining
equipments,
etc.,
have
been
built
with
the
aid
of
capital
derived
from
issues
and
reissues
of
long-term
bonds;
and
no
one
has
questioned
the
equity
of
thus
assuming
debts
to
be
paid
by
future
generations. It
is
a
curious
fact
that
the
establishment
of
institutions
for
neuropathic
persons
forms
almost
everywhere
a
unique
exception. No
state
as
yet
possesses
anything
like
adequate
facilities
for
the
care
of
its
neuropathic
persons. Yet
every
state
state
could,
without
any
added
expendi-
ture,
easily
double
or
treble
its
facilities
with
the
aid
of
bond
issues
and
thus
make
funds
that
are
now
expended
for
construc-
tion
available
for
the
maintenance
of
the
increased
number
of
its
wards. ‘Rosanoff,
A.
J.
“A
Program
of
Psychiatric
Progress.”
Medical
Record,
February
20,
1915.
‘Annual
Report
of
the
Comptroller
of
the
State
of
New
York,
January
s’
1913. ‘Annual
Report
of
the
Comptroller
of
the
State
of
New
York,
January
s’
1913. ‘Rosanoff,
A.
J.
“A
Program
of
Psychiatric
Progress.”
Medical
Record,
February
20,
1915. §6.
SIGNIFICANCE
OF
THE
DATA
EXHIBITED
IN
THE
PRECEDING
SECTIONS. In
New
York
State
alone
the
total
legislative
appropria-
tions
of
special
funds
for
new
construction
in
the
state
hospitals
and
charitable
institutions
in
the
year
1912
was
$3,210,304;
while
the
appropriations
desired
for
similar
purposes
at
the
1913
session
of
the
legislature
were
$5,747,849.50.’ If
the
present
generation
could
have
the
choice
between
having
to
pay
for
the
maintenance
of
a
large
number
of
neuropathic
persons
and
having
merely
to
pay
off
the
matured
bonds
issued
by
a
preceding
generation
for
the
establishment
of
facilities
of
pre- 1915] 24! A. J. ROSANOFF vention,
there
would
be
no
question
as
to
its
preference;
but
it
has
not
that
choice,
for
past
generations
acted
against
its
prefer-
ence. And
should
the
present
generation
decline
to
incur
a
bond
indebtedness
to
be
paid
off
in
the
future
and
thus
allow
neuro-
pathic
persons
to
remain
at
large
for
lack
of
room
in
the
institu-
tions,
its
action
would
be
similarly
against
the
preference
of
future
generations. No
other
enterprise
that
the
state
might
undertake
could
more
fully
justify
the
issuance
of
long-term
bonds
than
that
of
the
con-
struction
of
adequate
facilities
for
the
prevention,
treatment
and
custody
of
mental
disorder. The
Factor
of
Urban
Population.-Arnong
the
features
of
modern
civilization
are,
as
all
know,
a
stream
of
migration
from
country
to
city,
organization
of
new
urban
centers,
and
often
the
abandonment
of
rural
pursuits
for
those
of
manufacture,
com-
merce,
etc. The
special
advantages
and
opportunities
to
be
found
in
cities
have
lured
many
of
the
most
ambitious
and
most
capable
men
and
women
away
from
farms
and
villages. This
movement
has
aroused
considerable
alarm,
but
it
would
seem
clear
that,
from
the
eugenic
standpoint,
it
has
a
beneficial
aspect,
since,
as
has
been
shown
in
§2,
the
practice
of
segregating
neuropathic
persons
in
institutions
increases
with
the
percentage
of
urban
population. Are
rural
conditions,
then,
destined
forever
to
be
cacogenic? Are
rural
communities
to
continue
indefinitely
to
lose
so
many
of
their
best
forces? The
great
institutions
of
modern
civilization-public
schools,
newspapers,
improved
means
of
transportation
and
communica-
tion,
municipal
sanitation,
hospitals,
corporate
enterprises,
are
of
urban
origin,
and
it
is
not
surprising
that
rural
communities
have
remained
somewhat
behind. §6.
SIGNIFICANCE
OF
THE
DATA
EXHIBITED
IN
THE
PRECEDING
SECTIONS. But
the
very
growth
of
cities
must
bring
with
it
eventually
a
general
elevation
of
rural
standards
as
well;
the
more
disproportionate
this
growth
becomes,
the
higher
will
rise
the
prices
of
agricultural
products,
so
that
rural
pursuits
will
gradually
develop
a
new
attractiveness. Such,
at
least,
has
been
the
experience
of
the
past:
“A
large
part
of
the
extraordinary
increase
in
the
total
value
of
farm
crops
between
1899
and
1909
is attributable
to
higher
prices. While
the
acreage
of
crops
with
acreage
reports
increased
only
9.9
per
cent,
the
value [Oct. A
STUDY
OF
EUGENIC
FORCES 242 of
such
crops
increased
83.3
per
cent. The
percentages
of
in-
crease
in
the
quantity
of
the
various
individual
crops
were
in
nearly
all
cases
much
less
than
the
percentages
of
increase
in
value. Thus,
for
all
cereals
taken
together,
the
production
increased
only
1.7
per
cent,
while
the
value
increased
79.8
per
cent;
for
hay
and
forage
the
production
increased
23
per
cent
and
the
value
70.2
per
cent;
and
for
cotton
(including
cotton
seed),
the
production
increased
11.7
per
cent
and
the
value
122.5
per
cent.” Furthermore,
it
is
a
matter
not
alone
of
quantitative
relation
between
demand
and
supply:
urban
demands
are
growing
more
exacting
also
as
to
quality
of
agricultural
products. Milk
must
be
from
healthy
cows,
bacteriologically
innocuous,
carefully
pre-
pared
for
shipping,
chemically
up
to
certain
legal
specifications,
often
even
pasteurized;
it
will
command
a
higher
price
if
regu-
larly
inspected
by
experts
and
certified. Fruits
must
be
skilfully
cultivated,
carefully
selected,
and
attractively
packed
to
meet
the
demands
of
urban
markets. To
supply
products
in
ever
increasing
quantities
and
of
rising
quality
in
response
to
urban
demands
will
eventually
become
a task
no
longer
for
an
illiterate
peasantry
but
for
a
working
force
fully
as
intelligent,
as
enterprising
and
as
well
trained
as
any
that
is
required
for
the
production
of
the
finest
manufactured
products. ‘Abstract
of
the
Thirteenth
Census,
p. 363. §6.
SIGNIFICANCE
OF
THE
DATA
EXHIBITED
IN
THE
PRECEDING
SECTIONS. The
farmer,
like
the
manufacturer,
will
make
increasing
use
of
labor-saving
machinery
and
of
scientific
methods
of
work
and
management;
he
will
market
superior
products
and
receive
higher
prices;
he
will
demand
shorter
hours
of
labor,
and,
unlike
his
peasant
predecessor,
insist
on
standards
of
living
as
high
as
those
of
the
man
in
the
city;
he
will
refuse
to
get
along
without
a
safe
and
ample
water
supply,
sanitary
facilities
for
sewage
dis-
posal,
efficient
heating
and
lighting
equipments,
and
without
good
schools,
hospitals,
roads,
telephones,
means
of
recreation. The
insane
and
feeble-minded
will
not
be
at
large,
or
doing
chores
on
the
farmsteads,
but
will
be
cared
for
in
institutions
to
the
same
extent
as
those
of
urban
communities. A
great
deal
of
urban
growth,
however,
will
still
have
to
take
place
more
or
less
at
the
expense
of
the
country
before
the
attract-
iveness
of
rural
and
urban
pursuits,
regarded
from
the
economic 1915] 243 A. 5. ROSANOFF standpoint,
will
have
become
equalized,
and
rural
standards
of
living
and
civilization
are
raised
to
the
level
of
urban
standards. By
some,
the
continued,
almost
universal,
increase
of
the
cost
of
living
is
regarded
with
anxiety,
as
an
abnormal
social
phe-
nomenon;
but
it
seems
to
us
that
this
increase
is
but
a
sign
of
rural
progress. Heretofore
rural
labor
has
been,
perhaps
to
a
greater
extent
than
any
other
kind
of
labor,
unfairly
exploited
by
the
nation;
and
it
is
still
being
thus
exploited. In
ultimate
results
to
the
nation
such
exploitation
cannot
be
beneficial. We
shall
know
that
it
has
ceased,
that
agricultural
labor
is
as
well
rewarded
as
other
labor,
that
rural
living
conditions
are
as
attract-
ive
as
urban
ones,
that
a
fair
balance
has
established
itself,
when
the
stream
of
migration
cityward
has
ceased. The
Factor
of
Illiteracy.-Probably
no
other
factor
is
so
potent
in
its
influence
on
standards
of
living
and
civilization
as
is
that
of
public
education. An
undue
prevalence
of
illiteracy
in
a
com-
munity
is
sure
to
be
accompanied
by
a low
state
of
all
institutions
of
civilization,
including
facilities
for
the
segregation
of
neuro-
pathic
persons:
it
is
a
cacogenic
factor. Conversely,
money
spent
for
public
education
is
spent,
at
least
indirectly
or
in
ultimate
effect,
in
an
eugenic
cause. §6.
SIGNIFICANCE
OF
THE
DATA
EXHIBITED
IN
THE
PRECEDING
SECTIONS. The
Factors
of
Accessibility
of
Institutions
and
Intra-niural
Conditions.-The
factors
of
accessibility
of
institutions
and
intra-
mural
conditions
are
apt
to
work
against
each
other
in
a
peculiar
way. A
large
number
of
small
institutions
scattered
throughout
a
state
makes,
of
course,
for
accessibility. But
the
maintenance
of
small
institutions
is
expensive;
therefore,
given
a
certain
amount
of
money
available,
the
same
number
of
patients
can
be
maintained
under
better
intra-mural
conditions
in
a
small
number
of
large
institutions
than
in
a
larger
number
of
small
ones. In
densely
populated
areas
or
in
the
proximity
of
large
cities
the
choice
is
obviously
in
favor
of
large
institutions,
for
in
such
localities
multiplying
the
number
of
institutions
would
result
in
no
gain
in
accessibility,
but
only
in
loss
of
economy
in
administra-
tion. But
even
for
large
and
sparsely-settled
areas
the
factor
of
nearness
to
the
population
districts
is
becoming
less
important
with
the
improvement
and
extension
of
steam
and
electric
rail-
roads,
canals,
river
navigation,
highways,
automobile
travel,
etc. However,
even
with
the
best
transportation
facilities,
no
institu- [Oct. A
STUDY
OF
EUGENIC
FORCES 244 tion
can
be
considered
sufficiently
accessible
to
its
population
dis-
tricts
which
is
not
within
5o
miles
of
them. In
many
states
the
law
endeavors
to
distinguish
the
insane
from
idiots,
imbeciles,
and
epileptics,
often
making
separate
provisions
for
the
classes
of
neuropathic
persons
thus
distinguished. Much
can
be
said
in
favor
of
such
separate
provisions,
but
it may
readily
be
seen
that,
from
the
standpoint
of
accessibility
of
institutions,
the
disadvantages
would
outweigh
the
advantages
in
large
and
sparsely-settled
areas. In
densely-settled
states,
where
it
is
both
practicable
and
advan-
tageous
to
make
such
separate
provisions,
the
prevailing
custom
to
hold
by
legal
commitment
only
“insane”
patients
in
state
hos-
pitals
and
not
inmates
of
institutions
for
the
feeble-minded
or
epileptic,
constitutes
a
serious
drawback. This
is,
however,
not
unavoidable;
very
recently,
for
instance,
a
law
was
enacted
in
New
York
providing
for
the
commitment,
when
necessary,
of
epileptic
and
feeble-minded
persons
to
special
institutions
estab-
lished
for
their
care
by
a
procedure
similar
to
that
employed
for
the
commitment
of
insane
persons
to
the
state
hospitals. It
follows
from
the
above
that
a general
policy
could
hardly
be
formulated
which
would
best
govern
the
factors
of
accessibility
of
institutions,
intra-mural
conditions,
and
specialization
as
to
classes
of
cases
cared
for,
under
all
conditions. §6.
SIGNIFICANCE
OF
THE
DATA
EXHIBITED
IN
THE
PRECEDING
SECTIONS. In
some
cases,
a
number
of
small
institutions,
not
too
far
apart,
and
without
special
restrictions
as
to
classes
of
patients
admitted,
may
be
preferred
in
spite
of
high
expense
of
maintenance;
in
other
cases
a
smaller
number
of
large
institutions,
each
organized
for
the
care
of
special
classes
of
patients,
may
be
found
more
economical
and
in
every
way
more
desirable. Laws
Pertaining
to
the
Care
of
Neuropathic
Persons.-A
com-
parison
of
the
laws
of
the
various
states
reveals
the
general
fact
that
those
states
which
have
the
greatest
number
of
neuropathic
persons
in
institutions
in
proportion
to
their
general
population
have
also
the
simplest
legal
procedures
for
the
admission
of
patients,
and,
vice
versa,
those
which
have
the
least
numbers
have
also
the
most
cumbersome
legal
procedures
and
the
greatest
num-
ber
of
obstacles
and
restrictions. The
tendency
throughout
the
country
in
recent
times
has
been
towards
a
simplification
of
the
legal
procedure,
and
now,
in
the
leading
states,
cases
may
be 1915] 245 A. 5. ROSANOFF admitted
to
insane
hospitals
on
voluntary
application,
at
any
time,
without
special
formality. Psychiatrists
are
looking
forward
to
even
greater
facility
of
obtaining
treatment
for
cases
of
mental
derangement
in
the
future
in
psychopathic
wards
to
be
established
in
connection
with
general
hospitals: The
details
of
transfer
from
the
psychopathic
ward
to
the
large
state
institutions
should
be
made
as
simple
as
possible. Transfer
should
be
made
effective
on
a certificate
of
two
properly
qualified
physicians
and
the
matter
should
not
have
to
come
into
court
at
all
unless
it
is
brought
there
by
the
patient,
his
relatives,
or
some
friends
on
his
behalf. I
would
not
close
the
courts
to
the
so-called
insane
by
any
means,
but
I
would
not
insist
on
a
legal
process,
whether
the
patient
wanted
it
or
not;
I
would
not
insist,
so
to
speak,
on
cramming
an
alleged
constitutional
right
down
the
patient’s
throat
at the
expense
of
his
life. We
see
to-day
this
process
of
commitment
going
on
where
nobody
wants
it. The
patient
does
not
want
it,
the
patient’s
friends
and
relatives
do
not
want
it,
and
anybody
who
stands
and
watches
it proceed
recognizes
on
the
face
of
it that
it is a farce. I would,
therefore,
proceed
in
the
matter
of
commitment
in
the
simplest
way. ‘White,
Wm.
A.
“Dividing
Line
between
General
Hospital
and
Ho:-
pital
for
Insane.
The
Modern
Hospital,
March,
1914. §6.
SIGNIFICANCE
OF
THE
DATA
EXHIBITED
IN
THE
PRECEDING
SECTIONS. Leave
the
courts
accessible
to
the
patient
if
he
wants
to
appeal
for
relief,
and
it
will
be
surprising
how
rare
such
appeals
will
be.’ A
study
of
the
practice
of
segregating
neuropathic
persons
in
institutions,
as
revealed
by
its
growth
throughout
the
country
in
the
past
few
decades
and
by
its
extent
in
the
various
states,
shows
plainly
that
where
it
is
least
controlled
by
legislation
there
it
is
most
successful. A
state
needs
but
to
provide
ample,
well-con-
ducted
and
readily
accessible
institutions;
for
the
rest,
contrary
to
the
commonly
expressed
notion,
forced
segregation
is
far
less
effective
than
free
or
voluntary
segregation. The
interests
of
eugenics
are
best
served
where
detention
by
law
is
reduced
to
a
minimum. It
is
necessary,
above
all,
to
abolish
entirely
the
horror
of
insti-
tutions
that
still
lingers
in
the
minds
of
the
public
even
in
the
most
advanced
states. This
has
already
been
accomplished
in
part
by
the
general
improvement
of
intra-mural
conditions. Perhaps
of
equal
importance
is
a
liberal
practice
in
the
matter
of
discharge;
for,
in
spite
of
possibly
ideal
intra-mural
conditions,
any
grounds
for
fear
of
permanent
or
indefinite
detention
without
obvious [Oct. A
STUDY
OF
EUGENIC
FORCES 246 indications
will
naturally
serve
to
impede
the
stream
of
admis-
sions
to
institutions. Nobody
will
willingly
seek
admission
to
an
institution
or
send
a
relative
or
a
friend
there
if
there
is
any
likelihood
of
that
step
proving
to
be
an
irrevocable
one. Theo.-
retically
the
permanent
forced
segregation
of
every
neuropathic
person
might
be
assumed
to
be
most
effective;
but
practically
all
experience
shows
that
the
greatest
freedom
and
liberality,
the
least
force,
are
the
conditions
of
the
greatest
degree
of
success
as
regards
extent
of
segregation
accomplished. Unlike
other
eugenic
measures
that
have
been
proposed,
segre-
gation
is
an
old
practice
which
has
been
thoroughly
tried
out
and
to
which
no
objections
have
been
raised,
whether
on
religious,
legal,
or
humanitarian
grounds;
it
has
had
of
late
a
remark-
able
growth;
and
it
may
be
anticipated
that
improvement
of
methods
of
financing
will
create
vast
possibilities
of
further
growth. In
the
past
the
objects
of
segregation
have
been
to
protect
society
against
anti-social
conduct
of
neuropathic
persons,
to
organize
public
charity
on
an
efficient
basis,
and
to
provide
medical
treatment
for
cases
requiring
it. §6.
SIGNIFICANCE
OF
THE
DATA
EXHIBITED
IN
THE
PRECEDING
SECTIONS. That
it
also
serves
the
ends
of
eugenics
is
but
an
added
argument
in
its
favor;
but
whatever
it
accomplishes
for
eugenics
must
ever
remain
a
by-
product,
no
matter
how
highly
we
may
value
it. In
other
words,
in
no
case
could
forced
segregation
be
advocated
with
propriety
and
with
social
benefit
merely
because
a
diagnosis
of
some
neuro-
pathic
condition
has
been
made
and
on
eugenic
grounds
alone;
in
the
absence
of
a need
of
protection
for
society,
or
of
an
appeal
for
aid
or
for
medical
treatment,
indications
for
segregation
do
not
exist. This
point
of
view
would
eliminate
the
practically
unanswerable
question
in
eugenics,
What
persons
should
be
selected
for
segre-
gation? The
selection
is
made
automatically
according
to
the
standards
and
exactions
of
every
environment. There
can
be
no
doubt
that
many
persons,
say,
in
Arkansas,
New
Mexico
or
Oklahoma,
who
are
at
large
and
whom,
moreover,
their
fellow
citizens
do
not
consider
proper
subjects
for
an
insane
hospital,
would
be
promptly
committed
if
they
took
up
their
resi-
dence
in,
say,
Massachusetts,
New
York
or
Connecticut;
and
some
would
apply
for
voluntary
admission. And
it
is
equally
certain 1915] 247 A. J. ROSANOFF that
under
still
more
highly
developed
conditions
many
persons
who
are
now
at
large
even
in
the
last
mentioned
states
would
be
cared
for
in
institutions. Persons
are
placed
in
institutions
when,
by
reason
of
some
mental
defect
or
disturbance,
their
adaptation
to
their
environ-
ment
fails. The
environment
of
a
highly
organized
community
with
high
standards
of
living
is,
of
course,
more
exacting
than
that
of
a
community
characterized
by
a more
primitive
organiza-
tion
and
lower
standards. Whatever
may
be
one’s
theoretical
conception
of
neuropathic
conditions,
the
line
of
division
between
these
conditions
and
the
normal
condition,
as
it
is indicated
by
the
practice
of
communities,
is
a
shifting
one,
moving
from
the
abnormal
toward
the
normal
extreme
with
the
progress
of
civilization
and
the
concomitant
elevation
of
social
standards. One
may
well
question,
therefore,
whether
even
theoretically
any
but
a
provisional
and
temporary
definition
of
neuropathic
conditions
could
be
formulated. §6.
SIGNIFICANCE
OF
THE
DATA
EXHIBITED
IN
THE
PRECEDING
SECTIONS. One
who
believes
in
indefinite
possibilities
of
human
progress,
not
only
sociologically
but
also
by
organic
evolution,
may
well
imagine
a state
in
the
future
wherein
the
general
moral,
intellectual
and
artistic
standards
would
be
so
high
that
many
persons
such
as
are
to-day
active
and
even
influen-
tial
in
politics,
in
commerce,
or
in
society,
and
are
but
rarely
picked
out
for
segregation
in
asylums
or
penal
institutions,
would
then
be
so
far
below
the
average
as
to
be
considered
abnormal
and
not
to
be
tolerated
at
large. As
a
community
makes
progress
in
general
civilization,
so
will
it make
progress
in
eugenic
practice;
no
theoretical
standards
can
be
forced
on
any
community;
and
no
standards
of
one
community
can
be
suddenly
forced
on
any
other
that
is
in
a different
stage
of
social
organization. §7.
SUMMARY. The
number
of
the
insane
in
asylums
and
hospitals
has
been
on
the
increase
during
the
past
several
decades
at
a
more
rapid
rate
than
the
general
population. In
the
United
States
there
were
in
i88o,
86.
institution
inmates
per
ioo,ooo
of
the
general
population;
by
1910
the
number
had
risen
to
232.0. 248 [Oct. A
STUDY
OF
EUGENIC
FORCES At
any
given
time
the
number
differs
widely
in
the
various
states. At
any
given
time
the
number
differs
widely
in
the
various
states. In
1910
there
were
in
the
state
of
Oklahoma
67.0
institution
inmates
per
Ioo,ooo
of
the
general
population,
while
in
the
state
of
Massachusetts
there
were
413.4. To
attempt
an
analysis
of
conditions
which
have,
independently
of
any
eugenic
movement,
determined
in
the
various
states
of
the
Union
the
extent
of
the
practice
of
segregating
neuropathic
per-
sons
in
special
institutions
is
the
main
object
of
this
study. The
number
of
neuropathic
persons
in
institutions
per
Ioo,Ooo
of
the
general
population,
in
the
United
States
as
a whole
at
differ-
ent
times,
and
in
the
various
states
at
any
one
time,
is
in
direct
correlation
with
the
following
factors:
(i)
per
capita
wealth
production
;
(2)
percentage
of
urban
population;
(3)
accessi-
bility
of
institutions;
and
(4)
intra-mural
conditions,
as
indicated
by
per
capita
cost
of
maintenance
in
institutions. It
is
in
reverse
correlation
with
the
factor
of
percentage
of
illiteracy. The
controlling
influence
of
the
factor
of
per
capita
wealth
production
would
seem
to
be
not
inevitable,
in
the
nature
of
things,
but
rather
due
to
a fallacious
financial
policy
generally
in
vogue. The
benefits
of
segregation
are
to
be
felt
not
only
by
the
present
generation,
but
also,
and
probably
to
a
greater
extent,
by
genera-
tions
to
come;
it
would
seem
fair,
therefore,
that
a
part
of
the
large
expense
of
segregation
should
be
borne
by
them. Every
state
could
easily
double
or
treble
its
facilities
for
segre-
gation
with
the
aid
of
bond
issues
and
thus
make
funds
that
are
now
expended
for
construction
available
for
the
maintenance
of
the
increased
number
of
its
wards. §7.
SUMMARY. As
regards
the
factor
of
percentage
of
urban
population,
the
stream
of
migration
from
country
to
city,
which
is
so
character-
istic
of
modern
times
and
which
has
been
by
many
greatly
deplored,
seems,
from
the
eugenic
standpoint,
to
have
a
beneficial
aspect;
it
is
undoubtedly
a
cause
of
the
almost
universal
rise
in
prices
of
agricultural
products
and
thus
a
growing
influence
for
the
elevation
of
rural
standards. An
undue
prevalence
of
illiteracy
in
a community
is
a cacogenic
factor. 1915] 249 A. 5. ROSANOFF Conversely,
money
spent
for
public
education
is
spent,
at
least
indirectly
or
in
ultimate
effect,
in
an
eugenic
cause. Conversely,
money
spent
for
public
education
is
spent,
at
least
indirectly
or
in
ultimate
effect,
in
an
eugenic
cause. The
factors
of
accessibility
of
institutions
and
intra-mural
con-
ditions
are
apt
to
work
against
each
other
in
a
peculiar
way:
a
general
policy
could
hardly
be
formulated
that
would
best
govern
them. The
opinion
is offered
that
no
institution
is sufficiently
accessible
to
its
population
districts
which
is
not
within
5o
miles
of
them. In
some
cases
a number
of
small
institutions,
not
too
far
apart,
and
without
special
restrictions
as
to
classes
of
patients
admitted,
may
be
preferred
in
spite
of
high
expense
of
maintenance. In
other
cases,
especially
in
densely
populated
districts,
a
smaller
number
of
large
institutions,
each
organized
for
the
care
of
special
classes
of
patients,
may
be
found
more
economical
and
in
every
way
more
desirable. A
comparison
of
the
laws
of
the
various
states
reveals
the
general
fact
that
those
states
which
have
the
greatest
numbers
of
neuropathic
persons
in
institutions
in
proportion
to
their
general
population
have
also
the
simplest
legal
procedures
for
the
admis-
sion
of
patients,
and,
vice
versa,
those
which
have
the
least
num-
bers
have
also
the
most
cumbersome
legal
procedures
and
the
greatest
number
of
obstacles
and
restrictions. A
state
needs
but
to
provide
ample,
well
conducted
and
readily
accessible
institutions;
for
the
rest,
contrary
to
the
commonly
expressed
notion,
forced
segregation
is
far
less
effective
than
free
or
voluntary
segregation. The
interests
of
eugenics
are
best
served
where
detention
by
law
is
reduced
to
a minimum. §7.
SUMMARY. It
is
held
that
in
no
case
could
forced
segregation
be
advocated
with
propriety
and
with
social
benefit
merely
because
a
diagnosis
of
some
neuropathic
condition
has
been
made
and
on
eugenic
grounds
alone:
in
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https://openalex.org/W2129907187
|
https://zenodo.org/records/2409770/files/article.pdf
|
English
| null |
Caesar's Conquest of Gaul. Second edition, revised throughout and largely rewritten. By T. Rice Holmes, Litt.D. 9 × 6, xl + 872 pp. Frontispiece, 9 maps and 5 figures in the text. Oxford: Clarendon Press, 1911. 24s. n.
|
Journal of Roman studies
| 1,912
|
public-domain
| 2,935
|
NOTICES OF RECENT PUBLICATIONS. NOTICES OF RECENT PUBLICATIONS. CAESAR'S CONQUEST
OF GAUL. Second edition, revised throughout and largely rewritten. By
T. RICE HOLMES, LITT.D. 9 x 6, xl + 872 pp. Frontispiece, 9 maps and 5 figures in the text. Oxford:
Clarendon
Press, I9II. 24S. n. CAESAR'S CONQUEST
OF GAUL. Second edition, revised throughout and largely rewritten. By
T. RICE HOLMES, LITT.D. 9 x 6, xl + 872 pp. Frontispiece, 9 maps and 5 figures in the text. Oxford:
Clarendon
Press, I9II. 24S. n. M. Camille Jullian, reviewing Mr. Holmes' work on Ancient Britain and the Invasions
of Julius Caesar in the Revue des etudes anciennes, said that he should not be astonished
if England one day became proud of him. Those who can appreciate the thoroughness,
the honesty, the fine spirit, of Mr. Holmes' work, have long ago become proud of him;
and it is most satisfactory to find not only that the first edition of his earlier work has
been for some time exhausted, but that the Delegates of the Clarendon Press have been
willing to issue a second one on a large scale. In his preface to this second edition
Mr. Holmes tells us that he has spent the incessant labour of two years on the work
of revision, and apart from this, " the narrative is increased by thirty-seven pages, of
which twenty belong to the introduction. . . . Certain articles have been lengthened,
either because it had become necessary to take note of fresh information and recent
theories, or because the argument had not been completely thought out, while others
are entirely new. The instances in which conclusions reached in the old edition have
been altered, are, however, rare." ,
,
,
This extract from the new preface will suffice to indicate what the value of this
edition is. It is obvious that everyone who possesses the first edition must also procure
the second. True, its bulk is increased, and it has now become rather heavy and
unwieldy for convenient handling: 900 pages are just as much as frail human nature
can put up with in one volume. But whenever the owner feels equal to taking this
volume down from his shelf, such is the vigour and spirit of Mr. Holmes' writing, that
he will be certain to find abundance of interest and even of entertainment at every
page he turns to. page
If Mr. Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of
content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms
of scholarship. For more information about JSTOR, please contact support@jstor.org. Society for the Promotion of Roman Studies is collaborating with JSTOR to digitize, preserve and extend
access to The Journal of Roman Studies. This content downloaded from 194.29.185.247 on Fri, 9 May 2014 17:28:28 PM
All use subject to JSTOR Terms and Conditions This content downloaded from 194.29.185.247 on Fri, 9 May 2014 17:28:28 PM
All use subject to JSTOR Terms and Conditions NOTICES OF RECENT PUBLICATIONS. B. BURY. 8$ x 5i, I6 + 530 pp. London:
Macmillan
& Co. 191 z. i2s. n. English readers will heartily welcome this important volume, which is a continuation
of the author's well-known History of the Later Roman Empire (A.D. 395-800), published
nearly a quarter of a century ago. It lies, indeed, outside the limits which practical
considerations have assigned to this yournal, but it is fitting that it should be noticed
here, for there is no real break in the continuity of the Roman Empire between the
first and the fifteenth century. The earlier work, with all its merits, was planned on
too small a scale to supply the answers to many questions on which the student naturally
turned to it for help. The scale of the new instalment is four times as large, and the
increase affords a striking illustration of the progress of Byzantine studies in the interval,
the more so as this particular period has not attracted special attention. The same
tale is told by the eighteen pages of bibliography, a new and excellent feature, from
which the inexpert can cull two other facts. One is the relative smallness of the
contribution made by English research; the other is the desperately large linguistic
equipment which is now required for the task of rewriting the history of the Eastern
Empire. Among English historians Prof. Bury's range is unique, and we earnestly hope
English readers will heartily welcome this important volume, which is a continuation
of the author's well-known History of the Later Roman Empire (A.D. 395-800), published
nearly a quarter of a century ago. It lies, indeed, outside the limits which practical
considerations have assigned to this yournal, but it is fitting that it should be noticed
here, for there is no real break in the continuity of the Roman Empire between the
first and the fifteenth century. The earlier work, with all its merits, was planned on
too small a scale to supply the answers to many questions on which the student naturally
turned to it for help. The scale of the new instalment is four times as large, and the
increase affords a striking illustration of the progress of Byzantine studies in the interval,
the more so as this particular period has not attracted special attention. NOTICES OF RECENT PUBLICATIONS. 253
in doubting the " monstrous lie " which 0. E. Schmidt and his German followers have
attributed to Caesar in his account of the movements immediately following the
crossing of the Rubicon. These critics seem to fancy that in Caesar's mind the
occupation of Arretium and the other towns (B.C. i, 9-II) must necessarily have
needed most particular justification, and that his mistatement of facts was due to this
motive. But surely the occupation of Ariminum was the step that really needed
justification, and this of course he does not dream of mis-dating or excusing; the occu-
pation of the towns was, as Mr. Holmes says, " a justifiable measure of precaution,"
to protect his flanks from the attacks he expected, the inevitable result of the occupation
of Ariminum. I do not understand why Caesar should be supposed so anxious to justify
this step, and I am inclined to think that if our text is correct, which is by no means
certain, the mistake was unintentional. ,
One word more on a matter of personal interest. In a paper in The Classical Review
for April, 1903, I discussed Caesar's conception of Fortuna, and quoted a passage on
p. 22 of Mr. Holmes' first edition by way of illustrating the common view that Caesar
had a profound belief in his own good fortune. I see that on p. 41 of the new edition
Mr. Holmes is inclined to think that I misunderstood him. Perhaps to some extent
I did so, and I gladly take this opportunity of making my peace with him. But I
certainly did not take him to mean that Caesar believed in a personal goddess Fortuna,
as he suggests in his note. The fact is that it is very hard to say what the men of action
or the men of letters in that age really meant by Fortuna, and whether we should write
the word with or without a capital letter in many of the passages in which it occurs. I agree with Mr. NOTICES OF RECENT PUBLICATIONS. Holmes that in Caesar there is no certain sign of personification of
this mysterious power, " without whose aid " (to use his own words) " the strongest
judgment, the most diligent calculation, might fail." I only maintain that there is
not sufficient evidence in Caesar's own writings that he believed himself to be in any
way specially protected by this power, though he allows, as any general might, that
in the main he had been lucky in his campaigns. ,
One word more on a matter of personal interest. In a paper in The Classical Review
for April, 1903, I discussed Caesar's conception of Fortuna, and quoted a passage on
p. 22 of Mr. Holmes' first edition by way of illustrating the common view that Caesar
had a profound belief in his own good fortune. I see that on p. 41 of the new edition
Mr. Holmes is inclined to think that I misunderstood him. Perhaps to some extent
I did so, and I gladly take this opportunity of making my peace with him. But I
certainly did not take him to mean that Caesar believed in a personal goddess Fortuna,
as he suggests in his note. The fact is that it is very hard to say what the men of action
or the men of letters in that age really meant by Fortuna, and whether we should write
the word with or without a capital letter in many of the passages in which it occurs. I agree with Mr. Holmes that in Caesar there is no certain sign of personification of
this mysterious power, " without whose aid " (to use his own words) " the strongest
judgment, the most diligent calculation, might fail." I only maintain that there is
not sufficient evidence in Caesar's own writings that he believed himself to be in any
way specially protected by this power, though he allows, as any general might, that
in the main he had been lucky in his campaigns. W. WARDE FOWLER. W. WARDE FOWLER. A HISTORY OF THE EASTERN ROMAN EMPIRE FROM THE FALL OF IRENE TO THE
ACCESSION OF BASIL I (A.D. 802oz-867). By Prof. J. B. BURY. 8$ x 5i, I6 + 530 pp. London:
Macmillan
& Co. 191 z. i2s. n. A HISTORY OF THE EASTERN ROMAN EMPIRE FROM THE FALL OF IRENE TO THE
ACCESSION OF BASIL I (A.D. 802oz-867). By Prof. J. NOTICES OF RECENT PUBLICATIONS. Holmes is on the warpath, whether his antagonist be a third-rate German
scholar who has been impugning Caesar's veracity, or a warrior of renown like Prof. Ridgeway, or Signor Ferrero, who is attacked with refreshing vigour, he seems always
in earnest and almost always convincing. For a passage of arms with the brilliant Italian
historian, in which, so far as I can judge, the latter is completely pulverised, to use
Mr. Holmes' own word, the reader may turn to page 217, or to page 52I. I am tempted
to quote the concluding words of this latter section, with which few will disagree, as a
just criticism of Ferrero's peculiar methods. " Nothing is easier for a clever man than to
compose a plausible and interesting account of la haute politique in Gaul, as practised
by Caesar, if he is ready to ignore facts and give the rein to his imagination. But even
with all the help that we can get from Cicero's letters, we know too little of the course
of politics in Italy; and of the course of politics in Gaul we know hardly anything." p
y;
p
y
y
g
It is interesting to find in this edition a few pages added to the admirable chapter
on the credibility of Caesar's narrative, containing some account of the attacks made
recently in Germany on the veracity of the early chapters of the Bellum Civile. These
sadly mutilated chapters are easy enough to attack, and by no means easy to defend,
for Caesar wrote them, almost beyond doubt, some time after the events they describe,
and under the influence of strong indignation. Even Mr. Holmes is prepared to admit
that the narrative may be in certain respects disingenuous. I may be allowed to say,
knowing these chapters as intimately as I do, that I am disposed to think that in the
long run criticism will return to a generous view of Caesar's motives. The more closely
I examine the Bellum Civile as a whole, and especially the campaign of Dyrrhachium, This content downloaded from 194.29.185.247 on Fri, 9 May 2014 17:28:28 PM
All use subject to JSTOR Terms and Conditions NOTICES OF RECENT PUBLICATIONS. NOTICES OF RECENT PUBLICATIONS. NOTICES OF RECENT PUBLICATIONS. III
III the more convinced I am that it was natural to Caesar to tell the truth, and that the
furthest he went in the direction of falsehood was in suppression of details. I most heartily
wish, with many others, that Mr. Holmes may yet find time to devote himself to the
Civil war. I should be surprised if he did not go even further than he has on p. 253
in doubting the " monstrous lie " which 0. E. Schmidt and his German followers have
attributed to Caesar in his account of the movements immediately following the
crossing of the Rubicon. These critics seem to fancy that in Caesar's mind the
occupation of Arretium and the other towns (B.C. i, 9-II) must necessarily have
needed most particular justification, and that his mistatement of facts was due to this
motive. But surely the occupation of Ariminum was the step that really needed
justification, and this of course he does not dream of mis-dating or excusing; the occu-
pation of the towns was, as Mr. Holmes says, " a justifiable measure of precaution,"
to protect his flanks from the attacks he expected, the inevitable result of the occupation
of Ariminum. I do not understand why Caesar should be supposed so anxious to justify
this step, and I am inclined to think that if our text is correct, which is by no means
certain, the mistake was unintentional. the more convinced I am that it was natural to Caesar to tell the truth, and that the
furthest he went in the direction of falsehood was in suppression of details. I most heartily
wish, with many others, that Mr. Holmes may yet find time to devote himself to the
Civil war. I should be surprised if he did not go even further than he has on p. 253
in doubting the " monstrous lie " which 0. E. Schmidt and his German followers have
attributed to Caesar in his account of the movements immediately following the
crossing of the Rubicon. These critics seem to fancy that in Caesar's mind the
occupation of Arretium and the other towns (B.C. i, 9-II) must necessarily have
needed most particular justification, and that his mistatement of facts was due to this
motive. NOTICES OF RECENT PUBLICATIONS. But surely the occupation of Ariminum was the step that really needed
justification, and this of course he does not dream of mis-dating or excusing; the occu-
pation of the towns was, as Mr. Holmes says, " a justifiable measure of precaution,"
to protect his flanks from the attacks he expected, the inevitable result of the occupation
of Ariminum. I do not understand why Caesar should be supposed so anxious to justify
this step, and I am inclined to think that if our text is correct, which is by no means
certain, the mistake was unintentional. the more convinced I am that it was natural to Caesar to tell the truth, and that the
furthest he went in the direction of falsehood was in suppression of details. I most heartily
wish, with many others, that Mr. Holmes may yet find time to devote himself to the
Civil war. I should be surprised if he did not go even further than he has on p. 253
in doubting the " monstrous lie " which 0. E. Schmidt and his German followers have
attributed to Caesar in his account of the movements immediately following the
crossing of the Rubicon. These critics seem to fancy that in Caesar's mind the
occupation of Arretium and the other towns (B.C. i, 9-II) must necessarily have
needed most particular justification, and that his mistatement of facts was due to this
motive. But surely the occupation of Ariminum was the step that really needed
justification, and this of course he does not dream of mis-dating or excusing; the occu-
pation of the towns was, as Mr. Holmes says, " a justifiable measure of precaution,"
to protect his flanks from the attacks he expected, the inevitable result of the occupation
of Ariminum. I do not understand why Caesar should be supposed so anxious to justify
this step, and I am inclined to think that if our text is correct, which is by no means
certain, the mistake was unintentional. the more convinced I am that it was natural to Caesar to tell the truth, and that the
furthest he went in the direction of falsehood was in suppression of details. I most heartily
wish, with many others, that Mr. Holmes may yet find time to devote himself to the
Civil war. I should be surprised if he did not go even further than he has on p. This content downloaded from 194.29.185.247 on Fri, 9 May 2014 17:28:28 PM
All use subject to JSTOR Terms and Conditions NOTICES OF RECENT PUBLICATIONS. The same
tale is told by the eighteen pages of bibliography, a new and excellent feature, from
which the inexpert can cull two other facts. One is the relative smallness of the
contribution made by English research; the other is the desperately large linguistic
equipment which is now required for the task of rewriting the history of the Eastern
Empire. Among English historians Prof. Bury's range is unique, and we earnestly hope This content downloaded from 194.29.185.247 on Fri, 9 May 2014 17:28:28 PM
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Performance of the ATLAS RPC detector and Level-1 muon barrel trigger at √(s)=13 TeV
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Journal of Instrumentation PAPER • OPEN ACCESS Performance of the ATLAS RPC detector and
Level-1 muon barrel trigger at √(s)=13 TeV The ATLAS Fast TracKer system
The ATLAS collaboration, G. Aad, B. Abbott et al. - The ATLAS Fast TracKer system
The ATLAS collaboration, G. Aad, B. Abbott et al. - PAPER • OPEN ACCESS You may also like
Operation and performance of the ATLAS
semiconductor tracker in LHC Run 2
The ATLAS collaboration, Georges Aad,
Brad Abbott et al. -
The ATLAS Fast TracKer system
The ATLAS collaboration, G. Aad, B. Abbott et al. - c⃝2021 CERN for the benefit of the ATLAS collaboration. Published
by IOP Publishing Ltd on behalf of Sissa Medialab. Original content
from this work may be used under the terms of the Creative Commons Attribution 4.0
licence. Any further distribution of this work must maintain attribution to the author(s)
and the title of the work, journal citation and DOI. To cite this article: The ATLAS collaboration et al 2021 JINST 16 P07029 To cite this article: The ATLAS collaboration et al 2021 JINST 16 P07029 View the article online for updates and enhancements. This content was downloaded from IP address 129.240.84.21 on 21/02/2022 at 09:20 This content was downloaded from IP address 129.240.84.21 on 21/02/2022 at 09:20 Published by IOP Publishing for Sissa Medialab Received: March 2, 2021
Accepted: May 15, 2021
Published: July 19, 2021 Received: March 2, 2021
Accepted: May 15, 2021
Published: July 19, 2021 https://doi.org/10.1088/1748-0221/16/07/P07029 Performance of the ATLAS RPC detector and Level-1
muon barrel trigger at √𝒔= 13 TeV Keywords: Gaseous detectors; Muon spectrometers; Resistive-plate chambers; Trigger detector ArXiv ePrint: 2103.01029 https://doi.org/10.1088/1748-0221/16/07/P07029 Contents
1
Introduction
1
2
ATLAS detector and resistive-plate chambers
2
2.1
ATLAS muon spectrometer
3
2.2
ATLAS resistive-plate chambers
4
2.3
L1 muon barrel trigger
6
3
Dataset and event selection
8
4
RPC detector performance measurements
9
4.1
RPC single-module response
9
4.2
RPC detector response
11
4.3
RPC detector performance
14
4.4
Time resolution of RPC detector and readout system
16
5
Performance of L1 muon barrel trigger
19
5.1
Trigger roads
20
5.2
Trigger efficiency and timing
23
5.3
Trigger rates
26
5.4
Trigger composition
27
6
Measurements of RPC currents and counting rates
31
6.1
RPC current measurements
31
6.2
RPC counting rate measurements
34
6.3
RPC avalanche charge measurements
35
6.4
RPC efficiency as a function of counting rate
37
7
Expected performance of the existing RPCs at HL-LHC
38
7.1
Expected RPC currents at the HL-LHC
40
7.2
RPC detector performance using different operating voltage and FE threshold settings 40
8
Conclusions
43
The ATLAS collaboration
48 Contents
1
Introduction
1
2
ATLAS detector and resistive-plate chambers
2
2.1
ATLAS muon spectrometer
3
2.2
ATLAS resistive-plate chambers
4
2.3
L1 muon barrel trigger
6
3
Dataset and event selection
8
4
RPC detector performance measurements
9
4.1
RPC single-module response
9
4.2
RPC detector response
11
4.3
RPC detector performance
14
4.4
Time resolution of RPC detector and readout system
16
5
Performance of L1 muon barrel trigger
19
5.1
Trigger roads
20
5.2
Trigger efficiency and timing
23
5.3
Trigger rates
26
5.4
Trigger composition
27
6
Measurements of RPC currents and counting rates
31
6.1
RPC current measurements
31
6.2
RPC counting rate measurements
34
6.3
RPC avalanche charge measurements
35
6.4
RPC efficiency as a function of counting rate
37
7
Expected performance of the existing RPCs at HL-LHC
38
7.1
Expected RPC currents at the HL-LHC
40
7.2
RPC detector performance using different operating voltage and FE threshold settings 40
8
Conclusions
43
The ATLAS collaboration
48 Contents
1
Introduction
1
2
ATLAS detector and resistive-plate chambers
2
2.1
ATLAS muon spectrometer
3
2.2
ATLAS resistive-plate chambers
4
2.3
L1 muon barrel trigger
6
3
Dataset and event selection
8
4
RPC detector performance measurements
9
4.1
RPC single-module response
9
4.2
RPC detector response
11
4.3
RPC detector performance
14
4.4
Time resolution of RPC detector and readout system
16
5
Performance of L1 muon barrel trigger
19
5.1
Trigger roads
20
5.2
Trigger efficiency and timing
23
5.3
Trigger rates
26
5.4
Trigger composition
27
6
Measurements of RPC currents and counting rates
31
6.1
RPC current measurements
31
6.2
RPC counting rate measurements
34
6.3
RPC avalanche charge measurements
35
6.4
RPC efficiency as a function of counting rate
37
7
Expected performance of the existing RPCs at HL-LHC
38
7.1
Expected RPC currents at the HL-LHC
40
7.2
RPC detector performance using different operating voltage and FE threshold settings 40
8
Conclusions
43
The ATLAS collaboration
48 2021 JINST 16 P070 JINST 16 P07029 6
Measurements of RPC currents and counting rates
31
6.1
RPC current measurements
31
6.2
RPC counting rate measurements
34
6.3
RPC avalanche charge measurements
35
6.4
RPC efficiency as a function of counting rate
37
7
Expected performance of the existing RPCs at HL-LHC
38
7.1
Expected RPC currents at the HL-LHC
40
7.2
RPC detector performance using different operating voltage and FE threshold settings 40
8
Conclusions
43
The ATLAS collaboration
48 Performance of the ATLAS RPC detector and Level-1
muon barrel trigger at √𝒔= 13 TeV 2021 JINST 16 P07 JINST 16 P07029 E-mail: atlas.publications@cern.ch E-mail: atlas.publications@cern.ch Abstract: The ATLAS experiment at the Large Hadron Collider (LHC) employs a trigger system
consisting of a first-level hardware trigger (L1) and a software-based high-level trigger. The L1
muon trigger system selects muon candidates, assigns them to the correct LHC bunch crossing and
classifies them into one of six transverse-momentum threshold classes. The L1 muon trigger system
uses resistive-plate chambers (RPCs) to generate the muon-induced trigger signals in the central
(barrel) region of the ATLAS detector. The ATLAS RPCs are arranged in six concentric layers and
operate in a toroidal magnetic field with a bending power of 1.5 to 5.5 Tm. The RPC detector consists
of about 3700 gas volumes with a total surface area of more than 4000 m2. This paper reports on
the performance of the RPC detector and L1 muon barrel trigger using 60.8 fb−1 of proton-proton
collision data recorded by the ATLAS experiment in 2018 at a centre-of-mass energy of 13 TeV. Detector and trigger performance are studied using 𝑍boson decays into a muon pair. Measurements
of the RPC detector response, efficiency, and time resolution are reported. Measurements of the L1
muon barrel trigger efficiencies and rates are presented, along with measurements of the properties
of the selected sample of muon candidates. Measurements of the RPC currents, counting rates and
mean avalanche charge are performed using zero-bias collisions. Finally, RPC detector response and
efficiency are studied at different high voltage and front-end discriminator threshold settings in order
to extrapolate detector response to the higher luminosity expected for the High Luminosity LHC. 1
Introduction ATLAS [1–3] is a general-purpose detector that records high-energy collisions of protons and
heavy ions delivered by the Large Hadron Collider (LHC). The detector has been taking data since
its completion in 2008 and is scheduled to operate until approximately 2040, following extensive
accelerator and detector upgrades. These data have been used by the ATLAS Collaboration to
publish a diverse set of results that include the discovery of the Higgs boson [4] and measurements – 1 – of its properties [5–7], searches for new phenomena [8–10], and precision measurements of the
Standard Model (SM) properties [11–15]. Efficient online selection of events containing muons [16]
produced in collisions is essential for many of these measurements. The first-level (L1) hardware-based trigger system of ATLAS [17, 18] uses resistive-plate
chambers (RPCs) to identify muon candidates in the central (barrel) region of the muon spectrometer
(MS) [19]. RPCs are gaseous detectors with nanosecond-level time resolution [20, 21]. Excellent
performance of the RPC detector and its associated trigger system are therefore fundamental for
the ATLAS physics programme. This L1 muon barrel trigger system [22] selects muon candidates
produced in LHC collisions; it assigns the selected candidates to the correct LHC bunch crossing
and measures the transverse momentum (𝑝T) of the muon candidates using six predetermined
programmable thresholds. 2021 This paper reports measurements of the performance of the ATLAS RPC detector and L1
muon barrel trigger system using 60.8 fb−1 of proton-proton collision data recorded by the ATLAS
experiment in 2018 at a centre-of-mass energy of 13 TeV. The paper is organised as follows. Section 2 briefly describes the RPC detector and L1 muon barrel trigger system. Section 3 details
the dataset and tools used for the measurements presented in the subsequent sections. Section 4
presents measurements of RPC detector efficiency and time resolution. This section also describes
the RPC response to the passage of a muon. Section 5 presents measurements of the L1 muon barrel
trigger response, including measurements of the efficiency, rates and composition of the selected
muon candidates. Section 6 presents measurements of the currents in the gas volumes as a function
of the operating voltage, temperature and instantaneous luminosity. Measurements of RPC counting
rates are also reported in this section. The current and counting rate measurements are combined to
determine an average RPC avalanche charge using zero-bias collisions. 1ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in the centre of
the detector and the 𝑧-axis along the beam direction. The 𝑥-axis points from the IP to the centre of the LHC ring, and the
𝑦-axis points upward. Cylindrical coordinates (𝑟, 𝜙) are used in the (𝑥, 𝑦) plane, 𝜙being the azimuthal angle around the
𝑧-axis. The pseudorapidity is defined in terms of the polar angle 𝜃as 𝜂= −ln tan(𝜃/2). The distance Δ𝑅is defined as
Δ𝑅=
√︁
(Δ𝜂)2 + (Δ𝜙)2. 1
Introduction Section 7 presents studies
of expected RPC detector response at the High Luminosity LHC (HL-LHC). For these studies, the
measurements reported in section 6 are extrapolated to the HL-LHC design luminosity in order
to predict RPC detector response. In addition, studies of the RPC detector response at different
voltage working points and front-end (FE) electronics discriminator thresholds, corresponding to
expected RPC operational parameters for the HL-LHC, are presented. JINST 16 P07029 2
ATLAS detector and resistive-plate chambers ATLAS is a general-purpose detector at the LHC with a cylindrical geometry1 that provides nearly
full solid angle coverage around the collision point located at the centre of the detector. The
detector consists of an inner tracking detector (ID), electromagnetic and hadronic calorimeters,
and a muon spectrometer. The detector is subdivided into a barrel and two endcap sections, and
provides complete azimuthal angle coverage. The ID covers the pseudorapidity range of |𝜂| < 2.5
and is surrounded by a thin superconducting solenoid that provides a 2 T axial magnetic field. The ID consists of silicon pixel, silicon microstrip, and transition radiation tracking detectors. The – 2 – calorimeter system covers the pseudorapidity range |𝜂| < 4.9. High-granularity lead and liquid-
argon (LAr) sampling calorimeters provide electromagnetic calorimetry within the pseudorapidity
range |𝜂| < 3.2. An additional thin LAr presampler is used to correct for energy losses in the
material upstream of the calorimeters in the pseudorapidity range |𝜂| < 1.8. A steel and scintillator-
tile sampling calorimeter provides hadronic calorimetry within the pseudorapidity range |𝜂| < 1.7. Two copper/LAr endcap hadronic calorimeters cover the pseudorapidity range of 1.5 < |𝜂| < 3.2. The forward coverage is extended up to |𝜂| = 4.9 with copper/LAr and tungsten/LAr calorimeters,
which are optimised for electromagnetic and hadronic measurements, respectively. Large sector
Small sector
(a)
TGCs
(b)
Figure 1. (a) View of the ATLAS MS barrel detectors in the transverse (𝑥, 𝑦) plane. (b) View of the ATLAS
MS layout in the (𝑧, 𝑦) plane for a small azimuthal sector containing the barrel toroid coils. The green (blue)
chambers are MDT chambers in the barrel (endcap) regions of the spectrometer. The TGCs, RPCs, and
CSCs are shown in red, white, and yellow, respectively. Large sector
Small sector
(a) TGCs
(b) JINST 16 P07029 T 16 P07029 (b) (a) Figure 1. (a) View of the ATLAS MS barrel detectors in the transverse (𝑥, 𝑦) plane. (b) View of the ATLAS
MS layout in the (𝑧, 𝑦) plane for a small azimuthal sector containing the barrel toroid coils. The green (blue)
chambers are MDT chambers in the barrel (endcap) regions of the spectrometer. The TGCs, RPCs, and
CSCs are shown in red, white, and yellow, respectively. 2.1
ATLAS muon spectrometer The MS is designed to identify muon candidates and to measure the muon momentum and charge
independently from the ID. The MS is the outermost system of the ATLAS detector and it consists
of one barrel and two endcap sections. Each section incorporates an air-core magnet, with each
magnet consisting of eight superconducting coils. The barrel toroid magnet provides 1.5 to 5.5 Tm
of bending power for muon tracks in the pseudorapidity range |𝜂| < 1.4. The toroidal field causes
muon track deflections primarily in the (𝑟, 𝑧) plane, which are measured using the 𝜂coordinate. The MS is subdivided into eight large and eight small azimuthal sectors, with the small barrel
sectors containing the magnet coils. The muon barrel chambers are arranged in six concentric
cylindrical layers around the beam axis at radii of approximately 5 m, 7.5 m, and 10 m. In the
endcap regions, muon chambers form wheels positioned perpendicular to the beam axis at distances
of approximately 7.4 m, 10.8 m, 14 m, and 21.5 m from the interaction point. The MS barrel
geometry is illustrated in figure 1(a) in the (𝑥, 𝑦) plane and in figure 1(b) in the (𝑟, 𝑧) plane for the
small sectors. – 3 – The MS contains fast trigger detectors and precision tracking detectors that cover the pseudo-
rapidity ranges |𝜂| < 2.4 and |𝜂| < 2.7, respectively. The RPCs and thin-gap chambers (TGCs) are
used for triggering in the barrel and endcap regions, respectively. The RPC and TGC detectors are
also used to measure the muon 𝜙direction in the non-bending (𝑥, 𝑦) plane. The precision muon
tracking chambers are constructed from monitored drift tubes (MDTs). The MDT detector provides
an average resolution of about 80 μm per tube and 35 μm per chamber, when measured in the bend-
ing (𝑟, 𝑧) plane, which is approximately perpendicular to the magnetic field lines. Cathode-strip
chambers (CSCs) are used for precision tracking in the inner endcap region, 2 < |𝜂| < 2.7. The following nomenclature is used to identify different MS elements. The ATLAS detector
is divided into two halves along the 𝑧-axis, called side A (𝑧> 0) and side C (𝑧< 0). The barrel
and endcap regions are labelled as ‘B’ and ‘E’, respectively. Letters ‘I’ (inner), ‘M’ (middle) and
‘O’ (outer) are used to identify the corresponding MS layers. 2.1
ATLAS muon spectrometer Letters ‘S’ and ‘L’ specify whether a
chamber belongs to a small or large sector, respectively. For example, the ATLAS nomenclature for
a chamber located in a large sector of the middle barrel layer is ‘BML’, followed by: the chamber
position index along the z-direction, ranging from 1 to 8; detector side A or C; and the azimuthal
sector number, ranging from 1 to 16. In addition, special muon chambers were added in the barrel-
endcap transition region 1.05 < |𝜂| < 1.3 in order to close gaps and increase the overall detector
coverage. These chambers are referred to as ‘barrel endcap extra’ (BEE) and ‘extended endcap’
(EE) chambers. 2021 JINST 16 P07029 2.2
ATLAS resistive-plate chambers The ATLAS RPC detector [17, 19] provides up to six position measurements along the muon
trajectory in the MS, with a space-time resolution of the order of 2 cm × 2 ns. The RPC detector
covers the pseudorapidity range |𝜂| < 1.05. It consists of approximately 3700 gas volumes, with a
total surface area of more than 4000 m2, and operates in a toroidal magnetic field of about 0.5 T. Each RPC consists of two independent detector layers (referred to as a doublet), separated by about
2 cm. The RPCs are arranged in three concentric cylindrical doublet layers at radii of approximately
7.8 m (6.8 m), 8.4 m (7.5 m), and 10.2 m (9.8 m) for the small (large) azimuthal sectors. These three
doublet layers are referred to as the ‘middle confirm layer’ (RPC1), ‘middle pivot layer’ (RPC2) and
‘outer confirm layer’ (RPC3). Following the initial detector operations from late 2009 through early
2013, additional so-called feet and elevator chambers were installed during the first long shutdown
to increase the system acceptance before the resumption of operations in 2015 [18, 23]. Each single RPC detector layer is constructed from two parallel resistive electrodes which are
made of high-pressure phenolic-melaminic laminate (bakelite) with a high resistivity of approxi-
mately 1010 Ω cm, as illustrated in figure 2. The bakelite sheet prevents self-sustaining discharges
and limits the amount of charge produced in an ionisation event, thus allowing the high-rate opera-
tion of RPCs. A thin coat of linseed oil is applied to the inner surfaces of the electrodes in order to
ensure their smoothness. The two electrodes are separated by a distance of 2 mm using insulating
polycarbonate spacers, with a diameter of approximately 12 mm, which are placed every 10 cm. The spacers cover approximately 1% of the detector surface and therefore reduce the RPC efficiency
by the equivalent amount. The external sides of the resistive electrodes are coated with a graphite paint. A reference
voltage of 9.6 kV is typically applied across the two electrodes. The actual applied voltage is – 4 – Figure 2. A schematic drawing of an ATLAS RPC detector module. Figure 2. A schematic drawing of an ATLAS RPC detector module. 2.2
ATLAS resistive-plate chambers automatically adjusted according to continuous readings of environmental temperature and pres-
sure parameters in order to provide a muon detection efficiency equivalent to that obtained at a
temperature of 24◦C and a pressure of 970 mbar [24]. The average applied voltage across all
detector chambers was 9.6 kV, with the standard deviation of about 26 V. These variations were due
to changes in environmental temperature and pressure with respect to the standard conditions. 2021 The RPCs are continuously flushed with a gas mixture of C2H2F4(94.7%)–C4H10(5%)–
SF6(0.3%). This mixture includes a quencher component (C4H10) that helps to avoid propaga-
tion of the discharge and an electronegative component (SF6) that helps to limit the growth of
avalanches. This gas mixture has a strong greenhouse effect and it is currently being phased down
in the European Union, thereby also leading to rising cost. For these reasons, new gas mixtures are
under investigation for future RPC operation [25, 26]. JINST 16 P07029 The ATLAS RPCs are operated in avalanche mode, with the probability to produce streamers
during the gas multiplication of the primary ionisation kept below 2% [27]. The reduced pulse
charge in avalanche mode provides a high rate capability and enables stable operation over the
required detector lifetime. The lower gas amplification of the avalanche mode is compensated for
by a high signal amplification gain of the FE electronics [28]. Each single RPC layer measures 𝜂and 𝜙coordinates using orthogonal copper strips placed on
opposite sides of the electrodes, with the strip widths varying in a range between 24.5 and 33.3 mm. Muon sagittae due to the magnetic field are measured by 𝜂strips, aligned perpendicularly to the
bending (𝑟, 𝑧) plane. Strip foils are glued on 3 mm-thick low-density polyester foam plates. The
strips are isolated from the electrode with insulating polyethylene terephthalate (PET) foil. The
signal is recorded via capacitive coupling to the copper strips, which are connected to FE electronics. The RPC layout and readout functionality are not fully symmetric between 𝜂and 𝜙views. A second PET foil is placed between the 𝜂strips and the electrode where the positive voltage is
applied, resulting in the charge collected by the 𝜂strips being slightly smaller than that collected by
the 𝜙strips for the same avalanche. Moreover, FE boards for 𝜙strips include an additional circuit
which inverts the polarity of the positively charged signal induced in 𝜙strips. 2.2
ATLAS resistive-plate chambers After that, the same
circuit is used for 𝜂and 𝜙strip readout. The following nomenclature is used to refer to the RPC detector elements. One RPC gas volume
(gas gap) together with the 𝜂and 𝜙readout strips is called ‘module’ and is shown in figure 2. A
group of strips in one view (𝜂or 𝜙) belonging to one module is thereafter referred to as a strip
readout panel or simply a panel. Two layers, consisting of two or four modules each, assembled
into a common mechanical structure are referred to as a unit (or chamber). Each RPC unit therefore
forms one doublet layer. One or two RPC units, which are integrated with the corresponding MDT – 5 – chambers, make a (muon) station. One station in the MS middle layer integrates the RPC1 and
RPC2 doublet layers, while one station in the MS outer layer includes only the RPC3 doublet layer. There are 384 muon stations that contain RPCs, separated into 2 MS layers (middle and outer),
2 detector sides, 16 sectors and 6 locations along the 𝑧-axis for each side. In addition, there are a
small number of special RPCs that are described later. The position of each station is determined
by a coordinate along the 𝑧-axis (𝜂index ranging from 1 to 6, for |𝜂| values from 0.05 to 1.05) and
a coordinate along the 𝜙direction (𝜙sector ranging from 1 to 16). Modules within one station are
represented in figures using half-integer values of the 𝜂index and 𝜙sector. A signal recorded by the FE electronics in one strip is referred to as a hit. All RPC hits within
a 200 ns window, centred on the bunch crossing selected by the trigger system, are recorded for
offline analysis. After registering a hit in a channel, the FE electronics imposes the dead-time
window of 100 ns for that channel. LHC proton-proton collisions at an instantaneous luminosity of
1034 cm−2s−1 result in RPC counting rates in the range 10 to 30 Hz/cm2, depending on the chamber
location, as discussed in section 6. 2021 The ATLAS detector control system (DCS) [29, 30] is used to operate and monitor the RPC
detector. The DCS is used to ramp up/down the applied voltage and to set the thresholds of the FE
discriminators. 2.2
ATLAS resistive-plate chambers The DCS also provides continuous monitoring of configuration parameters (such as
threshold and voltage settings) and of operational detector parameters (such as temperature, pressure
and chamber currents). This monitoring information is available in real time in the ATLAS control
room during data-taking and is also recorded in a central database for subsequent offline analysis. JINST 16 P07029 The parameter 𝑉set is used by the DCS to control the threshold of the FE discriminators of
readout channels in a panel. The absolute threshold applied at the discriminator level is determined
by the FE electronics using the following expression: 𝑉thr = 𝑉reference −𝑉set, where 𝑉reference is a
reference voltage which is set to 2 V. The 𝑉thr corresponds to the physical threshold applied to the
RPC signal after the amplification stage [31]. Nearly all 𝑉thr parameters are typically in the range
0.8 V to 1.5 V, in steps of 0.1 V, and approximately 90% of panels use the nominal value of 1 V. This physical 𝑉thr threshold parameter is used for studies presented later. 2.3
L1 muon barrel trigger Interesting collision events are selected using a two-level trigger system [17, 18, 32]. The L1 trigger
processes events at a rate of 40 MHz, set by the LHC beam structure consisting of bunches separated
by 25 ns. The L1 trigger selects events at a rate of 100 kHz using data from the calorimeters and
muon trigger detectors. The high-level trigger (HLT) employs software algorithms with access to
the full detector information to analyse the accepted L1 events. The HLT selects events for offline
analysis at a rate of approximately 1 kHz. The L1 muon barrel trigger system [22] uses the RPCs to identify a region of interest (RoI)
containing a muon candidate in the pseudorapidity range |𝜂| < 1.05. A typical RoI has Δ𝜂× Δ𝜙
dimensions of approximately 0.1 × 0.1. The L1 trigger assigns muon candidates to the correct
LHC bunch crossing and determines the muon transverse momentum (𝑝T) using six programmable
thresholds. To compensate for different signal propagation times due to the different lengths of
readout cables, timing response of RPC electronics channels are calibrated using programmable
delays in steps of 3.125 ns, corresponding to an eighth of the LHC bunch spacing [22]. One
calibration constant is used for each group of eight channels. – 6 – The transverse momentum of muon candidates is measured by the L1 muon barrel trigger using
different algorithms for low-𝑝T and high-𝑝T triggers [22], as illustrated in figure 3. The low-𝑝T
algorithm starts with a signal in an RPC2 (pivot) strip and then checks for matching signals in
RPC1 (confirm) strips within a narrow cone pointing back to the collision point, shown in red in
figure 3. The low-𝑝T algorithm requires signals to be present in three out of four detector layers,
which results in a significant suppression of random coincidences due to background events. The
high-𝑝T algorithm starts with a muon candidate identified by the low-𝑝T algorithm and then checks
for the presence of matching signals in one of the two RPC3 (confirm) layers within a narrower
cone pointing back to the collision point, shown in blue in figure 3. Each trigger 𝑝T threshold
always satisfies the conditions of the lower ones. Therefore, only the highest 𝑝T threshold passed
by a muon candidate is reported by the L1 muon barrel trigger system. 3
Dataset and event selection The measurements presented here were performed using proton–proton collision data recorded by
the ATLAS experiment in 2018 at a centre-of-mass energy of 13 TeV, with 25 ns spacing between
LHC bunches. Only LHC fills with an integrated luminosity greater than 50 pb−1 were used for
these measurements. The total integrated luminosity of the analysed dataset amounts to 60.8 fb−1. Muon candidates are reconstructed by combining ID and MS information [33, 34]. The
precision MDT chambers are used to reconstruct muon trajectories in the (𝑟, 𝑧) plane. This plane
is approximately orthogonal to the magnetic field lines, thus allowing precise measurements of the
sagitta of the muon tracks. In the barrel region, the RPC detector is used to reconstruct the muon
trajectory in the non-bending azimuthal (𝑥, 𝑦) plane which is transverse to the beam direction. 2021 JINST 16 P07 2021 RPC 𝜙hits are used to build track patterns in the 𝜙view. For the pattern finding procedure
in the 𝜂view, both RPC and MDT hits are used. Three-dimensional track segments are recon-
structed in each station, then combined among different stations and fitted to form a final MS track
candidate. The fitting procedure takes into account effects of multiple scattering, magnetic field
inhomogeneities and inter-chamber misalignments. At least three MDT 𝜂hits are required to make
a track segment along the precision 𝜂view. In the 𝜙view, at least two RPC hits are required to form
a track candidate. JINST 16 P07029 Events were selected using several different trigger criteria which were based on the presence
of a muon candidate, a high-𝑝T hadronic jet, or significant missing transverse momentum [35]. The
majority of the selected events come from the Drell-Yan production of 𝑊and 𝑍bosons decaying
into a muon and neutrino or into a muon pair, respectively. A smaller fraction of events are due
to production of top quark pairs, electroweak vector-boson pairs and decays of hadrons containing
bottom or charm quarks. Selected events were recorded in a dedicated data stream during prompt data reconstruction
at CERN. This data includes the reconstructed muon candidates, the L1 muon trigger information
and the full information related to muon detector system, including all hits recorded by the RPC
detector in the 200 ns window centred on the selected bunch crossing. The offline reconstruction
selects only the first hit in time in each RPC strip, resulting in at most one hit associated with a
single strip. 2.3
L1 muon barrel trigger low p
T
high p
T
5
10
15 m
0
RPC 3
RPC 2
RPC 1
low pT
high p
T
MDT
MDT
MDT
M
D
T
TGC 1
TGC 2
TGC 3
M
D
T
M
D
T
TGC EI
TGC FI
XX-LL01V04
Tile Calorimeter
Figure 3. Illustration of the low-𝑝T and high-𝑝T L1 muon trigger algorithms in the barrel and endcap regions. JINST 16 P07029 gure 3. Illustration of the low-𝑝T and high-𝑝T L1 muon trigger algorithms in the barrel and endcap regions. Three low-𝑝T thresholds and three high-𝑝T thresholds were defined for the L1 muon barrel
trigger system [16]. In 2015–2018, the low-𝑝T trigger thresholds were 𝑝T = 4, 6 and 10 GeV, which
are referred to as MU4, MU6 and MU10 triggers, respectively. In 2015–2016, the high-𝑝T trigger
thresholds were 𝑝T = 10, 15 and 20 GeV, which are referred to as MU11, MU15 and MU20 triggers,
respectively. The MU11 nomenclature was used to distinguish this trigger from the low-𝑝T MU10
trigger. In 2017–2018, the MU15 trigger was removed, and the MU21 trigger was introduced. The
MU21 trigger was identical to the MU20 trigger except that the so-called new feet RPCs were not
included in its trigger logic. These new feet chambers were installed as a fourth RPC doublet layer
(RPC4) in 𝜙sectors 12 and 14, which contain the ATLAS detector support structures (feet). These new feet chambers increase the geometrical acceptance of the L1 muon barrel trigger
system for detecting muons with 𝑝T > 20 GeV from approximately 67% to around 70%, as described
in section 5.2. In these two sectors, some RPCs are absent in the middle MS layer (RPC1 and RPC2)
in order to make room for the detector feet. Therefore, muons travelling from the collision point
encounter only the two outermost RPC doublet layers in these sectors (RPC3 and RPC4). For this
reason, the trigger logic of the new feet trigger requires a geometrical matching only in the two
outermost doublet layers, for both the low- and high-𝑝T trigger thresholds. This leads to a larger
expected rate of background events for the high-𝑝T triggers, as discussed in section 5.4. The MU21
trigger was introduced as a backup trigger in case the acceptance rate from the new feet trigger
exceeded the allowed limit. – 7 – 3
Dataset and event selection Selected events were required to contain at least one reconstructed muon candidate
with 𝑝T > 10 GeV that satisfied the loose identification criteria [33, 34]. Muon candidates were
also required to originate from the reconstructed vertex with the highest sum of 𝑝2
T of the associated
reconstructed ID tracks. Requirements on the significance of the transverse impact parameter 𝑑0
(|𝑑0|/𝜎(𝑑0) < 3.0) and on the longitudinal impact parameter 𝑧0 (|𝑧0 sin 𝜃| < 0.5 mm) of the muon
are also imposed. Detector and trigger performance were evaluated using events containing a 𝑍boson decay
into two muons. Events containing a 𝑍boson candidate were first selected using the primary
single-muon trigger [16] with 𝑝T > 26 GeV. At least two reconstructed muon candidates were then
required to be present in each selected event. Among them, one muon (referred to as a tag) was
required to be matched with the muon candidate selected by the trigger system. The other muon
(referred to as a probe) was required to form an opposite-charge pair with the tag muon. Finally,
the invariant mass of each dimuon pair was required to be in the range 50 GeV < 𝑚𝜇𝜇< 150 GeV,
consistent with the 𝑍boson mass. Probe muons selected in this way are unbiased by the trigger
system and were used for the measurements presented in sections 4 and 5. – 8 – 4
RPC detector performance measurements This section presents studies of the RPC detector response to the passage of probe muons produced
in Z boson decays. A dedicated analysis algorithm was developed to study the response of RPCs to
the passage of muons. This algorithm extrapolates the trajectory of the muon track through the MS
and computes the expected position of the muon impact point on the surface of the RPC detector
modules near the trajectory. The algorithm takes into account the magnetic field configuration,
detector geometry and material density distribution. RPC modules with expected muon-induced
signals are selected for analysis following a two-step procedure. First, modules positioned within a
distance of Δ𝑅< 0.5 are retained, where Δ𝑅is computed between the muon track and the vector
pointing from the detector centre to the geometric centre of the module. Second, the extrapolated
muon track position on the module surface is required to be within 20 mm of the centre of at least
one strip belonging to that module. Each selected RPC module is therefore expected to contain
muon-induced hits because a muon trajectory is predicted to pass through the active surface of
the module. 2021 JINST 16 P07 2021 JINST 16 P07029 This section is organised as follows. Section 4.1 presents the response of one representative
RPC module in order to introduce the analysis technique. Section 4.2 reports the response of the
entire RPC detector, which is evaluated using probe muons. Section 4.3 shows the measurement
of the efficiency of all RPC detector modules, as well as the stability of the RPC detector response
as a function of time. Finally, section 4.4 presents measurements of the time resolution of the RPC
detector and the readout system. 4.1
RPC single-module response The response of one representative RPC module is evaluated using data recorded in one ATLAS
run. This module is located on side C in the RPC1 doublet layer in large sector 11, in the station
with 𝜂index 1. In this module, 𝜂and 𝜙strip widths are 27 mm and 25 mm, respectively. Time
distributions of 𝜂and 𝜙hits in this module are shown in figure 4 for events containing muon
candidates that are predicted to pass through this module. Hit times are calibrated according to the
procedure described in section 2.3. The zero time of the 𝑥-axis corresponds to the arrival time of
an ultra-relativistic particle produced at the interaction point in the bunch crossing selected by the
trigger system. The small timing offsets observed in figure 4 are partly due to imperfect timing
calibrations which are nevertheless still within required levels of precision. When considering all
hits recorded by this module, about 2% (5%) of 𝜂(𝜙) hits lie outside the 25 ns window centred at
zero, corresponding to the time interval that separates the selected bunch crossing from the previous
and subsequent bunch crossings. The fraction of hits outside this time window is reduced to less
than 0.5% when only the hits belonging to the strip containing the expected muon impact point are
considered (therefore selecting only those hits that lie on the muon trajectory). This small fraction
illustrates the typically good performance of the RPC time calibration for assigning muon-induced
hits to the correct bunch crossing. The timing response of the full RPC detector is presented in
section 4.2. The efficiency of detecting a muon-induced ionisation signal is evaluated by counting the hit
multiplicity in a module using events that contain muons that pass through that module. Three sets of
selection criteria are studied for counting RPC hits. 4.1
RPC single-module response Considering all hits recorded in a given module – 9 – Reconstructed hit time t [ns]
100
−
80
−
60
−
40
−
20
−
0
20
40
60
80
100
strips/3.125 ns
η
Hits from
500
1000
1500
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
view
η
probe muons, one RPC panel,
µ
µ
→
Z
All hits, fraction(|t| > 12.5 ns) = 0.021
On track hits, fraction(|t| > 12.5 ns) = 0.003
(a)
Reconstructed hit time t [ns]
100
−
80
−
60
−
40
−
20
−
0
20
40
60
80
100
strips/3.125 ns
φ
Hits from
500
1000
1500
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
view
φ
probe muons, one RPC panel,
µ
µ
→
Z
All hits, fraction(|t| > 12.5 ns) = 0.052
On track hits, fraction(|t| > 12.5 ns) = 0.004
(b)
Figure 4. Time distributions of the calibrated hits recorded by the (a) 𝜂panels and (b) 𝜙panels belonging to
a representative RPC module. Only events that contain a muon that is expected to pass through this module
are used. Dashed vertical lines correspond to the 25 ns time window centred at zero. The fractions of hits
with |𝑡| > 12.5 ns are also reported. Reconstructed hit time t [ns]
100
−
80
−
60
−
40
−
20
−
0
20
40
60
80
100
strips/3.125 ns
η
Hits from
500
1000
1500
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
view
η
probe muons, one RPC panel,
µ
µ
→
Z
All hits, fraction(|t| > 12.5 ns) = 0.021
On track hits, fraction(|t| > 12.5 ns) = 0.003
(a) Reconstructed hit time t [ns]
100
−
80
−
60
−
40
−
20
−
0
20
40
60
80
100
strips/3.125 ns
φ
Hits from
500
1000
1500
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
view
φ
probe muons, one RPC panel,
µ
µ
→
Z
All hits, fraction(|t| > 12.5 ns) = 0.052
On track hits, fraction(|t| > 12.5 ns) = 0.004
(b) strips/3.125 ns
φ
Hits from 2021 (b) (a) Figure 4. Time distributions of the calibrated hits recorded by the (a) 𝜂panels and (b) 𝜙panels belonging to
a representative RPC module. Only events that contain a muon that is expected to pass through this module
are used. 4.1
RPC single-module response Dashed vertical lines correspond to the 25 ns time window centred at zero. The fractions of hits
with |𝑡| > 12.5 ns are also reported. JINST 16 P07029 results in the maximum possible efficiency but also includes noise contributions. Hits within the
25 ns window centred at zero are referred to as in-time hits. In-time hits belonging to strips with
their centre within 30 mm of the extrapolated muon track position are referred to as signal hits. The hit multiplicity distributions obtained using these three selection criteria are shown in figure 5
for the 𝜂and 𝜙panels belonging to the representative module. The RPC module efficiency, 𝜖, is
computed as the number of muon candidates that generate at least one selected hit divided by the
total number of muon candidates that are predicted to pass through that module. This representative
module detects muon-induced ionisation signals with an efficiency of approximately 96%. The
measured efficiencies calculated using the three hit-selection criteria differ by less than 1%. strips hit multiplicity
η
0
1
2
3
4
5
6
7
8
9
10
Muons
0
200
400
600
800
1000
1200
1400
1600
1800
0.006
±
= 0.965
all
∈
All hits,
0.006
±
= 0.963
in-time
∈
In-time hits,
0.006
±
= 0.960
signal
∈
Signal hits,
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
view
η
One RPC panel,
(a)
strips hit multiplicity
φ
0
1
2
3
4
5
6
7
8
9
10
Muons
0
200
400
600
800
1000
1200
1400
0.005
±
= 0.968
all
∈
All hits,
0.005
±
= 0.967
in-time
∈
In-time hits,
0.006
±
= 0.965
signal
∈
Signal hits,
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
view
φ
One RPC panel,
(b)
Figure 5. Hit multiplicity and detector efficiency for the (a) 𝜂panels and (b) 𝜙panels belonging to the same
representative RPC module. Only events where a muon is expected to pass through this module are used. 4.1
RPC single-module response strips hit multiplicity
η
0
1
2
3
4
5
6
7
8
9
10
Muons
0
200
400
600
800
1000
1200
1400
1600
1800
0.006
±
= 0.965
all
∈
All hits,
0.006
±
= 0.963
in-time
∈
In-time hits,
0.006
±
= 0.960
signal
∈
Signal hits,
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
view
η
One RPC panel,
(a) strips hit multiplicity
φ
0
1
2
3
4
5
6
7
8
9
10
Muons
0
200
400
600
800
1000
1200
1400
0.005
±
= 0.968
all
∈
All hits,
0.005
±
= 0.967
in-time
∈
In-time hits,
0.006
±
= 0.965
signal
∈
Signal hits,
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
view
φ
One RPC panel,
(b) (b) (a) Figure 5. Hit multiplicity and detector efficiency for the (a) 𝜂panels and (b) 𝜙panels belonging to the same
representative RPC module. Only events where a muon is expected to pass through this module are used. As shown in figure 5, a muon passing through an RPC module typically generates one or two
hits. A cluster is defined as a set of hits in contiguous strips, with hit times that are separated by less – 10 – than 12.5 ns. The size of the muon-induced clusters is an important parameter of RPC performance
and is studied in section 4.2. Muon clusters are typically located close to the impact point of a
muon track. The cluster position residuals are defined as the difference between the cluster centre and the
extrapolated impact point of a reconstructed muon track on the module surface. These residuals
are shown in figure 6 for the representative RPC module for clusters with one or two strips. For
clusters with one strip, the cluster centre is given by the strip centre and the width of the 𝜂residual
distribution is therefore determined by the strip width. This is because the 𝜂coordinate of a muon
track was measured using the MDT detector, which has much more precise 𝜂position resolution
than that of the RPC detector. For clusters with two strips, the cluster centre is defined as the
midpoint between two strips. 4.1
RPC single-module response For these clusters the muon is more likely to pass between the two
strips and therefore the width of the 𝜂residual distribution is smaller because of the slightly better
estimate of the muon impact point compared to the clusters with a single hit. Muon tracks are more
likely to pass close to the 𝜙cluster centre because the 𝜙coordinate of the muon tracks in the MS
was measured using the RPC detector. Therefore, muon tracks are biased to be close to recorded
𝜙hits, resulting in the peak near zero for the 𝜙residual distributions. The 𝜙residual distribution
widths are similar for the clusters with one hit and with two hits because the muon track 𝜙position
uncertainty is dominated by the RPC hit position uncertainty. 2021 1 JINST 16 P07029 cluster residual [mm]
η
Closest
50
−
40
−
30
−
20
−
10
−
0
10
20
30
40
50
Muons
50
100
150
200
250
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
view
η
One RPC panel,
Clusters with 1 strip, RMS = 7.3 mm
Clusters with 2 strips, RMS = 3.7 mm
(a)
cluster residual [mm]
φ
Closest
50
−
40
−
30
−
20
−
10
−
0
10
20
30
40
50
Muons
50
100
150
200
250
300
Clusters with 1 strip, RMS = 6.3 mm
Clusters with 2 strips, RMS = 5.8 mm
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
view
φ
One RPC panel,
(b)
Figure 6. Cluster residuals for one representative RPC module for (a) 𝜂strips and (b) 𝜙strips. The residuals
were computed by subtracting the expected muon position from the centre of the cluster. Clusters with one
strip and with two strips are shown separately. The RMS values are computed using entries with residuals
between ±20 mm. No time requirements are used to select the individual RPC hits. For clusters made of two
strips, the hit times are required to be within 12.5 ns of each other. 4.1
RPC single-module response cluster residual [mm]
η
Closest
50
−
40
−
30
−
20
−
10
−
0
10
20
30
40
50
Muons
50
100
150
200
250
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
view
η
One RPC panel,
Clusters with 1 strip, RMS = 7.3 mm
Clusters with 2 strips, RMS = 3.7 mm
(a) cluster residual [mm]
φ
Closest
50
−
40
−
30
−
20
−
10
−
0
10
20
30
40
50
Muons
50
100
150
200
250
300
Clusters with 1 strip, RMS = 6.3 mm
Clusters with 2 strips, RMS = 5.8 mm
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
view
φ
One RPC panel,
(b) (b) (b) (a) Figure 6. Cluster residuals for one representative RPC module for (a) 𝜂strips and (b) 𝜙strips. The residuals
were computed by subtracting the expected muon position from the centre of the cluster. Clusters with one
strip and with two strips are shown separately. The RMS values are computed using entries with residuals
between ±20 mm. No time requirements are used to select the individual RPC hits. For clusters made of two
strips, the hit times are required to be within 12.5 ns of each other. 4.2
RPC detector response Fraction of on track RPC hits with |t| > 12.5 ns
0
0.02
0.04
0.06
0.08
0.1
RPC panels
0
500
1000
1500
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons, on track hits
µ
µ
→
Z
0.0002
±
view, mean = 0.0065
η
0.0003
±
view, mean = 0.0113
φ
(b) Fraction of all RPC hits with |t| > 12.5 ns
0
0.1
0.2
0.3
0.4
0.5
RPC panels
0
200
400
600
800
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons, all hits
µ
µ
→
Z
0.0010
±
view, mean = 0.0519
η
0.0013
±
view, mean = 0.0957
φ
(a) JINST 16 P07029 (b) (a) Figure 7. Fraction of RPC hits with a time |𝑡| > 12.5 ns, for (a) all hits and (b) only for hits belonging to
strips with an expected muon hit. Only active RPC modules were used for these plots. The passage of a muon through an RPC module can produce several hits and form a cluster. Two quantities are used to study cluster properties: the cluster hit multiplicity and the cluster size. The cluster hit multiplicity is an integer distribution, with one entry corresponding to one muon
candidate, where a given multiplicity bin is filled with the number of hits recorded in one event for
a muon candidate passing through a given panel. Distributions for individual panels are added up
to produce the cluster hit multiplicity distribution averaged over a collection of panels or over the
entire detector. These distributions can be computed using muon candidates recorded in one run
or in the entire dataset. The cluster size is defined as the mean value of the cluster hit multiplicity
distribution for one panel. The cluster hit multiplicity distribution is averaged over all RPC panels and shown in figure 8(a),
computed using the full 2018 dataset. The average RPC cluster is approximately 1.5 strips wide,
with the average taken separately over 𝜂and 𝜙panels and using all active RPC modules. The
average 𝜂panel has a cluster multiplicity which is slightly smaller than that of the average 𝜙panel,
due to the differences in the construction and readout as discussed in section 2.2. Figure 8(b) shows
the average cluster size distributions for all active RPC modules, which is computed as the mean of
the cluster hit multiplicity distribution for each 𝜂and 𝜙panel. 4.2
RPC detector response As discussed in section 4.1, a majority of the hits recorded in a module containing the muon impact
point are expected to be closely associated in time with the arrival time of that muon. RPC hit
times are calibrated in order to equalise the RPC timing response for ultra-relativistic particles
produced at the interaction point. A small number of out-of-time hits, defined as hits with time
|𝑡| > 12.5 ns, are also recorded, primarily due to noise and delayed signals, with a small contribution – 11 – from imperfect RPC timing calibration. Figure 7(a) shows the fractions of out-of-time hits for all
RPC modules. These fractions were computed for each module using only those events that include
a muon candidate predicted to pass through that module. The larger mean out-of-time fraction for 𝜙
hits is due to the differences between 𝜂and 𝜙panels described in section 2.2. The mean out-of-time
fraction is reduced to about 1% when only the hits lying on the extrapolated muon track are included
in the computation, as shown in figure 7(b). This result demonstrates that only a small fraction of
the out-of-time muon hits originate from imperfect RPC timing calibrations. Fraction of all RPC hits with |t| > 12.5 ns
0
0.1
0.2
0.3
0.4
0.5
RPC panels
0
200
400
600
800
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons, all hits
µ
µ
→
Z
0.0010
±
view, mean = 0.0519
η
0.0013
±
view, mean = 0.0957
φ
(a)
Fraction of on track RPC hits with |t| > 12.5 ns
0
0.02
0.04
0.06
0.08
0.1
RPC panels
0
500
1000
1500
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons, on track hits
µ
µ
→
Z
0.0002
±
view, mean = 0.0065
η
0.0003
±
view, mean = 0.0113
φ
(b)
Figure 7. Fraction of RPC hits with a time |𝑡| > 12.5 ns, for (a) all hits and (b) only for hits belonging to
strips with an expected muon hit. Only active RPC modules were used for these plots. 4.2
RPC detector response There are some 𝜂panels that have
larger-than-typical cluster sizes, seen as the tail of the 𝜂distribution in figure 8(b). The cluster hit multiplicity distribution averaged over the entire RPC detector is given in
table 1. Muons produce clusters with two or more hits in 30% and 36% of events for the 𝜂and 𝜙
panels, respectively. Larger clusters containing four or more hits are produced in 2.7% and 1.6% of
events for the 𝜂and 𝜙panels, respectively. Variations in the cluster hit multiplicity are due to the
differences between 𝜂and 𝜙panels described in section 2.2 and to small differences in strip widths. – 12 – RPC cluster multiplicity in response to muons [strips]
0
5
10
Fraction of muons
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
Combined cluster multiplicity over
all RPC modules
view, mean = 1.430
η
view, mean = 1.452
φ
(a)
Mean RPC module cluster size [strips]
1
2
3
4
Modules
0
500
1000
1500
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
All RPC modules
view, mean = 1.450
η
view, mean = 1.431
φ
(b)
Figure 8. (a) RPC cluster hit multiplicity distribution and (b) RPC cluster size distribution, both averaged
over all modules, in response to the passage of a muon through a module, with 𝜂and 𝜙panels shown
separately. RPC cluster multiplicity in response to muons [strips]
0
5
10
Fraction of muons
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
Combined cluster multiplicity over
all RPC modules
view, mean = 1.430
η
view, mean = 1.452
φ
(a) Mean RPC module cluster size [strips]
1
2
3
4
Modules
0
500
1000
1500
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
All RPC modules
view, mean = 1.450
η
view, mean = 1.431
φ
(b) 2021 (b) (a) Figure 8. (a) RPC cluster hit multiplicity distribution and (b) RPC cluster size distribution, both averaged
over all modules, in response to the passage of a muon through a module, with 𝜂and 𝜙panels shown
separately. JINST 16 P07029 Table 1. 4.2
RPC detector response Cluster hit multiplicity distribution in response to a passage of a muon, shown separately for 𝜂and
𝜙panels. These multiplicity distributions are summed over all active RPC modules, separately for 𝜂and 𝜙
panels. The statistical uncertainty for all table entries is smaller than 0.1%. Table 1. Cluster hit multiplicity distribution in response to a passage of a muon, shown separately for 𝜂and
𝜙panels. These multiplicity distributions are summed over all active RPC modules, separately for 𝜂and 𝜙
panels. The statistical uncertainty for all table entries is smaller than 0.1%. RPC cluster multiplicity in response to muons [strips]
0
5
10
Fraction of muons
0
0.2
0.4
0.6
0.8
1
1.2
1.4
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
= 1.0 V
thr
V
view,
η
= 1.0 V
thr
V
view,
φ
> 1.0 V
thr
V
view,
η
> 1.0 V
thr
V
view,
φ
Combined cluster multiplicity over
all selected RPC modules
(a)
Mean RPC module cluster size [strips]
1
2
3
4
Modules
500
1000
1500
2000
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
All RPC modules, Z
= 1.0 V
thr
V
view,
η
= 1.0 V
thr
V
view,
φ
> 1.0 V
thr
V
view,
η
> 1.0 V
thr
V
view,
φ
(b)
Figure 9. (a) Cluster hit multiplicity and (b) cluster size distributions for all selected modules shown
separately for 𝜂and 𝜙panels using nominal (𝑉thr = 1.0 V) and higher than nominal (𝑉thr > 1.0 V) FE
thresholds. 4.2
RPC detector response RPC cluster multiplicity in response to muons [strips]
0
5
10
Fraction of muons
0
0.2
0.4
0.6
0.8
1
1.2
1.4
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
= 1.0 V
thr
V
view,
η
= 1.0 V
thr
V
view,
φ
> 1.0 V
thr
V
view,
η
> 1.0 V
thr
V
view,
φ
Combined cluster multiplicity over
all selected RPC modules
(a) Mean RPC module cluster size [strips]
1
2
3
4
Modules
500
1000
1500
2000
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
All RPC modules, Z
= 1.0 V
thr
V
view,
η
= 1.0 V
thr
V
view,
φ
> 1.0 V
thr
V
view,
η
> 1.0 V
thr
V
view,
φ
(b) (b) (a) Figure 9. (a) Cluster hit multiplicity and (b) cluster size distributions for all selected modules shown
separately for 𝜂and 𝜙panels using nominal (𝑉thr = 1.0 V) and higher than nominal (𝑉thr > 1.0 V) FE
thresholds. To illustrate the effect of the FE thresholds on the RPC cluster size, the cluster hit multiplicity
distribution and cluster size were measured separately for panels with nominal FE thresholds and for
panels with FE thresholds higher than their nominal values. One typical ATLAS run was analysed
for this study. In this run, the majority of the panels utilised the default FE threshold setting of
𝑉thr = 1.0 V. A small fraction of the panels utilised higher-than-nominal FE thresholds that varied
in the range from 𝑉thr = 1.1 V to 𝑉thr = 1.5 V in steps of 0.1 V. These results are shown in figure 9 – 13 – Day/month in 2018
30/04 20/05 09/06 29/06 19/07 08/08 28/08 17/09 07/10 27/10
multiplicity [strips]
Mean combined RPC cluster
1.3
1.35
1.4
1.45
1.5
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
view
η
view
η
Run 358395
view
φ
view
φ
Run 358395
(a)
Mean number of pp interactions per event
10
20
30
40
50
60
multiplicity [strips]
Mean combined RPC cluster
1.36
1.38
1.4
1.42
1.44
1.46
1.48
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
view
η
view
φ
(b)
Figure 10. 4.2
RPC detector response Mean of the RPC detector cluster hit multiplicity distribution plotted (a) as a function of time and
(b) as a function of average number of proton-proton collisions per event. Mean values for 𝜂and 𝜙panels
are shown separately. The blue and red stars refer to the representative run used in most of the performance
studies shown in this paper. Mean number of pp interactions per event
10
20
30
40
50
60
multiplicity [strips]
Mean combined RPC cluster
1.36
1.38
1.4
1.42
1.44
1.46
1.48
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
view
η
view
φ
(b) Day/month in 2018
30/04 20/05 09/06 29/06 19/07 08/08 28/08 17/09 07/10 27/10
multiplicity [strips]
Mean combined RPC cluster
1.3
1.35
1.4
1.45
1.5
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
view
η
view
η
Run 358395
view
φ
view
φ
Run 358395
(a) 2021 (b) (a) Figure 10. Mean of the RPC detector cluster hit multiplicity distribution plotted (a) as a function of time and
(b) as a function of average number of proton-proton collisions per event. Mean values for 𝜂and 𝜙panels
are shown separately. The blue and red stars refer to the representative run used in most of the performance
studies shown in this paper. JINST 16 P07029 separately for the 𝜂and 𝜙panels. As expected, higher FE thresholds result in smaller cluster hit
multiplicity and smaller cluster sizes. The average cluster hit multiplicity was monitored as a function of time, as shown in figure 10(a)
where each entry corresponds to one ATLAS run. The small variations among runs are due to
changes in the FE thresholds and changes in the detector conditions, such as automatic adjustments
of the applied voltage. Overall, the mean RPC cluster multiplicity was stable in 2018 to within a
few percent. The small decrease in the cluster multiplicity in late April and early May was due to
operating the RPCs at nominal voltage with collisions following the winter LHC shutdown when
the RPC voltage was off. The small increase in August was due to adjustments of the FE thresholds,
leading to the slightly larger detector efficiency described in section 4.3. The average cluster hit multiplicity is plotted in figure 10(b) as a function of the number of
proton-proton interactions per event. 4.2
RPC detector response An instantaneous luminosity of 2×1034 cm−2s−1 corresponds
on average to about 56 collisions per event. A reduction of approximately 1% to 2% between the first
and last bins was observed. This small reduction is due to dead time imposed by the readout system
and to offline reconstruction in each individual strip, in addition to a small chamber inefficiency at
higher detector occupancy [36, 37]. If a strip records a hit due to a background event, then a later
muon hit within the dead-time window will be discarded, thereby introducing a small inefficiency
in that channel. These background hits would decrease both the mean cluster size and also the
muon detection efficiency, as shown in section 4.3. 4.3
RPC detector performance The RPC efficiency for muon-induced signals is shown in figure 11(a) for all RPC modules without
known defects using data from one representative ATLAS run. The efficiency is computed using
signal hits, defined in section 4.1. The efficiency for 𝜂and 𝜙panels belonging to the same module is
computed independently by requiring at least one hit in 𝜂or 𝜙strips, respectively. The module (gas
gap) efficiency is computed by requiring at least one hit either in 𝜂or 𝜙strips belonging to the same
module. The gap efficiency corresponds to the intrinsic RPC efficiency to detect a muon-induced
avalanche. The efficiency of the individual 𝜂or 𝜙panel within a module includes the additional – 14 – effect due to intrinsic efficiency of the FE electronics to register the avalanche signal. This FE
efficiency is estimated as the difference between the module efficiency and individual 𝜂or 𝜙panel
efficiency. The average efficiency of the FE electronics to record the avalanche signal is measured
to be approximately 97%. This number also includes small contributions from a few faulty strips
in some of the panels. The RPC detector efficiency is plotted as a function of time in figure 11(b), which shows the
𝜂/𝜙panel efficiency and gas gap efficiency. The mean detector efficiency in each ATLAS run
is computed by averaging over all active RPC modules with efficiency greater than 50%, which
represent about 90% of the total number of modules. A small increase in the average efficiency of
about 0.3% in August 2018 was due to adjustments of FE thresholds. No significant ageing effects
have been observed during 2018 while the ATLAS detector recorded approximately 60.8 fb−1 of
proton-proton collision data. 4.3
RPC detector performance 2021 Efficiency
0.5
0.6
0.7
0.8
0.9
1
Gaps, panels/0.01
200
400
600
800
1000
1200
panels, mean = 0.910
η
panels, mean = 0.914
φ
Gas gaps, mean = 0.948
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
|t| < 12.5 ns, |d| < 30 mm
(a) Efficiency
0.5
0.6
0.7
0.8
0.9
1
Gaps, panels/0.01
200
400
600
800
1000
1200
panels, mean = 0.910
η
panels, mean = 0.914
φ
Gas gaps, mean = 0.948
ATLAS
-1
= 13 TeV, Run 358395, 0.72 fb
s
probe muons
µ
µ
→
Z
|t| < 12.5 ns, |d| < 30 mm
(a)
Day/month in 2018
30/04 20/05 09/06 29/06 19/07 08/08 28/08 17/09 07/10 27/10
Mean RPC detector efficiency
0.82
0.84
0.86
0.88
0.9
0.92
0.94
0.96
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
Mean of all RPC modules
probe muons
µ
µ
→
Z
|t| < 12.5 ns, |d| < 30 mm
view
η
view
φ
gap
(b)
Figure 11. (a) Distribution of the muon detection efficiency for all active RPC modules. (b) Muon detection
efficiency for 𝜂panels, 𝜙panels and gaps averaged over all active RPC modules plotted as a function of time. Day/month in 2018
30/04 20/05 09/06 29/06 19/07 08/08 28/08 17/09 07/10 27/10
Mean RPC detector efficiency
0.82
0.84
0.86
0.88
0.9
0.92
0.94
0.96
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
Mean of all RPC modules
probe muons
µ
µ
→
Z
|t| < 12.5 ns, |d| < 30 mm
view
η
view
φ
gap
(b) JINST 16 P07029 T 16 P07029 (a) (b) Figure 11. (a) Distribution of the muon detection efficiency for all active RPC modules. (b) Muon detection
efficiency for 𝜂panels, 𝜙panels and gaps averaged over all active RPC modules plotted as a function of time. The module efficiency is shown in figure 12 for all modules in the first and second layers of
the RPC1 doublet layer. The red bins indicate modules with low efficiency or modules with the
operating voltage offdue to gas leaks. The fraction of the chambers with gas leaks was around 5%
in 2015 and increased to about 10% in 2018 at the end of the most recent data-taking period. 4.3
RPC detector performance These
less efficient modules had only a small impact on the overall muon trigger efficiency because muons
producing a single hit in the two layers can still satisfy the trigger logic requirements, as discussed
in section 5.1. Approximately two thirds of the chambers with gas leaks are expected to be repaired
during the 2019–2021 LHC shutdown. The muon detection efficiency is shown in figure 13 as a function of the mean number of
proton-proton interactions per event. The detector efficiency is reduced by approximately 1% over
the range considered for the reasons discussed in section 4.2. The detector efficiency linearly
extrapolated to an instantaneous luminosity of 7.5 × 1034 cm−2s−1 is approximately 87%. The
dependence of the detector efficiency on detector occupancy is investigated further in section 6.4. One potential bias in measuring the efficiency of 𝜙panels can arise from the offline recon-
struction of muon candidates, which uses RPCs to measure the 𝜙coordinates of muon tracks in
the MS. The RPC detector provides up to six 𝜂and 𝜙position measurements for each muon track. – 15 – index
η
8
−
7
−
6
−
5
−
4
−
3
−
2
−
1
−
0 1 2 3 4 5 6 7 8
sector
φ
L1
S2
L3
S4
L5
S6
L7
S8
L9
S10
L11
FG12
L13
FG14
L15
S16
gap
∈
0
0.2
0.4
0.6
0.8
1
-1
= 13 TeV, 0.72 fb
s
, Run 358395,
ATLAS
probe muons, RPC1, gas gap 1
µ
µ
→
Z
(a)
index
η
8
−
7
−
6
−
5
−
4
−
3
−
2
−
1
−
0 1 2 3 4 5 6 7 8
sector
φ
L1
S2
L3
S4
L5
S6
L7
S8
L9
S10
L11
FG12
L13
FG14
L15
S16
gap
∈
0
0.2
0.4
0.6
0.8
1
-1
= 13 TeV, 0.72 fb
s
, Run 358395,
ATLAS
probe muons, RPC1, gas gap 2
µ
µ
→
Z
(b)
Figure 12. Muon detection efficiency in the 𝜂–𝜙plane for all modules in (a) the first layer and (b) the
second layer of the RPC1 doublet layer. Modules within one station are represented using half-integer values
of 𝜂index and 𝜙sector. Empty bins correspond to logical combinations of indices that do not represent
installed RPCs. 4.3
RPC detector performance index
η
8
−
7
−
6
−
5
−
4
−
3
−
2
−
1
−
0 1 2 3 4 5 6 7 8
sector
φ
L1
S2
L3
S4
L5
S6
L7
S8
L9
S10
L11
FG12
L13
FG14
L15
S16
gap
∈
0
0.2
0.4
0.6
0.8
1
-1
= 13 TeV, 0.72 fb
s
, Run 358395,
ATLAS
probe muons, RPC1, gas gap 1
µ
µ
→
Z
(a) index
η
8
−
7
−
6
−
5
−
4
−
3
−
2
−
1
−
0 1 2 3 4 5 6 7 8
sector
φ
L1
S2
L3
S4
L5
S6
L7
S8
L9
S10
L11
FG12
L13
FG14
L15
S16
gap
∈
0
0.2
0.4
0.6
0.8
1
-1
= 13 TeV, 0.72 fb
s
, Run 358395,
ATLAS
probe muons, RPC1, gas gap 2
µ
µ
→
Z
(b) 2021 (b) (a) Figure 12. Muon detection efficiency in the 𝜂–𝜙plane for all modules in (a) the first layer and (b) the
second layer of the RPC1 doublet layer. Modules within one station are represented using half-integer values
of 𝜂index and 𝜙sector. Empty bins correspond to logical combinations of indices that do not represent
installed RPCs. JINST 16 P07029 Mean number of pp interactions per event
10
20
30
40
50
60
Mean RPC detector efficiency
0.895
0.9
0.905
0.91
0.915
0.92
0.925
0.93
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
|t| < 12.5 ns, |d| < 30 mm
view
η
view
φ
Figure 13. Muon detection efficiency for 𝜂and 𝜙panels, averaged over all active RPC modules, plotted as a
function of average number of proton-proton collisions per event. Mean number of pp interactions per event
10
20
30
40
50
60
Mean RPC detector efficiency
0.895
0.9
0.905
0.91
0.915
0.92
0.925
0.93
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
probe muons
µ
µ
→
Z
|t| < 12.5 ns, |d| < 30 mm
view
η
view
φ Figure 13. Muon detection efficiency for 𝜂and 𝜙panels, averaged over all active RPC modules, plotted as a
function of average number of proton-proton collisions per event. Therefore, the impact of a single RPC detector layer on the muon detection efficiency is expected
to be negligible. This hypothesis was first checked by extrapolating ID tracks to the RPC detector
surfaces. 4.3
RPC detector performance No detectable biases were observed for the measurements reported in this section when
using this alternative extrapolation algorithm. This effect was also checked by comparing the ef-
ficiencies of the 𝜂panel and 𝜙panel belonging to the same RPC module. The average difference
between the 𝜂and 𝜙efficiencies is consistent with zero, confirming that no significant bias was
introduced by using RPCs for reconstructing 𝜙positions of muon tracks in the MS. 4.4
Time resolution of RPC detector and readout system The RPC technology was chosen by the ATLAS experiment for the L1 muon barrel trigger because
of its fast response, good time and position resolution, and relatively low cost [19]. Before the
construction of the ATLAS detector, the RPC time resolution was measured to be between 1.1 ns
and 1.4 ns, using full-size prototype units of the ATLAS RPCs equipped with the final version of the
FE electronics [38]. The time resolution of the installed RPC detectors was measured using muons – 16 – produced in proton-proton collisions recorded in 2011 at a centre-of-mass energy of 7 TeV. A time
resolution value of around 2 ns was obtained [39]. This higher value, compared to the results from
test-beam facilities, was due to combined effects of the RPC intrinsic resolution and the readout
system resolution. In this section, the intrinsic time resolution of the RPC detector and the time resolution of RPC
electronics are evaluated separately using newly developed procedures for the analysis of the RPC
timing response to probe muons produced in the decays of 𝑍bosons, with the selection criteria
detailed in section 3. These results aim to quantify the RPC intrinsic time resolution for detecting
a signal induced by an ionising particle, excluding effects related to the resolution of its absolute
arrival time. The intrinsic RPC time resolution depends on fluctuations in the location of the first
ionisation event along the muon path through the RPC gas gap and on the statistical fluctuations
inherent in the subsequent avalanche amplification process [40]. The electronics time resolution
component includes several sources, such as the 320 MHz sampling frequency and the associated
jitter [22]. The total time resolution (𝜎total) of an RPC is defined as: 2021 1 JINST 16 P07029 𝜎2
total = 𝜎2
intrinsic + 𝜎2
electronics
(4.1) (4.1) JINST 16 P07029 where 𝜎intrinsic is the component corresponding to the intrinsic RPC time resolution and 𝜎electronics
is the electronics component corresponding to the time resolution of the readout system. The total RPC time resolution was estimated using time differences between detector signals
generated by the same muon passing through two parallel single RPC layers belonging to the same
module. These two layers are separated by a distance of approximately 20 mm. 4.4
Time resolution of RPC detector and readout system – 17 – 10
−
5
−
0
5
10
[ns]
η
layer 1
- t
η
layer 0
RPC t
0
100
200
300
400
Muons/3.125 ns
ATLAS
-1
= 13 TeV, 60.8 fb
s
0.12 ns
±
Fitted mean = 0.26
0.09 ns
±
= 1.92
fit, total
σ
Fitted width =
Data
Gaussian fit
Expected entries from fit
(a)
0
2
4
6
8
10
[ns]
fit, total
σ
0
2000
4000
6000
8000
10000
RPC readout strip pairs/0.1 ns
ATLAS
-1
= 13 TeV, 60.8 fb
s
fit, total
σ
Fitted width =
view, mean = 2.06 ns
η
view, mean = 2.21 ns
φ
(b)
Figure 14. (a) Time-difference distribution between signals generated by the same muon in a single pair of
𝜂strips, matched with the muon track, in two parallel RPC detector layers. The bin width corresponds to the
3.125 ns sampling time. (b) Distribution of the total time differences for the all selected RPC strip pairs. 0
2
4
6
8
10
[ns]
fit, total
σ
0
2000
4000
6000
8000
10000
RPC readout strip pairs/0.1 ns
ATLAS
-1
= 13 TeV, 60.8 fb
s
fit, total
σ
Fitted width =
view, mean = 2.06 ns
η
view, mean = 2.21 ns
φ
(b) 10
−
5
−
0
5
10
[ns]
η
layer 1
- t
η
layer 0
RPC t
0
100
200
300
400
Muons/3.125 ns
ATLAS
-1
= 13 TeV, 60.8 fb
s
0.12 ns
±
Fitted mean = 0.26
0.09 ns
±
= 1.92
fit, total
σ
Fitted width =
Data
Gaussian fit
Expected entries from fit
(a) 2021 (a) (b) Figure 14. (a) Time-difference distribution between signals generated by the same muon in a single pair of
𝜂strips, matched with the muon track, in two parallel RPC detector layers. The bin width corresponds to the
3.125 ns sampling time. (b) Distribution of the total time differences for the all selected RPC strip pairs. JINST 16 P07029 When the time differences were measured using only those muons that produce clusters with a single
hit in both layers, no difference between 𝜂and 𝜙strips was observed for the time difference widths. When the time differences were measured using only those muons that produce clusters with a single
hit in both layers, no difference between 𝜂and 𝜙strips was observed for the time difference widths. 4.4
Time resolution of RPC detector and readout system The time difference
was computed using hits produced in the two parallel strips (either a pair of 𝜂strips or a pair
of 𝜙strips) that are closest to the muon track in each of the two detector layers. A relativistic
muon travelling nearly at the speed of light will produce a time-of-flight difference of about 0.07 ns
when travelling perpendicular to the two detector layers. Since the contribution from the muon
time-of-flight component is negligible, the time differences between signals produced in the two
layers are dominated by the total RPC time resolution. The time-difference distributions were obtained for all geometrically possible combinations of
𝜂strip pairs and 𝜙strip pairs in each doublet layer of the RPC detector. The same timing circuit was
used to measure these two signals so no differences are introduced due to clock synchronisation. Only distributions with at least 100 entries were selected for the analysis in order to remove strips
with low efficiency. The width of the time-difference distribution was determined from a binned
maximum-likelihood fit of a Gaussian function to the observed distribution of time differences. An
example fit is shown in figure 14(a) for a pair of 𝜂strips from the two parallel layers of one RPC
chamber. The Kolmogorov-Smirnov (KS) test was performed to assess the goodness of fit. To
perform this test, a new histogram was generated with the number of entries in each bin equal to the
area of the Gaussian curve in that bin divided by the bin width. Only strip pair distributions with
a KS probability greater than 0.1 and with at least three non-empty bins were retained for the final
analysis. These selection criteria have a combined efficiency of about 98%. The best-fit Gaussian widths for all selected 𝜂strip pairs and 𝜙strip pairs are shown in
figure 14(b) for the full RPC system. The mean width of the time differences is approximately
2.1 ns and 2.2 ns for 𝜂and 𝜙strips, respectively. The small difference between the 𝜂and 𝜙strips is
due to the different cluster size distributions for 𝜂and 𝜙panels, which is discussed in section 4.2. 4.4
Time resolution of RPC detector and readout system 10
−
5
−
0
5
10
[ns]
φ
layer 0
- t
η
layer 0
RPC t
0
50
100
Muons/3.125 ns
ATLAS
-1
= 13 TeV, 60.8 fb
s
0.22 ns
±
Fitted mean = 0.04
0.17 ns
±
= 1.61
fit, electronics
σ
Fitted width =
Data
Gaussian fit
Expected entries from fit
(a)
1
2
3
4
[ns]
fit, electronics
σ
Weighted
0
10000
20000
30000
40000
RPC readout strip pairs/0.1 ns
ATLAS
-1
= 13 TeV, 60.8 fb
s
= fitted width
fit, electronics
σ
Layer0, mean = 1.48 ns
Layer1, mean = 1.48 ns
Combined, mean = 1.47 ns
(b)
Figure 15. (a) Time-difference distribution between signals generated by the same muon in a single pair of 𝜂
and 𝜙strips, matched with the muon track, belonging to one detector layer. (b) Distribution of the electronics
component of the time resolution. 10
−
5
−
0
5
10
[ns]
φ
layer 0
- t
η
layer 0
RPC t
0
50
100
Muons/3.125 ns
ATLAS
-1
= 13 TeV, 60.8 fb
s
0.22 ns
±
Fitted mean = 0.04
0.17 ns
±
= 1.61
fit, electronics
σ
Fitted width =
Data
Gaussian fit
Expected entries from fit
(a) 1
2
3
4
[ns]
fit, electronics
σ
Weighted
0
10000
20000
30000
40000
RPC readout strip pairs/0.1 ns
ATLAS
-1
= 13 TeV, 60.8 fb
s
= fitted width
fit, electronics
σ
Layer0, mean = 1.48 ns
Layer1, mean = 1.48 ns
Combined, mean = 1.47 ns
(b) (b) (a) Figure 15. (a) Time-difference distribution between signals generated by the same muon in a single pair of 𝜂
and 𝜙strips, matched with the muon track, belonging to one detector layer. (b) Distribution of the electronics
component of the time resolution. The electronics component of the time resolution was estimated by taking the difference
between time measurements of simultaneous 𝜂and 𝜙hits from the same avalanche event induced
by a muon passing through a single detector layer. The intrinsic RPC resolution cancels out in this
measurement because the same avalanche event is observed by a pair of 𝜂and 𝜙strips. Two different
timing circuits were used to measure these two signals but since their clocks are synchronised to
the same LHC reference clock, any differences in clock synchronisation would produce a shift of
the mean of the time-difference distribution but leave its width unchanged. 4.4
Time resolution of RPC detector and readout system The time-difference distribution was computed for each pair of orthogonal 𝜂and 𝜙strips
observing the same avalanche event in the common gas volume. Delays due to different strip and
cable lengths result in a constant offset of the mean value of this time-difference distribution. The – 18 – width of this time-difference distribution is then proportional to the electronics time resolution. A binned maximum-likelihood fit to a Gaussian function was performed for each time-difference
distribution. Only distributions with at least 20 entries were selected in order to remove strip pairs
with low efficiency. An example fit is shown in figure 15(a). The KS test was performed to assess
the goodness of fit. Only strip pair distributions with a KS probability greater than 0.4 and with at
least three non-empty bins were retained for the final analysis, with the later criterion removing the
majority of strip pairs. These selection criteria have a combined efficiency of about 75%. 0
2
4
6
8
10
[ns]
2
fit, electronics
σ
-
2
fit, total
σ
0
2000
4000
6000
8000
RPC readout strip pairs/0.1 ns
ATLAS
-1
= 13 TeV, 60.8 fb
s
view, mean = 1.44 ns
η
view, mean = 1.64 ns
φ
Figure 16. Distribution of the intrinsic component of the time resolution for 𝜂and 𝜙panels. JINST 16 P07029 Figure 16. Distribution of the intrinsic component of the time resolution for 𝜂and 𝜙panels. All possible combinations of 𝜂and 𝜙strip pairs were considered in each layer of each module
for the full RPC system. For each 𝜂(𝜙) strip, several combinations with orthogonal 𝜙(𝜂) strips
can be made. The electronics time resolution component associated with each single strip is
therefore estimated as the statistically weighted average among all possible combinations with the
orthogonal strips, where each individual measurement was weighted by the inverse of the square
of the uncertainty in the fitted value of the Gaussian width parameter. Figure 15(b) shows the
distribution of the best-fit Gaussian widths of the time-difference distributions for the full RPC
system. The results for two layers for each module were combined for the estimate of the intrinsic
time resolution component using the same weighting procedure, since both layers were used in the
total time resolution measurements. Finally, the intrinsic component of the time resolution was estimated using eq. (4.1). The
resulting distributions are shown in figure 16 for all selected 𝜂and 𝜙strip pairs. 4.4
Time resolution of RPC detector and readout system The mean values
of the distributions should be divided by
√
2 to extract the intrinsic time resolution of one RPC
module. The mean value of the resulting intrinsic time resolution is consistent with the previous
measurements obtained at test-beam facilities. 5.1
Trigger roads Processing of signals (hits) produced by the RPC FE electronics is first performed by the on-detector
electronics contained in the processor box (PAD) [22]. Each PAD contains four coincidence
matrices, with each matrix implemented in one application-specific integrated circuit, referred to
as CMA. These custom-designed circuits perform digital signal shaping, set a programmable dead
time, mask channels and perform trigger logic operations. The CMA trigger logic aligns the FE
signals in time, checks the time coincidence of RPC hits, and applies the geometrical matching
criteria for selecting one of the three programmable 𝑝T thresholds. Two types of PADs are deployed:
one is responsible for the low-𝑝T trigger and another is responsible for the high-𝑝T trigger. In each
PAD, two CMAs collect the signals from the 𝜂view strips and another two CMAs collect the signals
from the 𝜙view strips. Each RPC PAD covers an 𝜂× 𝜙detector region of approximately 0.2 × 0.2,
with the overlap of one 𝜙CMA and one 𝜂CMA corresponding to a single RoI of approximately
0.1 × 0.1. Two PADs, one for the low-𝑝T trigger and one for the high-𝑝T trigger, make one trigger
tower. A set of six, seven or eight trigger towers, placed along the 𝑧-axis at a fixed 𝜙position, makes
one trigger sector. There are in total 432 trigger towers, divided in 64 azimuthal trigger sectors,
with 32 sectors on each ATLAS side. 2021 JINST 16 P07 2021 JINST 16 P07029 CMAs identify muon candidates and measure their momentum by searching for geometrical
matching of RPC hits inside programmable windows, called trigger roads, defined using the detector
strips, as illustrated in figure 3 of ref. [41]. A muon candidate satisfies the L1 barrel trigger logic
conditions if it generates RPC hits inside the trigger roads for a matching pair of 𝜂and 𝜙CMAs. The low-𝑝T (high-𝑝T) trigger checks for coincident RPC signals between the pivot RPC2 and RPC1
(RPC3) doublet layers within the corresponding trigger road. The low-𝑝T (high-𝑝T) trigger also
requires signals in three out of four (one out of two) RPC detector layers, in both the 𝜂and 𝜙
views. The trigger roads were defined to contain 95% of positively and negatively charged muons,
simulated using the ATLAS simulation infrastructure [42] with a fixed 𝑝T value equal to the trigger
𝑝T threshold. 5
Performance of L1 muon barrel trigger This section reports measurements of the L1 muon barrel trigger performance obtained using
proton-proton collision data. Section 5.1 studies the performance of individual trigger towers. Section 5.2 presents measurements of the L1 muon barrel trigger efficiency as a function of several
quantities. The measurements in these two sections were performed using events containing a 𝑍
boson decay into a pair of muons. Section 5.3 presents measurements of the L1 muon trigger’s – 19 – event selection rate as a function of the instantaneous luminosity. Finally, section 5.4 studies the
composition of the events selected by the L1 muon barrel trigger. 5.1
Trigger roads Trigger roads encode the RPC detector layout, magnetic field configuration, and
geometric relationships among strips in the different layers, as seen by a muon travelling from the
interaction region. 2. List of the hit selection criteria used to evaluate the performance of each individual CMA. Table 2. List of the hit selection criteria used to evaluate the performance of each individual CMA. Type
CMA type
Selection criteria
Muon kinematics
Low 𝑝T
𝑝T ≥10 GeV, |𝜂| ≤1.05
High 𝑝T
𝑝T ≥20 GeV, |𝜂| ≤1.05
Time
| 𝑡pivot layer channel −𝑡confirm layer channel| ≤12.5 ns
Layer
Low 𝑝T
𝑁pivot layers with hits + 𝑁confirm layers with hits ≥3
High 𝑝T
𝑁pivot layers with hits ≥1, 𝑁confirm layers with hits ≥1
Hit multiplicity
Low 𝑝T
𝑁hits in pivot layer ≤4, 𝑁hits in confirm layer ≤4
High 𝑝T
𝑁hits in pivot layer ≤2, 𝑁hits in confirm layer ≤4 – 20 – The performance of each individual CMA was evaluated using probe muons produced in
decays of the 𝑍bosons. Probe muons were matched with the four closest CMAs by requiring the
angular Δ𝑅distance between the muon track and the centre of the CMA to be less than 0.15. The
MU10 (MU20) trigger roads were evaluated using offline muon candidates with 𝑝T > 10 (20) GeV. Signals in the confirm layer were required to be within the 25 ns time window centred at the
pivot signal time, thereby requiring the selected signals to belong to the same bunch crossing. To
reduce contributions from background events due to random coincidences, at least three out of four
layers of RPC1 and RPC2 were required to contain signals in the selected low-𝑝T CMA. Similarly,
at least one of the two layers of RPC3 was required to contain signals in the selected high-𝑝T
CMA. These two selection criteria approximate similar conditions applied by the on-detector PAD
electronics [22]. Finally, events with more than four signals in one detector layer were removed to
reduce the number of combinations between the pivot and confirm layers. These selection criteria
are summarised in table 2. 5.1
Trigger roads 2021 0
5
10
15
20
25
30
Pivot channel
0
10
20
30
40
50
60
Confirm channel
ATLAS
-1
= 13 TeV, 60.8 fb
s
>10 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU10
CMA
T
region low p
η
Sector 0 PAD 2 CMA 3
= 98.16%
All
Muon hits
N
Inside trigger road
Muon hits
N
Muon hits
Trigger road
(a)
0
5
10
15
20
25
30
Pivot channel
0
10
20
30
40
50
60
Confirm channel
ATLAS
-1
= 13 TeV, 60.8 fb
s
>10 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU10
CMA
T
region low p
φ
Sector 0 PAD 2 CMA 1
= 95.66%
All
Muon hits
N
Inside trigger road
Muon hits
N
Muon hits
Trigger road
(b)
0
5
10
15
20
25
30
Pivot channel
0
10
20
30
40
50
60
Confirm channel
ATLAS
-1
= 13 TeV, 60.8 fb
s
>20 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU20
CMA
T
region high p
η
Sector 0 PAD 2 CMA 6
= 98.26%
All
Muon hits
N
Inside trigger road
Muon hits
N
Muon hits
Trigger road
(c)
0
5
10
15
20
25
30
Pivot channel
0
10
20
30
40
50
60
Confirm channel
ATLAS
-1
= 13 TeV, 60.8 fb
s
>20 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU20
CMA
T
region high p
φ
Sector 0 PAD 2 CMA 4
= 97.10%
All
Muon hits
N
Inside trigger road
Muon hits
N
Muon hits
Trigger road
(d)
Figure 17. L1 muon barrel trigger roads for four example CMAs: (a) MU10 low-𝑝T 𝜂CMA, (b) MU10
low-𝑝T 𝜙CMA, (c) MU20 high-𝑝T 𝜂CMA, (d) MU20 high-𝑝T 𝜙CMA. 5.1
Trigger roads 0
5
10
15
20
25
30
Pivot channel
0
10
20
30
40
50
60
Confirm channel
ATLAS
-1
= 13 TeV, 60.8 fb
s
>10 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU10
CMA
T
region low p
η
Sector 0 PAD 2 CMA 3
= 98.16%
All
Muon hits
N
Inside trigger road
Muon hits
N
Muon hits
Trigger road 0
5
10
15
20
25
30
Pivot channel
0
10
20
30
40
50
60
Confirm channel
ATLAS
-1
= 13 TeV, 60.8 fb
s
>10 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU10
CMA
T
region low p
φ
Sector 0 PAD 2 CMA 1
= 95.66%
All
Muon hits
N
Inside trigger road
Muon hits
N
Muon hits
Trigger road JINST 16 P07029 (b) (a) (a)
0
5
10
15
20
25
30
Pivot channel
0
10
20
30
40
50
60
Confirm channel
ATLAS
-1
= 13 TeV, 60.8 fb
s
>20 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU20
CMA
T
region high p
η
Sector 0 PAD 2 CMA 6
= 98.26%
All
Muon hits
N
Inside trigger road
Muon hits
N
Muon hits
Trigger road
(c) 0
5
10
15
20
25
30
Pivot channel
0
10
20
30
40
50
60
ATLAS
-1
= 13 TeV, 60.8 fb
s
>20 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU20
CMA
T
region high p
φ
Sector 0 PAD 2 CMA 4
= 97.10%
All
Muon hits
N
Inside trigger road
Muon hits
N
Muon hits
Trigger road
(d) Confirm channel (d) (c) Figure 17. L1 muon barrel trigger roads for four example CMAs: (a) MU10 low-𝑝T 𝜂CMA, (b) MU10
low-𝑝T 𝜙CMA, (c) MU20 high-𝑝T 𝜂CMA, (d) MU20 high-𝑝T 𝜙CMA. Figure 17 shows the selected RPC hits in the pivot and confirm layers for four representative
CMAs belonging to the same PAD: low- and high-𝑝T CMAs in both the 𝜂and 𝜙views. For each hit
in the pivot layer, all possible combinations with the selected hits in the confirm layer are reported. The trigger roads used for 2018 data-taking are shown in yellow. As expected, the majority of hits
are produced by the probe muons and are contained within the trigger roads. 5.1
Trigger roads Low-𝑝T CMA
numbers range from 0 to 3 and high-𝑝T CMA numbers range from 4 to 7. The trigger roads employed for
this study correspond to the MU10 and MU20 triggers for the low-𝑝T and high-𝑝T CMAs, respectively. 1 JINST 16 P07029 The fractions of the selected RPC hit pairs inside the trigger roads (referred to as trigger road
hit fractions) were computed for all CMAs in order to study the trigger road performance using the
actual detector. Figures 18(a) and 18(b) show the number of probe muons passing through each
CMA and the trigger road hit fractions for each CMA, respectively. Typically, several hundred
probe muons pass through each CMA, providing sufficient precision to study the performance of
the trigger roads. The fractions of the selected RPC hit pairs inside the trigger roads (referred to as trigger road
hit fractions) were computed for all CMAs in order to study the trigger road performance using the
actual detector. Figures 18(a) and 18(b) show the number of probe muons passing through each
CMA and the trigger road hit fractions for each CMA, respectively. Typically, several hundred
probe muons pass through each CMA, providing sufficient precision to study the performance of
the trigger roads. 16 P07029 50
55
60
65
70
75
80
85
90
95
100
Muon hits inside trigger road (%)
1
10
2
10
Number of CMA
region CMAs
η
region CMAs
φ
ATLAS
-1
= 13 TeV, 60.8 fb
s
>10 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU10
CMAs
T
Low p
(a)
50
55
60
65
70
75
80
85
90
95
100
Muon hits inside trigger road (%)
1
10
2
10
Number of CMA
region CMAs
η
region CMAs
φ
ATLAS
-1
= 13 TeV, 60.8 fb
s
>20 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU20
CMAs
T
High p
(b)
Figure 19. Distributions of the fraction of selected RPC hits inside the trigger road for the (a) MU10 and (b)
MU20 triggers, shown separately for 𝜂and 𝜙CMAs. 5.1
Trigger roads – 21 – 0
10
20
30
40
50
60
Sector number
0
10
20
30
40
50
60
PAD number x 10 + CMA number
0
500
1000
1500
2000
2500
3000
3500
4000
pivot-confirm pair of trigger road
Average muon hits in single
occupancy for MU10/MU20 trigger roads
L1 muon barrel trigger, average muon
-1
= 13 TeV, 60.8 fb
s
ATLAS
(a)
0
10
20
30
40
50
60
Sector number
0
10
20
30
40
50
60
PAD number x 10 + CMA number
0
10
20
30
40
50
60
70
80
90
100
Fraction of muon hits inside trigger road [%]
L1 muon barrel trigger, MU10/MU20 trigger road
-1
= 13 TeV, 60.8 fb
s
ATLAS
(b)
Figure 18. (a) Average number of the probe muons passing through each CMA and (b) fraction of the
selected RPC hits inside the trigger road for each CMA. The horizontal axis corresponds to the trigger sector
number. The vertical axis shows the PAD number multiplied by 10 plus the CMA number. Low-𝑝T CMA
numbers range from 0 to 3 and high-𝑝T CMA numbers range from 4 to 7. The trigger roads employed for
this study correspond to the MU10 and MU20 triggers for the low-𝑝T and high-𝑝T CMAs, respectively. 0
10
20
30
40
50
60
Sector number
0
10
20
30
40
50
60
PAD number x 10 + CMA number
0
500
1000
1500
2000
2500
3000
3500
4000
pivot-confirm pair of trigger road
Average muon hits in single
occupancy for MU10/MU20 trigger roads
L1 muon barrel trigger, average muon
-1
= 13 TeV, 60.8 fb
s
ATLAS
(a) 0
10
20
30
40
50
60
Sector number
0
10
20
30
40
50
60
PAD number x 10 + CMA number
0
10
20
30
40
50
60
70
80
90
100
Fraction of muon hits inside trigger road [%]
L1 muon barrel trigger, MU10/MU20 trigger road
-1
= 13 TeV, 60.8 fb
s
ATLAS
(b) 2021 (a) (b) Figure 18. (a) Average number of the probe muons passing through each CMA and (b) fraction of the
selected RPC hits inside the trigger road for each CMA. The horizontal axis corresponds to the trigger sector
number. The vertical axis shows the PAD number multiplied by 10 plus the CMA number. 5.1
Trigger roads 50
55
60
65
70
75
80
85
90
95
100
Muon hits inside trigger road (%)
1
10
2
10
Number of CMA
region CMAs
η
region CMAs
φ
ATLAS
-1
= 13 TeV, 60.8 fb
s
>20 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU20
CMAs
T
High p
(b) 50
55
60
65
70
75
80
85
90
95
100
Muon hits inside trigger road (%)
1
10
2
10
Number of CMA
region CMAs
η
region CMAs
φ
ATLAS
-1
= 13 TeV, 60.8 fb
s
>10 GeV
µ
T
, p
µ
µ
→
Z
Trigger road MU10
CMAs
T
Low p
(a) (b) (a) Figure 19. Distributions of the fraction of selected RPC hits inside the trigger road for the (a) MU10 and (b)
MU20 triggers, shown separately for 𝜂and 𝜙CMAs. Figure 19 shows the distributions of the trigger road hit fractions for the MU10 and MU20
triggers for all CMAs. In events with probe muons passing through the CMA, more than 95% of
the selected pivot and confirm hit pairs are contained within the trigger road. The trigger road hit
fractions are larger than 95% for the majority of CMAs, indicating good performance of the MU10 – 22 – and MU20 triggers. These fractions are slightly larger for the 𝜂CMAs than for the 𝜙CMAs because
the trigger roads were defined to contain 95% of muons with 𝑝T = 20 GeV while the majority of the
probe muons have 𝑝T between 30 and 50 GeV. Therefore, probe muon trajectories in the bending
𝜂view have a slighter higher probability to fall within the trigger road than in the non-bending 𝜙
view. Approximately 2% of 𝜂CMAs and 4% of 𝜙CMAs have trigger road hit fractions below 90%. These lower fractions are due to residual differences between the actual and simulated detector
geometries. 5.2
Trigger efficiency and timing 2021 JINST 16 P0 2021 The efficiency of the L1 muon barrel trigger to detect a probe muon was evaluated as a function of
several parameters. Figure 20 shows the trigger efficiency as a function of probe muon 𝜂and 𝜙for
the MU10 and MU20 triggers. Only muons with 𝑝T > 25 GeV were used for these measurements;
this removes the dependence of the trigger efficiency on the muon 𝑝T. Regular features in the trigger
efficiency distributions correspond to the ATLAS detector support structures and service elements. The drop in the trigger efficiency at 𝜂∼0 in figure 20(a) is due to the presence of detector services,
such as gas pipes, water pipes, cryogenic lines, and cables, which cause the region |𝜂| < 0.1 to be
only sparsely instrumented with RPCs. Other, smaller efficiency drops, mainly at |𝜂| ∼0.4 and
|𝜂| ∼0.8, are due to the presence of the detector support and service structures. The eight barrel
magnet coils are located in the small RPC sectors and their presence is visible in figure 20(b), where
a regular eightfold structure can be observed. JINST 16 P07029 1
−
0.5
−
0
0.5
1
η
Offline muon
0
0.2
0.4
0.6
0.8
1
L1 muon barrel trigger efficiency
L1 MU10
L1 MU20
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
> 25 GeV
T
µ
, p
µ
µ
→
Z
(a)
2
−
0
2
φ
Offline muon
0
0.2
0.4
0.6
0.8
1
L1 muon barrel trigger efficiency
L1 MU10
L1 MU20
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
> 25 GeV
T
µ
, p
µ
µ
→
Z
(b)
Figure 20. L1 muon barrel trigger efficiency as a function of (a) muon pseudorapidity and (b) muon
azimuthal angle. 2
−
0
2
φ
Offline muon
0
0.2
0.4
0.6
0.8
1
L1 muon barrel trigger efficiency
L1 MU10
L1 MU20
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
> 25 GeV
T
µ
, p
µ
µ
→
Z
(b) 1
−
0.5
−
0
0.5
1
η
Offline muon
0
0.2
0.4
0.6
0.8
1
L1 muon barrel trigger efficiency
L1 MU10
L1 MU20
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
> 25 GeV
T
µ
, p
µ
µ
→
Z
(a) (b) (a) Figure 20. 5.2
Trigger efficiency and timing They also require that the low-𝑝T muon candidate is matched with at least
one 𝜂and one 𝜙hit in the two outer RPC layers. This additional requirement reduces the ∼76%
efficiency of the MU10 trigger to approximately 70% for the MU20 trigger. JINST 16 P07029 The MU20 trigger was the primary L1 muon trigger for data-taking in 2015–2018 [16]. The
MU20 barrel trigger selects muon candidates using the entire RPC detector, including the new
chambers in the ATLAS feet region which were commissioned during 2015 and 2016. The new feet
trigger chambers were not included in the MU21 trigger logic, as demonstrated by the 3% higher
efficiency of the MU20 trigger in figure 22(a) compared to that of MU21. In the feet region, the
trigger logic for the high-𝑝T thresholds is similar to the low-𝑝T trigger logic because only two RPC
doublet layers are traversed by a muon produced at the collision point. This leads to a higher overall
trigger rate and to a higher fraction of trigger candidates due to background events, as discussed in
sections 5.3 and 5.4. The MU21 trigger was included as a backup in case the event acceptance rate
by the MU20 trigger exceeded the allowed limit. The overall efficiency of the L1 muon barrel trigger system was measured as a function of the
mean number of proton-proton interactions per event using probe muons with 𝑝T > 25 GeV, as
shown in figure 22(b). The barrel trigger efficiency decreases by about 1% between the lowest and
highest instantaneous luminosity values recorded in 2018. This decrease corresponds to a similar
decrease observed for the RPC detector, as discussed in section 4.3. The overall efficiency of the L1 muon barrel trigger was measured in individual runs recorded
by the ATLAS experiment in 2018. The efficiency to detect muons with 𝑝T > 25 GeV is plotted as
a function of time in figure 23(a) for all six trigger thresholds. Each point corresponds to a separate
ATLAS run, where only runs with an integrated luminosity greater than 50 pb−1 were included. The
small variations in the trigger efficiency between individual runs were due to changes in detector
conditions. For example, a small fraction of the detector elements may have been disabled in a
given run due to transient problems with the RPC modules or with the data acquisition system of
the RPC detector. 5.2
Trigger efficiency and timing L1 muon barrel trigger efficiency as a function of (a) muon pseudorapidity and (b) muon
azimuthal angle. The overall efficiency of the L1 muon barrel trigger system was measured as a function of the
probe muon 𝑝T for six thresholds, as shown in figure 22(a) and quantified in table 3. This overall
efficiency includes the geometrical acceptance effects illustrated in figure 21, the RPC efficiency
to detect a muon signal, and the efficiency of trigger logic conditions. Probe muons falling in the
region |𝜂| < 0.1 were removed from the analysis because this region is not instrumented with RPCs. The three low-𝑝T triggers (MU4, MU6, MU10) require a geometrical and timing coincidence of
at least three 𝜂hits and three 𝜙hits in the four innermost RPC layers. The three high-𝑝T triggers
(MU11, MU20, MU21) require that the highest threshold of the three low-𝑝T triggers is satisfied – 23 – 1
−
0.5
−
0
0.5
1
η
Offline muon
2
−
0
2
φ
Offline muon
0
0.2
0.4
0.6
0.8
1
MU10
∈
>25 GeV
µ
T
, p
µ
µ
→
L1 barrel trigger efficiency, MU10, Z
-1
= 13 TeV, 60.8 fb
s
ATLAS
(a)
1
−
0.5
−
0
0.5
1
η
Offline muon
2
−
0
2
φ
Offline muon
0
0.2
0.4
0.6
0.8
MU20
∈
>25 GeV
µ
T
, p
µ
µ
→
L1 barrel trigger efficiency, MU20, Z
-1
= 13 TeV, 60.8 fb
s
ATLAS
(b)
Figure 21. L1 muon barrel trigger efficiency as a function of the muon pseudorapidity and azimuthal angle
for the (a) MU10 and (b) MU20 triggers. 1
−
0.5
−
0
0.5
1
η
Offline muon
2
−
0
2
0
0.2
0.4
0.6
0.8
MU20
∈
>25 GeV
µ
T
, p
µ
µ
→
L1 barrel trigger efficiency, MU20, Z
-1
= 13 TeV, 60.8 fb
s
ATLAS
(b) 1
−
0.5
−
0
0.5
1
η
Offline muon
2
−
0
2
φ
Offline muon
0
0.2
0.4
0.6
0.8
1
MU10
∈
>25 GeV
µ
T
, p
µ
µ
→
L1 barrel trigger efficiency, MU10, Z
-1
= 13 TeV, 60.8 fb
s
ATLAS
(a) φ
Offline muon 2021 (b) (a) Figure 21. L1 muon barrel trigger efficiency as a function of the muon pseudorapidity and azimuthal angle
for the (a) MU10 and (b) MU20 triggers. by the muon candidate. 5.2
Trigger efficiency and timing The removed elements, which impact the trigger efficiency by at most 4%, were
usually recovered and included in the next run. The assignment of an L1 muon trigger candidate to the correct bunch crossing requires accurate
timing calibration of the RPC readout system. These timing calibrations [22] correct for the different – 24 – 0
20
40
60
80
[GeV]
T
Offline muon p
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
L1 muon barrel trigger efficiency
L1 MU4
L1 MU6
L1 MU10
L1 MU11
L1 MU20
L1 MU21
ATLAS
Data 2018
-1
= 13 TeV, 60.8 fb
s
< 1.05
µ
η
, 0.1 <
µ
µ
→
Z
(a)
10
20
30
40
50
60
70
Mean number of pp interactions per event
0.5
0.55
0.6
0.65
0.7
0.75
0.8
L1 muon barrel trigger efficiency
L1 MU4
L1 MU6
L1 MU10
L1 MU11
L1 MU20
L1 MU21
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
> 25 GeV
µ
T
, p
µ
µ
→
Z
< 1.05
µ
η
0.1 <
(b)
Figure 22. (a) L1 muon barrel trigger efficiency plotted as a function of the probe muon 𝑝T. (b) Overall L1
muon barrel trigger efficiency as a function of the mean number of proton-proton interactions per event. 0
20
40
60
80
[GeV]
T
Offline muon p
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
L1 muon barrel trigger efficiency
L1 MU4
L1 MU6
L1 MU10
L1 MU11
L1 MU20
L1 MU21
ATLAS
Data 2018
-1
= 13 TeV, 60.8 fb
s
< 1.05
µ
η
, 0.1 <
µ
µ
→
Z
(a) 10
20
30
40
50
60
70
Mean number of pp interactions per event
0.5
0.55
0.6
0.65
0.7
0.75
0.8
L1 muon barrel trigger efficiency
L1 MU4
L1 MU6
L1 MU10
L1 MU11
L1 MU20
L1 MU21
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
> 25 GeV
µ
T
, p
µ
µ
→
Z
< 1.05
µ
η
0.1 <
(b) 2021 (a) (b) Figure 22. (a) L1 muon barrel trigger efficiency plotted as a function of the probe muon 𝑝T. (b) Overall L1
muon barrel trigger efficiency as a function of the mean number of proton-proton interactions per event. JINST 16 P07029 Table 3. 5.2
Trigger efficiency and timing L1 muon barrel trigger efficiency in each 𝑝T bin of figure 22(a) for different L1 trigger thresholds. The statistical uncertainty for all table entries is smaller than 0.02%. Threshold
2–3 GeV
3–4 GeV
4–5 GeV
5–6 GeV
6–7 GeV
7–8 GeV
8–10 GeV
10–12 GeV
12–14 GeV
14–16 GeV
16–18 GeV
L1 MU4
5.7%
33.2%
58.6%
67.5%
71.4%
73.3%
74.5%
75.3%
76.1%
76.4%
76.6%
L1 MU6
2.35%
5.2%
17.8%
43.1%
62.1%
70.5%
73.4%
74.8%
75.7%
76.1%
76.3%
L1 MU10
1.54%
3.1%
5.2%
11.6%
25.7%
44.9%
63.3%
72.5%
74.9%
75.7%
76.0%
L1 MU11
0.72%
1.10%
1.58%
2.25%
3.6%
11.4%
39.5%
62.2%
67.5%
68.9%
69.5%
L1 MU20
0.54%
0.68%
1.04%
1.31%
1.66%
2.28%
3.7%
10.1%
26.8%
46.5%
59.4%
L1 MU21
0.54%
0.65%
0.98%
1.21%
1.47%
1.75%
2.56%
8.1%
24.5%
44.0%
56.9%
Threshold
18–20 GeV
20–25 GeV
25–30 GeV
30–35 GeV
35–40 GeV
40–50 GeV
50–60 GeV
60–70 GeV
70–80 GeV
80–85 GeV
L1 MU4
76.9%
77.2%
77.6%
77.5%
77.1%
76.8%
76.8%
76.8%
76.8%
76.9%
L1 MU6
76.5%
76.8%
77.2%
77.1%
76.7%
76.4%
76.4%
76.4%
76.4%
76.5%
L1 MU10
76.3%
76.6%
77.1%
77.0%
76.5%
76.3%
76.2%
76.2%
76.2%
76.3%
L1 MU11
69.8%
70.2%
70.7%
70.7%
70.3%
70.1%
70.0%
70.1%
70.2%
70.3%
L1 MU20
65.7%
69.0%
70.3%
70.4%
70.1%
69.9%
69.8%
69.9%
69.9%
70.1%
L1 MU21
63.1%
66.5%
67.7%
67.7%
67.4%
67.2%
67.1%
67.1%
67.2%
67.3% signal propagation times of different RPC channels. These calibrations were initially performed in
2010–2012 using timing corrections in steps of 3.125 ns. The stability of the timing calibrations was
monitored by measuring the fraction of hits belonging to trigger candidates that were associated
with the correct bunch crossing. This fraction, averaged over the entire detector, is plotted in
figure 23(b) as a function of time for hits associated with high-𝑝T trigger candidates. This fraction
was around 99.6% and remained stable during the data-taking period. In some runs, this fraction
was slightly lower due to the absence of a few trigger towers that lost synchronisation with the
readout system. The impact of removing one tower is of the order of 1/432 ≈0.23%, where 432
is the total number of the RPC trigger towers. Trigger towers removed during a run were usually
recovered in the next run. 5.3
Trigger rates The ATLAS data acquisition and trigger system [43] has an upper limit of approximately 100 kHz
for the total rate of standard physics events accepted by the L1 trigger system [18]. Muon signatures
play an important role in the ATLAS physics programme and therefore a significant fraction of this
L1 bandwidth is allocated to muon triggers [16]. The L1 muon trigger was designed to be efficient
in detecting muon candidates while keeping L1 muon rates within acceptable limits. In this section,
rates of the L1 muon trigger as a function of the instantaneous luminosity are presented for the full
L1 muon trigger and separately for the barrel and endcap triggers. Several representative runs recorded in 2018 were used for measuring L1 muon trigger rates. Small random fractions of MU10 and MU20 candidates were recorded at the constant rate of about
1 Hz each, without any additional selections applied by the HLT. The constant rate was maintained
by varying the sampling fraction as a function of the instantaneous luminosity. Trigger rates were
computed by counting the number of MU10 or MU20 candidates in fixed time intervals of about
60 s and using the known sampling fraction to obtain the full rate. The instantaneous luminosity
was assumed to be constant during each 60 s time interval. The trigger rates shown here were
averaged over several time intervals that belong to a single instantaneous luminosity bin with a
width of 1032 cm−2s−1 and plotted as a function of the instantaneous luminosity. The rates of events containing one or more MU10 or MU20 trigger candidates are plotted as
a function of the instantaneous luminosity in figure 24. The total L1 muon trigger rate and the
L1 barrel muon trigger rate are shown separately. The rate of MU10 triggers was well beyond the
L1 bandwidth allocated to muon triggers. Therefore, only a small, random fraction of the events
selected by the MU10 trigger was accepted by the L1 trigger system. On average, one MU10
event out of approximately 200 000 (50 000) was selected at the start (end) of a typical ATLAS
run. Moreover, MU10 and other low-𝑝T L1 muon triggers were used in combination with other L1
trigger objects in order to reduce L1 rates to acceptable levels, for example by requiring presence
of two or more MU10 trigger candidates in a single event [16]. 5.2
Trigger efficiency and timing – 25 – 30/4
20/5
9/6
29/6
19/7
8/8
28/8
18/9
8/10 28/10
Day/Month in 2018
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
L1 muon barrel trigger efficiency
L1 MU4
L1 MU6
L1 MU10
L1 MU11
L1 MU20
L1 MU21
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
> 25 GeV
µ
T
, p
µ
µ
→
Z
< 1.05
µ
η
0.1 <
(a)
Day/Month in 2018
0.988
0.989
0.99
0.991
0.992
0.993
0.994
0.995
0.996
0.997
0.998
0.999
Fraction of trigger hits in correct BC
07/05 28/05 18/06 09/07 30/07 20/0810/09 01/10 22/10
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
thresholds
T
High p
(b)
Figure 23. (a) The overall measured L1 muon barrel trigger efficiency as a function of time. (b) The
fraction of high-𝑝T trigger hits associated with the correct bunch crossing as a function of time. Each point
corresponds to a separate run recorded by the ATLAS experiment in 2018. Day/Month in 2018
0.988
0.989
0.99
0.991
0.992
0.993
0.994
0.995
0.996
0.997
0.998
0.999
07/05 28/05 18/06 09/07 30/07 20/0810/09 01/10 22/10
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
thresholds
T
High p
(b) 30/4
20/5
9/6
29/6
19/7
8/8
28/8
18/9
8/10 28/10
Day/Month in 2018
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
L1 muon barrel trigger efficiency
L1 MU4
L1 MU6
L1 MU10
L1 MU11
L1 MU20
L1 MU21
ATLAS
-1
= 13 TeV, 60.8 fb
s
Data 2018,
> 25 GeV
µ
T
, p
µ
µ
→
Z
< 1.05
µ
η
0.1 <
(a) 2021 (b) (a) Figure 23. (a) The overall measured L1 muon barrel trigger efficiency as a function of time. (b) The
fraction of high-𝑝T trigger hits associated with the correct bunch crossing as a function of time. Each point
corresponds to a separate run recorded by the ATLAS experiment in 2018. JINST 16 P07029 5.3
Trigger rates Since the MU20 trigger was a primary L1 muon trigger, all MU20 candidates were accepted
by the L1 system for further processing by the HLT. This corresponds to a rate of approximately – 26 – 0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
50
100
150
200
250
300
L1 trigger rate [kHz]
ATLAS
-1
= 13 TeV, 2.15 fb
s
Total L1 MU10 rate
Barrel L1 MU10 rate
(a)
0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
5
10
15
20
25
30
L1 trigger rate [kHz]
ATLAS
-1
= 13 TeV, 2.15 fb
s
Total L1 MU20 rate
Barrel L1 MU20 rate
(b)
Figure 24. Rates of (a) MU10 and (b) MU20 L1 triggers, plotted as a function of the instantaneous luminosity. The fits demonstrate the linearity of the trigger rates as a function of the instantaneous luminosity. 0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
50
100
150
200
250
300
L1 trigger rate [kHz]
ATLAS
-1
= 13 TeV, 2.15 fb
s
Total L1 MU10 rate
Barrel L1 MU10 rate
(a) 0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
5
10
15
20
25
30
L1 trigger rate [kHz]
ATLAS
-1
= 13 TeV, 2.15 fb
s
Total L1 MU20 rate
Barrel L1 MU20 rate
(b) 2021 (b) (a) Figure 24. Rates of (a) MU10 and (b) MU20 L1 triggers, plotted as a function of the instantaneous luminosity. The fits demonstrate the linearity of the trigger rates as a function of the instantaneous luminosity. 16 kHz and 5 kHz for the full and barrel-only MU20 trigger, respectively, when evaluated at
the instantaneous luminosity of 2 × 1034 cm−2s−1. The MU20 barrel trigger rate is substantially
lower than the MU10 barrel trigger rate due to the higher 𝑝T threshold value, implemented as the
additional hit coincidence requirement in the outermost doublet layer (RPC3). This requirement
leads to a more precise 𝑝T measurement due to the longer muon trajectory in the barrel magnetic
field between the RPC2 and RPC3 layers. The different relative contributions of the barrel triggers
to the total MU10 and MU20 trigger rates are due to different definitions of trigger roads for the
RPC and TGC detectors. 5.3
Trigger rates For the low-𝑝T MU10 threshold, the trigger rate in the barrel region is
higher than the endcap rate. This is because the barrel trigger uses a looser requirement, based
on the coincidence of signals between the two innermost RPC doublet layers, than is used in the
endcap trigger, where the coincidence of three layers of TGC chambers [17, 18] is required. JINST 16 P07029 The muon barrel trigger rates were also measured separately for the MU20 and MU21 triggers,
and also for the new feet trigger component of MU20, as shown in figure 25. New feet trigger
candidates were selected by requiring that an L1 muon barrel candidate satisfies the MU20 trigger
logic and fails the MU21 trigger logic. The contribution from the new feet chambers is approximately
30% of the MU20 barrel trigger rate, while these chambers account for only ∼3% of the L1 muon
barrel trigger coverage. This higher relative rate was due to using only two out of four coincidences
for high-𝑝T L1 muon triggers in the feet chambers, as discussed in section 5.2. 5.4
Trigger composition This section presents the results of studying the composition of events accepted by the L1 muon
barrel trigger system. High rates of proton-proton collisions produce high levels of ionising radiation
in the ATLAS cavern, resulting in background (non-muon) candidates being selected by the L1
muon trigger system. These background events originate from high-momentum charged hadrons
that punch through the calorimeter system or from secondary particles produced in interactions of
high-momentum particles with the detector material. This study estimates the fraction of muon
candidates accepted by the L1 muon barrel trigger system that are due to background events. This
fraction was measured for an inclusive sample of selected L1 muon trigger candidates and for – 27 – 0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
1
2
3
4
5
6
7
8
9
10
L1 trigger rate [kHz]
ATLAS
-1
= 13 TeV, 2.15 fb
s
Barrel L1 MU20
Barrel L1 MU21
Barrel L1 MU20 - new feet trigger only
Figure 25. Rates of MU20 and MU21 triggers, and the contribution due to the new feet trigger component of
MU20, plotted as a function of the instantaneous luminosity. The fits demonstrate the linearity of the trigger
rates as a function of the instantaneous luminosity. 0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
1
2
3
4
5
6
7
8
9
10
L1 trigger rate [kHz]
ATLAS
-1
= 13 TeV, 2.15 fb
s
Barrel L1 MU20
Barrel L1 MU21
Barrel L1 MU20 - new feet trigger only L1 trigger rate [kHz] 2021 ]
-1
s
-2
cm
30
Instantaneous luminosity [10 Figure 25. Rates of MU20 and MU21 triggers, and the contribution due to the new feet trigger component of
MU20, plotted as a function of the instantaneous luminosity. The fits demonstrate the linearity of the trigger
rates as a function of the instantaneous luminosity. candidates selected in different detector regions. This fraction was also estimated as a function of
the mean number of interactions per event. JINST 16 P07029 This study uses MU10 and MU20 candidates that were recorded using only the criteria imposed
in the L1 trigger system, as detailed in section 5.3, without any additional selections in the HLT. One representative run recorded in 2018 with an integrated luminosity of 0.51 fb−1 was analysed
for this study. 5.4
Trigger composition Inclusive reconstructed muons with 𝑝T > 3 GeV and |𝜂| < 1.05 were used for
this study (offline muons), without requiring the selection criteria described in section 3. RoIs
selected by the L1 muon barrel trigger were matched to the closest offline muon candidate using
a geometrical requirement of Δ𝑅< 0.3 between the muon track and centre of the RoI. The muon
reconstruction is highly efficient for selecting muons produced in collisions and has a small rate for
misidentifying other particles as muons. Therefore, it is assumed that any RoI not matched with an
offline muon candidate was associated with a background candidate traversing the RPC system, as
described above. Table 4. Fractions of RoIs that were not matched to offline muon candidates with 𝑝T > 3 GeV or wi
𝑝T > 20 GeV. Fractions are shown for RoIs selected in the barrel region by MU10, MU20, MU21, and t
new feet trigger component of MU20. The statistical uncertainty for all table entries is smaller than 0.7% Table 4. Fractions of RoIs that were not matched to offline muon candidates with 𝑝T > 3 GeV or with
𝑝T > 20 GeV. Fractions are shown for RoIs selected in the barrel region by MU10, MU20, MU21, and the
new feet trigger component of MU20. The statistical uncertainty for all table entries is smaller than 0.7%. Table 4. Fractions of RoIs that were not matched to offline muon candidates with 𝑝T > 3 GeV or with
𝑝T > 20 GeV. Fractions are shown for RoIs selected in the barrel region by MU10, MU20, MU21, and the
new feet trigger component of MU20. The statistical uncertainty for all table entries is smaller than 0.7%. L1 trigger
Unmatched RoI fraction for 𝑝T > 3 GeV
Unmatched RoI fraction for 𝑝T > 20 GeV
MU10
71.1%
—
MU20
27.8%
88.3%
MU21
6.7%
82.7%
MU20 new feet trigger
66.8%
98.6% Table 4 shows fractions of RoIs selected by the L1 muon barrel trigger system that were not
matched to offline muon candidates with 𝑝T > 3 GeV. These unmatched RoIs are assumed to be
due to background events. The unmatched RoI fraction of the MU10 trigger is higher than that of
the MU20 trigger due to its looser trigger coincidence criteria, in which only four out of six RPC
layers are used. 5.4
Trigger composition |
η
Region of Interest |
0
0.2
0.4
0.6
0.8
1
Unmatched trigger fraction
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
ATLAS
-1
= 13 TeV, Run 358615, 0.51 fb
s
> 3 GeV
T
Match muons with p
Barrel trigger, threshold MU20
Feet sectors
Small sectors
Large sectors
(a) Mean number of pp interactions per event
20
30
40
50
60
Unmatched trigger fraction
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
ATLAS
-1
= 13 TeV, Run 358615, 0.51 fb
s
> 3 GeV
T
Match muons with p
Barrel muon trigger
MU10
MU20
MU21
MU20 - new feet trigger only
(b) JINST 16 P07029 (a) (b) 16 P07029 Figure 26. (a) Fractions of MU20 RoIs not matched to offline muon candidates with 𝑝T > 3 GeV plotted as
a function of the RoI pseudorapidity and shown separately for the small, large and feet sectors of the RPC
system. (b) Fractions of RoIs not matched to offline muon candidates with 𝑝T > 3 GeV plotted as a function
of the mean number of interactions per event for different L1 muon barrel trigger thresholds. Table 4 also shows fractions of RoIs selected by the L1 muon barrel trigger system that were
not matched to offline muon candidates with 𝑝T > 20 GeV. The unmatched RoI fraction for the
MU20 trigger increases from 27.8% to 88.3% when the minimum offline muon 𝑝T requirement is
raised from 3 GeV to 20 GeV. Table 4 also shows fractions of RoIs selected by the L1 muon barrel trigger system that were
not matched to offline muon candidates with 𝑝T > 20 GeV. The unmatched RoI fraction for the
MU20 trigger increases from 27.8% to 88.3% when the minimum offline muon 𝑝T requirement is
raised from 3 GeV to 20 GeV. Figure 27 shows the 𝑝T spectra of the offline muons matching the L1 muon barrel RoIs for
two different cases. Figure 27(a) shows the exclusive case where muons are matched only to those
RoIs that satisfy the specific trigger threshold and fail higher thresholds. Figure 27(b) shows the
inclusive case where muons are matched to the RoIs that satisfy either the specific trigger threshold
or any higher threshold. For the inclusive muon 𝑝T distributions, only events with one muon RoI
are considered in order to simplify analysis procedures. 5.4
Trigger composition For muon candidates with 𝑝T > 3 GeV, the unmatched RoI fractions for the MU20 – 28 – and MU21 triggers are 27.8% and 6.7%, respectively. The new feet trigger is excluded from the
MU21 trigger, which is otherwise identical to the MU20 trigger. The unmatched RoI fraction for
the new feet trigger is high because only four RPC layers are used by the new feet trigger component
of the MU20 trigger. This effect is illustrated in figure 26(a) where the unmatched RoI fraction is
plotted as a function of the RoI pseudorapidity separately for large, small and feet RPC sectors. The
new feet trigger is installed in the region |𝜂| ≳0.35, resulting in the large increase of unmatched
RoIs in this region. The unmatched RoI fraction is also plotted in figure 26(b) as a function of the mean number
of interactions per event for several L1 muon barrel triggers. The stability of the unmatched trigger
fraction over the full range of the mean number of interactions per event implies that the relative
contribution of background events to the trigger rate is independent of instantaneous luminosity. 2021 |
η
Region of Interest |
0
0.2
0.4
0.6
0.8
1
Unmatched trigger fraction
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
ATLAS
-1
= 13 TeV, Run 358615, 0.51 fb
s
> 3 GeV
T
Match muons with p
Barrel trigger, threshold MU20
Feet sectors
Small sectors
Large sectors
(a)
Mean number of pp interactions per event
20
30
40
50
60
Unmatched trigger fraction
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
ATLAS
-1
= 13 TeV, Run 358615, 0.51 fb
s
> 3 GeV
T
Match muons with p
Barrel muon trigger
MU10
MU20
MU21
MU20 - new feet trigger only
(b)
Figure 26. (a) Fractions of MU20 RoIs not matched to offline muon candidates with 𝑝T > 3 GeV plotted as
a function of the RoI pseudorapidity and shown separately for the small, large and feet sectors of the RPC
system. (b) Fractions of RoIs not matched to offline muon candidates with 𝑝T > 3 GeV plotted as a function
of the mean number of interactions per event for different L1 muon barrel trigger thresholds. 5.4
Trigger composition 0
10
20
30
40
50
[GeV]
T
Offline muon p
6
10
7
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8
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9
10
10
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11
10
Muons
ATLAS
-1
= 13 TeV, 2.15 fb
s
MU4
≥
Match
MU6
≥
Match
MU10
≥
Match
MU11
≥
Match
MU20
≥
Match
(b) 0
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[GeV]
T
Offline muon p
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Muons
ATLAS
-1
= 13 TeV, 2.15 fb
s
Match = MU4
Match = MU6
Match = MU10
Match = MU11
Match = MU20
Match = MU20 - new feet trigger only
(a) 2021 (b) (a) Figure 27. The 𝑝T distribution of offline muon candidates matching the L1 muon barrel RoIs for the (a)
exclusive and (b) inclusive cases. The L1 muon barrel trigger system reports the highest 𝑝T threshold
passed by a muon candidate. For high muon 𝑝T values, muon candidates are therefore more likely to pass a
corresponding high-𝑝T threshold, thus leading to a smaller fraction of muons selected by the low-𝑝T triggers. The rightmost bin includes the overflow entries. JINST 16 P07029 [GeV]
T
Offline muon p
0
20
40
60
80
100
120
multiplicity [strips]
Mean combined RPC cluster
1.4
1.5
1.6
1.7
1.8
1.9
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
RPC clusters along muon track
view
η
> 3 GeV,
µ
T
probe muons, p
µ
µ
→
Z
All muons
Muons matching MU20 RoI
(a)
[GeV]
T
Offline muon p
0
20
40
60
80
100
120
multiplicity [strips]
Mean combined RPC cluster
1.4
1.5
1.6
1.7
1.8
1.9
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
RPC clusters along muon track
view
φ
> 3 GeV,
µ
T
probe muons, p
µ
µ
→
Z
All muons
Muons matching MU20 RoI
(b)
Figure 28. Mean cluster hit multiplicity for (a) 𝜂panels and (b) 𝜙panels averaged over all RPC modules
plotted as a function of muon 𝑝T using 𝑍→𝜇𝜇probe muons. The mean cluster hit multiplicity is shown
for all probe muons and separately for probe muons matched to the MU20 trigger RoI. The rightmost bin
includes the overflow entries. 5.4
Trigger composition The selected RoI is required to be generated
in the muon barrel region. For the exclusive muon 𝑝T distributions, this requirement is not applied. The majority of muon candidates selected by the MU20 trigger are muons with 𝑝T < 20 GeV. This
mismeasurement of the muon 𝑝T by the RPC detector is due to its position resolution being less
precise than that of the MDT detector, which is used for offline muon reconstruction in the barrel
region. This is expected because the RPC detector was optimised for fast triggering with sufficient
𝑝T resolution to keep trigger rates within allowed limits, as discussed in section 5.3. – 29 – 0
10
20
30
40
50
[GeV]
T
Offline muon p
5
10
6
10
7
10
8
10
9
10
10
10
11
10
Muons
ATLAS
-1
= 13 TeV, 2.15 fb
s
Match = MU4
Match = MU6
Match = MU10
Match = MU11
Match = MU20
Match = MU20 - new feet trigger only
(a)
0
10
20
30
40
50
[GeV]
T
Offline muon p
6
10
7
10
8
10
9
10
10
10
11
10
Muons
ATLAS
-1
= 13 TeV, 2.15 fb
s
MU4
≥
Match
MU6
≥
Match
MU10
≥
Match
MU11
≥
Match
MU20
≥
Match
(b)
Figure 27. The 𝑝T distribution of offline muon candidates matching the L1 muon barrel RoIs for the (a)
exclusive and (b) inclusive cases. The L1 muon barrel trigger system reports the highest 𝑝T threshold
passed by a muon candidate. For high muon 𝑝T values, muon candidates are therefore more likely to pass a
corresponding high-𝑝T threshold, thus leading to a smaller fraction of muons selected by the low-𝑝T triggers. The rightmost bin includes the overflow entries. 6
Measurements of RPC currents and counting rates Nearly all of the RPC counting rates and ionisation currents are induced by secondary particles,
mostly photons and neutrons, that are produced in interactions of primary particles, i.e., those
that originate in proton-proton collisions, with the detector and beam-pipe material. The flux of
the secondary particles in the MS was computed using simulation and presented in appendix A
of ref. [44]. These secondary particles induce a dominant fraction of RPC avalanche events,
which causes the resulting RPC ionisation currents and counting rates to be proportional to the
instantaneous luminosity. 2021 JINST 16 P07 In the previous two sections, muons were used to study the detector and trigger performance. In this section, the RPC detector response is evaluated using zero-bias proton-proton collisions. Sections 6.1 and 6.2 present measurements of the RPC counting rates and RPC ionisation currents,
respectively. These measurements are performed as a function of the instantaneous luminosity for
RPCs located in different detector regions in order to study their dependence on the distance to the
collision point. In section 6.3, these measurements are combined in order to measure the average
avalanche charge of the ATLAS RPC detector. 2021 JINST 16 P07029 5.4
Trigger composition [GeV]
T
Offline muon p
0
20
40
60
80
100
120
multiplicity [strips]
Mean combined RPC cluster
1.4
1.5
1.6
1.7
1.8
1.9
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
RPC clusters along muon track
view
φ
> 3 GeV,
µ
T
probe muons, p
µ
µ
→
Z
All muons
Muons matching MU20 RoI
(b) [GeV]
T
Offline muon p
0
20
40
60
80
100
120
multiplicity [strips]
Mean combined RPC cluster
1.4
1.5
1.6
1.7
1.8
1.9
ATLAS
-1
= 13 TeV, Data 2018, 60.8 fb
s
RPC clusters along muon track
view
η
> 3 GeV,
µ
T
probe muons, p
µ
µ
→
Z
All muons
Muons matching MU20 RoI
(a) T 16 P07029 (b) (a) Figure 28. Mean cluster hit multiplicity for (a) 𝜂panels and (b) 𝜙panels averaged over all RPC modules
plotted as a function of muon 𝑝T using 𝑍→𝜇𝜇probe muons. The mean cluster hit multiplicity is shown
for all probe muons and separately for probe muons matched to the MU20 trigger RoI. The rightmost bin
includes the overflow entries. A muon with 𝑝T < 20 GeV would bend outside a geometrical matching window of the MU20
trigger and therefore normally would be expected to fail the MU20 trigger logic. In addition to
intrinsic limitations of the RPC position resolution, low-𝑝T muons can also satisfy the MU20
trigger requirement by generating larger than typical clusters because such muons are more likely
to produce hits within the trigger coincidence window. To illustrate this effect, the mean cluster hit
multiplicity is plotted in figure 28 as a function of muon 𝑝T for all probe muons and only for probe
muons matching the MU20 RoIs. The mean cluster hit multiplicity is approximately constant within
the considered 𝑝T range for the inclusive muon sample. A small increase is observed at high-𝑝T
values, and is due to muon bremsstrahlung radiation. For probe muons matching the MU20 RoIs,
the mean cluster hit multiplicity increases by up to 30% at low muon 𝑝T values. This observation
confirms that the low-𝑝T muons that produce larger-than-typical clusters are more likely to satisfy
the MU20 trigger requirement. – 30 – 6.1
RPC current measurements These results confirm the expected linear
dependence of the RPC current density as a function of the instantaneous luminosity within the
currently accessible luminosity range. Therefore, it is assumed that the RPC current density can be
extrapolated linearly to the HL-LHC instantaneous luminosity of 7.5 × 1034 cm−2s−1. 10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
2
4
6
8
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14
]
2
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µ
Mean gap current density [
ATLAS
= 13 TeV
s
Data 2018,
BML1A01
BML3A01
BML6A01
(a)
0
5000
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20000
]
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s
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30
Instantaneous luminosity [10
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1.5
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2.5
3
3.5
4
4.5
5
]
2
A/m
µ
Mean gap current density [
ATLAS
= 13 TeV
s
Data 2018,
BML1A01
BMS1A02
BOL1A01
BOS1A02
(b)
Figure 29. RPC current density shown as a function of instantaneous luminosity for several representative
modules. The nominal voltage setting of 9.6 kV was used for these measurements. (a) Each set of points
corresponds to one individual module belonging to one of the three stations located at different positions
along the z-direction. (b) Each line corresponds to the current averaged over all the modules in each RPC
station. The four stations belong to small and large sectors in the RPC1 and RPC3 layers. 0
5000
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20000
]
-1
s
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5
]
2
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µ
Mean gap current density [
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= 13 TeV
s
Data 2018,
BML1A01
BMS1A02
BOL1A01
BOS1A02
(b) 10000
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]
2
A/m
µ
Mean gap current density [
ATLAS
= 13 TeV
s
Data 2018,
BML1A01
BML3A01
BML6A01
(a) 2021 JINST 16 P07029 (b) (a) Figure 29. RPC current density shown as a function of instantaneous luminosity for several representative
modules. The nominal voltage setting of 9.6 kV was used for these measurements. (a) Each set of points
corresponds to one individual module belonging to one of the three stations located at different positions
along the z-direction. (b) Each line corresponds to the current averaged over all the modules in each RPC
station. The four stations belong to small and large sectors in the RPC1 and RPC3 layers. 6.1
RPC current measurements The total ionisation current for each RPC gas volume is inferred by measuring the voltage across a
100 kΩ resistor placed between the RPC readout panel and ground [24]. Current measurements are
recorded and time-stamped by the DCS [30]. The current measurements used in this analysis were
recorded during periods of proton-proton collisions with stable LHC and detector conditions. The
baseline current measured for each chamber using the nominal voltage settings, but without beams
in the LHC, is subtracted in order to study the net effect of collisions. The current measurements
are normalised to the active surface area of each module (current density) to allow comparisons
between modules of different size. About 90% of the RPC modules without known problems were
selected for this analysis. The RPCs that were excluded comprise modules disconnected due to gas
leaks, modules where the DCS current readout had problems, and modules that had low efficiency
for detecting muons. The measured current density is shown in figure 29 as a function of the instantaneous luminosity
for several representative modules in order to illustrate the typical RPC response in different detector
regions. The nominal voltage setting of 9.6 kV was used for these measurements. Figure 29(a)
shows the current density for the individual RPC modules located in the stations with 𝜂indices 1, 3
and 6; only the BML RPCs belonging to the large 𝜙sector number 1 of ATLAS side A were used
for these plots. The current density increases at higher 𝜂values due to the higher flux of hadrons in
the regions outside the endcap calorimeters, as shown in appendix A of ref. [44]. Figure 29(b) shows the current density measurements averaged over the modules belonging
to the stations with 𝜂index 1, plotted as a function of the instantaneous luminosity for the BML,
BMS, BOL and BOS sectors. Only the RPCs located in 𝜙sectors 1 and 2 of side A were used for
these measurements. The current density decreases at larger radii due to the reduced particle flux
density in the BOL and BOS layers. The differences between small and large chambers are due to
the presence of the barrel magnet coils which shadow the small sectors from the collision point. In addition, the small chambers are located at slightly larger radii than the large chambers in the – 31 – same layer and therefore detect smaller particles fluxes. 6.1
RPC current measurements Figure 30 shows the current density distributions for all selected RPCs at an instantaneous
luminosity of 1.8×1034 cm−2s−1 for voltages of 9.2 kV and 9.6 kV. Since the voltages applied to the
detector modules can be corrected for temperature and pressure effects, the actual applied voltage
could differ from the set value. These corrections were taken into account to allow comparisons
between different modules. The current density for each module was scaled to obtain current density
values at 9.2 kV and 9.6 kV, using the linear relationship between current and applied voltage that
was measured for that particular module. 0
2
4
6
8
10
12
14
16
18
20
]
2
A/m
µ
Current [
0
500
1000
1500
Gas gaps
ATLAS
-1
s
-2
cm
34
10
×
= 1.8
inst
L
= 13 TeV
s
Data 2018,
HV = 9.2 kV
HV = 9.6 kV
Figure 30. Distributions of the measured current density for the selected RPCs obtained at an instantaneous
luminosity of 1.8 × 1034 cm−2s−1. The measurements were taken during one representative run in 2018. Figure 30. Distributions of the measured current density for the selected RPCs obtained at an instantaneous
luminosity of 1.8 × 1034 cm−2s−1. The measurements were taken during one representative run in 2018. – 32 – The current density in each selected RPC was measured as a function of the operating voltage at
the beginning of several proton-proton collision runs recorded in 2018. These measurements were
performed while the RPC operational voltage was increasing from the standby setting of 9.0 kV to
the nominal setting of 9.6 kV. The standby voltage setting was used in order to protect the RPCs
during periods with unstable LHC beam conditions. The current density measurements for each
individual module were linearly extrapolated to an instantaneous luminosity of 1.8 × 1034 cm−2s−1. Figure 31(a) shows the current density as a function of the applied voltage for representative BML
modules located in 𝜂stations 1, 3, and 6, and belonging to 𝜙sector 1. The current density
has an approximately linear dependence on voltage within the measurement range of 9.0 kV to
9.6 kV. Each current measurement was taken twice in order to estimate the uncertainty arising from
variations in the instantaneous luminosity within the approximately one minute interval during which
the instantaneous luminosity measurement was performed; the analysis assumes the instantaneous
luminosity was constant within this interval. 6.1
RPC current measurements This uncertainty was estimated as half of the difference
between the two measurements taken at the same voltage and separated by approximately 60 s. 2021 The temperature gradient in the ATLAS cavern is about 10◦C, as illustrated in figure 8 of
ref. [45]. This gradient is due to the air circulation in the cavern, with lower temperatures at the
bottom of the detector. Figure 31(b) shows the extrapolated current density measured as a function
of temperature for BML modules located in 𝜂stations 1, 4, and 6. The measured current density
in each module is extrapolated to the value expected at the nominal voltage of 9.6 kV using the
procedure described earlier. The current density measurements were averaged over modules from
all 𝜙sectors that belong to the same temperature bin. An approximately linear dependence of the
current density as a function of temperature is observed. The small deviation from linearity for
the BOL temperature at around 26◦C is likely to be due to the local module temperatures differing
from the temperature recorded by the nearest temperature sensor, which is spatially separated from
the modules. JINST 16 P07029 9
9.2
9.4
9.6
Voltage [kV]
0
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16
]
2
A/m
µ
Current [
ATLAS
-1
s
-2
cm
34
10
×
= 1.8
inst
= 13 TeV, L
s
Data 2018,
sector = 1
φ
index = 1,
η
BML,
sector = 1
φ
index = 3,
η
BML,
sector = 1
φ
index = 6,
η
BML,
(a)
20
22
24
26
28
30
C]
°
Temperature [
0
2
4
6
8
10
12
14
]
2
A/m
µ
Current [
ATLAS
= 13 TeV
s
Data 2018,
, HV = 9.6 kV
-1
s
-2
cm
34
10
×
= 1.8
inst
L
index = 1
η
BOL,
index = 4
η
BOL,
index = 6
η
BOL,
(b)
Figure 31. (a) RPC current density shown as a function of the applied voltage for representative BML
modules located in 𝜂stations 1, 3, and 6, and belonging to the large 𝜙sector 1. (b) RPC current density
shown as a function of temperature for BOL modules located in 𝜂stations 1, 4, and 6. The current density is
averaged over 𝜙sector modules included for each temperature bin. 6.2
RPC counting rate measurements Counting rates were measured using collision events selected online with a primary single-muon
trigger [16] that requires the presence of an isolated muon with 𝑝T > 26 GeV. This single-muon
trigger selects energetic collisions that typically contain a 𝑊or 𝑍boson, or a heavy-flavour jet
with high 𝑝T. Because such energetic events include a hard-scattering process that produces many
secondary hadrons and photons, they are not representative of an average LHC bunch crossing. Since
the production rates of 𝑊and 𝑍bosons are directly proportional to the instantaneous luminosity,
sampling the three bunch crossings preceding the trigger provides an unbiased sample of LHC
collisions at a given instantaneous luminosity. 2021 RPC hits were required to belong to the 75 ns time window preceding the bunch crossing
selected by the trigger. Selected hits have times between −87.5 ns and −12.5 ns, where the zero
time corresponds to the bunch crossing selected by the muon trigger. This criterion selects the
three bunch crossings immediately preceding the triggered bunch crossing. Counting rates were
computed over a time period of approximately one minute during which the instantaneous luminosity
was assumed to be constant. A statistically weighted average of the counting rates from several such
time intervals was used to compute the counting rate as a function of the instantaneous luminosity. This averaging was performed using an instantaneous luminosity binning of 1032 cm−2s−1. The
resulting counting rates were then normalised to the active module surface area. Only modules
with detector efficiency larger than 50% and with a nominal voltage of 9.6 kV were used for the
measurements presented in this section. 6.1
RPC current measurements 9
9.2
9.4
9.6
Voltage [kV]
0
2
4
6
8
10
12
14
16
]
2
A/m
µ
Current [
ATLAS
-1
s
-2
cm
34
10
×
= 1.8
inst
= 13 TeV, L
s
Data 2018,
sector = 1
φ
index = 1,
η
BML,
sector = 1
φ
index = 3,
η
BML,
sector = 1
φ
index = 6,
η
BML,
(a) 20
22
24
26
28
30
C]
°
Temperature [
0
2
4
6
8
10
12
14
]
2
A/m
µ
Current [
ATLAS
= 13 TeV
s
Data 2018,
, HV = 9.6 kV
-1
s
-2
cm
34
10
×
= 1.8
inst
L
index = 1
η
BOL,
index = 4
η
BOL,
index = 6
η
BOL,
(b) (b) (a) Figure 31. (a) RPC current density shown as a function of the applied voltage for representative BML
modules located in 𝜂stations 1, 3, and 6, and belonging to the large 𝜙sector 1. (b) RPC current density
shown as a function of temperature for BOL modules located in 𝜂stations 1, 4, and 6. The current density is
averaged over 𝜙sector modules included for each temperature bin. – 33 – 6.2
RPC counting rate measurements 6.2
RPC counting rate measurements JINST 16 P07029 0
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20000
]
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Instantaneous luminosity [10
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10
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40
]
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RPC counting rate [Hz/cm
ATLAS
= 13 TeV
s
HV = 9.6 kV
used during data-taking
thr
V
Standard
Zero-bias collisions
-3
10
×
0.015)
±
RPC1 BM, slope = (1.200
-3
10
×
0.011)
±
RPC2 BM, slope = (0.782
-3
10
×
0.007)
±
RPC3 BO, slope = (0.536
(a)
0
10
20
30
40
50
60
]
2
RPC counting rate [Hz/cm
0
100
200
300
400
500
600
700
800
RPC panels
RPC1 BM, mean = 18.0
RPC2 BM, mean = 14.8
RPC3 BO, mean = 11.5
ATLAS
= 13 TeV
s
HV = 9.6 kV
used during data-taking
thr
V
Standard
Zero-bias collisions
-1
s
-2
cm
34
10
×
= 1.8
inst
L
(b)
Figure 32. (a) RPC counting rates measured per unit surface area plotted as a function of the instantaneous
luminosity for three RPC 𝜙panels located at different distances from the collision point, corresponding to
RPC1, RPC2 and RPC3 layers. All 𝜙panels used for this study belong to the large 𝜙sector 1 and are located
at 𝜂station 2. The slope parameters were extracted from a linear fit to the data points. (b) Distribution of the
counting rates for all RPC panels, separated into three sets of chambers in different detector doublet layers,
at an instantaneous luminosity of 1.8 × 1034 cm−2s−1. Nominal voltage and FE threshold settings were used
in this study. 6.2
RPC counting rate measurements 0
5000
10000
15000
20000
]
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s
-2
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30
Instantaneous luminosity [10
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10
20
30
40
]
2
RPC counting rate [Hz/cm
ATLAS
= 13 TeV
s
HV = 9.6 kV
used during data-taking
thr
V
Standard
Zero-bias collisions
-3
10
×
0.015)
±
RPC1 BM, slope = (1.200
-3
10
×
0.011)
±
RPC2 BM, slope = (0.782
-3
10
×
0.007)
±
RPC3 BO, slope = (0.536
(a) 0
10
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]
2
RPC counting rate [Hz/cm
0
100
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400
500
600
700
800
RPC panels
RPC1 BM, mean = 18.0
RPC2 BM, mean = 14.8
RPC3 BO, mean = 11.5
ATLAS
= 13 TeV
s
HV = 9.6 kV
used during data-taking
thr
V
Standard
Zero-bias collisions
-1
s
-2
cm
34
10
×
= 1.8
inst
L
(b) ]
2
RPC counting rate [Hz/cm (b) (a) Figure 32. (a) RPC counting rates measured per unit surface area plotted as a function of the instantaneous
luminosity for three RPC 𝜙panels located at different distances from the collision point, corresponding to
RPC1, RPC2 and RPC3 layers. All 𝜙panels used for this study belong to the large 𝜙sector 1 and are located
at 𝜂station 2. The slope parameters were extracted from a linear fit to the data points. (b) Distribution of the
counting rates for all RPC panels, separated into three sets of chambers in different detector doublet layers,
at an instantaneous luminosity of 1.8 × 1034 cm−2s−1. Nominal voltage and FE threshold settings were used
in this study. Figure 32(a) shows the RPC counting rates as a function of instantaneous luminosity for three
representative RPC panels located at radii of approximately 6.8 m, 7.4 m and 9.8 m from the
beams. As expected, the counting rates depend linearly on the instantaneous luminosity in all – 34 – detector regions. This observation confirms that secondary particles, produced in interactions of
the primary particles with the detector and beam-pipe material, are the main source of RPC hits. Figure 32(b) shows the distribution of counting rates measured at an instantaneous luminosity of
1.8 × 1034 cm−2s−1 for the RPC panels located in three different detector layers. Panels located
further from the collision point see smaller counting rates because the flux of secondary particles
decreases with increasing distance from the centre of the detector. 6.2
RPC counting rate measurements 0
10
20
30
40
50
60
]
2
RPC counting rate [Hz/cm
0
100
200
300
400
500
600
700
800
900
1000
RPC panels
indices = 1-2, mean = 10.4
η
indices = 3-4, mean = 13.2
η
indices = 5-6, mean = 21.1
η
ATLAS
= 13 TeV
s
HV = 9.6 kV
used during data-taking
thr
V
Standard
Zero-bias collisions
-1
s
-2
cm
34
10
×
= 1.8
inst
L
(a)
8
−
6
−
4
−
2
−
0
2
4
6
8
η
Gap index along
2
4
6
8
10
12
14
16
φ
Gap index along
0
10
20
30
40
50
60
]
2
RPC counting rate [Hz/cm
= 13 TeV
s
,
ATLAS
-1
s
-2
cm
34
10
×
= 1.8
inst
HV = 9.6 kV, L
RPC1 BM layer, zero-bias collisions
(b)
Figure 33. RPC counting rates per unit surface area at an instantaneous luminosity of 1.8 × 1034 cm−2s−1
for nominal voltage and FE threshold settings. (a) Distribution of the counting rates for all RPC panels,
separated into three sets of chambers with different 𝜂indices. (b) Counting rates for the RPC1 layer shown
as a function of RPC 𝜂and 𝜙coordinates. The values were averaged over the two individual sub-layers. Empty bins correspond to logical combinations of indices that do not represent installed RPCs or correspond
to modules with efficiencies less than 50%. 8
−
6
−
4
−
2
−
0
2
4
6
8
η
Gap index along
2
4
6
8
10
12
14
16
φ
Gap index along
0
10
20
30
40
50
60
]
2
RPC counting rate [Hz/cm
= 13 TeV
s
,
ATLAS
-1
s
-2
cm
34
10
×
= 1.8
inst
HV = 9.6 kV, L
RPC1 BM layer, zero-bias collisions
(b) 0
10
20
30
40
50
60
]
2
RPC counting rate [Hz/cm
0
100
200
300
400
500
600
700
800
900
1000
RPC panels
indices = 1-2, mean = 10.4
η
indices = 3-4, mean = 13.2
η
indices = 5-6, mean = 21.1
η
ATLAS
= 13 TeV
s
HV = 9.6 kV
used during data-taking
thr
V
Standard
Zero-bias collisions
-1
s
-2
cm
34
10
×
= 1.8
inst
L
(a) JINST 16 P07029 (a) (b) Figure 33. 6.2
RPC counting rate measurements RPC counting rates per unit surface area at an instantaneous luminosity of 1.8 × 1034 cm−2s−1
for nominal voltage and FE threshold settings. (a) Distribution of the counting rates for all RPC panels,
separated into three sets of chambers with different 𝜂indices. (b) Counting rates for the RPC1 layer shown
as a function of RPC 𝜂and 𝜙coordinates. The values were averaged over the two individual sub-layers. Empty bins correspond to logical combinations of indices that do not represent installed RPCs or correspond
to modules with efficiencies less than 50%. The distributions of RPC counting rates at an instantaneous luminosity of 1.8 × 1034 cm−2s−1
are presented in figure 33(a), where RPC panels in each station are grouped by their 𝜂indices. Figure 33(b) shows the counting rates at an instantaneous luminosity of 1.8 × 1034 cm−2s−1 for the
RPC1 doublet layer as a function of RPC 𝜂and 𝜙coordinates. Because the flux of ionising particles
increases at higher |𝜂| values, panels located in the more forward regions of the MS measure higher
counting rates. 6.3
RPC avalanche charge measurements The mean avalanche charge is a key performance parameter of the RPC detector [40]. Its value
depends on the properties of the ionising particle and on the gain of the electron amplification
process in the gas. The gain of the amplification process is determined by the electrical field across
the gas gap and by the gas mixture. The readout system of the ATLAS RPC detector only measures
pulse time when the leading edge of the pulse is above a predetermined threshold. Therefore, the
avalanche charge cannot be measured directly by the ATLAS RPC detector. Instead, the mean
avalanche charge is estimated for each RPC module by combining simultaneous measurements of – 35 – the current and counting rate for that module. Since both of these measurements integrate over
many ionisation events, only the mean avalanche charge can be assessed by this procedure. The mean avalanche charge, 𝑄, was measured using the following expression: 𝑄=
Current
Counting rate 𝑄=
Current
Counting rate where the current and counting rate were measured simultaneously for each RPC module following
the procedures described in sections 6.1 and 6.2, respectively. These measurements were performed
on the same ATLAS run recorded in October 2018, using standard operating voltage and FE
threshold values. In this run, about 90% of the FE channels had thresholds set to the nominal value
of 1.0 V, while the majority of the remaining channels used higher-than-nominal thresholds. 2021 JINST 16 P07 2021 Counting rate measurements include only those ionisation events that produce signals above the
FE thresholds in a given module, while current measurements account for the total ionisation charge
in that module. Therefore, the present study overestimates the mean avalanche charge since the
ionisation signals below the FE thresholds contribute to the measured current but not to the counting
rate. The fraction of ionisation events below the FE thresholds is expected to remain constant as
a function of the instantaneous luminosity. This hypothesis was verified by checking that the
measurements of the mean avalanche charge do not depend on the specific instantaneous luminosity
used to obtain the measurements. This is expected since both the currents and counting rates depend
linearly on the instantaneous luminosity as discussed in sections 6.1 and 6.2, respectively. JINST 16 P07029 The fraction of ionisation events produced by muons is underestimated by approximately 3%,
due to the inefficiency of the readout chain, which is discussed in section 4.3. 6.3
RPC avalanche charge measurements This implies that
the mean avalanche charge produced by minimum-ionising particles is overestimated by a similar
amount. Most RPC hits are due to photons and neutrons and their avalanche spectra cannot be
estimated in situ. Therefore, the precise fraction of the missing ionisation events due to these
particles cannot be determined in this analysis. This implies that the measured mean avalanche
charge is probably overestimated. In addition, the mean avalanche charge measurements depend on
environmental parameters and on the specific FE threshold values used to obtain the counting rates. Despite these limitations, the mean avalanche charge measurements provide useful benchmarks
for studies of detector performance as a function of time and for studies of the expected detector
lifetime for the HL-LHC programme. The counting rate measurements were performed independently for each 𝜂and 𝜙panel. The
average of the 𝜂and 𝜙counting rates for each module was used for avalanche charge measurements. The current and rate measurements were also averaged over the two parallel modules of the doublet
layer. The distance between the parallel modules of the RPC doublet layer is approximately 2 cm,
so the counting rates are expected to be the same in these two modules. The distribution of the mean avalanche charge is shown in figure 34(a) for all active RPC
modules. The same distribution shape was observed when it was computed using only the modules
with the nominal FE thresholds. Therefore, the width and shape of this distribution is likely due to
natural differences among the modules. The average of the mean avalanche charge distribution is
30.4 pC and is in good agreement with previous measurements performed using a gamma irradiation
source, when the same FE thresholds are used, as shown in figure 7 of ref. [46]. Those measurements – 36 – 0
20
40
60
80
100
Mean avalanche charge [pC]
0
50
100
150
200
250
RPC modules
ATLAS
-1
s
-2
cm
34
10
×
= 1.8
inst
= 13 TeV, L
s
HV = 9.6 kV, zero-bias collisions
mean = 30.4 pC
(a)
5
−
0
5
η
Gap index along
5
10
15
φ
Gap index along
0
20
40
Mean avalanche charge [pC]
= 13 TeV
s
,
ATLAS
-1
s
-2
cm
34
10
×
= 1.8
inst
HV = 9.6 kV, L
RPC1 BM layer, zero-bias collisions
(b)
Figure 34. 6.3
RPC avalanche charge measurements (a) Distribution of the mean avalanche charge of the ATLAS RPC detector. (b) Mean avalanche
charge measured as a function of the 𝜂and 𝜙positions of the RPCs belonging to the RPC1 doublet layer. Empty bins correspond to logical combinations of indices that do not represent installed RPCs or to modules
with efficiency less than 50%. 5
−
0
5
η
Gap index along
5
10
15
φ
Gap index along
0
20
40
Mean avalanche charge [pC]
= 13 TeV
s
,
ATLAS
-1
s
-2
cm
34
10
×
= 1.8
inst
HV = 9.6 kV, L
RPC1 BM layer, zero-bias collisions
(b) 0
20
40
60
80
100
Mean avalanche charge [pC]
0
50
100
150
200
250
RPC modules
ATLAS
-1
s
-2
cm
34
10
×
= 1.8
inst
= 13 TeV, L
s
HV = 9.6 kV, zero-bias collisions
mean = 30.4 pC
(a) φ
Gap index along 2021 (b) (a) Figure 34. (a) Distribution of the mean avalanche charge of the ATLAS RPC detector. (b) Mean avalanche
charge measured as a function of the 𝜂and 𝜙positions of the RPCs belonging to the RPC1 doublet layer. Empty bins correspond to logical combinations of indices that do not represent installed RPCs or to modules
with efficiency less than 50%. JINST 16 P07029 were obtained using RPCs of the same design as the ATLAS RPCs, with the same FE electronics,
gas mixture and operating voltage. Figure 34(b) shows the mean avalanche charge as a function of the 𝜂and 𝜙positions of the
RPCs belonging to the RPC1 doublet layer. The observed variations between different chambers are
due to intrinsic variations among modules and to variations in operating voltages and FE threshold
settings. The operating voltages are expected to affect the measured current values, while the
thresholds influence the measured counting rates. 6.4
RPC efficiency as a function of counting rate As discussed in sections 4.3 and 5.2, the mean RPC detector efficiency and L1 muon barrel
trigger efficiency decrease by approximately 1% as the number of proton-proton collisions per
event increases from 10 to 60 collisions. In this section, this effect is investigated further by
correlating the RPC module efficiency with its counting rate. It is assumed that an avalanche
multiplication process would deplete available charge on the RPC surface in the vicinity of the
avalanche, resulting in a decreased efficiency in that localised area to detect a subsequent avalanche
event shortly thereafter. The results presented in this section aim to correlate the muon detection
efficiency with the mean counting rate for each individual RPC module and for the detector as
the whole. The full dataset of 60.8 fb−1 was used for the efficiency measurements in order to maximise
the number events with a 𝑍boson decaying into two muons. A smaller dataset of 3.6 fb−1 was used
for the counting rate measurements in order to simplify analysis procedures. Sufficient statistical
precision was obtained using this smaller dataset. The mean RPC detector efficiency and counting
rates were measured using only RPC panels with an efficiency greater than 50%. – 37 – The mean RPC detector counting rate was measured in seven bins of the number of collisions
per event, shown in figure 35(a). A linear dependence of the counting rate was observed, in
agreement with the measurements of the RPC counting rates as a function of the instantaneous
luminosity presented in section 6.2. The mean RPC detector efficiency in the same seven bins were
presented in section 4.3. Figure 35(b) shows the mean RPC detector efficiency plotted as a function
of the mean RPC detector counting rate computed separately for the seven bins of the number of
collisions per event. A decrease of approximately 1% is observed within the considered range. 20
40
60
Mean number of pp interactions per event
6
8
10
12
14
16
18
20
]
2
Mean RPC counting rate [Hz/cm
ATLAS
-1
= 13 TeV, 3.6 fb
s
All RPC modules
view
η
view
φ
(a)
8
10
12
14
16
18
]
2
Mean RPC counting rate [Hz/cm
0.895
0.9
0.905
0.91
0.915
Mean RPC detector efficiency
ATLAS
-1
= 13 TeV, 60.8 - 3.6 fb
s
All RPC modules
view
η
view
φ
(b)
Figure 35. 6.4
RPC efficiency as a function of counting rate (a) Mean RPC counting rates measured in seven different intervals of the number of proton-proton
collisions per event. The counting rate was averaged over all active RPC modules and is shown separately
for 𝜂and 𝜙panels. (b) Correlation between the mean RPC detector efficiency and the mean RPC detector
counting rate, measured in seven different intervals of the number of proton-proton collisions per event,
shown separately for 𝜂and 𝜙panels. Two different datasets, corresponding to 60.8 fb−1 and 3.6 fb−1, are
used for the efficiency measurements and the counting rate measurements, respectively. Both figures were
obtained by averaging over all active 𝜂and 𝜙panels. Only RPC panels with an average efficiency larger than
50% were used for these figures. 20
40
60
Mean number of pp interactions per event
6
8
10
12
14
16
18
20
]
2
Mean RPC counting rate [Hz/cm
ATLAS
-1
= 13 TeV, 3.6 fb
s
All RPC modules
view
η
view
φ 8
10
12
14
16
18
]
2
Mean RPC counting rate [Hz/cm
0.895
0.9
0.905
0.91
0.915
Mean RPC detector efficiency
ATLAS
-1
= 13 TeV, 60.8 - 3.6 fb
s
All RPC modules
view
η
view
φ
(b) JINST 16 P07029 Mean number of pp interactions per event ]
2
Mean RPC counting rate [Hz/cm (b) (a) (a) Mean RPC counting rates measured in seven different intervals of the number of proton-proton Figure 35. (a) Mean RPC counting rates measured in seven different intervals of the number of proton-proton
collisions per event. The counting rate was averaged over all active RPC modules and is shown separately
for 𝜂and 𝜙panels. (b) Correlation between the mean RPC detector efficiency and the mean RPC detector
counting rate, measured in seven different intervals of the number of proton-proton collisions per event,
shown separately for 𝜂and 𝜙panels. Two different datasets, corresponding to 60.8 fb−1 and 3.6 fb−1, are
used for the efficiency measurements and the counting rate measurements, respectively. Both figures were
obtained by averaging over all active 𝜂and 𝜙panels. Only RPC panels with an average efficiency larger than
50% were used for these figures. The muon detection efficiency and mean counting rate were also measured for each individual
RPC panel in seven bins of the number of collisions per event. The mean module efficiency is
plotted as a function of the mean counting rate in figure 36(a) for one representative RPC module. 6.4
RPC efficiency as a function of counting rate The slope of the efficiency versus the counting rate for each RPC panel was determined from a
linear fit. The distribution of the linear slope parameter is shown in figure 36(b) for all selected
RPC panels. Only RPC panels with a fit 𝜒2 per degree of freedom less than 5.0 were retained for
the analysis. The negative mean value of the distribution indicates clearly that a majority of the
RPC panels show a decrease in muon detection efficiency as a function of the counting rate. This
result supports the hypothesis that the observed decrease in the RPC detector efficiency at higher
instantaneous luminosity is due to the increase in RPC counting rates in individual modules. 7
Expected performance of the existing RPCs at HL-LHC The ATLAS detector is scheduled to operate with the HL-LHC until approximately 2040. An
extensive set of upgrades will be installed in 2025–2026. These include upgrades of the MS and
its electronics [44], and of the trigger system [47]. In particular, three new layers of RPCs will be – 38 – 8
10
12
14
16
]
2
Mean RPC counting rate [Hz/cm
0.93
0.94
0.95
0.96
0.97
0.98
Mean RPC module efficiency
ATLAS
-1
= 13 TeV, 60.8 - 3.6 fb
s
One RPC module
view
η
view
φ
(a)
0.01
−
0.005
−
0
0.005
0.01
/Hz]
2
Linear fit slope [cm
0
100
200
300
400
RPC modules
ATLAS
-1
= 13 TeV, 60.8 - 3.6 fb
s
All RPC modules
/Hz
2
mean = -0.00116 cm
η
/Hz
2
mean = -0.00123 cm
φ
view
η
view
φ
(b)
Figure 36. (a) Correlation between the mean RPC module efficiency and the mean RPC counting rate,
measured in seven different intervals of the number of proton-proton collisions per event, for one RPC
module in both the 𝜂and 𝜙views. (b) Distribution of the slope parameters extracted from a linear fit of
the efficiency versus counting rate dependence of each RPC panel. Two different datasets, corresponding
to 60.8 fb−1 and 3.6 fb−1, are used for the efficiency measurements and the counting rate measurements,
respectively. Only RPC panels with an average efficiency larger than 50% were used for this plot. 8
10
12
14
16
]
2
Mean RPC counting rate [Hz/cm
0.93
0.94
0.95
0.96
0.97
0.98
Mean RPC module efficiency
ATLAS
-1
= 13 TeV, 60.8 - 3.6 fb
s
One RPC module
view
η
view
φ
(a) 0.01
−
0.005
−
0
0.005
0.01
/Hz]
2
Linear fit slope [cm
0
100
200
300
400
RPC modules
ATLAS
-1
= 13 TeV, 60.8 - 3.6 fb
s
All RPC modules
/Hz
2
mean = -0.00116 cm
η
/Hz
2
mean = -0.00123 cm
φ
view
η
view
φ
(b) 2021 (b) (a) Figure 36. (a) Correlation between the mean RPC module efficiency and the mean RPC counting rate,
measured in seven different intervals of the number of proton-proton collisions per event, for one RPC
module in both the 𝜂and 𝜙views. (b) Distribution of the slope parameters extracted from a linear fit of
the efficiency versus counting rate dependence of each RPC panel. 7
Expected performance of the existing RPCs at HL-LHC Two different datasets, corresponding
to 60.8 fb−1 and 3.6 fb−1, are used for the efficiency measurements and the counting rate measurements,
respectively. Only RPC panels with an average efficiency larger than 50% were used for this plot. JINST 16 P07029 installed in the innermost region of the barrel MS in order to improve redundancy in the RPC system,
to increase muon trigger efficiency and to reduce trigger rates. More sophisticated, customisable
trigger algorithms will be deployed utilising field-programmable gate array (FPGA) devices. The
MDT detector will be also used by the hardware-level muon trigger [44], allowing more effective
rejection of muons with 𝑝T below a target trigger threshold. The ATLAS RPC system was originally designed to operate at an instantaneous luminosity of
1034 cm−2s−1 for a total integrated luminosity of 300 fb−1. The same RPCs and FE electronics will
be used for HL-LHC operations at an instantaneous luminosity of 7.5 × 1034 cm−2s−1 for a total
integrated luminosity of more than 3000 fb−1. The majority of RPCs are expected to stay within
safe operating limits at the HL-LHC, as detailed in ref. [44]. Nevertheless, the integrated charge
collected by the chambers located at |𝜂| ≈1 will exceed the design specifications by more than a
factor of three. In order to operate these RPCs safely during HL-LHC data-taking, the operating
voltage will be lowered from the nominal value of 9.6 kV to approximately 9.2 kV, which will lead
to a reduction of the muon detection efficiency. This section presents several studies that extrapolate the performance of the existing RPCs
to the conditions expected during HL-LHC operations. Section 7.1 presents extrapolations of
RPC currents to the expected HL-LHC instantaneous luminosity for both the default and reduced
operating voltage settings. Section 7.2 presents measurements of the RPC counting rates measured
using zero-bias collisions, obtained at different operating voltage and FE threshold settings. This
section also presents measurements of RPC detector efficiency and cluster size, obtained at different
operating voltage and FE threshold settings, using muons produced in proton-proton collisions. – 39 – 7.1
Expected RPC currents at the HL-LHC In order to extrapolate RPC currents to the expected HL-LHC instantaneous luminosity of 7.5 ×
1034 cm−2s−1, the measured current density of each module was scaled by a factor of 4.2. Figure 37
shows the distributions of the extrapolated current density for all selected RPCs at the instantaneous
luminosity of 7.5 × 1034 cm−2s−1. For a fraction of the RPCs operating at 9.6 kV, the extrapolated
current density exceeds the safety limit of about 30 μA/m2 required for stable RPC operations at the
HL-LHC [44]. The expected current density in the affected RPCs can be reduced by lowering the
operating voltage to 9.2 kV, thereby allowing safe operation of nearly all RPCs. The lower voltage
also leads to a decrease of the muon detection efficiency that can be partially recovered by adjusting
the thresholds of the FE electronics, as discussed in section 7.2. 0
10
20
30
40
50
60
]
2
A/m
µ
Extrapolated current [
0
100
200
300
400
500
600
Gas gaps
ATLAS
Extrapolation from 2018 data
-1
s
-2
cm
34
10
×
= 7.5
inst
L
= 13 TeV
s
HV = 9.2 kV
HV = 9.6 kV
Figure 37. Distributions of the expected current density for the selected RPCs extrapolated to an instantaneous
luminosity of 7.5 × 1034 cm−2s−1. JINST 16 P07029 Figure 37. Distributions of the expected current density for the selected RPCs extrapolated to an instantaneous
luminosity of 7.5 × 1034 cm−2s−1. Figures 38(a) and 38(b) show the expected current density measurements at an instantaneous
luminosity of 7.5 × 1034 cm−2s−1 for operating voltage values of 9.2 kV and 9.6 kV, respectively. The current density measurements are shown as a function of 𝜂and 𝜙position for the RPCs
belonging to the RPC1 doublet layer. The values were averaged over the two individual layers of
each RPC chamber. The expected current density for each module was obtained using the linear
extrapolation method described previously. The RPCs with current density higher than the safety
limit of 30 μA/m2 are located primarily at high values of |𝜂|. The expected current density is slightly
lower for the RPCs located at the bottom of the ATLAS detector, corresponding to the sectors with
higher 𝜙indices (from 10 up to 16). This effect is consistent with the temperature gradient in the
ATLAS cavern, where lower temperatures are measured in the bottom part of the detector. 7.2
RPC detector performance using different operating voltage and FE threshold setting ATLAS 8
−
6
−
4
−
2
−
0
2
4
6
8
η
Gap index along
2
4
6
8
10
12
14
16
φ
Gap index along
0
5
10
15
20
25
30
35
40
]
2
A/m
µ
Extrapolated current [
S
Extrapolation from 2018 data, HV = 9.2 kV
-1
s
-2
cm
34
10
×
= 7.5
inst
= 13 TeV, L
s
(a) 8
−
6
−
4
−
2
−
0
2
4
6
8
η
Gap index along
2
4
6
8
10
12
14
16
φ
Gap index along
0
5
10
15
20
25
30
35
40
]
2
A/m
µ
Extrapolated current [
Extrapolation from 2018 data, HV = 9.6 kV
-1
s
-2
cm
34
10
×
= 7.5
inst
= 13 TeV, L
s φ
Gap index along 2021 (b) (a) JINST 16 P07029 Figure 38. Expected current density at an instantaneous luminosity of 7.5×1034 cm−2s−1 shown as a function
of the 𝜂and 𝜙position for the barrel RPCs belonging to the RPC middle doublet layer. The current density
values were averaged over the two individual sub-layers and are shown for operating voltages of (a) 9.2 kV
and (b) 9.6 kV. Empty bins correspond to logical combinations of indices that do not represent installed RPCs
or to modules that failed the selection criteria. applied to only one layer of the outermost RPC doublet layer (RPC3) of the large 𝜙sector 13 in
ATLAS side A. 7.2
RPC detector performance using different operating voltage and FE threshold setting 0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
10
20
30
40
]
2
RPC counting rate [Hz/cm
ATLAS
= 13 TeV
s
HV = 9.2 kV
scan
thr
V
Zero-bias collisions
-3
10
×
0.008)
±
= 1.0 V, slope = (0.640
thr
V
-3
10
×
0.012)
±
= 0.9 V, slope = (0.875
thr
V
-3
10
×
0.012)
±
= 0.8 V, slope = (1.287
thr
V
(a) 0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
10
20
30
40
]
2
RPC counting rate [Hz/cm
ATLAS
= 13 TeV
s
HV = 9.2 kV
scan
thr
V
Zero-bias collisions
-3
10
×
0.008)
±
= 1.0 V, slope = (0.640
thr
V
-3
10
×
0.012)
±
= 0.9 V, slope = (0.875
thr
V
-3
10
×
0.012)
±
= 0.8 V, slope = (1.287
thr
V
(a)
0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
10
20
30
40
50
]
2
RPC counting rate [Hz/cm
ATLAS
= 13 TeV
s
HV = 9.6 kV
scan
thr
V
Zero-bias collisions
-3
10
×
0.010)
±
= 1.0 V, slope = (0.927
thr
V
-3
10
×
0.039)
±
= 0.9 V, slope = (1.478
thr
V
-3
10
×
0.015)
±
= 0.8 V, slope = (1.714
thr
V
(b)
Figure 39. RPC counting rates per unit surface area as a function of instantaneous luminosity for one
representative 𝜂panel operating at (a) 9.2 kV and (b) 9.6 kV, using different FE threshold settings. This
module belongs to the BOL doublet layer and is located in large 𝜙sector 1 and 𝜂station 5. Slope parameters
were extracted from a linear fit to data points. 0
5000
10000
15000
20000
]
-1
s
-2
cm
30
Instantaneous luminosity [10
0
10
20
30
40
50
]
2
RPC counting rate [Hz/cm
ATLAS
= 13 TeV
s
HV = 9.6 kV
scan
thr
V
Zero-bias collisions
-3
10
×
0.010)
±
= 1.0 V, slope = (0.927
thr
V
-3
10
×
0.039)
±
= 0.9 V, slope = (1.478
thr
V
-3
10
×
0.015)
±
= 0.8 V, slope = (1.714
thr
V
(b) ]
2
RPC counting rate [Hz/cm ]
2
RPC counting rate [Hz/cm (b) (a) Figure 39. 7.2
RPC detector performance using different operating voltage and FE threshold setting As discussed in section 7.1, the operating voltage will be lowered to approximately 9.2 kV for
some RPCs to permit safe operation at the HL-LHC. This reduction will be necessary to keep
ionisation currents below the safety limit for those chambers. To check the resulting effects on RPC
performance, three runs were recorded in 2018 with several modules operating at 9.2 kV and using
three different FE threshold settings 𝑉thr: 0.8 V, 0.9 V and 1.0 V. In order to minimise the impact
on the muon trigger efficiency during ATLAS physics data-taking, these non-standard settings were – 40 – 8
−
6
−
4
−
2
−
0
2
4
6
8
η
Gap index along
2
4
6
8
10
12
14
16
φ
Gap index along
0
5
10
15
20
25
30
35
40
]
2
A/m
µ
Extrapolated current [
ATLAS
Extrapolation from 2018 data, HV = 9.2 kV
-1
s
-2
cm
34
10
×
= 7.5
inst
= 13 TeV, L
s
(a)
8
−
6
−
4
−
2
−
0
2
4
6
8
η
Gap index along
2
4
6
8
10
12
14
16
φ
Gap index along
0
5
10
15
20
25
30
35
40
]
2
A/m
µ
Extrapolated current [
ATLAS
Extrapolation from 2018 data, HV = 9.6 kV
-1
s
-2
cm
34
10
×
= 7.5
inst
= 13 TeV, L
s
(b)
Figure 38. Expected current density at an instantaneous luminosity of 7.5×1034 cm−2s−1 shown as a function
of the 𝜂and 𝜙position for the barrel RPCs belonging to the RPC middle doublet layer. The current density
values were averaged over the two individual sub-layers and are shown for operating voltages of (a) 9.2 kV
and (b) 9.6 kV. Empty bins correspond to logical combinations of indices that do not represent installed RPCs
or to modules that failed the selection criteria. 7.2
RPC detector performance using different operating voltage and FE threshold setting RPC counting rates per unit surface area as a function of instantaneous luminosity for one
representative 𝜂panel operating at (a) 9.2 kV and (b) 9.6 kV, using different FE threshold settings. This
module belongs to the BOL doublet layer and is located in large 𝜙sector 1 and 𝜂station 5. Slope parameters
were extracted from a linear fit to data points. Figure 39 shows the area-normalised RPC counting rates as a function of instantaneous lumi-
nosity for one representative RPC 𝜂panel operating at 9.2 kV and 9.6 kV, and using three different
FE threshold settings. Decreasing the FE threshold from 1.0 V to 0.8 V results in an increase of – 41 – the RPC counting rates by about a factor of two. This effect is also demonstrated in figure 40,
which shows the distributions of the counting rates measured at an instantaneous luminosity of
1.8 × 1034 cm−2s−1 for 36 tested panels operating at 9.2 kV and using three different FE thresholds. 0
10
20
30
]
2
RPC counting rate [Hz/cm
0
5
10
15
20
25
RPC panels
= 1.0 V, mean = 10.2
thr
V
= 0.9 V, mean = 11.5
thr
V
= 0.8 V, mean = 16.9
thr
V
ATLAS
= 13 TeV
s
HV = 9.2 kV
scan
thr
V
Zero-bias collisions
-1
s
-2
cm
34
10
×
= 1.8
inst
L
Figure 40. Distribution of the measured RPC counting rates at an instantaneous luminosity of 1.8 ×
1034 cm−2s−1 for panels operating at 9.2 kV using three different FE threshold settings. Figure 40. Distribution of the measured RPC counting rates at an instantaneous luminosity of 1.8 ×
1034 cm−2s−1 for panels operating at 9.2 kV using three different FE threshold settings. JINST 16 P07029 Figure 41 shows the muon detection efficiency as a function of 𝑉thr values for the representative
𝜂and 𝜙panels belonging to the same module operating at 9.2 kV and 9.6 kV. When the voltage
was lowered to 9.2 kV and the FE threshold was kept at the nominal value of 1.0 V, the absolute
efficiency was reduced by about 24% and 30% for the 𝜂and 𝜙panel, respectively. Then decreasing
the FE threshold from 1.0 V to 0.8 V leads to an absolute efficiency gain of about 17% for the
𝜂panel and 20% for the 𝜙panel. 7.2
RPC detector performance using different operating voltage and FE threshold setting The efficiency gain is computed as the difference between the
muon detection efficiencies computed at the 𝑉thr of 1.0 V and 0.8 V, when operating at 9.2 kV. A constant detector efficiency is observed while varying the FE thresholds at 9.6 kV because the
RPC is operating at the efficiency plateau and therefore it is not sensitive to changes of the FE
threshold values. 0.8
0.9
1
[V]
thr
V
Front-end discriminator setting
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
RPC detector efficiency
HV = 9.6 kV
HV = 9.2 kV
ATLAS
= 13 TeV
s
side view, high gain
η
One RPC module,
(a)
0.8
0.9
1
[V]
thr
V
Front-end discriminator setting
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
RPC detector efficiency
HV = 9.6 kV
HV = 9.2 kV
ATLAS
= 13 TeV
s
side view, high gain
φ
One RPC module,
(b)
Figure 41. RPC detector efficiency plotted as a function of the FE threshold setting 𝑉thr for the (a) 𝜂panel
and (b) 𝜙panel of one representative RPC module, measured at the operating voltage values of 9.2 kV and
9.6 kV. 0.8
0.9
1
[V]
thr
V
Front-end discriminator setting
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
RPC detector efficiency
HV = 9.6 kV
HV = 9.2 kV
ATLAS
= 13 TeV
s
side view, high gain
η
One RPC module,
(a) 0.8
0.9
1
[V]
thr
V
Front-end discriminator setting
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
RPC detector efficiency
HV = 9.6 kV
HV = 9.2 kV
ATLAS
= 13 TeV
s
side view, high gain
φ
One RPC module,
(b) [V]
thr
V
Front-end discriminator setting (b) (a) Figure 41. RPC detector efficiency plotted as a function of the FE threshold setting 𝑉thr for the (a) 𝜂panel
and (b) 𝜙panel of one representative RPC module, measured at the operating voltage values of 9.2 kV and
9.6 kV. – 42 – Figure 42(a) shows the distribution of the panel efficiencies at different 𝑉thr threshold values
for the 9.2 kV voltage setting. The average absolute efficiency gain is around 8% when lowering
the 𝑉thr from 1.0 V to 0.8 V. The corresponding distribution for the mean cluster size is shown in
figure 42(b). 7.2
RPC detector performance using different operating voltage and FE threshold setting 0.5
0.6
0.7
0.8
0.9
1
Detector efficiency
0
5
10
15
20
25
30
RPC panels
= 1.0 V, mean = 0.81
thr
V
= 0.9 V, mean = 0.82
thr
V
= 0.8 V, mean = 0.89
thr
V
ATLAS
= 13 TeV
s
HV = 9.2 kV
scan
thr
V
(a)
0
0.5
1
1.5
2
2.5
3
Mean cluster size [strips]
0
5
10
15
20
25
RPC panels
= 1.0 V, mean = 1.23
thr
V
= 0.9 V, mean = 1.48
thr
V
= 0.8 V, mean = 2.08
thr
V
ATLAS
= 13 TeV
s
HV = 9.2 kV
scan
thr
V
(b)
Figure 42. Distributions of (a) the panel efficiencies and (b) the mean cluster size measured at different 𝑉thr
threshold values with the operating voltage set to 9.2 kV. 0.5
0.6
0.7
0.8
0.9
1
Detector efficiency
0
5
10
15
20
25
30
RPC panels
= 1.0 V, mean = 0.81
thr
V
= 0.9 V, mean = 0.82
thr
V
= 0.8 V, mean = 0.89
thr
V
ATLAS
= 13 TeV
s
HV = 9.2 kV
scan
thr
V
(a) 0
0.5
1
1.5
2
2.5
3
Mean cluster size [strips]
0
5
10
15
20
25
RPC panels
= 1.0 V, mean = 1.23
thr
V
= 0.9 V, mean = 1.48
thr
V
= 0.8 V, mean = 2.08
thr
V
ATLAS
= 13 TeV
s
HV = 9.2 kV
scan
thr
V
(b) 2021 JINST 16 P07029 (a) (b) Figure 42. Distributions of (a) the panel efficiencies and (b) the mean cluster size measured at different 𝑉thr
threshold values with the operating voltage set to 9.2 kV. Despite the RPC efficiency decrease due to the lower applied voltage, the overall efficiency of the
L1 muon barrel trigger is expected to increase after the ATLAS MS and trigger upgrades [44, 47]
planned for the HL-LHC. After these upgrades, three new layers of RPCs will increase the
redundancy of the muon barrel trigger system and lead to a higher overall trigger efficiency. A study
of the expected muon trigger performance after these upgrades is outside the scope of the present
work, which instead aims to evaluate the expected performance of the RPC detector. 7.2
RPC detector performance using different operating voltage and FE threshold setting The present
studies demonstrate that lowering the operating voltage to 9.2 kV will allow safe RPC operation
and provide an RPC detector efficiency similar to that obtained using the nominal voltage if the FE
thresholds are lowered. Acknowledgments We thank CERN for the very successful operation of the LHC, as well as the support stafffrom our
institutions without whom ATLAS could not be operated efficiently. We thank CERN for the very successful operation of the LHC, as well as the support stafffrom our
institutions without whom ATLAS could not be operated efficiently. 2021 JINST 16 P07 2021 We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia;
BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil;
NSERC, NRC and CFI, Canada; CERN; ANID, Chile; CAS, MOST and NSFC, China; COL-
CIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC,
Denmark; IN2P3-CNRS and CEA-DRF/IRFU, France; SRNSFG, Georgia; BMBF, HGF and
MPG, Germany; GSRT, Greece; RGC and Hong Kong SAR, China; ISF and Benoziyo Center, Is-
rael; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; NWO, Netherlands; RCN, Norway;
MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; JINR; MES of Russia and NRC
KI, Russian Federation; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF,
South Africa; MICINN, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Can-
tons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom;
DOE and NSF, United States of America. In addition, individual groups and members have re-
ceived support from BCKDF, CANARIE, Compute Canada, CRC and IVADO, Canada; Beijing
Municipal Science & Technology Commission, China; COST, ERC, ERDF, Horizon 2020 and
Marie Skłodowska-Curie Actions, European Union; Investissements d’Avenir Labex, Investisse-
ments d’Avenir Idex and ANR, France; DFG and AvH Foundation, Germany; Herakleitos, Thales
and Aristeia programmes co-financed by EU-ESF and the Greek NSRF, Greece; BSF-NSF and GIF,
Israel; La Caixa Banking Foundation, CERCA Programme Generalitat de Catalunya and PROME-
TEO and GenT Programmes Generalitat Valenciana, Spain; Göran Gustafssons Stiftelse, Sweden;
The Royal Society and Leverhulme Trust, United Kingdom. JINST 16 P07029 The crucial computing support from all WLCG partners is acknowledged gratefully, in partic-
ular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway,
Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands),
PIC (Spain), ASGC (Taiwan), RAL (U.K.) and BNL (U.S.A.), the Tier-2 facilities worldwide and
large non-WLCG resource providers. Major contributors of computing resources are listed in
ref. [48]. 8
Conclusions Measurements of the performance of the ATLAS RPC detector and L1 muon barrel trigger were
obtained using data from 60.8 fb−1 of √𝑠= 13 TeV proton-proton collisions at the LHC recorded
in 2018. The response of individual RPC modules was measured using muons produced in decays
of 𝑍bosons, including measurements of efficiency, cluster hit multiplicity and time resolution. The RPC response was also measured as a function of time and as a function of the mean number
of proton-proton collisions per bunch crossing. Performance of the L1 muon barrel trigger was
also measured using muons produced in decays of 𝑍bosons, including measurements of trigger
efficiency as a function of several variables. Acceptance rates were measured for the L1 muon
trigger thresholds and the composition of accepted events was studied using reconstructed muon
candidates. Measurements of RPC counting rates and currents were performed using zero-bias
collisions, which are dominated by background events from neutron and photon interactions with
the RPCs. The avalanche charge was measured for all RPC modules and was found to be consistent – 43 – with earlier measurements in test beams. The RPC detector response was measured as a function
of the instantaneous luminosity and several possible scenarios were tested for operating the RPCs
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K. Asai125, S. Asai162, T. Asawatavonvanich163, N. Asbah59, E.M. Asimakopoulou170, L. Asquith155,
J. Assahsah35e, K. Assamagan29, R. Astalos28a, R.J. Atkin33a, M. Atkinson171, N.B. Atlay19, H. Atmani64,
P.A. Atmasiddha105, K. Augsten140, V.A. Austrup180, G. Avolio36, M.K. Ayoub15c, G. Azuelos109,ak,
D. Babal28a, H. Bachacou143, K. Bachas161, F. Backman45a,45b, P. Bagnaia72a,72b, M. Bahmani84,
H. Bahrasemani151, A.J. Bailey172, V.R. Bailey171, J.T. Baines142, C. Bakalis10, O.K. Baker181,
PJ B kk
119 E B k
16 D B k hi G
8 S B l ji156 R B l
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119 E M B ldi 121b 121a Y. Amaral Coutinho80b, A. Ambler103, L. Ambroz133, C. Amelung36, D. Amidei105, Y. Amaral Coutinho
, A. Ambler
, L. Ambroz
, C. Amelung
, D. Amidei
,
S.P. Amor Dos Santos138a, S. Amoroso46, C.S. Amrouche54, C. Anastopoulos148, N. Andari143,
T. Andeen11, J.K. Anders20, S.Y. Andrean45a,45b, A. Andreazza68a,68b, V. Andrei61a, C.R. Anelli174,
S. Angelidakis9, A. Angerami39, A.V. Anisenkov121b,121a, A. Annovi71a, C. Antel54, M.T. Anthony148,
E. Antipov128, M. Antonelli51, D.J.A. Antrim18, F. Anulli72a, M. Aoki81, J.A. The ATLAS collaboration Aparisi Pozo172,
M.A. Aparo155, L. Aperio Bella46, N. Aranzabal36, V. Araujo Ferraz80a, R. Araujo Pereira80b,
C. Arcangeletti51, A.T.H. Arce49, J-F. Arguin109, S. Argyropoulos52, J.-H. Arling46, A.J. Armbruster36,
A. Armstrong169, O. Arnaez165, H. Arnold119, Z.P. Arrubarrena Tame113, G. Artoni133, H. Asada116,
K. Asai125, S. Asai162, T. Asawatavonvanich163, N. Asbah59, E.M. Asimakopoulou170, L. Asquith155,
J. Assahsah35e, K. Assamagan29, R. Astalos28a, R.J. Atkin33a, M. Atkinson171, N.B. Atlay19, H. Atmani64,
P.A. Atmasiddha105, K. Augsten140, V.A. Austrup180, G. Avolio36, M.K. Ayoub15c, G. Azuelos109,ak,
D Babal28a H Bachacou143 K Bachas161 F Backman45a,45b P Bagnaia72a,72b M Bahmani84 JINST 16 P07029 y
y
P.J. Bakker119, E. Bakos16, D. Bakshi Gupta8, S. Balaji156, R. Balasubramanian119, E.M. Baldin1 p
j
P. Balek178, F. Balli143, W.K. Balunas133, J. Balz99, E. Banas84, M. Bandieramonte137, p
j
Balek178, F. Balli143, W.K. Balunas133, J. Balz99, E. Banas84, M. Bandieramonte137, A. Bandyopadhyay19, L. Barak160, W.M. Barbe38, E.L. Barberio104, D. Barberis55b,55a, M. Barbe A. Bandyopadhyay19, L. Barak160, W.M. Barbe38, E.L. Barberio104, D. Barberis55b,55a, M. Barbero101, G. Barbour94, T. Barillari114, M-S. Barisits36, J. Barkeloo130, T. Barklow152, B.M. Barnett142, R.M. Barnett18, Z. Barnovska-Blenessy60a, A. Baroncelli60a, G. Barone29, A.J. Barr133, L. Barranco Navarro45a,45b, F. Barreiro98, J. Barreiro Guimarães da Costa15a, U. Barron160, S. Barsov F. Bartels61a, R. Bartoldus152, G. Bartolini101, A.E. Barton89, P. Bartos28a, A. Basalaev46, A. Basan99, B. Batool150, M. Battaglia144, M. Bauce72a,72b, F. Bauer143,*, P. Bauer24, H.S. Bawa31, A. Bayirli12c,
J.B. Beacham49, T. Beau134, P.H. Beauchemin168, F. Becherer52, P. Bechtle24, H.P. Beck20,q, K. Becker176,
C. Becot46, A.J. Beddall12a, V.A. Bednyakov79, C.P. Bee154, T.A. Beermann180, M. Begalli80b, M. Begel29,
A. Behera154, J.K. Behr46, F. Beisiegel24, M. Belfkir5, G. Bella160, L. Bellagamba23b, A. Bellerive34,
P. Bellos21, K. Beloborodov121b,121a, K. Belotskiy111, N.L. Belyaev111, D. Benchekroun35a, N. Benekos10,
Y. Benhammou160, D.P. Benjamin6, M. Benoit29, J.R. Bensinger26, S. Bentvelsen119, L. Beresford133,
M. Beretta51, D. Berge19, E. Bergeaas Kuutmann170, N. Berger5, B. Bergmann140, L.J. Bergsten26,
J. Beringer18, S. Berlendis7, G. Bernardi134, C. Bernius152, F.U. Bernlochner24, T. Berry93, P. Berta99,
A. Berthold48, I.A. Bertram89, O. Bessidskaia Bylund180, S. Bethke114, A. Betti42, A.J. Bevan92,
S. Bhatta154, D.S. Bhattacharya175, P. Bhattarai26, V.S. Bhopatkar6, R. Bi137, R.M. Bianchi137,
O. Biebel113, D. Biedermann19, R. Bielski36, K. Bierwagen99, N.V. Biesuz71a,71b, M. Biglietti74a,
T.R.V. Billoud140, M. Bindi53, A. Bingul12d, C. Bini72a,72b, S. Biondi23b,23a, C.J. Birch-sykes100,
M. Birman178, T. Bisanz36, J.P. Biswal3, D. Biswas179,j, A. Bitadze100, C. Bittrich48, K. Bjørke132,
T. Blazek28a, I. Bloch46, C. Blocker26, A. Blue57, U. Blumenschein92, G.J. Bobbink119, – 48 – S. Bobrovnikov121b,121a, D. Bogavac14, A.G. The ATLAS collaboration Bogdanchikov121b,121a, C. Bohm45a, V. Boisvert93, ,
g
,
g
,
,
,
Bokan170,53, T. Bold83a, M. Bomben134, M. Bona92, J.S. Bonilla130, M. Boonekamp143, C.D. Booth93, p
G. Borbély57, H.M. Borecka-Bielska90, L.S. Borgna94, A. Borisov122, G. Borissov89, D. Bortoletto133, y
g
D. Boscherini23b, M. Bosman14, J.D. Bossio Sola103, K. Bouaouda35a, J. Boudreau137, 2021 Calvente Lopez98, D. Calvet38, S. Calvet38, T.P. Calvet101, M. Calvetti71a,71b, R. Camacho Toro134,
Camarda36, D. Camarero Munoz98, P. Camarri73a,73b, M.T. Camerlingo74a,74b, D. Cameron132, JINST 16 P07029 g
Camincher36, M. Campanelli94, A. Camplani40, V. Canale69a,69b, A. Canesse103, M. Cano Bret77, Cantero128, Y. Cao171, M. Capua41b,41a, R. Cardarelli73a, F. Cardillo172, G. Carducci41b,41a, T. Carli36, Y-H. Chen46, H.C. Cheng62a, H.J. Cheng15a, A. Cheplakov79, E. Cheremushkina122, Cherkaoui El Moursli35f, E. Cheu7, K. Cheung63, T.J.A. Chevalérias143, L. Chevalier143, V. Chiarella51, R. Cherkaoui El Moursli35f, E. Cheu7, K. Cheung63, T.J.A. Chevalérias143, L. Chevalier143, V. Chiarella51,
G. Chiarelli71a, G. Chiodini67a, A.S. Chisholm21, A. Chitan27b, I. Chiu162, Y.H. Chiu174, M.V. Chizhov79,
K Ch i11 A R Ch
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K. Choi11, A.R. Chomont72a,72b, Y. Chou102, Y.S. Chow119, L.D. Christopher33e, M.C. Chu62a, p
X. Chu15a,15d, J. Chudoba139, J.J. Chwastowski84, D. Cieri114, K.M. Ciesla84, V. Cindro91, I.A. Cioară2 A. Ciocio18, F. Cirotto69a,69b, Z.H. Citron178,k, M. Citterio68a, D.A. Ciubotaru27b, B.M. Ciungu165, g
A. Clark54, P.J. Clark50, S.E. Clawson100, C. Clement45a,45b, L. Clissa23b,23a, Y. Coadou101, ,
,
,
,
,
,
M. Cobal66a,66c, A. Coccaro55b, J. Cochran78, R. Coelho Lopes De Sa102, H. Cohen160, A.E.C. Coimbra36,
B. Cole39, J. Collot58, P. Conde Muiño138a,138h, S.H. Connell33c, I.A. Connelly57, F. Conventi69a,al,
A.M. Cooper-Sarkar133, F. Cormier173, L.D. Corpe94, M. Corradi72a,72b, E.E. Corrigan96,
F. Corriveau103,aa, M.J. Costa172, F. Costanza5, D. Costanzo148, G. Cowan93, J.W. Cowley32, J. Crane100,
K. Cranmer124, R.A. Creager135, S. Crépé-Renaudin58, F. Crescioli134, M. Cristinziani24,
M. Cristoforetti75a,75b, V. Croft168, G. Crosetti41b,41a, A. Cueto5, T. Cuhadar Donszelmann169,
H. Cui15a,15d, A.R. Cukierman152, W.R. Cunningham57, S. Czekierda84, P. Czodrowski36,
M.M. Czurylo61b, M.J. Da Cunha Sargedas De Sousa60b, J.V. Da Fonseca Pinto80b, C. Da Via100,
W. Dabrowski83a, F. Dachs36, T. Dado47, S. Dahbi33e, T. Dai105, C. Dallapiccola102, M. Dam40,
G. D’amen29, V. D’Amico74a,74b, J. Damp99, J.R. Dandoy135, M.F. Daneri30, M. Danninger151, V. Dao36,
G. Darbo55b, O. Dartsi5, A. Dattagupta130, S. D’Auria68a,68b, C. David166b, T. Davidek141, D.R. Davis49,
I. Dawson148, K. De8, R. De Asmundis69a, M. De Beurs119, S. De Castro23b,23a, N. The ATLAS collaboration Della Pietra69a,69b, G. Di Gregorio71a,71b, A. Di Luca75a,75b, B. Di Micco74a,74b, R. Di Nardo74a,74b, R. Di Sipio165, M. Didenko164, E.B. Diehl105, J. Dietrich19, S. Díez Cornell46, C. Diez Pardos150, A. Dimitrievska18, y
y
y
A. du Pree119, Y. Duan60d, F. Dubinin110, M. Dubovsky28a, A. Dubreuil54, E. Duchovni178, y
Duckeck113, O.A. Ducu36,27b, D. Duda114, A. Dudarev36, A.C. Dudder99, M. D’uffizi100, L. Duflot64, M. Dührssen36, C. Dülsen180, M. Dumancic178, A.E. Dumitriu27b, M. Dunford61a, S. Dungs47, JINST 16 P07029 A. Duperrin101, H. Duran Yildiz4a, M. Düren56, A. Durglishvili158b, B. Dutta46, D. Duvnjak1, G.I. Dyckes135, M. Dyndal36, S. Dysch100, B.S. Dziedzic84, M.G. Eggleston49, T. Eifert8, G. Eigen17, K. Einsweiler18, T. Ekelof170, H. El Jarrari35f, A. El Moussaouy35a, V. Ellajosyula170, M. Ellert1 K. Einsweiler18, T. Ekelof170, H. El Jarrari35f, A. El Moussaouy35a, V. Ellajosyula170, M. Ellert170, F. Ellinghaus180, A.A. Elliot92, N. Ellis36, J. Elmsheuser29, M. Elsing36, D. Emeliyanov142, F. Ellinghaus180, A.A. Elliot92, N. Ellis36, J. Elmsheuser29, M. Elsing36, D. Emeliyanov142, Emerman39, Y. Enari162, J. Erdmann47, A. Ereditato20, P.A. Erland84, M. Errenst180, M. Escalier64, A. Emerman39, Y. Enari162, J. Erdmann47, A. Ereditato20, P.A. Erland84, M. Errenst180, M. Esca C. Escobar172, O. Estrada Pastor172, E. Etzion160, G. Evans138a, H. Evans65, M.O. Evans155, C. Escobar172, O. Estrada Pastor172, E. Etzion160, G. Evans138a, H. Evans65, M.O. Evans155, A. Ezhilov136, F. Fabbri57, L. Fabbri23b,23a, V. Fabiani118, G. Facini176, R.M. Fakhrutdinov122, S. Falciano72a, P.J. Falke24, S. Falke36, J. Faltova141, Y. Fang15a, Y. Fang15a, G. Fanourakis44, S. Falciano72a, P.J. Falke24, S. Falke36, J. Faltova141, Y. Fang15a, Y. Fang15a, G. Fanourakis44, Falciano
, P.J. Falke
, S. Falke
, J. Faltova
, Y. Fang
, Y. Fang
, G. Fanourakis
Fanti68a,68b, M. Faraj60c, A. Farbin8, A. Farilla74a, E.M. Farina70a,70b, T. Farooque106, g
g
M. Fanti68a,68b, M. Faraj60c, A. Farbin8, A. Farilla74a, E.M. Farina70a,70b, T. Farooque106, S.M. Farrington50, P. Farthouat36, F. Fassi35f, D. Fassouliotis9, M. Faucci Giannelli50, W.J. Fawcett32, S.M. Farrington50, P. Farthouat36, F. Fassi35f, D. Fassouliotis9, M. Faucci Giannelli50, W.J. Fawcett32, g
Fayard64, O.L. Fedin136,p, A. Fehr20, M. Feickert171, L. Feligioni101, A. Fell148, C. Feng60b, M. Feng49, L. Fayard64, O.L. Fedin136,p, A. Fehr20, M. Feickert171, L. Feligioni101, A. Fell148, C. Feng60b, M. Feng49,
M.J. Fenton169, A.B. Fenyuk122, S.W. Ferguson43, J. Ferrando46, A. Ferrari170, P. Ferrari119, R. Ferrari70a, y
g
g
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M.J. Fenton169, A.B. Fenyuk122, S.W. Ferguson43, J. Ferrando46, A. Ferrari170, P. Ferrari119, R. Ferrari70a y
g
g
g
J. Fenton169, A.B. Fenyuk122, S.W. Ferguson43, J. Ferrando46, A. Ferrari170, P. Ferrari119, R. The ATLAS collaboration De Groot118, Cobal66a,66c, A. Coccaro55b, J. Cochran78, R. Coelho Lopes De Sa102, H. Cohen160, A.E.C. Coimbra36,
Cole39 J Collot58 P Conde Muiño138a,138h S H Connell33c I A Connelly57 F Conventi69a,al y
M. Cooper-Sarkar133, F. Cormier173, L.D. Corpe94, M. Corradi72a,72b, E.E. Corrigan96, A.M. Cooper Sarkar
, F. Cormier
, L.D. Corpe
, M. Corradi
, E.E. Corrigan
,
F. Corriveau103,aa, M.J. Costa172, F. Costanza5, D. Costanzo148, G. Cowan93, J.W. Cowley32, J. Crane100,
K. Cranmer124, R.A. Creager135, S. Crépé-Renaudin58, F. Crescioli134, M. Cristinziani24, F. Corriveau103,aa, M.J. Costa172, F. Costanza5, D. Costanzo148, G. Cowan93, J.W. Cowley32, J. Crane100, F. Corriveau103,aa, M.J. Costa172, F. Costanza5, D. Costanzo148, G. Cowan93, J.W. Cowley32, J. Crane100,
K. Cranmer124, R.A. Creager135, S. Crépé-Renaudin58, F. Crescioli134, M. Cristinziani24,
M. Cristoforetti75a,75b, V. Croft168, G. Crosetti41b,41a, A. Cueto5, T. Cuhadar Donszelmann169,
H. Cui15a,15d, A.R. Cukierman152, W.R. Cunningham57, S. Czekierda84, P. Czodrowski36,
M.M. Czurylo61b, M.J. Da Cunha Sargedas De Sousa60b, J.V. Da Fonseca Pinto80b, C. Da Via100,
W. Dabrowski83a, F. Dachs36, T. Dado47, S. Dahbi33e, T. Dai105, C. Dallapiccola102, M. Dam40,
G. D’amen29, V. D’Amico74a,74b, J. Damp99, J.R. Dandoy135, M.F. Daneri30, M. Danninger151, V. Dao36,
G. Darbo55b, O. Dartsi5, A. Dattagupta130, S. D’Auria68a,68b, C. David166b, T. Davidek141, D.R. Davis49,
I Dawson148 K De8 R De Asmundis69a M De Beurs119 S De Castro23b,23a N De Groot118 ,
g
,
p
,
,
,
M. Cristoforetti75a,75b, V. Croft168, G. Crosetti41b,41a, A. Cueto5, T. Cuhadar Donszelmann169,
H. Cui15a,15d, A.R. Cukierman152, W.R. Cunningham57, S. Czekierda84, P. Czodrowski36,
M.M. Czurylo61b, M.J. Da Cunha Sargedas De Sousa60b, J.V. Da Fonseca Pinto80b, C. Da Via100, M. Cristoforetti75a,75b, V. Croft168, G. Crosetti41b,41a, A. Cueto5, T. Cuhadar Donszelmann169,
15a 15d
152
57
84
36 M.M. Czurylo61b, M.J. Da Cunha Sargedas De Sousa60b, J.V. Da Fonseca Pinto80b, C. Da Via100,
W. Dabrowski83a, F. Dachs36, T. Dado47, S. Dahbi33e, T. Dai105, C. Dallapiccola102, M. Dam40,
G. D’amen29, V. D’Amico74a,74b, J. Damp99, J.R. Dandoy135, M.F. Daneri30, M. Danninger151, V. Dao36,
G. Darbo55b, O. Dartsi5, A. Dattagupta130, S. D’Auria68a,68b, C. David166b, T. Davidek141, D.R. Davis49,
I. Dawson148, K. De8, R. De Asmundis69a, M. De Beurs119, S. De Castro23b,23a, N. De Groot118, – 49 – P. de Jong119, H. De la Torre106, A. De Maria15c, D. De Pedis72a, A. De Salvo72a, U. De Sanctis73a,7 g
M. De Santis73a,73b, A. De Santo155, J.B. De Vivie De Regie58, D.V. Dedovich79, A.M. Deiana42, g
J. Del Peso98, Y. Delabat Diaz46, D. Delgove64, F. Deliot143, C.M. Delitzsch7, M. The ATLAS collaboration Ferrari70a, y
g
D. Ferrere54, C. Ferretti105, F. Fiedler99, A. Filipčič91, F. Filthaut118, K.D. Finelli25, D. Ferrere54, C. Ferretti105, F. Fiedler99, A. Filipčič91, F. Filthaut118, K.D. Finelli25, M.C.N. Fiolhais138a,138c,a, L. Fiorini172, F. Fischer113, J. Fischer99, W.C. Fisher106, T. M.C.N. Fiolhais138a,138c,a, L. Fiorini172, F. Fischer113, J. Fischer99, W.C. Fisher106, T. Fitschen21, I. Fleck150, P. Fleischmann105, T. Flick180, B.M. Flierl113, L. Flores135, L.R. Flores Castillo62a, I. Fleck150, P. Fleischmann105, T. Flick180, B.M. Flierl113, L. Flores135, L.R. Flores Castillo62a, F.M. Follega75a,75b, N. Fomin17, J.H. Foo165, G.T. Forcolin75a,75b, B.C. Forland65, A. Formica143, A. Förster14, A.C. Forti100, E. Fortin101, M.G. Foti133, D. Fournier64, H. Fox89, P. Francavilla71a,71b, Francescato72a,72b, M. Franchini23b,23a, S. Franchino61a, D. Francis36, L. Franco5, L. Franconi20, Franklin59, G. Frattari72a,72b, P.M. Freeman21, B. Freund109, W.S. Freund80b, E.M. Freundlich47, M. Franklin
, G. Frattari
, P.M. Freeman
, B. Freund
, W.S. Freund
, E.M. Freundlich
,
D.C. Frizzell127, D. Froidevaux36, J.A. Frost133, M. Fujimoto125, E. Fullana Torregrosa172, T. Fusayasu115
J. Fuster172, A. Gabrielli23b,23a, A. Gabrielli36, P. Gadow114, G. Gagliardi55b,55a, L.G. Gagnon109,
G.E. Gallardo133, E.J. Gallas133, B.J. Gallop142, R. Gamboa Goni92, K.K. Gan126, S. Ganguly178,
J. Gao60a, Y. Gao50, Y.S. Gao31,m, F.M. Garay Walls145a, C. García172, J.E. García Navarro172,
J.A. García Pascual15a, M. Garcia-Sciveres18, R.W. Gardner37, S. Gargiulo52, C.A. Garner165,
V. Garonne132, S.J. Gasiorowski147, P. Gaspar80b, G. Gaudio70a, P. Gauzzi72a,72b, I.L. Gavrilenko110,
A. Gavrilyuk123, C. Gay173, G. Gaycken46, E.N. Gazis10, A.A. Geanta27b, C.M. Gee144, C.N.P. Gee142,
J. Geisen96, M. Geisen99, C. Gemme55b, M.H. Genest58, C. Geng105, S. Gentile72a,72b, S. George93,
T. Geralis44, L.O. Gerlach53, P. Gessinger-Befurt99, G. Gessner47, M. Ghasemi Bostanabad174,
M. Ghneimat150, A. Ghosh64, A. Ghosh77, B. Giacobbe23b, S. Giagu72a,72b, N. Giangiacomi165,
P. Giannetti71a, A. Giannini69a,69b, G. Giannini14, S.M. Gibson93, M. Gignac144, D.T. Gil83b,
B.J. Gilbert39, D. Gillberg34, G. Gilles180, N.E.K. Gillwald46, D.M. Gingrich3,ak, M.P. Giordani66a,66c, ,
,
,
,
,
,
D.C. Frizzell127, D. Froidevaux36, J.A. Frost133, M. Fujimoto125, E. Fullana Torregrosa172, T. Fusayasu115,
J. Fuster172, A. Gabrielli23b,23a, A. Gabrielli36, P. Gadow114, G. Gagliardi55b,55a, L.G. Gagnon109, D.C. Frizzell127, D. Froidevaux36, J.A. Frost133, M. Fujimoto125, E. Fullana Torregrosa172, T. Fusayasu115,
J. Fuster172, A. Gabrielli23b,23a, A. Gabrielli36, P. Gadow114, G. Gagliardi55b,55a, L.G. Gagnon109,
G.E. Gallardo133, E.J. Gallas133, B.J. Gallop142, R. Gamboa Goni92, K.K. Gan126, S. Ganguly178,
J. Gao60a, Y. Gao50, Y.S. Gao31,m, F.M. Garay Walls145a, C. García172, J.E. García Navarro172,
J.A. García Pascual15a, M. Garcia-Sciveres18, R.W. Gardner37, S. Gargiulo52, C.A. Garner165,
V. Garonne132, S.J. Gasiorowski147, P. Gaspar80b, G. Gaudio70a, P. Gauzzi72a,72b, I.L. Gavrilenko110,
A. The ATLAS collaboration Gavrilyuk123, C. Gay173, G. Gaycken46, E.N. Gazis10, A.A. Geanta27b, C.M. Gee144, C.N.P. Gee142,
J. Geisen96, M. Geisen99, C. Gemme55b, M.H. Genest58, C. Geng105, S. Gentile72a,72b, S. George93,
T. Geralis44, L.O. Gerlach53, P. Gessinger-Befurt99, G. Gessner47, M. Ghasemi Bostanabad174,
M. Ghneimat150, A. Ghosh64, A. Ghosh77, B. Giacobbe23b, S. Giagu72a,72b, N. Giangiacomi165,
P. Giannetti71a, A. Giannini69a,69b, G. Giannini14, S.M. Gibson93, M. Gignac144, D.T. Gil83b,
B.J. Gilbert39, D. Gillberg34, G. Gilles180, N.E.K. Gillwald46, D.M. Gingrich3,ak, M.P. Giordani66a,66c, G.E. Gallardo133, E.J. Gallas133, B.J. Gallop142, R. Gamboa Goni92, K.K. Gan126, S. Ganguly178,
J. Gao60a, Y. Gao50, Y.S. Gao31,m, F.M. Garay Walls145a, C. García172, J.E. García Navarro172,
J.A. García Pascual15a, M. Garcia-Sciveres18, R.W. Gardner37, S. Gargiulo52, C.A. Garner165,
V. Garonne132, S.J. Gasiorowski147, P. Gaspar80b, G. Gaudio70a, P. Gauzzi72a,72b, I.L. Gavrilenko110,
A. Gavrilyuk123, C. Gay173, G. Gaycken46, E.N. Gazis10, A.A. Geanta27b, C.M. Gee144, C.N.P. Gee142,
J. Geisen96, M. Geisen99, C. Gemme55b, M.H. Genest58, C. Geng105, S. Gentile72a,72b, S. George93,
T. Geralis44, L.O. Gerlach53, P. Gessinger-Befurt99, G. Gessner47, M. Ghasemi Bostanabad174,
M. Ghneimat150, A. Ghosh64, A. Ghosh77, B. Giacobbe23b, S. Giagu72a,72b, N. Giangiacomi165, G.E. Gallardo
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J.A. García Pascual15a, M. Garcia-Sciveres18, R.W. Gardner37, S. Gargiulo52, C.A. Garner165,
V. Garonne132, S.J. Gasiorowski147, P. Gaspar80b, G. Gaudio70a, P. Gauzzi72a,72b, I.L. Gavrilenko110, J.A. García Pascual
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V. Garonne132, S.J. Gasiorowski147, P. Gaspar80b, G. Gaudio70a, P. Gauzzi72a,72b, I.L. Gavrilenko110,
A. Gavrilyuk123, C. Gay173, G. Gaycken46, E.N. Gazis10, A.A. Geanta27b, C.M. Gee144, C.N.P. Gee142,
J. Geisen96, M. Geisen99, C. Gemme55b, M.H. Genest58, C. Geng105, S. Gentile72a,72b, S. George93,
T. Geralis44, L.O. Gerlach53, P. Gessinger-Befurt99, G. Gessner47, M. Ghasemi Bostanabad174,
M. Ghneimat150, A. Ghosh64, A. Ghosh77, B. Giacobbe23b, S. Giagu72a,72b, N. Giangiacomi165,
P. Giannetti71a, A. Giannini69a,69b, G. Giannini14, S.M. Gibson93, M. Gignac144, D.T. Gil83b,
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D.M. Mac Donell174, G. Maccarrone51, C.M. Macdonald148, J.C. MacDonald148, J. Machado Miguens135,
R. Madar38, W.F. Mader48, M. Madugoda Ralalage Don128, N. Madysa48, J. Maeda82, T. Maeno29,
M. Maerker48, V. Magerl52, J. Magro66a,66c,r, D.J. Mahon39, C. Maidantchik80b, A. Maio138a,138b,138d,
K. Maj83a, O. Majersky28a, S. Majewski130, N. Makovec64, B. Malaescu134, Pa. Malecki84, V.P. Maleev136,
F. Malek58, D. Malito41b,41a, U. Mallik77, C. Malone32, S. Maltezos10, S. Malyukov79, J. Mamuzic172,
G. Mancini51, J.P. Mandalia92, I. Mandić91, L. Manhaes de Andrade Filho80a, I.M. Maniatis161,
J. Manjarres Ramos48, K.H. Mankinen96, A. Mann113, A. Manousos76, B. Mansoulie143, I. Manthos161,
S. Manzoni119, A. Marantis161, L. Marchese133, G. Marchiori134, M. Marcisovsky139, L. Marcoccia73a,73b,
C. Marcon96, M. Marjanovic127, Z. Marshall18, M.U.F. Martensson170, S. Marti-Garcia172, T.A. Martin176,
V.J. Martin50, B. Martin dit Latour17, L. Martinelli74a,74b, M. Martinez14,w, P. Martinez Agullo172,
V.I. Martinez Outschoorn102, S. Martin-Haugh142, V.S. Martoiu27b, A.C. Martyniuk94, A. Marzin36,
S.R. Maschek114, L. Masetti99, T. Mashimo162, R. Mashinistov110, J. Masik100, A.L. Maslennikov121b,121a,
L Massa23b,23a P Massarotti69a,69b P Mastrandrea71a,71b A Mastroberardino41b,41a T Masubuchi162 g
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F. Malek58, D. Malito41b,41a, U. Mallik77, C. Malone32, S. Maltezos10, S. Malyukov79, J. Mamuzic172,
G. Mancini51, J.P. Mandalia92, I. Mandić91, L. Manhaes de Andrade Filho80a, I.M. Maniatis161,
J. Manjarres Ramos48, K.H. Mankinen96, A. Mann113, A. Manousos76, B. Mansoulie143, I. Manthos161,
S. Manzoni119, A. Marantis161, L. Marchese133, G. Marchiori134, M. Marcisovsky139, L. Marcoccia73a,73b,
C. Marcon96, M. Marjanovic127, Z. Marshall18, M.U.F. Martensson170, S. Marti-Garcia172, T.A. Martin176,
V.J. Martin50, B. Martin dit Latour17, L. Martinelli74a,74b, M. Martinez14,w, P. Martinez Agullo172,
V.I. Martinez Outschoorn102, S. Martin-Haugh142, V.S. Martoiu27b, A.C. Martyniuk94, A. Marzin36,
S.R. Maschek114, L. Masetti99, T. Mashimo162, R. Mashinistov110, J. Masik100, A.L. Maslennikov121b,121a, 2021 ,
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K.J.C. Leney42, T. Lenz24, S. Leone71a, C. Leonidopoulos50, A. Leopold134, C. Leroy109, R. Les106,
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B.P. Nachman18, O. Nackenhorst47, A.Nag Nag48, K. Nagai133, K. Nagano81, J.L. Nagle29, E. Nagy101,
A.M. Nairz36, Y. Nakahama116, K. Nakamura81, H. Nanjo131, F. Napolitano61a, R.F. Naranjo Garcia46,
R. Narayan42, I. Naryshkin136, M. Naseri34, T. Naumann46, G. Navarro22a, J. Navarro-Gonzalez172,
P.Y. Nechaeva110, F. Nechansky46, T.J. Neep21, A. Negri70a,70b, M. Negrini23b, C. Nellist118, C. Nelson103,
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F. Prokoshin79, S. Protopopescu29, J. Proudfoot6, M. Przybycien83a, D. Pudzha136, A. Puri171, P. Puzo64,
D. Pyatiizbyantseva111, J. Qian105, Y. Qin100, A. Quadt53, M. Queitsch-Maitland36, G. Rabanal Bolanos59,
M. Racko28a, F. Ragusa68a,68b, G. Rahal97, J.A. Raine54, S. Rajagopalan29, K. Ran15a,15d, D.F. Rassloff61a,
D.M. Rauch46, S. Rave99, B. Ravina57, I. Ravinovich178, M. Raymond36, A.L. Read132, N.P. Readioff148,
M. Reale67a,67b, D.M. Rebuzzi70a,70b, G. Redlinger29, K. Reeves43, D. Reikher160, A. Reiss99, A. Rej150,
C. Rembser36, A. Renardi46, M. Renda27b, M.B. Rendel114, A.G. Rennie57, S. Resconi68a,
E.D. Resseguie18, S. Rettie94, B. Reynolds126, E. Reynolds21, O.L. Rezanova121b,121a, P. Reznicek141,
E. Ricci75a,75b, R. Richter114, S. Richter46, E. Richter-Was83b, M. Ridel134, P. Rieck114, O. Rifki46,
M. Rijssenbeek154, A. Rimoldi70a,70b, M. Rimoldi46, L. Rinaldi23b, T.T. Rinn171, G. Ripellino153, I. Riu14,
P. Rivadeneira46, J.C. Rivera Vergara174, F. Rizatdinova128, E. Rizvi92, C. Rizzi36, S.H. Robertson103,aa,
M. Robin46, D. Robinson32, C.M. Robles Gajardo145d, M. Robles Manzano99, A. Robson57, M.M. Prapa44, S. Prell78, D. Price100, M. Primavera67a, M.L. Proffitt147, N. Proklova111, K. Prokofiev62c,
F. Prokoshin79, S. Protopopescu29, J. Proudfoot6, M. Przybycien83a, D. Pudzha136, A. Puri171, P. Puzo64,
D. Pyatiizbyantseva111, J. Qian105, Y. Qin100, A. Quadt53, M. Queitsch-Maitland36, G. Rabanal Bolanos59,
M. Racko28a, F. Ragusa68a,68b, G. Rahal97, J.A. Raine54, S. Rajagopalan29, K. Ran15a,15d, D.F. Rassloff61a,
D.M. Rauch46, S. Rave99, B. Ravina57, I. Ravinovich178, M. Raymond36, A.L. Read132, N.P. Readioff148,
M. Reale67a,67b, D.M. Rebuzzi70a,70b, G. Redlinger29, K. Reeves43, D. Reikher160, A. Reiss99, A. Rej150,
C. Rembser36, A. Renardi46, M. Renda27b, M.B. Rendel114, A.G. Rennie57, S. Resconi68a,
E.D. Resseguie18, S. Rettie94, B. The ATLAS collaboration Oh100, C.C. Ohm153, H. Oide163, R. Oishi162, M.L. Ojeda165, Y. Okazaki85, A. Ogrodnik83a, A. Oh100, C.C. Ohm153, H. Oide163, R. Oishi162, M.L. Ojeda165, Y. Okazaki85, D. Oliveira Damazio29, J.L. Oliver1, M.J.R. Olsson169, A. Olszewski84, J. Olszowska84, Ö.O. Öncel24, D.C. O’Neil151, A.P. O’neill133, A. Onofre138a,138e, P.U.E. Onyisi11, H. Oppen132, 2021 R.G. Oreamuno Madriz120, M.J. Oreglia37, G.E. Orellana88, D. Orestano74a,74 R.G. Oreamuno Madriz120, M.J. Oreglia37, G.E. Orellana88, D. Orestano74a,74b, N. Orlando14, R.S. Orr165, O’Shea57, R. Ospanov60a, G. Otero y Garzon30, H. Otono87, P.S. Ott61a, G.J. Ottino18, M. Ouchrif35e, p
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S.V. Peleganchuk121b,121a, O. Penc139, C. Peng62b, H. Peng60a, B.S. Peralva80a, M.M. Perego64, y
S.V. Peleganchuk121b,121a, O. Penc139, C. Peng62b, H. Peng60a, B.S. Peralva80a, M.M. Perego64, p
L. Perini68a,68b, H. Pernegger36, S. Perrella36, A. Perrevoort119, K. Peters46, R.F.Y. Peters100, L. Perini68a,68b, H. Pernegger36, S. Perrella36, A. Perrevoort119, K. Peters46, R.F.Y. Peters100, F. Petrucci74a,74b, M. Pettee181, N.E. Pettersson102, K. Petukhova141, A. Peyaud143, R. Pezoa145d, F. Petrucci74a,74b, M. Pettee181, N.E. Pettersson102, K. Petukhova141, A. Peyaud143, R. Pezoa145d, y
L. Pezzotti70a,70b, G. Pezzullo181, T. Pham104, P.W. Phillips142, M.W. Phipps171, G. Piacquadio15 E. Pianori18, A. Picazio102, R. Piegaia30, D. Pietreanu27b, J.E. Pilcher37, A.D. Pilkington100, nori18, A. Picazio102, R. Piegaia30, D. Pietreanu27b, J.E. Pilcher37, A.D. Pilkington100, M. The ATLAS collaboration Salt172,
A. Salvador Salas14, D. Salvatore41b,41a, F. Salvatore155, A. Salzburger36, D. Sammel52,
D. Sampsonidis161, D. Sampsonidou60d,60c, J. Sánchez172, A. Sanchez Pineda66a,36,66c, H. Sandaker132,
C.O. Sander46, I.G. Sanderswood89, M. Sandhoff180, C. Sandoval22b, D.P.C. Sankey142, M. Sannino55b,55a,
Y. Sano116, A. Sansoni51, C. Santoni38, R. Santonico73b, H. Santos138a,138b, S.N. Santpur18, A. Santra178,
K.A. Saoucha148, A. Sapronov79, J.G. Saraiva138a,138d, O. Sasaki81, K. Sato167, F. Sauerburger52,
E. Sauvan5, P. Savard165,ak, R. Sawada162, C. Sawyer142, L. Sawyer95, I. Sayago Galvan172, C. Sbarra23b,
A. Sbrizzi66a,66c, T. Scanlon94, J. Schaarschmidt147, P. Schacht114, D. Schaefer37, L. Schaefer135,
U. Schäfer99, A.C. Schaffer64, D. Schaile113, R.D. Schamberger154, E. Schanet113, C. Scharf19, g
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L. Roos134, S. Rosati72a, G. Rosin102, B.J. Rosser135, E. Rossi46, E. Rossi74a,74b, E. Rossi69a,69b, L.P. Rossi55b, L. Rossini46, R. Rosten126, M. Rotaru27b, B. Rottler52, D. Rousseau64, G. Rovelli70a,70b, A. Roy11, A. Rozanov101, Y. Rozen159, X. Ruan33e, A.J. Ruby90, T.A. Ruggeri1, F. Rühr52, JINST 16 P07029 ,
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U. Schäfer99, A.C. Schaffer64, D. Schaile113, R.D. Schamberger154, E. Schanet113, C. Scharf19, N. Scharmberg100, V.A. Schegelsky136, D. Scheirich141, F. Schenck19, M. Schernau169, C. Schiavi55b,55 L.K. Schildgen24, Z.M. Schillaci26, E.J. Schioppa67a,67b, M. Schioppa41b,41a, K.E. Schleicher52, g
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S. Schlenker36, K.R. Schmidt-Sommerfeld114, K. Schmieden99, C. Schmitt99, S. Schmitt46, L. Schoeffel143, A. Schoening61b, P.G. Scholer52, E. Schopf133, M. Schott99, J.F.P. Schouwenberg118 g
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J. Schovancova36, S. Schramm54, F. Schroeder180, A. Schulte99, H-C. Schultz-Coulon61a, M. Schumacher52, B.A. Schumm144, Ph. Schune143, A. Schwartzman152, T.A. Schwarz105, Ph. Schwemling143, R. Schwienhorst106, A. Sciandra144, G. Sciolla26, F. Scuri71a, F. Scutti104, L.M. Scyboz114, C.D. Sebastiani90, K. Sedlaczek47, P. Seema19, S.C. Seidel117, A. Seiden144, B.D. Seidlitz29, T. Seiss37, C. Seitz46, J.M. Seixas80b, G. Sekhniaidze69a, S.J. Sekula42, B.D. Seidlitz29, T. Seiss37, C. Seitz46, J.M. Seixas80b, G. Sekhniaidze69a, S.J. Sekula42, N. Semprini-Cesari23b,23a, S. Sen49, C. Serfon29, L. Serin64, L. Serkin66a,66b, M. Sessa60 N. Semprini-Cesari23b,23a, S. Sen49, C. Serfon29, L. Serin64, L. Serkin66a,66b, M. Sessa60a, H. Sever S. Sevova152, F. Sforza55b,55a, A. Sfyrla54, E. Shabalina53, J.D. Shahinian135, N.W. Shaikh45a,45b, D. Shaked Renous178, L.Y. Shan15a, M. Shapiro18, A. Sharma36, A.S. Sharma1, P.B. Shatalov123, Shaw155, S.M. Shaw100, M. Shehade178, Y. Shen127, P. Sherwood94, L. Shi94, C.O. Shimmin181, Shimogama177, M. Shimojima115, J.D. Shinner93, I.P.J. Shipsey133, S. Shirabe163, M. Shiyakova79,y, g
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E. Ricci75a,75b, R. Richter114, S. Richter46, E. Richter-Was83b, M. Ridel134, P. Rieck114, O. Rifki46,
M. Rijssenbeek154, A. Rimoldi70a,70b, M. Rimoldi46, L. Rinaldi23b, T.T. Rinn171, G. Ripellino153, I. Riu14,
P. Rivadeneira46, J.C. Rivera Vergara174, F. Rizatdinova128, E. Rizvi92, C. Rizzi36, S.H. Robertson103,aa,
M. Robin46, D. Robinson32, C.M. Robles Gajardo145d, M. Robles Manzano99, A. Robson57, M.M. Prapa44, S. Prell78, D. Price100, M. Primavera67a, M.L. Proffitt147, N. Proklova111, K. Prokofiev62c,
F. Prokoshin79, S. Protopopescu29, J. Proudfoot6, M. Przybycien83a, D. Pudzha136, A. Puri171, P. Puzo64,
D. Pyatiizbyantseva111, J. Qian105, Y. Qin100, A. Quadt53, M. Queitsch-Maitland36, G. Rabanal Bolanos59,
M. Racko28a, F. Ragusa68a,68b, G. Rahal97, J.A. Raine54, S. Rajagopalan29, K. Ran15a,15d, D.F. Rassloff61a,
D.M. Rauch46, S. Rave99, B. Ravina57, I. Ravinovich178, M. Raymond36, A.L. Read132, N.P. Readioff148,
M. Reale67a,67b, D.M. Rebuzzi70a,70b, G. Redlinger29, K. Reeves43, D. Reikher160, A. Reiss99, A. Rej150,
C. Rembser36, A. Renardi46, M. Renda27b, M.B. Rendel114, A.G. Rennie57, S. Resconi68a,
E.D. Resseguie18, S. Rettie94, B. Reynolds126, E. Reynolds21, O.L. Rezanova121b,121a, P. Reznicek141,
E. Ricci75a,75b, R. Richter114, S. Richter46, E. Richter-Was83b, M. Ridel134, P. Rieck114, O. Rifki46,
M. Rijssenbeek154, A. Rimoldi70a,70b, M. Rimoldi46, L. Rinaldi23b, T.T. Rinn171, G. Ripellino153, I. Riu14,
P. Rivadeneira46, J.C. Rivera Vergara174, F. Rizatdinova128, E. Rizvi92, C. Rizzi36, S.H. Robertson103,aa,
M. Robin46, D. Robinson32, C.M. Robles Gajardo145d, M. Robles Manzano99, A. Robson57, – 54 – A. Rocchi73a,73b, C. Roda71a,71b, S. Rodriguez Bosca172, A. Rodriguez Rodriguez52, A. Rocchi73a,73b, C. Roda71a,71b, S. Rodriguez Bosca172, A. Rodriguez Rodriguez52, ,
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C.P.A. Roland65, J. Roloff29, A. Romaniouk111, M. Romano23b,23a, N. Rompotis90, M. Ronzani124,
L. Roos134, S. Rosati72a, G. Rosin102, B.J. Rosser135, E. Rossi46, E. Rossi74a,74b, E. Rossi69a,69b,
L.P. Rossi55b, L. Rossini46, R. Rosten126, M. Rotaru27b, B. Rottler52, D. Rousseau64, G. Rovelli70a,70b,
A. Roy11, A. Rozanov101, Y. Rozen159, X. Ruan33e, A.J. Ruby90, T.A. Ruggeri1, F. Rühr52,
A. Ruiz-Martinez172, A. Rummler36, Z. Rurikova52, N.A. Rusakovich79, H.L. Russell103, L. Rustige38,47,
J.P. Rutherfoord7, E.M. Rüttinger148, M. Rybar141, E.B. Rye132, A. Ryzhov122, J.A. Sabater Iglesias46,
P. Sabatini172, L. Sabetta72a,72b, S. Sacerdoti64, H.F-W. Sadrozinski144, R. Sadykov79, F. Safai Tehrani72a,
B. Safarzadeh Samani155, M. Safdari152, P. Saha120, S. Saha103, M. Sahinsoy114, A. Sahu180,
M. Saimpert36, M. Saito162, T. Saito162, D. Salamani54, G. Salamanna74a,74b, A. Salnikov152, J. The ATLAS collaboration Sidoti23b,23a, g
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F. Siegert48, Dj. Sijacki16, M.V. Silva Oliveira36, S.B. Silverstein45a, S. Simion64, R. Simoniello99, F. Siegert48, Dj. Sijacki16, M.V. Silva Oliveira36, S.B. Silverstein45a, S. Simion64, R. Simoniello99,
C.J. Simpson-allsop21, S. Simsek12b, P. Sinervo165, V. Sinetckii112, S. Singh151, S. Sinha33e,
M. Sioli23b,23a, I. Siral130, S.Yu. Sivoklokov112, J. Sjölin45a,45b, A. Skaf53, E. Skorda96, P. Skubic127,
M. Slawinska84, K. Sliwa168, V. Smakhtin178, B.H. Smart142, J. Smiesko28b, N. Smirnov111,
S.Yu. Smirnov111, Y. Smirnov111, L.N. Smirnova112,s, O. Smirnova96, E.A. Smith37, H.A. Smith133,
M. Smizanska89, K. Smolek140, A. Smykiewicz84, A.A. Snesarev110, H.L. Snoek119, I.M. Snyder130,
S. Snyder29, R. Sobie174,aa, A. Soffer160, A. Søgaard50, F. Sohns53, C.A. Solans Sanchez36,
E.Yu. Soldatov111, U. Soldevila172, A.A. Solodkov122, A. Soloshenko79, O.V. Solovyanov122,
V. Solovyev136, P. Sommer148, H. Son168, A. Sonay14, W.Y. Song166b, A. Sopczak140, A.L. Sopio94,
F. Sopkova28b, S. Sottocornola70a,70b, R. Soualah66a,66c, A.M. Soukharev121b,121a, D. South46,
S. Spagnolo67a,67b, M. Spalla114, M. Spangenberg176, F. Spanò93, D. Sperlich52, T.M. Spieker61a,
G. Spigo36, M. Spina155, D.P. Spiteri57, M. Spousta141, A. Stabile68a,68b, B.L. Stamas120, R. Stamen61a,
M. Stamenkovic119, A. Stampekis21, E. Stanecka84, B. Stanislaus133, M.M. Stanitzki46, M. Stankaityte133, – 55 – B. Stapf119, E.A. Starchenko122, G.H. Stark144, J. Stark58, P. Staroba139, P. Starovoitov61a, S. Stärz103 p
R. Staszewski84, G. Stavropoulos44, P. Steinberg29, A.L. Steinhebel130, B. Stelzer151,166a, H.J. Stelzer p
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X. Sun100, C.J.E. Suster156, M.R. Sutton155, M. Svatos139, M. Swiatlowski166a, S.P. Swift2, T. Swirski175 ,
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A. Sydorenko99, I. Sykora28a, M. Sykora141, T. Sykora141, D. Ta99, K. Tackmann46,x, J. Taenzer160, y
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A. Taffard169, R. Tafirout166a, E. Tagiev122, R.H.M. Taibah134, R. Takashima86, K. Takeda82, T. Takeshita149, E.P. Takeva50, Y. Takubo81, M. Talby101, A.A. Talyshev121b,121a, K.C. Tam62b 2021 N.M. Tamir160, J. Tanaka162, R. Tanaka64, S. Tapia Araya171, S. Tapprogge99, A. Tarek Abouelfadl Mohamed106, S. Tarem159, K. Tariq60b, G. Tarna27b,e, G.F. Tartarelli68a, P. Tas141,
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M. Tasevsky139, E. Tassi41b,41a, G. Tateno162, Y. Tayalati35f, G.N. Taylor104, W. The ATLAS collaboration Taylor166b, H. Teagle90, A.S. Tee89, R. Teixeira De Lima152, P. Teixeira-Dias93, H. Ten Kate36, J.J. Teoh119, K. Terashi162, J. Terron98, S. Terzo14, M. Testa51, R.J. Teuscher165,aa, N. Themistokleous50, T. Theveneaux-Pelzer19, D.W. Thomas93, J.P. Thomas21, E.A. Thompson46, P.D. Thompson21, E. Thomson135, E.J. Thorpe92, JINST 16 P07029 V.O. Tikhomirov110,ag, Yu.A. Tikhonov121b,121a, S. Timoshenko111, P. Tipton181, S. Tisserant101, ,
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R. Tombs32, M. Tomoto81,116, L. Tompkins152, P. Tornambe102, E. Torrence130, H. Torres48, Torró Pastor172, M. Toscani30, C. Tosciri37, J. Toth101,z, D.R. Tovey148, A. Traeet17, C.J. Treado124, E. Torró Pastor172, M. Toscani30, C. Tosciri37, J. Toth101,z, D.R. Tovey148, A. Traeet17, C.J. Treado124, Trefzger175, F. Tresoldi155, A. Tricoli29, I.M. Trigger166a, S. Trincaz-Duvoid134, D.A. Trischuk173, W. Trischuk165, B. Trocmé58, A. Trofymov64, C. Troncon68a, F. Trovato155, L. Truong33c, M. Trzebinski84 W. Trischuk165, B. Trocmé58, A. Trofymov64, C. Troncon68a, F. Trovato155, L. Truong33c, M. Trzebinski84, M. Tsopoulou161, I.I. Tsukerman123, V. Tsulaia18, S. Tsuno81, D. Tsybychev154, Y. Tu62b, A. Tudorache27b, M. Tsopoulou161, I.I. Tsukerman123, V. Tsulaia18, S. Tsuno81, D. Tsybychev154, Y. Tu62b, A. Tudorache27b, R. Turra68a, P.M. Tuts39, S. Tzamarias161, E. Tzovara99, K. Uchida162, F. Ukegawa167, G. Unal36, Unal11, A. Undrus29, G. Unel169, F.C. Ungaro104, K. Uno162, J. Urban28b, P. Urquijo104, G. Usai8, Uysal12d, V. Vacek140, B. Vachon103, K.O.H. Vadla132, T. Vafeiadis36, A. Vaidya94, C. Valderanis113, Valdes Santurio45a,45b, M. Valente166a, S. Valentinetti23b,23a, A. Valero172, L. Valéry46, A. Vallance21, A. Vallier36, J.A. Valls Ferrer172, T.R. Van Daalen14, P. Van Gemmeren6, Van Stroud94, I. Van Vulpen119, M. Vanadia73a,73b, W. Vandelli36, M. Vandenbroucke143, R. Vandewall128, D. Vannicola72a,72b, R. Vari72a, E.W. Varnes7, C. Varni55b,55a, T. Varol157, – 56 – C. Weber29, M.S. Weber20, S.A. Weber34, S.M. Weber61a, Y. Wei133, A.R. Weidberg133, J. Weingarten47,
M. Weirich99, C. Weiser52, P.S. Wells36, T. Wenaus29, B. Wendland47, T. Wengler36, S. Wenig36,
N. Wermes24, M. Wessels61a, T.D. Weston20, K. Whalen130, A.M. Wharton89, A.S. White105, A. White8,
M.J. White1, D. Whiteson169, B.W. Whitmore89, W. Wiedenmann179, C. Wiel48, M. Wielers142,
N. Wieseotte99, C. Wiglesworth40, L.A.M. Wiik-Fuchs52, H.G. Wilkens36, L.J. Wilkins93,
D.M. Williams39, H.H. Williams135, S. Williams32, S. Willocq102, P.J. Windischhofer133,
I. Wingerter-Seez5, E. Winkels155, F. Winklmeier130, B.T. Winter52, M. Wittgen152, M. Wobisch95,
A. Wolf99, R. Wölker133, J. Wollrath52, M.W. Wolter84, H. Wolters138a,138c, V.W.S. Wong173,
A.F. Wongel46, N.L. Woods144, S.D. Worm46, B.K. Wosiek84, K.W. Woźniak84, K. Wraight57, S.L. Wu179,
X. Wu54, Y. Wu60a, J. Wuerzinger133, T.R. Wyatt100, B.M. Wynne50, S. Xella40, J. The ATLAS collaboration Xiang62c, X. Xiao105,
X. Xie60a, I. Xiotidis155, D. Xu15a, H. Xu60a, H. Xu60a, L. Xu29, R. Xu135, T. Xu143, W. Xu105, Y. Xu15b,
Z. Xu60b, Z. Xu152, B. Yabsley156, S. Yacoob33a, D.P. Yallup94, N. Yamaguchi87, Y. Yamaguchi163,
M. Yamatani162, H. Yamauchi167, T. Yamazaki18, Y. Yamazaki82, J. Yan60c, Z. Yan25, H.J. Yang60c,60d,
H.T. Yang18, S. Yang60a, T. Yang62c, X. Yang60a, X. Yang15a, Y. Yang162, Z. Yang105,60a, W-M. Yao18,
Y.C. Yap46, H. Ye15c, J. Ye42, S. Ye29, I. Yeletskikh79, M.R. Yexley89, P. Yin39, K. Yorita177,
K. Yoshihara78, C.J.S. Young36, C. Young152, R. Yuan60b,i, X. Yue61a, M. Zaazoua35f, B. Zabinski84,
G. Zacharis10, E. Zaffaroni54, J. Zahreddine134, A.M. Zaitsev122,af, T. Zakareishvili158b, N. Zakharchuk34,
S. Zambito36, D. Zanzi52, S.V. Zeißner47, C. Zeitnitz180, G. Zemaityte133, J.C. Zeng171, O. Zenin122,
T. Ženiš28a, S. Zenz92, S. Zerradi35a, D. Zerwas64, M. Zgubič133, B. Zhang15c, D.F. Zhang15b,
G. Zhang15b, J. Zhang6, K. Zhang15a, L. Zhang15c, L. Zhang60a, M. Zhang171, R. Zhang179, S. Zhang105,
X. Zhang60c, X. Zhang60b, Y. Zhang15a,15d, Z. Zhang64, P. Zhao49, Y. Zhao144, Z. Zhao60a,
A. Zhemchugov79, Z. Zheng105, D. Zhong171, B. Zhou105, C. Zhou179, H. Zhou7, M. Zhou154, N. Zhou60c,
Y. Zhou7, C.G. Zhu60b, C. Zhu15a,15d, H.L. Zhu60a, H. Zhu15a, J. Zhu105, Y. Zhu60a, X. Zhuang15a,
K. Zhukov110, V. Zhulanov121b,121a, D. Zieminska65, N.I. Zimine79, S. Zimmermann52,*, Z. Zinonos114,
M. Ziolkowski150, L. Živković16, A. Zoccoli23b,23a, K. Zoch53, T.G. Zorbas148, R. Zou37, L. Zwalinski36. 2021 JINST 16 P07029 A. Zhemchugov79, Z. Zheng105, D. Zhong171, B. Zhou105, C. Zhou179, H. Zhou7, M. Zhou154, N. Zhou60c,
Y. Zhou7, C.G. Zhu60b, C. Zhu15a,15d, H.L. Zhu60a, H. Zhu15a, J. Zhu105, Y. Zhu60a, X. Zhuang15a,
K. Zhukov110, V. Zhulanov121b,121a, D. Zieminska65, N.I. Zimine79, S. Zimmermann52,*, Z. Zinonos114,
M. Ziolkowski150, L. Živković16, A. Zoccoli23b,23a, K. Zoch53, T.G. Zorbas148, R. Zou37, L. Zwalinski36. 18 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United
States of America The ATLAS collaboration 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany NY, United States of America
3 3 Department of Physics, University of Alberta, Edmonton AB, Canada 4 Department of Physics(𝑎), Ankara University, Ankara; Istanbul Aydin University(𝑏), Application and Research
Center for Advanced Studies, Istanbul; Division of Physics(𝑐), TOBB University of Economics and Technology,
Ankara, Turkey 5 LAPP, Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS/IN2P3, Annecy, France 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States of America 7 Department of Physics, University of Arizona, Tucson AZ, United States of America 8 Department of Physics, University of Texas at Arlington, Arlington TX, United States of America 9 Physics Department, National and Kapodistrian University of Athens, Athens, Greece 11 Department of Physics, University of Texas at Austin, Austin TX, United State 12 Bahcesehir University(𝑎), Faculty of Engineering and Natural Sciences, Istanbul; Istanbul Bilgi University(𝑏),
Faculty of Engineering and Natural Sciences, Istanbul; Department of Physics(𝑐), Bogazici University, 12 Bahcesehir University(𝑎), Faculty of Engineering and Natural Sciences, Istanbul; Istanbul Bilgi University(𝑏),
Faculty of Engineering and Natural Sciences, Istanbul; Department of Physics(𝑐), Bogazici University,
Istanbul; Department of Physics Engineering(𝑑), Gaziantep University, Gaziantep, Turkey Istanbul; Department of Physics Engineering(𝑑), Gaziantep University, Gaziantep, Turkey
13 13 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 13 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 14 Institut de Física d’Altes Energies (IFAE), Barcelona Institute of Science and Technology, Barcelona, Spain g
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15 Institute of High Energy Physics(𝑎), Chinese Academy of Sciences, Beijing; Physics Department(𝑏), Tsinghua
University, Beijing; Department of Physics(𝑐), Nanjing University, Nanjing; University of Chinese Academy of
Science (UCAS)(𝑑), Beijing, China 15 Institute of High Energy Physics(𝑎), Chinese Academy of Sciences, Beijing; Physics Department(𝑏), Tsinghua
University, Beijing; Department of Physics(𝑐), Nanjing University, Nanjing; University of Chinese Academy of
Science (UCAS)(𝑑), Beijing, China 16 Institute of Physics, University of Belgrade, Belgrade, Serbia 16 Institute of Physics, University of Belgrade, Belgrade, Serbia 16 Institute of Physics, University of Belgrade, Belgrade, Serbia 17 Department for Physics and Technology, University of Bergen, Bergen, Norway 17 Department for Physics and Technology, University of Bergen, Bergen, Norway 18 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United
States of America 18 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United
States of America – 57 – 19 Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany 20 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern,
Bern, Switzerland 21 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdo 22 Facultad de Ciencias y Centro de Investigaciónes(𝑎), Universidad Antonio Nariño, Bogotá; Departamento de
Física(𝑏), Universidad Nacional de Colombia, Bogotá, Colombia, Colombia 23 INFN Bologna and Universita’ di Bologna(𝑎), Dipartimento di Fisica; INFN Sezione di Bologna(𝑏), Italy 24 Physikalisches Institut, Universität Bonn, Bonn, Germany 24 Physikalisches Institut, Universität Bonn, Bonn, Germany 25 Department of Physics, Boston University, Boston MA, United States of America
26 Department of Physics, Boston University, Boston MA, Un 26 Department of Physics, Brandeis University, Waltham MA, United States of America 27 Transilvania University of Brasov(𝑎), Brasov; Horia Hulubei National Institute of Physics and Nuclear
Engineering(𝑏), Bucharest; Department of Physics(𝑐), Alexandru Ioan Cuza University of Iasi, Iasi; National
Institute for Research and Development of Isotopic and Molecular Technologies(𝑑), Physics Department,
Cluj-Napoca; University Politehnica Bucharest(𝑒), Bucharest; West University in Timisoara( 𝑓), Timisoara,
Romania 2021 JINST 16 P07 28 Faculty of Mathematics(𝑎), Physics and Informatics, Comenius University, Bratislava; Department of
Subnuclear Physics(𝑏), Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak
Republic JINST 16 P07029 30 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina epartamento de Física, Universidad de Buenos Aires, Buen 31 California State University, CA, United States of America 32 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 33 Department of Physics(𝑎), University of Cape Town, Cape Town; iThemba Labs(𝑏), Western Cape; Department
of Mechanical Engineering Science(𝑐), University of Johannesburg, Johannesburg; University of South
Africa(𝑑), Department of Physics, Pretoria; School of Physics(𝑒), University of the Witwatersrand,
Johannesburg, South Africa 34 Department of Physics, Carleton University, Ottawa ON, Canada 35 Faculté des Sciences Ain Chock(𝑎), Réseau Universitaire de Physique des Hautes Energies — Université
Hassan II, Casablanca; Faculté des Sciences(𝑏), Université Ibn-Tofail, Kénitra; Faculté des Sciences
Semlalia(𝑐), Université Cadi Ayyad, LPHEA-Marrakech; Moroccan Foundation for Advanced Science
Innovation and Research (MAScIR)(𝑑), Rabat; LPMR(𝑒), Faculté des Sciences, Université Mohamed Premier,
Oujda; Faculté des sciences( 𝑓), Université Mohammed V, Rabat, Morocco 35 Faculté des Sciences Ain Chock(𝑎), Réseau Universitaire de Physique des Hautes Energies — Université
Hassan II, Casablanca; Faculté des Sciences(𝑏), Université Ibn-Tofail, Kénitra; Faculté des Sciences
Semlalia(𝑐), Université Cadi Ayyad, LPHEA-Marrakech; Moroccan Foundation for Advanced Science
Innovation and Research (MAScIR)(𝑑), Rabat; LPMR(𝑒), Faculté des Sciences, Université Mohamed Premier,
Oujda; Faculté des sciences( 𝑓), Université Mohammed V, Rabat, Morocco Innovation and Research (MAScIR)(𝑑), Rabat; LPMR(𝑒), Faculté des Sciences, Université Mohamed Premier, 36 CERN, Geneva, Switzerland 37 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of 37 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of Ameri 38 LPC, Université Clermont Auvergne, CNRS/IN2P3, Clermont-Ferrand, France 39 Nevis Laboratory, Columbia University, Irvington NY, United States of America 39 Nevis Laboratory, Columbia University, Irvington NY, United States of America 40 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 40 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 41 Dipartimento di Fisica(𝑎), Università della Calabria, Rende; INFN Gruppo Collegato di Cosenza(𝑏),
Laboratori Nazionali di Frascati, Italy 41 Dipartimento di Fisica(𝑎), Università della Calabria, Rende; INFN Gruppo Collegato di Cosenza(𝑏)
Laboratori Nazionali di Frascati, Italy 43 Physics Department, University of Texas at Dallas, Richardson TX, United 43 Physics Department, University of Texas at Dallas, Richardson TX, Un 44 National Centre for Scientific Research "Demokritos", Agia Paraskevi, Greece 45 Department of Physics(𝑎), Stockholm University; Oskar Klein Centre(𝑏), Stock 46 Deutsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany 49 Department of Physics, Duke University, Durham NC, United States of America
50 50 SUPA — School of Physics and Astronomy, University of Edinburgh, 50 SUPA — School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 51 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 52 Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 53 II. The ATLAS collaboration Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, German 54 Département de Physique Nucléaire et Corpusculaire, Université de Genève, Genève, Switzerland – 58 – 55 Dipartimento di Fisica(𝑎), Università di Genova, Genova; INFN Sezione di Genova(𝑏), Italy 56 II. The ATLAS collaboration Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 57 SUPA — School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 58 LPSC, Université Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 60 Department of Modern Physics and State Key Laboratory of Particle Detection and Electronics(𝑎), University
of Science and Technology of China, Hefei; Institute of Frontier and Interdisciplinary Science and Key 60 Department of Modern Physics and State Key Laboratory of Particle Detection and Electronics(𝑎), University
of Science and Technology of China, Hefei; Institute of Frontier and Interdisciplinary Science and Key
Laboratory of Particle Physics and Particle Irradiation (MOE)(𝑏), Shandong University, Qingdao; School of
Physics and Astronomy(𝑐), Shanghai Jiao Tong University, Key Laboratory for Particle Astrophysics and
Cosmology (MOE), SKLPPC, Shanghai; Tsung-Dao Lee Institute(𝑑), Shanghai, China Laboratory of Particle Physics and Particle Irradiation (MOE)(𝑏), Shandong University, Qingdao; School of Physics and Astronomy(𝑐), Shanghai Jiao Tong University, Key Laboratory for Particle Astrophysics and Cosmology (MOE), SKLPPC, Shanghai; Tsung-Dao Lee Institute(𝑑), Shanghai, China 61 Kirchhoff-Institut für Physik(𝑎), Ruprecht-Karls-Universität Heidelberg, Heidelberg; Physikalisches Institut(𝑏
Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 2021 62 Department of Physics(𝑎), Chinese University of Hong Kong, Shatin, N.T., Hong Kong; Department of
Physics(𝑏), University of Hong Kong, Hong Kong; Department of Physics and Institute for Advanced Study(𝑐),
Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 62 Department of Physics(𝑎), Chinese University of Hong Kong, Shatin, N.T., Hong Kong; Department of
Physics(𝑏), University of Hong Kong, Hong Kong; Department of Physics and Institute for Advanced Study(𝑐), Physics(𝑏), University of Hong Kong, Hong Kong; Department of Physics and Institute for Advanced Study(𝑐
Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 63 Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 64 IJCLab, Université Paris-Saclay, CNRS/IN2P3, 91405, Orsay, France 65 Department of Physics, Indiana University, Bloomington IN, United States of America p
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66 INFN Gruppo Collegato di Udine(𝑎), Sezione di Trieste, Udine; ICTP(𝑏), Trieste; Dipartimento Politecnico di
Ingegneria e Architettura(𝑐), Università di Udine, Udine, Italy JINST 16 P07029 67 INFN Sezione di Lecce(𝑎); Dipartimento di Matematica e Fisica(𝑏), Università del Salento, Lecce, Italy 68 INFN Sezione di Milano(𝑎); Dipartimento di Fisica(𝑏), Università di Milano, Milano, Italy 69 INFN Sezione di Napoli(𝑎); Dipartimento di Fisica(𝑏), Università di Napoli, Napoli, Italy INFN Sezione di Napoli(𝑎); Dipartimento di Fisica(𝑏), Uni 70 INFN Sezione di Pavia(𝑎); Dipartimento di Fisica(𝑏), Università di Pavia, Pavia, Italy 71 INFN Sezione di Pisa(𝑎); Dipartimento di Fisica E. The ATLAS collaboration Petersburg, Russia
137 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of America
138 Laboratório de Instrumentação e Física Experimental de Partículas — LIP(𝑎), Lisboa; Departamento de
Física(𝑏), Faculdade de Ciências, Universidade de Lisboa, Lisboa; Departamento de Física(𝑐), Universidade
de Coimbra, Coimbra; Centro de Física Nuclear da Universidade de Lisboa(𝑑), Lisboa; Departamento de
Física(𝑒), Universidade do Minho, Braga; Departamento de Física Teórica y del Cosmos( 𝑓), Universidad de 95 Louisiana Tech University, Ruston LA, United States of America 96 Fysiska institutionen, Lunds universitet, Lund, Sweden 97 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3),
Villeurbanne, France Departamento de Física Teorica C-15 and CIAFF, Universi 99 Institut für Physik, Universität Mainz, Mainz, Germany 99 Institut für Physik, Universität Mainz, Mainz, Germany 100 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 00 School of Physics and Astronomy, University of Manche 101 CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 102 Department of Physics, University of Massachusetts, Amherst MA, United States of America epartment of Physics, University of Massachusetts, Amherst 103 Department of Physics, McGill University, Montreal QC, Canada 104 School of Physics, University of Melbourne, Victoria, Australia 106 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United 106 Department of Physics and Astronomy, Michigan State Univ 107 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belar 108 Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Bel 109 Group of Particle Physics, University of Montreal, Montreal QC, Canada Particle Physics, University of Montreal, Montreal QC, Can 110 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia 111 National Research Nuclear University MEPhI, Moscow, Russia V. Skobeltsyn Institute of Nuclear Physics, M.V. The ATLAS collaboration Fermi(𝑏), Università di Pisa, Pisa, Italy 73 INFN Sezione di Roma Tor Vergata(𝑎); Dipartimento di Fisica(𝑏), Università di Roma Tor Vergata, R 74 INFN Sezione di Roma Tre(𝑎); Dipartimento di Matematica e Fisica(𝑏), Università Roma Tre, Ro 75 INFN-TIFPA(𝑎); Università degli Studi di Trento(𝑏), Trento, Italy 75 INFN-TIFPA(𝑎); Università degli Studi di Trento(𝑏), Trento, Italy Institut für Astro- und Teilchenphysik, Leopold-Franzens- 76 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 77 University of Iowa, Iowa City IA, United States of America 78 Department of Physics and Astronomy, Iowa State University, Ames IA, United States of America 78 Department of Physics and Astronomy, Iowa State University, Ames IA, United States of America 79 Joint Institute for Nuclear Research, Dubna, Russia 80 Departamento de Engenharia Elétrica(𝑎), Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora;
Universidade Federal do Rio De Janeiro COPPE/EE/IF(𝑏), Rio de Janeiro; Instituto de Física(𝑐), Universidade
de São Paulo, São Paulo, Brazil 80 Departamento de Engenharia Elétrica(𝑎), Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora;
Universidade Federal do Rio De Janeiro COPPE/EE/IF(𝑏), Rio de Janeiro; Instituto de Física(𝑐), Universidade
de São Paulo, São Paulo, Brazil 81 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 81 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 82 Graduate School of Science, Kobe University, Kobe, Japan 82 Graduate School of Science, Kobe University, Kobe, Japan 83 AGH University of Science and Technology(𝑎), Faculty of Physics and Applied Computer Science, Krakow;
Marian Smoluchowski Institute of Physics(𝑏), Jagiellonian University, Krakow, Poland 84 Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 84 Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 84 Institute of Nuclear Physics Polish Academy of Science 85 Faculty of Science, Kyoto University, Kyoto, Japan 86 Kyoto University of Education, Kyoto, Japan 87 Research Center for Advanced Particle Physics and Department of Physics, Kyushu University 88 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 89 Physics Department, Lancaster University, Lancaster, United Kingdom 90 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 91 Department of Experimental Particle Physics, Jožef Stefan Institute and Department of Physics, University of
Ljubljana, Ljubljana, Slovenia 92 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 93 Department of Physics, Royal Holloway University of London, Egham, United Kingdom 94 Department of Physics and Astronomy, University College London, London, United Kingdom – 59 – 95 Louisiana Tech University, Ruston LA, United States of America
96 Fysiska institutionen, Lunds universitet, Lund, Sweden
97 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3),
Villeurbanne, France
98 Departamento de Física Teorica C-15 and CIAFF, Universidad Autónoma de Madrid, Madrid, Spain
99 Institut für Physik, Universität Mainz, Mainz, Germany
100 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom
101 CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France
102 Department of Physics, University of Massachusetts, Amherst MA, United States of America
103 Department of Physics, McGill University, Montreal QC, Canada
104 School of Physics, University of Melbourne, Victoria, Australia
105 Department of Physics, University of Michigan, Ann Arbor MI, United States of America
106 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States of America
107 B.I. The ATLAS collaboration Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus
108 Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Belarus
109 Group of Particle Physics, University of Montreal, Montreal QC, Canada
110 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia
111 National Research Nuclear University MEPhI, Moscow, Russia
112 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia
113 Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany
114 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany
115 Nagasaki Institute of Applied Science, Nagasaki, Japan
116 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan
117 Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States of America
118 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University/Nikhef, Nijmegen, Netherlands
119 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands
120 Department of Physics, Northern Illinois University, DeKalb IL, United States of America
121 Budker Institute of Nuclear Physics and NSU(𝑎), SB RAS, Novosibirsk; Novosibirsk State University
Novosibirsk(𝑏), Russia
122 Institute for High Energy Physics of the National Research Centre Kurchatov Institute, Protvino, Russia
123 Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of National Research Centre
"Kurchatov Institute", Moscow, Russia
124 Department of Physics, New York University, New York NY, United States of America
125 Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan
126 Ohio State University, Columbus OH, United States of America
127 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of
America
128 Department of Physics, Oklahoma State University, Stillwater OK, United States of America
129 Palacký University, RCPTM, Joint Laboratory of Optics, Olomouc, Czech Republic
130 Institute for Fundamental Science, University of Oregon, Eugene, OR, United States of America
131 Graduate School of Science, Osaka University, Osaka, Japan
132 Department of Physics, University of Oslo, Oslo, Norway
133 Department of Physics, Oxford University, Oxford, United Kingdom
134 LPNHE, Sorbonne Université, Université de Paris, CNRS/IN2P3, Paris, France
135 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America
136 Konstantinov Nuclear Physics Institute of National Research Centre "Kurchatov Institute", PNPI, St. The ATLAS collaboration Lomonosov JINST 16 P07029 113 Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 113 Fakultät für Physik, Ludwig-Maximilians-Universität Mün 114 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 115 Nagasaki Institute of Applied Science, Nagasaki, Japan 116 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoy Graduate School of Science and Kobayashi-Maskawa Ins 118 Institute for Mathematics, Astrophysics and Particle Physics, Ra 120 Department of Physics, Northern Illinois University, DeKalb
( ) 120 Department of Physics, Northern Illinois University, DeKalb IL, United States of Ame
( ) 121 Budker Institute of Nuclear Physics and NSU(𝑎), SB RAS, Novosibirsk; Novosibirsk State University
Novosibirsk(𝑏), Russia 122 Institute for High Energy Physics of the National Research Centre Kurchatov Institute, Protvino, Russia 123 Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of National Research Centre
"Kurchatov Institute", Moscow, Russia 125 Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan 125 Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan 126 Ohio State University, Columbus OH, United States of America 127 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of
America 128 Department of Physics, Oklahoma State University, Stillwater OK, United States of America 129 Palacký University, RCPTM, Joint Laboratory of Optics, Olomouc, Czech Republic 130 Institute for Fundamental Science, University of Oregon, Eugene
131 131 Graduate School of Science, Osaka University, Osaka, Japan 131 Graduate School of Science, Osaka University, Osaka, Japan 132 Department of Physics, University of Oslo, Oslo, Norway 133 Department of Physics, Oxford University, Oxford, United Kingdom 134 LPNHE, Sorbonne Université, Université de Paris, CNRS/IN2P3, Paris, France 135 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America
136 136 Konstantinov Nuclear Physics Institute of National Research Centre "Kurchatov Institute", PNPI, St. The ATLAS collaboration Petersburg, Russia 137 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, 138 Laboratório de Instrumentação e Física Experimental de Partículas — LIP(𝑎), Lisboa; Departamento de
Física(𝑏), Faculdade de Ciências, Universidade de Lisboa, Lisboa; Departamento de Física(𝑐), Universidade
de Coimbra, Coimbra; Centro de Física Nuclear da Universidade de Lisboa(𝑑), Lisboa; Departamento de
Física(𝑒), Universidade do Minho, Braga; Departamento de Física Teórica y del Cosmos( 𝑓), Universidad de – 60 – Granada, Granada (Spain); Dep Física and CEFITEC of Faculdade de Ciências e Tecnologia(𝑔), Universida Nova de Lisboa, Caparica; Instituto Superior Técnico(ℎ), Universidade de Lisboa, Lisboa, Portugal 139 Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 140 Czech Technical University in Prague, Prague, Czech Republic ch Technical University in Prague, Prague, Czech Republic rles University, Faculty of Mathematics and Physics, Pragu 142 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 143 IRFU, CEA, Université Paris-Saclay, Gif-sur-Yvette, France 144 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, United States of
America 145 Departamento de Física(𝑎), Pontificia Universidad Católica de Chile, Santiago; Universidad Andres Bello(𝑏),
Department of Physics, Santiago; Instituto de Alta Investigación(𝑐), Universidad de Tarapacá; Departamento
de Física(𝑑), Universidad Técnica Federico Santa María, Valparaíso, Chile 146 Universidade Federal de São João del Rei (UFSJ), São João del Rei, Brazil 147 Department of Physics, University of Washington, Seattle WA, United States of America 148 Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 149 Department of Physics, Shinshu University, Nagano, Japan 150 Department Physik, Universität Siegen, Siegen, Germany 151 Department of Physics, Simon Fraser University, Burnaby BC, Canada ional Accelerator Laboratory, Stanford CA, United States o JINST 16 P07029 153 Physics Department, Royal Institute of Technology, Stockholm, Sweden 153 Physics Department, Royal Institute of Technology, Stockholm, Sweden 154 Departments of Physics and Astronomy, Stony Brook University, Stony Brook NY, United States of America 154 Departments of Physics and Astronomy, Stony Brook University, Stony Bro 155 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 156 School of Physics, University of Sydney, Sydney, Australia 157 Institute of Physics, Academia Sinica, Taipei, Taiwan 158 E. Andronikashvili Institute of Physics(𝑎), Iv. The ATLAS collaboration Javakhishvili Tbilisi State University, Tbilisi; High Energy
Physics Institute(𝑏), Tbilisi State University, Tbilisi, Georgia 159 Department of Physics, Technion, Israel Institute of Technology, Haifa, I 160 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University,
6 161 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece
6 162 International Center for Elementary Particle Physics and Department of Physics, University of Tokyo, Tokyo,
Japan 163 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 164 Tomsk State University, Tomsk, Russia 165 Department of Physics, University of Toronto, Toronto ON, Canada 166 TRIUMF(𝑎), Vancouver BC; Department of Physics and Astronomy(𝑏), York University, Toronto ON, Canada 166 TRIUMF(𝑎), Vancouver BC; Department of Physics and Astronomy(𝑏), 167 Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Applied Sciences,
University of Tsukuba, Tsukuba, Japan 168 Department of Physics and Astronomy, Tufts University, Medford MA, United States of America 169 Department of Physics and Astronomy, University of California Irvine, Irvine CA, United States of America 171 Department of Physics, University of Illinois, Urbana IL, United States of America 172 Instituto de Física Corpuscular (IFIC), Centro Mixto Universidad de Valencia — CSIC, Valencia, Spain 173 Department of Physics, University of British Columbia, Vancouver BC, Canada 174 Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 175 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
176 176 Department of Physics, University of Warwick, Coventry, United Kingdom 177 Waseda University, Tokyo, Japan 178 Department of Particle Physics and Astrophysics, Weizmann Institute of Science, Rehovot, Israel 179 Department of Physics, University of Wisconsin, Madison WI, United States of America 180 Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal,
Wuppertal, Germany 181 Department of Physics, Yale University, New Haven CT, United States of America – 61 – 𝑎Also at Borough of Manhattan Community College, City University of New York, New York NY, United States of
America 𝑎Also at Borough of Manhattan Community College, City University of New York, New York NY, United States of
America 𝑏Also at Center for High Energy Physics, Peking University, China 𝑏Also at Center for High Energy Physics, Peking University, China 𝑐Also at Centro Studi e Ricerche Enrico Fermi, Italy 𝑑Also at CERN, Geneva, Switzerland 𝑒Also at CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 𝑒Also at CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 𝑓
Also at Département de Physique Nucléaire et Corpusculaire, Université de Genève, Genève, Switzerland 𝑓
Also at Département de Physique Nucléaire et Corpusculaire, Université de Genève, Genève, Switzerland 𝑔Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain ℎAlso at Department of Financial and Management Engineering, University of the Aegean, Chios, Greece 𝑖Also at Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States of
America 2021 JINST 16 P070 𝑗Also at Department of Physics and Astronomy, University of Louisville, Louisville, KY, United St 𝑜Also at Department of Physics, King’s College London, London, United Kingdom JINST 16 P07029 𝑞Also at Department of Physics, University of Fribourg, Fribourg, Switzerland 𝑟Also at Dipartimento di Matematica, Informatica e Fisica, Università di Udine, Udine, Italy 𝑠Also at Faculty of Physics, M.V. The ATLAS collaboration Lomonosov Moscow State University, Moscow, Russia 𝑡Also at Giresun University, Faculty of Engineering, Giresun, Turkey 𝑢Also at Graduate School of Science, Osaka University, Osaka, Japan 𝑣Also at Hellenic Open University, Patras, Greece 𝑤Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain 𝑤Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain 𝑥Also at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germ 𝑥Also at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany 𝑦Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences,
Sofia, Bulgaria 𝑧Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary 𝑎𝑏Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 𝑎𝑏Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 𝑎𝑑Also at Istanbul University, Dept. of Physics, Istanbul, Turkey 𝑎𝑒Also at Joint Institute for Nuclear Research, Dubna, Russia 𝑎𝑒Also at Joint Institute for Nuclear Research, Dubna, Russia 𝑎𝑓
Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia 𝑎𝑓
Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia 𝑎𝑔Also at National Research Nuclear University MEPhI, Moscow, Russia 𝑎𝑔Also at National Research Nuclear University MEPhI, Moscow, Russia 𝑎ℎAlso at Physics Department, An-Najah National University, Nablus, Palestine 𝑎ℎAlso at Physics Department, An-Najah National University, Nablus, Palestine 𝑎𝑖Also at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 𝑎𝑖Also at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 𝑎𝑘Also at TRIUMF, Vancouver BC, Canada 𝑎𝑙Also at Universita di Napoli Parthenope, Napoli, Italy 𝑎𝑙Also at Universita di Napoli Parthenope, Napoli, Italy 𝑎𝑚Also at University of Chinese Academy of Sciences (UCAS), Beijing, China 𝑎𝑚Also at University of Chinese Academy of Sciences (UCAS), Beijing, China ∗Deceased ∗Deceased ∗Deceased – 62 –
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Acceleration of sarcopenia in elderly patients who develop asymptomatic pneumonia shadow within one year after surgery for early gastric cancer
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BMC surgery
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RESEARCH Open Access Abstract Background Although early gastric cancer is curable with local treatment, the overall survival in elderly patients
did not reach 80% at five years after surgery. The major cause of death in elderly patients with early gastric cancer
is not cancer itself but is related to postoperative sarcopenia. Elderly patients frequently develop postoperative
asymptomatic pneumonia shadow, which is associated with a poor prognosis. However, why asymptomatic
pneumonia shadow worsens the prognosis remains unclear. We investigated whether sarcopenia is accelerated in
patients who developed asymptomatic pneumonia shadow. Methods We retrospectively examined patients of > 75 years of age who underwent R0 gastrectomy for gastric
cancer and were diagnosed with T1 disease at National Cancer Center Hospital between 2005 and 2012. The
diagnosis of asymptomatic pneumonia shadow was defined by diagnostic findings of pneumonia (consolidation
type, reticular type, and nodular type) which were newly observed on chest computed tomography performed one
year after surgery in comparison to preoperative computed tomography. Postoperative muscle loss was assessed by
a computed tomography-based analysis using the L3 skeletal muscle index before and two years after surgery and
the rate of decrease was calculated. Patients were classified into two groups according to the rate of decrease (cut-off
value: 10%). Results Of the 3412 patients who underwent gastrectomy in our hospital during the study period, 142 were
included in this study. Asymptomatic pneumonia shadow was found in 26 patients (18%). Patients who developed
asymptomatic pneumonia shadow showed a significantly greater loss of muscle volume in comparison to patients
who did not develop asymptomatic pneumonia shadow. In the multivariate analysis, total gastrectomy and
asymptomatic pneumonia shadow were the independent risk factors for severe muscle loss. However, there was no
significant difference in prognosis between the two groups. Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. BMC Surgery BMC Surgery Kamiya et al. BMC Surgery (2023) 23:232
https://doi.org/10.1186/s12893-023-02096-2 Abstract If material is not included
in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data. Acceleration of sarcopenia in elderly patients
who develop asymptomatic pneumonia
shadow within one year after surgery for early
gastric cancer Ayako Kamiya1, Tsutomu Hayashi1, Ryota Sakon1, Kenichi Ishizu1, Takeyuki Wada1, Sho Otsuki1, Yukinori Yamagata1,
Hitoshi Katai1 and Takaki Yoshikawa1* © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included
in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data. Background In Japan, gastric cancer is characterized by early stage at
the diagnosis and elderly age [1, 2]. Although early gas
tric cancer is curable with local treatment, the overall
survival in elderly patients did not reach 80% at five years
after surgery [3]. It would be explained by the high pro
portion of deaths from causes other than gastric cancer
itself [4].i To clarify whether sarcopenia is accelerated in patients
who develop APS, we compared the change of muscle
volume in the 2 years after gastrectomy between elderly
patients who developed APS at 1 year after surgery and
those who did not. Moreover, we also clarified whether
APS is a significant risk factor for sarcopenia in the 2
years after surgery. Sarcopenia has been defined as the loss of muscle mass
and strength that occurs with aging [5]. Sarcopenia is
associated with increased adverse outcomes including
falls, functional decline, frailty, and mortality [6]. The
prevalence of sarcopenia was reported to be approxi
mately 22% of men and women aged 75–79 years old
and 32.4% of men and 47.7% of women aged 80 years and
older [7]. Loss of body weight is a common and causes
serious outcome in patients with gastric cancer who have
undergone gastrectomy. Takahashi et al. reported that
6% of the elderly patients were diagnosed with sarcope
nia preoperatively and it increased to 22% by 1 year after
gastrectomy [8]. Sarcopenia is gradually being recognized
as a resistance factor in cancer treatment. Some studies
have shown that skeletal muscle loss after gastrectomy
could predict a poor prognosis in gastric cancer patients
[9–11]. Methods
Patients Patients were selected from a clinical database of con
secutive patients who received gastrectomy for gastric
cancer at the National Cancer Center Hospital between
January 2005 and December 2012, and who met the fol
lowing criteria: (1) a pathological diagnosis of T1, (2)
age ≥ 75 years, (3) R0 resection achieved, and (4) chest to
abdominal CT performed before surgery and within two
years after surgery. In the elderly population, pneumonia is one of the most
frequent causes of death [12]. We previously investigated
asymptomatic pneumonia shadow (APS) on chest com
puted tomography (CT) at regular follow-up in elderly
patients who received surgical treatment for early gas
tric cancer [13, 14]. Surprisingly, this pneumonia shadow
was detected in approximately one-quarter of the elderly Page 2 of 8 (2023) 23:232 Kamiya et al. BMC Surgery (2023) 23:232 Kamiya et al. BMC Surgery Conclusions Sarcopenia was accelerated in elderly patients who developed asymptomatic pneumonia shadow
after surgery for early gastric cancer. However, the poor prognosis in these patients may not be related to accelerated
sarcopenia. Keywords Elderly, Gastrectomy, Pneumonia, Sarcopenia, Skeletal muscle patients [13] and APS was significantly associated with
poor survival [14]. However, it remains unclear why APS
worsens the prognosis despite being a silent shadow
without active symptoms. If APS accelerates sarcopenia,
it could worsen the prognosis. Surgery and follow-uph The treatment strategy for early-stage gastric cancer was
determined by the Japanese Gastric Cancer Treatment
Guideline version 2 or version 3 depending on the date of
the surgery [15, 16]. In summary, gastrectomy with D1 or
D1 + lymphadenectomy was performed according to the Fig. 1 Definition of the diagnosis of pneumonia based on CT consolidation type, b. reticular type, c. nodular type Fig. 1 Definition of the diagnosis of pneumonia based on CT consolidation type, b. reticular type, c. nodular type Page 3 of 8 Kamiya et al. BMC Surgery (2023) 23:232 (2023) 23:232 Kamiya et al. BMC Surgery Fig. 3 Flow diagram of the present study
Fig. 2 Axial computed tomography slice of the third lumbar vertebra (L3). Green areas indicate skeletal muscle were newly observed on chest CT performed 1 year after
surgery in comparison to preoperative CT. After certi
fied radiologists confirmed the radiological findings, two
surgeons evaluated the images to determine the pres
ence or absence of APS. If the two surgeons’ diagnoses
differed, the images were evaluated again to make a final
determination. Evaluation of decreased muscle mass We performed a CT-based analysis and used the muscle
area measured on CT [18]; axial slices of the third lum
bar vertebrae (L3) were evaluated [19]. The sum of the
cross-sectional areas of the skeletal muscles including the
psoas, paraspinal, and abdominal wall muscles was cal
culated using the SYNAPSE VINCENT system (Fig. 2). The skeletal muscle area in a single abdominal image is
proportional to the whole-body muscle mass [20]. The
muscle area normalized by the square of the height [m] is
called the L3 skeletal muscle index (SMI, [cm2/m2]) [21]. We investigated the L3 SMI before surgery and at 2 years
after surgery and calculated the rate of decrease in the
L3 SMI (%SMI-dec) as follows: %SMI-dec = 100 x (SMI
before surgery - SMI 2 years after surgery) / SMI before
surgery. Considering the clinical significance of %SMI-
dec, we set the cut-off value as 10%. Then, the patients
were classified into those with high %SMI-dec (H group)
and those with low %SMI-dec (L group). Fig. 2 Axial computed tomography slice of the third lumbar vertebra (L3). Green areas indicate skeletal muscle Fig. 2 Axial computed tomography slice of the third lumbar vertebra (L3). Green areas indicate skeletal muscle Fig. 3 Flow diagram of the present study Fig. 3 Flow diagram of the present study Statistical analysis
ll
l Fig. 3 Flow diagram of the present study All statistical analyses were performed using the SPSS
software program (Statistical Package for the Social
Sciences version 15.0; SPSS, Chicago, IL, USA). Sta
tistical comparisons of the differences in the age, body
mass index (BMI = body weight [kg]/height [m]2), Geri
atric Nutritional Risk Index (GNRI = 4.89×Alb [g/
dl] + 41.7×(body weight [kg] / ideal body weight [kg]),
vital capacity (%VC) and forced expiratory volume in
1 s (FEV1%) were analyzed by Student’s t-test, and other
variables including the Charlson score [22] were analyzed
by the chi-squared test. tumor characteristics, without adjuvant chemotherapy,
regardless of the patient’s age. Postoperative follow-up
evaluations, including physical examinations, blood tests,
and CT or ultrasound, were basically performed every six
months for the first year and then every year for the next
four years. As routine oncologic follow-up, CT of the
chest to the abdomen was basically performed every year
for at least five years postoperatively. Additional imaging
studies were performed if recurrence was suspected. To identify risk factors for high %SMI-dec, age, surgi
cal procedure, Charlson score, BMI and GNRI were con
verted to binary data. The median GNRI was used as the
cut-off value. Statistical comparisons of differences in
each variable between the L and the H groups were per
formed using the chi-squared test. Variables were also
investigated by a multivariate logistic regression analysis
to assess the risk factors associated with high %SMI-dec. P values of < 0.05 were considered to indicate statistical
significance. Diagnostic criteria for asymptomatic pneumonia shadow
A S
d
d
f ll
CT
h fi
f Diagnostic criteria for asymptomatic pneumonia shadow
APS was diagnosed on follow-up CT in the first year after
surgery. According to the guidelines for diagnostic imag
ing of adult community-acquired pneumonia 2007, diag
nostic findings of pneumonia on CT were classified into
three types: consolidation type, reticular type, and nodu
lar type, and those presenting with two or more types at
the same time were called mixed type (Fig. 1) [17]. The
diagnosis of APS was defined by these findings which Page 4 of 8 Kamiya et al. BMC Surgery (2023) 23:232 Page 4 of 8 Kamiya et al. BMC Surgery h
CT images taken before and after surgery. No patients
had treated by neoadjuvant chemotherapy or postop
erative adjuvant chemotherapy. Figure 3 shows a con
sort diagram of the present study. APS was found in 26
patients (18%) by chest CT: consolidation type (n = 11),
reticular type (n = 3), nodular type (n = 10), and mixed
type (n = 2). Table 1 summarizes the clinicopathological character
istics of the patients. All patients who underwent distal
gastrectomy were reconstructed with Roux-en-Y, and
all patients who underwent proximal gastrectomy were
reconstructed with jejunal interposition. In comparison
to patients without APS, the patients with APS included
a high proportion of male and a high proportion of
patients with preoperative pneumonia shadow. The other
background characteristics of the groups were similar. Figure 4 shows the %SMI-dec of the patients with and
Fig. 5 Over-all survival curve of the patients with APS and those without
APS
Fig. 4 %SMI-dec of the patients with and without APS Fig. 4 %SMI-dec of the patients with and without APS Fig. 4 %SMI-dec of the patients with and without APS CT images taken before and after surgery. No patients
had treated by neoadjuvant chemotherapy or postop
erative adjuvant chemotherapy. Figure 3 shows a con
sort diagram of the present study. APS was found in 26
patients (18%) by chest CT: consolidation type (n = 11),
reticular type (n = 3), nodular type (n = 10), and mixed
type (n = 2). Table 1 summarizes the clinicopathological character
istics of the patients. All patients who underwent distal
gastrectomy were reconstructed with Roux-en-Y, and
all patients who underwent proximal gastrectomy were
reconstructed with jejunal interposition. Diagnostic criteria for asymptomatic pneumonia shadow
A S
d
d
f ll
CT
h fi
f In comparison
to patients without APS, the patients with APS included
a high proportion of male and a high proportion of
patients with preoperative pneumonia shadow. The other
background characteristics of the groups were similar. Fig. 5 Over-all survival curve of the patients with APS and those without
APS Fig. 5
APS Figure 4 shows the %SMI-dec of the patients with and
without APS. The median %SMI-dec in patients with and
without APS was 17.4 and 11.6, respectively (p = 0.035). The overall survival (OS) curves were calculated based
on the Kaplan–Meier curves and were compared by the
log-rank test. The data for patients who did not experi
ence an event by the date of the final observation were
treated as censored cases. Risk factors for high %SMI-dec To identify risk factors for high %SMI-dec, variables were
converted to binary data (Table 2). Eighty-nine patients
(63%) were included in the H group. The surgical pro
cedures were classified into two groups by the presence
or absence of the residual stomach (distal gastrectomy,
pylorus preservastrectomy and proximal gastrectomy). In our study population, 15% of patients received total Patients’ demographics Among the 3412 patients who underwent gastrectomy
for gastric cancer at National Cancer Center Hospital, we
reviewed the records of 142 patients who had adequate Page 5 o
Kamiya et al. BMC Surgery (2023) 23:232
Fig. 6 Over-all survival curve of the patients with high %SMI-dec (H group) and those with low %SMI-dec (L group) Page 5 of 8 Page 5 of 8 Kamiya et al. BMC Surgery Fig. 6 Over-all survival curve of the patients with high %SMI-dec (H group) and those with low %SMI-dec (L group) gastrectomy. Among the variables included in the uni
variate analyses, APS was the only factor that showed
a significant difference between the L and H groups
(p = 0.043). In the multivariate analysis, the surgical pro
cedure and APS were independent risk factors for high
%SMI-dec (p = 0.025 and p = 0.043, respectively). with muscle loss exceeding 10% in the 2 years after gas
trectomy. The patients who had accelerated sarcopenia
2 years after surgery had not so poor prognosis as com
pared with those who had not.i Patients who developed APS showed a significant loss
of muscle volume in the 2 years after surgery in compari
son to those who did not. Moreover, APS was a signifi
cant risk factor related with postoperative muscle loss. Why was sarcopenia accelerated in patients having APS
despite APS being a silent shadow without active symp
toms? APS might reflect the presence of mild pneumonia. Even mild inflammation may result in a loss of muscle
mass. Pneumonia has been reported to induce muscle
atrophy in an animal model and in the elderly human
population [23–25]. The skeletal muscle loss induced by
pneumonia might cause a vicious cycle with the repeated
episodes of pneumonia and further muscle atrophy.i Survival outcomesh The median follow-up period from the date of surgery
was 77 months (range, 30–132 months). In the Kaplan-
Meier survival analysis, patients with APS experienced
significantly shorter OS (p = 0.0005) than those with
out APS (Fig. 5). However, comparing H group and L
group there was no significant difference in the survival
between the two groups. (p = 0.742, Fig. 6). Discussion In this study, we could confirm that patients who devel
oped APS had the poor prognosis as compared with
those who did not as described in the previous study
[14]. However, the patients who have accelerated sar
copenia had not so poor prognosis as compared with
those who had not. So, accelerated sarcopenia was not
directly related to poor prognosis, which is contradic
tory to the previous studies [9–11]. This difference could
be explained by the difference of the cohort. In the pre
vious reports, the cohort was not limited to the elderly
patients. In the elderly patients, sarcopenia is frequently
observed. When the sarcopenia was defined as the cut-
off values at 49.2 cm2 /m2 for males and 35.7 cm2 /m2for Among elderly patients with Stage I gastric cancer,
deaths due to other diseases were frequently observed in
the long period after surgery [4]. The prevalence of sar
copenia was reported to be approximately 22% of men
and women aged 75–79 years old and 32.4% of men and
47.7% of women aged 80 years and older [7]. In the present study, we examined the relationships
between postoperative muscle loss and APS in elderly
patients after gastrectomy for early gastric cancer. We
found that muscle loss in the 2 years after surgery was
significantly greater in patients who developed APS in
comparison to those who did not. Moreover, APS and
total gastrectomy were independent risk factors related Kamiya et al. Discussion BMC Surgery (2023) 23:232 Page 6 of 8 Page 6 of 8 Table 1 Clinicopathological characteristics
Variables
Total
N (%)*
APS† (-) (n = 116)
N (%)*
APS† (+) (n = 26)
N (%)*
p value**
Age (Median, range)
77 (75–87)
77 (75–87)
77 (75–83)
0.808
Sex
0.123
Male
90 (63)
70 (60)
20 (77)
Female
52 (37)
46 (40)
6 (23)
Surgical approach
0.695
Open
130 (92)
105 (91)
25 (96)
Laparoscopy
12 (8)
11 (9)
1 (4)
Surgical procedure
0.861
Distal gastrectomy
65 (46)
53 (46)
12 (46)
Pylorus preserving gastrectomy
43 (30)
35 (30)
8 (30)
Proximal gastrectomy
12 (8)
9 (8)
3 (12)
Total gastrectomy
22 (16)
19 (16)
3 (12)
BMI (Median, range) ††
22.4 (16.5–32.2)
22.5 (16.7–32.2)
22.1 (16.5–30.5)
0.652
GNRI (Median, range) †††
104.5 (89.0-128.0)
104.5 (89.0-125.3)
104.0(89.9–128.0)
0.791
Charlson Score
0.726
0
82 (58)
69 (59)
13 (50)
1
27 (19)
22 (19)
5 (20)
2
20 (14)
15 (13)
5 (20)
3
7 (5)
5 (4)
2 (7)
4
2 (1)
2 (2)
0 (0)
5
0 (0)
0 (0)
0 (0)
6
3 (2)
2 (2)
1 (3)
7
1 (1)
1 (1)
0 (0)
Current smoking history
0.695
+
57 (40)
48 (41)
9 (35)
-
85 (60)
68 (59)
17 (65)
%VC (Median, range) ††††
104 (53–175)
104 (53–175)
103 (75–132)
0.99
FEV1% (Median, range)†††††
73.5 (34–96)
73.5 (34–92)
74 (58–96)
0.386
Sliding hernia
1.000
+
44 (31)
36 (31)
8 (31)
-
98 (69)
80 (69)
18 (69)
Preoperative pneumonia
0.129
+
62 (44)
47 (41)
15 (58)
-
80 (56)
69 (59)
11 (42)
Pathological TNM stage
0.346
T1aN0
59(41)
49 (42)
10 (38)
T1aN1
1 (1)
0(0)
1(4)
T1bN0
71 (50)
58 (50)
13 (50)
T1bN1
11 (8)
9 (8)
2 (8)
*: Number and percentage of patients in each category in each group (except age, BMI, GNRI, %VC and FEV1%)
**: Age, BMI, GNRI, %VC and FEV1% were analyzed by Student’s t-test, and other variables were analyzed by the chi-squared test
APS†: asymptomatic pneumonia shadow sarcopenia 2 years after surgery. Degree of sarcopenia
may not be related with prognosis.i females, respectively, as described in the previous study
[13], 69% of the patients in this cohort had sarcopenia
before surgery. Moreover, 89% of the patients had sarco
penia 2 years after surgery. Discussion BMC Surgery Table 2 Risk factors for high %SMI†-dec†† according to univariate and multivariate analyses
Variables
Total
N (%)*
L group
N (%)*
H group
N (%)*
Univariate analysis
Multivariate analysis
p**
Odds ratio
p***
95% confidence interval
Age
0.187
1.850
0.136
0.824–4.170
<80
100 (70)
41 (77)
59 (66)
80≤
42 (30)
12 (23)
30 (34)
Sex
0.593
Male
90 (63)
32 (60)
58 (65)
Female
52 (37)
21 (40)
31 (35)
Surgical procedure
0.055
3.802
0.025
1.181–12.20
DG†††+PPG††††+PG†††††
120 (85)
49 (92)
71 (80)
TG††††††
22 (15)
4 (8)
18 (20)
Charlson score
1.000
<3
129 (91)
48 (91)
81 (91)
3≤
13 (9)
5 (9)
8 (9)
BMI†††††††
0.488
<22.5
71 (50)
29 (55)
42(47)
22.5≤
71 (50)
24 (45)
47(53)
GNRI††††††††
0.862
<104.5
67 (47)
26 (49)
41 (46)
104.5≤
75 (53)
27 (51)
48 (54)
APS†††††††††
0.043
2.990
0.043
1.030–8.660
+
26 (18)
5 (9)
21 (24)
-
116 (82)
48 (91)
68 (76)
*: The number and percentage of patients in each category in each group
**: Variables were analyzed by a chi-squared test
***: Variables were analyzed by a logistic regression analysis
SMI†: skeletal muscle index = L3 skeletal muscle area (cm2)/ height (m)2
%SMI-dec††: rate of decrease in L3 SMI = 100 x (SMI before surgery - SMI 2 years after surgery) / SMI before surgery
DG†††: distal gastrectomy
PPG††††: pylorus preserving gastrectomy
PG†††††: proximal gastrectomy
TG††††††: total gastrectomy
BMI†††††††: Body Mass Index = body weight (kg)/ height (m)2
GNRI††††††††: Geriatric Nutritional Risk Index = 14.89×Alb (g/dl) + 41.7×(body weight (kg)/ ideal body weight (kg))
APS†††††††††: asymptomatic pneumonia shadow Table 2 Risk factors for high %SMI†-dec†† according to univariate and multivariate analyses ***: Variables were analyzed by a logistic regression analysis
SMI†: skeletal muscle index = L3 skeletal muscle area (cm2)/ height (m)2
%SMI-dec††: rate of decrease in L3 SMI = 100 x (SMI before surgery - SMI 2 years after surgery) / SMI before surgery
DG†††: distal gastrectomy
PPG††††: pylorus preserving gastrectomy
PG†††††: proximal gastrectomy
TG††††††: total gastrectomy
BMI†††††††: Body Mass Index = body weight (kg)/ height (m)2
GNRI††††††††: Geriatric Nutritional Risk Index = 14.89×Alb (g/dl) + 41.7×(body weight (kg)/ ideal body weight (kg))
APS†††††††††: asymptomatic pneumonia shadow the muscle function. Third, this was a cross-sectional
study, which thereby makes it difficult to clarify the tem
poral or causal relationship between skeletal muscle loss
and APS. Discussion muscle loss [9, 10, 26, 27]. Kim et al. reported that female
sex, preoperative weight loss, proximal location of the
tumor and differentiated tumor were significant risk fac
tors for post-gastrectomy sarcopenia [9]. Yamazaki et al. also demonstrated that total gastrectomy was indepen
dently associated with severe muscle loss [26]. The severe
muscle loss after total gastrectomy might be attributed
to reduced oral intake. Two reasons for this reduced oral
intake could be considered, namely loss of retention abil
ity and/or decreased ghrelin (an appetizing hormone that
is released from the stomach) [28].h Conclusions Sarcopenia was accelerated in elderly patients who devel
oped APS after surgery for early gastric cancer. APS was
a significant risk factor associated with severe muscle
loss after surgery. However, the poor prognosis in these
patients may not be related to accelerated sarcopenia. The present study was associated with some limitations. First, this was a retrospective study based on data from a
single institution that analyzed 142 elderly patients with
early gastric cancer. Longitudinal larger data are required
to confirm the present results. Second, we only examined
muscle volume and did not measure muscle strength. Therefore, we could not evaluate whether APS reduced Discussion Thus, even the elderly patients
who did not have accelerated sarcopenia had been in females, respectively, as described in the previous study
[13], 69% of the patients in this cohort had sarcopenia
before surgery. Moreover, 89% of the patients had sarco
penia 2 years after surgery. Thus, even the elderly patients
who did not have accelerated sarcopenia had been in In addition to APS, total gastrectomy was also a signifi
cant risk factor for muscle depletion in the 2 years after
surgery. Previously, several investigators also showed
that total gastrectomy was a risk factor for postoperative Page 7 of 8 Kamiya et al. BMC Surgery (2023) 23:232 Kamiya et al. References References
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outcomes of elderly patients with stage I gastric cancer from the nation
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2016. 2. Foundation for Promotion of Cancer Research. CANCER STATISTICS IN JAPAN
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Not applicable. 11. Park HS, Kim HS, Beom SH, Rha SY, Chung HC, Kim JH, et al. Marked loss of
muscle, visceral fat, or subcutaneous fat after gastrectomy predicts poor
survival in advanced gastric Cancer: single-center study from the CLASSIC
trial. Ann Surg Oncol. 2018;25(11):3222–30. Received: 6 February 2023 / Accepted: 3 July 2023 23. Komatsu R, Okazaki T, Ebihara S, Kobayashi M, Tsukita Y, Nihei M, et al. Aspira
tion pneumonia induces muscle atrophy in the respiratory, skeletal, and
swallowing systems. J Cachexia Sarcopenia Muscle. 2018;9:643–53. 24. Iwai-Saito K, Shobugawa Y, Aida J, Kondo K. Frailty is associated with
susceptibility and severity of pneumonia in older adults (a JAGES multilevel
cross-sectional study). Sci Rep. 2021;11(1):7966. Competing interests 21. Martin L, Birdsell L, Macdonald N, Reiman T, Clandinin MT, McCargar LJ,
et al. Cancer cachexia in the age of obesity: skeletal muscle depletion is a
powerful prognostic factor, independent of body mass index. J Clin Oncol. 2013;31:1539–47. Authors’ contributions Authors contributions
All authors made substantial contributions to conception and design, and/
or acquisition of data, and/or analysis and interpretation of data. A.K., T.H. and
T.Y. participated in drafting the article or revising it critically for important
intellectual content. All authors gave final approval of the version to be
published. 12. The Ministry of. Health Labour and Welfare in Japan. Vital statistics of popula
tion in Japan. 13. Kamiya A, Hayashi T, Sakon R, Ishizu K, Wada T, Otsuki S, et al. Long-term
postoperative pneumonia in elderly patients with early gastric cancer. BMC
Surg. 2022;22(1):220. 13. Kamiya A, Hayashi T, Sakon R, Ishizu K, Wada T, Otsuki S, et al. Long-term
postoperative pneumonia in elderly patients with early gastric cancer. BMC
Surg. 2022;22(1):220. Funding 14. Kamiya A, Hayashi T, Sakon R, Ishizu K, Wada T, Otsuki S, et al. Prognostic
impact of long-term postoperative pneumonia in Elderly patients with early
gastric Cancer. J Cancer. 2022;13(9):2905–11. This study was carried out without funding. Consent for publication
N
li
bl 20. Shen W, Punyanitya M, Wang Z, Gallagher D, St-Onge MP, Albu J, et al. Total
body skeletal muscle and adipose tissue volumes: estimation from a single
abdominal cross-sectional image. J Appl Physiol. 2004;97:2333–38. Ethics approval and consent to participate This study was conducted with the approval of the National Cancer Center
Hospital Ethics Committee (No: 2017-077) and it conforms to the provisions of
the Declaration of Helsinki. Informed consent was obtained from all subjects
and guardians. 18. Malietzis G, Aziz O, Bagnall NM, Johns N, Fearon KC, Jenkins JT. The role of
body composition evaluation by computerized tomography in determining
colorectal cancer treatment outcomes: a systematic review. Eur J Surg Oncol. 2015;41:186–96. 19. Giani M, Rezoagli E, Grassi A, Porta M, Riva L, Famularo S et al. Low skeletal
muscle index and myosteatosis as predictors of mortality in critically ill surgi
cal patients. Nutrition. 2022;101. List of Abbreviations List of Abbreviations
APS
Asymptomatic pneumonia shadow
CT
Computed tomography
L3
The third lumbar vertebrae
SMI
Skeletal muscle index
%SMI-dec
The rate of decrease in the L3 SMI
BMI
Body mass index
GNRI
Geriatric Nutritional Risk Index List of Abbreviations
APS
Asymptomatic pneumonia shadow
CT
Computed tomography
L3
The third lumbar vertebrae
SMI
Skeletal muscle index
%SMI-dec
The rate of decrease in the L3 SMI
BMI
Body mass index
GNRI
Geriatric Nutritional Risk Index Page 8 of 8 Page 8 of 8 Kamiya et al. BMC Surgery (2023) 23:232 Kamiya et al. BMC Surgery % VC
% Vital capacity
FEV1%
Forced expiratory volume % in 1 s
OS
Overall survival % VC
% Vital capacity
FEV1%
Forced expiratory volume % in 1 s
OS
Overall survival 9. Kim HJ, Lee ES, Kim BJ, Kim WS, Park JY, Kimet JG. al. Risk factors and clinical
outcomes of postgastrectomy sarcopenia newly developed after curative
resection for gastric cancer. Medicine. 2022;101(6). 10. Kugimiya N, Harada E, Oka K, Kawamura D, Suehiro Y, Takemoto Y, et al. Loss
of skeletal muscle mass after curative gastrectomy is a poor prognostic factor. Oncol Lett. 2018;16(1):1341–47. Author details
1 1Department of Gastric Surgery, National Cancer Center Hospital, 5-1-1
Tsukiji, Chuo-ku, Tokyo 104-0045, Japan 1Department of Gastric Surgery, National Cancer Center Hospital, 5-1-1
Tsukiji, Chuo-ku, Tokyo 104-0045, Japan 22. Charlson ME, Pompei P, Ales KL, MacKenzie CR. A new method of classifying
prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis. 1987;40(5):373–83. Received: 6 February 2023 / Accepted: 3 July 2023 Received: 6 February 2023 / Accepted: 3 July 2023 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 8. Takahashi S, Shimizu S, Nagai S, Watanabe H, Nishitani Y, Kurisu Y. Character
istics of sarcopenia after distal gastrectomy in elderly patients. PLoS ONE. 2019;11:149.
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Testing for shifts in a time trend panel data model with serially correlated error component disturbances
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Testing for Shifts in a Time Trend Panel Data Model with Serially
Testing for Shifts in a Time Trend Panel Data Model with Serially
Correlated Error Component Disturbances
Correlated Error Component Disturbances Badi Baltagi
bbaltagi@maxwell.syr.edu Follow this and additional works at: https://surface.syr.edu/cpr Part of the Economic Policy Commons, and the Economics Commons Syracuse University
Syracuse University
SURFACE at Syracuse University
SURFACE at Syracuse University Maxwell School of Citizenship and Public
Affairs Center for Policy Research Recommended Citation
Recommended Citation Baltagi, Badi; Kao, Chihwa; and Liu, Long, "Testing for Shifts in a Time Trend Panel Data Model with
Serially Correlated Error Component Disturbances" (2019). Center for Policy Research. 245. https://surface.syr.edu/cpr/245 This Working Paper is brought to you for free and open access by the Maxwell School of Citizenship and Public
Affairs at SURFACE at Syracuse University. It has been accepted for inclusion in Center for Policy Research by an
authorized administrator of SURFACE at Syracuse University. For more information, please contact
surface@syr.edu. Paper No. 213
February 2019 Paper No. 213
February 2019 SENIOR RESEARCH ASSOCIATES Badi Baltagi, ECON
Robert Bifulco, PAIA
Leonard Burman, PAIA Stuart Rosenthal, ECON
Michah Rothbart, PAIA
Alexander Rothenberg, ECON
Rebecca Schewe, SOC
Amy Ellen Schwartz, PAIA/ECON
Saba Siddiki, PAIA
Perry Singleton, ECON
Yulong Wang, ECON
Michael Wasylenko, ECON
Peter Wilcoxen, PAIA Hugo Jales, ECON
Jeffrey Kubik, ECON
Yoonseok Lee, ECON
Amy Lutz, SOC
Yingyi Ma, SOC
Katherine Michelmore, PAIA
Jerry Miner, ECON
Shannon Monnat, SOC
Jan Ondrich, ECON
David Popp, PAIA Robert Bifulco, PAIA
Jeffrey Kubik, ECON
Michah Rothbart, PAIA
Leonard Burman, PAIA
Yoonseok Lee, ECON
Alexander Rothenberg, ECON
Carmen Carrión-Flores, ECON
Amy Lutz, SOC
Rebecca Schewe, SOC
Alfonso Flores-Lagunes, ECON
Yingyi Ma, SOC
Amy Ellen Schwartz, PAIA/EC
Sarah Hamersma, PAIA
Katherine Michelmore, PAIA
Saba Siddiki, PAIA
Madonna Harrington Meyer, SOC
Jerry Miner, ECON
Perry Singleton, ECON
Colleen Heflin, PAIA
Shannon Monnat, SOC
Yulong Wang, ECON
William Horrace, ECON
Jan Ondrich, ECON
Michael Wasylenko, ECON
Yilin Hou, PAIA
David Popp, PAIA
Peter Wilcoxen, PAIA
GRADUATE ASSOCIATES
Rhea Acuña, PAIA
Hyoung Kwon, PAIA
David Schwegman, PAIA
Jun Cai, ECON
Judith Liu, ECON
Iuliia Shybalkina, PAIA
Ziqiao Chen, PAIA
Rob Loomis, PAIA
Stephanie Spera, PAIA
Yoon Jung Choi, PAIA
Mattie Mackenzie-Liu, PAIA
Saied Toossi, PAIA
Stephanie Coffey, ECON
Maeve Maloney, ECON
Huong Tran, ECON
Emily Gutierrez, PAIA
Qasim Mehdi, PAIA
Joaquin Urrego, ECON
Jeehee Han, PAIA
Christine Marie Parsons, PAIA
Rebecca Wang, SOC
Dan Hiller, PAIA
Jonathan Presler, ECON
Sean Withington, PAIA
Yusun Kim, PAIA
Christopher Rick, PAIA
Yi Yang, ECON
STAFF
Joanna Bailey, Research Associate
Kathleen Nasto, Administrative Assistant
Joseph Boskovski, Maxwell X Lab
Candi Patterson, Computer Consultant
Katrina Fiacchi, Administrative Specialist
Laura Walsh, Administrative Assistant
Emily Minnoe, Administrative Assistant Alfonso Flores-Lagunes, ECON
Sarah Hamersma, PAIA
Madonna Harrington Meyer, SOC
Colleen Heflin, PAIA
William Horrace, ECON
Yilin Hou, PAIA Rhea Acuña, PAIA
Jun Cai, ECON
Ziqiao Chen, PAIA
Yoon Jung Choi, PAIA
Stephanie Coffey, ECON
Emily Gutierrez, PAIA
Jeehee Han, PAIA
Dan Hiller, PAIA
Yusun Kim, PAIA STAFF
Joanna Bailey, Research Associate
Kathleen Nasto, Administrative Assistant
Joseph Boskovski, Maxwell X Lab
Candi Patterson, Computer Consultant
Katrina Fiacchi, Administrative Specialist
Laura Walsh, Administrative Assistant
Emily Minnoe, Administrative Assistant Keywords: Non-Stationary Panels, Time Trends, Serial Correlation, Wald Type Tests Abstract This paper studies testing of shifts in a time trend panel data model with serially correlated error
component disturbances, without any prior knowledge of whether the error term is stationary or
nonstationary. This is done in case the shift is known as well as unknown. Following Vogelsang (1997)
in the time series literature, we propose a Wald type test statistic that uses a fixed effects feasible
generalized least squares (FE-FGLS) estimator derived in Baltagi, et al. (2014). The proposed test has a
Chi-square limiting distribution and is valid for both I (0) and I (1) errors. The finite sample size and
power of this Wald test is investigated using Monte Carlo simulations. Abstract This paper studies testing of shifts in a time trend panel data model with serially correlated
error component disturbances, without any prior knowledge of whether the error term is sta-
tionary or nonstationary. This is done in case the shift is known as well as unknown. Following
Vogelsang (1997) in the time series literature, we propose a Wald type test statistic that uses
a fixed effects feasible generalized least squares (FE-FGLS) estimator derived in Baltagi, et al. (2014). The proposed test has a Chi-square limiting distribution and is valid for both I(0) and
I(1) errors. The finite sample size and power of this Wald test is investigated using Monte Carlo
simulations. JEL C23 C3 JEL: C23, C3 JEL No. C23, C33 Keywords: Non-Stationary Panels, Time Trends, Serial Correlation, Wald Type Tests Authors: Badi H. Baltagi, Center for Policy Research, Department of Economics, Syracuse University;
Chihwa Kao, Department of Economics, University of Connecticut; Long Liu, Department of
Economics, College of Business, University of Texas at San Antonio Testing for Shifts in a Time Trend Panel Data Model with
Serially Correlated Error Component Disturbances∗ Badi H. Baltagi†, Chihwa Kao‡, Long Liu§
This version: February 18, 2019 Badi H. Baltagi†, Chihwa Kao‡, Long Liu§ This version: February 18, 2019 †Address correspondence to: Badi H. Baltagi, Department of Economics and Center for Policy Research, 426
Eggers Hall, Syracuse University, Syracuse, NY 13244-1020; tel: 315-443-1630; fax: 315-443-1081; e-mail: bbalt-
agi@maxwell.syr.edu. ‡Chihwa Kao: Department of Economics, University of Connecticut, 365 Fairfield Way, U-1063, Storrs, CT 06269-
1063; tel: 860-486-4669; e-mail: chih-hwa.kao@uconn.edu §Long Liu: Department of Economics, College of Business, University of Texas at San Antonio, One UTSA Circle,
TX 78249-0633; tel: 210-458-6169; fax: 210-458-5837; e-mail: long.liu@utsa.edu. ∗This paper extends the results of Baltagi, Kao and Liu (2014) to allow for possible structural change. Both papers
are dedicated in honour of Peter C.B. Phillips’s many contributions to econometrics and in particular non-stationary
time series analysis and panel data. 1
Introduction Testing for structural change in a time trend model has been an important research topic in the
econometrics literature. In a pure time series framework, it has been well studied by Vogelsang
(1997), Perron and Zhu (2005), Perron and Yabu (2009a, 2009b), to name a few. Emerson and Kao
(2001) extend the Wald test for structural change of Vogelsang (1997) to a panel data setting. They
show that the asymptotic distribution of the Wald statistic is different depending on whether the
panel data is stationary or nonstationary. Kim (2011) extends the Perron and Zhu (2005) article
to large (n, T) panel data with cross-sectional dependence. ∗This paper extends the results of Baltagi, Kao and Liu (2014) to allow for possible structural change. Both papers
are dedicated in honour of Peter C.B. Phillips’s many contributions to econometrics and in particular non-stationary
time series analysis and panel data. 1 This paper focuses on the testing for structural change in a panel data time trend model
with a stationary or nonstationary error term. In the time series literature, various estimation
methods have been discussed in Phillips and Lee (1996). In the panel data literature, various
estimation methods have been discussed in Baltagi, Kao and Liu (2014), hereafter denoted by BKL. In particular, BKL derive a fixed effects feasible generalized least squares (FE-FGLS) procedure and
propose a super-efficient estimate of the autoregressive parameter. Note that BKL did not consider
the possibility of a structural change. This is the subject of this paper. Testing for structural
change in a panel data model with a stationary or nonstationary regressor and error term has
been discussed in Baltagi, Kao and Liu (2017) but not for the time trend panel data model with
serial correlation. Also, Baltagi, Kao and Liu (2017) did not discuss the FE-FGLS procedure. Based on the FE-FGLS procedure derived in BKL, this paper proposes a Wald test for structural
change for the time trend panel data model with serial correlation that is robust to stationary or
nonstationary error terms. We derive the asymptotic distribution of this test and check its finite
sample performance using Monte Carlo simulations. The article is organized as follows. Section 2 introduces the model and the test with an unknown
change point. Asymptotic properties of the proposed test are derived. Section 3 discusses some
extensions of the model. 1The limiting distribution of double indexed integrated processes has been extensively studied by Phillips and
Moon (1999, 2000). Under the additional condition, n/T →0, they show that sequential asymptotic results for their
pooled estimators would be equivalent to the joint ones. Our sequential limit results can be extended to joint limit
following Phillips and Moon (1999, 2000). 1
Introduction When additional stationary or nonstationary regressors are included in
the regression, we show that the asymptotic distribution of the Wald test stays the same. We
also study the case when individual effects are not included in the model. Simulation results are
presented in Section 4 and provide the concluding remarks. Mathematical proofs are contained in
the supplemental appendix available upon request from the authors. A few words on notation. All
limits are taken sequentially as T →∞and n →∞unless otherwise specified. We use (n, T) →∞
to denote the sequential limit.1 Convergence in probability and distribution are denoted as →
p and
d , respec
→
tively. 1The limiting distribution of double indexed integrated processes has been extensively studied by Phillips and
Moon (1999, 2000). Under the additional condition, n/T →0, they show that sequential asymptotic results for their
pooled estimators would be equivalent to the joint ones. Our sequential limit results can be extended to joint limit
following Phillips and Moon (1999, 2000). 2 2
The Model and the Tests Consider the following panel data time trend model with structural change: Consider the following panel data time trend model with structural change: yit = δ + βt + γDT + µi + vit,
(1) (1) yit = δ + βt + γDT + µi + vit, for i = 1, . . . , n, t = 1, . . . T, where DT = 1 (t > k) (t −k) for a change point k = [λT] for some λ ∈
(0, 1), where [·] denotes the largest integer that is less than or equal to the argument and 1 (·) is the
indicator function. The change point k is unknown. Of course, δ, β and γ are unknown parameters
to be estimated. We assume that µi are the unobservable individual effects with µi ∼iid 0, σ2
µ
and vit are AR(1) stationary disturbance terms with νit = ρνit−1 + eit
(2) (2) with |ρ| ≤1, where eit is a white noise process with variance σ2
e. The µi are assumed to be
independent of vit for all i and t. The null hypothesis is H0 : γ = 0. Emerson and Kao (2001)
derive the Wald statistic based on Ordinary Least Squares (OLS) estimators as
βb
b
2
1k −β2k
βb
b
1k
WOLS(k) =
−β
2k
,
P
σ2
n
P
1
k
n
P
−1
P
T
−
v
i=1
t=1(t −t
2
1k)2
+
i=1
t=k+1(t −t2k) OLS(k) =
,
P
σ2
n
P
1
k
n
P
−1
P
T
−
v
i=1
t=1(t −t
2
1k)2
+
i=1
t=k+1(t −t2k)
,
P
2
n
P
1
k
n
P
−1
P
T
−
v
i=1
t=1(t −t
2
1k)2
+
i=1
t=k+1(t −t2k) P
P
b
n
Pk
−
b
n
PT
where
(t
t1k)yit ,
(t
t
)y
P
PT
β
=
Pi=1
t=1
k
n
Pk
β
=
Pi=1 Pt=k+1
1k
2
2
T
−2k
it
k
n
, t1k = 1
t=1 t, and t
=
1
2k
. 2
The Model and the Tests (t−
t=k+1 t
t1k)
(t
t
)2
k
T
k
i=1
t=1
i=1
t=k+1
−2k
−
Emerson and Kao (2001) show that under H0 : γ = 0, if |ρ| < 1, then P
P
b
n
Pk
−
b
n
PT
where
(t
t1k)yit ,
(t
t
)y
P
PT
β
=
Pi=1
t=1
k
n
Pk
β
=
Pi=1 Pt=k+1
1k
2
2
T
−2k
it
k
n
, t1k = 1
t=1 t, and t
=
1
2k
. (t−
t=k+1 t
t1k)
(t
t
)2
k
T
k
i=1
t=1
i=1
t=k+1
−2k
−
Emerson and Kao (2001) show that under H0 : γ = 0, if |ρ| < 1, then (1 −
2
ρ) σ2
v
d
P(λ)(1 −λ)3 −λ3 [P(1)
WOLS(k)
2
h
h
−
2
P(λ) + W(λ) −λW(1)]
3σε
→
ii 1
,
λ3(1 −λ)3 (1 −
3
λ) + λ3
2 R
where
−
λ
P(λ) = λW(λ)
2
W(s)ds
0
. However, Emerson and Kao (2001) show that under H0 : γ =
0, if ρ = 1, h
3
R
R
i
2
2
1
λ
Q(λ) (1 −λ) −Q(1) −Q(λ)
)ds +
W
ds r3
σ
d
−λ
W(s
(s)
0
0
v
WOLS(k)
3T 2σ2ε
→
h
h
ii
,
1/2
λ3 (1 −
3
3
λ)
(1 −λ) + λ3 R
h
i
where
λ
R
h
i
Q(λ) = 2
s W(s) + W
f(π) ds −
λ
λ
W(s) + W
f(π) ds
0
0
and W
f(π) is a different Wiener
process from W(s). These imply that, under the null, WOLS(k) = Op (1) when vit is I (0) and
WOLS(k)
when vit is I (1). In view of this and given that the order of integration of vit is not
→∞ 3 known in practice, it is natural to consider a robust test procedure. In a pure time series model,
Perron and Yabu (2009b) suggest a generalized least squares (GLS) procedure using a super-efficient
estimate of the autoregressive parameters as a robust test. Following the idea of Perron and Yabu
(2009b), we propose a robust test using the FE-FGLS estimator for the panel data model discussed
in BKL and modified for the case of structural change in the next section. 2.1
The FE-FGLS Estimator Rewrite equation (1) in matrix notation as y = διnT + Z′Ψ + u
(3) (3) where u = Zµµ + ν, µ is an n × 1 vector of µi, ν
is an nT × 1 vector of νit, Zµ = In ⊗ιT ,
where In is an identity matrix of dimension n, ιT is a vector of ones of dimension T and ⊗denotes
Kronecker product. y is an nT × 1 vector of yit, Z = ιn ⊗Zi where ιn is a vector of ones of
dimension n and Zi = (xi, DTi) where xi is a T × 1 vector of (1, 2, . . . , T) and DTi is a T × 1
vector of (0, . . . , 0, 1, . . . , T −k). ιnT is a vector of ones of dimension nT. Ψ = (β, γ)′. In order to
save space, the FE-GLS estimator of
, ˆ
Ψ ΨFE−GLS, is given in Equation (A5) in the supplemental
Appendix. Define R = (0, 1), the null hypothesis H0 can be rewritten into RΨ = 0. Therefore, the
Wald statistic of the FE-GLS estimator is h
i h
i
i
ˆ
′
ˆ
−1 h
ˆ
WFE−GLS(k) = R ΨFE
−Ψ
RV ar ΨFE
R′
−GLS
R Ψ
Ψ
. (4)
−GLS
FE−GLS − (4) The Wald statistic based on the FE-GLS depends on ρ. A popular estimator of ρ suggested in
Baltagi and Li (1991) is given by Pn
PT
i=1 P=2 νˆitνˆ
P
t
i,t−1
ρ˜ =
,
(5)
n
T
i=1
t=2 νˆ2
i,t−1 (5)
where νˆit is the within residual in (3). For a panel trend model, BKL show that
√
1+ρ
d
nT
ρ˜
ρ +
−
T
→
N 0, 1 −ρ2
if |ρ < 1 and √
|
,
nT ρ˜ −1 + 3
→
d N 0, 51
T
5
if ρ = 1 as (n, T) →∞. When |ρ| < 1,
a bias-corrected estimator of ρ is ρ˜ + 1+ρ˜. When ρ = 1, a bias-corrected estimator of ρ is ρ˜ + 3
T
T . Therefore, we use the bias-corrected estimator ρˆ suggested by BKL as follows:
1+ρˆ
ρ˜ +
1
T
if
ρˆ =
−ρ˜ > 3
T
. 2.1
The FE-FGLS Estimator λ∗
2 Theorem 1 implies that WFE−GLS(k) converges to a χ2 (1) for both ρ < 1
| |
and ρ = 1. 2.1
The FE-FGLS Estimator (6)
1
if 1 −ρ˜ ≤3
T (6) Since change point k is unknown, following Vogelsang (1997), we consider three statistics: supWFE−GLS(k),
MeanWFE−GLS(k), and ExpWFE−GLS(k), where supWFE−GLS(k) =
sup
WFE
(k),
[Tλ∗]
k
T
[Tλ∗]
−GLS
≤≤−
T−[Tλ∗]
1
X
MeanWFE−GLS(k) =
WFE
k
T
−GLS( ),
k=[Tλ∗]
T
1
−X
[Tλ∗]
1
ExpWFE−GLS(k) = log
exp
WFE
) ,
T
2
−GLS(k
k=[Tλ∗] supWFE−GLS(k) =
sup
WFE
(k),
[Tλ∗]
k
T
[Tλ∗]
−GLS
≤≤− T−[Tλ∗]
1
X
MeanWFE−GLS(k) =
WFE
k
T
−GLS( ),
k=[Tλ∗]
T
1
−X
[Tλ∗]
1
ExpWFE−GLS(k) = log
exp
WFE
) ,
T
2
−GLS(k
k=[Tλ∗] and λ∗is the fraction of trimming. In a pure time series, the trimming parameter λ∗is chosen
to be 0.01 or 0.15 so that there are enough observations before and after the break dates. In a
panel data, the break date k could be chosen between 2 and T −1. The asymptotic properties are
summarized in the following Theorem: Theorem 1 For ρˆ, under the null hypothesis
→
d
H0, we have WFE
FGLS(k)
G(λ)
−
∼χ2 (1) for
both |ρ| < 1 and ρ = 1 as (n, T) →∞, where Theorem 1 For ρˆ, under the null hypothesis
→
d
H0, we have WFE
FGLS(k)
G(λ)
−
∼χ2 (1) for
both |ρ| < 1 and ρ = 1 as (n, T) →∞, where
L2
0 (λ)
if ρ < 1,
G(λ) =
| |
L2
1 (λ)
if ρ = 1,
L2
0 (λ)
if ρ < 1,
G(λ) =
| |
L2
1 (λ)
if ρ = 1, h
i
√
where
3
0
2
R
0
0
1
L0 (λ) =
λ3/2
3/2 H (λ)
(1
λ)
2
−
−(1 + 2λ) (1 −λ) H (0)
with H (λ) =
(r
λ
λ
−
) dW −
(1−λ)
R
W (1) and H0 (0) =
rdW −1W (1). L1 (λ) =
1
(1)
2
2
λ1/2
−
1/2 [λW
(1
λ)
−W (λ)]. Furthermore, supWFE
GLS(k) →
d
sup
G(λ),
−
λ∗≤λ≤1−λ∗
Z 1
λ
d
−
∗
MeanWFE
(k)
G(λ)dλ,
−GLS
→
λ
∗
! Z 1
λ
d
−
∗
1
ExpWFE−GLS(k) →log
exp
G(λ)
dλ
. 2.2
Local Asymptotic Power In order to compare the power of the statistics, the following local alternatives were used. When
|ρ| < 1, the local alternatives are given by H1 : γ = n−1/2T −3/2γ0, where γ0 is a nonzero constant. When ρ = 1, the local alternatives are given by H
1/2
1/2
1 : γ = n−
T −
γ0. The local asymptotic
power properties are given in the following Theorem. 5 Theorem 2 When |ρ| < 1, under the local alternative hypothesis H1 : γ = n−1/2T −3/2γ0, where γ0
is a nonzero constant, we have
d
2
WFE
FGLS(k) →H0 (λ) ≡[L0 (λ) + M0 (λ)] as (n, T) →∞, where
−
L0 (λ) is defined in Theorem 1 and M0 (λ) = 3−1/2σ−1
e
(1 −ρ) λ3/2 (1 −
3/2
λ)
γ0. Furthermore, Theorem 2 When |ρ| < 1, under the local alternative hypothesis H1 : γ = n−1/2T −3/2γ0, where γ0
is a nonzero constant, we have
d
2
WFE
FGLS(k) →H0 (λ) ≡[L0 (λ) + M0 (λ)] as (n, T) →∞, where
−
L0 (λ) is defined in Theorem 1 and M0 (λ) = 3−1/2σ−1
e
(1 −ρ) λ3/2 (1 −
3/2
λ)
γ0. Furthermore, →
d
supWFE−GLS(k)
sup
H0 (λ) ,
λ∗≤λ≤1−λ∗
Z 1
d
−λ∗
MeanWFE−GLS(k) →
H0 (λ) dλ,
λ
∗
Z 1
λ
! −
∗
xp
→
d
1
E
WFE
GLS(k)
log
exp
H (
−
0 λ)
dλ
. λ∗
2 →
d
supWFE−GLS(k)
sup
H0 (λ) ,
λ∗≤λ≤1
λ∗ Z 1
d
−λ∗
MeanWFE−GLS(k) →
H0 (λ) dλ,
λ
∗
Z 1
λ
! −
∗
xp
→
d
1
E
WFE
GLS(k)
log
exp
H (
−
0 λ)
dλ
. λ∗
2 When ρ = 1, under the local alternative hypothesis H1 : γ = n−1/2T −1/2γ0, where γ0 is a nonzero
constant, we have
d
2
WFE
FGLS(k) →H1 (λ) ≡[L1 (λ) + M1 (λ)]
as (n, T)
−
→∞, where L1 (λ) is
defined in Theorem 1 and M1 (λ) = σ−1
1/2
e λ1/2 (1 −λ)
γ0. Furthermore, d
supWFE−GLS(k) →
sup
H1 (λ) ,
λ∗≤λ≤1−λ∗
Z 1
d
−λ∗
MeanWFE
GLS(k) →
H1 (λ) dλ,
−
λ
∗
Z 1
! −λ∗
d
1
ExpWFE−GLS(k) →log
exp
H1 (λ)
dλ
. 2.2
Local Asymptotic Power λ∗
2 d
supWFE−GLS(k) →
sup
H1 (λ) ,
λ∗≤λ≤1−λ∗ As we can see from Theorem 2, the local asymptotic power depends on σ2
e, γ0 and λ. To be
specific, both M0 (λ) and M1 (λ) are functions of λ (1 −λ), which is maximized at λ = 0.5. If λ is
close to 0 or 1, the break date is close to the boundary and is harder to detect. When |ρ| < 1, the
local asymptotic power also depends on the value of ρ. When ρ increases towards 1, 1−ρ decreases
and hence the asymptotic power decreases, too. Furthermore, when |ρ| < 1, the local alternatives
are given by H1 : γ = n−1/2T −3/2γ0. Hence large T improves the asymptotic power by a bigger
margin than n does. When ρ = 1, the local alternatives are given by H
1/2
1/2
1 : γ = n−
T −
γ0. Comparing to the case of |ρ| < 1, we know that for the same sample sizes n and T, the asymptotic
power is smaller when ρ = 1. Theorem 3 When |ρ| < 1,
n1/2T 3/2
ˆΨFE
GLS −Ψ
=
n−1T −3F1,Z
−1
n−1/2T −3/2F
−
2,Z
+ op (1) ,
nT 3
ˆ
1
V ar Ψ
1
FE
=
n−T −3F
−GLS
1,Z
−+ op (1) . When ρ = 1, When ρ = 1,
ˆ
n1/2T 1/2
ΨFE
GLS −Ψ
=
n−1T −1F1,Z
−1
n−1/2T −1/2F
−
2,Z
+ op (1) ,
ˆ
1
nTV ar Ψ
1
1
FE
GLS
=
n−T −F1,Z
−+ o
−
p (1) . Therefore, the asymptotic distribution ˆΨFE−GLS is not changed when additional stationary or
nonstationary regressors are included in the regression. Hence, for the hypothesis H0: RΨ = 0, the
corresponding Wald test has the same asymptotic properties given in Theorems 1 and 2. 3.1
Generalization of the Independent Variables When other regressors are included in the model, Equation (1) becomes yit = δ + βt + γDT + θ1w1,it + θ2w2,it + µi + vit,
(7) (7) 6 where w1,it and w2,it are nonstationary and stationary variables, respectively. Without loss of
generality, let us assume w1,it = w1,i,t
+ ϵ
−1
it with ϵit ∼iid 0, σ2
1
and w2,it ∼iid 0, σ2
2 . Rewrite
in matrix notation as y = διnT + Z′Ψ + W ′Θ + u
(8) (8) where W = (W1, W2, . . . , Wn)′, Wi = (Wi1, Wi2, . . . , WiT )′, Wit = (w1,it, w2,it) and Θ = (θ1, θ2)′. ιnT is a vector of ones of dimension nT. Ψ = (β, γ)′. The FE-GLS estimators of Ψ and Θ are given
by where W = (W1, W2, . . . , Wn)′, Wi = (Wi1, Wi2, . . . , WiT )′, Wit = (w1,it, w2,it) and Θ = (θ1, θ2)′. ιnT is a vector of ones of dimension nT. Ψ = (β, γ)′. The FE-GLS estimators of Ψ and Θ are given
by
ˆ
1
ΨFE−GLS −Ψ
Z∗
′
−
Z∗
′
=
(I
α
W ∗
(I
α
ˆ
n
ET ) (Z∗,
)
n
ET ) v∗
(9)
ΘFE−GLS −Θ
W ∗
⊗
W ∗
⊗
−1
F
1,Z
F1,ZW
F
=
2,Z ,
(10)
F1,WZ
F1,W
F2,W (9) (10) where F
= Z∗′ (I ⊗Eα) Z∗, F
= Z∗′ (I ⊗Eα
α
1,Z
n
T
2,Z
n
T ) v∗, F1,W = W ∗′ (In ⊗ET ) W ∗, F1,ZW =
Z∗′ (In ⊗Eα
α
α
T ) W ∗, F2,W = W ∗′ (In ⊗ET ) y∗and W ∗= (In ⊗C) W with ET and C defined in
Equations (A4) and (A5) in the supplemental Appendix. Therefore, we have
1
ˆ
−
ΨFE−GLS −Ψ =
F1,Z −F
1
−1
1,ZW F ,W F ,ZW
F
1
1
2,Z −F1,ZW F1
−
,W F2,W
. (11) (11) Theorem 3 When |ρ| < 1, Theorem 3 When |ρ| < 1, 3.2
Generalization of the Error Component We now consider an extension of the analysis to the case where the error term νit is allowed to have
a more general structure. Following Perron and Yabu (2009a, 2009b), we can modify Equation (2) 7 7 into νit
=
ρνi,t−1 + ϵit
(12)
ϵit
=
d (L) eit
(13) νit
=
ρνi,t−1 + ϵit
(12)
ϵit
=
d (L) eit
(13) (12) (13) ϵit
=
d (L) eit
(13) P
P
with d (L) =
∞
=0 dkLk
k
,
∞
k=0 k |dk| < ∞, d (1)
0
= ,
ei
and
t is a white noise process with variance
σ2
e. Under these conditions, νit has an autoregressive representation, say A (L) vit = eit, where
P
A (L) = 1−
∞
k=0 akLk. Let the parameter ρ represent the sum of the AR coefficients. Accordingly,
we have the representation
̸ νit = ρνi,t−1 + A∗(L) ∆vi,t−1 + eit, νit = ρνi,t−1 + A∗(L) ∆vi,t−1 + eit, P
P
where A∗(L) =
∞
k=0 a∗
kLk with a∗
k = −
∞
j=k+2 aj. Based on a truncated autoregression of order
K, an estimate ˜
˜
θ =
ρ,˜ ζ1, · · · ˜
′
, ζK
can be obtained from the following regression P
P
where A∗(L) =
∞
k=0 a∗
kLk with a∗
k = −
∞
j=k+2 aj. Based on a truncated autoregression of order
K, an estimate ˜
˜
θ =
ρ,˜ ζ1, · · · ˜
′
, ζK
can be obtained from the following regression P
P
where A∗(L) =
∞
k=0 a∗
kLk with a∗
k = −
∞
j=k+2 aj. Based on a truncated autoregression of order
K, an estimate ˜
˜
θ =
ρ,˜ ζ1, · · · ˜
′
, ζK
can be obtained from the following regression X
K
νˆit = ρνˆi,t
1 +
ζk∆νˆi,t
k + eit,
−
−
k=1 where νˆit is the within residual in (3). If K →∞and K3/T →0 as T →∞, we have the following
theorem: where νˆit is the within residual in (3). If K →∞and K3/T →0 as T →∞, we have the following
theorem: Theorem 4 Assume (n, T) →∞, 3.3
A Special Case: Without Individual Effects Let us consider a special case where µi = 0 for all i, i.e., there are no individual effects in the panel
data model. The variance-covariance matrix of equation (1) is
Φ = E uu′ = σ2
e (In ⊗A) . The least squares estimator of the transformed equation yields the GLS estimator: The least squares estimator of the transformed equation yields the GLS estimator: h
i
1
ˆ
−
ΨGLS = Z∗′Mι∗Z
Z
nT
∗
∗′Mι∗y
nT
∗,
(14) (14) where
¯
Mι∗
= InT −
¯
ι∗
−
α
nT (ι∗′
nT ι∗
nT ) ι∗′
nT = I
J
α
nT
n ⊗JT using the fact that ι∗
nT = (1 −ρ) (ιn ⊗ι )
nT
T .
It is easy to see that Mι∗Z
nT
∗
¯
=
−¯Jn ⊗¯
I
Jα
T
(ιn ⊗zi
∗) = ι
α
nT
n ⊗zi
∗−JT zi
∗= (In ⊗Eα
T ) z∗. This
proves that ˆΨGLS and ˆΨFE−GLS are the same if there are no individual effects in the model. The
Wald-statistic based on ˆΨGLS is in turn the same as the one based on ˆΨFE−GLS in equation (4). Similar to equation (5) for the general model with individual effects, let where
¯
Mι∗
= InT −
¯
ι∗
−
α
nT (ι∗′
nT ι∗
nT ) ι∗′
nT = I
J
α
nT
n ⊗JT using the fact that ι∗
nT = (1 −ρ) (ιn ⊗ι )
nT
T .
It is easy to see that Mι∗Z
nT
∗
¯
=
−¯Jn ⊗¯
I
Jα
T
(ιn ⊗zi
∗) = ι
α
nT
n ⊗zi
∗−JT zi
∗= (In ⊗Eα
T ) z∗. This
proves that ˆΨGLS and ˆΨFE−GLS are the same if there are no individual effects in the model. The
ˆ
ˆ where
¯
Mι∗
= InT −
¯
ι∗
−
α
nT (ι∗′
nT ι∗
nT ) ι∗′
nT = I
J
α
nT
n ⊗JT using the fact that ι∗
nT = (1 −ρ) (ιn ⊗ι )
nT
T .
It is easy to see that Mι∗Z
nT
∗
¯
=
−¯Jn ⊗¯
I
Jα
T
(ιn ⊗zi
∗) = ι
α
nT
n ⊗zi
∗−JT zi
∗= (In ⊗Eα
T ) z∗. This
proves that ˆΨGLS and ˆΨFE−GLS are the same if there are no individual effects in the model. Theorem 4 Assume (n, T) →∞, 1. If |ρ| < 1,
√
T (ρ˜ −ρ) →
p 0. 1. If |ρ| < 1, 1. If |ρ| < 1, √
T (ρ˜ −ρ) →
p 0. 2. If ρ = 1,
T (ρ˜ −
p
3
1) →−
. 1 −ζ1 −· · · −ζK 2. If ρ = 1, T (ρ˜ −
p
3
1) →−
. 1 −ζ1 −· · · −ζK In Theorem 4, the asymptotic bias of ρ˜ depends on ζ1, . . . , ζK when ρ = 1. When ζ1 = . . . =
ζK = 0, the asymptotic bias reduces to -3 as in BKL. Similar to Equation (6), a bias-corrected
h
i
estimator ρˆ can be obtained replacing the threshold by
˜
˜
3/ T
1 −ζ1 −· · · −ζK
. Using ρˆ, the
Wald test statistics can be obtained from Equation (4). To calculate the Wald test statistics, we
need an estimate of σ2
ϵ, i.e., the variance of the error term ϵit. When ν
2
it is I(0) or I(1), σϵ can be
estimated following Equations (12) and (13) in Perron and Yabu (2009b), respectively. 8 4
Monte Carlo Results This section reports the results of Monte Carlo experiments designed to investigate the finite sample
size and power properties of the proposed test statistic. The model is generated by yit = 5 + 10t + γDT + µi + vit,
i = 1, . . . , n,
t = 1, . . . , T,
(16) (16) where DT = 1 (t > k) (t −k) with a change point k = 0.5T. iid
µi ∼N (0, 5), νit = ρνit−1 + eit
with vi0 = 0 and
iid
eit ∼N (0, 5). γ varies over the range (0, 0.02, 0.04, 0.06, 0.08, 0.1). ρ varies
over the range (0, 0.2, 0.4, 0.6, 0.8, 0.9, 1). The sample sizes considered are n = (50, 200, 500) and
T = (20, 50). For each experiment, we perform 1, 000 replications. For each replication we estimate
the model using: (i) FD: first-difference ignoring serial correlation; (ii) FE: fixed-effects ignoring
serial correlation; (iii) FE-GLS: FE-GLS estimator using the true value of ρ; (iv) FE-FGLS1: FE-
FGLS estimator using ρ˜ calculated by the method suggested in Baltagi and Li (1991); and (v)
FE-FGLS2: FE-FGLS estimator using a bias-corrected estimator ρˆ. Tables 1 and 6 report the size
and size-adjusted power of the Wald-test for H0 : γ = 0 corresponding to each estimator of γ. In
Table 1 for example, the size of the Wald-test based on the FD estimator is 0.000 when the true
value of ρ = 0 and the size of the Wald-test based on the FE estimator is 0.251 when the true
value of ρ = 1. By comparison, the size of the Wald-test based on the FE-GLS estimator is always
close to 0.05. Using ρ˜ in Baltagi and Li (1991), the size of the Wald-test based on the FE-FGLS1
estimator is larger than 0.05 when the true value of ρ is large. For example, when ρ = 1, the size of
the Wald-test based on the FE-FGLS1 estimator is 0.060 when (n, T) = (500, 20) and 0.087 when
(n, T) = (500, 50). Using the bias-corrected estimator ρˆ, the size of the Wald-test based on the
FE-FGLS2 estimator performs better. For example, when ρ = 1, the size of the Wald-test based on
the FE-FGLS2 estimator is 0.042 when (n, T) = (500, 20) and 0.053 when (n, T) = (500, 50). 2We thank a referee pointing out that the Wald-test based on true FE-GLS has size distortion when ρ is large.
The size performance is actually worse than the Wald-test based on FE-FGLS2. In a pure time series frame work,
Theorem 2 of Perron and Yabu (2009b) derived the asymptotic distribution of the Wald test based on GLS estimator
in case of local to unity. They find that “a conservative test may be expected for values of α (ρ in our notation) close
to 1, relative to the sample size.” The FGLS estimator has the same asymptotic distribution as the FD estmiator
in the local to unity case. Hence FGLS performs better. Our simulations find similar results in a panel data frame
work. 3.3
A Special Case: Without Individual Effects The
Wald-statistic based on ˆΨGLS is in turn the same as the one based on ˆΨFE−GLS in equation (4). Similar to equation (5) for the general model with individual effects, let Pn
PT
uˆituˆi,t
1
ρˆ =
Pi=1
t=2
−
n
P
,
(15)
T
i=1
t=2 uˆ2
i,t−1 (15) where uˆit denotes the OLS residuals. Define uˆ∗as an nT × 1 vector of OLS residuals from the
Prais-Winsten transformed regression using ρˆ. An estimator of σ2 is σˆ2
1
e
e =
uˆ
nT
∗′uˆ∗. Substituting
ρˆ and σˆ2
e, the Wald-statistic corresponding to the FGLS estimator can be obtained from equation
(4). BKL show that then
√
−
→
d
→
d
nT (ρˆ
ρ)
N 0, 1 −ρ2
if |ρ| < 1, and √nT (ρˆ −1)
N (0, 3) if
ρ = 1 as (n, T) →∞. Therefore, when µi = 0 for all
→
d
i, for ρˆ, WFGLS(k)
χ2 (1) when |ρ| < 1
or ρ = 1 as (n, T) →∞. Hence WFGLS(k) converges to χ2 (1) whether the error term is I(0)
or I(1) when there are no individual effects in the model. This is an interesting result, i.e., the
Wald test based on FGLS effectively bridges the gap between the I(0) and I(1) error terms, if
there are no individual effects in the model. This implies that inference on the slope parameter
can be performed using the standard normal distribution if there are no individual effects. This is
different from the pure time series model discussed in Perron and Yabu (2009b) which requires a
super-efficient estimate in order to achieve this goal. 9 9 4
Monte Carlo Results This
finding is consistent with the asymptotic results in Theorem 1.2 The size-adjusted power increases
as γ increases. 10 Finally, we examine the performance of the proposed Wald test that is based on the FE-FGLS
estimator when the change point is treated as unknown. Tables 7—12 report the size and size-
adjusted power of the supWFE
GLS, MeanWFE
GLS and ExpW
−
−
FE−GLS tests. As discussed
before, the break date k is chosen between 2 and T −1. Following Perron and Yabu (2009b),
asymptotic critical values are calculated by simulations using random standard normal variables to
approximate the Wiener process. The integrals are approximated by normalized sums with 2,000
steps, and 10,000 replications are used. The Monte Carlo results show that the MeanWFE−GLS
test performs the best. Its size is always close to 0.05 for different values of ρ. When the sample
sizes are small, ExpWFE
GLS has relatively large size distortion. However, when the sample sizes
−
increase to (n, T) = (500, 50), the size of ExpWFE
GLS shown in Table 12 is also close to 0.05. In
−
contrast, Table 12 for example shows that the size of supWFE
GLS is still as large as 0.095 when
−
ρ = 0.9.3 In conclusion, this paper recommends the MeanWFE−GLS test for testing structural change in
a time trend panel data model when the change point is treated as unknown. In case, the change
point is known, we recommend the Wald-test based on the FE-FGLS2 estimator. 3Perron and Yabu (2009b), page 375, also showed that sup diverges to infinity as T →∞. Our simulations find
similar results in a panel data frame work. References [1] Baltagi, B. H., and Li, Q. (1991), “A Transformation that will Circumvent the Problem of
Autocorrelation in an Error-Component Model,” Journal of Econometrics, 48, 385-393. [2] Baltagi, B. H., Kao, C., and Liu, L. (2014), “Test of Hypotheses in a Time Trend Panel Data
Model with Serially Correlated Error Component Disturbances,”Advances in Econometrics,
Essays in Honor of Peter C.B. Phillips, 33, 347-394. [3] Baltagi, B. H., Kao, C., and Liu, L. (2017), “Estimation and Identification of Change Points
in Panel Models with Nonstationary or Stationary Regressors and Error Term,”Econometric
Reviews, 36, 85-102. [4] Emerson, J., and Kao, C. (2001),“Testing for Structural Change of a Time Trend Regression
in Panel Data: Part I,” Journal of Propagations in Probability and Statistics, 2, 57-75. 3Perron and Yabu (2009b), page 375, also showed that sup diverges to infinity as T →∞. Our simulations find
similar results in a panel data frame work. 11 11 [5] Kim, D. (2011), “Estimating a Common Deterministic Time Trend Break in Large Panels with
Cross Sectional Dependence,” Journal of Econometrics, 164, 310-330. [6] Perron, P., and Yabu, T. (2009a), “Estimating Deterministic Trends with an Integrated or
Stationary Noise Component,” Journal of Econometrics, 151, 56-69. [7] Perron, P., and Yabu, T. (2009b), “Testing for Shifts in Trend With an Integrated or Stationary
Noise Component,” Journal of Business & Economic Statistics, 27, 369-395. [8] Perron, P., and Zhu, X. (2005), “Structural Breaks with Deterministic and Stochastic Trends,”
Journal of Econometrics, 129, 65—119. [9] Phillips, P.C.B., and Lee, C.C. (1996), “Efficiency Gains From Quasi-Differencing Under Non-
stationarity,” in Athens Conference on Applied Probability and Time Series, Volume II: Time
Series Analysis in Memory of E.J. Hannan, eds. P. M. Robinson and M. Rosenblatt. Lecture
Notes in Statistics, New York: Springer-Verlag, 115, 300—313. [10] Phillips, P.C.B., and Moon, H. (1999), “Linear Regression Limit Theory for Nonstationary
Panel Data,” Econometrica 67, 1057—1111. [11] Phillips, P.C.B., and Moon, H. (2000), “Nonstationary Panel Data Analysis: An overview of
Some Recent Developments,” Econometric Reviews 19, 263—286. [12] Vogelsang, T. J. (1997), “Wald-Type Tests for Detecting Breaks in the Trend Function of a
Dynamic Time Series,” Econometric Theory, 13, 818-849. References 12 12 Table 1: Size and Size-Adjusted Power of the Wald Test at a Known Date (n = 50, T = 20) ρ
γ
FD
FE
FE-GLS
FE-FGLS1
FE-FGLS2
0
0.00
0.000
0.043
0.043
0.042
0.030
0
0.02
0.055
0.082
0.082
0.088
0.084
0
0.04
0.081
0.149
0.149
0.150
0.150
0
0.06
0.117
0.267
0.267
0.272
0.272
0
0.08
0.161
0.405
0.405
0.408
0.407
0
0.10
0.227
0.579
0.579
0.582
0.582
0.2
0.00
0.000
0.099
0.043
0.048
0.031
0.2
0.02
0.044
0.075
0.066
0.069
0.068
0.2
0.04
0.064
0.121
0.122
0.120
0.120
0.2
0.06
0.099
0.191
0.200
0.202
0.198
0.2
0.08
0.138
0.304
0.305
0.312
0.311
0.2
0.10
0.200
0.430
0.425
0.434
0.430
0.4
0.00
0.000
0.167
0.039
0.053
0.033
0.4
0.02
0.049
0.064
0.059
0.061
0.061
0.4
0.04
0.071
0.088
0.097
0.101
0.105
0.4
0.06
0.098
0.150
0.142
0.144
0.144
0.4
0.08
0.131
0.217
0.225
0.220
0.226
0.4
0.10
0.193
0.293
0.304
0.305
0.307
0.6
0.00
0.000
0.248
0.033
0.066
0.031
0.6
0.02
0.044
0.063
0.059
0.058
0.060
0.6
0.04
0.064
0.074
0.076
0.078
0.077
0.6
0.06
0.087
0.114
0.115
0.119
0.117
0.6
0.08
0.112
0.156
0.161
0.165
0.163
0.6
0.10
0.166
0.214
0.224
0.228
0.219
0.8
0.00
0.006
0.317
0.036
0.090
0.047
0.8
0.02
0.058
0.057
0.050
0.062
0.050
0.8
0.04
0.059
0.067
0.063
0.071
0.068
0.8
0.06
0.069
0.084
0.077
0.089
0.082
0.8
0.08
0.090
0.115
0.100
0.118
0.100
0.8
0.10
0.127
0.148
0.132
0.144
0.137
0.9
0.00
0.032
0.334
0.040
0.090
0.057
0.9
0.02
0.056
0.053
0.052
0.052
0.055
0.9
0.04
0.059
0.061
0.058
0.057
0.059
0.9
0.06
0.064
0.072
0.067
0.073
0.072
0.9
0.08
0.085
0.085
0.085
0.092
0.086
0.9
0.10
0.106
0.101
0.113
0.122
0.121
1
0.00
0.043
0.251
0.043
0.067
0.043
1
0.02
0.052
0.050
0.052
0.044
0.052
1
0.04
0.057
0.053
0.057
0.056
0.060
1
0.06
0.065
0.064
0.065
0.061
0.064
1
0.08
0.077
0.078
0.077
0.074
0.078
1
0.10
0.097
0.097
0.097
0.097
0.098 13 13 Table 2: Size and Size-Adjusted Power of the Wald Test at a Known Date (n = 50, T = 50) ρ
γ
FD
FE
FE-GLS
FE-FGLS1
FE-FGLS2
0
0.00
0.000
0.045
0.045
0.044
0.037
0
0.02
0.090
0.392
0.392
0.392
0.390
0
0.04
0.209
0.906
0.906
0.908
0.908
0
0.06
0.468
1.000
1.000
1.000
1.000
0
0.08
0.719
1.000
1.000
1.000
1.000
0
0.10
0.902
1.000
1.000
1.000
1.000
0.2
0.00
0.000
0.116
0.045
0.048
0.043
0.2
0.02
0.087
0.284
0.288
0.289
0.288
0.2
0.04
0.209
0.755
0.763
0.761
0.759
0.2
0.06
0.460
0.986
0.989
0.985
0.985
0.2
0.08
0.709
1.000
1.000
1.000
1.000
0.2
0.10
0.895
1.000
1.000
1.000
1.000
0.4
0.00
0.000
0.194
0.045
0.051
0.041
0.4
0.02
0.085
0.187
0.188
0.173
0.171
0.4
0.04
0.201
0.532
0.544
0.529
0.528
0.4
0.06
0.415
0.873
0.883
0.873
0.878
0.4
0.08
0.671
0.990
0.993
0.989
0.990
0.4
0.10
0.865
1.000
1.000
1.000
1.000
0.6
0.00
0.000
0.296
0.038
0.056
0.036
0.6
0.02
0.081
0.124
0.117
0.118
0.119
0.6
0.04
0.174
0.322
0.335
0.328
0.331
0.6
0.06
0.348
0.581
0.623
0.614
0.612
0.6
0.08
0.563
0.827
0.863
0.855
0.860
0.6
0.10
0.767
0.949
0.965
0.965
0.964
0.8
0.00
0.000
0.455
0.027
0.055
0.033
0.8
0.02
0.081
0.072
0.080
0.081
0.079
0.8
0.04
0.153
0.146
0.173
0.173
0.171
0.8
0.06
0.250
0.267
0.306
0.315
0.304
0.8
0.08
0.406
0.413
0.475
0.471
0.472
0.8
0.10
0.559
0.571
0.656
0.653
0.658
0.9
0.00
0.007
0.510
0.029
0.076
0.044
0.9
0.02
0.066
0.066
0.069
0.066
0.064
0.9
0.04
0.111
0.108
0.099
0.106
0.100
0.9
0.06
0.179
0.159
0.181
0.186
0.190
0.9
0.08
0.267
0.243
0.268
0.264
0.272
0.9
0.10
0.374
0.353
0.383
0.384
0.387
1
0.00
0.048
0.482
0.048
0.077
0.046
1
0.02
0.055
0.061
0.055
0.058
0.056
1
0.04
0.068
0.089
0.068
0.077
0.073
1
0.06
0.103
0.127
0.103
0.108
0.106
1
0.08
0.143
0.171
0.143
0.153
0.146
1
0.10
0.209
0.219
0.209
0.217
0.214 14 14 Table 3: Size and Size-Adjusted Power of the Wald Test at a Known Date (n = 200, T = 20) ρ
γ
FD
FE
FE-GLS
FE-FGLS1
FE-FGLS2
0
0.00
0.000
0.048
0.048
0.047
0.042
0
0.02
0.071
0.119
0.119
0.122
0.119
0
0.04
0.146
0.353
0.353
0.352
0.350
0
0.06
0.291
0.689
0.689
0.688
0.683
0
0.08
0.481
0.909
0.909
0.908
0.904
0
0.10
0.671
0.976
0.976
0.976
0.976
0.2
0.00
0.000
0.096
0.048
0.053
0.042
0.2
0.02
0.070
0.094
0.096
0.095
0.098
0.2
0.04
0.158
0.253
0.256
0.247
0.255
0.2
0.06
0.285
0.531
0.533
0.526
0.526
0.2
0.08
0.469
0.786
0.783
0.775
0.779
0.2
0.10
0.670
0.933
0.936
0.932
0.932
0.4
0.00
0.000
0.162
0.048
0.060
0.042
0.4
0.02
0.083
0.083
0.083
0.083
0.088
0.4
0.04
0.149
0.169
0.174
0.179
0.175
0.4
0.06
0.278
0.332
0.373
0.373
0.379
0.4
0.08
0.441
0.563
0.605
0.606
0.612
0.4
0.10
0.640
0.778
0.795
0.804
0.802
0.6
0.00
0.002
0.236
0.037
0.069
0.039
0.6
0.02
0.068
0.071
0.079
0.076
0.079
0.6
0.04
0.128
0.124
0.136
0.131
0.135
0.6
0.06
0.229
0.222
0.255
0.245
0.259
0.6
0.08
0.350
0.357
0.420
0.414
0.415
0.6
0.10
0.521
0.544
0.594
0.586
0.594
0.8
0.00
0.010
0.308
0.037
0.082
0.051
0.8
0.02
0.062
0.058
0.064
0.058
0.061
0.8
0.04
0.115
0.093
0.114
0.101
0.108
0.8
0.06
0.170
0.146
0.171
0.164
0.165
0.8
0.08
0.266
0.225
0.265
0.259
0.259
0.8
0.10
0.382
0.326
0.389
0.370
0.383
0.9
0.00
0.036
0.337
0.047
0.100
0.062
0.9
0.02
0.060
0.073
0.059
0.057
0.059
0.9
0.04
0.092
0.088
0.090
0.086
0.084
0.9
0.06
0.132
0.131
0.133
0.126
0.129
0.9
0.08
0.212
0.184
0.212
0.202
0.209
0.9
0.10
0.304
0.271
0.312
0.296
0.304
1
0.00
0.060
0.246
0.060
0.084
0.059
1
0.02
0.055
0.059
0.055
0.055
0.055
1
0.04
0.078
0.077
0.078
0.074
0.079
1
0.06
0.113
0.104
0.113
0.115
0.113
1
0.08
0.162
0.157
0.162
0.162
0.162
1
0.10
0.252
0.223
0.252
0.242
0.248 15 Table 4: Size and Size-Adjusted Power of the Wald Test at a Known Date (n = 200, T = 50) ρ
γ
FD
FE
FE-GLS
FE-FGLS1
FE-FGLS2
0
0.00
0.000
0.038
0.038
0.040
0.033
0
0.02
0.245
0.917
0.917
0.915
0.914
0
0.04
0.722
1.000
1.000
1.000
1.000
0
0.06
0.962
1.000
1.000
1.000
1.000
0
0.08
0.999
1.000
1.000
1.000
1.000
0
0.10
1.000
1.000
1.000
1.000
1.000
0.2
0.00
0.000
0.100
0.037
0.040
0.035
0.2
0.02
0.240
0.769
0.779
0.775
0.770
0.2
0.04
0.703
1.000
1.000
1.000
1.000
0.2
0.06
0.960
1.000
1.000
1.000
1.000
0.2
0.08
0.999
1.000
1.000
1.000
1.000
0.2
0.10
1.000
1.000
1.000
1.000
1.000
0.4
0.00
0.000
0.182
0.041
0.045
0.037
0.4
0.02
0.226
0.528
0.562
0.564
0.566
0.4
0.04
0.657
0.989
0.991
0.991
0.990
0.4
0.06
0.943
1.000
1.000
1.000
1.000
0.4
0.08
0.997
1.000
1.000
1.000
1.000
0.4
0.10
1.000
1.000
1.000
1.000
1.000
0.6
0.00
0.000
0.295
0.038
0.051
0.037
0.6
0.02
0.186
0.309
0.339
0.339
0.338
0.6
0.04
0.566
0.828
0.852
0.851
0.854
0.6
0.06
0.895
0.994
0.996
0.996
0.996
0.6
0.08
0.988
1.000
1.000
1.000
1.000
0.6
0.10
1.000
1.000
1.000
1.000
1.000
0.8
0.00
0.000
0.437
0.029
0.059
0.031
0.8
0.02
0.124
0.136
0.158
0.159
0.162
0.8
0.04
0.374
0.412
0.483
0.480
0.486
0.8
0.06
0.715
0.725
0.801
0.805
0.807
0.8
0.08
0.917
0.916
0.966
0.963
0.966
0.8
0.10
0.983
0.991
0.998
0.998
0.999
0.9
0.00
0.004
0.534
0.032
0.077
0.042
0.9
0.02
0.097
0.107
0.102
0.108
0.107
0.9
0.04
0.241
0.256
0.259
0.267
0.268
0.9
0.06
0.494
0.447
0.517
0.513
0.517
0.9
0.08
0.727
0.672
0.756
0.749
0.759
0.9
0.10
0.894
0.837
0.903
0.902
0.906
1
0.00
0.048
0.477
0.048
0.081
0.048
1
0.02
0.078
0.058
0.078
0.075
0.077
1
0.04
0.151
0.135
0.151
0.147
0.150
1
0.06
0.264
0.235
0.264
0.260
0.264
1
0.08
0.454
0.376
0.454
0.440
0.451
1
0.10
0.613
0.522
0.613
0.605
0.611 16 Table 5: Size and Size-Adjusted Power of the Wald Test at a Known Date (n = 500, T = 20) ρ
γ
FD
FE
FE-GLS
FE-FGLS1
FE-FGLS2
0
0.00
0.000
0.051
0.051
0.051
0.044
0
0.02
0.126
0.240
0.240
0.246
0.241
0
0.04
0.353
0.728
0.728
0.726
0.721
0
0.06
0.658
0.969
0.969
0.969
0.969
0
0.08
0.882
0.999
0.999
0.999
1.000
0
0.10
0.972
1.000
1.000
1.000
1.000
0.2
0.00
0.000
0.105
0.050
0.053
0.047
0.2
0.02
0.132
0.184
0.188
0.194
0.193
0.2
0.04
0.353
0.563
0.570
0.579
0.575
0.2
0.06
0.652
0.898
0.895
0.898
0.896
0.2
0.08
0.876
0.992
0.993
0.994
0.994
0.2
0.10
0.973
1.000
1.000
1.000
1.000
0.4
0.00
0.000
0.180
0.042
0.059
0.040
0.4
0.02
0.118
0.147
0.161
0.152
0.156
0.4
0.04
0.311
0.415
0.434
0.431
0.423
0.4
0.06
0.598
0.736
0.764
0.760
0.758
0.4
0.08
0.832
0.923
0.938
0.935
0.936
0.4
0.10
0.960
0.995
0.994
0.995
0.994
0.6
0.00
0.001
0.259
0.036
0.068
0.035
0.6
0.02
0.099
0.118
0.114
0.121
0.115
0.6
0.04
0.260
0.273
0.312
0.310
0.312
0.6
0.06
0.499
0.512
0.581
0.582
0.589
0.6
0.08
0.729
0.758
0.814
0.815
0.817
0.6
0.10
0.897
0.901
0.939
0.936
0.939
0.8
0.00
0.009
0.336
0.031
0.080
0.043
0.8
0.02
0.092
0.083
0.091
0.096
0.095
0.8
0.04
0.218
0.189
0.216
0.215
0.216
0.8
0.06
0.391
0.353
0.403
0.403
0.404
0.8
0.08
0.589
0.528
0.600
0.606
0.599
0.8
0.10
0.753
0.716
0.769
0.770
0.772
0.9
0.00
0.027
0.356
0.038
0.093
0.056
0.9
0.02
0.078
0.074
0.075
0.078
0.086
0.9
0.04
0.182
0.147
0.174
0.171
0.181
0.9
0.06
0.329
0.293
0.318
0.312
0.323
0.9
0.08
0.496
0.431
0.489
0.483
0.495
0.9
0.10
0.660
0.589
0.656
0.643
0.661
1
0.00
0.044
0.270
0.044
0.060
0.042
1
0.02
0.088
0.093
0.088
0.086
0.088
1
0.04
0.160
0.171
0.160
0.166
0.159
1
0.06
0.271
0.274
0.271
0.283
0.271
1
0.08
0.442
0.422
0.442
0.448
0.440
1
0.10
0.611
0.576
0.611
0.617
0.610 17 17 Table 6: Size and Size-Adjusted Power of the Wald Test at a Known Date (n = 500, T = 50) ρ
γ
FD
FE
FE-GLS
FE-FGLS1
FE-FGLS2
0
0.00
0.000
0.052
0.052
0.051
0.049
0
0.02
0.524
0.999
0.999
0.999
0.999
0
0.04
0.981
1.000
1.000
1.000
1.000
0
0.06
1.000
1.000
1.000
1.000
1.000
0
0.08
1.000
1.000
1.000
1.000
1.000
0
0.10
1.000
1.000
1.000
1.000
1.000
0.2
0.00
0.000
0.093
0.052
0.053
0.047
0.2
0.02
0.518
0.984
0.984
0.986
0.986
0.2
0.04
0.979
1.000
1.000
1.000
1.000
0.2
0.06
1.000
1.000
1.000
1.000
1.000
0.2
0.08
1.000
1.000
1.000
1.000
1.000
0.2
0.10
1.000
1.000
1.000
1.000
1.000
0.4
0.00
0.000
0.183
0.047
0.053
0.046
0.4
0.02
0.472
0.881
0.900
0.897
0.899
0.4
0.04
0.964
1.000
1.000
1.000
1.000
0.4
0.06
1.000
1.000
1.000
1.000
1.000
0.4
0.08
1.000
1.000
1.000
1.000
1.000
0.4
0.10
1.000
1.000
1.000
1.000
1.000
0.6
0.00
0.000
0.291
0.043
0.056
0.044
0.6
0.02
0.356
0.631
0.646
0.647
0.651
0.6
0.04
0.904
0.995
0.996
0.996
0.996
0.6
0.06
0.999
1.000
1.000
1.000
1.000
0.6
0.08
1.000
1.000
1.000
1.000
1.000
0.6
0.10
1.000
1.000
1.000
1.000
1.000
0.8
0.00
0.000
0.450
0.030
0.069
0.037
0.8
0.02
0.234
0.271
0.295
0.302
0.297
0.8
0.04
0.714
0.756
0.833
0.832
0.831
0.8
0.06
0.964
0.976
0.992
0.991
0.991
0.8
0.08
1.000
0.999
1.000
1.000
1.000
0.8
0.10
1.000
1.000
1.000
1.000
1.000
0.9
0.00
0.001
0.523
0.041
0.083
0.054
0.9
0.02
0.140
0.165
0.159
0.159
0.158
0.9
0.04
0.456
0.461
0.513
0.502
0.507
0.9
0.06
0.814
0.781
0.847
0.843
0.845
0.9
0.08
0.962
0.955
0.976
0.976
0.975
0.9
0.10
0.999
0.995
0.999
0.999
0.999
1
0.00
0.053
0.499
0.053
0.087
0.053
1
0.02
0.097
0.102
0.097
0.112
0.100
1
0.04
0.279
0.261
0.279
0.304
0.278
1
0.06
0.547
0.488
0.547
0.566
0.552
1
0.08
0.786
0.718
0.786
0.798
0.786
1
0.10
0.929
0.894
0.929
0.935
0.929 18 Table 7: Size and Size-Adjusted Power of the Wald Test at an Unknown Date (n = 50, T = 20) ρ
γ
sup-W
Mean-W
Exp-W
0
0.00
0.044
0.069
0.045
0
0.02
0.054
0.049
0.060
0
0.04
0.080
0.098
0.140
0
0.06
0.129
0.152
0.190
0
0.08
0.226
0.269
0.300
0
0.10
0.329
0.388
0.415
0.2
0.00
0.042
0.051
0.045
0.2
0.02
0.053
0.058
0.050
0.2
0.04
0.066
0.086
0.100
0.2
0.06
0.103
0.128
0.160
0.2
0.08
0.158
0.217
0.215
0.2
0.10
0.237
0.311
0.305
0.4
0.00
0.052
0.050
0.055
0.4
0.02
0.052
0.053
0.025
0.4
0.04
0.056
0.071
0.055
0.4
0.06
0.077
0.094
0.095
0.4
0.08
0.104
0.126
0.135
0.4
0.10
0.147
0.186
0.175
0.6
0.00
0.077
0.047
0.065
0.6
0.02
0.047
0.052
0.045
0.6
0.04
0.047
0.062
0.050
0.6
0.06
0.053
0.070
0.065
0.6
0.08
0.068
0.090
0.100
0.6
0.10
0.082
0.108
0.135
0.8
0.00
0.093
0.043
0.090
0.8
0.02
0.050
0.057
0.050
0.8
0.04
0.051
0.061
0.075
0.8
0.06
0.054
0.068
0.095
0.8
0.08
0.063
0.069
0.100
0.8
0.10
0.065
0.077
0.095
0.9
0.00
0.108
0.032
0.105
0.9
0.02
0.050
0.052
0.055
0.9
0.04
0.055
0.053
0.065
0.9
0.06
0.060
0.054
0.075
0.9
0.08
0.062
0.061
0.080
0.9
0.10
0.066
0.065
0.085
1
0.00
0.017
0.044
0.035
1
0.02
0.052
0.055
0.050
1
0.04
0.055
0.057
0.045
1
0.06
0.060
0.063
0.060
1
0.08
0.068
0.073
0.080
1
0.10
0.076
0.089
0.095 19 Table 8: Size and Size-Adjusted Power of the Wald Test at an Unknown Date (n = 50, T = 50) ρ
γ
sup-W
Mean-W
Exp-W
0
0.00
0.110
0.113
0.050
0
0.02
0.039
0.039
0.250
0
0.04
0.025
0.025
0.785
0
0.06
0.023
0.023
0.990
0
0.08
0.015
0.015
1.000
0
0.10
0.006
0.006
1.000
0.2
0.00
0.046
0.043
0.050
0.2
0.02
0.155
0.246
0.180
0.2
0.04
0.551
0.677
0.595
0.2
0.06
0.894
0.954
0.925
0.2
0.08
0.996
1.000
0.995
0.2
0.10
1.000
1.000
1.000
0.4
0.00
0.057
0.041
0.050
0.4
0.02
0.104
0.167
0.095
0.4
0.04
0.328
0.464
0.390
0.4
0.06
0.640
0.749
0.680
0.4
0.08
0.889
0.954
0.925
0.4
0.10
0.989
0.999
0.985
0.6
0.00
0.065
0.041
0.050
0.6
0.02
0.066
0.088
0.065
0.6
0.04
0.133
0.234
0.180
0.6
0.06
0.314
0.448
0.380
0.6
0.08
0.506
0.668
0.600
0.6
0.10
0.716
0.837
0.795
0.8
0.00
0.102
0.038
0.060
0.8
0.02
0.056
0.060
0.040
0.8
0.04
0.074
0.092
0.050
0.8
0.06
0.108
0.148
0.085
0.8
0.08
0.161
0.234
0.145
0.8
0.10
0.247
0.346
0.235
0.9
0.00
0.129
0.031
0.075
0.9
0.02
0.053
0.052
0.045
0.9
0.04
0.054
0.065
0.055
0.9
0.06
0.063
0.087
0.065
0.9
0.08
0.081
0.108
0.080
0.9
0.10
0.110
0.138
0.095
1
0.00
0.023
0.032
0.040
1
0.02
0.051
0.056
0.045
1
0.04
0.053
0.072
0.050
1
0.06
0.063
0.082
0.055
1
0.08
0.078
0.105
0.080
1
0.10
0.091
0.135
0.095 20 Table 9: Size and Size-Adjusted Power of the Wald Test at an Unknown Date (n = 200, T = 20) and Size-Adjusted Power of the Wald Test at an Unknown Date (n = 200, T = 20) ρ
γ
sup-W
Mean-W
Exp-W
0
0.00
0.036
0.058
0.035
0
0.02
0.091
0.087
0.105
0
0.04
0.234
0.261
0.315
0
0.06
0.469
0.526
0.565
0
0.08
0.730
0.782
0.760
0
0.10
0.907
0.921
0.950
0.2
0.00
0.042
0.055
0.035
0.2
0.02
0.077
0.069
0.090
0.2
0.04
0.158
0.170
0.215
0.2
0.06
0.317
0.373
0.420
0.2
0.08
0.509
0.579
0.615
0.2
0.10
0.728
0.775
0.755
0.4
0.00
0.048
0.051
0.035
0.4
0.02
0.067
0.056
0.070
0.4
0.04
0.107
0.112
0.140
0.4
0.06
0.188
0.211
0.275
0.4
0.08
0.296
0.377
0.405
0.4
0.10
0.451
0.526
0.575
0.6
0.00
0.059
0.048
0.045
0.6
0.02
0.058
0.053
0.065
0.6
0.04
0.078
0.072
0.095
0.6
0.06
0.111
0.113
0.140
0.6
0.08
0.164
0.170
0.210
0.6
0.10
0.235
0.260
0.305
0.8
0.00
0.089
0.041
0.055
0.8
0.02
0.054
0.055
0.055
0.8
0.04
0.060
0.056
0.055
0.8
0.06
0.067
0.068
0.065
0.8
0.08
0.080
0.090
0.090
0.8
0.10
0.098
0.110
0.115
0.9
0.00
0.094
0.039
0.055
0.9
0.02
0.048
0.054
0.050
0.9
0.04
0.054
0.051
0.065
0.9
0.06
0.059
0.057
0.080
0.9
0.08
0.061
0.059
0.080
0.9
0.10
0.073
0.079
0.080
1
0.00
0.022
0.068
0.085
1
0.02
0.057
0.057
0.060
1
0.04
0.066
0.062
0.075
1
0.06
0.083
0.093
0.070
1
0.08
0.107
0.127
0.115
1
0.10
0.151
0.173
0.180 21 Table 10: Size and Size-Adjusted Power of the Wald Test at an Unknown Date (n = 200, T = 50) Table 10: Size and Size-Adjusted Power of the Wald Test at an Unknown Date (n = 200, T = 50) ρ
γ
sup-W
Mean-W
Exp-W
0
0.00
0.082
0.080
0.035
0
0.02
0.396
0.571
0.785
0
0.04
0.998
1.000
1.000
0
0.06
1.000
1.000
1.000
0
0.08
1.000
1.000
1.000
0
0.10
1.000
1.000
1.000
0.2
0.00
0.043
0.034
0.035
0.2
0.02
0.548
0.669
0.530
0.2
0.04
0.997
0.999
1.000
0.2
0.06
1.000
1.000
1.000
0.2
0.08
1.000
1.000
1.000
0.2
0.10
1.000
1.000
1.000
0.4
0.00
0.055
0.035
0.035
0.4
0.02
0.325
0.467
0.360
0.4
0.04
0.899
0.961
0.900
0.4
0.06
1.000
1.000
1.000
0.4
0.08
1.000
1.000
1.000
0.4
0.10
1.000
1.000
1.000
0.6
0.00
0.069
0.034
0.040
0.6
0.02
0.158
0.242
0.190
0.6
0.04
0.541
0.688
0.515
0.6
0.06
0.899
0.964
0.895
0.6
0.08
0.996
0.999
1.000
0.6
0.10
1.000
1.000
1.000
0.8
0.00
0.080
0.030
0.055
0.8
0.02
0.081
0.102
0.095
0.8
0.04
0.190
0.257
0.185
0.8
0.06
0.372
0.480
0.330
0.8
0.08
0.604
0.702
0.520
0.8
0.10
0.802
0.880
0.740
0.9
0.00
0.107
0.025
0.065
0.9
0.02
0.052
0.066
0.055
0.9
0.04
0.085
0.115
0.090
0.9
0.06
0.127
0.191
0.140
0.9
0.08
0.234
0.306
0.205
0.9
0.10
0.347
0.434
0.295
1
0.00
0.023
0.032
0.070
1
0.02
0.059
0.058
0.055
1
0.04
0.073
0.103
0.060
1
0.06
0.123
0.172
0.075
1
0.08
0.213
0.253
0.115
1
0.10
0.292
0.349
0.175 22 22 Table 11: Size and Size-Adjusted Power of the Wald Test at an Unknown Date (n = 500, T = 20) ρ
γ
sup-W
Mean-W
Exp-W
0
0.00
0.036
0.048
0.040
0
0.02
0.139
0.188
0.185
0
0.04
0.504
0.603
0.605
0
0.06
0.873
0.917
0.945
0
0.08
0.985
0.990
0.995
0
0.10
1.000
1.000
1.000
0.2
0.00
0.043
0.048
0.040
0.2
0.02
0.111
0.125
0.145
0.2
0.04
0.318
0.418
0.460
0.2
0.06
0.653
0.749
0.780
0.2
0.08
0.909
0.946
0.960
0.2
0.10
0.986
0.990
0.995
0.4
0.00
0.050
0.042
0.045
0.4
0.02
0.080
0.089
0.105
0.4
0.04
0.189
0.247
0.275
0.4
0.06
0.414
0.504
0.555
0.4
0.08
0.651
0.746
0.775
0.4
0.10
0.873
0.915
0.925
0.6
0.00
0.074
0.041
0.055
0.6
0.02
0.065
0.068
0.065
0.6
0.04
0.120
0.135
0.130
0.6
0.06
0.201
0.260
0.235
0.6
0.08
0.327
0.439
0.415
0.6
0.10
0.508
0.609
0.585
0.8
0.00
0.093
0.037
0.070
0.8
0.02
0.056
0.060
0.055
0.8
0.04
0.075
0.083
0.075
0.8
0.06
0.112
0.115
0.110
0.8
0.08
0.154
0.177
0.155
0.8
0.10
0.213
0.263
0.230
0.9
0.00
0.113
0.032
0.070
0.9
0.02
0.056
0.055
0.050
0.9
0.04
0.074
0.069
0.060
0.9
0.06
0.096
0.091
0.075
0.9
0.08
0.118
0.111
0.110
0.9
0.10
0.154
0.153
0.140
1
0.00
0.022
0.073
0.065
1
0.02
0.066
0.067
0.065
1
0.04
0.107
0.115
0.105
1
0.06
0.173
0.193
0.140
1
0.08
0.263
0.303
0.230
1
0.10
0.388
0.457
0.345 23 Table 12: Size and Size-Adjusted Power of the Wald Test at an Unknown Date (n = 500, T = 50) ρ
γ
sup-W
Mean-W
Exp-W
0
0.00
0.052
0.048
0.040
0
0.02
0.992
0.997
1.000
0
0.04
1.000
1.000
1.000
0
0.06
1.000
1.000
1.000
0
0.08
1.000
1.000
1.000
0
0.10
1.000
1.000
1.000
0.2
0.00
0.054
0.047
0.040
0.2
0.02
0.916
0.962
0.965
0.2
0.04
1.000
1.000
1.000
0.2
0.06
1.000
1.000
1.000
0.2
0.08
1.000
1.000
1.000
0.2
0.10
1.000
1.000
1.000
0.4
0.00
0.059
0.047
0.040
0.4
0.02
0.667
0.792
0.805
0.4
0.04
1.000
1.000
1.000
0.4
0.06
1.000
1.000
1.000
0.4
0.08
1.000
1.000
1.000
0.4
0.10
1.000
1.000
1.000
0.6
0.00
0.077
0.046
0.040
0.6
0.02
0.303
0.458
0.500
0.6
0.04
0.909
0.964
0.965
0.6
0.06
1.000
1.000
1.000
0.6
0.08
1.000
1.000
1.000
0.6
0.10
1.000
1.000
1.000
0.8
0.00
0.110
0.045
0.060
0.8
0.02
0.124
0.151
0.155
0.8
0.04
0.345
0.485
0.475
0.8
0.06
0.710
0.813
0.800
0.8
0.08
0.935
0.971
0.975
0.8
0.10
0.997
0.999
1.000
0.9
0.00
0.127
0.035
0.060
0.9
0.02
0.068
0.088
0.095
0.9
0.04
0.135
0.174
0.190
0.9
0.06
0.272
0.367
0.355
0.9
0.08
0.483
0.565
0.565
0.9
0.10
0.689
0.762
0.740
1
0.00
0.034
0.044
0.045
1
0.02
0.057
0.083
0.080
1
0.04
0.131
0.181
0.175
1
0.06
0.269
0.353
0.375
1
0.08
0.463
0.519
0.590
1
0.10
0.638
0.647
0.730 24
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https://openalex.org/W3014605053
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https://www.nature.com/articles/s41419-020-2692-9.pdf
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English
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Correction: Generation of novel affibody molecules targeting the EBV LMP2A N-terminal domain with inhibiting effects on the proliferation of nasopharyngeal carcinoma cells
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Cell death and disease
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C O R R E C T I O N O p e n A c c e s s © The Author(s) 2020
OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction
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https://doi.org/10.1038/s41419-020-2692-9 Zhu et al. Cell Death and Disease (2020) 11:494
https://doi.org/10.1038/s41419-020-2692-9 Cell Death & Disease Cell Death & Disease Correction: Generation of novel affibody molecules
targeting the EBV LMP2A N-terminal domain with
inhibiting effects on the proliferation of
nasopharyngeal carcinoma cells Jinshun Zhu, Saidu Kamara, Danwei Cen, Wanlin Tang, Meiping Gu, Xingyuan Ci, Jun Chen, Lude Wang, Shanli Zhu,
Pengfei Jiang, Shao Chen, Xiangyang Xue and Lifang Zhang is LGF18H160030. This has now been corrected in the
PDF and HTML versions of the Article. Correction to: Cell Death and Disease
https://doi.org/10.1038/s41419-020-2410-7
published online 01 April 2020 Correction to: Cell Death and Disease
https://doi.org/10.1038/s41419-020-2410-7
published online 01 April 2020 The original version of this Article incorrectly gave the
foundation number of the Public Welfare Foundation of
Zhejiang Province as GF18H160070. The correct number Official journal of the Cell Death Differentiation Association
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English
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Use of a 12 months’ self-referral reminder to facilitate uptake of bowel scope (flexible sigmoidoscopy) screening in previous non-responders: a London-based feasibility study
|
British journal of cancer
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cc-by
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Use of a 12 months’ self-referral reminder to
facilitate uptake of bowel scope (flexible
sigmoidoscopy) screening in previous
non-responders: a London-based feasibility
study 1, Lesley M McGregor1, Sarah Marshall2, John Isitt3, Nicholas Counsell1, Jane Wardle
W
1 Robert S Kerrison*,1, Lesley M McGregor1, Sarah Marshall2, John Isitt3, Nicholas Counsell1, Jane Wardle1
and Christian von Wagner1 Robert S Kerrison*,1, Lesley M McGregor1, Sarah Marshall2, John Isitt3, Nicholas Counsell1, Jane Wardle1
and Christian von Wagner1 1Health Behaviour Research Centre, Department of Epidemiology and Public Health, University College London, 1-19 Torrington
Place, London WC1E 7HB, UK; 2St. Mark’s Bowel Cancer Screening Centre, St. Mark’s Hospital, Watford Road, Harrow, Middlesex
HA1 3UJ, UK and 3Resonant Behaviour Change and Social Marketing, Canterbury Court, 1-3 Brixton Road, London, SW9 6DE, UK Background: In March 2013, NHS England extended its national Bowel Cancer Screening Programme to include ‘one-off’ Flexible
Sigmoidoscopy screening (NHS Bowel Scope Screening, BSS) for men and women aged 55. With less than one in two people
currently taking up the screening test offer, there is a strong public health mandate to develop system-friendly interventions to
increase uptake while the programme is rolling out. This study aimed to assess the feasibility of sending a reminder to previous
BSS non-responders, 12 months after the initial invitation, with consideration for its potential impact on uptake. Method: This study was conducted in the ethnically diverse London Boroughs of Brent and Harrow, where uptake is below the
national average. Between September and November 2014, 160 previous non-responders were randomly selected to receive a
reminder of the opportunity to self-refer 12 months after their initial invitation. The reminder included instructions on how to book
an appointment, and provided options for the time and day of the appointment and the gender of the endoscopist performing
the test. To address barriers to screening, the reminder was sent with a brief locally tailored information leaflet designed
specifically for this study. Participants not responding within 4 weeks were sent a follow-up reminder, after which there was no
further intervention. Self-referral rates were measured 8 weeks after the delivery of the follow-up reminder and accepted as final. Results: Of the 155 participants who received the 12 months’ reminder (returned to sender, n¼ 5), 30 (19.4%) self-referred for an
appointment, of which 24 (15.5%) attended and were successfully screened. Attendance rates differed by gender, with significantly more
women attending an appointment than men (20.7% vs 8.8%, respectively; OR¼ 2.73, 95% CI¼ 1.02–7.35, P¼ 0.05), but not by area (Brent
vs Harrow) or area-level deprivation. British Journal of Cancer (2016) 114, 751–758 | doi: 10.1038/bjc.2016.43 Keywords: colorectal cancer screening; flexible sigmoidoscopy screening; reminders; patient education; leaflet FULL PAPER FULL PAPER FULL PAPER *Correspondence: RS Kerrison; E-mail: robert.kerrison.13@ucl.ac.uk
Received 15 September 2015; revised 7 December 2015; accepted 1 February 2016;
published online 15 March 2016
& 2016 Cancer Research UK. All rights reserved 0007 – 0920/16
www.bjcancer.com | DOI:10.1038/bjc.2016.43
751 Use of a 12 months’ self-referral reminder to
facilitate uptake of bowel scope (flexible
sigmoidoscopy) screening in previous
non-responders: a London-based feasibility
study Of the 30 people who self-referred for an appointment, 27 (90%) indicated a preference for a same-
sex practitioner, whereas three (10%) gave no preference. Preference for a same-sex practitioner was higher among women than men
(w2¼ 7.78, Po0.05), with only 67% of men (six of nine) requesting a same-sex practitioner, compared with 100% of women (n¼ 21). Conclusions: Sending previous non-responders a 12 months’ reminder letter with a brief information leaflet is a feasible and
efficacious intervention, which merits further investigation in a randomised controlled trial. *Correspondence: RS Kerrison; E-mail: robert.kerrison.13@ucl.ac.uk
Received 15 September 2015; revised 7 December 2015; accepted 1 February 2016;
published online 15 March 2016
& 2016 Cancer Research UK. All rights reserved 0007 – 0920/16
www.bjcancer.com | DOI:10.1038/bjc.2016.43
751 & 2016 Cancer Research UK. All rights reserved 0007 – 0920/16 www.bjcancer.com | DOI:10.1038/bjc.2016.43 751 Reminders for bowel scope screening non-responders BRITISH JOURNAL OF CANCER Colorectal cancer (CRC) is the fourth most common cancer in the
United Kingdom and the second leading cause of cancer deaths
(Cancer Research UK, 2015a, b). Screening can improve clinical
outcomes by detecting cases early, when they are easier to treat
(Logan et al, 2012; Cancer Research UK, 2014; Cancer Research
UK, 2015c. In addition, screening can prevent cases through the
timely detection and removal of adenomas (the precancerous
lesions from which most CRCs develop) (Leslie et al, 2002),
improving outcomes even further (Atkin et al, 2010; Whyte et al,
2012). yield greater results than they do if used individually (Hewitson
et al, 2011), additional barriers (e.g., anticipated pain) were
addressed with the inclusion of a FS information leaflet, designed
by a social marketing company, with a focus on engaging
individuals with low literacy using social cognitive approaches to
address misconceptions about screening (Bandura, 2004). This study set out to test whether this strategy could be
implemented in a centre with below average uptake and, if so,
whether it met a minimum level of efficacy that would merit
further investigation in a randomised controlled trial (RCT). A review of the methods and invitation process employed
was included in order to help refine the strategy in any
subsequent RCT. MATERIALS AND METHODS The sample size also allowed us to test for a
minimum level of efficacy within the intervention arm, to check
whether investigation in a RCT was merited (minimum number of
self-referred appointments needed ¼ 3), as well as obtain estimates
to inform the statistical parameters required to design such a study
(which would be powered to confirm the efficacy of the
intervention via a direct comparison with a control arm). The
A’Hern single-stage design, which is commonly used in smaller
studies with such aims (A’hern, 2001), confirmed that our sample
(n ¼ 160) gave us acceptable levels for power (b40.8) and
significance (ao0.05) to test for a minimum level of efficacy,
with the unacceptable response rate based on the self-referral rate
of non-responders of the previous year (i.e., 0.35%), and the
desired response rate based on a 5% improvement (i.e., 5.35%). Similar to other screening programmes, the BSS programme
structure incorporates specific strategies to maximise uptake
(i.e., pre-notification letters, reminder letters and so on) (Baron
et al, 2008; Libby et al, 2011), but the test itself is unique in that it is
a ‘one-off’ procedure. Other screening programmes in England
invite people to be screened every few years (e.g., FOBt every 2
years, mammography every 3 years), allowing additional oppor-
tunities for people to take part (Steele et al, 2010; Lo et al, 2014). In
BSS, all adults are eligible to self-refer up to the age of 60, offering a
similar window of opportunity for those who do not take up the
initial invitation; however, there is no direct ‘reminder’ of this
possibility during the 5-year time frame. Procedures. Eligible participants (n ¼ 844) were identified on
the
National
Bowel
Cancer
Screening
System
(BCSS):
an
electronic system which provides an up-to-date record of
individual-uptake data for patients enrolled in the national
screening programme. For a period of 10 weeks (September 2014
to November 2014), 16 people per week were randomly selected
for inclusion in the study from a variable weekly total of non-
responders using simple computerised pseudo-random selection
methods (Babbie, 2011). This study, therefore aimed to examine the potential of a mailed
12 months’ self-referral reminder that addressed key barriers by
including an opportunity to choose the day and time of the
appointment and the gender of the endoscopist performing the
screening procedure. MATERIALS AND METHODS As with all screening programmes, the extent to which the cost
and public health benefits of including BSS are realised will depend
largely on uptake (Whyte et al, 2012; Geurts et al, 2015). In a
London-based pilot study, uptake of FS screening among the
invited population was 50% (Robb et al, 2010); if representative of
attendance in the national programme, it is expected that the
addition of FS would result in an additional 2000 CRC deaths and
10 000 CRC cases being prevented in England by mid-2030 (Geurts
et al, 2015). However, a recent analysis of uptake over the first 14
months of the programmes introduction revealed that only 43% of
the invited population attended a BSS appointment, and thereby
currently falls shy of the 50% benchmark (McGregor et al, 2015a). Study population. Eligible participants were men and women
registered with a General Practice in the London Boroughs of Brent
and Harrow who had not attended BSS within one year of their
initial invitation. Responders who had initially confirmed an
appointment but failed to attend were excluded from the study. Commonly cited reasons for not attending a FS appointment
include a lack of current health problems; practical barriers (i.e.,
inconvenient appointment time, difficulties travelling to the
appointment and so on); worry about pain, discomfort, or injury
associated with the examination; and not wanting to know about
any health issues (e.g., Vernon, 1997). For women specifically, the
gender of the practitioner performing the test has also been
reported as a potential barrier (Menees et al, 2005). In contrast to
the FOBt-screening programme, women have been found to be less
likely than men to have a FS (McGregor et al, 2015a), and the
anticipated gender of the endoscopist is considered to be a
contributing factor (Robb et al, 2010). Women have been found to
be more willing to have FS if the endoscopist is female (Farraye
et al, 2004) and are willing to wait longer for the test to ensure this
happens (Varadarajulu et al, 2002). Sample size. To explore the feasibility of including a 12 months’
reminder in the routine operations of the centre, we set out to run
the study for a minimum of 10 weeks, with 16 previous non-
responders sent a reminder per week, giving a total sample size of
160 participants. Use of a 12 months’ self-referral reminder to
facilitate uptake of bowel scope (flexible
sigmoidoscopy) screening in previous
non-responders: a London-based feasibility
study A recent appraisal of the options for CRC screening in England
highlighted that a combination of ‘once-only’ flexible sigmoido-
scopy (FS) at the age of 55, delivered in conjunction with the
current biennial faecal occult blood-testing strategy offered to
60–74-year olds, would be highly cost-effective, and would provide
significant reductions the incidence, mortality and treatment costs
associated with the disease (Whyte et al, 2012). As a result, NHS
England subsequently introduced ‘once-only’ FS screening for 55-
year olds (known as Bowel Scope Screening, BSS) to the National
Bowel Cancer Screening Programme in March 2013 (Department
of Health, 2012). www.bjcancer.com | DOI:10.1038/bjc.2016.43 MATERIALS AND METHODS Study design and trial setting. We performed a single-centre
feasibility study at St. Mark’s Hospital in London. St. Mark’s
Hospital serves an ethnically diverse population with a high level of
socioeconomic deprivation; the majority (56%) of people invited to
take part in screening at St. Mark’s during the first 14 months were
living within either the most deprived areas in England (calculated
using census data for individual postcodes), or the most ethnically
diverse areas (74%; also calculated using census data for individual
postcodes) and, in total, only 40.5% of invitees attended an
appointment, which was significantly less than the national average
(43.5%) (McGregor et al, 2015a). ,
)
As with all screening programmes, the extent to which the cost
and public health benefits of including BSS are realised will depend
largely on uptake (Whyte et al, 2012; Geurts et al, 2015). In a
London-based pilot study, uptake of FS screening among the
invited population was 50% (Robb et al, 2010); if representative of
attendance in the national programme, it is expected that the
addition of FS would result in an additional 2000 CRC deaths and
10 000 CRC cases being prevented in England by mid-2030 (Geurts
et al, 2015). However, a recent analysis of uptake over the first 14
months of the programmes introduction revealed that only 43% of
the invited population attended a BSS appointment, and thereby
currently falls shy of the 50% benchmark (McGregor et al, 2015a). Commonly cited reasons for not attending a FS appointment
include a lack of current health problems; practical barriers (i.e.,
inconvenient appointment time, difficulties travelling to the
appointment and so on); worry about pain, discomfort, or injury
associated with the examination; and not wanting to know about
any health issues (e.g., Vernon, 1997). For women specifically, the
gender of the practitioner performing the test has also been
reported as a potential barrier (Menees et al, 2005). In contrast to
the FOBt-screening programme, women have been found to be less
likely than men to have a FS (McGregor et al, 2015a), and the
anticipated gender of the endoscopist is considered to be a
contributing factor (Robb et al, 2010). Women have been found to
be more willing to have FS if the endoscopist is female (Farraye
et al, 2004) and are willing to wait longer for the test to ensure this
happens (Varadarajulu et al, 2002). MATERIALS AND METHODS Participants were given a total of 12 weeks
(from receipt of the 12 months’ reminder) to respond before their
‘episode’ was closed: any self-referrals made after this time were
not included in the study results. thereby initiating a call from a member of the administrative team
at the centre to confirm an appointment date, or by calling the
St. Mark’s Bowel Cancer Screening Centre directly on the Freephone
telephone number provided. Participants were able to indicate
their preferences for the gender of the endoscopist and the day and
time of the appointment, either on the appointment–request slip or
during the phone call to the Freephone number. Participants not
responding within 4 weeks of the 12 months’ reminder were sent a
second reminder (henceforth
referred
to as the ‘follow-up
reminder’), which again included an appointment–request slip,
the information leaflet and a freepost return envelope. After the
follow-up reminder, no further attempt to contact non-responding
participants was made. Participants were given a total of 12 weeks
(from receipt of the 12 months’ reminder) to respond before their
‘episode’ was closed: any self-referrals made after this time were
not included in the study results. Intervention development. The materials used in this study
were designed in conjunction with ‘Resonant’: a social marketing
company which specialises in the development of health
behaviour change interventions (Resonant, 2015). The initial
content of the leaflet and reminder letter was informed by work
conducted by the UCL Research Team, which included a review
of the literature on patient-specific factors for non-attendance
(e.g., Vernon, 1997; Jones et al, 2010), and semi-structured
telephone interviews with people who had recently taken part in
the programme (n ¼ 5; three female, two male). The semi-
structured telephone interviews were conducted with previously
screened adults to learn more about the key factors which
influenced the decision to be screened. Statements from the
interviews were then selected for use in the leaflet, with
permission from interviewees. Initial designs of the reminder
letter and leaflet were developed by Resonant and then tested in a
co-design workshop, facilitated by the company, in which
screening eligible adults from the London Boroughs of Brent
and Harrow (n ¼ 4; three male, one female; ages 55–58) gave
feedback to inform future iterations. MATERIALS AND METHODS Because barriers to FS screening are unlikely
to
exist
in
isolation
(Jones
et
al,
2010),
and
combining
interventions that target CRC screening at a number of levels Participants were mailed a ‘self-referral reminder letter’, along
with an ‘appointment–request slip’, information leaflet and
freepost return envelope B12 months after their initial invitation
(12 months reminder) (see Figure 1). Participants could book an
appointment either by returning their appointment–request slip, www.bjcancer.com | DOI:10.1038/bjc.2016.43 752 Reminders for bowel scope screening non-responders BRITISH JOURNAL OF CANCER BSS pre-invitation
letter sent
Appointment letter
sent
Appointment
confirmed
Appointment
made
Appointment
made
Appointment not
made
Appointment not
made
No appointment
confirmed
No appointment
confirmed
Appointment
confirmed
Appointment not
attended
Appointment
attended
Enema
preparation sent
Programme procedure
Reminder procedure
Reminder letter
sent
Self-referral
reminder letter and
leaflet sent
Self-referral
reminder letter
and leaflet sent
2 weeks
2 weeks to respond
2 weeks to respond
8 weeks to respond
4 weeks to respond
11 months
2 weeks
2 weeks
Key:
Programme procedures
Reminder procedures
Figure 1. BSS invitation flowchart with self-referral reminder added. Reminder procedure Self-referral
reminder letter and
leaflet sent 4 weeks to respond Appointment letter
sent Appointment not
made Appointment
made Reminder letter
sent Appointment not
made Appointment
made No appointment
confirmed Appointment
confirmed Enema
preparation sent 2 weeks Appointment
attended Figure 1. BSS invitation flowchart with self-referral reminder added. The study was approved by the South Central Oxford B
Research Ethics Service (Ref: 14/SC/1246). The study was approved by the South Central Oxford B
Research Ethics Service (Ref: 14/SC/1246). thereby initiating a call from a member of the administrative team
at the centre to confirm an appointment date, or by calling the
St. Mark’s Bowel Cancer Screening Centre directly on the Freephone
telephone number provided. Participants were able to indicate
their preferences for the gender of the endoscopist and the day and
time of the appointment, either on the appointment–request slip or
during the phone call to the Freephone number. Participants not
responding within 4 weeks of the 12 months’ reminder were sent a
second reminder (henceforth
referred
to as the ‘follow-up
reminder’), which again included an appointment–request slip,
the information leaflet and a freepost return envelope. After the
follow-up reminder, no further attempt to contact non-responding
participants was made. www.bjcancer.com | DOI:10.1038/bjc.2016.43 MATERIALS AND METHODS Mark’s Bowel Cancer Screening
Centre; (2) reminded participants that they had previously been
invited for an appointment 1 year earlier and were eligible to make
an appointment up until their 60th birthday; (3) gave participants
the opportunity to select a preference for the day and time of the
appointment and the gender of the practitioner performing the test
and; (4) highlighted three key messages: i) that the risk of
developing bowel cancer is highest in the patients’ age group
(55 þ years), ii) screening is for people who do not have any signs
or symptoms of bowel cancer and iii) screening can help prevent
bowel cancer by removing small asymptomatic growths (called
polyps), which have the potential to become malignant over time
(see Supplementary Appendix 2). Follow-up reminder. A follow-up reminder letter was sent to
individuals not responding to the 12 months’ reminder within 4
weeks. This follow-up reminder repeated the information included
in the 12 months’ reminder, but also highlighted individuals had
recently received a reminder letter (see Supplementary Appendix 3). Measures. Routinely available data stored on the BCSS were used
to verify self-referral and attendance 4 weeks following the
distribution of the 12 months’ reminder, and 8 weeks following
the distribution of the follow-up reminder. The BCSS was also
consulted to obtain the gender, area (Harrow or Brent) and an
area-based socioeconomic deprivation score for each participant. Ethnicity is not routinely collected or available on the BCSS, and so
this information could not be extracted. Leaflet. The development of the leaflet was guided by two
psychological models of health behaviour that have previously
been used to explain individual level factors associated with
screening (e.g., perceived barriers and benefits) (Kiviniemi et al,
2011): the Health Belief Model (Rosenstock, 1974) and Social
Cognitive Theory (Bandura, 2004). In addition, the leaflet was
tailored to the London areas served by St. Mark’s hospital (i.e.,
included a map with information about local transport links to the
hospital). The leaflet also included an educational/knowledge-
building component to reinforce messages regarding the benefits of Socioeconomic deprivation was obtained by converting each
individual’s postcode to a score on the 2010 Index of Multiple
Deprivation (IMD) (Department for Communities and Local
Government, 2010). The IMD scores obtained were categorised
into tertiles of their national distributions. MATERIALS AND METHODS The IMD uses census-
derived indicators of income, education, employment, environ-
ment, health and disability, barriers to housing and services, and Table 1. Characteristics of the reminder leaflet
Description
Motivational characteristics
Easy to read
The Flesch formula was used to assess the readability of the leaflet, which gave it a score of 68.7
(equivalent of a 13–15-year reading age). Pilot participants indicated that the leaflet was ‘the right
length’, not ‘too positive’ and ‘included enough information to make a decision about screening’. General Practitioner cancer lead endorsement
The BSS programme was endorsed by the local general practitioner cancer lead for Hillingdon. A
photograph and quote of the GP were included: ‘I would urge anyone aged 55–59 to take this quick,
potentially lifesaving, one-off test that significantly reduces your risk of getting bowel cancer’. Provincial social norms message
The leaflet included a descriptive provincial social norms message: ‘About 270 people take up the
Bowel Scope Screening test at St. Mark’s Hospital every month’. Effective communication of risk
The antecedents and consequences of bowel cancer were used to communicate risk and explain the
preventative mechanisms of bowel scope screening: ‘Bowel cancer develops from polyps, which are
small growths in your bowel. Most polyps are harmless, but some can turn into cancer if left
untreated. By removing any polyps in your bowel during the test, bowel scope screening is a very
effective way of reducing the chance that you will get bowel cancer in the future’. Patient narratives
The leaflet included two patient narratives (one male, one female). Narratives have been associated
with a reduction in the perceived impact of barriers and increased perceived risk of CRC (Dillard et al,
2010), increased intention to be screened (McGregor et al, 2015b) and improved attendance at
colonoscopy screening (Jensen et al, 2014). Female narrative: ‘I must admit I was nervous, but the specialist nurse explained everything very clearly. It wasn’t painful at all. I was told I had no polyps and given the all clear, which was a huge relief. My
friend died from bowel cancer 5 years ago, so I was determined this wouldn’t happen to me!’
Male narrative: ‘The staff at St. Mark’s Hospital were great. The doctor found a polyp, which he
removed. I didn’t feel a thing. The doctor explained that polyps often don’t have any symptoms, so
people don’t always know if they have them. MATERIALS AND METHODS A revised version was then
presented to individuals who were either the eligible age or
approaching the eligible age for screening (n ¼ 20; 12 female,
8 male, aged 50–59 years) and feedback obtained through
interviews conducted by a member of the UCL Research Team. The final leaflet (see Supplementary Appendix 1) had a Flesch
readability score of 68.7, indicating that it was suitable for use For those participants who made an appointment, a confirma-
tion letter and BSS consent form was sent to their home address. The BSS consent form and confirmation letter used in this study
were the same as those used for routine appointments in the
national screening programme. Participants were asked to read
the BSS consent form, which contained information regarding the
risks and benefits of the procedure, before attending their
appointment, and to call the screening centre if they had any
questions. Participants were asked to bring the BSS consent form to
their appointment, where a specialist screening practitioner/
endoscopy nurse would discuss the risks of the procedure with
the individual to ensure an informed decision to be screened
was made. www.bjcancer.com | DOI:10.1038/bjc.2016.43 753 Reminders for bowel scope screening non-responders BRITISH JOURNAL OF CANCER within the general population (easily understood by 13–15-year
olds) (Kincaid et al, 1975). within the general population (easily understood by 13–15-year
olds) (Kincaid et al, 1975). screening (effectiveness and rationale), a descriptive social norms
message outlining uptake of BSS at St. Mark’s Hospital (‘270 people
screened every month’), and several practical components designed
to improve self-efficacy (i.e., instructions on how to book an
appointment and directions to the hospital). In addition, factors
previously found to increase screening intentions and participation
were incorporated into the design, for example, male/female
patient narratives (Jensen et al, 2014; McGregor et al, 2015b) (See
Table 1). 12 months’ reminder letter. The 12 months’ reminder letter was
a personally addressed letter from St. Mark’s Hospital, which:
(1) invited participants to make a screening appointment by
returning an ‘appointment–request slip’ or by calling the Free-
phone telephone number for St. MATERIALS AND METHODS I’m glad they found the polyp before it had a chance to
become something more serious’. Reducing worry about pain, discomfort and
embarrassment associated with the procedure
The leaflet was designed to reduce worry about pain, discomfort and embarrassment associated with
the procedure. Statements addressing pain were based on patient reported outcomes from the UK
FS pilot study: ‘The test is done in private and nearly everyone says it’s not embarrassing’; ‘Most
people say they felt no pain, or only mild pain’ (Robb et al, 2012). Practical characteristics
Map and local transport options
The leaflet was designed to address practical barriers to screening. A map of the area, and
description of local transport links to the hospital was included to help patients plan their journey. Instructions on how to make an appointment
Instructions on how to make an appointment by telephone referral were reiterated in the leaflet. Patients were also informed they could call the St. Mark’s Freephone telephone number for further
information about the test. The leaflet included a descriptive provincial social norms message: ‘About 270 people take up the
Bowel Scope Screening test at St. Mark’s Hospital every month’. The antecedents and consequences of bowel cancer were used to communicate risk and explain the
preventative mechanisms of bowel scope screening: ‘Bowel cancer develops from polyps, which are
small growths in your bowel. Most polyps are harmless, but some can turn into cancer if left
untreated. By removing any polyps in your bowel during the test, bowel scope screening is a very
effective way of reducing the chance that you will get bowel cancer in the future’. The antecedents and consequences of bowel cancer were used to communicate risk and explain the
preventative mechanisms of bowel scope screening: ‘Bowel cancer develops from polyps, which are
small growths in your bowel. Most polyps are harmless, but some can turn into cancer if left untreated. By removing any polyps in your bowel during the test, bowel scope screening is a very
effective way of reducing the chance that you will get bowel cancer in the future’. The leaflet was designed to reduce worry about pain, discomfort and embarrassment associated with
the procedure. MATERIALS AND METHODS Statements addressing pain were based on patient reported outcomes from the UK
FS pilot study: ‘The test is done in private and nearly everyone says it’s not embarrassing’; ‘Most
people say they felt no pain, or only mild pain’ (Robb et al, 2012). The leaflet was designed to address practical barriers to screening. A map of the area, and
description of local transport links to the hospital was included to help patients plan their journey. Instructions on how to make an appointment by telephone referral were reiterated in the leaflet. Patients were also informed they could call the St. Mark’s Freephone telephone number for further
information about the test. www.bjcancer.com | DOI:10.1038/bjc.2016.43 754 BRITISH JOURNAL OF CANCER Reminders for bowel scope screening non-responders crime at small-area level to generate a scale ranging from 0 (least
deprived) to 80 (most deprived) (Department for Communities
and Local Government, 2010). BSS-screening referrals and attendance. A total of 30 (19.4%)
adults self-referred for BSS. Of these, 24 (80%) attended their
appointment and were screened, 3 (10%) did not attend, 2
cancelled and 1 did attend but was not screened owing to high
blood pressure (Figure 2). The overall attendance rate was
therefore 15.5% (24/155). Analysis. Descriptive statistics were used to test whether the
number of self-referred appointments exceeded the threshold for
further investigation in a RCT. To explore possible variations of
the impact of the intervention in relation to deprivation, area and
gender, a multivariate logistic regression analysis was performed
(Engel, 1988). Differences in patient preferences for a same-sex
practitioner were examined by gender using the X2-test of
independence (Pearson, 1900); the data were analysed using SPSS
Statistics (version 22). Attendance differed significantly by gender, with more women
attending an appointment than men (n ¼ 21 (20.7%) vs n ¼ 9
(8.8%), respectively; OR ¼ 2.73, 95% CI ¼ 1.02–7.35, P ¼ 0.05). There were no statistically significant differences between localities
(Brent vs Harrow) or tertiles of area-level deprivation (Table 3). The self-referral attendance rate of eligible adults not included
in this feasibility trial during the study period was 1.2% (8/684). RESULTS Sample characteristics of the reminder population
n
%
Gender
Male
68
43.9
Female
87
56.1
Area
Brent
76
49.0
Harrow
79
51.0
Tertile of deprivation (IMD score)
Tertile 1 (least deprived)
31
20
Tertile 2
62
40
Tertile 3 (most deprived)
62
40 our results would have exceeded the minimum level of efficacy
even assuming this higher rate (A’Hern, 2001). g
g
The finding that this intervention is feasible and has the
potential to improve uptake by this group is highly important; if
the findings of this study were replicated in a large RCT, then this
simple intervention could have a considerable impact on uptake at
St. Mark’s Hospital. A self-referral rate of 15% among previous
non-responders would equate to an increase in overall uptake of
B9% (estimated by multiplying the proportion of adults not
responding to the initial invitation (0.6) by the proportion of adults
attending screening in response to the 12 months’ reminder
(0.15)). This would increase overall uptake at St. Mark’s to almost
50%. If similar rates are observed in a multicentre study, the
implementation of a 12 months’ reminder in the national
programme
could
have
considerable
public
health
benefits
(Geurts et al, 2015). Furthermore, additional reminders, possibly
at 24, 36 and 48 months’, have the potential to increase overall
uptake even further. Finally by a process of elimination such
additional reminders would target the most deprived population
and could ultimately reduce the socioeconomic gradient in
screening attendance. period. No responses were received beyond the 12-week cut-off
Table 3. Uptake following the reminders by gender, tertiles
of the Index of Multiple Deprivation and location in the
eligible sample (n ¼ 155)
Comparisons
n attended (%)
OR (95% CI)
P-value
Overall (n ¼ 155)
24 (15.5)
—
—
Gender
Male (n ¼ 68)
6 (8.8)
—
—
Female (n ¼ 87)
18 (20.7)
2.73 (1.02–7.35)
0.05
Location
Harrow (n ¼ 79)
12 (15.2 )
—
—
Brent (n ¼ 76)
12 (15.8)
1.10 (0.35–3.40)
0.87
Tertile of deprivation
1 (n ¼ 32, least deprived )
4 (12.5)
—
—
2 (n ¼ 64)
11 (17.2)
1.46 (0.39–5.51)
0.57
3 (n ¼ 63, most deprived)
9 (14.3)
1.08 (0.22–5.37)
0.93 Our study found that women were more likely to attend in
response to the reminder than men. RESULTS The 12 months’ reminder,
therefore, has the potential to reduce the gender gap that has been
observed in response to the first invitation (McGregor et al, 2015a). Previous research has indicated that for women particularly, the
possibility of having a male endoscopist leading the procedure is a
barrier to uptake (Menees et al, 2005), and so the option to allow
participants to communicate a preference for the gender of the
endoscopist is likely to have explicitly and directly addressed this
barrier, thereby encouraging women to re-consider BSS atten-
dance. In addition, it is also likely that the leaflet had a role in
facilitating uptake in women specifically, given that it was designed
to reduce barriers to FS, and women have been found to report
more barriers to the test than men (Wardle et al, 2005). period. No responses were received beyond the 12-week cut-off
period. Although the proportions requesting a same-sex practitioner
between men and women were significantly different in our study,
the number of men and women self-referring in this study was
small (n ¼ 9 and n ¼ 21 respectively) and may not be representa-
tive of the proportions of men and women who would request a
same-sex practitioner in the general population. For instance, the
current study was set in an ethnically diverse area and so the
impact of the gender preference option may have been all the more
apparent here, as previous research has found the gender of the
endoscopist to be a pertinent barrier for black and ethnic minority
women (Varadarajulu et al, 2002). Future work should aim to
explore individual ethnicity when examining preferences for a
same-sex practitioner. Preference for day and time slot of appointment. Of the 30
people who self-referred for a BSS appointment, 24 (80%)
expressed a preference for a specific day and/or time. It was not
possible to accommodate preferences for 12 people (50%); only one
went on to cancel. Preference for gender of practitioner. Of the 30 people who self-
referred for a BSS appointment, 27 (90%) indicated a preference
for a same-sex practitioner; none (0%) indicated a preference for a
practitioner of the opposite sex; and three (10%) gave no
preference. It was not possible to accommodate the preference of
eight (30%) people; however, no-one asked to be rescheduled. RESULTS Preferences for the sex of the practitioner were examined by
gender: women were significantly more likely to request a same-sex
practitioner than men, with all of the women who self-referred for
an appointment requesting a same-sex practitioner, compared with
two-thirds of men (100% vs 67%; w2 ¼ 7.78, Po0.05). Previous research examining uptake of BSS in response to the
initial invitation has identified a strong socioeconomic gradient in
participation, with rates varying from 33% in the most deprived
areas to 53% in the least deprived (McGregor et al, 2015a). In this
study, we found no significant differences in participation between
tertiles of area-level deprivation (McGregor et al, 2015a); however,
it is important to note that the study was not designed to test for
differences between tertiles of area-level deprivation, and so may
have been underpowered to detect such differences. If the finding
were reproduced in a larger trial, the intervention examined in this
study may represent a potential strategy to reduce socioeconomic
inequalities within the national programme. www.bjcancer.com | DOI:10.1038/bjc.2016.43 RESULTS Self-referral method. Of the 30 people who self-referred for a BSS
appointment, 28 (93.3%) did so by returning the ‘appointment–
request slip’; the remaining two (6.7%) did so by calling the
provided Freephone telephone number. Sample characteristics. A total of 160 people (male ¼ 71, 44.4%;
female ¼ 89, 55.6%) were randomly selected to receive a 12
months’ self-referral reminder; however, five (3.1%) reminders
were found to be undeliverable and were ‘returned to sender’. Subsequently, 155 people (male ¼ 68, 43.9%, female ¼ 87, 56.1%)
were monitored as part of this study (Figure 2). Variation by
locality and IMD score tertile are shown in Table 2. Follow-up reminder reminder. A total of 21 people (13.5%)
responded to the self-referral reminder within 4 weeks. Subse-
quently, 134 follow-up reminders were sent with a further nine
(6.7%) responses received within the remaining 8-week response 844
Non-participants
identified via BCSS
16
Randomly selected each week to
receive a 12 months self-referral
reminder
684
Randomly
excluded
5
‘Returned to
sender’
24
Completed
screening in total
8
Completed screening
16
Completed screening
0
Cancelled
2
Cancelled
0
Not screened
1
Not screened
21
Self-referrals made
9
Self-referrals made
30
Self-referrals made
in total
134
follow-up reminder
letters sent
160
Sent a 12 months
self-referral reminder
2
DNA*
1
DNA*
*DNA = did not attend
Figure 2. Basic design of the study. 844
Non-participants
identified via BCSS 16
Randomly selected each week to
receive a 12 months self-referral
reminder 5
‘Returned to
sender’
24
Completed
screening in total
8
Completed screening
16
Completed screening
0
Cancelled
2
Cancelled
0
Not screened
1
Not screened
21
Self-referrals made
9
Self-referrals made
30
Self-referrals made
in total
134
follow-up reminder
letters sent
2
DNA*
1
DNA*
*DNA = did not attend
Figure 2. Basic design of the study. 5
‘Returned to
sender’ 2
Cancelled 30
Self-referrals made
in total Completed
screening in total Figure 2. Basic design of the study. www.bjcancer.com | DOI:10.1038/bjc.2016.43 www.bjcancer.com | DOI:10.1038/bjc.2016.43 755 BRITISH JOURNAL OF CANCER Reminders for bowel scope screening non-responders Table 2. Sample characteristics of the reminder population
n
%
Gender
Male
68
43.9
Female
87
56.1
Area
Brent
76
49.0
Harrow
79
51.0
Tertile of deprivation (IMD score)
Tertile 1 (least deprived)
31
20
Tertile 2
62
40
Tertile 3 (most deprived)
62
40 Table 2. Reminders for bowel scope screening non-responders BRITISH JOURNAL OF CANCER One potential concern regarding the leaflet used in this study is
that information within was not balanced. It was specifically
designed to promote uptake by addressing the barriers and
highlighting the benefits associated with the test. However, as the
leaflet supplemented the existing information (i.e., the standard
leaflet–which is balanced–, the consent form–which outlines the
risks of the procedure– and the face to face counseling–during
which the risks are discussed with a specialist screening practi-
tioner and which the individual must undergo prior to the
screening procedure–), the requirements of consent when making
a screening decision were still met in accordance with the General
Medical Council guidelines (General Medical Council, 2008). Nonetheless, to examine satisfaction with the reminder leaflet, we
will use waiting room questionnaires and compare responses with
individuals receiving the standard NHS leaflet in the subsequent
RCT. Bandura A (2004) Health promotion by social cognitive means. Health Educ
Behav 31(2): 143–164. Baron RC, Rimer BK, Breslow RA, Coates RJ, Kerner J, Melillo S, Habarta N,
Kalra GP, Chattopadhyay S, Wilson KM (2008) Client-directed
interventions to increase community demand for breast, cervical, and
colorectal cancer screening: a systematic review. Am J Prev Med 35(1):
S34–S55. Cancer Research UK (2014) Saving lives, averting costs: an analysis of the
financial mplications of achieving earlier diagnosis of colorectal, lung and
ovarian cancer. Available from http://www.incisivehealth.com/uploads/
Saving%20lives%20averting%20costs.pdf (accessed 14 September 2015). Cancer Research UK (2015a) Bowel Cancer Incidence Statistics. Available from http://www.cancerresearchuk.org/health-professional/
cancer-statistics/statistics-by-cancer-type/bowel-cancer/incidence
(accessed 14 September 2015). Cancer Research UK (2015b) Bowel Cancer Mortality Statistics. Available from http://www.cancerresearchuk.org/health-professional/
cancer-statistics/statistics-by-cancer-type/bowel-cancer/mortality
(accessed 14 September 2015). At last, it is important to note that in the UK, one in six people
have a reading level below that expected of an 11-year old (Harding
et al, 2012); therefore, the leaflet may still have been too difficult
for some people to read. Reducing the readability of written
information further may have benefits over and above those of the
current interventions used within our study; however, for these
adults written materials may not be the most suitable. Researchers
seeking to reduce inequalities should focus on alternative channels
of engaging these adults, such as through community outreach and
telephone intervention. Cancer Research UK (2015c) Bowel Cancer Survival Statistics. Available from http://www.cancerresearchuk.org/health-professional/
cancer-statistics/statistics-by-cancer-type/bowel-cancer/survival
(accessed 14 September 2015). Department for Communities and Local Government (2010) English indices
of deprivation 2010. Available from https://www.gov.uk/government/
statistics/english-indices-of-deprivation-2010 (accessed: 10 July 2015). ACKNOWLEDGEMENTS We dedicate this article in memory of Professor Jane Wardle
(1950–2015). We would like to acknowledge funding support from
St. Mark’s Hospital, University College London and Cancer
Research UK. RSK has a doctoral studentship funded by St. Mark’s Hospital and UCL; LMM is funded by a CRUK Project
Grant (C27064/A17326) Awarded to CVW. We would also like to
thank acknowledge St. Mark’s Hospital for supporting this project. In particular, we would like to acknowledge the contributions of
Lorraine Gorman and Shaila Kumar, whose advice and support
from initial conception to completion were invaluable to this study. Harding C, Romanou E, Williams J, Peters M (2012) The 2011 Skills for Life
Survey: A Survey of Literacy, Numeracy and ICT Levels in England:
Report for BIS, BIS Research Paper. Available from https://www.gov.uk/
government/uploads/system/uploads/attachment_data/file/36000/
12-p168-2011-skills-for-life-survey.pdf (accessed 7 December 2012). Hewitson P, Ward A, Heneghan C, Halloran S, Mant D (2011) Primary care
endorsement letter and a patient leaflet to improve participation in
colorectal cancer screening: results of a factorial randomised trial. Br J Cancer 105(4): 475–480. Jensen JD, King AJ, Carcioppolo N, Krakow M, Samadder NJ, Morgan S
(2014) Comparing tailored and narrative worksite interventions at
increasing colonoscopy adherence in adults 50–75: a randomized
controlled trial. Soc Sci Med 104: 31–40. Reminders for bowel scope screening non-responders Department of Health (2012) NHS Bowel Cancer Screening Programme –
Advice to the NHS Bidding Process. Available from https://www.gov.uk/
government/uploads/system/uploads/attachment_data/file/215205/
dh_132468.pdf (accessed 29 May 2015). Dillard AJ, Fagerlin A, Dal Cin S, Zikmund-Fisher BJ, Ubel PA (2010)
Narratives that address affective forecasting errors reduce perceived
barriers to colorectal cancer screening. Soc Sci Med 71(1): 45–52. The authors declare no conflict of interest. The authors declare no conflict of interest. Kincaid JP, Fishburne Jr RP, Rogers RL, Chissom BS (1975) Derivation of new
readability formulas (automated readability index, fog count and flesch
reading ease formula) for navy enlisted personnel: Research Branch Report
8-75. Memphis, TN: Naval Air Station. Available from http://
www.dtic.mil/dtic/tr/fulltext/u2/a006655.pdf (accessed: 7 December 2015). CONFLICT OF INTEREST Jones RM, Woolf SH, Cunningham TD, Johnson RE, Krist AH, Rothemich SF,
Vernon SW (2010) The relative importance of patient-reported barriers to
colorectal cancer screening. Am J Prev Med 38(5): 499–507. ACKNOWLEDGEMENTS Geurts SME, Massat NJ, Duffy SW (2015) Likely effect of adding flexible
sigmoidoscopy to the English NHS Bowel Cancer Screening Programme:
impact on colorectal cancer cases and deaths. Br J Cancer 113(1): 142–149. DISCUSSION This feasibility study was initiated to test the format and potential
efficacy of incorporating a mailed self-referral reminder and locally
tailored information leaflet into the current BSS invitation process. The reminder, when sent with the locally tailored information
leaflet 1 year after the participants’ initial invitation, facilitated
uptake in 15.5% of recipients, thereby exceeding the threshold for
further investigation in a RCT (n ¼ 24 vs n ¼ 3). The self-referral
rate for individuals not sent a 12 months’ reminder during the
study period was higher than anticipated (1.2% vs 0.35%); however, This feasibility study was initiated to test the format and potential
efficacy of incorporating a mailed self-referral reminder and locally
tailored information leaflet into the current BSS invitation process. The reminder, when sent with the locally tailored information
leaflet 1 year after the participants’ initial invitation, facilitated
uptake in 15.5% of recipients, thereby exceeding the threshold for
further investigation in a RCT (n ¼ 24 vs n ¼ 3). The self-referral
rate for individuals not sent a 12 months’ reminder during the
study period was higher than anticipated (1.2% vs 0.35%); however, This feasibility study was initiated to test the format and potential
efficacy of incorporating a mailed self-referral reminder and locally
tailored information leaflet into the current BSS invitation process. The main limitation of this study is that the intervention used
contained multiple components (including gender preference,
appointment preference and a locally tailored information leaflet),
and so without the appropriate control groups the contribution of
each factor to the success of the reminder could not be teased
apart. The next step would be to perform a RCT testing each of
these components with appropriate controls. The reminder, when sent with the locally tailored information
leaflet 1 year after the participants’ initial invitation, facilitated
uptake in 15.5% of recipients, thereby exceeding the threshold for
further investigation in a RCT (n ¼ 24 vs n ¼ 3). The self-referral
rate for individuals not sent a 12 months’ reminder during the
study period was higher than anticipated (1.2% vs 0.35%); however, www.bjcancer.com | DOI:10.1038/bjc.2016.43 756 CONCLUSION This study found that a locally tailored information leaflet and
mailed reminder letter, with options for the day and time of the
appointment and the gender of the practitioner performing the
test, was feasible, efficacious and exceeded a minimum level of
efficacy needed to merit further investigation in a RCT. Engel J (1988) Polytomous logistic regression. Stat Neerl 42(4): 233–252. Farraye FA, Wong M, Hurwitz S, Puleo E, Emmons K, Wallace MB,
Fletcher RH (2004) Barriers to endoscopic colorectal cancer screening: are
women different from men? Am J Gastroenterol 99(2): 341–349. General Medical Council (2008) Consent: patients and doctors making
decisions together. Available from http://www.gmc-uk.org/Consent___
English_1015.pdf_48903482.pdf (accessed 18 November 2015). REFERENCES A’Hern R (2001) Sample size tables for exact single-stage phase II designs. Stat Med 20(6): 859–866. Kiviniemi MT, Bennett A, Zaiter M, Marshall JR (2011) Individual-level
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Resonant (2015) About us. Available from http://resonant.agency/about/
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Steele R, Kostourou I, McClements P, Watling C, Libby G, Weller D, Brewster
D, Black R, Carey F, Fraser C (2010) Effect of repeated invitations on
uptake of colorectal cancer screening using faecal occult blood testing:
analysis of prevalence and incidence screening. BMJ 341: c5531. Varadarajulu S, Petruff C, Ramsey WH (2002) Patient preferences for gender
of endoscopists. Gastrointest Endosc 56(2): 170–173. Vernon SW (1997) Participation in colorectal cancer screening: a review. J Natl Cancer Inst 89(19): 1406–1422. Wardle J, Miles A, Atkin W (2005) Gender differences in utilization of
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(accessed 10 July 2015). Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc) Supplementary Information accompanies this paper on British Journal of Cancer website (http://ww 758 www.bjcancer.com | DOI:10.1038/bjc.2016.43
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Do family physicians advise younger people on cardiovascular disease prevention? A cross-sectional study from Slovenia
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Abstract Background: One of the main family practice interventions in the younger healthy population is advice on how to
keep or develop a healthy lifestyle. In this study we explored the level of counselling regarding healthy lifestyle by
family physicians and the factors associated with it. Methods: A cross-sectional study with a random sample of 36 family practices, stratified by size and location. Each
practice included up to 40 people aged 18–45 with low/medium risk for cardiovascular disease (CVD). Data were
obtained by patient and practice questionnaires and semi-structured interviews. Several predictors on the patient
and practice level for received advice in seven areas of CVD prevention were applied in corresponding models
using a two-level logistic regression analysis. Results: Less than half of the eligible people received advice for the presented risk factors and the majority of
them found it useful. Practices with medium patient list-sizes showed consistently higher level of advice in all areas
of CVD prevention. Independent predictors for receiving advice on cholesterol management were patients’ higher
weight (regression coefficient 0.04, p=0.03), urban location of practice (regression coefficient 0.92, p=0.04),
organisation of education by the practice (regression coefficient 0.47, p=0.01) and practice list size (regression
coefficient 6.04, p=0.04). Patients who self-assessed their health poorly more frequently received advice on smoking
(regression coefficient −0.26, p=0.03). Hypertensive patients received written information more often (regression
coefficient 0.66, p=0.04). People with increased weight more often received advice for children’s lifestyle (regression
coefficient 0.06, p=0.03). We did not find associations with patient or practice characteristics and advice regarding
weight and physical activity. We did not find a common pattern of predictors for advice. Conclusions: Counselling for risk diseases such as increased cholesterol is more frequently provided than basic
lifestyle counselling. We found some doctors and practice factors associated with counselling behaviour, but the
majority has to be explained by further studies. Keywords: Primary prevention, Cardiovascular diseases, Family practice, Counselling, Multilevel analysis still not unanimous [2] and is the subject of extensive re-
search activity [3]. Petek et al. BMC Family Practice 2013, 14:82 Petek et al. BMC Family Practice 2013, 14:82 Petek et al. BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 Open Access Do family physicians advise younger people
on cardiovascular disease prevention?
A cross-sectional study from Slovenia Davorina Petek1*, Rok Platinovsek2, Zalika Klemenc-Ketis1,3 and Janko Kersnik1,3 Davorina Petek1*, Rok Platinovsek2, Zalika Klemenc-Ketis1,3 and Janko Kersnik1,3 * Correspondence: davorina.petek@gmail.com
1Department of Family Medicine, Medical Faculty, University of Ljubljana,
Poljanski nasip 58, 1000, Ljubljana, Slovenia
Full list of author information is available at the end of the article © 2013 Petek et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Subjects A random sample of 36 family medicine practices, strati-
fied by size (small: up to two full-time equivalent (FTE)
working family physicians on the same location, large:
more than two FTE family physicians) and location
(urban: more than 30,000 habitants, rural: 30,000 habi-
tants or less) were included in the study. Out of 56 in-
vited practices, 36 agreed to participate (response rate of
64.3%). According to European guidelines [4], primary preven-
tion in the broad sense of counselling to retain a healthy
lifestyle and improve risky behaviour should also be di-
rected at healthy people without any known risk factors. This is a feature of the holistic and comprehensive ap-
proach typical for family medicine. Furthermore, advice
on healthy lifestyle cannot be given without assessing
one’s lifestyle first. Therefore, GPs should advise all pa-
tients on a healthy lifestyle - regardless of their health
status and the presence of risk factors. In each practice, we aimed to include a random sam-
ple of 40 people (age 18 to 45) from their list of regis-
tered patients. Eligible people were those without any
chronic cardiovascular diseases. Patients with a diag-
nosed arterial hypertension or hypercholesterolemia but
not assessed by the family physician as being at high risk
for cardiovascular diseases were eligible for inclusion as
well. Cardiovascular risk assessment was defined by
using the Framingham score system, which is by na-
tional agreement a compulsory tool for CVD risk assess-
ment in the country. It provides scores from 0 to over
40 on the basis of patients’ age, sex, smoking history,
systolic blood pressure and cholesterol levels. The scores
are calculated automatically by a computer programme
as part of each patient’s record keeping system after en-
tering these data. Patients with scores from 0 to 20 on a
Framingham scale were eligible for inclusion. It is mostly
useful in age groups over 40. People were invited to take
part in the study by phone and mail. An introductory
letter, the letter of agreement to participate in the study,
signed by their physician and practicing nurse were
sent out. For the younger population some national guidelines
even suggest to intervene at a lower risk threshold [5]. Despite recommendations, many physicians feel that
healthcare’s resources and budget are insufficient for all
primary prevention activities and cannot meet the needs
of this group of people [6]. Methods been focused on the prevention of transmissible dis-
eases and was typically provided through public health
agencies. In recent years, cardiovascular prevention
also became the task of family physicians who can
provide individual counselling and use other strategies
for lifestyle modification. European guidelines on car-
diovascular prevention emphasise objectives of cardio-
vascular prevention in the younger population: to
retain a healthy lifestyle and to improve risky beha-
viour where necessary [4]. The study was a part of the international European
Practice Assessment of Cardiovascular risk manage-
ment (Epa-Cardio) study, which involved nine European
countries. Here, we present results of the Slovenian
sample. The detailed description of methodology has
already been published elsewhere [14]. Background Prevention of cardiovascular diseases for high risk popu-
lation is a traditional task of family physicians as part of
the comprehensive approach and community orientation
[1]. There is ample evidence that multiple interventions
lower mortality in high risk groups but the evidence for
health-promoting activities in the general population is In primary cardiovascular diseases (CVD) preven-
tion, a substantial part of the older generation is con-
sidered to have high cardiovascular risk even with only
a moderate elevation of modifiable risk factors. As a
consequence, interventions are focused on this gener-
ation and not on the younger population, for whom
cardiovascular risk is also assessed less often. Primary
prevention of the younger population has traditionally * Correspondence: davorina.petek@gmail.com
1Department of Family Medicine, Medical Faculty, University of Ljubljana,
Poljanski nasip 58, 1000, Ljubljana, Slovenia
Full list of author information is available at the end of the article Page 2 of 9 Petek et al. BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 Petek et al. BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 Subjects Many studies on cardiovascular prevention have focused
on the population older than 40 or 50 years [7,8] or on a
wide age range population [9] and have dealt with their
risk assessment. Only a few studies have addressed cardio-
vascular prevention for young people [10]. Attitudes of
people towards lifestyle change, the role of their family
doctor [11], patient expectations and received counselling
[12] have been assessed in the whole population, not tak-
ing into account any age differences. Younger groups of people usually come for a consult-
ation when they need a health service for acute health
problem and are classified as less frequent visitors. Young people are usually not included in routine pre-
ventive programs. Slovenia is one of the few European
countries that launched a National Programme of Car-
diovascular Prevention already in 2001. One of its main
critiques was that eligible people groups were too old
(men 35–65, women 45–70 years old) in terms of long-
term primary prevention impact. Out of 1,440 invited people, 953 of them returned the
questionnaires. Later on we excluded 16 people because
of missing data and performed a final analysis on 937
questionnaires by list-wise exclusion (response rate of
65.0%). Attitudes and practice toward preventive activities dif-
fer also between the family physicians themselves; they
depend on their own lifestyle [13] and on their practice
characteristics such as a heavy workload. Questionnaire Each enrolled participant filled in the questionnaire that
consisted of basic demographic data (gender, age, educa-
tion, marital and employment status), self-assessment of
health (using a five-point Likert-type scale ranging from
excellent (5 points) to poor (1 point), length of attach-
ment to the practice and frequency of yearly attendance
of the practice. In the second part of the questionnaire,
the participants stated whether they received advice on In our study, we aimed to explore the level of counsel-
ling that young people receive in family medicine prac-
tices and their experience with the received counselling,
and to determine factors that can influence healthy life-
style counselling provision. Petek et al. BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 Page 3 of 9 Page 3 of 9 risk factors for CVD and lifestyle and gave their opinion
on the usefulness of this advice. Some questions on
whether CVD advice was received were not administered
to all respondents but rather to a smaller subgroup. The
question on smoking advice was only administered to
smokers, ex-smokers or occasional smokers and the ques-
tion on children’s lifestyle was only administered to re-
spondents with children. All patients also filled out the
questionnaire on their lifestyle. The following validated
questionnaires were used for this purpose: physical ac-
tivity (RAPA questionnaire) [15], eating habits [16,17] and
smoking status (MID-SIZED Model questionnaire) [18]. lifestyle more often than the rest of the population. A
corresponding dummy-variable was included in the
analyses. The software used to perform the estimation was the
package lme4 [21] for R (R Development Core Team
2011) [22]. Statistical methods 312 respondents (33.3%) were overweight (BMI 25–30
kg/m2) and 138 (14.7%) were obese (BMI >30 kg/m2). 89
(9.5%) patients claimed that they had hypertension and 87
(9.3%) patients stated that they had hypercholesterolemia. The Epa-Cardio data cannot be analyzed with classical
methods because these methods assume that the units
were sampled independently. This assumption is violated
in our case because people were sampled in clusters
(practices). People attending the same practice are likely
to be more similar than people attending different prac-
tices. This clustering requires the use of multilevel ana-
lysis [19,20], which allows for correct estimation of the
standard errors of predictors in the explanatory model. This is especially important since we have a number of
predictors that are measured at the practice level whose
standard errors would be grossly underestimated with
classical methods of analysis. The statistical analysis is
focused on whether the participant received advice in
one of the seven cardiovascular diseases prevention
areas. Because the response was dichotomous (advice
received/not received), we applied a two-level logistic re-
gression to the data. We fit the so-called random inter-
cept model seven times – to each of the areas of advice
that the people could receive. The response variables
and patient-level predictors are described in the follow-
ing section. A specific part of our sample - males be-
tween 35 and 45 years are included in the Slovenian
National Preventive Programme, which is why we hy-
pothesized that this group would receive advice on 424 (45.2%) of respondents stated that they never
smoked cigarettes; others were current, occassional or
past smokers. Lifestyle/ risk factors of the respondents According to the RAPA questionnaire, less than half of
the sample of 850 people had adequate aerobic physical
activity (384 respondents, 45.2%) and an even lower per-
cent of the people were also performing exercises for
stretching and for muscular strength (240 respondents,
28.5% of the sample). The average score on the REAP-S questionnaire for
healthy diets of the respondents was 26.5 (SD=4.0). The
lowest possible score on this item was 12 points and the
highest score 39 with higher scores indicating a health-
ier diet. The study was approved by the Slovenian National
Committee on Medical Ethics (No. 87/11/07). Description of the sample The majority (23 or 63.9%) of the participating practices
were small practices (employed no more than two GPs)
and located in rural areas (26 practices or 72.2%). The
mean age of people was 35.2 (SD 8,1) years. The basic
demographic structure of the participating people is
given in Table 1. Family physicians from each included practice filled out
the questionnaire on practice characteristics. Additionally,
the main researcher (DP) performed a semi-structured
interview with all of the physicians. The questionnaire in-
cluded questions on practice-led contacts for prevention
(system for recalling people for CVD prevention), clinical
information system (computer-supported patient file sys-
tem), case finding methods to detect people with cardio-
vascular risk factors, existing procedures for smoking
cessation, work in community (participation of physicians
and nurses in public healthcare programmes on lifestyle),
education of GPs and nurses on CVD in the last year and
registered patients’ list-size. Advice on lifestyle Figure 1 shows how many respondents recall being given
advice in seven CVD areas. Less than half of the eligible
respondents received advice for the presented risk fac-
tors. In the two cases where the subgroups of (ex)
smokers/occasional smokers and people with children
were analysed, the percentages in Figure 1 pertain to the
narrower subgroup as is also reflected in the lower num-
ber of cases. The questionnaire also inquired whether the respon-
dents found the advice to be helpful or not. Because the
great majority of respondents reported the advice to be
helpful, we performed the analysis by merging the cat-
egories “not helpful” and “helpful” and performed the
multilevel analyses on the resulting dichotomous (rather
that multinomial) variable discriminating only between
whether advice was given or not. Page 4 of 9 Petek et al. BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 Table 1 Demographic, behavioural and health self-
assessment characteristics of 937 patients aged from
18-45 years with low/medium cardiovascular risk
Demographic characteristics
No. (%) of patients
Gender
Men
384 (41.0)
Women
528 (56.4)
Missing
25 (2.7)
Age
18-30
266 (28.4)
31-40
388 (41.4)
41-45
261 (27.9)
Missing
22 (2.3)
Education
Primary school or less
134 (14.3)
Secondary school
453 (48.3)
University
313 (33.4)
Missing
37 (3.9)
Employment status
Unemployed
116 (12.4)
Employed
788 (84.1)
Missing
33 (3.5)
Marital status
Married, cohabiting
649 (69.3)
Single, divorced, widowed
264 (28.2)
Missing
24 (2.6)
Self assessment of health
Poor
37 (3.9)
Fair
139 (14.8)
Good
386 (41.2)
Very good
276 (29.5)
Excellent
71 (7.6)
Missing
28 (3.0)
Practice attendance (years)
≤2
112 (12.0)
3-7
226 (24.1)
8-12
169 (18.0)
>13
401 (42.8)
Missing
29 (3.1)
Visit frequency/year (n=918)
0-1
212 (22.6)
2-3
384 (41.0)
4-5
177 (18.9)
6-7
68 (7.3)
≥8
77 (8.2)
Missing
19 (2.0) Table 1 Demographic, behavioural and health self-
assessment characteristics of 937 patients aged from
18-45 years with low/medium cardiovascular risk
Demographic characteristics
No. Factors related to received advice for prevention of CVD Factors related to received advice for prevention of CVD
Table 2 shows association of several predictor variables
on patient and practice level with received preventive
advice. We could not find any statistical prediction of the
following variables on received CVD advice: gender,
employment, marital status, practice size, case-finding
methods, community resources. Some variables were
borderline statistically significant. Advice on lifestyle (%) of patients
Gender
Men
384 (41.0)
Women
528 (56.4)
Missing
25 (2.7)
Age
18-30
266 (28.4)
31-40
388 (41.4)
41-45
261 (27.9)
Missing
22 (2.3)
Education
Primary school or less
134 (14.3)
Secondary school
453 (48.3)
University
313 (33.4)
Missing
37 (3.9)
Employment status
Unemployed
116 (12.4)
Employed
788 (84.1)
Missing
33 (3.5)
Marital status
Married, cohabiting
649 (69.3)
Single, divorced, widowed
264 (28.2)
Missing
24 (2.6)
Self assessment of health
Poor
37 (3.9)
Fair
139 (14.8)
Good
386 (41.2)
Very good
276 (29.5)
Excellent
71 (7.6)
Missing
28 (3.0)
Practice attendance (years)
≤2
112 (12.0)
3-7
226 (24.1)
8-12
169 (18.0)
>13
401 (42.8)
Missing
29 (3.1)
Visit frequency/year (n=918)
0-1
212 (22.6)
2-3
384 (41.0)
4-5
177 (18.9)
6-7
68 (7.3) Table 1 Demographic, behavioural and health self-
assessment characteristics of 937 patients aged from
18-45 years with low/medium cardiovascular risk While conducting preliminary bivariate analyses, we
encountered a curvilinear association between the pa-
tient list size (the total number of patients on the prac-
tice list of one GP) and the proportion of respondents
who reported receiving CVD advice. Figures 2 and 3 de-
pict the proportion of respondents who received advice
as a function of the list size categorized into four cat-
egories. The proportion of respondents who received ad-
vice is lowest in the high and low numbers of people on
the list, while this proportion is higher in the middle two
categories of the list size. This trend is remarkably con-
sistent across all eight areas of CVD advice. Summary of main findings and their contextualisation Figure 1 Advice and avowed helpfulness of advice- valid row percentages in the sample of younger people in national part of the
Epa-Cardio study. Legend: Number of given advice in different areas differs due to missing values in the questionnaire and due to analysis of
two subgroups of patients (smokers/ex-smokers, people with children). list size. With only one exception (advice on cigarette
smoking in respondents with poor self-assessment of
health), we did not find any meaningful (or the signifi-
cance was borderline) association with patient/practice
characteristics and advice in three models: for cigarette
smoking, weight and physical activity. More often,
provision of advice on healthy lifestyle for respondents
who assessed their health worse was also found in other
studies, athough the results were not consistent across
all types of advice [26]. On the contrary to our results,
other studies found that a rural location was associated
with better provision of preventive services [32]. information possibilities. Another less often provided ad-
vice was the advice on children’s lifestyle. In Slovenia,
paediatricians are included in primary care and serve as
children’s personal doctors. Regardless of this, family
physicians should advise their patients – parents about
children’s lifestyle too. Similarly, advice on community
resources was not common, despite the fact that our
country has a long tradition in community organisation
of healthcare [30]. The percentage of advice on smoking
was also surprisingly low, a classical field for education
on healthy lifestyle. It was also perceived as the least
helpful by respondents. We found it very interesting to receive a stable result
in the association of middle-sized patient lists with more
regularly given advice for all types of healthy living life-
styles. Large list sizes of registered patients are an obs-
tacle for preventive work, especially counselling [31]. Small list size practices are difficult to analyse, but they
might experience difficulties in the organisation of pre-
ventive activities as the doctors from such practices
might have been involved in other primary care services
or do not work full-time in the practice. Surprisingly, in the subsample of males from 35 to 45
years old, who are included in the Slovenian National
Preventive Programme, our analyses could not confirm
any higher figures on counselling for this group of
people. Summary of main findings and their contextualisation Summary of main findings and their contextualisation The lifestyle of the studied younger family practice
population showed disappointing results regarding the
level of physical activity, healthy food habits, the num-
ber of people who are currently regular or occasional
smokers or who were smokers in the past and increased
body weight/obesity. The figures on obesity were similar
to the figures in a national epidemiological study, while
the percentage of people exercising adequately showed
a lower level of physical activity in our study [23]. In a group of healthy people 18–45 years of age
(around 10% having isolated health problems like arterial
hypertension or hypercholesterolemia), advice on any as-
pect of cardiovascular prevention was given in only 50%
of the people. Similar to other studies [24], we found that advice on
physical exercise was the most common advice given
(49.1%). Complex counselling on more aspects of life-
style was also relatively common (45.3%). Percentages
found in our study are slightly higher than in most of
the other studies, where the figures in these studies were
low in the early nineties [25], and higher in recent years
[24] and varied considerably – advice on diet around
40% [26-28], on physical exercise 25-42% [26,27] and on
smoking 30% or 40% for the adult population [27,29]. On the other hand, we found many »missing oppor-
tunities«, such as a low level of given written informa-
tion or advice to visit educational websites. The latter
might be especially important because this was a young
generation, which is familiar with the internet and its Page 5 of 9 Petek et al. BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 education: children's lifestyle (n=592)
local sports club or physical exercise (n=862)
educational websites (n=859)
written information on life style (n=866)
two or more consultations on lifestyle (n=855)
children's lifestyle (n=552)
cholesterol (n=858)
blood preassure (n=865)
physical exercise/activity (n=858)
weight (n=859)
smoking (n=513)
valid percent
0
20
40
60
80
100
no
advice
not
helpful
helpful
Figure 1 Advice and avowed helpfulness of advice- valid row percentages in the sample of younger people in national part of the
Epa-Cardio study. Legend: Number of given advice in different areas differs due to missing values in the questionnaire and due to analysis of
two subgroups of patients (smokers/ex-smokers, people with children). Discussion on methodology The methodology has already been tested in the pilot
study for the EPA-Cardio study. The questionnaire for
participants contained questions, which were the results
of previous work in the EPA-Cardio study [34]. Validated
questionnaires were used to obtain information on diet,
physical activity and smoking status. Questions on prac-
tice characteristics were based on previous research on
practice assessment [35]. A further strength of this study is a large sample, a
good response rate and multi-level statistical analyses. The response rate was actually one of the highest com-
pared to other countries in the Epa-Cardio study. The inclusion of the practices followed strict stratifica-
tion rules and every practice that refused to participate
was replaced by a practice from the same stratification
field. This in fact could be a source of selection bias. Figure 2 Proportion of advice on smoking, blood pressure,
physical activity and children’s lifestyle according to patient
list size. As we can observe in other studies, the number of
women in the sample was substantially higher, which can
be attributed to the effect of self-choice: women are more
frequent attenders of the practice than men [36] and more
interested in participation in the survey than men [37]. Most of the respondents were long-term patients in the
same practice, which is also in line with other European
practices that have a patient list system [38]. The education of providers on preventive activities in-
creased counselling on cholesterol. The result is interest-
ing because it can show that education is too concentrated
on secondary prevention and the management of risk dis-
eases for CVD and does not emphasize lifestyle modifica-
tion in general. We can compare our results with the systematic review
of Bock et al. [33]: some practice characteristics that were
related to higher levels of counselling in the review were
the size of the practice, protocols for prevention and We also have to address problems of multiple testing. The statistical models include many predictor variables:
nine on the patient level and nine on the practice level. We do not, however, consider multiple testing to be an
important issue as the inclusion of each predictor was the-
oretically well-grounded as described in the introduction. Summary of main findings and their contextualisation This goes in line with other studies – the group
of younger people with low risk of cardiovascular disease
is known to be rather neglected in the provision of pre-
ventive activities [10,26]. We could not prove the positive relationship of elec-
tronic support system on advice; on the opposite, prac-
tices with computer-supported patient file systems gave
less advice on several topics of lifestyle; we cannot ex-
plain the result but it can be connected with the lack of
systematic recording in e-patient files. Independent determinants for provided advice on
managing blood cholesterol levels were the most numer-
ous: patients’ weight and the following practice charac-
teristics: urban location, practice education and patient Petek et al. BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 Page 6 of 9 proportion
0
0.2
0.4
0.6
list size (categorized)
−1500
1500−2000
2000−2500
2500+
phisical activity
smoking
blood preassure
children's lifestyle
Figure 2 Proportion of advice on smoking, blood pressure,
physical activity and children’s lifestyle according to patient
list size. printed materials in the practice. The first one was proven
also in our study. proportion
0
0.2
0.4
0.6
list size (categorized)
−1500
1500−2000
2000−2500
2500+
phisical activity
smoking
blood preassure
children's lifestyle Discussion on methodology proportion
0
0.2
0.4
0.6
list size (categorized)
−1500
1500−2000
2000−2500
2500+
lifestyle
weight
cholesterol
written information
Figure 3 Proportion of complex lifestyle advice, advice on
weight, cholesterol and given written information according to
patient list size. y
g
The information on received advice came from the
respondents and was not compared with their record
and was therefore prone to subjectivity. We also have
to be aware of possible social desirability in assessing
the helpfulness of advice. However, other studies used
the same methodology [39,40]. We also know that re-
cords on advice are not very consistent either [41], and
that the agreement between self-reports and medical
records varies [42,43]. The delivery and recording of
advice was much higher in the systems with adequate
incentives, such as in the Quality and Outcomes Frame-
work programme in the United Kingdom [44,45]. In
Slovenian paper or electronic records, counselling has
no structured recording form and we believe that re-
cording is forgotten on many occasions, although we do
not have any statistical data to prove it. No incentives
are given for the recording of counselling in this age
group. Moreover, respondents’ information/perception
of received advice is the information that we were
looking for in this study. Figure 3 Proportion of complex lifestyle advice, advice on
weight, cholesterol and given written information according to
patient list size. Petek et al. References
W 1. Wonca Europe: The European Definition of Primary Care. http://www. woncaeurope.org/gp-definitions. 1. Wonca Europe: The European Definition of Primary Care. http://www. woncaeurope.org/gp-definitions. Discussion on methodology BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 Page 7 of 9 Page 7 of 9 Table 2 Multilevel logistic regression analysis of patient and practice factors on received advice for prevention of CVD
in the sample of younger people (regression coefficient above; statistical significance below in each cell)
Smoking
Weight
Physical activity
Cholesterol
Lifestyle
Children lifestyle
Written in
N= 478
N=775
N=779
N=770
N=777
N=501
N=774
intercept
−3.89
-3.52
-3.45
-9.69
-3.12
-20.56
-3.32
0.16
0.17
0.12
0.00
0.22
0.00
0.29
Gender (female)
0.36
-0.07
0.07
-0.01
-0.20
0.14
0.23
0.11
0.68
0.67
0.98
0.26
0.60
0.27
age
-0.01
0.01
0.00
-0.01
0.01
-0.06
-0.02
0.75
0.29
0.89
0.70
0.21
0.01
0.19
Male 35-45 years
1.42
1.17
0.81
-1.08
-0.04
1.04
-0.36
0.05
0.05
0.14
0.23
0.95
0.66
0.05
education
-0.03
0.08
0.04
0.27
-0.15
-0.06
-0.20
0.87
0.54
0.77
0.09
0.25
0.74
0.09
Employment (not employed)
0.14
0.05
-0.07
0.46
0.34
-0.10
0.36
0.65
0.85
0.76
0.10
0.17
0.81
0.10
Marital status (not married)1
'0.06
'0.01
0.00
0.09
-0.11
-0.56
-0.15
0.82
0.97
0.99
0.71
0.58
0.07
0.51
Self assessment of health2
-0.26
-0.08
0.00
-0.06
0.14
0.11
0.08
0.03
0.36
0.96
0.60
0.12
0.47
0.47
BMI3
0.03
0.01
0.00
0.04
-0.01
0.06
0.03
0.14
0.53
0.99
0.03
0.47
0.03
0.15
Hypertension4
0.16
0.20
-0.19
-0.08
0.32
0.37
0.66
0.67
0.50
0.50
0.82
0.27
0.38
0.04
Practice size (large)
-0.03
-0.04
0.01
0.06
0.33
0.04
0.00
0.93
0.89
0.97
0.87
0.33
0.94
1.00
Practice loca- tion (urban)
-0.04
-0.21
-0.13
0.92
-0.18
-0.02
0.41
0.91
0.60
0.71
0.04
0.66
0.96
0.42
Recall system CVD5
-0.74
-0.93
-0.53
-0.76
-0.34
0.33
-0.60
0.14
0.05
0.22
0.16
0.49
0.61
0.32
E pt file6
-0.48
-0.48
-0.75
-2.14
-1.29
-1.09
-0.92
0.27
0.26
0.05
0.00
0.00
0.06
0.09
Case finding methods7
0.21
0.25
0.20
0.00
0.29
-1.19
0.84
0.73
0.67
0.70
1.00
0.62
0.15
0.29
Community resources8
-0.06
-0.21
-0.07
-0.13
-0.07
-0.05
0.01
0.70
0.15
0.58
0.42
0.63
0.82
0.96
Education organisation9
-0.03
0.10
0.22
0.47
0.12
0.00
0.09
0.88
0.55
0.14
0.01
0.47
0.99
0.67
List size10 linear
3.99
3.16
2.99
6.04
2.44
21.21
1.19
0.12
0.20
0.15
0.04
0.31
0.00
0.70 Table 2 Multilevel logistic regression analysis of patient and practice factors on received advice for prevention of CVD
in the sample of younger people (regression coefficient above; statistical significance below in each cell) c regression analysis of patient and practice factors on received advice for prevention of CVD
people (regression coefficient above; statistical significance below in each cell) Petek et al. Discussion on methodology BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 Page 8 of 9 Table 2 Multilevel logistic regression analysis of patient and practice factors on received advice for prevention of CVD
in the sample of younger people (regression coefficient above; statistical significance below in each cell) (Continued) Table 2 Multilevel logistic regression analysis of patient and practice factors on received advice for prevention of CVD
in the sample of younger people (regression coefficient above; statistical significance below in each cell) (Continued)
List size quadratic
-0.88
-0.72
-0.62
-1.19
-0.46
-5.30
-0.24
0.13
0.19
0.19
0.07
0.40
0.01
0.72
Legend
1Marital status: 1= Married / cohabiting, 2= Single, Separated / divorced, Widowed. 2Self-assessment of health: How would you estimate your health status in general: 1= excellent, 2= very good and 3= good were aggregated to “good”, 4= fair
and 5= poor to “poor”. 3Value of BMI (kg/m2). 4Yes/No answer of the patient if he/she has hypertension. 5Use of system for recall of people at risk for CVD. 6Computer-supported patient file system. 7If the practice uses case-finding methods to detect people with cardiovascular risk factors. 8Do practice physicians and nurses cooperate in local/community campaigns or actions? 9Education of providers: education of all nurses/GPs in the last five years on CVD, education of at least one nurse/GP on CVD in the last 15 months. 10Number of people on the patient list. As this was a cross-sectional study, we could not as-
sess the effect of the advice given. We also did not in-
clude advice on alcohol consumption in the study. Funding for the international EPA-Cardio study was supported by the
Bertelsmann foundation (http://www.bertelsmann-stiftung.de/cps/rde/xchg/
bst/hs.xsl/prj_8519.htm), which had no involvement in the study design,
analyses or interpretation of the data. In Slovenia it was supported also by
the Slovenian Family Medicine Society. Funding for the international EPA-Cardio study was supported by the
Bertelsmann foundation (http://www.bertelsmann-stiftung.de/cps/rde/xchg/
bst/hs.xsl/prj_8519.htm), which had no involvement in the study design,
analyses or interpretation of the data. In Slovenia it was supported also by
the Slovenian Family Medicine Society. Authors’ contributions
DP
i i
d i
h d y
8. Guallar E, Banegas JR, Blasco-Colmenares E, Jiménez FJ, Dallongeville J,
Halcox JP, et al: Excess risk attributable to traditional cardiovascular risk
factors in clinical practice settings across Europe - The EURIKA Study. BMC Publ Health 2011, 11:704. DP participated in the design of the study, acquisition and interpretation of
the data and drafted the manuscript. RP performed the statistical analysis
and interpretation and participated in manuscript writing. ZKK participated in
the acquisition of the data and manuscript writing. JK coordinated the study,
participated in the acquisition and interpretation of the data and manuscript
writing. All authors read and approved the final manuscript. 9. Thio SL, Twickler TB, Cramer MJ, Giral P: National differences in screening
programmes for cardiovascular risks could obstruct understanding of
cardiovascular prevention studies in Europe. Neth Heart J 2011,
19:458–463. Conclusion Competing interests
All authors declare that they have no competing interest. Author details
1 As the research in the field of giving health advice to the
young and healthy population is scarce, more studies are
needed to determine the best possible methodology for
providing advice in order to achieve maximum effective-
ness. Also, there is a need for larger international studies
in this subject, which will use prospective methodology
to assess the success of such advice. 1Department of Family Medicine, Medical Faculty, University of Ljubljana,
Poljanski nasip 58, 1000, Ljubljana, Slovenia. 2Faculty of Social Sciences,
University of Ljubljana, Kardeljeva ploscad 5, 1000, Ljubljana, Slovenia. 3Department of Family Medicine, Medical Faculty, University of Maribor,
Slomskov trg 15, 2000, Maribor, Slovenia. Conclusion In our study we could relate some people and practice
characteristics to certain forms of cardiovascular preven-
tion advice provided. It seems that basic counselling for
lifestyle (smoking, weight and physical activity) is not
provided widely enough to the younger population and
we could prove only some associations in each model. It
seems that the providers’ education is still too concen-
trated on what they perceive as a disease. The recording
of advice in electronic files needs to be standardized and
carried out systematically. Policy makers should be
aware that list size matters in the uptake of preventive
activities. Neither too small nor to large practices per-
form on a desirable level regarding provision of advice
on healthy lifestyle for young people. 2. Ebrahim S, Taylor F, Ward K, Beswick A, Burke M, Davey Smith G: Multiple
risk factor interventions for primary prevention of coronary heart
disease. Cochrane Database Syst Rev 2011, 1:CD001561. y
y
p
risk factor interventions for primary prevention of coronary heart
disease. Cochrane Database Syst Rev 2011, 1:CD001561. y
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characteristics, health behavior, and reported stage of change. Subst Abuse Treat Prev Policy 2010, 5:30. Acknowledgments Submit your next manuscript to BioMed Central
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• Immediate publication on acceptance
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and take full advantage of:
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Submit your manuscript at
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Cite this article as: Petek et al.: Do family physicians advise younger
people on cardiovascular disease prevention? A cross-sectional study
from Slovenia. BMC Family Practice 2013 14:82. Petek et al. BMC Family Practice 2013, 14:82
http://www.biomedcentral.com/1471-2296/14/82 Submit your next manuscript to BioMed Central
and take full advantage of: 30. Klancar D, Svab I, Kersnik J: Vizijaprihodnostizdravstvenihdomov v
Sloveniji. The vision of health centers in Slovenia. ZdravVar 2010,
49:37–43. In slovenian. • Convenient online submission • Thorough peer review 31. Pelletier-Fleury N, Le Vaillant M, Hebbrecht G, Boisnault: Determinants of
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Community Health SNAP Project Team: Explaining the variation in the
management of lifestyle risk factors in primary health care: a multilevel
cross sectional study. BMC Publ Health 2009, 9:165.
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Plasma Retinol Concentrations and Dietary Intakes of Mother–Infant Sets in Singleton versus Twin Pregnancy
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University of Nebraska Medical Center
University of Nebraska Medical Center
DigitalCommons@UNMC
DigitalCommons@UNMC
Journal Articles: Pediatrics
Pediatrics
2023
Plasma Retinol Concentrations and Dietary Intakes of Mother-
Plasma Retinol Concentrations and Dietary Intakes of Mother-
Infant Sets in Singleton versus Twin Pregnancy
Infant Sets in Singleton versus Twin Pregnancy
Anum Akbar
Sarah Duvall
Matthew Van Ormer
Rebecca Slotkowski
Taija Hahka
See next page for additional authors
Tell us how you used this information in this short survey.
Follow this and additional works at: https://digitalcommons.unmc.edu/com_peds_articles
Part of the Pediatrics Commons University of Nebraska Medical Center
University of Nebraska Medical Center
DigitalCommons@UNMC
DigitalCommons@UNMC Plasma Retinol Concentrations and Dietary Intakes of Mother-
Plasma Retinol Concentrations and Dietary Intakes of Mother-
Infant Sets in Singleton versus Twin Pregnancy
Infant Sets in Singleton versus Twin Pregnancy See next page for additional authors Tell us how you used this information in this short survey. Follow this and additional works at: https://digitalcommons.unmc.edu/com_peds_articles
Part of the Pediatrics Commons Anum Akbar, Sarah Duvall, Matthew Van Ormer, Rebecca Slotkowski, Taija Hahka, Thiago Genaro-Mattos,
Zeljka Korade, Corrine K. Hanson, Ann Anderson-Berry, and Melissa K. Thoene Citation: Akbar, A.; Duvall, S.;
VanOrmer, M.; Slotkowski, R.; Hahka
T.; Genaro-Mattos, T.; Korade, Z.;
Hanson, C.; Anderson Berry, A.;
Thoene, M. Plasma Retinol
Concentrations and Dietary Intakes
of Mother–Infant Sets in Singleton
versus Twin Pregnancy. Nutrients
2023, 15, 2553. https://doi.org/
10.3390/nu15112553 Keywords: pregnancy; twins; retinol; RAE; vitamin A; maternal; infant; newborn Article
Plasma Retinol Concentrations and Dietary Intakes of
Mother–Infant Sets in Singleton versus Twin Pregnancy Anum Akbar 1
, Sarah Duvall 2, Matthew VanOrmer 1
, Rebecca Slotkowski 1
, Taija Hahka 1,
Thiago Genaro-Mattos 3
, Zeljka Korade 1
, Corrine Hanson 2, Ann Anderson Berry 1
and Melissa Thoene 1,* Anum Akbar 1
, Sarah Duvall 2, Matthew VanOrmer 1
, Rebecca Slotkowski 1
, Taija Hahka 1,
Thiago Genaro-Mattos 3
, Zeljka Korade 1
, Corrine Hanson 2, Ann Anderson Berry 1
and Melissa Thoene 1,* 1
Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE 68198, USA;
anum.akbar@unmc.edu (A.A.); rebecca.slotkowski@unmc.edu (R.S.)
2
College of Allied Health Professions, University of Nebraska Medical Center, Omaha, NE 68198, USA
3
Munroe Meyer Institute, University of Nebraska Medical Center, Omaha, NE 68198, USA
*
Correspondence: melissak.thoene@unmc.edu *
Correspondence: melissak.thoene@unmc.edu Abstract: Vitamin A (retinol) is essential for normal fetal development, but the recommendation
for maternal dietary intake (Retinol Activity Equivalent, RAE) does not differ for singleton vs. twin pregnancy, despite the limited evaluation of retinol status. Therefore, this study aimed to
evaluate plasma retinol concentrations and deficiency status in mother–infant sets from singleton
vs. twin pregnancies as well as maternal RAE intake. A total of 21 mother–infant sets were included
(14 singleton, 7 twin). The HPLC and LC-MS/HS evaluated the plasma retinol concentration, and
data were analyzed using the Mann–Whitney U test. Plasma retinol was significantly lower in twin vs. singleton pregnancies in both maternal (192.2 vs. 312.1 vs. mcg/L, p = 0.002) and umbilical cord (UC)
samples (102.5 vs. 154.4 vs. mcg/L, p = 0.002). The prevalence of serum-defined vitamin A deficiency
(VAD) <200.6 mcg/L was higher in twins vs. singletons for both maternal (57% vs. 7%, p = 0.031) and
UC samples (100% vs. 0%, p < 0.001), despite a similar RAE intake (2178 vs. 1862 mcg/day, p = 0.603). Twin pregnancies demonstrated a higher likelihood of vitamin A deficiency in mothers, with an
odds ratio of 17.3 (95% CI: 1.4 to 216.6). This study suggests twin pregnancy may be associated with
VAD deficiency. Further research is needed to determine optimal maternal dietary recommendations
during twin gestation. nutrients nutrients nutrients 1. Introduction Vitamin A (retinol) is a fat-soluble vitamin produced in the body from preformed
vitamin A and provitamin A carotenoids found in food [1]. Preformed vitamin A, mainly
in the form of retinyl palmitate, is exclusively derived from animal sources [2]. Foods
such as milk, meat, and eggs are primary sources of this type of vitamin A. On the other
hand, provitamin A carotenoids such α-carotene, β-carotene, and β-cryptoxanthin can
be found in both plant-based foods and animal products [3]. The body converts pro-
vitamin A carotenoids into retinol. Among these carotenoids, β-carotene has full biological
activity, while α-carotene and β-cryptoxanthin exhibit partial biological activity, compared
to retinol [4]. Vitamin A in the form of retinoic acid carries out its functions primarily by
interacting with nuclear receptors known as retinoic acid receptors (RAR) and retinoid X
receptors (RXR) [5]. Upon binding to these receptors, a complex forms between the retinoic
acid and its receptors, which subsequently activates the retinoic acid response elements
(RAREs) located in the promoter region of target genes. This mechanism allows vitamin A
to regulate gene expression, influencing various biological processes. Academic Editor: Jaume Amengual Academic Editor: Jaume Amengual Received: 10 May 2023
Revised: 26 May 2023
Accepted: 29 May 2023
Published: 30 May 2023 Received: 10 May 2023
Revised: 26 May 2023
Accepted: 29 May 2023
Published: 30 May 2023 Authors
Authors Anum Akbar, Sarah Duvall, Matthew Van Ormer, Rebecca Slotkowski, Taija Hahka, Thiago Genaro-Mattos,
Zeljka Korade, Corrine K. Hanson, Ann Anderson-Berry, and Melissa K. Thoene nutrients nutrients Copyright:
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). g
g
p
g
g
p
Vitamin A is an essential micronutrient that plays a vital role in early embryo de-
velopment, fetal lung development and maturation, cellular differentiation, and fetal
organ formation during pregnancy [6,7]. Studies have shown that vitamin A plays a role https://www.mdpi.com/journal/nutrients Nutrients 2023, 15, 2553. https://doi.org/10.3390/nu15112553 Nutrients 2023, 15, 2553 2 of 10 in regulating cell differentiation and proliferation through epigenetic modifications of
DNA methylation and histone modification [8]. However, vitamin A deficiency (VAD)
remains one of the leading nutritional deficiencies worldwide, especially among preg-
nant women [9]. VAD during pregnancy can have various adverse consequences for both
mothers and infants [10–13]. Studies have found an association between VAD and two
adverse outcomes in mothers: maternal anemia [11] and night blindness [10]. One possible
explanation for the anemia is that vitamin A plays a role in the absorption and utilization of
non-heme iron, and a deficiency in vitamin A can lead to anemia [11]. Furthermore, VAD
increases susceptibility to infections, and maternal infections may be a contributing factor
to preterm delivery in individuals with VAD [12]. In infants, VAD can result in intrauterine
growth restriction (IUGR), preterm birth, and increased risk of sepsis [9,12,13]. g
(
) p
p
Maternal metabolic demand during pregnancy is heightened, due to fetal growth. In the case of twin pregnancy, this metabolic demand is further amplified, potentially
leading to alterations in nutrient requirements for optimal pregnancy outcomes and a
greater probability of encountering nutritional deficiencies in comparison to singleton preg-
nancy [14]. Worldwide, approximately 2% of all gestations are twin pregnancies, and rates
are continuing to rise, due to the growing use of assisted reproductive techniques [15,16]. Twin pregnancy confers several risks to infants and mothers [17,18]. Infants are more prone
to preterm birth, leading to potential developmental challenges and health issues. Growth
alterations are also common, with an increased likelihood of low birth weight or IUGR [19]. Mothers of twins face a higher susceptibility to gestational diabetes and preeclampsia,
posing risks to both mother and babies [17,18]. Additionally, there is a greater chance of
maternal or infant morbidity and mortality, due to several factors including the intricate
nature of managing multiple pregnancies [15,17]. However, to our knowledge, no study
has evaluated the status of retinol in singleton and twin pregnancy and no specific recom-
mendations have been made for vitamin A dietary intake requirements in multiparous
pregnancies. Studies comparing the status of vitamin D, folic acid, and energy requirements
in singleton vs. twin pregnancy show that the demands for folic acid and vitamin D are
higher in twin pregnancies [20]. Notably, however, current maternal dietary recommenda-
tions for vitamin A do not differ between singleton and multiparous pregnancies, implying
that vitamin A requirements are not increased during twin pregnancy [20,21]. Fetal retinol
status is impacted by maternal retinol status and dietary intake [10]; however, there is a gap
in our knowledge of how pregnancy type (singleton vs. twin) affects infant retinol status. Therefore, this study aims to analyze maternal RAE intake and retinol concentrations in the
umbilical cord (UC) and maternal blood plasma of mother–infant sets from both singleton
and twin pregnancies. 2. Materials and Methods 2.1. Study Design and Participants 2.5. Electronic Data Collection The electronic medical record was used to gather data for all mothers and infants
in the identified subcohort. From the mother’s medical record, we collected maternal
race/ethnicity, delivery mode, pregnancy type (singleton vs. twin), smoking status (current
vs. former/never smoker), and mass index (BMI) in kilograms/meters2 (kg/m2) measured
at <10 weeks of gestation. From the infant medical record, we collected infant sex, corrected
gestational age at birth (CGA), birth length (centimeters, cm), birth weight (kg) and birth
head circumference (cm). CGA groups were defined as preterm (<37 weeks CGA) and term
(≥37 weeks CGA), and maternal pre-pregnancy BMI categories were defined using CDC
guidelines [26,27]. 2.3. Retinol Laboratory Analysis A subset (n = 22) of the biological samples was analyzed at the Nutritional Biomarker
Lab at Harvard T. H. Chan School of Public Health using high-performance liquid chro-
matography (HPLC) [22]. The other subset (n = 6) of samples was analyzed at the University
of Nebraska Medical Center using liquid chromatography–tandem mass spectrometry (LC-
MS/MS). Plasma retinol concentrations were measured in 100 µL aliquots. The antioxidants
and internal standards were added, followed by Folch extraction, separation of the organic
phase, and reconstitution in ethanol and acetonitrile. Samples were then placed in an Ac-
quity ultra-performance liquid chromatography (UPLC) system with an ANSI-compliant
well plate holder coupled to a Thermo Scientific TSQ Quantis mass spectrometer equipped
with an atmospheric pressure chemical ionization (APCI) source. Ten microliters of the
sample was injected onto the Phenomenex Luna Omega C18 column using water (0.1% v/v
acetic acid) as solvent A and methanol (0.1% v/v acetic acid) as solvent B. The total run time
was 15 min, at a flow rate of 500 µL/min. The retinol was analyzed using selective reaction
monitoring (SRM). Quantitation was achieved using a cocktail of internal standards, and
the concentrations were normalized to the amount of sample and reported as mcg/L. Quality control in both analyzing labs was ensured by using NIST standards, and each
batch of samples run included several replicates of a plasma pool sample set. 2.4. Retinol Status Classification Maternal plasma retinol concentrations were categorized based on the guidelines pro-
vided by the World Health Organization (WHO) into adequate (>300.9 mcg/L), insufficient
(≥200.6–300.9 mcg/L), or deficient (<200.6 mcg/L) [23]. For infant UC plasma, there is no
clear consensus on the cut-off concentrations for VAD. Thus, in this study, the values used
to categorize retinol status in the infant were based on the cut-off points identified by the
WHO for extreme cases, which is consistent with other research [24]. Infant UC plasma
retinol concentrations were therefore defined as similar to maternal classifications [25]. 2.1. Study Design and Participants After IRB approval, this study performed a secondary analysis of mothers and their
infants previously enrolled in a cohort of 687 maternal-infant dyads. Inclusion criteria for
the overall cohort included having at least one live-born infant, maternal age of 19 years or
older, and admission to the Labor and Delivery Unit at Nebraska Medicine (Omaha, NE,
USA) between June 2015 and September 2020. Exclusion criteria included gastrointestinal,
liver, or kidney disease affecting nutrient metabolism in mother or infant, inborn errors of
metabolism, congenital abnormalities, or infants deemed wards of the state. g
After identifying seven twin pregnancies among this initial cohort, we conducted a
2:1 matching process to pair newborns from singleton pregnancies with those from twin
pregnancies, resulting in a total sample size of 21 mothers and 28 infants in this subcohort. In the matching process, participants were primarily grouped according to gestational age. In the case of multiple births of the same age, matching was completed stepwise by birth
weight, followed by sex, and lastly, race/ethnicity. It should be noted that not all pairs were
a perfect match, and some variables were not identical between paired infants. Nutrients 2023, 15, 2553 3 of 10 3 of 10 2.2. Biospecimen Collection 2.2. Biospecimen Collection Maternal blood plasma and infant umbilical cord plasma samples were collected in
K2 EDTA tubes. The maternal blood samples were obtained during regular laboratory
procedures when the mother was admitted for delivery. UC blood samples were collected
from every newborn during routine cord blood collection at time of delivery. To maintain
the nutrient integrity, all blood samples were kept protected from light and heat, and
processed and frozen within 12 h of collection at a temperature of −80 ◦C. 2.6. Maternal Dietary Intake and Recommended Daily Allowance 2.6. Maternal Dietary Intake and Recommended Daily Allowance Maternal dietary intake during pregnancy was evaluated using the Harvard Food
Frequency Questionnaire (FFQ), which was completed by the mothers during their hospital
stay at the time of delivery [28]. The questionnaire was used to calculate average daily
vitamin A intake as measured by retinol activity equivalents (RAE, mcg/day), a standard
measure for vitamin A that accounts for pre-formed retinol and provitamin A carotenoid
dietary sources [29]. Nutrients 2023, 15, 2553 4 of 10 2.7. Statistical Analysis 2.7. Statistical Analysis Non-parametric statistical tests were employed for the analysis of continuous and
categorical data, due to the small sample size and non-normal data distribution, as con-
firmed by the Shapiro–Wilk test (p < 0.05). The Mann–Whitney U test was used to compare
continuous data within and between categorical groups, and Spearman’s rank correlation
coefficients were used to evaluate the relationship between continuous variables within
and between groups with significant differences. Fisher’s exact and Pearson’s chi-square
tests were utilized to determine the association between categorical data. Linear regres-
sion analyses were employed to further assess the relationship between variables. The
statistical analyses were conducted using IBM SPSS Statistics software (Version 28.0) and a
p-value < 0.05 was considered significant. ** For singleton pregnancies, pre-conception BMI was available for 8 mothers. For twin pregnancies, pre
conception BMI was available for 6 mothers. *** RAE: Retinol Activity Equivalent. 3.1. Study Population ** Characteristics of twins are noted as individuals, not as sets, so each twin infant’s characteristics are represented. Table 3 displays the plasma retinol concentrations for the maternal and UC samples,
as categorized by pregnancy type. Mothers of twins had significantly lower plasma retinol
concentrations than mothers of singletons (192.2 mcg/L vs. 312 mcg/L, p = 0.012). Likewise,
infant UC plasma retinol concentrations were significantly lower in twins compared to
singletons (103 mcg/L vs. 154 mcg/L, p = 0.002). However, no significant difference was
found between infant UC plasma retinol concentrations for twin 1 and twin 2 (106.9 mcg/L
vs 100.9 mcg/, p = 0.383) (not shown in Table 3) and most of the mothers in our study
cohort had pregnancies with diamniotic and dichorionic twins. Table 3. Differences in Maternal and Infant Plasma Retinol Concentrations Between Singleton and
Twin Pregnancies. Singletons (n = 14)
Twins (n = 14)
p-Value
Median
IQR **
Median
IQR **
Maternal Plasma Retinol
(mcg/L)
312.1
262.5–396.5
192.2
181.2–297.6
0.012
Infant UC *** Plasma Retinol
(mcg/L)
154.4
123.1–211.2
102.5
96.4–137.1
0.002
** IQR: Interquartile Range. *** UC: Umbilical Cord. Table 3. Differences in Maternal and Infant Plasma Retinol Concentrations Between Singleton and
Twin Pregnancies. 3.1. Study Population Maternal and infant characteristics are summarized in Tables 1 and 2, respectively. The
results showed no significant differences between singleton- and twin-pregnancy types, in
terms of maternal age at delivery, BMI, race/ethnicity, mode of delivery, or smoking habits
(p > 0.05). Additionally, there were no significant differences in infant characteristics, includ-
ing birth CGA, birthweight, birth length, head circumference, or sex, between singleton-
and twin-pregnancy types (p > 0.05). All mothers from both groups met the recommended
intake of 770 mcg/d of RAE, with one mother in the singleton-pregnancy type exceed-
ing the maximum dose or upper limit (UL) of 3000 mcg/d, by consuming 3206 mcg/d
(107% of UL) [29]. Moreover, there was no significant difference found in maternal RAE
intake between the groups (p = 0.603), with a median RAE intake of 1862 mcg/day and
2178 mcg/day in mothers of singletons vs. mothers of twins, respectively. Table 1. Baseline Characteristics of Mothers by Pregnancy Type. Maternal Characteristics (n = 21)
Singleton Mothers
(n = 14)
Twin Mothers
(n = 7)
p-Value
Median
IQR
Median
IQR
Age at delivery (years)
31
26.25–36.25
32
28.0–36.0
0.946
BMI ** (kg/m2)
30.9
24.7–34.6
31.2
26.0–38.9
0.202
Maternal RAE intake *** (mcg/day)
1862
1256.5–2371.0
2178
1606.3–2342.8
0.603
n (%)
n (%)
Race
White
9 (64%)
5 (57%)
1.00
Non-white
5 (36%)
3 (43%)
Mode of Delivery
Vaginal
6 (43%)
3 (43%)
1.00
Cesarean
8 (57%)
4 (57%)
Smoking Status
Current smoker
3 (21%)
1 (14%)
1.00
Former/Never smoker
11 (79%)
6 (86%)
** For singleton pregnancies pre conception BMI was available for 8 mothers
For twin pregnancies pre Table 1. Baseline Characteristics of Mothers by Pregnancy Type. Table 1. Baseline Characteristics of Mothers by Pregnancy Type. Nutrients 2023, 15, 2553 5 of 10 Table 2. Baseline Characteristics of Singleton and Twin Newborns. Infant Characteristics (n = 28)
Singleton Newborns
(n = 14)
Twin Newborns
(n = 14) **
p-Value
Median
IQR
Median
IQR
Corrected Gestational Age (CGA) (weeks)
36.6
32.7–37.6
36.6
32.9–37.6
0.804
Birth weight (kg)
2.4
2.2–3.0
2.4
1.8–3.2
0.874
Birth length (cm)
45.3
44.4–48.5
46.8
43.4–48.3
0.804
Head circumference (cm)
32.9
31.9–34.5
32.8
30.8–34.5
0.804
n (%)
n (%)
Sex
Male
12 (86%)
8 (57%)
0.209
Female
2 (14%)
6 (43%)
** Characteristics of twins are noted as individuals, not as sets, so each twin infant’s characteristics are represented. Table 2. Baseline Characteristics of Singleton and Twin Newborns. 3.3. Vitamin A Status between Pregnancy Types Similarly, correlations between birth CGA and infant UC
plasma retinol or maternal plasma retinol were both found to be insignificant (p = 0.895
and p = 0.130). Table 5. Correlations of Maternal and Infant Retinol Plasma Concentrations with Other Variables. Retinol Concentration
Correlations with
Single Pregnancy
Twin Pregnancy
Spearman’s R
p-Value
Spearman’s R
p-Value
Maternal Plasma Retinol
(mcg/L)
UC *** Plasma Retinol
0.574
0.032
0.408
0.148
Maternal RAE **
0.288
0.318
0.071
0.808
UC *** Plasma Retinol
(mcg/L)
Maternal RAE **
−0.108
0.714
−0.266
0.358
** RAE: Retinol Activity Equivalents. *** UC: Umbilical Cord. The results of linear regression analysis indicate that singleton pregnancy is associated Table 4. Categorizations of Retinol Status of Mothers and Infants Between Pregnancy Type. Singleton Pregnancy (n = 14)
Twin Pregnancy (n = 7)
p-Value
Mothers (n = 21)
n (%)
n (%)
0.031
Deficient (<200.55 mcg/L)
1 (7%)
4 (57%)
Insufficient (≥200.55–300.82 mcg/L)
5 (36%)
2 (29%)
Adequate (>300.82 mcg/L)
8 (57%)
1 (14%)
Singleton Pregnancy (n = 14)
Twin Pregnancy (n = 14)
p-Value
Infants (n = 28)
n (%)
n (%)
<0.001
Deficient (<100.27 mcg/L)
0 (0%)
14 (100%)
Insufficient (≥100.27–300.82 mcg/L)
13 (93%)
0 (0%)
Adequate (>300.82 mcg/L)
1 (7%)
0 (0%)
3.4. Relationship between Maternal Retinol Concentrations and Pregnancy Type on Infant
Retinol Concentrations Table 4. Categorizations of Retinol Status of Mothers and Infants Between Pregnancy Type. 3.4. Relationship between Maternal Retinol Concentrations and Pregnancy Type on Infant
Retinol Concentrations 3.4. Relationship between Maternal Retinol Concentrations and Pregnancy Type on Infant
Retinol Concentrations A significant positive correlation was found between maternal plasma and infant UC
plasma retinol concentrations in singleton pregnancies (r = 0.574, p = 0.032). However,
there was no significant correlation found between the same variables in twin pregnancies
(r = 0.408, p = 0.148). No significant correlations were identified between maternal plasma
retinol and maternal RAE intake in singleton or twin pregnancies (r = 0.288, p = 0.318 and
r = 0.071, p = 0.808, respectively) or between infant UC plasma retinol and maternal RAE
intake in singleton or twin pregnancies (r = −0.018, p = 0.714 and r = −0.266, p = 0.358, re-
spectively) (Table 5). Spearman’s rank correlation coefficients showed a lack of a significant
relationship between the UC plasma retinol concentration of twin 1 and twin 2 (r = 0.643
and p = 0.119) in a singular set. 3.3. Vitamin A Status between Pregnancy Types 3.3. Vitamin A Status between Pregnancy Types Table 4 presents the proportions of adequate, insufficient, and deficient categorizations
of plasma retinol for both mothers and infants. The results demonstrate significant differ-
ences in the proportion of classifications by pregnancy type for both mothers (p = 0.031)
and infants (p < 0.001). In mothers, only 7% of singletons were VAD, compared to 57%
of twin pregnancies (p = 0.025). However, there were no significant differences between
classifications of adequacy (p = 1.00) or insufficiency (p = 0.159). q
y p
y p
Among infants, all 14 twin newborns but no singleton newborns met the criteria for
VAD (p < 0.001). As such, there was a significant difference in the number of singleton
newborns meeting the criteria for insufficiency, compared to twin newborns (p < 0.001). Nutrients 2023, 15, 2553 6 of 10 Table 4. Categorizations of Retinol Status of Mothers and Infants Between Pregnancy Type. Singleton Pregnancy (n = 14)
Twin Pregnancy (n = 7)
p-Value
Mothers (n = 21)
n (%)
n (%)
0.031
Deficient (<200.55 mcg/L)
1 (7%)
4 (57%)
Insufficient (≥200.55–300.82 mcg/L)
5 (36%)
2 (29%)
Adequate (>300.82 mcg/L)
8 (57%)
1 (14%)
Singleton Pregnancy (n = 14)
Twin Pregnancy (n = 14)
p-Value
Infants (n = 28)
n (%)
n (%)
<0.001
Deficient (<100.27 mcg/L)
0 (0%)
14 (100%)
Insufficient (≥100.27–300.82 mcg/L)
13 (93%)
0 (0%)
Adequate (>300.82 mcg/L)
1 (7%)
0 (0%)
3.4. Relationship between Maternal Retinol Concentrations and Pregnancy Type on Infant
Retinol Concentrations
A significant positive correlation was found between maternal plasma and infant UC
plasma retinol concentrations in singleton pregnancies (r = 0.574, p = 0.032). However,
there was no significant correlation found between the same variables in twin pregnancies
(r = 0.408, p = 0.148). No significant correlations were identified between maternal plasma
retinol and maternal RAE intake in singleton or twin pregnancies (r = 0.288, p = 0.318 and
r = 0.071, p = 0.808, respectively) or between infant UC plasma retinol and maternal RAE
intake in singleton or twin pregnancies (r = −0.018, p = 0.714 and r = −0.266, p = 0.358, re-
spectively) (Table 5). Spearman’s rank correlation coefficients showed a lack of a significant
relationship between the UC plasma retinol concentration of twin 1 and twin 2 (r = 0.643
and p = 0.119) in a singular set. 4.1. Retinol Status Twin pregnancy was identified as a possible risk factor for VAD in infants. To our
knowledge, this is the first study to report this finding. No singleton infants had VAD, which
conflicts with other studies. We have previously reported high rates of VAD in singleton
infants [30]. However, this study observed striking VAD among the twin participants
in this cohort. While there is limited information on plasma retinol concentrations in
twin pregnancies, similar results have been found in the case of other micronutrients [31]. A prior study found that twin newborns and their mothers had higher rates of vitamin
D deficiency compared to singletons. The transfer of nutrients through the placenta is
believed to contribute to VAD, particularly in the context of twin pregnancies, where there
may be two placentas [32]. This is because each placenta needs to fulfill the nutritional
needs of one fetus with a singular maternal nutrient supply, and each placenta also retains
retinol within its tissue, combining to result in low plasma retinol levels in twin infants. In
this study, it was found that 6 out of 7 twin sets had diamniotic dichorionic pregnancies
(the most common type of twin placentation), which could have also contributed to the
observed low concentrations of plasma retinol in infants. Similarly, mothers of twins had a higher prevalence of VAD than singleton mothers,
which is theorized to be due to increased nutrient requirements during twin compared to
singleton pregnancy, depleting retinol stores more quickly by the time of delivery [33]. It
is possible that having to nourish two babies instead of one contributes to this difference. In addition, the difference in plasma retinol concentration between twin and singleton
pregnancies may also be due to variations in the concentration of the retinol binding
protein (RBP4), which has been shown in previous research to affect retinol concentrations
in singleton pregnancies [34]. Not surprisingly, twin newborns did not have significantly
different plasma retinol concentrations within individual twin sets (twin 1 vs. twin 2). Even though RAE intake was within recommended limits in both groups, twin mothers
and infants were still found to be deficient in vitamin A. In addition, the study found that
there was no significant difference in maternal RAE intake between mothers of singletons
and twin pregnancies, and no significant correlations were identified between maternal
and infant UC plasma retinol and maternal RAE intake in singleton or twin pregnancies. 3.3. Vitamin A Status between Pregnancy Types Similarly, correlations between birth CGA and infant UC
plasma retinol or maternal plasma retinol were both found to be insignificant (p = 0.895
and p = 0.130). Table 5. Correlations of Maternal and Infant Retinol Plasma Concentrations with Other Variables. Retinol Concentration
Correlations with
Single Pregnancy
Twin Pregnancy
Spearman’s R
p-Value
Spearman’s R
p-Value
Maternal Plasma Retinol
(mcg/L)
UC *** Plasma Retinol
0.574
0.032
0.408
0.148
Maternal RAE **
0.288
0.318
0.071
0.808
UC *** Plasma Retinol
(mcg/L)
Maternal RAE **
−0.108
0.714
−0.266
0.358
** RAE: Retinol Activity Equivalents. *** UC: Umbilical Cord. Table 5. Correlations of Maternal and Infant Retinol Plasma Concentrations with Other Variables. The results of linear regression analysis indicate that singleton pregnancy is associated
with significantly higher UC plasma retinol levels, with a beta value of 63.73 (p < 0.05). This suggests that, on average, singleton pregnancy UC plasma retinol is 63.73 higher
when compared to twin pregnancy. The pregnancy-type variable was found to account for
21.7% of the variance in the UC plasma retinol levels (r2 = 0.217, p < 0.05), indicating that
pregnancy type is a significant predictor of retinol levels. Moreover, the odds of vitamin
A deficiency in mothers with twin pregnancies were found to be significantly higher
compared to singleton pregnancies, with a 17.3-fold increase in odds (95% confidence
interval: 1.4–216.6). This suggests that twin pregnancy is associated with a substantially
greater risk of VAD in mothers. 7 of 10 Nutrients 2023, 15, 2553 7 of 10 4.1. Retinol Status These findings suggest that there may be differences in the relationship between maternal
and infant retinol concentrations in singleton versus twin pregnancies, and that maternal
RAE intake may not be the only contributor to infant UC plasma retinol concentrations. In addition, it should be noted that the status of maternal retinol does not always have an
impact on infant retinol stores, as there are several factors that can affect this status [35]. Therefore, increasing dietary retinol intake may not necessarily result in an immediate
increase in maternal plasma or infant umbilical cord plasma retinol concentrations, since the
metabolism and distribution of the nutrient can vary throughout the body [36]. Nonetheless,
it is crucial for pregnant women to meet the recommended intake of retinol to fulfill their
physiological needs, decrease the risk of VAD, and provide sufficient supply to their
newborn infants during breastfeeding [6]. It is also important to note that excessive exposure to vitamin A could lead to vitamin
A toxicity, although such instances are rare in the US. Vitamin A toxicity can be categorized
into two main forms: acute and chronic [37]. Acute vitamin A toxicity predominantly
affects the mucocutaneous system, while chronic vitamin A toxicity can impact multiple
organ systems. Of particular concern during fetal development is the teratogenicity caused
by excessive vitamin A, which can lead to various congenital anomalies affecting multiple
systems, including the central nervous system (CNS), cardiovascular system (CVS), and
genitourinary system [38]. Conversely, mothers experiencing acute vitamin A toxicity
may exhibit symptoms such as nausea, vomiting, increased cerebrospinal fluid pressure,
headaches, blurred vision, and impaired muscular coordination [39]. Consequently, inter-
ventions aimed at increasing maternal RAE intake should consider focusing on provitamin
A carotenoids as a safer alternative to preformed vitamin A [38]. Nutrients 2023, 15, 2553 8 of 10 Unlike preformed vitamin A, provitamin A carotenoids are generally considered
safe, even at high intake levels. A possible reason for this disparity is a difference in the
intestinal absorption of preformed vitamin A via passive diffusion, whereas provitamin
A carotenoids are absorbed via protein-mediated transport [40]. With passive diffusion, a
higher intake of vitamin A will lead to more absorption of vitamin A, hence increasing the
chances of vitamin A toxicity. However, with protein-mediated transport, absorption is
limited once the transport proteins are saturated, preventing high intakes of provitamin
A carotenoids from causing vitamin A toxicity. 4.2. Strengths and Limitations This study has several notable strengths, including its novel exploration of retinol
status among mother–infant sets of singletons vs. twin pregnancies and the use of a match-
ing process to control confounding variables. However, there are also several limitations
to consider. The sample size was relatively small, and certain samples were analyzed at
different labs—although NIST standards were used in each. Additionally, factors such as
socioeconomic status and smoking were not adjusted for in the analysis, despite potentially
affecting retinol levels. Furthermore, the matching process based on CGA may have skewed
the singleton group towards premature infants—factors such as group B streptococcus
infection, advanced maternal age, and a history of alcohol and smoking were found to be
associated with preterm delivery in singleton pregnancies—which could be influenced by
the shorter gestational period of twin pregnancies. It is important to note that retinol status
assessment is challenging in infants, due to low sample volumes, and the WHO criteria for
VAD is targeted towards older populations [23,41]. Therefore, some limitations need to be
considered when interpreting the study’s findings. 4.1. Retinol Status This characteristic renders provitamin A
carotenoids a potentially beneficial strategy for preventing VAD during pregnancy. By
promoting the consumption of provitamin A carotenoid-rich foods, such as fruits and
vegetables, interventions can help ensure an adequate vitamin A supply to pregnant
women while minimizing the risk of excessive retinol intake. Until more information
becomes available, women with multiparous pregnancies should continue to adhere to the
current recommendations for dietary RAE intake. References 1. Takahashi, N.; Saito, D.; Hasegawa, S.; Yamasaki, M.; Imai, M. Vitamin A in health care: Suppression of growth and induction of
differentiation in cancer cells by vitamin A and its derivatives and their mechanisms of action. Pharmacol. Ther. 2022, 230, 107942. [CrossRef] 1. Takahashi, N.; Saito, D.; Hasegawa, S.; Yamasaki, M.; Imai, M. Vitamin A in health care: Suppression of growth and induction of
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2. Knapik, J.J.; Hoedebecke, S.S. Vitamin A and Bone Fractures. J. Spec. Oper. Med. 2021, 21, 115–119. [CrossRef] 2. Knapik, J.J.; Hoedebecke, S.S. Vitamin A and Bone Fractures. J. Spec. Oper. Med. 2021, 21, 115–119. 3. do Tatiele Casagrande, N.; Eduardo, J.-L.; de Veridiana Vera, R.; Leila Queiroz, Z. Introductory Chapter
Vitamin A. In Vitamin A; Leila Queiroz, Z., de Veridiana Vera, R., Eduardo, J.-L., Eds.; IntechOpen: Rijek 3. do Tatiele Casagrande, N.; Eduardo, J.-L.; de Veridiana Vera, R.; Leila Queiroz, Z. Introductory Chapter: A Global Perspective on
Vitamin A. In Vitamin A; Leila Queiroz, Z., de Veridiana Vera, R., Eduardo, J.-L., Eds.; IntechOpen: Rijeka, Croatia, 2019; p. Ch. 1. 4. Perveen, S.; Zafar, S.; Iqbal, N.; Riaz, M. Provitamin A Carotenoids. In Carotenoids: Structure and Function in the Human Body;
Zia-Ul-Haq, M., Dewanjee, S., Riaz, M., Eds.; Springer International Publishing: Cham, Switzerland, 2021; pp. 775–797. g
y
p
p
Vitamin A. In Vitamin A; Leila Queiroz, Z., de Veridiana Vera, R., Eduardo, J.-L., Eds.; IntechOpen: Rijeka, Croatia, 2019; p. Ch. 1. 4. Perveen, S.; Zafar, S.; Iqbal, N.; Riaz, M. Provitamin A Carotenoids. In Carotenoids: Structure and Function in the Human Body;
Zi
Ul H
M D
j
S Ri
M Ed
S
i
I
i
l P bli hi
Ch
S
i
l
d 2021
775 797 j
4. Perveen, S.; Zafar, S.; Iqbal, N.; Riaz, M. Provitamin A Carotenoids. In Carotenoids: Structure and Function in the Human Body; 4. Perveen, S.; Zafar, S.; Iqbal, N.; Riaz, M. Provitamin A Carotenoids. In Carotenoids: Structure and Function in the Human Body;
Zia-Ul-Haq M Dewanjee S Riaz M Eds ; Springer International Publishing: Cham Switzerland 2021; pp 775–797 Zia-Ul-Haq, M., Dewanjee, S., Riaz, M., Eds.; Springer International Publishing: Cham, Switzerland, 2021; pp. 775–797. 5. Conaway, H.H.; Henning, P.; Lerner, U.H. Vitamin a metabolism, action, and role in skeletal homeostasis. Endocr. Rev. 5. Conclusions This study has revealed that the plasma retinol concentrations of mother–infant sets at
the time of delivery are impacted by multiparity, as twin mothers and infants demonstrated
a higher prevalence of VAD. Although the study did not establish a link between maternal
vitamin A consumption and plasma retinol concentrations, it highlights the need for
additional research to identify age-specific plasma retinol values for infants, which may
indicate health risks associated with VAD. Future studies could also evaluate whether
dietary RAE intake recommendations for singleton vs. multiparous pregnancies differ,
and analyze larger populations of term versus preterm individuals in singleton versus
twin pregnancies. Author Contributions: Conceptualization, M.T., C.H., A.A. and S.D..; methodology, C.H., M.V. and
S.D.; software, S.D., A.A. and R.S.; validation, C.H., M.T. and A.A.B.; formal analysis, S.D., A.A. and
R.S.; investigation, A.A., S.D., Z.K. and T.G.-M.; resources, M.T., C.H., M.V., Z.K., T.G.-M. and A.A.B.;
data curation, A.A., S.D. and R.S.; writing—original draft preparation, A.A. and S.D.; writing—review
and editing, A.A., S.D., M.V., R.S., T.H., T.G.-M., Z.K., C.H., A.A.B. and M.T.; visualization, S.D., A.A. and R.S.; supervision, M.T., C.H. and A.A.B.; project administration, M.V.; funding acquisition, A.A.B. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, M.T., C.H., A.A. and S.D..; methodology, C.H., M.V. and
S.D.; software, S.D., A.A. and R.S.; validation, C.H., M.T. and A.A.B.; formal analysis, S.D., A.A. and
R.S.; investigation, A.A., S.D., Z.K. and T.G.-M.; resources, M.T., C.H., M.V., Z.K., T.G.-M. and A.A.B.;
data curation, A.A., S.D. and R.S.; writing—original draft preparation, A.A. and S.D.; writing—review
and editing, A.A., S.D., M.V., R.S., T.H., T.G.-M., Z.K., C.H., A.A.B. and M.T.; visualization, S.D., A.A. and R.S.; supervision, M.T., C.H. and A.A.B.; project administration, M.V.; funding acquisition, A.A.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was internally funded by UNMC/Children’s Hospital Omaha Child Health
Research Institute. Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the University
of Nebraska Medical Center (112-15-EP, approved April 2015). Nutrients 2023, 15, 2553 9 of 10 9 of 10 Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data is available upon request to the corresponding author. 5. Conclusions Acknowledgments: We would like to thank Jeremy Furtado and the Harvard Nutrition Biomarkers
Lab for assistance in quantifying the carotenoid concentrations. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data is available upon request to the corresponding author. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data is available upon request to the corresponding author. Acknowledgments: We would like to thank Jeremy Furtado and the Harvard Nutrition Biomarkers
Lab for assistance in quantifying the carotenoid concentrations. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results. References 2013, 34,
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Concentrations Are Altered in Pregnant Women. Nutrients 2022, 14, 1365. [CrossRef] 35. van Stuijvenberg, M.E.; Schoeman, S.E.; Nel, J.; Lombard, C.J.; Dhansay, M.A. Serum retinol in post-partum mothers and
newborns from an impoverished South African community where liver is frequently eaten and vitamin A deficiency is absent. Matern Child. Nutr. 2017, 13, 3–6. [CrossRef] 36. Thoene, M.; Haskett, H.; Furtado, J.; Thompson, M.; Van Ormer, M.; Hanson, C.; Anderson-Berry, A. Effect of Maternal Retinol
Status at Time of Term Delivery on Retinol Placental Concentration, Intrauterine Transfer Rate, and Newborn Retinol Status. Biomedicines 2020, 8, 321. [CrossRef] 37. Olson, J.M.; Ameer, M.A.; Goyal, A. Vitamin A Toxicity. In StatPearls; StatPearls Publishing LLC.: Treasure Island, FL, USA, 2023. 38. Rothman, K.J.; Moore, L.L.; Singer, M.R.; Nguyen, U.S.; Mannino, S.; Milunsky, A. Teratogenicity of high vitamin A intake. N. Engl. J. Med. 1995, 333, 1369–1373. [CrossRef] y
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38. Rothman, K.J.; Moore, L.L.; Singer, M.R.; Nguyen, U.S.; Mannino, S.; Milunsky, A. Teratogenicity of high vitamin A intake. N. Engl. J. Med. 1995, 333, 1369–1373. [CrossRef] g
39. Allen, L.H.; Haskell, M. Estimating the potential for vitamin A toxicity in women and young children. J. Nutr. 2002, 132,
2907s–2919s. [CrossRef] 40. von Lintig, J.; Moon, J.; Lee, J.; Ramkumar, S. Carotenoid metabolism at the intestinal barrier. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2020, 1865, 158580. [CrossRef] 41. Allen, L.H.; Haskell, M. Vitamin A Requirements of Infants under Six Months of Age. Food Nutr. Bull. 2001, 22, 214–234. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
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people or property resulting from any ideas, methods, instructions or products referred to in the content.
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SplitSlider: A Tangible Interface to Input Uncertainty
Miriam Greis, Hyunyoung Kim, Andreas Korge, Albrecht Schmidt, Céline
Coutrix
To cite this version:
Miriam Greis, Hyunyoung Kim, Andreas Korge, Albrecht Schmidt, Céline Coutrix. SplitSlider: A
Tangible Interface to Input Uncertainty.
17th IFIP Conference on Human-Computer Interaction
(INTERACT), Sep 2019, Paphos, Cyprus. pp.493-510, 10.1007/978-3-030-29390-1. hal-02413628 SplitSlider: A Tangible Interface to Input Uncertainty
Miriam Greis, Hyunyoung Kim, Andreas Korge, Albrecht Schmidt, Céline
Coutrix To cite this version: Miriam Greis, Hyunyoung Kim, Andreas Korge, Albrecht Schmidt, Céline Coutrix. SplitSlider: A
Tangible Interface to Input Uncertainty. 17th IFIP Conference on Human-Computer Interaction
(INTERACT), Sep 2019, Paphos, Cyprus. pp.493-510, 10.1007/978-3-030-29390-1. hal-02413628 Distributed under a Creative Commons Attribution 4.0 International License SplitSlider: A Tangible Interface to Input Uncertainty
Miriam Greis1, Hyunyoung Kim1,2, Andreas Korge1,
Albrecht Schmidt1, Céline Coutrix1,2 Miriam Greis1, Hyunyoung Kim1,2, Andreas Korge1,
Albrecht Schmidt1, Céline Coutrix1,2 1 University of Stuttgart, Stuttgart, Germany
2 Université Grenoble Alpes, CNRS, Grenoble, France 1 University of Stuttgart, Stuttgart, Germany
2 Université Grenoble Alpes, CNRS, Grenoble, France miriam.greis@codecentric.de, hyunyoung.kim@imag.fr,
andi-korge@web.de, albrecht.schmidt@um.ifi.lmu.de,
celine.coutrix@imag.fr miriam.greis@codecentric.de, hyunyoung.kim@imag.fr,
andi-korge@web.de, albrecht.schmidt@um.ifi.lmu.de,
celine.coutrix@imag.fr Abstract. Experiencing uncertainty is common when answering questionnaires. E.g., users are not always sure to answer how often they use trains. Enabling users
to input their uncertainty is thus important to increase the data’s reliability and to
make better decision based on the data. However, few interfaces have been
explored to support uncertain input, especially with TUIs. TUIs are more
discoverable than GUIs and better support simultaneous input of multiple
parameters. It motivates us to explore different TUI designs to input users’ best
estimate answer (value) and uncertainty. In this paper, we first generate 5 TUI
designs that can input both value and uncertainty and build low-fidelity
prototypes. We then conduct focus group interviews to evaluate the prototypes
and implement the best design, SplitSlider, as a working prototype. A lab study
with SplitSlider shows that one third of the participants (4/12) were able to
discover the uncertainty input function without any explanation, and once
explained, all of them could easily understand the concept and input uncertainty. Keywords: Tangible User Interface; Uncertainty; Input Modality; Dial; Slider. HAL Id: hal-02413628
https://hal.science/hal-02413628v1
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abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1
Introduction Inputting data into a system while being uncertain about it is a common task. For
instance, in a usage survey at the train station, we might get asked about how often we
take the train. If we do not take the train regularly, it is difficult to answer a precise
value between like “daily” and “never”. Our answer might rather be “between every 6
months and weekly, and most often every 2 months”. Other examples range from
satisfaction surveys in airports to availability inquiry or medical questionnaires, where
we might hesitate between 54kg and 56kg to input our weight. Currently, respondents
are forced to answer a single, precise value even when they are uncertain. However,
allowing respondents to express their uncertainty about their answer enables the people
who requested the questionnaire to have precise and reliable data. For example, intra-
participant uncertainty can increase transparency and reliability of the data [22, 25],
produce relevant results [2, 10] and help to make better decisions [15, 25], e.g., when 2 designing a fidelity program for a company. Therefore, input mechanisms should allow
expressing uncertainty together with the most probable input value. However, there is a tradeoff between the usage simplicity and the ability to express
the uncertainty. Interactions with questionnaires are recommended to be easy to use and
discoverable while providing sufficient information [7]. Respondents often stay
novices when responding to a survey, as they rarely answer to the exact same
questionnaires twice. Hence survey companies either choose to offer a simple interface
to encourage as many respondents as possible, or a more complex interface able to
gather richer data from fewer participants but with the help of surveyors. Corresponding
examples can be found in public transportation surveys, where clients are offered either
happy/angry faces buttons in airports, or a surveyor approaching respondents with
tablets and multiple questions. Allowing respondents to input uncertainty can increase
data reliability, but it can also increase the survey’s interface complexity and cost. This motivates us to balance the simplicity and the ability to express the uncertainty. To achieve this goal, we explore Tangible User Interfaces (TUIs) as a way to offer users
with a flexible compromise between simplicity and the ability to express the
uncertainty. 1
Introduction TUIs are a good candidate to offer a flexible compromise between
simplicity and expressiveness, as they were found more discoverable than GUIs [14,
19] and better foster the simultaneous adjustments of parameters than GUIs, even more
than multitouch GUIs [20]. TUIs are already used to answer questionnaires, e.g.,
VoxBox [9] and happy/angry faces tangible buttons in satisfaction surveys in airports
[12]. However, these TUIs do not yet allow users to express their uncertainty about
their answer. To balance the simplicity and the ability to express the uncertainty, we explore the
design of physical dials and sliders that can capture users’ value and uncertainty on one
device. We first use Morphees+ [17] features to design five controls based on standard
tangible dials and sliders and that can input both value and uncertainty. We then present
low-fidelity prototypes and conduct a focus group study to find design requirements for
uncertain, tangible input. Following these findings, we present the most promising
design: the SplitSlider. The SplitSlider’s thumb supports entering one value (1-thumb
slider) and can be split to additionally enter a probability distribution (2- and 3-thumb
slider). We implemented and evaluated a functional tangible prototype. The results of
our study show that the use of SplitSlider as 1-thumb slider is discoverable. The use of
SplitSlider as 3-thumb slider is not as discoverable as the 1-thumb mode, but after its
explanation, it was found easy to use to express the uncertainty. SplitSlider allows users
to choose on the fly between its discoverable use as a standard 1-thumb slider or the
ability to express their uncertainty as a 3-thumb slider. 2.1
Uncertainty and Its Communication Uncertainty, as described by Pang et al. [23], includes statistical variations or spread,
errors and differences, minimum and maximum range values, noise, or missing data. Studies show that decision-making is better supported by capturing, modeling, and
visualizing uncertain data [2, 15, 22, 25]. For instance, communicating uncertainty can
increase transparency and reliability of weather forecasts [22, 25]. Pang et al. [23] also name three steps of data processing in which uncertainty can be
introduced: acquisition, transformation and visualization. In data acquisition,
uncertainty is inevitable due to inexact measurements. During transformation, the
original data can be altered by a human or an algorithm. Lastly, visualization may
introduce uncertainty as it does not usually use the same media as the original data. We
focus on the transformation step, where humans alter the data when answering
questionnaires. We especially tackle the input of the possible spread of an input value. Users answer questionnaires in many different ways. Among the most common
methods, we find pen and paper, e.g., in trains, and computer systems. The interfaces
to computer systems include TUIs, e.g., in airports with happy/angry buttons to give
feedback about a service, or GUIs, e.g., to fill forms on the Web. Previous work
proposed GUIs for users to express their uncertainty [10]. Among others, they
compared 1-thumb, 2-thumb and 3-thumb sliders to input uncertain data: •
The 1-thumb slider allows users to input a probability distribution by moving the
single thumb: users move the peak of the distribution, while the standard deviation,
skew and kurtosis are fixed. •
The 2-thumb slider allows users to input a probability distribution by
independently moving two thumbs: users move the minimum and maximum
values, while the peak of the distribution stays in the middle of these two values. •
The 3-thumb slider allows users to input a probability distribution by
independently moving three thumbs: users move the minimum, the maximum and
the peak values of the distribution. Our study focuses on gradual transition between certain and uncertain TUI inputs in
questionnaire settings. We consider the discoverability of interactions and the
compromise between ease of use and expressiveness. The previous study did not evaluate the discoverability of each slider, as each slider
was first explained to participants. The study rather focused on ease of use. 2
Related Work In the following, we present related work addressing the communication of uncertainty,
graphical interfaces supporting users to input uncertainty and tangible user interfaces
(TUIs) that could support uncertain input. 3 2.2
Communicating Input Uncertainty with TUIs Even though TUIs have not explicitly addressed the problem of inputting uncertain
data, prior work could be used for this purpose. Even though TUIs have not explicitly addressed the problem of inputting uncertain
data, prior work could be used for this purpose. Coutrix et al. suggest a resizable tangible slider [5] to compromise between travel
time of the thumb and input accuracy. The design could be also used to input uncertain
continuous data. By interpreting the size of the slider as the certainty (smaller size
means less precision and thus more uncertainty), this slider would be able to represent
a Gaussian distribution (uncertainty ≈ standard deviation; value ≈ mean). The Inflatable mouse [18] could be also used for uncertain input. The mouse has an
elastic chamber, and users can squeeze it to zoom a map in/out or to change their scroll
settings. For uncertain input, users could squeeze the mouse to express their certainty. The device can be used when users are inputting 2D data (e.g., X-Y coordinate values)
with 1D uncertainty (same standard deviation in both dimensions). In our work, we choose to systematically explore the design space of uncertain input
TUIs from scratch, rather than starting from the previous work. By doing so, we
consider more interactions than extending [5] and squeezing [18] only. 3
Design Exploration for Uncertain Input TUIs To design deformable tangible input controls that allow the quantitative input of
uncertainty, we generated variations of conventional continuous interfaces: dials and
sliders. We keep their rotational or linear control to input value and added other
modalities to input uncertainty. As an idea generation tool, we used the Morphees+
taxonomy [17]. The taxonomy describes all possible system-actuated shape-changes
and manual deformations in a systematic way. The generated designs can express
uncertainty in two ways: 1) all the dials and Expandable Slider (Fig. 1 and Fig. 2-left)
can input uncertainty as an amount centered around the value (i.e., 0-100%), and 2)
Split Slider (Fig. 2-right) can input a range of estimated values, independently of the
central value. When the device was to input an amount of uncertainty, we mapped the
larger surface or open space with more uncertainty and less pressure with more
uncertainty for design coherency. 2.1
Uncertainty and Its Communication Even though
the graphical 3-thumb slider was experimentally found the best compromise between
easiness and the ability to express uncertainty [10], it offers a fixed compromise
between easiness and the ability to express uncertainty. Moving the three thumbs in
sequence was found cumbersome by the participants, compared to the 1-thumb slider. In addition, the 1-thumb slider was better suited for users with little knowledge in
statistics, while the 3-thumb slider was better suited for users with more expertise in
statistics. We aim at supporting all levels of expertise in statistics. To support all levels of expertise in statistics, we explore deformable TUIs that allow
both certain and uncertain inputs. Instead of using the one-, two-, three- thumbed
sliders, we suggest different deformation of both dials and sliders to learn the
advantages and disadvantages of the interactions with them. 4 2.2 3.1
Dial-Based Designs Dials are common TUIs for continuous variables. They can have bounded and
unbounded input ranges depending on parameters and user needs. In this paper, we
choose to use unbounded ranges to emphasis their difference with sliders, which have
bounded input ranges. The usual interaction of turning dials changes the best estimate
value (≈ mean of a desired input). The extended interactions inspired by Morphees+
change uncertainties (≈ standard deviation of a desired input, see Fig. 1). 5 Expandable Dial (Fig. 1-left) was driven by Morphees+’ Area feature, which describes
changes in area size on a surface. The dial’s diameter can be increased or decreased,
resulting in the area change at the top of the dial. The larger diameter the dial has, the
more uncertainty the users are inputting. When the users are 100% sure (no
uncertainty), they can decrease the diameter to the minimum. Pinch Dial (Fig. 1-middle) includes an open space between the center and one side of
the circumference of the dial. The open space can be closed or opened, making the
dial looks as a full circle or a sector from the top of the dial. This is inspired by the
Closure feature in Morphees+. A larger open space allows to input more uncertainty. Pressure Dial (Fig. 1-right) can be pressed downwards in addition to the rotation,
similarly to Button+ [27]. It was driven by the Strength feature of Morphees+, i.e. the
force needed to move a control point to another position. The stronger the dial is
pressed, the more certain the input is. Fig. 1. The dial-based interfaces, enabling both value and uncertainty input. The usual rotation
interactions are to input value and the extended inputs (grey arrows) are to input uncertainty. Expandable Dial: users increase the diameter to express more uncertainty. Pinch Dial: users
increase the open space – between the center and one side of the circumference – for more
uncertainty. Pressure Dial: users enter their uncertainty by adjusting the pressure at the top of the
dial (less pressure = more uncertainty). Fig. 1. The dial-based interfaces, enabling both value and uncertainty input. The usual rotation
interactions are to input value and the extended inputs (grey arrows) are to input uncertainty. Expandable Dial: users increase the diameter to express more uncertainty. Pinch Dial: users
increase the open space – between the center and one side of the circumference – for more
uncertainty. 3.1
Dial-Based Designs Pressure Dial: users enter their uncertainty by adjusting the pressure at the top of the
dial (less pressure = more uncertainty). 3.2
Slider-Based Designs Sliders are widely used in both graphical and tangible user interfaces for ranged
variables. The standard interaction of sliding the (central) thumb changes the best
estimate value (≈ mean of a desired input). The extended interactions inspired by
Morphees+ change uncertainty (≈ standard deviation of a desired input or range of a
desired input). Expandable Slider (Fig. 2-left) was inspired by Morphees+’s Area feature as was the
Expandable Dial. It has one thumb that can be expanded along the slider axis to 6 communicate uncertainty. The center of the thumb represents the best estimate value,
and the size of the thumb represents the amount of uncertainty. Split Slider (Fig. 2-right) was driven by Morphees+’s Modularity, which describes an
object’s ability to be split into multiple pieces. It has a thumb that can be split into two
or three thumbs. In the one-thumb mode, it functions as a standard slider and can input
a single, precise value. When the thumb is split into two, the two thumbs input a range
of uncertainty, and the center of the range is the input value. When the thumb is split
into three, the middle thumb inputs a value while the outer thumbs enter the range of
uncertainty. Fig. 2. The slider-based interfaces, enabling both value and uncertainty input. The usual
interaction of sliding thumbs is to input value, and the additional interactions (grey arrows) are
to input uncertainty. Expandable Slider: users increase the width of slider thumb to increase
uncertainty. Split Slider: users split the thumbs into two or three, to input the range of uncertainty. Fig. 2. The slider-based interfaces, enabling both value and uncertainty input. The usual
interaction of sliding thumbs is to input value, and the additional interactions (grey arrows) are
to input uncertainty. Expandable Slider: users increase the width of slider thumb to increase
uncertainty. Split Slider: users split the thumbs into two or three, to input the range of uncertainty. 4
Study Evaluating Our Designs In this section, we aim at learning design criteria for uncertain input TUIs. We first
implement low-fidelity prototypes of the explored design. We then conduct a focus
group study where the participants evaluate the low-fidelity prototypes. 4.1
Low-Fidelity Prototype Implementation We implemented low-fidelity prototypes of all the design explorations, except for the
pressure dial, where we used a consumer product [24] (Fig. 3d). We had six prototypes
as we had two different prototypes for the Expandable Dial, one that could be stretched
(Fig. 3a) and one that could be squeezed (Fig. 3b). Here we briefly describe how they
are implemented, and how they work. Expandable Dial: Stretching Design. (Fig. 3a) We use a Hoberman mechanism [13]
to create an expandable circular shape. The center of the mechanism is connected to a
rotational axis for value input. At the external corners, there are concave disks where
users put their fingers on. Users can slightly press them and stretch the fingers inwards
or outwards for inputting uncertainty. 7 Expandable Dial: Squeezing Design. (Fig. 3b) We use an aerospace design [11] to
fold a paper sheet into a condensed shape. The tension of the paper keeps the dial
expanded, and users squeeze it to change the diameter. Pinch Dial. (Fig. 3c) We create six equilateral prisms from a laser cutter and connect
them into a hexagon, roughly resembling the round shape of a dial. They are held
together with adhesive tape on the outer faces. We place a bent piece of plastic sheet
between two prisms to act as a spring to open the dial. Users can close the dial by
pinching the device. Pinch Dial. (Fig. 3c) We create six equilateral prisms from a laser cutter and connect
them into a hexagon, roughly resembling the round shape of a dial. They are held
together with adhesive tape on the outer faces. We place a bent piece of plastic sheet
between two prisms to act as a spring to open the dial. Users can close the dial by
pinching the device. Pressure Dial. (Fig. 3d) We used a PowerMate Bluetooth [24] for the design. It has a
spring inside to push the upper rotational part back up when pressed. The device has
only two states of height – pressed and not-pressed. Expandable Slider. (Fig. 3e) We place a bent piece of plastic sheet between two
thumbs of a laser-cut slider. The plastic sheet forced the thumbs to separate. Users can
squeeze the thumbs for more certain input. Split Slider. (Fig. 3f) There are three thumbs on a laser-cut slider. 4.1
Low-Fidelity Prototype Implementation The thumbs have
grooves on them, and users can place a U-shaped plastic piece to combine two thumbs
together. Hence, the number of thumbs can be changed between one (certain input),
two (range of uncertainty), and three (e.g., range of uncertainty and median). a
b
c
d
d
f
Fig. 3. Low-fidelity prototypes of the explored uncertain input dials/sliders designs. A)
Expandable Dial – Stretching Design, B) Expandable Dial – Squeezing Design, C) Pinch Dial,
D) Pressure Dial, E) Expandable Slider, F) Split Slider. b
d
f a b c d c f d d Fig. 3. Low-fidelity prototypes of the explored uncertain input dials/sliders designs. A)
Expandable Dial – Stretching Design, B) Expandable Dial – Squeezing Design, C) Pinch Dial,
D) Pressure Dial, E) Expandable Slider, F) Split Slider. 8 4.2
Focus Group Study Design To evaluate our design explorations, we conducted two focus group interviews. For
this, we provided the low-fidelity prototypes for the explored designs. Participants. We recruited twelve participants (10 male, 2 female) between 20 and 34
years old (M = 24.92, SD = 3.65) via personal invitations in order to have varying
backgrounds such as social sciences and natural sciences. Their degrees ranged from
A-level to M.Sc. Task and Procedure. We had two participant groups. Each focus group interview
lasted about 30 minutes. We took pictures and recorded the audio during the interviews. p
g
The participants were asked to sign the consent form and provide their demographic
information. The task aimed to evaluate the low-fidelity prototypes when inputting
value and uncertainty. The participants were grouped in three pairs, and we introduced
our prototypes to the participants. Each pair was assigned a scenario to work for, e.g.,
inputting an uncertain time. Then each pair was randomly given one of the low-fidelity
prototypes, so that three prototypes were given in total. They were asked to write down
advantages, disadvantages, improvements and suitability of their respective prototypes
for their scenario. Each pair then presented their findings to the others. We then
repeated this step with the remaining three prototypes. 4.3
Results Design Requirements of Uncertain Input TUIs. Using thematic analysis [3], we
identified preliminary requirements for designing tangible devices for uncertain input. The requirements confirm previous work for rotational and linear input [16]. It shows
that the general purposes of the input interfaces are similar. However, introducing
uncertain input to these devices revealed a new requirement that were not considered
for dials and sliders and that we present here. There are in total seven requirements,
which we grouped in four categories. Fast manipulation.
Si
i R1 Simultaneous input. In general, the participants liked to input both value and
uncertainty simultaneously as this allowed fast manipulation. For instance, they
could change the diameter of both Expandable Dial prototypes while rotating them. However, they complained that the Pinch Dial was difficult to pinch when the
opening is no longer between the thumbs and index fingers. Precise manipulation. p
R2 Small intervals. Dials were preferred for small intervals in value inputs, because
they could have small control display gain. On the other hand, the input ranges of
the sliders were limited by the slider sizes, hence a larger control display gain. p
R2 Small intervals. Dials were preferred for small intervals in value inputs, because
they could have small control display gain. On the other hand, the input ranges of
the sliders were limited by the slider sizes, hence a larger control display gain. R2 Small intervals. Dials were preferred for small intervals in value inputs, because
they could have small control display gain. On the other hand, the input ranges of
the sliders were limited by the slider sizes, hence a larger control display gain. 9 R3 No interference between inputs. Participants preferred not to have interference
between the value and uncertainty inputs, i.e., sometimes they wanted to change
either value or uncertainty and did not want to change the other by accident. E.g.,
when participants changed uncertainty (stretching or squeezing) with Expandable
Dial, it could rotate the device slightly. When users grasped Pinch Dial, it applied
some pressure on it, and it could cause unwanted change on uncertainty. p
g
y
R4 Easy finalization. The participants needed to finalize the inputs easily. With all
designs, users needed another input interface (e.g., button) to finalize their inputs
after changing value or uncertainty. It would require users either: to use another
hand to press the button while the users are manipulating the devices, or to take off
their hands from the devices and then press the button. We considered the second
interaction was easier, as users do not have to keep the tension on the device (e.g.,
squeezing or pressing the devices) while pressing the button. It allows users to take
off their hands from the devices and rest between manipulating the devices. For
instance, with the Stretching Design of the Expandable Dial or Split Slider, the
participants were able to take off their hands from it and then finalize their inputs. Observable uncertainty for speed and precision. R5 Visual feedback. The participants preferred explicit visual information on the
amount of uncertainty. For instance, the participants liked that Split Slider’s thumb
intervals supported fast and precise interaction through clear visual feedback on the
uncertainty. R6 Force feedback. Precise manipulation. Participants also liked that Pinch Dial and Expandable Slider
supported fast and eyes-free interaction through force feedback on uncertainty. The
force feedback provided additional information on the visual feedback. The
participants also mentioned that using more pressure for more certainty feels
intuitive. Supporting statistical knowledge. It could be improved by replacing the
spring to a mechanism that allows a wider range of pressure such as memory foam. The Split Slider did not allow simultaneous input of value and uncertainty,
because it had separate thumbs for them. – Squeezing Design (Fig. 3b) were both through squeezing, it was difficult for the
participants to squeeze the Pinch Dial when the open space was not between the
thumb of the index finger. With the Pressure Dial, it was not easy to adjust the
level of the pressure on it while rotating. It could be improved by replacing the
spring to a mechanism that allows a wider range of pressure such as memory foam. The Split Slider did not allow simultaneous input of value and uncertainty,
because it had separate thumbs for them. R2 Small intervals. The dial-based designs had boundless input. It allows inputting
small intervals and thus users can enter precise values. The slider-based designs
had bounded ranges and resulted inevitably in bigger intervals than the dial-based
designs. This can be mitigated with a longer input axis for the slider. R2 Small intervals. The dial-based designs had boundless input. It allows inputting
small intervals and thus users can enter precise values. The slider-based designs
had bounded ranges and resulted inevitably in bigger intervals than the dial-based
designs. This can be mitigated with a longer input axis for the slider. R3 No interference between inputs. Only the Split Slider fulfilled this requirement,
because it had separated thumbs. Both prototypes of the Expandable Dials could
cause unwanted change in the value or uncertainty, when the users were changing
uncertainty (stretching or squeezing), or value (rotating). It was the same with the
Pinch Dial (squeezing and rotating), Pressure Dial (pressing and rotating) and
Expandable Slider (squeezing and sliding). R4 Easy finalization. The Expandable Dial – Stretching Design (Fig. 3a) could keep
both the angle and the diameter even when the users took off their hands. The
Split Slider thumbs stayed still when the users were not manipulating them. They
allowed the users to rest their hands while finalizing the inputs (e.g., pressing
another button for finalization). On the other hand, the prototypes with spring
mechanisms – the Squeezing Design of the Expandable Dial, Pinch Dial, Pressure
Dial, and Expandable Slider changed the amount of uncertainty to an initial
amount when the users were not manipulating them. Supporting statistical knowledge. R7 Supporting both experts and novices. The participants liked that the Split Slider
supported both novice and expert users through the input mode change between 1
and three thumbs. Novice users with limited knowledge of statistics could use the
one- or two-thumb modes. Expert users could use the three-thumb mode, allowing
input of probability distributions such as normal distribution. R7 Supporting both experts and novices. The participants liked that the Split Slider
supported both novice and expert users through the input mode change between 1
and three thumbs. Novice users with limited knowledge of statistics could use the
one- or two-thumb modes. Expert users could use the three-thumb mode, allowing
input of probability distributions such as normal distribution. Evaluation of the Designs. We evaluated the 5 designs based on the design
requirements and user comments from the focus group study (see Table 1). The
fulfillment of the requirements was sometimes dependent on implementations. For
instance, the spring of the Pressure Dial was strong, and the participants complained
that it is hard to keep the pressure while rotating it. In this case, we clarify that the
advantages and disadvantages are related to the implementation methods and discuss
other implementation methods that could fulfill the requirements and used for future
implementations. 10 Table 1. Evaluation of the designs based on the design requirements. Expandable
Dial
Pinch Dial
Pressure Dial
Expandable
Slider
Split Slider
R1. Simultaneous input
✓
✓
R2. Small intervals
✓
✓
✓
R3. No interference
between inputs
✓
R4. Easy finalization
✓
✓
R5. Visual feedback
✓
✓
R6. Force feedback
✓
✓
✓
R7. Supporting both
experts and novices
✓
R1 Simultaneous input. The Expandable Dial and Expandable Slider fulfilled the
requirement. The participants could change the diameter of the Expandable Dial
and thumb size of the Expandable Slider while changing the value. Although the
interactions of changing uncertainty with the Pinch Dial and the Expandable Dial
– Squeezing Design (Fig. 3b) were both through squeezing, it was difficult for the
participants to squeeze the Pinch Dial when the open space was not between the
thumb of the index finger. With the Pressure Dial, it was not easy to adjust the
level of the pressure on it while rotating. It could be improved by replacing the
spring to a mechanism that allows a wider range of pressure such as memory foam. Supporting statistical knowledge. The Split Slider did not allow simultaneous input of value and uncertainty,
because it had separate thumbs for them. R2 Small intervals The dial based designs had boundless input It allows inputting Table 1. Evaluation of the designs based on the design requirements. Expandable
Dial
Pinch Dial
Pressure Dial
Expandable
Slider
Split Slider
R1. Simultaneous input
✓
✓
R2. Small intervals
✓
✓
✓
R3. No interference
between inputs
✓
R4. Easy finalization
✓
✓
R5. Visual feedback
✓
✓
R6. Force feedback
✓
✓
✓
R7. Supporting both
experts and novices
✓ Table 1. Evaluation of the designs based on the design requirements. Evaluation of the designs based on the design requirements. R1 Simultaneous input. The Expandable Dial and Expandable Slider fulfilled the
requirement. The participants could change the diameter of the Expandable Dial
and thumb size of the Expandable Slider while changing the value. Although the
interactions of changing uncertainty with the Pinch Dial and the Expandable Dial
– Squeezing Design (Fig. 3b) were both through squeezing, it was difficult for the
participants to squeeze the Pinch Dial when the open space was not between the
thumb of the index finger. With the Pressure Dial, it was not easy to adjust the
level of the pressure on it while rotating. It could be improved by replacing the
spring to a mechanism that allows a wider range of pressure such as memory foam. The Split Slider did not allow simultaneous input of value and uncertainty,
because it had separate thumbs for them. R1 Simultaneous input. The Expandable Dial and Expandable Slider fulfilled the
requirement. The participants could change the diameter of the Expandable Dial
and thumb size of the Expandable Slider while changing the value. Although the
interactions of changing uncertainty with the Pinch Dial and the Expandable Dial R1 Simultaneous input. The Expandable Dial and Expandable Slider fulfilled the
requirement. The participants could change the diameter of the Expandable Dial
and thumb size of the Expandable Slider while changing the value. Although the
interactions of changing uncertainty with the Pinch Dial and the Expandable Dial
– Squeezing Design (Fig. 3b) were both through squeezing, it was difficult for the
participants to squeeze the Pinch Dial when the open space was not between the
thumb of the index finger. With the Pressure Dial, it was not easy to adjust the
level of the pressure on it while rotating. Supporting statistical knowledge. Hence the users needed to
keep the tension on the device to preserve the wanted amount of uncertainty and
would need another hand to press a button for finalization. 11 R5 Visual feedback. The slider-based designs could clearly show the relative
amounts of uncertainty. The users could see the ranges of the possible input on
the axis of the sliders. The users could see the uncertainty between the thumbs of
the Expandable Slider or between the outer thumbs of the Split Slider. The users
could see the amount of uncertainty relatively to the ranges. The boundless dials
did not provide such clear visual feedback. For instance, it was not clear for the
users whether Pinch Dial was 50% or 60% open. R6 Force feedback. This requirement was mutually exclusive with the R4, easy
finalization of inputs. The spring mechanisms of the Pinch Dial, Pressure Dial and
Expandable Slider could give force feedback but also caused unwanted movement
when finalizing input. R7 Supporting both experts and novices. The Split Slider was the only design that
had two input modes for uncertainty: ranged input with 2-thumb mode and
probabilistic distribution input with 3-thumb mode. The evaluation shows that the Split Slider performs the best as an uncertain input TUI. It allowed no interference between the value and uncertainty input, and the finalization
of the input was easy. It provided a clear visual feedback on the amount of uncertainty. The feature that the slider could have between one and three thumb(s) supported users
with both limited and good knowledge of statistics. We decide to build a high-fidelity
prototype of the Split Slider to evaluate its ability to capture uncertain input. 5
SplitSlider: A Tangible Slider for Uncertain Input 5 We implement SplitSlider, a tangible interface that allows both certain and uncertain
input (Fig. 4, Fig. 5). We use three off-the-shelf sliders as sensors (Bourns PSM
100mm). They are placed next to each other in a box, with dimensions
W168×L68×D52.5 mm. This gives an illusion that the device is a single slider with
three thumbs. We use 3D-printed thumbs to cover the width of all the three sliders. We
insert small magnets (Ø5 mm, height 2 mm, strength 520g) on both sides of the thumbs
to give haptic feedback on the (un)combination of the thumbs, and to hold the thumbs
together. g
The outer thumbs are slightly higher than the middle thumb. When the three thumbs
are combined, they create a concave shape that most of slider thumbs have. Each
thumb’s long edges are cut in order to let users easily put fingers between combined
thumbs and split them. Each thumb can travel around 88 mm. We use an Arduino UNO
to connect the prototype to a computer. 12 Fig. 4. The schematic of the SplitSlider device with dimensions. Fig. 5. Working prototype of SplitSlider. Left: One-thumb mode to input a single value. Right:
Separated thumbs for communicate both a value and a range of uncertainty. Fig. 4. The schematic of the SplitSlider device with dimensions. Fig. 4. The schematic of the SplitSlider device with dimensions. Fig. 4. The schematic of the SplitSlider device with dimensions. Fig. 4. The schematic of the SplitSlider device with dimensions. Fig. 5. Working prototype of SplitSlider. Left: One-thumb mode to input a single value. Right:
Separated thumbs for communicate both a value and a range of uncertainty. Fig. 5. Working prototype of SplitSlider. Left: One-thumb mode to input a single value. Right:
Separated thumbs for communicate both a value and a range of uncertainty. 6
Evaluation of the SplitSlider 6 The SplitSlider aims at balancing simplicity and ability to express the uncertainty. In
order to measure how discoverable it is, we first asked participants to use it without
being provided any explanation. Then, in a second phase, after having explained
SplitSlider to participants, we measured its perceived ease of use and its ability to
express the participants’ uncertainty. Note that we decided to focus on the 1-thumb
and 3-thumb modes only in this study to keep the study simple. 6.1
Study Design We asked participants to use the working SplitSlider to answer public transportation
survey questions (see Table 2). The questions were selected to have continuous and
quantitative answers, which our interface accommodates. 13 Table 2. Questions used to evaluate the SplitSlider, with their respective min and max values. Table 2. Questions used to evaluate the SplitSlider, with their respective min and max values. Question
Min
Max
1. How often do you use the train? Never
Daily
2. How full is the train in general? Empty
Very crowded
3. How much do you like traveling with the train? Not at all
Very much
4. How do you perceive the hygiene within trains? Very dirty
Very Clean
5. How secure do you feel in trains? At risk
Very safe
6. How do you find train ticket prices? Very cheap
Very Expensive
7. How is the comfort of the train chairs? Very uncomfortable
Very comfortable
8. How do you perceive the noise level in the train? Very loud
Silent
9. How is the timeliness of the trains? Always late
Always on time
10. How fast do you get to your destination using the train? Very slowly
Very fast
11. How reliable do you perceive the arrival time displays? Very unreliable
Very reliable
12. How modern do you find the trains? Very old-fashioned
Very modern As an independent variable, we had the two phases of the study: the phase before and
the phase after the explanation of the prototype, to see if the use of the slider is
discoverable and participants can use the uncertain input function without explanation. At the end of each phase, the participants filled in a feedback questionnaire, including
a “Usability Metric for User Experience” (UMUX) questionnaire [8]. We used the same
Likert scale for all these feedback questions, ranging from 1 (Strongly Disagree) to 7
(Strongly Agree). The participants then answered the Berlin Numeracy Test [4]. We
did it at the end of the experiment not to bias the participants. 6.2
Participants For the study, we recruited 12 participants (7 males, 5 females). Their ages ranged from
16 to 59 years (M = 35.25, SD = 16.09). None of them had seen or heard of the prototype
prior to their participation. The participation was voluntary, and no compensation was
given. 6.4
Tasks and Procedure The participants signed a consent form and completed a demographic questionnaire. They were given a brief overview of the study procedure. The study was divided into
two phases, and the participants answered six questions of the transportation survey
per phase. The questions’ order was pseudo-randomized. The study took around 30
minutes per participant. • Phase 1: Participants used the prototype to answer the first six transportation survey
questions. No explanation of the prototype was provided but they were asked to
explore it. The three thumbs were combined at the beginning of the study. Afterwards, they filled in the feedback questionnaire. • Prototype explanation: The instructor explained the operating mode of the prototype
to the participants. This included deterministic input with the one-slider mode, the
meaning of each individual thumb when split, and an example of how these options
could be used. • Phase 2: The participants answered the last six transportation survey questions with
the prototype. Following the task, the participants filled in the identical feedback
questionnaire that they already completed after Phase 1. 6.3
Apparatus We implemented the software using C++ and openFrameworks. It showed a user
interface that displays the questions for the user study and gives feedback of the device
interpretation in a form of a gradient visualization on a distant screen (Fig. 6). Fig. 6. (a) Experimental setup used by the participants. (b) Close-up of the distant visual
feedback. Fig. 6. (a) Experimental setup used by the participants. (b) Close-up of the distant visual
feedback. 14 The slider was taped on the table to prevent it from moving. We placed a keyboard on
the left side to use its spacebar to finalize the input on the prototype. A 1440×900px
(~287×180mm) laptop was positioned approximately 50 cm behind the slider,
displaying the questions of the transportation survey and a graphical representation of
the participant’s answer below the questions. The slider on the screen had 1000px width
(~199 mm), and 10mm movement of a SplitSlider thumb moved a cursor on the screen
around 114px (~23mm). One question was displayed at once. The feedback
questionnaire was prepared on a separate laptop. The participants swapped between a
block of public transportation survey and a block of feedback questionnaire. As advised
in previous work [21], landmarks were not present on the slider’s scale. 6.5
Results The SplitSlider as a 3-thumb slider is not discoverable without explanation to most
people. Only one third of the participants were able to discover the splitting function
and used it to input uncertainty in Phase 1 (before explanation) (4/12, 33.3%, Fig. 7a). Among the other eight participants, only one managed to split the thumb into three at
the last question of Phase 1, but he tried them for ~8 seconds and then put them back
together to answer the question. In Phase 2 (after explanation), all participants used
three thumbs to answer questions. Among the other eight participants, only one managed to split the thumb into three at
the last question of Phase 1, but he tried them for ~8 seconds and then put them back
together to answer the question. In Phase 2 (after explanation), all participants used
three thumbs to answer questions. Although the questions were calling for similar amount of uncertainty in Phase 1 and
in Phase 2, the participants expressed different amount of uncertainty in both phases. Fig. 7b shows how much variance (i.e., uncertainty) was expressed in the two phases
in a range of [0, 1000]. In Phase 1 the participants used the 3-thumb mode less than in Phase 2. Fig. 7c shows
how often the different thumb modes were used in the two phases. A chi-square test of 15 15
a
b
Fig. 7. Thumb usage of the SplitSlider, comparing Phase 1: before explanation and Phase 2:
after explanation of the three thumbs. (a) Number of participants that answered the
questionnaires with three thumbs in each phase. (b) Amount of variance the participants used to
answer the questionnaire. 15
b a a Fig. 7. Thumb usage of the SplitSlider, comparing Phase 1: before explanation and Phase 2:
after explanation of the three thumbs. (a) Number of participants that answered the
questionnaires with three thumbs in each phase. (b) Amount of variance the participants used to
answer the questionnaire. a
b
Fig. 8. (a) Counted usage of the 1-thumb and 3-thumb modes in Phase 1: before explanation and
Phase 2: after explanation of the three thumbs. (b) UMUX scores comparing Phase 1: before
explanation and Phase 2: after explanation of the three-thumb mode for uncertainty input. b a a Fig. 8. (a) Counted usage of the 1-thumb and 3-thumb modes in Phase 1: before explanation and
Phase 2: after explanation of the three thumbs. 7
Discussion and Future Work The impact of the low fidelity of the prototypes on the results of a qualitative study
should be further investigated. The aim of the first study was to find the design
requirements and evaluate the low-fidelity prototypes, and we decided not to make
obvious improvements, such as making the pressure of the Pressure Dial gradual to
make the pressing interaction easy. Although the participants did not make any
comments about the gradual pressing and the use of low-fidelity prototypes essential in
HCI to evaluate the designs [6, 19], it would be interesting to explore if different
prototypes would have resulted different design requirements. The suggested design requirements are preliminary and should be evaluated. For
example, R4 Easy finalization and R6 Force feedback were mutually exclusive, and we
do not know which one is more important to novice or expert users. A future evaluation
of the requirements can give priority to one over the other requirements. The UMUX scores of the SplitSlider allow indirect comparison between the
SplitSlider and its graphical counterpart by Greis et al. [10]. The UMUX questionnaire
has strong correlation with SUS scores (higher than 0.8 [8]), and the SUS was used in
[10]. The mean UMUX score of the SplitSlider was 85.75 in phase 2, and the mean
SUS score of the graphical 3-thumb slider was 72.5. It shows the possibility that the
SplitSlider may offer better usability than the graphical slider. A future study should
investigate this comparison and explore different strengths of the two interfaces, such
as discoverability, ease of use, accuracy and precision of user inputs. The results of this work can be improved, refined and used. Our results could be
improved by conducting further studies. First, the visualization of the user study was
based on [21]. We chose not to mention the meaning of the three thumbs, in order to let
users freely interpret them, and include users with limited statistical knowledge. We
could have added minimum value, most probable value and maximum value to balance
between confusion and simplicity. Our results can be refined in three directions. First, future work can check if
participants’ answers to the transportation survey were accurate by interviewing them
before or after the study. Second, the SplitSlider can be compared to future alternative
techniques to express uncertainty. Third, future work can explore how to improve its
discoverability. 6.5
Results (b) UMUX scores comparing Phase 1: before
explanation and Phase 2: after explanation of the three-thumb mode for uncertainty input. independence showed that participants used the 3-thumb mode significantly more often
after the explanation (p < 0.001), showing that the prototype is not self-explanatory. The SplitSlider is easy to use, both in 1-thumb and 3-thumb modes. The median
UMUX score was 87.5 (Q0=33.3, Q1=75, Q3=92.7, Q4=95.83, mean=82.29) in the
first phase, with a minor increase to 89.58 in the second phase (Q0=29.17, Q1=83.33,
Q3=96.88, Q4=100, mean=85.75, see Fig. 8). Both scores are interpreted as excellent
[1], and there was no significant difference between the scores (p=0.366). This shows
that the SplitSlider was found easy to use, whether expressing uncertainty or not. The major criticisms in the qualitative feedback were the lack of smoothness of the
thumb movement and the too large minimum interval between the thumbs. Removing
the magnets could solve the problem. 16 Conclusion The SplitSlider allows users to choose on the fly between its
use as a standard 1-thumb slider or the ability to express their uncertainty as a 3-thumb
slider. We envision that the SplitSlider to be the next standard TUI for uncertain input. In future work we are thus interested in its ability to accurately input uncertainty and
compare it with traditional TUIs and other designs such as dial-based ones in different
context. 7
Discussion and Future Work Further design cues such as feedforward and affordances should be
explored to better invite the user to explore the possibilities of the SplitSlider. Our results could be used as an inspiration for future design of TUIs supporting the
expression of uncertainty together with the input value. Sliders and dials have each their
own benefits and drawbacks and are both widely used [15]. Future work should further
explore how to introduce the ability to express uncertainty in other common widgets
such as buttons or dials. Our dials designed were not preferred in our focus group study. Further work can explore other deformation features of Morphees+ and also consider
using uncertain input dials in different tasks, such as tasks that require more accuracy
in value input (primary input through precise rotation [16]) and less accuracy in
uncertainty input (secondary input). 17 8 Conclusion In this paper, we aimed at balancing the simplicity and the ability to express the
uncertainty. We explored the design of physical dials and sliders that can capture users’
value and uncertainty on one device. We first used Morphees+ [17] features to design
5 controls that can input both value and uncertainty, which are based on standard
tangible dials and sliders. We then presented low-fidelity prototypes and conducted a
focus group study to find design requirements for uncertain, tangible input. Following
these findings, we presented the most promising design: the SplitSlider. The
SplitSlider’s thumb supports entering one value (1-thumb slider) and can be split to
additionally enter a probability distribution (2- and 3-thumb slider). We implemented
and evaluated a functional tangible prototype. The results of our study show that the
use of the SplitSlider as 1-thumb slider is discoverable. The use of the SplitSlider as 3-
thumb slider is not discoverable, but after explanation, it was found easy to use to
express the uncertainty. The SplitSlider allows users to choose on the fly between its
use as a standard 1-thumb slider or the ability to express their uncertainty as a 3-thumb
slider. We envision that the SplitSlider to be the next standard TUI for uncertain input. In future work we are thus interested in its ability to accurately input uncertainty and
compare it with traditional TUIs and other designs such as dial-based ones in different
context. In this paper, we aimed at balancing the simplicity and the ability to express the
uncertainty. We explored the design of physical dials and sliders that can capture users’
value and uncertainty on one device. We first used Morphees+ [17] features to design
5 controls that can input both value and uncertainty, which are based on standard
tangible dials and sliders. We then presented low-fidelity prototypes and conducted a
focus group study to find design requirements for uncertain, tangible input. Following
these findings, we presented the most promising design: the SplitSlider. The
SplitSlider’s thumb supports entering one value (1-thumb slider) and can be split to
additionally enter a probability distribution (2- and 3-thumb slider). We implemented
and evaluated a functional tangible prototype. The results of our study show that the
use of the SplitSlider as 1-thumb slider is discoverable. The use of the SplitSlider as 3-
thumb slider is not discoverable, but after explanation, it was found easy to use to
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One-year outcomes in patients undergoing very high-power short-duration ablation for atrial fibrillation
|
Journal of interventional cardiac electrophysiology
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cc-by
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Abstract Background The very high-power short-duration (vHPSD) temperature-controlled ablation (vHPSD) improves the efficiency
of pulmonary vein isolation (PVI) procedures. We evaluated the procedural and 12-months outcomes in atrial fibrillation (AF) patients undergoing PVI by means of vHPSD
ablation. In patients with AF or atrial tachyarrythmia (AT) recurrence undergoing a redo procedure the durability of the
PVI was investigated. Methods Consecutive paroxysmal/persistent AF patients undergoing PVI with the vHPSD ablation strategy (90 W, for
4 s) were enrolled. The rate of PVI, first-pass isolation, acute reconnection, and procedural complications were evaluated. Follow-up examinations and EKG were scheduled at 3,6, and 12 months. In case of AF/AT recurrence, patients underwent
a redo procedure. Results Overall, 163 AF patients (29 persistent and 134 paroxysmal) were enrolled. The PVI was reached in 100% of patients
(88% at the first pass). The rate of acute reconnection was 2%. The radiofrequency, fluoroscopy and procedural times were
respectively 5.5 ± 1 min, 9 ± 1 min and 75 ± 20 min. No death, tamponade nor steam pops occurred; however, 5 patients
had vascular complications. The 12-months freedom from AF/AT recurrence was 86% in both paroxysmal and persistent
patients. Overall, 9 patients underwent a redo procedure, and in 4 all veins were still isolated, whereas in 5 pulmonary vein
reconnections were found. The PVI durability was 78%. No overt clinical complications were observed in the follow-up. Conclusions The vHPSD ablation represents an effective and safe ablation strategy to achieve PVI. The 12-months follow-up
showed high freedom from AF/AT recurrence and a good safety profile. Keywords Atrial fibrillation · Pulmonary vein isolation · Very high-power short-duration · Catheter ablation ·
Radiofrequency ablation Keywords Atrial fibrillation · Pulmonary vein isolation · Very high-power short-duration · Catheter ablation ·
Radiofrequency ablation One‑year outcomes in patients undergoing very high‑power
short‑duration ablation for atrial fibrillation Francesco Solimene1 · Teresa Strisciuglio1,2 · Vincenzo Schillaci1 · Alberto Arestia1 · Gergana Shopova1 ·
Armando Salito1 · Giuseppe Bottaro1 · Giovanni Marano1 · Fernando Coltorti1 · Giuseppe Stabile1,3,4,5 Francesco Solimene1 · Teresa Strisciuglio1,2 · Vincenzo Schillaci1 · Alberto Arestia1 · Gergana Shopova1 ·
Armando Salito1 · Giuseppe Bottaro1 · Giovanni Marano1 · Fernando Coltorti1 · Giuseppe Stabile1,3,4,5 Received: 25 January 2023 / Accepted: 27 February 2023 / Published online: 10 March 2023
© The Author(s) 2023 Journal of Interventional Cardiac Electrophysiology (2023) 66:1911–1917
https://doi.org/10.1007/s10840-023-01520-0 Journal of Interventional Cardiac Electrophysiology (2023) 66:1911–1917
https://doi.org/10.1007/s10840-023-01520-0 * Teresa Strisciuglio
teresastri@hotmail.it 1 Introduction force (CF) catheter optimized for temperature-controlled
ablation with microelectrodes and 6 thermocouples for real-
time temperature monitoring during ablation. The associ-
ated vHPSD algorithm modulates power to maintain target
temperature during lesion formation (maximum temperature
65 °C by using 90 W for 4 s) [2]. A vHPSD strategy of RF
ablation aims to minimize conductive heating and increase
resistive heating in order to deliver targeted heating to the
atrial wall, achieving uniform and transmural lesion while
reducing the risk of collateral tissue damages [3, 4]. The
vHPSD ablation produces much less conductive heat leading
to wider, shallower lesion formation, and much less revers-
ible injury. So far, preliminary data on the novel vHPSD
ablation mode showed promising results and demonstrated
its safety and efficacy for pulmonary vein isolation (PVI) in Radiofrequency (RF) energy is the widest energy source
used for catheter ablation of arrhythmias [1]. The very high-
power short-duration (vHPSD) catheter is a novel contact * Teresa Strisciuglio
teresastri@hotmail.it
1
Clinica Montevergine, Mercogliano, AV, Italy
2
Department of Advanced Biomedical Sciences, University
of Naples Federico II, Naples, NA, Italy
3
Mediterranea Cardiocentro, Naples, NA, Italy
4
Clinica San Michele, Maddaloni, CE, Italy
5
Anthea Hospital, Bari, BA, Italy * Teresa Strisciuglio
teresastri@hotmail.it
1
Clinica Montevergine, Mercogliano, AV, Italy
2
Department of Advanced Biomedical Sciences, University
of Naples Federico II, Naples, NA, Italy
3
Mediterranea Cardiocentro, Naples, NA, Italy
4
Clinica San Michele, Maddaloni, CE, Italy
5
Anthea Hospital, Bari, BA, Italy (0121 3456789)
3 1912 Journal of Interventional Cardiac Electrophysiology (2023) 66:1911–1917 The encirclement of the veins was performed in a point-
by-point fashion, by delivering RF energy (90 Watts for 4 s)
when a contact force (CF) ≥ 5 g was reached. The target
interlesion distance was ≤ 4 mm at the anterior wall, in order
to have overlapping of the tags, whereas at the posterior wall
was ≤ 6 mm [7]. This strategy aimed to create deeper lesions
on the anterior wall that is thicker. The intervenous ablation
line was performed at the discretion of the operator. patients undergoing atrial fibrillation (AF) ablation [5–8]. However, these studies included only a limited number of
patients and long follow-up data are missing. The aim of our
study is to evaluate the efficacy and safety of the vHPSD
ablation on a larger cohort of patients and to investigate the
12-months outcomes of this strategy. 2.1 Study population This prospective study enrolled consecutive AF patients
undergoing their first pulmonary vein ablation between
September 2020 and December 2021. The study protocol
conforms to the ethical guidelines of the 1975 Declaration of
Helsinki and was approved by the Institutional Ethics Com-
mittees. Participants included patients who were ≥ 18 years
old at the time of presentation with drug-resistant AF,
regardless of its duration. Each patient provided informed
consent to participate in the study. The exclusion criteria were: inability to provide informed
consent, AF potentially attributable to a cardiac or noncar-
diac reversible cause (dyselectrolytaemia, dysthyroidism,
severe anaemia, sepsis, acute cardiac ischemia and others),
previous AF ablation, patients waiting for heart transplant
or other cardiac surgery, previous cardiac surgery in the last
3 months, known atrial myxoma, unstable angina, unstable
heart failure, life expectancy < 12 months, pregnancy and
breastfeeding. Once the encirclement of the vein was completed the PVI
was assessed by entrance block and by remapping with the
Pentaray catheter.i 1 Introduction The QDOT Micro™ catheter has an improved irrigation
system that includes backward flow toward the proximal
electrode, allowing increased irrigation during ablation in
a parallel orientation. Ablation has been performed using a
RF generator capable of delivering power up to 100 W with
a rapid ramp-up time of ≤ 0.5 s that also provides real-time
temperature feedback every 33 ms (nGEN RF Generator,
Biosense Webster). The vHPSD algorithm rapidly cycles
power based on the hottest surface thermocouple (tempera-
ture target at 60 °C, cutoff at 65 °C). During the mapping,
the flow is low at 2 ml/min. When a RF application starts,
a 2-s pre-RF delay begins for the high flow rate (8 ml/s) to
cool the surface tissue prior to the onset of RF delivery. After the 2-s delay, the energy quickly ramps up to 90 W
and is automatically terminated after 4 s (Fig. 1). At the
end of each application, the high flow continues for 4 s. The
energy of 90W is delivered for the entire 4 s if the target
temperature is not reached. If, during the 4-s application, the
target temperature is reached, the power is modulated down
to prevent overheating. The power continues to be modulated
in response to the temperature measured for the duration of
the application. If the temperature cut-off is reached, the RF
application is stopped immediately. 2.2.1 Pulmonary veins ablation In case the PVs were not isolated at first pass, the earliest
signal on the mapping catheter was targeted for ablation until
the PVI was reached. After a waiting time of 20 min, the
PVI was reassessed and in case of reconnection touch-up,
RF applications were delivered, until re-isolation. The cavo-
tricuspid isthmus (CTI) line was performed only in case the
patient had a documented typical atrial flutter. All procedures were performed under general anaesthesia or
under deep sedation. Vitamin K antagonist treatment (with
a target INR of 2.0–3.0 on the day of the procedure) or non-
vitamin K anticoagulants were uninterrupted. Through femoral venous access, a diagnostic catheter was
placed in the coronary sinus (CS) and was used as a timing
reference for the creation of high-density electroanatomic
maps. After a single transseptal puncture, an open-irrigated
tip catheter (the QDOT Micro™ catheter, Biosense Webster,
Inc, CA, USA) was inserted into the LA through the hole
created, and the fixed-curve sheath (SL0, Abbott) was used
to insert the mapping catheter (Pentaray, Biosense Webster,
Inc, CA, USA). The left atrial geometry was reconstructed
using a 3D electro-anatomic mapping system (Carto Bio-
sense Webster, USA) during pacing from the CS. In patients
in AF, electrical cardioversion was performed to restore
sinus rhythm before starting the acquisition of the map. 2.2.4 Statistics In case of PV
reconnections, the ablations were delivered at the sites of
earliest signals until complete PVI. An AF/AT recurrence was considered when documented by
an ECG or when clinical symptoms potentially related to AF/
AT were reported by the patient. As early relapse within the
first 3 months after RF ablation may be a transient phenome-
non, this transition period was excluded from the final analysis. i
A redo procedure was scheduled in case of AF/AT recur-
rence out of the blanking period. During \the redo proce-
dure a careful remap was performed in order to depict PV
reconnections. In case all the veins were still isolated a more
antral ablation was performed or extra PV ablations were
targeted, depending on the operators’ choice. In case of PV
reconnections, the ablations were delivered at the sites of
earliest signals until complete PVI. 3.1 Population Overall, 163 patients were enrolled in the study (29 persistent
AF, 134 paroxysmal AF). Clinical characteristics are shown
in Table 1. The cohort included 105 men and 58 women with
a mean age of 61 ± 8 and a CHA2DVAS2 score of 2 ± 1. 2.2.3 Study endpoints The primary outcomes we evaluated were (1) the rate of
“First Pass” isolation and the rate of complete PVI at the
end of the procedure, (2) the safety of the procedure, (3)
the 12-months outcomes including freedom from AF/AT
recurrence and the occurrence of any complication, and (4)
the PVI durability in redo procedures. 2.2.4 Statistics 6 months, after the initial treatment. A Holter monitoring was
performed at 3, 6, and 12 months and in case of symptoms as
palpitations. In case the patients were unable to come to the
visits, trans-telephonic interviews were performed. Continuous variables are expressed as mean ± standard deviation
or median and interquartile range according to their distribution. Normality of data distribution was tested with the Shapiro–Wilk
test. Categorical variables are expressed as absolute number
with percentage (%). The rate of freedom from any atrial tach-
yarrhythmias was assessed by using the Kaplan–Meier curve. Statistical significance was set at a 2-tailed probability level
of < 0.05. All statistical analyses were performed using SPSS
software (Version 24.0, IBM, Armonk, NY, US). Continuous variables are expressed as mean ± standard deviation
or median and interquartile range according to their distribution. Normality of data distribution was tested with the Shapiro–Wilk
test. Categorical variables are expressed as absolute number
with percentage (%). The rate of freedom from any atrial tach-
yarrhythmias was assessed by using the Kaplan–Meier curve. Statistical significance was set at a 2-tailed probability level
of < 0.05. All statistical analyses were performed using SPSS
software (Version 24.0, IBM, Armonk, NY, US). Continuous variables are expressed as mean ± standard deviation
or median and interquartile range according to their distribution. Normality of data distribution was tested with the Shapiro–Wilk
test. Categorical variables are expressed as absolute number
with percentage (%). The rate of freedom from any atrial tach-
yarrhythmias was assessed by using the Kaplan–Meier curve. Statistical significance was set at a 2-tailed probability level
of < 0.05. All statistical analyses were performed using SPSS
software (Version 24.0, IBM, Armonk, NY, US). An AF/AT recurrence was considered when documented by
an ECG or when clinical symptoms potentially related to AF/
AT were reported by the patient. As early relapse within the
first 3 months after RF ablation may be a transient phenome-
non, this transition period was excluded from the final analysis. A redo procedure was scheduled in case of AF/AT recur-
rence out of the blanking period. During \the redo proce-
dure a careful remap was performed in order to depict PV
reconnections. In case all the veins were still isolated a more
antral ablation was performed or extra PV ablations were
targeted, depending on the operators’ choice. 2.2.2 Post‑ablation management and follow‑up An interrupted oral anticoagulation strategy was pursued
in all patients. After ablation, the oral anticoagulation was
administered for at least 3 months and long life in patients
with a CHA2DS2-VASC score ≥ 2. Patients with parox-
ysmal AF were discharged without antiarrhythmic drugs. Patients with persistent AF were discharged with or with-
out antiarrhythmic drugs according to the clinician’s pref-
erence. Patients were scheduled for clinical and ECG fol-
low-up examinations at 3,6, and 12 months, and then every 3 1913 Journal of Interventional Cardiac Electrophysiology (2023) 66:1911–1917 toring was
mptoms as
ome to the
d. mented by
ted to AF/
within the
phenome-i
al analysis. /AT recur-
do proce-
depict PV
2.2.4 Statistics
Continuous variables are expressed as mean ± standard deviation
or median and interquartile range according to their distribution. Normality of data distribution was tested with the Shapiro–Wilk
test. Categorical variables are expressed as absolute number
with percentage (%). The rate of freedom from any atrial tach-
yarrhythmias was assessed by using the Kaplan–Meier curve. Statistical significance was set at a 2-tailed probability level
of < 0.05. All statistical analyses were performed using SPSS
software (Version 24.0, IBM, Armonk, NY, US). Fig. 1 Radiofrequency delivery
with very high-power short
duration (90 W for 4 s). The left
panel shows a tilted postero-
anterior view of the left atrium
with the ablation catheter
delivering the RF energy pos-
teriorly at the ostium of the left
inferior pulmonary vein and the
Pentaray catheter at the ostium
of the left superior pulmonary
vein. The right panel shows
superiorly a latero-lateral view
of the left atrium with the bipo-
lar voltage map, and inferiorly
there is a graph showing the
temperature curve (in yellow),
the impedance curve (in green)
and the contact force (in blue)
during the ablation 3.2 Procedural data The procedures were performed under general anaesthesia
in all patients except for 5 that received deep sedation. With
a median number of 80 RF tags (IQR 74–90), the PVI could
be reached in all patients and in 88% of cases at the first pass. The median impedance drop was 10.8 (IQR 8.5–13) Ohms, The secondary outcomes we evaluated included over-
all procedural duration (defined as the time from the first
venous puncture to the withdrawal of the sheath), radiof-
requency time and fluoroscopy time. 1 3 Journal of Interventional Cardiac Electrophysiology (2023) 66:1911–1917 1914 3.4 Redo procedures Nine patients underwent a redo procedure for AF/AT recur-
rence. In 4 patients all the veins were still isolated, whereas
in the remaining 5 patients, one reconnected vein was
observed in 4 patients and 2 reconnected veins in 1 patient. Thus, 28/36 veins were still isolated and the PVI durability
was 78%. The sites of reconnection are described in Table 2. TIA, transient ischemic attack; LA, left atrium the median contact force was 9.2 (IQR 5.9–14) g, and the
median maximum temperature was 47 (IQR 44–50.3) °C. In
27 patients an additional CTI was performed for a documented
typical atrial flutter. In 2% of patients, acute PV reconnections
(one at anterior left superior PV, one at anterior left inferior
PV, one at posterior right inferior PV) were observed and the
earliest signal on the Pentaray catheter was targeted for abla-
tion until complete signal disappearance. The procedural time
(skin to skin) was 75 ± 20 min, the RF time was 5.5 ± 1 min
and the fluoroscopy time was 9 ± 6 min. No steam pops, tam-
ponade, death nor stroke occurred, however 5 patients expe-
rienced an access site-related vascular complication. 3.3 Follow‑up Table 1 Clinical characteristics of the study population
TIA, transient ischemic attack; LA, left atrium
N = 163
Age (years)
61 ± 8
Male sex (%)
105 (64%)
AF type
○ Paroxysmal
○ Persistent
134 (82)
29 (18)
Dyslipidemia (%)
24 (15)
Hypertension (%)
98 (60)
Diabetes mellitus (%)
10 (6)
Obesity (%)
5 (3)
Ischemic heart disease (%)
4 (3)
TIA/stroke (%)
1 (1%)
CHA2-DS2-VASc score (mean)
2 ± 1
LA diameter, mm
43 ± 10
Left common ostium (%)
24 (15%) All patients completed the 12-months follow-up. After a
mean follow-up of 19 ± 2 months the freedom from any
atrial tachyarrhythmias recurrence was 86% in both cohorts
of paroxysmal (18 AF recurrence and 1 patient had electro-
cardiographic evidence of both AF and atrial flutter recur-
rence) and persistent AF patients (3 AF recurrence and 1
atrial flutter /tachycardia) (Fig. 2). The mean time to recur-
rence was 14 ± 4 months. No overt clinical complications
were reported during the follow-up. 4 Discussion This study demonstrated that the use of the vHPSD ablation
(90W, 4 s) for PVI is effective and increases the efficiency
with short procedural and RF times, without compromising
procedural safety. Furthermore, in our cohort of patients,
the freedom from AF/AT recurrence at 12 months was high
and also the safety profile was confirmed by the absence of Fig. 2 Kaplan–Meier curves
showing 12-months freedom
from AF/AT recurrence in
paroxysmal and persistent AF
patients
100
80
60
40
20
3
6
9
12
Months
0
N. at risk
Paroxysmal AF patients
(blue curve)
Persistent AF patients
(green curve)
134
134
128
124
115
29
29
28
27
25 1 3 Journal of Interventional Cardiac Electrophysiology (2023) 66:1911–1917 Journal of Interventional Cardiac Electrophysiology (2023) 66:1911–1917 1915 ite of PV
ions at redo
s
PV, pulmonary vein; RSPV, right superior pulmonary vein; RIPV, right inferior pulmonary vein; LSPV, left
superior pulmonary vein; LIPV, left inferior pulmonary vein
Patient
Reconnected PV
Sites of reconnection
RSPV
RIPV
LSPV
LIPV
RSPV
RIPV
LSPV
LIPV
1
0
0
0
0
0
0
0
0
2
0
0
1
0
0
0
Ridge; Anterior roof
0
3
0
0
0
0
0
0
0
0
4
1
0
1
0
Anterior
0
Ridge
0
5
0
0
0
0
0
0
0
0
6
0
0
0
0
0
0
0
0
7
1
0
1
0
Posterior roof
0
Ridge
0
8
0
0
1
0
0
0
Anterior roof
0
9
1
0
1
0
Anterior
0
Ridge
0 Table 2 Site of PV
reconnections at redo
procedures PV, pulmonary vein; RSPV, right superior pulmonary vein; RIPV, right inferior pulmonary vein; LSPV, left
superior pulmonary vein; LIPV, left inferior pulmonary vein overt complications during the follow-up. The PVI durabil-
ity evaluated at redo procedures was 83%. lesion dimensions compared to conventional 50 W-10 s or
30 W-30 s applications; however, this study also suggested
that there is a sort of thermal latency, indeed after RF ter-
mination the tissue temperature increases due to a slower
conductive heating, and thus the lesion size is influenced by
the neighbouring RF application and this may create deeper
lesions. 4.1 Efficacy data In recent years, higher-power RF delivery has been proposed
as a strategy to improve the efficiency of PVI procedures, by
shortening their duration [9]. The point-by-point ablation,
indeed, may be time-consuming, and increasing the power
enables to reach faster the target ablation index. However,
the safety window is narrower, especially for the risk of col-
lateral damage to the oesophagus, when ablating the poste-
rior wall. Indeed, some data on the use of 45/50 W reported
the occurrence of steam pops and also oesophageal lesions
detected by endoscopy [9, 10]. The QDOT catheter is optimized for temperature-con-
trolled radiofrequency ablation, as it has embedded at the tip
microelectrodes and 6 thermocouples that enable a real-time
monitoring of the temperature at the tip-tissue interface dur-
ing the ablation. In the QMODE + modality, the new cath-
eter delivers RF energy with 90 Watts in 4 s (vHPSD), and
during the ablation the power is modulated based on the
temperature, and this reduces the risk of overheating [11]. Another advantage is that the RF application is so short that
catheter stability may not be a concern anymore. 4 Discussion For this reason, we always aim to create overlap-
ping lesions (interlesion distance ≤ 4 mm) at the anterior
wall of the left atrium, which is thicker compared to the
posterior wall, and this may account for the high rate of first
pass isolation that we report in this study, which is compa-
rable to that reported for point-by-point AI-guided ablation
in the multicentric VISTAX study [13]. Furthermore, the
general anaesthesia that we systematically perform almost
in every patient, increases the catheter stability during the
RF delivery thus increasing the probability to reach an effec-
tive lesion. Patient consent Each patient provided informed consent to participate
in the study. The good safety profile previously described for the QDOT
catheter [5–8] has been confirmed by our data. No deaths,
cardiac tamponade, perforation, stroke, or atrio-oesophageal
fistula were observed acutely and during the 12 months follow-
up. Previous studies reported the occurrence of silent cerebral
events [6, 8] detected by cerebral MRI; however, we did not
investigate this aspect. Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 4.4 Limitations The study has the following several limitations: (1) this is a pro-
spective mono-centric non-randomized study; (2) the number
of enrolled patients is relatively small; however, to our knowl-
edge, there are no other data regarding the vHPSD with 90W
for 4 s for PVI in larger cohorts; (3) the adenosine/isoproter-
enol challenge was not performed at the end of the PVI, and
thus, the dormant PV conduction has not been excluded; (4)
the impact of the vHPSD on the oesophagus and the brain was
not investigated with a oesophagogastroduodenoscopy nor a
cerebral MRI; however, in the follow-up, none of the patients
had symptoms suggestive of oesophageal nor cerebral injury;
(5) no intra-cardiac monitors were used for the assessment of
the AF recurrences, thus, the absence of a continuous rhythm
monitoring may have underestimated the rate of recurrences. References 1. Habibi M, Berger RD, Calkins H. Radiofrequency ablation:
technological trends, challenges, and opportunities. Europace. 2021;23:511–9. https://doi.org/10.1093/europace/euaa328. 2. Stabile G, Schillaci V, Strisciuglio T, et al. In vivo biophysical
characterization of very high power, short duration, temperature-
controlled lesions. Pacing Clin Electrophysiol. 2021;44:1717–23. https://doi.org/10.1111/pace.14358. 3. Leshem E, Zilberman I, Tschabrunn CM, et al. High-power and
short-duration ablation for pulmonary vein isolation: biophysi-
cal characterization. JACC Clin Electrophysiol. 2018;4:467–79. https://doi.org/10.1016/j.jacep.2017.11.018. 4. Barkagan M, Contreras-Valdes FM, Leshem E, Buxton AE,
Nakaagawa H, Anter E. High-power and short-duration ablation
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bility. J Cardiovasc Electrophysiol. 2018;29:1287–96. https://doi.
org/10.1111/jce.13651. 4. Barkagan M, Contreras-Valdes FM, Leshem E, Buxton AE,
Nakaagawa H, Anter E. High-power and short-duration ablation
for pulmonary vein isolation: safety, efficacy, and long-term dura-
bility. J Cardiovasc Electrophysiol. 2018;29:1287–96. https://doi.
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Randomized Evaluation of High Power during CLOSE-Guided
Pulmonary Vein Isolation: The POWER-AF Study. Circ Arrhythm
Electrophysiol. 2021;14:e009112. https://doi.org/10.1161/CIR-
CEP.120.009112. 4.2 Twelve‑months freedom from AF/AT recurrence
and PVI durability Our data also provide insights into the long-term outcomes
of the vHPSD ablation, which seems to be highly effective
with 84% of paroxysmal AF patients free from AT/AF recur-
rence, in line with previous studies reporting on HPSD [10]. As for the PVI durability, in our cohort of patients undergo-
ing a redo procedure, 78% of PVs were still isolated, with
most of the reconnections occurring at the superior veins. These data raise enthusiasm on the long-term effectiveness
of the vHPSD ablation strategy, also because the rate of
durable PVI is much higher than the previously reported
with moderate power moderate duration (MPMD) [14]. A
similar result was reported also by Yavin et al., that reported
a lower incidence of chronic PV reconnection at redo pro-
cedures for the HPSD compared to the MPMD (16.6% vs. 52.2%) [15]. Data on the use of vHPSD for PVI are still limited; how-
ever, they confirm the increased procedural efficiency with
reduced procedural, fluoroscopy and RF times [5–8]. Our
results confirm the shortened procedural times compared to
the ablation index-guided procedures [5].i As for the acute procedural success, a low rate of first-
pass isolation has been reported by previous studies [6–8]
and this has raised concerns also for the durability of the
PVI. The study by Nakagawa et al. [12] reported that the
applications delivered with the QDOT result in smaller 1 3 1916 Journal of Interventional Cardiac Electrophysiology (2023) 66:1911–1917 5 Conclusion The vHPSD ablation represents an effective and safe ablation
strategy to achieve PVI. The 12-months follow-up showed
high freedom from AF/AT recurrence and a good safety pro-
file with the absence of overt clinical complications. 5. Reddy VY, Grimaldi M, De Potter T, et al. Pulmonary Vein Isola-
tion With Very High Power, Short Duration, Temperature-Con-
trolled Lesions: The QDOT-FAST Trial. JACC Clin Electrophysiol. 2019;5:778–86. https://doi.org/10.1016/j.jacep.2019.04.009. 5. Reddy VY, Grimaldi M, De Potter T, et al. Pulmonary Vein Isola-
tion With Very High Power, Short Duration, Temperature-Con-
trolled Lesions: The QDOT-FAST Trial. JACC Clin Electrophysiol. 2019;5:778–86. https://doi.org/10.1016/j.jacep.2019.04.009. 6. Halbfass P, Wielandts JY, Knecht S, et al. Safety of very high-
power short-duration radiofrequency ablation for pulmonary vein
isolation: a two-centre report with emphasis on silent oesophageal
injury. Europace. 2022;24(3):400–5. https://doi.org/10.1093/europ
ace/euab261. 6. Halbfass P, Wielandts JY, Knecht S, et al. Safety of very high-
power short-duration radiofrequency ablation for pulmonary vein
isolation: a two-centre report with emphasis on silent oesophageal
injury. Europace. 2022;24(3):400–5. https://doi.org/10.1093/europ
ace/euab261. Author contribution All authors have read and approved the manu-
script and they have substantially contributed to the manuscript:
Concept/design: Solimene F, Stabile G, Schillaci V, Strisciuglio T. Data analysis/interpretation: Marano G, Bottaro G, Salito A, Stabile G. Drafting article: Strisciuglio T, Schillaci V, Salito A, Shopova G. Critical revision of article: Arestia A, Shopova G, Salito A, Coltorti F. Approval of article: Strisciuglio T, Stabile G, Solimene F, Schillaci V. Statistics: Strisciuglio T, Solimene F, Coltorti F. Data collection: Shopova G Marano G Arestia A Bottaro G Author contribution All authors have read and approved the manu-
script and they have substantially contributed to the manuscript: 7. Tilz R, Sano M, Vogler J, et al. Very high-power short-duration
temperature-controlled ablation versus conventional power-con-
trolled ablation for pulmonary vein isolation: The fast and furious
- AF study. Int J Cardiol Heart Vasc. 2021;35:100847. https://doi.
org/10.1016/j.ijcha.2021.100847. 7. Tilz R, Sano M, Vogler J, et al. Very high-power short-duration
temperature-controlled ablation versus conventional power-con-
trolled ablation for pulmonary vein isolation: The fast and furious
- AF study. Int J Cardiol Heart Vasc. 2021;35:100847. https://doi.
org/10.1016/j.ijcha.2021.100847. Data collection: Shopova G, Marano G, Arestia A, Bottaro G. 8. Mueller J, Halbfass P, Sonne K, et al. Safety aspects of very
high power very short duration atrial fibrillation ablation using a
modified radiofrequency RF-generator: Single-center experience. J Cardiovasc Electrophysiol. 2022;33:920–7. https://doi.org/10.
1111/jce.15433. 8. 4.3 Safety data Patient consent Each patient provided informed consent to participate
in the study. 5 Conclusion Mueller J, Halbfass P, Sonne K, et al. Safety aspects of very
high power very short duration atrial fibrillation ablation using a
modified radiofrequency RF-generator: Single-center experience. J Cardiovasc Electrophysiol. 2022;33:920–7. https://doi.org/10.
1111/jce.15433. Funding Open access funding provided by Università degli Studi di
Napoli Federico II within the CRUI-CARE Agreement. Funding Open access funding provided by Università degli Studi di
Napoli Federico II within the CRUI-CARE Agreement. Data availability Data are available upon reasonable request. 15. Yavin HD, Leshem E, Shapira-Daniels A, et al. Impact of High- Power
Short-Duration Radiofrequency Ablation on Long-Term Lesion
Durability for Atrial Fibrillation Ablation. JACC Clin Electrophysiol.
2020;6:973–85. https://doi.org/10.1016/j.jacep.2020.04.023. Declarations Ethics approval The study protocol conforms to the ethical guidelines
of the 1975 Declaration of Helsinki and was approved by the Institu-
tional Ethics Committees. 10. Chen S, Schmidt B, Bordignon S, et al. Catheter ablation of atrial
fibrillation using ablation index-guided high-power technique: 10. Chen S, Schmidt B, Bordignon S, et al. Catheter ablation of atrial
fibrillation using ablation index-guided high-power technique: 10. Chen S, Schmidt B, Bordignon S, et al. Catheter ablation of atrial
fibrillation using ablation index-guided high-power technique: 1 3 1917 Journal of Interventional Cardiac Electrophysiology (2023) 66:1911–1917 Frankfurt AI high-power 15-month follow-up. J Cardiovasc Elec-
trophysiol. 2021;32(3):616–24. https://doi.org/10.1111/jce.14912. (Epub 2021 Feb 1). 14. De Pooter J, Strisciuglio T, El Haddad M, et al. Pulmonary Vein
Reconnection No Longer Occurs in the Majority of Patients
After a Single Pulmonary Vein Isolation Procedure. JACC Clin
Electrophysiol. 2019;5:295–305. https://doi.org/10.1016/j.jacep.
2018.11.020. p
11. Wielandts JY, Almorad A, Hilfiker G, et al. Biosense Web-
ster’s QDOT Micro™ radiofrequency ablation catheter. Future Cardiol. 2021;17(5):817–25. https://doi.org/10.2217/
fca-2021-0002. 15. Yavin HD, Leshem E, Shapira-Daniels A, et al. Impact of High- Power
Short-Duration Radiofrequency Ablation on Long-Term Lesion
Durability for Atrial Fibrillation Ablation. JACC Clin Electrophysiol. 2020;6:973–85. https://doi.org/10.1016/j.jacep.2020.04.023. 12. Nakagawa H, Ikeda A, Sharma T, et al. Comparison of in vivo
tissue temperature profile and lesion geometry for radiofre-
quency ablation with high power- short duration and moder-
ate power-moderate duration effects of thermal latency and
contact force on lesion formation. Circ Arrhythmia Electro-
physiol. 2021;14:e009899. https://doi.org/10.1161/CIRCEP.
121.009899. Publisher's note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. 13. Duytschaever M, Vijgen J, De Potter T, et al. Standardized pul-
monary vein isolation workflow to enclose veins with contiguous
lesions: The multicentre VISTAX trial. Europace. 2020;22:1645–
52. https://doi.org/10.1093/europace/euaa157. 1 3
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Nme Gene Family Evolutionary History Reveals Pre-Metazoan Origins and High Conservation between Humans and the Sea Anemone, Nematostella vectensis
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PloS one
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Nme Gene Family Evolutionary History Reveals
Pre-Metazoan Origins and High Conservation between
Humans and the Sea Anemone, Nematostella vectensis
Thomas Desvignes, Pierre Pontarotti, Julien Bobe Nme Gene Family Evolutionary History Reveals
Pre-Metazoan Origins and High Conservation between
Humans and the Sea Anemone, Nematostella vectensis
Thomas Desvignes, Pierre Pontarotti, Julien Bobe To cite this version: Thomas Desvignes, Pierre Pontarotti, Julien Bobe. Nme Gene Family Evolutionary History Reveals
Pre-Metazoan Origins and High Conservation between Humans and the Sea Anemone, Nematostella
vectensis. PLoS ONE, 2010, 5:11 (e15506), pp.1-12. 10.1371/journal.pone.0015506. hal-01204995 Distributed under a Creative Commons Attribution 4.0 International License Introduction Nme-related genes have been sporadically reported in Archaea
[5], Eubacteria [6,7], and in several eukaryotic lineages including
fungi [8], plants [9], and bilaterians [10,11]. However, the
evolutionary history of the family that has led to a repertoire of 5
Nme genes in the chordate ancestor remains poorly understood. Indeed, the complexity of the Nme gene repertoire outside the
chordate lineage was previously uncharacterized and existing
literature suggested that the complexity of the Nme gene family
was much more limited in non-chordate species. In Dictyostelium
discoideum, two Nme-related proteins, named NdkC-2 and NdkM,
and expressed in the cytosol and in the mitochondria, respectively,
were used for biochemical and structural studies [12–14]. In
Drosophila melanogaster, only one Nme-related gene, named awd, had
been reported and intensively studied for its role in aberrant
development [10]. In Caenorhabditis elegans, one Nme-related gene
had been shown to be associated with severe developmental Nme-related genes have been sporadically reported in Archaea
[5], Eubacteria [6,7], and in several eukaryotic lineages including
fungi [8], plants [9], and bilaterians [10,11]. However, the
evolutionary history of the family that has led to a repertoire of 5
Nme genes in the chordate ancestor remains poorly understood. Indeed, the complexity of the Nme gene repertoire outside the
chordate lineage was previously uncharacterized and existing
literature suggested that the complexity of the Nme gene family
was much more limited in non-chordate species. In Dictyostelium
discoideum, two Nme-related proteins, named NdkC-2 and NdkM,
and expressed in the cytosol and in the mitochondria, respectively,
were used for biochemical and structural studies [12–14]. In
Drosophila melanogaster, only one Nme-related gene, named awd, had
been reported and intensively studied for its role in aberrant
development [10]. In Caenorhabditis elegans, one Nme-related gene
had been shown to be associated with severe developmental The Nme family, initially called NDPK or Nm23, was named
after the identification of a novel gene associated with low
metastatic potential [1]. In humans, NME genes are involved in a
wide variety of physiological or pathological cellular processes
including development, metastatic potential, ciliary functions, and
cell differentiation and proliferation at various tissular and
subcellular localization (see [2] for recent review). Despite their
critical role in key developmental and pathological processes, the
molecular functions of Nme genes remain poorly documented [3]. Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an INRA - IFREMER PhD fellowship to TD. The research leading to these results has received funding from the European
Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 222719 - LIFECYCLE. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript. ooooo Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Julien.Bobe@rennes.inra.fr * E-mail: Julien.Bobe@rennes.inra.fr Abstract Background: The Nme gene family is involved in multiple physiological and pathological processes such as cellular
differentiation, development, metastatic dissemination, and cilia functions. Despite the known importance of Nme genes
and their use as clinical markers of tumor aggressiveness, the associated cellular mechanisms remain poorly understood. Over the last 20 years, several non-vertebrate model species have been used to investigate Nme functions. However, the
evolutionary history of the family remains poorly understood outside the vertebrate lineage. The aim of the study was thus
to elucidate the evolutionary history of the Nme gene family in Metazoans. Methodology/Principal Findings: Using a total of 21 eukaryote species including 14 metazoans, the evolutionary history of
Nme genes was reconstructed in the metazoan lineage. We demonstrated that the complexity of the Nme gene family,
initially thought to be restricted to chordates, was also shared by the metazoan ancestor. We also provide evidence
suggesting that the complexity of the family is mainly a eukaryotic innovation, with the exception of Nme8 that is likely to
be a choanoflagellate/metazoan innovation. Highly conserved gene structure, genomic linkage, and protein domains were
identified among metazoans, some features being also conserved in eukaryotes. When considering the entire Nme family,
the starlet sea anemone is the studied metazoan species exhibiting the most conserved gene and protein sequence features
with humans. In addition, we were able to show that most of the proteins known to interact with human NME proteins were
also found in starlet sea anemone. Conclusion/Significance: Together, our observations further support the association of Nme genes with key cellular
functions that have been conserved throughout metazoan evolution. Future investigations of evolutionarily conserved Nme
gene functions using the starlet sea anemone could shed new light on a wide variety of key developmental and cellular
processes. Citation: Desvignes T, Pontarotti P, Bobe J (2010) Nme Gene Family Evolutionary History Reveals Pre-Metazoan Origins and High Conservation between Humans
and the Sea Anemone, Nematostella vectensis. PLoS ONE 5(11): e15506. doi:10.1371/journal.pone.0015506 Citation: Desvignes T, Pontarotti P, Bobe J (2010) Nme Gene Family Evolutionary History Reveals Pre-Metazo
and the Sea Anemone, Nematostella vectensis. PLoS ONE 5(11): e15506. doi:10.1371/journal.pone.0015506 Editor: Sergios-Orestis Kolokotronis, American Museum of Natural History, United States of America Received July 27, 2010; Accepted October 5, 2010; Published November 11, 2010 Copyright: 2010 Desvignes et al. Nme Gene Family Evolutionary History Reveals Pre-
Metazoan Origins and High Conservation between
Humans and the Sea Anemone, Nematostella vectensis Thomas Desvignes1,2, Pierre Pontarotti3, Julien Bobe1* Thomas Desvignes
, Pierre Pontarotti , Julien Bobe
1 UMR 6632/IFR48, Universite´ de Provence Aix Marseille 1/CNRS, F-13000, Marseille, France, 2 IFREMER, LALR, F-34250, Palavas les flots, France, 3 UMR 6632/IFR48,
Universite´ de Provence Aix Marseille 1/CNRS, F-13000, Marseille, France R48, Universite´ de Provence Aix Marseille 1/CNRS, F-13000, Marseille, France, 2 IFREMER, LALR, F-34250, Palavas les flots, France,
Provence Aix Marseille 1/CNRS, F-13000, Marseille, France HAL Id: hal-01204995
https://hal.science/hal-01204995v1
Submitted on 4 Oct 2018 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License PLoS ONE | www.plosone.org Results/Discussion y
All studied metazoan species displayed a full set of group II Nme
genes with the exception of the 4 ecdysozoan species and T. adhaerens (Figures 2–5). The 2 other group II Nme genes, Nme9 and
Nme10, that have been shown to be eutherian and vertebrate
innovations, respectively [4], will not be discussed here. No Nme7
homolog could be identified in the C. elegans genome thus
indicating a possible gene loss after the nematode radiation. In
M. brevicollis the Nme7 protein is structurally highly divergent as
shown but the topology of the phylogenetic tree (Figure 4A) and
displays a unique and incomplete domain (Figure 4B). In insects,
Nme7 proteins also displayed specific domain structure (Figure 4B)
resulting in a divergent position of the corresponding group in the
phylogenetic tree (Figure 4A). As for all phylogenetic analyses
reported here, the tree topology remained unchanged whether we
used the full length protein sequence or only domains for the
phylogenetic reconstruction. Similarly, very divergent Nme8-
related sequences were identified in insects and T. adhaerens
(Figure 5). In these 4 species, the Nme8-related proteins have lost
the 3 NDPK_TX domains that are found in all other metazoan
species, including the starlet sea anemone (Figure 5). In M. brevicollis, the Nme8 protein does not display typical Nme8
NDPK_TX domains but 2 different domains of the NDPk
superfamily. It is also noteworthy that the exon/intron structure
corresponding to the Thioredoxin TRX_NDPK domain is very
well conserved among all studied choanoflagellate and metazoan
species. In contrast, the exon/intron structure corresponding to
the NDPK domains is highly divergent in insects, placozoans and
choanoflagellates (Figure S2). Together, our data demonstrate that
Nme5, Nme6, Nme7, and Nme8 genes were already present in the
genome of the common ancestor of choanoflagellates and
metazoans. In non-choanoflagellate/metazoan species, all Nme
proteins of the group II were found in low branching eukaryotic
lineages such as heteroloboseans, green plants, amoebozoans,
alveolates, and fungi (Figures 2–4, Table 2) with the exception of
Nme8. In contrast, we failed to identify Nme genes of the group
II outside the eukaryotic lineage. Together, our results strongly
suggest that Nme5, Nme6, and Nme7 emerged around Eukaryote
radiation. Interestingly, none of the studied non-choanoflagel-
late/metazoan species display all 3 proteins suggesting lineage
specific loss of Nme 5, Nme6, and Nme7 genes. The Nme8 protein
typically displays 1 Thioredoxin (TRX) domain followed by 2 or
3 complete NDPk domains. Nme gene family complexity predates Metazoan
radiation Using available sequenced genomes of 21 eukaryote species
ranging
from
amoebozoans
to
humans,
we
were
able
to
reconstruct, in opisthokonts – the metazoan/choanoflagellate/
fungi phylum – the evolutionary history of the Nme genes that had
previously been identified in the chordate ancestor [4]. We were
able to show that the metazoan and chordate ancestors share a
similar Nme gene repertoire. A similar repertoire was also found in
Monosiga brevicollis, a choanoflagellate, but not in fungi (Figures 1–5). Monosiga brevicollis, a choanoflagellate, but not in fungi (Figures 1–5). All non-vertebrate metazoans species displayed 2 Nme genes of
the group I with the exception of Trichoplax adhaerens, Drosophila
melanogaster, Aedes aegypti, Tribolium castaneum, and Lottia gigantea, in
which a single gene could be identified (Figure 1A–B). In all
studied non-metazoan eukaryotes, two Nme genes of the group I
could also be identified with the exception of Saccharomyces cerevisiae
and Tetrahymena thermophila. In starlet sea anemone Nematostella
vectensis, Caenorhabditis elegans and Branchiostoma floridae, the two genes
are located tandemly and thus originate from a cis-duplication of
an ancestral gene (Table 1). In addition, a surprisingly well
conserved gene synteny of group I Nme sequences was found
between sea anemone and humans (Figure 1C). The location of
the Sox8/9 ancestor gene in the vicinity of Nme genes in the sea
anemone and the location of SOX8 and SOX9 on human
chromosomes 17 and 16, respectively, are consistent with the first
round of whole genome duplication (1R) that gave rise to Nme2
and Nme3/4 in vertebrates [4]. In Ciona intestinalis, we have
obtained evidence suggesting a duplication of a large portion of
genomic DNA resulting in duplicated genes on two different
chromosomes (Table 1, Figure S1). Two duplicated Nme genes of
the group I originating from a common group I ancestral gene
were found on chromosomes 2q and 8q (Figure S1). In all studied
non-metazoan eukaryotes, Capitela teleta, and Strongylocentrotus
purpuratus, two Nme genes of the group I can be found on different
scaffolds. Because of the limited size of these scaffolds and the level
of assembly of corresponding draft genomes, it is not currently
possible to speculate on the nature of the duplication event that
has led to 2 genes. In Dictyostelium discoideum, 2 Nme genes of the
group I could be identified on different chromosomes. Results/Discussion In non-choanoflagellates/metazoan
species investigated, very few sequences displaying one thior-
edoxin domain could be identified but were never associated with
an NDPk domain. We thus hypothesize that Thioredoxin
domains already existed in the opisthokont ancestor and that
Nme8 emerged in the choanoflagellate/metazoan ancestor by
domain shuffling of 2 or 3 NDPk domains. This would, however,
require further analysis. Pre-Metazoan origins of the Nme family Pre-Metazoan origins of the Nme family brevicollis, and C. elegans one sequence is highly divergent in
comparison to the other one. Altogether, this strongly suggests
independent and lineage specific gene duplications. It was
previously shown that a single Nme gene of the group I was
present in the vertebrate ancestor [4]. This ancestor gene
subsequently duplicated differently in the different vertebrates
lineages and resulted in 2 to 5 genes, depending on the species [4]. Together, our data indicate that a single Nme gene of the group I
was present in the opisthokont ancestor. Our observations also
suggest that a single Nme gene of the group I was present in the
eukaryote ancestor. Understanding the selective factors that have
led to multiple independent duplications events in the Nme family
and the role of these proteins in non-vertebrate species would
provide major insights into the evolution and functions of the
family. defects [15]. As recently stressed, the use of model species to
decipher Nme gene functions is extremely beneficial and needs to
be further supported [16]. However, a better understanding of the
evolutionary links between the Nme genes that are found in non-
vertebrate model species and their mammalian counterparts is
required to allow this comparative biology approach. In order to
gain insight into putatively conserved key functions of Nme genes
that would have been retained throughout evolution, the aim of
the present study was thus to characterize gene family complexity
and protein features among metazoans. We were able to show that
the complexity of the Nme family predates the metazoan
radiation. We also provided evidence supporting the association
of Nme genes with key cellular functions that have been conserved
throughout metazoan evolution. Introduction In vertebrates, Nme genes can be separated in 2 groups – group I
and group II – based on their evolutionary history and protein
domains [4]. Nme genes of the group I (Nme1-4) originate from a
unique gene of the chordate ancestor while Nme genes of the
group II (Nme 5-8) are present throughout chordate evolution [4]. PLoS ONE | www.plosone.org November 2010 | Volume 5 | Issue 11 | e15506 1 Nme gene family complexity predates Metazoan
radiation The
topology of the phylogenetic tree displaying – for Chlamydomonas
reinhardtii, Nematostella vectensis, Capitela teleta, Strongylocentrotus purpur-
atus, Branchiostoma floridae, and Ciona intestinalis – the group I Nme
sequences more closely related within a species than between
species strongly suggests that corresponding duplication events are
independent and lineage-specific. It is noteworthy that for the
above species, duplicated sequences remained closely related,
whereas in other species, Naegleria gruberi, Ustilago maydis, M. PLoS ONE | www.plosone.org November 2010 | Volume 5 | Issue 11 | e15506 PLoS ONE | www.plosone.org 2 Pre-Metazoan origins of the Nme family PLoS ONE | www.plosone.org
3
November 2010 | Volume 5 | Issue 11 | e15506 November 2010 | Volume 5 | Issue 11 | e15506 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org 3 Pre-Metazoan origins of the Nme family Pre-Metazoan origins of the Nme family Figure 1. Phylogenetic tree and exon-intron structure of Group I Nme in Eukaryotes and synteny conservation between human and
starlet sea anemone Group I Nme. A. The phylogenetic tree was constructed from a single multiple sequence alignment. Bootstrap values for
neighbor joining, maximum parsimony, and maximum likelihood methods, respectively, are indicated for each node. Asterisks (*) indicate that the
node was not recovered by the corresponding phylogenetic method. The consensus midpoint-rooted tree was calculated using the FIGENIX
automated phylogenomic annotation pipeline [41]. For each sequence, species and corresponding name are shown. B. Corresponding exon/intron
structures of genes coding for the proteins used for the phylogenetic tree estimation. Exon/intron structure was obtained through Ensembl, NCBI or
JGI databases. When exon boundaries correspond to identical amino acid positions, the exons are displayed in color. Otherwise, exons are displayed
in black. Non-coding exons are shown in grey. Exon size (in nucleotides) is indicated above each box. C. Synteny analysis between Nematostella
vectensis NmeGp1 genes and human group I NME genes. Nematostella vectensis gene names are NCBI Entrez gene symbols. For clarity reasons
‘‘NEMVEDRAFT_’’ was removed from all gene symbols. Duplication time estimations are from TimeTree [46]. doi:10.1371/journal.pone.0015506.g001 be consistent with their known involvement in key biological
processes such as cell proliferation and development. Here, we have shown that the Nme gene repertoire of the
metazoan ancestor was similar to that of the vertebrate ancestor
(Figure 6). Nme gene family complexity predates Metazoan
radiation In agreement with prior studies reporting major gene
losses and genomic rearrangement experienced by ecdysozoans
[17–20] we show that Nme genes have highly diverged in this
phylogenetic group, resulting in the loss of either functional
NDPK domains or entire genes. Furthermore, we demonstrate
that the complexity of the family predates metazoan radiation. We
also provide evidence suggesting that the complexity of the family
is mainly a eukaryotic innovation, with the exception of Nme8 that
is likely to be a choanoflagellate/metazoan innovation. This
unexpectedly ancient complexity of the eukaryotic Nme gene
family
is
in
striking
contrast
with the
existing
hypothesis
associating the emergence of Nme family complexity with the
differentiation of Bilateralian [21] lineage. It should be stressed
that this burst of complexity in the Nme family is much more
ancient than what has been reported for many genes in which the
expansion of the family is thought to have occurred around the
metazoan radiation [18,19,22,23]. This would be in favor of the
participation of Nme genes in ancestral functions and would also Highly conserved Nme sequence features exist among
Metazoans and Eukaryotes The topology of the phylogenetic tree (Figure 1A) obtained
using group I Nme proteins does not match the topology of the
tree of life. Indeed, N. vectensis sequences appear, along with B. floridae and C. teleta sequences, as a sister group of the vertebrate
and insect sequences, in contrast to T. adhaerens, S. purpuratus, and
C. intestinalis sequences that appear much more divergent. The
topology of the tree is, however, consistent with the genomic
structure of group I Nme genes reported on Figure 1B. A highly
conserved exon/intron structure is observed between T. adhaerens,
N. vectensis, L. gigantea, and vertebrates, while ecdysozoans, C. teleta,
C. intestinalis, and S. purpuratus exhibit a totally different genomic
organization that reflects the high gene divergence observed in
these species. Interestingly, the D. discoideum mitochondrial NdkM
shows gene and protein features that are similar (Figure 1B) to its
cytosolic paralog NdkC-2, but displays a longer N-terminus Figure 2. Phylogenetic tree, protein domains, and exon-intron structure of Nme5 in Eukaryotes. A. The phylogenetic midpoint-rooted
tree was constructed as described in Figure 1A. B. Corresponding protein domain structure of Nme proteins. Protein domain information was
obtained using Genbank Conserved Domain Database [40]. Parentheses indicate that the domain type is the best hit given by NCBI CDD but is not a
specific hit according to CDD default parameters. C. Exon/intron gene structure was obtained as described in Figure 1B. doi:10.1371/journal.pone.0015506.g002 Figure 2. Phylogenetic tree, protein domains, and exon-intron structure of Nme5 in Eukaryotes. A. The phylogenetic midpoint-rooted
tree was constructed as described in Figure 1A. B. Corresponding protein domain structure of Nme proteins. Protein domain information was
obtained using Genbank Conserved Domain Database [40]. Parentheses indicate that the domain type is the best hit given by NCBI CDD but is not a
specific hit according to CDD default parameters. C. Exon/intron gene structure was obtained as described in Figure 1B. doi:10.1371/journal.pone.0015506.g002 November 2010 | Volume 5 | Issue 11 | e15506 PLoS ONE | www.plosone.org 4 Pre-Metazoan origins of the Nme family Figure 3. Phylogenetic tree, protein domains, and exon-intron structure of Nme6 in Eukaryotes. A. The phylogenetic midpoint-rooted
tree was constructed as described in Figure 1A. B. Corresponding protein domain structure was obtained as described in Figure 2A. C. Exon/intron
gene structure was obtained as described in Figure 1B. doi:10.1371/journal.pone.0015506.g003 Figure 3. Phylogenetic tree, protein domains, and exon-intron structure of Nme6 in Eukaryotes. A. Highly conserved Nme sequence features exist among
Metazoans and Eukaryotes The phylogenetic midpoint-rooted
tree was constructed as described in Figure 1A. B. Corresponding protein domain structure was obtained as described in Figure 2A. C. Exon/intron
gene structure was obtained as described in Figure 1B. doi:10.1371/journal.pone.0015506.g003 Figure 4. Phylogenetic tree and protein domains of Nme7 in Eukaryotes. A. The phylogenetic midpoint-rooted tree was constructed as
described in Figure 1A. B. Corresponding protein domain structure was obtained as described in Figure 2A. doi:10.1371/journal.pone.0015506.g004 Figure 4. Phylogenetic tree and protein domains of Nme7 in Eukaryotes. A. The phylogenetic midpoint-rooted tree was constructed as
described in Figure 1A. B. Corresponding protein domain structure was obtained as described in Figure 2A. doi:10.1371/journal.pone.0015506.g004 November 2010 | Volume 5 | Issue 11 | e15506 November 2010 | Volume 5 | Issue 11 | e15506 PLoS ONE | www.plosone.org 5 Pre-Metazoan origins of the Nme family Figure 5. Phylogenetic tree and protein domains of Nme8 in Metazoans. A. The phylogenetic midpoint-rooted tree was constructed as
described in Figure 1A. B. Corresponding protein domain structure was obtained as described in Figure 2A. C. Synteny analysis between Nematostella
vectensis Nme8 genes and human TXNDC3 (NME8) genes. Nematostella vectensis gene names correspond to the NCBI Entrez Gene identification. For
clarity reasons the unchanged part ‘‘NEMVEDRAFT_’’ was removed. doi:10.1371/journal.pone.0015506.g005 Figure 5. Phylogenetic tree and protein domains of Nme8 in Metazoans. A. The phylogenetic midpoint-rooted tree was constructed as
described in Figure 1A. B. Corresponding protein domain structure was obtained as described in Figure 2A. C. Synteny analysis between Nematostella
vectensis Nme8 genes and human TXNDC3 (NME8) genes. Nematostella vectensis gene names correspond to the NCBI Entrez Gene identification. For
clarity reasons the unchanged part ‘‘NEMVEDRAFT_’’ was removed. doi:10.1371/journal.pone.0015506.g005 in terms of genomic exon/intron structure, protein size, and
functional domains (Figures 4B and S4). No synteny analysis could
be performed between starlet sea anemone and humans for Nme5,
Nme6, and Nme7 genes due to the limited number of genes present
on corresponding sea anemone scaffolds (Table 2). Similarly to
Nme7, no Nme8 ortholog was identified in C. elegans. In insects
and T. adhaerens, the Nme8-related sequences that could be
identified were extremely divergent (Figure 5A, Figure S2) and
lacked the 3 NDPK_TX domains found in all eumetazoan Nme8
proteins, including the starlet sea anemone (Figure 5B). A
conserved synteny was also identified between human and sea
anemone (Figure 5C). Highly conserved Nme sequence features exist among
Metazoans and Eukaryotes Together our data suggest that, in contrast
to Nme5 and Nme6 that have been identified in all investigated
metazoan species, Nme7 and Nme8 have been lost in C. elegans. In
insects, very divergent Nme7 and Nme8 genes remain. For Nme8,
the high divergence associated with the loss of the 3 NDPK_TX
domains in insects and T. adhaerens suggests a fast evolution of the
gene possibly associated with a loss of ancestral metazoan Nme8
function. sequence
containing a mitochondrial assignation signal. In
vertebrates, the Nme4 protein also displays a mitochondrial
assignation signal in N-terminus sequence and was shown to be a
gnathostome innovation [4]. In contrast, no mitochondrial
assignation signal is found in any other Nme protein. This feature
is thus likely to be a functional convergence between amoebozoan
NdkM and vertebrate Nme4. As indicated above, orthologs of Human Nme5, Nme6, Nme7, and
Nme8 are found in metazoans (Figure S3). The topology of the
phylogenetic tree (Figure 2A) obtained using Nme5 proteins
showed that ecdysozoan sequences are highly divergent. Ecdy-
sozoan Nme5 proteins appear to be even more divergent than the
early diverging eukaryotes N. gruberi and M. pusilla. Similarly to
group I, gene exon/intron structure and protein domains of N. vectensis, B. floridae, S. purpuratus, C. teleta, and L. gigantea Nme 5
proteins (Figures 2B and 2C) were highly similar to their human
counterpart, while ecdysozoan sequences exhibited different
protein
length
and/or
domains. In
agreement
with
these
observations was the remarkably conserved exon/intron structure
observed between humans and sea anemone Nme5 genomic
sequences (Figure 2C). Similar observations on tree topology,
protein domains, and genomic structure were made for Nme6
(Figure 3). For Nme7, no ortholog could be identified in C. elegans,
while very divergent Nme7 sequences were identified in non-
metazoan eukaryote species, M. brevicollis, and insects as shown by
the topology of the phylogenetic tree (Figure 4A). In contrast to all
other studied species, no NDPk7A domain was found (Figure 4B)
in M. brevicollis and insects sequences whereas they could be
identified in several non-opisthokont eukaryote species, thus
suggesting a high divergence of ecdysozoan genes within the
metazoan lineage. In addition, an incomplete NDPk7B domain
was found in A. aegypti (Figure 4B). It should be stressed that, in
contrast to insects, the Nme7 sequences of T. adhaerens, N. vectensis,
and B. Highly conserved Nme sequence features exist among
Metazoans and Eukaryotes floridae were remarkably similar to their human counterpart We have shown that, in addition to the complexity of the Nme
family, several highly conserved gene structures and protein
domains are also conserved throughout metazoan evolution, some
features being also conserved throughout the eukaryotic lineage. When considering the entire Nme family, the starlet sea anemone
is the metazoan species exhibiting the most conserved gene and
protein sequence features with humans. Starlet sea anemone as a model to investigate
evolutionarily conserved Nme functions Several non vertebrate model species, mainly C. elegans and D. melanogaster, have been used to investigate Nme functions. This
fruitful approach has shed light on evolutionary conserved
mechanisms involved in Alzheimer’s disease [15], ciliary function
[24], or epithelial integrity [25]. Nevertheless, the need for studies of November 2010 | Volume 5 | Issue 11 | e15506 PLoS ONE | www.plosone.org 6 Pre-Metazoan origins of the Nme family Table 1. GroupI Nme proteins: names and symbols by species, accession numbers and corresponding chromosomal location. Table 1. GroupI Nme proteins: names and symbols by species, accession numbers and corresponding chromosomal location. Species
Name
GenBank Acc. no. Ensembl or JGI Acc. no. Localisation
Position
N. gruberi
NmeGp1NgA
XP_002672581
72250
Scaffold_52
70,213–70,711
N. gruberi
NmeGp1NgB
XP_002678151
60551
Scaffold_19
18,232–18,965
T. thermophila
NmeGp1
XP_001018488
Scaffold_8254597
472,318–473,033
D. discoideum
NdkC-2
XP_644519
DDB0238334
Chr 2
3,427,961–3,428,694
D. discoideum
NdkM
XP_641417
DDB0214817
Chr 3
2,740,248–2,741,470
C. reinhardtii
NmeGp1CrA
XP_001698246
58944
Chr 16
365,385–367,897
C. reinhardtii
NmeGp1CrB
XP_001702884
292075
Scaffold_26
140,833–142,142
U. maydis
NmeGp1UmA
XP_759114
2967
Contig_1_101
162,394–163,065
U. maydis
NmeGp1UmB
XP_758923
2776
Contig_1_94
34,001–34,701
S. cerevisiae
Ynk1
NP_012856
YKL067W
Chromosome XI
314,456–314,917
M. brevicollis
NmeGp1MbA
XP_001742597
19073
Scaffold_2
878,808–880,474
M. brevicollis
NmeGp1MbB
XP_001749207
28652
Scaffold_28
66,018–71,920
T. adhaerens
NmeGp1
XP_002115688
49508
Scaffold_11
2,334,568–2,336,179
N. vectensis
NmeGp1NvA
XP_001630018
169568
Scaffold_129
58,094–60,224
N. vectensis
NmeGp1NvB
XP_001630017
115087
Scaffold_129
51,034–52,881
C. elegans
NmeGp1CeA
NP_492761
F25H2.5.1
Chr I
10,553,902–10,553,101
C. elegans
NmeGp1CeB
NP_492819
F55A3.6
Chr I
10,784,780–10,783,989
T. castaneum
Awd
XP_967503
Linkage Group 3
32,141,388–32,142,070
Group1
D. melanogaster
Awd
NP_476761
FBpp0085223
Chr 3R
27,570,894–27,571,706
Nme
A. aegypti
Awd
XP_001662512
AAEL012359-PA
SuperContig1.684
87,333–88,186
L. gigantea
NmeGp1
205662
Scaffold_3
3,942,249–3,946,053
C. teleta
NmeGp1CtA
21725
Scaffold_758
80,222–84,888
C. teleta
NmeGp1CtB
116635
Scaffold_527
188,815–189,048
S. purpuratus
NmeGp1SpA
XP_799145
Scaffold71291
17,770–21,754
S. purpuratus
NmeGp1SpB
XP_785384
Scaffold42444
298,840–301,211
C. intestinalis
NmeGp1CiA
XP_002123476
ENSCINP00000011619
Chr 8q
5,908,347–5,908,808
C. intestinalis
NmeGp1CiB
XP_002121438
ENSCINP00000002194
Chr 2q
7,888,026–7,888,562
B. floridae
NmeGp1BfA
XP_002598285
57540
Scaffold_24
2,074,549–2,076,844
B. floridae
NmeGp1BfB
XP_002598283
69640
Scaffold_24
2,064,578–2,066,435
H. sapiens
NME1
NP_937818
ENSP00000337060
Chr 17
49,230,937–49,239,422
H. sapiens
NME2
NP_001018149
ENSP00000376888
Chr 17
49,243,639–49,249,108
X. tropicalis
Nme2
NP_001005140
ENSXETP00000024764
Scaffold_673
77,640–81,451
Nme2
D. rerio
Nme2a
NP_956264
ENSDARP00000064338
Scaffold Zv8_scaffold3117
528,950–537,135
D. rerio
Nme2b1
NP_571001
ENSDARP00000099319
Chr 19
48,501,402–48,504,320
D. rerio
Nme2b2
NP_571002
ENSDARP00000098065
Chr 19
48,507,488–48,510,647
H. sapiens
NME3
NP_002504
ENSP00000219302
Chr 16
1,820,321–1,821,710
Nme3
X. tropicalis
Nme3
NP_001005115
ENSXETP00000022770
Scaffold_27
933,363–937,018
D. rerio
Nme3
NP_571003
ENSDARP00000075112
Chr 3
11,995,596–12,045,134
H. PLoS ONE | www.plosone.org November 2010 | Volume 5 | Issue 11 | e15506 Starlet sea anemone as a model to investigate
evolutionarily conserved Nme functions sapiens
NME4
NP_005000
ENSP00000219479
Chr 16
447,209–450,759
Nme4
X. tropicalis
Nme4
NP_001039239
ENSXETP00000022726
Scaffold_27
1,305,654–1,312,714
D. rerio
Nme4
NP_957489
ENSDARP00000103207
Chr 3
10,909,410–10,922,804
Protein names were retrieved from Ensembl and NCBI or proposed according to the evolutionary history of the genes. Chromosomal/genomic location was obtained
using Ensembl genome browser, JGI databases, or NCBI Entrez Gene when not available on Ensembl or JGI. Protein names were retrieved from Ensembl and NCBI or proposed according to the evolutionary history of the genes. Chromosomal/genomic location was obtained
using Ensembl genome browser, JGI databases, or NCBI Entrez Gene when not available on Ensembl or JGI. doi:10.1371/journal.pone.0015506.t001 as separate sexes, inducible spawning, flagellated sperm, and
external fertilization. This model species thus offers significant
opportunities to investigate Nme gene functions and thus shed new
light on Nme functions that remain poorly understood [16]. Nme functions in non-vertebrate model species was recently stressed
by the scientific community [16]. Here, we show that the starlet sea
anemone exhibits a full set of metazoan Nme genes and shares
remarkably conserved gene and protein sequence features with
humans that were lost in flies and C. elegans. The starlet sea anemone
is an emerging model [26] offering several biological features such Nme functions in non-vertebrate model species was recently stressed
by the scientific community [16]. Here, we show that the starlet sea
anemone exhibits a full set of metazoan Nme genes and shares
remarkably conserved gene and protein sequence features with
humans that were lost in flies and C. elegans. The starlet sea anemone
is an emerging model [26] offering several biological features such Nme proteins of the group I are involved in a wide variety of
cellular and physiological processes including tumor metastatic PLoS ONE | www.plosone.org November 2010 | Volume 5 | Issue 11 | e15506 7 Pre-Metazoan origins of the Nme family Table 2. GroupII Nme proteins: names and symbols by species, accession numbers and corresponding chromosomal location. Species
Name
GenBank Acc. no. Ensembl or JGI Acc. no. Localisation
Position
Nme5
N. gruberi
Nme5
XP_002682323
29950
Scaffold_3
679,761–680,378
M. pusilla
Nme5
XP_003056664
15249
Scaffold_3
169,341–169,943
M. brevicollis
Nme5
XP_001749876
38924
Scaffold_34
201,106–202,764
T. adhaerens
Nme5
XP_002112439
50304
Scaffold_5
3,451,699–3,452,497
N. vectensis
Nme5
XP_001631264
244029
Scaffold_105
447,049–449,367
C. elegans
Nme5
NP_501212
R05G6.5
Chr IV
7,508,567–7,509,459
T. castaneum
Nme5
XP_001811234
Linkage Group 2
13,741,320–13,742,593
D. melanogaster
Nme5
NP_651833
FBpp0085077
Chr 3R
26,707,514–26,709,225
A. aegypti
Nme5
XP_001662993
AAEL003030-PA
SuperContig1.75
2,400,537–2,515,468
L. November 2010 | Volume 5 | Issue 11 | e15506 Starlet sea anemone as a model to investigate
evolutionarily conserved Nme functions gigantea
Nme5
226200
Scaffold_168
228,601–233,346
C. teleta
Nme5
177417
Scaffold_116
97,523–98,941
S. purpuratus
Nme5
XP_790390
Scaffold25557
320,279–323,900
C. intestinalis
Nme5
NP_001154961
ENSCINP00000008954
Chr 7q
1,031,987–1,036,756
B. floridae
Nme5
XP_002596479
61845
Scaffold_430
334,501–336,787
H. sapiens
NME5
NP_003542
ENSP00000265191
Chr 5
137,450,866–137,475,104
X. tropicalis
Nme5
NP_001072619
ENSXETP00000008322
Scaffold_65
2,613–8,494
D. rerio
Nme5
NP_001002516
ENSDARP00000060997
Chr 14
6,455,589–6,462,855
Nme6
D. discoideum
Nme6
XP_629447
DDB0191701
Chr 6
2,282,811–2,283,293
C. reinhardtii
Nme6
XP_001698136
139197
Chr 16
825,573–828,346
U. maydis
Nme6
XP_760135
3988
Contig_1_139
15,223–16,271
M. brevicollis
Nme6
XP_001744091
15435
Scaffold_5
413,751–414,530
T. adhaerens
Nme6
XP_002114036
58087
Scaffold_7
2,675,108–2,676,755
N. vectensis
Nme6
XP_001622626
140474
Scaffold_525
60,519–64,497
C. elegans
Nme6
NP_001021779
Y48G8AL.15
Chr I
1,254,771–1,258,597
D. melanogaster
Nme6
NP_572965
FBpp0073750
Chr X
14,477,710–14,478,649
T. castaneum
Nme6
XP_972639
Linkage Group 8
6,083,034–6,083,586
A. aegypti
Nme6
XP_001648448
AAEL004107-PA
SuperContig1.107
2,016,668–2,033,334
L. gigantea
Nme6
112601
Scaffold_15
3,040,725–3,042,846
C. teleta
Nme6
91319
Scaffold_32
665,868–668,578
S. purpuratus
Nme6
XP_001200902
Scaffold35436
11,834–18,064
C. intestinalis
Nme6
XP_002129729
ENSCINP00000027945
Scaffold_1779
5,509–6,021
B. floridae
Nme6
XP_002589414
279926
Scaffold_243
2,320,842–2,322,329
H. sapiens
NME6
NP_005784
ENSP00000416658
Chr 3
48,334,754–48,342,848
X. tropicalis
Nme6
NP_001123709
ENSXETP00000034257
Scaffold_857
287,305–293,502
D. rerio
Nme6
NP_571672
ENSDARP00000094574
Chr 20
18,803,906–18,812,975
Nme7
N. gruberi
Nme7
XP_002677897
33146
Scaffold_20
272,036–273,256
C. reinhardtii
Nme7
XP_001702841
180221
Chr 12
8,939,035–8,941,564
T. thermophila
Nme7
XP_001015884
Scaffold_8254649
122,957–124,735
M. brevicollis
Nme7
XP_001749106
11267
Scaffold_27
519,462–520,004
T. adhaerens
Nme7
XP_002108466
51403
Scaffold_1
1,598,731–1,601,855
N. vectensis
Nme7
XP_001626602
125694
Scaffold_222
17,379–24,092
T. castaneum
Nme7
XP_974333
Linkage Group 7
18,619,008–18,620,171
D. melanogaster
Nme7
NP_649926
FBpp0081561
Chr 3R
5,505,663–5,507,224
A. aegypti
Nme7
XP_001661412
AAEL011098-PA
SuperContig1.541
206,988–220,408
L. gigantea
Nme7
187020
Scaffold_18
1,179,880–1,185,739
C. teleta
Nme7
160391
Scaffold_422
28,192–33,650
S. purpuratus
Nme7
XP_795051
Scaffold9766
108,673–117,014
C. intestinalis
Nme7
NP_001155162
ENSCINP00000025129
Chr 1p
3,423,461–3,424,231
B. floridae
Nme7
XP_002588622
287848
Scaffold_254
342,459–356,708
H. sapiens
NME7
NP_037462
ENSP00000356785
Chr 1
169,101,769–169,337,205
PLoS ONE | www.plosone.org
8
November 2010 | Volume 5 | Issue 11 | e15506 PLoS ONE | www.plosone.org 8 Pre-Metazoan origins of the Nme family Table 2. Cont. Species
Name
GenBank Acc. no. Ensembl or JGI Acc. no. Localisation
Position
X. tropicalis
Nme7
NP_988903
ENSXETP00000005150
Scaffold_169
1,646,165–1,680,298
D. rerio
Nme7
NP_571004
ENSDARP00000073091
Chr 6
31,135,867–31,196,533
Nme8
M. brevicollis
Nme8
XP_001746342
32671
Scaffold_12
606,463–609,773
T. adhaerens
Nme8
XP_002110931
22954
Scaffold_3
3,392,460–3,393,060
N. vectensis
Nme8
XP_001634297
101462
Scaffold_61
602,301–615,863
T. castaneum
TRX-Nme8
XP_972627
Linkage Group 5
12,145,420–12,148,468
D. melanogaster
TRX-Nme8
NP_572772
FBpp0073425
Chr X
11,884,151–11,886,801
A. aegypti
TRX-Nme8
XP_001652618
AAEL007253-PA
SuperContig1.245
180,440–198,050
L. Starlet sea anemone as a model to investigate
evolutionarily conserved Nme functions Interestingly, c-MYC and p53
homologs could also be identified in the starlet sea anemone
genome thus suggesting that at least some down-stream targets of
Nme proteins are also present in this species (Table S1). Interestingly, the importance of p53 in sea anemone development
was recently stressed and found to be similar to its known function
in vertebrate development [32]. the starlet sea anemone we were able to show that most proteins
known to interact with human NME were also found in the
eumetazoan ancestor. Together, these observations suggest a
participation of Nme genes in key cellular functions that have been
conserved throughout evolution. In this context, the starlet sea
anemone that exhibits a full set of highly conserved Metazoan
group II Nme genes and appropriate biological features - such as
separate sex, flagellated sperm, and asymmetrical expression
patterns during development -offers major opportunities to
investigate Nme functions. Materials and Methods As indicated above, functional evidence exist demonstrating the
importance of Nme gene for key biological processes, including
development, cell proliferation, ciliary function, and cancer. We
have shown here that the complexity of the Nme family predates
metazoan radiation and that all Nme proteins display functional
domains that have been conserved throughout evolution. Using Conclusion In summary, we demonstrated that the complexity of the Nme
gene family initially thought to be restricted to chordates was also
shared by the Metazoan ancestor. We also provide evidence
suggesting that the complexity of the family is mainly a eukaryotic
innovation, with the exception of Nme8 that is likely to be a
choanoflagellate/metazoan innovation. Remarkably conserved
gene structures, genomic linkage, and protein domains were
identified among metazoans, some features being also conserved in
eukaryotes. When considering the entire Nme family, the starlet
sea anemone is the studied metazoan species exhibiting the most
conserved gene and protein sequence features with humans. In
addition, we were able to show that most of the proteins known to
interact with human NME proteins were also found in the starlet
sea anemone. Together, our observations further support the
association of Nme genes with key cellular functions that have
been conserved throughout metazoan evolution. As previously documented, most Nme proteins of the group II,
with the exception of Nme6, have been associated with ciliary
functions. They have been shown to play critical roles in
spermatogenesis [33–35], sperm motility [2], development [36],
and human conditions associated with primary ciliary dyskinesia
[37]. The existence of orthologs in non-metazoan species was
however unsuspected with the exception of the report of Nme7 in
C. reinhardtii and T. thermophila [24]. In the present study, we have
demonstrated that Nme5, Nme6, Nme7, and Nme8 genes were
present in the metazoan ancestor, while Nme5, Nme6, and Nme7
were most likely present in the eukaryote ancestor. To date, only 7
proteins are known to interact with NME proteins of the group II. We were however able to identify homologs for 6 of these
interacting partners in starlet sea anemone (Table S1). PLoS ONE | www.plosone.org Starlet sea anemone as a model to investigate
evolutionarily conserved Nme functions gigantea
Nme8
107502
Scaffold_7
645,416–655,143
C. teleta
Nme8
96991
Scaffold_940
8,079–11,527
S. purpuratus
Nme8
XP_001181827
Scaffold66657
21,472–41,768
C. intestinalis
Nme8
NP_001027618
ENSCINP00000013583
Chr 9q
3,694,058–3,703,935
B. floridae
Nme8
XP_002597926
280761
Scaffold_152
66,164–77,826
H. sapiens
NME8
NP_057700
ENSP00000199447
Chr 7
37,888,199–37,940,003
X. tropicalis
Nme8
NP_001121456
ENSXETP00000002355
Scaffold_664
444,670–467,036
D. rerio
Nme8
NP_001082944
ENSDARP00000103107
Chr9
51,493,472–51,569,898
Protein names were retrieved from Ensembl and NCBI or proposed according to the evolutionary history of the genes. Chromosomal/genomic location was obtained
using Ensembl genome browser, JGI databases, or NCBI Entrez Gene when not available on Ensembl or JGI. doi:10.1371/journal.pone.0015506.t002 Protein names were retrieved from Ensembl and NCBI or proposed according to the evolutionary history of the genes. Chromosomal/genomic location was obtained
using Ensembl genome browser, JGI databases, or NCBI Entrez Gene when not available on Ensembl or JGI. doi:10.1371/journal.pone.0015506.t002 potential. Using a reciprocal BLASTP strategy, the starlet sea
anemone genome was searched for homologs of human proteins
known to interact with group I NME proteins. Among the 48
human proteins known to interact with NME1, NME2, NME3, or
NME4, 44 had a homolog in sea anemone (Table S1). For
instance, homologs of proteins involved in cancer and cell cycle
control, such as MIF [27] and Rac1 [28], were clearly identified in
sea anemone (Table S1). In addition, NME1 and NME2 have
been demonstrated to regulate the expression of specific genes
such as c-MYC [29,30] and p53 [31]. Interestingly, c-MYC and p53
homologs could also be identified in the starlet sea anemone
genome thus suggesting that at least some down-stream targets of
Nme proteins are also present in this species (Table S1). Interestingly, the importance of p53 in sea anemone development
was recently stressed and found to be similar to its known function
in vertebrate development [32]. potential. Using a reciprocal BLASTP strategy, the starlet sea
anemone genome was searched for homologs of human proteins
known to interact with group I NME proteins. Among the 48
human proteins known to interact with NME1, NME2, NME3, or
NME4, 44 had a homolog in sea anemone (Table S1). For
instance, homologs of proteins involved in cancer and cell cycle
control, such as MIF [27] and Rac1 [28], were clearly identified in
sea anemone (Table S1). In addition, NME1 and NME2 have
been demonstrated to regulate the expression of specific genes
such as c-MYC [29,30] and p53 [31]. Sequence analysis All Nme sequences were identified using the following genome
assemblies: human (Homo sapiens, Assembly GRCh37), xenopus
(Xenopus tropicalis, Assembly V.4.1), zebrafish (Danio rerio, Assembly
ZV8), tunicate (Ciona intestinalis, Assembly V.2.0), florida lancelet PLoS ONE | www.plosone.org November 2010 | Volume 5 | Issue 11 | e15506 9 Pre-Metazoan origins of the Nme family Figure 6. Schematic depiction of Nme genes evolution in Opisthokonts. The gene repertoire
opisthokont ancestor. For all lineages, duplication events are shown and cis-duplications (CD) indicated. Fo
genome duplications events are shown and duplication events of group I Nme genes redrawn from [4]. Nm
be vertebrate innovations [4] are not displayed here for clarity reasons. The gene repertoire is given for
complete name of the gene is not given and only the gene number is given and the color kept consis
opisthokont ancestor. doi:10 1371/journal pone 0015506 g006 Figure 6. Schematic depiction of Nme genes evolution in Opisthokonts. The gene repertoire is shown at the root of the tree for the
opisthokont ancestor. For all lineages, duplication events are shown and cis-duplications (CD) indicated. For the vertebrate lineage, 1R and 3R whole
genome duplications events are shown and duplication events of group I Nme genes redrawn from [4]. Nme 9 and Nme10 that have been shown to
be vertebrate innovations [4] are not displayed here for clarity reasons. The gene repertoire is given for all studied species. For clarity reasons, the
complete name of the gene is not given and only the gene number is given and the color kept consistent with the corresponding gene in the
opisthokont ancestor. doi:10.1371/journal.pone.0015506.g006 Figure 6. Schematic depiction of Nme genes evolution in Opisthokonts. The gene repertoire is shown at the root of the tree for the
opisthokont ancestor. For all lineages, duplication events are shown and cis-duplications (CD) indicated. For the vertebrate lineage, 1R and 3R whole
genome duplications events are shown and duplication events of group I Nme genes redrawn from [4]. Nme 9 and Nme10 that have been shown to
be vertebrate innovations [4] are not displayed here for clarity reasons. The gene repertoire is given for all studied species. For clarity reasons, the
complete name of the gene is not given and only the gene number is given and the color kept consistent with the corresponding gene in the
opisthokont ancestor. Sequence analysis doi:10 1371/journal pone 0015506 g006 doi:10.1371/journal.pone.0015506.g006 (Branchiostoma floridae, Assembly V.2.0), purple sea urchin (Strongy-
locentrotus purpuratus, Assembly NCBI V.2.1), fruit fly (Drosophila
melanogaster, Assembly BDGP5), yellow fever mosquito (Aedes aegypti,
Assembly AaegL1), red flour beetle (Tribolium castaneum, Assembly
Tcas 3.0), nematode (Caenorhabditis elegans, Assembly WS214),
polychaete worm (Capitella teleta, Assembly V1.0), owl limpet (Lottia
gigantea, Assembly V.1.0), starlet sea anemone (Nematostella vectensis,
Assembly V.1.0), placozoan (Trichoplax adhaerens, Assembly Grell-
BS-1999 V.1.0), marine choanoflagellate (Monosiga brevicollis,
Assembly V1.0), fungi (Saccharomyces cerevisiae, Assembly EF 2;
and Ustilago maydis, Assembly 1), amoebozoa (Dictyostelium dis-
coideum, Assembly V.2.1), alveolate (Tetrahymena thermophila, Assembly
1.1), green plants (Micromonas pusilla, Assembly V.2.0; and
Chlamydomonas reinhardtii, Assembly V.4.0) and heterolobosean
(Naegleria gruberi, Assembly V.1.0). A large number of sequences
were obtained from the NCBI NR database using human or
zebrafish protein sequences as a query. When more than one
sequence was obtained, the RefSeq one was preferentially selected. When no RefSeq sequence was available, the longest sequence was
used. When no sequences were available in the NR database,
BLASTP was used on the Ensembl [38] and DoE Joint Genome
Institute databases. The chromosomal localization of Nme genes
was established using the Ensembl genome browser or JGI gene
information, or when not available, using the UCSC Genome
Bioinformatics BLAT [39] and the NCBI Sequence Viewer. Exon/intron structure was obtained from the Ensembl, NCBI, or
JGI databases. The protein domain structure of Nme proteins was
obtained from the GenBank Conserved Domain Database [40]. (Branchiostoma floridae, Assembly V.2.0), purple sea urchin (Strongy-
locentrotus purpuratus, Assembly NCBI V.2.1), fruit fly (Drosophila
melanogaster, Assembly BDGP5), yellow fever mosquito (Aedes aegypti,
Assembly AaegL1), red flour beetle (Tribolium castaneum, Assembly
Tcas 3.0), nematode (Caenorhabditis elegans, Assembly WS214),
polychaete worm (Capitella teleta, Assembly V1.0), owl limpet (Lottia
gigantea, Assembly V.1.0), starlet sea anemone (Nematostella vectensis,
Assembly V.1.0), placozoan (Trichoplax adhaerens, Assembly Grell-
BS-1999 V.1.0), marine choanoflagellate (Monosiga brevicollis,
Assembly V1.0), fungi (Saccharomyces cerevisiae, Assembly EF 2;
and Ustilago maydis, Assembly 1), amoebozoa (Dictyostelium dis-
coideum, Assembly V.2.1), alveolate (Tetrahymena thermophila, Assembly
1.1), green plants (Micromonas pusilla, Assembly V.2.0; and
Chlamydomonas reinhardtii, Assembly V.4.0) and heterolobosean
(Naegleria gruberi, Assembly V.1.0). A large number of sequences
were obtained from the NCBI NR database using human or
zebrafish protein sequences as a query. When more than one
sequence was obtained, the RefSeq one was preferentially selected. References 1. Steeg PS, Bevilacqua G, Kopper L, Thorgeirsson UP, Talmadge JE, et al. (1988)
Evidence for a novel gene associated with low tumor metastatic potential. J Natl
Cancer Inst 80: 200–204. 12. Troll H, Winckler T, Lascu I, Muller N, Saurin W, et al. (1993) Separate nuclear
genes encode cytosolic and mitochondrial nucleoside diphosphate kinase in
Dictyostelium discoideum. J Biol Chem 268: 25469–25475. 1. Steeg PS, Bevilacqua G, Kopper L, Thorgeirsson UP, Talmadge JE, et al. (1988)
Evidence for a novel gene associated with low tumor metastatic potential. J Natl
Cancer Inst 80: 200–204. 2. Boissan M, Dabernat S, Peuchant E, Schlattner U, Lascu I, et al. (2009) The
mammalian Nm23/NDPK family: from metastasis control to cilia movement. Mol Cell Biochem 329: 51–62. 13. Cherfils J, Morera S, Lascu I, Veron M, Janin J (1994) X-ray structure of
nucleoside diphosphate kinase complexed with thymidine diphosphate and
Mg2+ at 2-A resolution. Biochemistry (Mosc) 33: 9062–9069. 3. Postel EH (2003) Multiple biochemical activities of NM23/NDP kinase in gene
regulation. J Bioenerg Biomembr 35: 31–40. g
y (
)
14. Lascu L, Giartosio A, Ransac S, Erent M (2000) Quaternary structure of
nucleoside diphosphate kinases. J Bioenerg Biomembr 32: 227–236. nucleoside diphosphate kinases. J Bioenerg Biomembr 32: 227–2 4. Desvignes T, Pontarotti P, Fauvel C, Bobe J (2009) Nme protein family
evolutionary history, a vertebrate perspective. BMC Evol Biol 9: 256. 15. Napolitano F, D’Angelo F, Bimonte M, Perrina V, D’Ambrosio C, et al. (2008) A
differential proteomic approach reveals an evolutionary conserved regulation of
Nme proteins by Fe65 in C. elegans and mouse. Neurochem Res 33: 2547–2555. 5. Polosina YY, Jarrell KF, Fedorov OV, Kostyukova AS (1998) Nucleoside
diphosphate kinase from haloalkaliphilic archaeon Natronobacterium magadii:
purification and characterization. Extremophiles 2: 333–338. 16. Mehta A, Orchard S (2009) Nucleoside diphosphate kinase (NDPK, NM23,
AWD): recent regulatory advances in endocytosis, metastasis, psoriasis, insulin
release, fetal erythroid lineage and heart failure; translational medicine
exemplified. Mol Cell Biochem 329: 3–15. 6. Hama H, Almaula N, Lerner CG, Inouye S, Inouye M (1991) Nucleoside
diphosphate kinase from Escherichia coli; its overproduction and sequence
comparison with eukaryotic enzymes. Gene 105: 31–36. 17. Takahashi T, McDougall C, Troscianko J, Chen WC, Jayaraman-Nagarajan A,
et al. (2009) An EST screen from the annelid Pomatoceros lamarckii reveals patterns
of gene loss and gain in animals. BMC Evol Biol 9: 240. 7. Authors thank Alexis Fostier for helpful discussions. Figure S1
Ciona intestinalis genomic region paralogy
relationships between chromosomes 2q and 8q. For Ciona
intestinalis paralogy analysis, synteny relationships were inquired
using the Synteny Database [45] and putative paralogs were
validated by reciprocal BLASTP on NCBI NR databases. (TIF) Authors thank Alexis Fostier for helpful discussions. Identification of Nme partners homologs Validated human NME partners were obtained though NCBI
Entrez Gene Interactions (http://www.ncbi.nlm.nih.gov/gene) in-
formation. Homologous genes in Nematostella vectensis were identified
by reciprocal BLASTP on the NCBI NR database. Only BLASTP
hits with an E-value lower than 10210 were considered significant. Phylogenetic analysis of Nme proteins Only Homo sapiens, Danio rerio, Ciona intestinalis
and Nematostella vectensis sequences were used in this phylogenetic
tree reconstruction because of the highly divergent ecdysozoans
sequences greatly modifying the tree topology. (TIF) Author Contributions Conceived and designed the experiments: TD JB PP. Performed the
experiments: TD. Analyzed the data: TD JB PP. Contributed reagents/
materials/analysis tools: JB PP. Wrote the paper: TD JB. Performed in
silico analyses and prepared the figures: TD. Conceived and designed the experiments: TD JB PP. Performed the
experiments: TD. Analyzed the data: TD JB PP. Contributed reagents/
materials/analysis tools: JB PP. Wrote the paper: TD JB. Performed in
silico analyses and prepared the figures: TD. Figure S2
Exon/intron structure of Nme8 genes. Exon/
intron structure was obtained through Ensembl, NCBI, or JGI
databases. When exon boundaries correspond to similar amino Synteny analysis The synteny relationships of starlet sea anemone and human
Nme genes were analyzed by reciprocal BLASTP on the NCBI NR
database using surrounding genes of Nme genes in Nematostella
vectensis. Homologous genes were considered in the analysis only
when reciprocal BLASTP returned the couple as best hit. For the
Ciona intestinalis paralogy analysis, synteny relationships were
obtained using the Synteny Database [45] and putative paralogs
were validated by reciprocal BLASTP on the NCBI NR database. Figure S4
Exon/intron structure of Nme7 genes. Exon/
intron structure was obtained through Ensembl, NCBI, or JGI
databases. When exon boundaries correspond to similar amino
acid positions, the exons are displayed in color. Otherwise, exons
are displayed in black. Non-coding exons are shown in grey. Numbers indicate exon size in nucleotides. (TIF)
Table S1
BLASTP hits of human NME partners against
starlet sea anemone sequences. Partners of human NME
proteins
were
listed
from
NCBI
Entrez
Gene
interaction
information. BLASTP hits were considered significant for e-values
lower than 10210. Accession numbers and BLASTP e-values are
also given for c-Myc and p53, two downstream targets of human
NME1 and NME2 proteins. (XLS) Figure S4
Exon/intron structure of Nme7 genes. Exon/
intron structure was obtained through Ensembl, NCBI, or JGI
databases. When exon boundaries correspond to similar amino
acid positions, the exons are displayed in color. Otherwise, exons
are displayed in black. Non-coding exons are shown in grey. Numbers indicate exon size in nucleotides. (TIF) Table S1
BLASTP hits of human NME partners against
starlet sea anemone sequences. Partners of human NME
proteins
were
listed
from
NCBI
Entrez
Gene
interaction
information. BLASTP hits were considered significant for e-values
lower than 10210. Accession numbers and BLASTP e-values are
also given for c-Myc and p53, two downstream targets of human
NME1 and NME2 proteins. (XLS) Phylogenetic analysis of Nme proteins Phylogenetic reconstructions were performed using the auto-
mated genomic annotation platform FIGENIX [41]. For each
phylogenetic tree reconstruction, all selected protein sequences
were added to a single multiple sequence alignment. Sequence
alignment was performed automatically by the FIGENIX pipeline
using MUSCLE v3.6 [42,43]. The pipeline used is based on three PLoS ONE | www.plosone.org PL November 2010 | Volume 5 | Issue 11 | e15506 PLoS ONE | www.plosone.org 10 Pre-Metazoan origins of the Nme family different
methods
of
phylogenetic
tree
reconstruction
(i.e. neighbour-joining (NJ), maximum parsimony (MP), and maximum
likelihood (ML)). The substitution model was calculated from data
for ML while BLOSUM was used for NJ. Bootstrapping was
carried out to assess node support with 1000 pseudoreplicates [44]. Support values were mapped onto a midpoint-rooted 50%
majority rule consensus tree for each optimality criterion. Bootstrap values are reported for the nodes that are present in
all three phylogenetic reconstruction methods. Asterisks denote the
absence of a node for a given phylogenetic method. acid positions, the exons are displayed in color. Otherwise, exons
are displayed in black. Non-coding exons are shown in grey. Numbers indicate exon size in nucleotides. (TIF)
Figure S3
Phylogenetic reconstruction of the Nme pro-
tein family in eumetazoans. Phylogenetic tree was constructed
from a single multiple alignment. Bootstrap values for neighbor
joining, maximum parsimony, and maximum likelihood methods,
respectively, are indicated for each node. * indicates that the node
does not exist in the corresponding tree. The consensus tree was
calculated using the FIGENIX [41] automated phylogenomic
annotation pipeline. Only Homo sapiens, Danio rerio, Ciona intestinalis
and Nematostella vectensis sequences were used in this phylogenetic
tree reconstruction because of the highly divergent ecdysozoans
sequences greatly modifying the tree topology. (TIF) acid positions, the exons are displayed in color. Otherwise, exons
are displayed in black. Non-coding exons are shown in grey. Numbers indicate exon size in nucleotides. (TIF)
Figure S3
Phylogenetic reconstruction of the Nme pro-
tein family in eumetazoans. Phylogenetic tree was constructed
from a single multiple alignment. Bootstrap values for neighbor
joining, maximum parsimony, and maximum likelihood methods,
respectively, are indicated for each node. * indicates that the node
does not exist in the corresponding tree. The consensus tree was
calculated using the FIGENIX [41] automated phylogenomic
annotation pipeline. Acknowledgments Figure S1
Ciona intestinalis genomic region paralogy
relationships between chromosomes 2q and 8q. For Ciona
intestinalis paralogy analysis, synteny relationships were inquired
using the Synteny Database [45] and putative paralogs were
validated by reciprocal BLASTP on NCBI NR databases. (TIF)
Figure S2
Exon/intron structure of Nme8 genes. Exon/
intron structure was obtained through Ensembl, NCBI, or JGI
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