1
Methods
Study species. The four study colonies of Monomorium pharaonis (L.) each had
approximately 1500 workers, 15-30 queens and brood of all stages. M. pharaonis is
unicolonial and showed no nestmate discrimination1 among our colonies. Colonies were
housed in wooden nest boxes (13 x 8 x 2 cm) within plastic foraging arenas (56 x 36 x
11 cm). The experimental room was maintained at 24±2˚, LD 12:12h. Colonies were
given water ad libitum, mealworm larvae (Tenebrio molitor) daily and dried liver
monthly. Sugar syrup was withheld for a week prior to experiments. Colonies received
1M sucrose during trials which took place on alternate days.
Experiment 1. Branch choice at trial bifurcations. Two colonies were used per trial:
a trail-laying colony (A) and a test colony (B). We placed a Y-bifurcation apparatus into
the foraging arena of each colony 24 hours prior to the trial (apparatus as in reference
8). The upper surface of each apparatus was covered with Eucalyptus Chlorine-free
(ECF) paper as trail substrate and a control piece of ECF paper was placed in the
foraging arena of colony A, away from foraging trails to water and food but where the
ants could still mark it with territorial pheromones2.
We began each trial by placing 1M sucrose feeders3 at the end of one branch (alternated
between trials) in colony A and both branches in colony B. Foraging took place for 20
minutes. The feeders were removed and a 10mm x 20mm section of paper was cut from
the unrewarding side, 3mm after the bifurcation (Fig. S1a). This section and the control
section were transferred to the bifurcation region of the trail apparatus in the test colony,
B (Fig. S1b). Ants choosing one branch had to walk over a 20mm piece of paper
substrate from the unrewarding branch of the foraging trail system. To choose the other
branch, ants had to walk over a 20mm piece of control paper. The control was marked
with the ants’ territorial pheromones, but not trail pheromones attracting to a current
food source. We did not use fresh paper as a neutral control because Pharaoh’s ants
actively recruit to new territory.2 Each ant was brushed from the apparatus after it
passed the bifurcation to prevent further pheromone deposition4. After the first 25 ants
had reached the bifurcation, the locations of the two paper sections were swapped and a
further 25 ants were observed. This swap eliminated the possibility that ants were
following pheromones laid by the previous ants or visual cues. A trial lasted 8-10
minutes. Two pairs of colonies were used, alternating between days, yielding four trials.
Ant choice was determined from video recordings. High traffic flow has been shown to
increase the tendency of some ants to search for a new route to food5. To ensure that
variation in traffic flow between colonies was not affecting results, ant traffic was
counted during the last minute of foraging for each trial. There was no significant
difference in ant flow rate among trail-laying colonies (Mann-Whitney Test: w=5.0,
n=4, NS) or test colonies (Mann-Whitney Test: w=7.0, n=4, NS).
a, Number of ants that U-turned while walking on different test sections, relative to
controls. Chi-squared tests: n=200, d.f.=1; S: 2=21.7, P<0.001; Fb: 2=5.9, P<0.05; Nb:
2=31.2, P<0.001; Fe: 2=20.2, P<0.001; Ne: 2=0.04, NS. b, Percentage of straightwalking (left bars) or zigzagging (right bars) ants, relative to controls. Chi-squared tests:
d.f.=1; S: 2=8.4, P<0.01; Fb: 2=5.5, P<0.05; Nb: 2=9.8, P<0.01; Fe: 2=3.9, P<0.05;
2
Ne: 2=6.3, P<0.05.Experiment 2. U-turning and zigzagging on unbranched trails.
This experiment was performed as for Experiment 1, except that the test colony (A) was
also the trail-laying colony and contained the control section. The second colony (B)
was used only to prime an unbranched apparatus with a foraging trail. This unbranched
apparatus was placed in colony B’s foraging arena 24 hours prior to a trial, and was
covered in ECF paper. Feeders were placed at the end of the unbranched apparatus and
the end of one branch (alternated between trials) of the bifurcating apparatus in colony
A. After foraging, the feeders were removed and five substrate sections cut from paper
on the bifurcating apparatus: S, 1mm prior to bifurcation; Fb and Nb, 3mm after
bifurcation on feeder and non-feeder branches; Fe and Ne, 60mm from bifurcation at
ends of feeder and non-feeder branches (Fig. S2). The unbranched foraging apparatus
was transferred to colony A, and sections were each tested on it. The order in which
sections were tested was randomised. Each section was tested with 50 ants. To avoid
effects of trail decay, the number of trail sections used per trial was limited to three: Fb
and Nb were tested for four trials; Fe, Ne and S for four, each set with a control. Two
pairs of colonies were used, alternating between days. All trials were video recorded. Uturns were counted and the straightness of each ant’s path was quantified by counting
the number of edges of the unbranched apparatus. We defined an ant as zigzagging if it
contacted both sides of the 10mm wide trail apparatus at least once during the last
30mm (15 body lengths) of its approach to a test substrate section. We defined an ant as
walking straight if it did not contact the sides of the apparatus at all during this 30mm.
To eliminate any possible effects on path straightness of encountering another ant, ants
which contacted returning U-turners were excluded from the zigzag study (Fig. 1a).
Ants were brushed off the apparatus once they had walked over the trail section. There
was no significant difference in ant flow rate during section testing between test
colonies (Mann-Whitney Test: w=47, n=28, NS).
1.
Hölldobler, B. & Wilson, E. O. The Ants (The Belknap Press of Harvard University Press,
Cambridge, MA, 1990).
2.
Fourcassié, V. & Deneubourg, J. L. The dynamics of collective exploration and trailformation in Monomorium pharaonis - experiments and model. Physiological Entomology 19, 291300 (1994).
3.
Jackson, D. E., Holcombe, M. & Ratnieks, F. L. W. Knowing which way to go - trail
geometry gives polarity to ant foraging trails. Nature 432, 907-909 (2004).
4.
Jeanson, R., Deneubourg, J. L. & Ratnieks, F. L. W. Pheromone trail decay rates on
different substrates in the Pharaoh's ant, Monomorium pharaonis. Physiological Entomology 28,
192-198 (2003).
5.
Dussutour, A., Fourcassié, V., Helbing, D. & Deneubourg, J. L. Optimal traffic
organization in ants under crowded conditions. Nature 428, 70-73 (2004).
3
Supplementary Table 1. U-turning ants
Test
section
(see Fig 2a)
U-turns
Control
Difference
S
25/200
54/200
- 29
Fb
24/200
37/200
- 13
Fe
26/200
54/200
- 28
Nb
68/200
37/200
+ 31
Ne
53/200
54/200
-1
Data for Experiment 2: U-turning. Numbers of U-turns on control substrate varied
between trials so expected values for each test section were calculated from the control
values of trials including that section (see methods). Mean proportion of ants which Uturned on control substrate: 22%. Chi-squared tests: n=200, d.f.=1; S: 2=21.7,
P<0.001; Fb: 2=5.9, P<0.05; Nb: 2=31.2, P<0.001; Fe: 2=20.2, P<0.001; Ne: 2=0.04,
NS.
Supplementary Table 2. Zigzagging ants
Test
section
(see Fig 2a)
Zigzagging
ants
Control
Difference
(%)
S
5/199
12/190
-8
Fb
12/152
25/170
- 14
Fe
6/192
12/190
-6
Nb
40/200
25/170
+ 11
Ne
18/157
12/190
+ 10
Data for Experiment 2: Zigzagging. Mean proportion approaching control that were
zigzagging ants: 10%. The 28% of ants that could not be allocated to either the
zigzagging or straight walking category (see methods) were excluded from the analysis.
To eliminate the possible effects on path straightness of encountering another ant, ants
that contacted returning U-turners were excluded from the zigzag study, giving variable
4
values of n. To correct for variable sample size, differences in percentages rather than
numbers of ants are given here and used in Fig. 2c. Chi-squared tests: see below.
Supplementary Table 3. Straight walking ants
Test
section
(see Fig 2a)
Straight
walking
ants
Control
Difference
(%)
S
160/199
122/190
+ 32
Fb
72/152
71/170
+ 11
Fe
134/192
122/190
+ 11
Nb
61/200
71/170
- 23
Ne
90/157
122/190
- 14
Data for Experiment 2: Straight walking ants. Mean proportion approaching control
which were straight walking ants: 62%.
Chi-squared tests for zig-zagging and straight walking ants: d.f.=1; S: 2=8.4, P<0.01;
Fb: 2=5.5, P<0.05; Nb: 2=9.8, P<0.01; Fe: 2=3.9, P<0.05; Ne: 2=6.3, P<0.05.
5
Supplemental figure S1
a) Trail sections
b) Test positions
Colony A
10mm
of sections
Colony B
feeder
80m
m
Nb
Nb
10mm
10mm
90mm
20mm
10mm
from nest
from nest
Figure S1. Experiment 1. Branch choice at trial bifurcations.
Experimental setup of trail bifurcation apparatus for trail laying
and substrate section transfer for choice test.
6