Electronic Supplementary Material
Supplementary Materials and Methods:
Methods-Whirligig Experiments
Whirligig beetles (Gyrinidae: Dineutes discolor) were collected by long-handled dip nets
from groups along the Raquette River in Potsdam, NY, USA in mid July 2013. Approximately
200 whirligigs were collected each week for two weeks. They were kept in aged tap water at a
temperature of 20° C on a 12L:12D cycle. All beetles were fed 4 mg/beetle/day freeze-dried
bloodworms split into two daily doses throughout their time in the laboratory. Beetles were
marked and manipulated, then given three days to recover before filming.
We created “EYE ̶ ” beetles by painting their dorsal and ventral eye on one side
(unilaterally) with black Faber-Castell PaintPens. To do this, we restrained each whirligig
between a wire screen and a foam pad under a dissecting microscope. We then allowed the paint
to dry for 30 seconds. Then a paint mark was placed half on the right, half on the left elytra.
Twenty males and 20 females were marked and placed in replicated treatment tanks for three
days before filming. In half the trials (the first week) left eyes were painted, and the second
week right eyes were painted. We chose to make unilateral sensory manipulations: 1) so beetles
could see the visual predator stimulus, 2) to assess if their responses were unilateral, moving in
one direction or the other, and 3) to make sure they did not stop grouping entirely.
We created “ANT ̶ ” beetles by removing the top part of their right or left antenna. In
whirligigs the top part of the antennae (flagellum) goes above the water, and the lower feathered
part (pedicel) rests on the water’s surface [14]. We removed their flagellum with fine tipped
tweezers while restraining the beetles between foam and a screen under a dissecting microscope.
Equal numbers of males and females were prepared in this way and held in replicated treatment
tanks to recover. In previous studies, whirligigs behaved and survived normally after the
procedure [14]. There was no mortality in the current experiment due to antenna removal. We
chose to make unilateral rather than bilateral cuts for similar reasons as those stated above. We
did not have a control with both types of sensory deprivation in individual beetles though this
would be useful in the future to see if they could exhibit a FE.
We treated the control beetles the same, by picking them up and trapping them between
the foam and disturbing them with predator models at an equal rate and feeding them equally to
the two treatment conditions. We did not mark the control beetles for the current study.
However previous studies [12,6] found that marking itself or the color used, produced no change
in a beetle’s NND or positioning (outside/inside) within the group.
After the beetles had been taken from the stock tank and prepared (sensory deprivation),
they were put into multiple “treatment” tanks. 80 beetles for each treatment (no eye, no
antennae, or intact) were each split into four groups of 20 and held in their own 24x50 cm tank
on a given week. To construct a group for filming, 12 impaired and 12 normal beetles were
netted at random from the treatment tanks and placed together in a round filming tank (1 m
diameter). Previous studies have found that this is a good density so that beetles act in a natural
way and it can be seen whether they are in a group or have left a group. Tanks were filled with
aged tap water to a depth of 8 cm. Beetles were allowed one hour to acclimate while being
stimulated by a visual fright stimulus (30 x 60 cm black flag waved over the tank at 2 m/s from 1
m above them every 20 minutes). This causes them to group into the typical tight formation seen
in the wild. An equal sex ratio (as determined by microscopic examination) was placed into each
of the treatment tanks from which the experimental groups were selected at random.
Groups were then filmed (at 30 frames per second) from directly above with a Canon
60D digital camera (1920 x 1080 pixels, 100 ISO, 1/120 speed, F 3.5, Canon Zoom Lens 18-135
mm) mounted 190 cm above the tank on a rolling cart. We lit the tanks with two 500 watt
equivalent compact fluorescent light bulbs to produce 800 lumens at the surface of the water.
The camera was positioned on a tripod attached to a rolling cart above a given tank and the
camera was operated with a remote control. Each group was filmed for 30 seconds while at rest,
then a flash expansion was triggered in the group by an aerial stimulus (30 x 30 cm black plastic
flag passed swung by hand at 2 m/s from 1 m above them) until a clear flash expansion occurred.
Then we filmed the next three minutes and rolled the filming apparatus to the next tank. After
filming these, another batch of six groups was prepared and filmed on a given day. No beetle
was used more than twice overall and if it was used twice it was in a different group. Overall, 72
groups were filmed during two weeks (24 groups for each of the three treatments). The order in
which the six tanks were filmed in a set was stratified so that the three treatments were equally
likely to be first, second, or third.
Methods--Video Analysis:
The MOV format videos were transformed into AVI format using VirtualDub. Then
ImageJ [17] was used to calculate individual and emergent properties of the beetles/group.
Beetles more than ten body lengths from the other beetles were not included in the analysis. On
average, 22 of 24 beetles met the criteria for grouping, and no treatment bias was seen in those
that did not join the group.
The following parameters were measured on each video.
1. Collision rate: Collisions per frame (CPF) is the collision number divided by the number
of frames (30 frames/second). Collision number is the total number of times in which
any beetle made contact with another from the time that the flash expansion started to the
maximal extension of the group diameter (usually less than 2 seconds). A subset of eight
beetles per group (for 72 groups) was chosen randomly for this measure in a way similar
to an ecological line transect. Specifically, the center of the group was determined
visually, then the positive and negative X and Y axes was followed to each side of the
middle to determine 4 rays. The two beetles closest to each ray were then chosen. For
example, if a beetle was touching the ray it was one of those chosen.
2. Turn direction: Whether a beetle turned to its right or left when it was about to hit another
beetle. These were the same beetles analyzed for collision rate as described in #1 above.
3. NND: the average nearest neighbor distance of beetles before the flash expansion was
calculated for all 72 groups. 16 beetles (8 treatments and 8 controls) were selected
equally among the four quadrants of the group. Then ImageJ was used to measure the
head-to-head distance to the nearest beetle.
4. First Responder: The identity (treatment vs. control) and location (outside vs. inside of
the group) of the first beetle to expand, as part of the flash expansion, were identified.
For this measure, outside was qualitatively defined as being closest to the pool edge
along an axes going through the middle. There were approximately the same numbers of
outside and inside beetles in several representative groups examined.
5. Distance to the center (DTC). The DTC and identity of each beetle (control vs. treatment)
was calculated for all beetles at the frame prior to the start of the FE and at the frame at
which the maximum expanse of the FE had been reached for the first 16 groups for each
treatment (48 groups total). DTC was obtained by first obtaining the XY coordinates
using ImageJ [17], then calculating the distance of each beetle from the geometric center
of the group (See Romey [6] for details).
6. We measured the diameter of the 72 groups before and at the maximum expanse of the
FE using ImageJ. To do this we took an average of the horizontal and vertical diameter.
7. Time to Flash Expand: the number of frames it took a group to go from starting FE to
maximum expanse was taken. The filming rate was 30 frames per second.
Methods--Statistical Analysis:
We transformed the following non-normal dependent variables before statistical tests
were applied. DTC was square root transformed. NND was log(10) transformed. For the
GLMM models, group was included as a random variable. For the independent variables: time
(morning, noon, afternoon), days in the lab (three catagories-3,4,5), week (two categories), and
order of video (six categories) were initially included with interaction effects but some were
dropped from the model when it was found that they were not significant. It was found that the
date and order of video had a significant influence in many of the analyses and were kept in the
model with interaction terms. In the results section, we identify and report the dependent
variables without indicating the above transformations. Marginal means are reported.
Methods--Simulation
The Control simulation (Table S1) follows the Romey model [3, also see Romey 1996,
Ecol. Model. 92:65-77. doi:10.1016/0304-3800(95)00202-2] in which the formula for force
between particle i and j is equal to the unit vector (eij) multiplied by [(distance between i and j)a –
b) / (distance between i and j)c – d). The “default” attraction-repulsion (AR) parameters (a,b,c,d)
is set to 2, 4, 2.5, 4 (Figure S1). In order to reduce wall effects (particles reflecting off of
simulation boundary) and to make sure all members joined one large group, we used periodic
boundary conditions (the “wrap” option in Netlogo). The default settings for the control and
simulations, unless otherwise mentioned are: speed = 0.5 units per move, viewing angle = 360o
(180o to left and 180o to right), viewing distance = 100 units, 24 individuals with random starting
positions, with a random move [3] added every 20 moves. Movement parameters for the eight
simulated groups (Table S1) were chosen so that the group centroid would remain relatively
stationary [3]. Parameters were also chosen for the simulations so that: x-intercept ≈ 2,
maximum attraction ≈ +0.34, and maximum repulsion ≈ -1.0. Since the x-intercept is the point at
which there is neither attraction nor repulsion, the NND is related to this, so it is important to
keep it the same for individuals. For Ant-H1, we created an AR function with reduced repulsion
(Figure S1). For Ant-H2 we created a mixed group of 12 individuals following Ant-H1 rules
and 12 individuals following the default settings (to mimic the beetle experiments had half
deprived and half control beetles). For Ant-H3 we examined the effects of unilateral decrease in
vision and repulsion. We programmed the simulation to only respond (AR) to those on the right
hand side (180 degrees) of the object and ignore those on the left (similar to one of our unilateral
treatments of the whirligigs). This resembles the blind angles studied in Romey and Vidal [3]
but in the present study the blind angle is on one side, not behind. Ant-H4 was composed of a
mix of Ant-H3 and Control individuals to determine the effect of mixed groups on the emergent
group properties.
We produced three models simulating a decrease in vision. Eye-H1 was produced by
reducing the total distance at which they could see from 100 to four units. Pure groups of these
“myopic” individuals took long periods to coalesce, so only a mix with control beetles was
simulated, and we allowed them 3000 time units to stabilize rather than the standard 500. For
Eye-H2 we changed the AR function to half the overall attraction from 0.34 to 0.17, while
leaving the maximum repulsion and x-intercept the same (-1 and 2 respectively, Figure S4).
Eye-H2 was a pure group of 24 following the same rules, whereas Eye-H3 was a mixed group
composed of half C individuals.
For each of the eight types of groups, 500 time units were simulated (except Eye-H1 with
3000, giving them time to stabilize). One hundred simulations were run for each of the eight
group types and data were collected on the final frame. On the last frame the average NND and
the diameter of the group was calculated. NND was calculated to the closest nearest neighbor.
Diameter was calculated by finding the longest linear dimension across the convex polygon
surrounding each group [3]. Mean and S.E. for each of the eight kinds of groups were calculated
and an ANOVA and individual LSD-posthoc comparisons were made comparing each
simulation to the control.
Electronic Supplementary Materials
Supplementary Results and Discussion
Table S1: Description of the eight different kinds of simulations. See Figure S1 for the three
different AR functions and methods section for more details. The intercept is the distance at
which there is no attraction or repulsion.
Simulation
Name
Control (C)
Ant-H1
AR-function
Other differences
Description
Default AR: 2,4,2.5,4
Default settings:
intercept = 2
Viewing distance =100
attraction max = 0.34
Viewing angle=360o
repulsion max = -1
AR: 2, 4, 2.4, 16
intercept = 2
attraction max = 0.36
repulsion max = -0.3
pure group
Ant-H2
Same as Ant-H1
Mixed H1 and C
Ant-H3
Default AR
View angle = 180o
on right only
Ant-H4
Default AR
Eye-H1
Default AR
Eye-H2
AR: 1.5, 2.8, 2.4,2.8
intercept = 2.0
attraction max = 0.17
repulsion max = -1
Pure
Eye-H3
Same as Eye-H2
Mixed Eye-H2 and
C
Mixed H3 and C
Viewing distance
=4
(and half C)
Bilateral decrease in
repulsion
Truncated attraction
Reduced maximum
attraction
0.6
Force of Attraction (+)
Repulsion (-)
0.4
0.2
0
-0.2
0
5
10
15
Control
-0.4
Ant-H1
-0.6
Eye-H2
-0.8
-1
-1.2
Distance between simulated organisms
Figure S1. Attraction-Repulsion forces for select hypotheses in Table S1 simulations. Each of
these functions is defined by the parameters a-d [3, and Romey 1996, Ecol. Model.]. The
default x-intercept is 2.0, default repulsion maximum is -1.0 (when two individuals are almost
touching), and default attraction maximum is 0.36.
100%
80%
60%
40%
20%
0%
Right -
Left ANT-
Right -
Left EYE-
Figure S2: Percentage of beetles with right or left unilateral sensory deprivation turning left or
right prior to colliding during flash expansion. (N = 74 ANT- beetles, N = 66 EYE- beetles).
Gray bars are those turning right, whereas solid bars are those that turned left. For example,
“ANT-, Right-” are beetles in which the right antenna was removed.
2.20
2.10
NND
2.00
1.90
1.80
1.70
1.60
Simulation
Figure S3: Mean NND of group for simulations. Standard Error bars are not shown because
they are minimal (approximately 0.026). The black bar is the control simulation, striped bars are
the results simulating antenna deprivation and gray bars are the results simulating sight
deprivation. See Table S1 for the key to these labels. Bars with asterisks are significantly
different (p < 0.05, t-test) from the control bar in black.
Frequency
25
20
15
10
5
0
Control
Ant -
Eye -
Figure S4: Frequency of first beetle to respond to an aerial predator stimulus in relation to
position in three types of group. Stippled bars on the left represent beetles on the inside of the
group while gray bars on the right represent those on the outside of the group (N = 24 groups for
each treatment).
Results and Discussion for Outside-Inside Positions
Whirligig Results for Group Position
The first beetle to respond to an aerial stimulus was an outside beetle 90% of the time,
regardless of the treatment type (electronic supplementary material, figure S4). There was no
significant difference between the three treatment types in whether the first beetle to respond was
on the inside or outside of the group (X2 = 1.62, p = 0.44). In the ANT ̶ groups, ANT ̶ beetles
were significantly less likely to respond first than control beetles within that group (6 vs. 17
times, X2 = 5.26, p = 0.022). However, in the EYE ̶ groups, there was no significant difference
in whether the EYE ̶ beetles or control beetles responded first (16 vs. 8, X2 = 2.67, p = 0.102).
Discussion for Group Position
Typically, there are asymmetric fitness costs and benefits in animal groups. For example,
in many stationary groups, the centre is safer from predators whereas the outside is better for
foraging [6]. Individual differences in state determine preferred positions in some cases: with
hungry individuals going to the outside whereas risk prone individuals prefer the inside [6].
Therefore, it is surprising that in our experiment, sensory deprived individuals (both ANT ̶ and
EYE ̶ whirligigs), who would be more at risk to predators, were more likely to be on the outside
of groups. Alternative explanations for this include: 1) healing beetles did not eat as much
during the days post-surgery and move to the outside of a group in search of food, 2) choosing
outside positions allows them to detect predators earlier, 3) the marking procedure led them to be
on the outside, or 4) they cannot assess what an inside or outside position is and are displaced by
other beetles seeking the center. The first explanation could be tested by including another
control group in the experiment that had a sham operation such as a wing removal. There is
partial support for the second explanation, in that the outside individuals were the first to respond
to the fright stimulus, regardless of the treatment. Hypothetically, in our experiment, all
organisms could see the aerial stimulus at the same time, just that the ones on the outside could
move away from the group first. Edge positions in whirligig groups are attacked more often by
fish than center positions (Romey, Walston, and Watt, 2007, Behav. Ecol. 19:74-78.
doi:10.1093/beheco/arm105). Further studies would be helpful in understanding the influence of
different combinations of dorsal and ventral eye obstructions on emergent group responses. The
third explanation for position preference cannot be disproven by our experiment since we did not
mark control beetles. Future studies with various types of controls (marked/unmarked and with
sham operations) would help to clarify the data on positioning within the group.