BRIEF COMMUNICATIONS NATURE|Vol 438|24 November 2005
442
*Biophysics Program, University of California,
San Francisco, California 94143, USA
e-mail: cavoigt@picasso.ucsf.edu
Center for Systems and Synthetic Biology
and Institute for Cell and Molecular
Biology, and Department of Chemistry and
Biochemistry, University of Texas, Austin,
Texas 78712, USA
§Department of Synthetic Biology, Lawrence
Berkeley National Laboratory, Berkeley,
California 94720, USA
||Department of Pharmaceutical Chemistry,
University of California, San Francisco,
California 94107, USA
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Supplementary information accompanies this
communication on Nature's website.
Competing financial interests: declared none.
doi:10.1038/nature04405
used by Pharaoh's ants in trail choice, or they
could prevent strong positive feedback by
attractive pheromones from locking the sys-
tem into suboptimal solutions1,8
.
Elva J. H. Robinson*, Duncan E. Jackson,
Mike Holcombe, Francis L. W. Ratnieks*
*Laboratory of Apiculture and Social Insects,
Department of Animal and Plant Sciences,
University of Sheffield, Sheffield S10 2TN, UK
e-mail: e.robinson@dcs.sheffield.ac.uk
Department of Computer Science, University of
Sheffield, Sheffield S1 4DP, UK
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(2003).
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InsectesSoc.37,258­267(1990).
Supplementary information accompanies this
communication on Nature's website.
Competing financial interests: declared none.
doi:10.1038/438442a
BRIEF COMMUNICATIONS ARISING online
 www.nature.com/bca see Nature contents.
Forager ants lay attractive trail pheromones to
guide nestmates to food1,2
, but the effective-
ness of foraging networks might be improved
if pheromones could also be used to repel
foragers from unrewarding routes3,4
. Here we
present empirical evidence for such a negative
trail pheromone, deployed by Pharaoh's ants
(Monomorium pharaonis) as a `no entry' signal
to mark an unrewarding foraging path. This
finding constitutes another example of the
sophisticated control mechanisms used in self-
organized ant colonies.
To investigate whether foragers lay a nega-
tive signal on the unrewarding branch of a trail
bifurcation, we removed paper substrate from
immediately after the fork on the unrewarding
branch (the other branch led to a sucrose
feeder) after it had been used by a trail-laying
colony of ants. This paper substrate was trans-
ferred to the entrance of one branch of a
similar set-up, in which both branches had
previously led to sucrose and had been used by
a second colony of ants. The other branch of
the second set-up received a neutral control
paper substrate (for details, see supplementary
information). Foragers walking from the nest
could choose either of the test branches or
make a U-turn.
We found that 69% continued to walk away
from the nest and make a branch choice. Of
these, most (71%) chose the branch with the
control substrate ( 2
=22.1, d.f.=1, n=137,
P<0.001); the remainder U-turned towards
the nest on reaching the trail bifurcation. U-
turns were more than four times as likely if the
ant had contacted the unrewarding-branch
substrate (55%) as opposed to the neutral-con-
trol substrate (13%) ( 2
=40.9, d.f.=1, n=200,
P<0.0001). Neither substrate came from a
previously rewarding trail, so this result can-
not be attributed to differences in positive-trail
pheromone concentrations.
We next investigated the negative signal's
location by taking substrate from five locations
on a bifurcating trail that had one rewarding
and one unrewarding branch. These sections,
along with neutral controls, were tested on
unbranched foraging trails (see supplemen-
tary information) by noting whether individ-
ual foragers walking over them did a U-turn.
Compared with ants on the control substrate,
almost twice as many ants U-turned when
walking on substrate from the unrewarding
branch near the bifurcation (Nb) (19% and
34%, respectively; P<0.001) (Fig. 1a). How-
ever, U-turns were as frequent on substrate
from the unrewarding branch end (Ne) (27%)
as on the control (27%) (NS) (Fig. 1a). Ants U-
turned less often on sections from the reward-
ing trail (stem S, 12%; feeder branch close to
the bifurcation Fb, 12%; and feeder-branch end
Fe, 13%). These values are significantly lower
than those for the relevant control (S,
P<0.001; Fb, P<0.05; Fe, P<0.001) (Fig. 1a).
In the same experiment, we also determined
whether foragers could detect the negative
signal before reaching the substrate on which
it had been laid, using walking behaviour
(zigzagging versus walking straight) as a bio-
assay. Our results show that significantly more
ants zigzagged when approaching substrate
from an unrewarding branch just after the
bifurcation (P<0.01) or at the branch end
(P<0.05) than did controls (Fig. 1b). Con-
versely, significantly fewer zigzagged when
approaching substrate leading to the feeder (S,
P<0.01; Fb, P<0.05; Fe, P<0.05) (Fig. 1b).
Our results show that Pharaoh's ants use a
sophisticated trail system with a negative,
repellent pheromone to mark unrewarding
branches. The signal is concentrated at deci-
sion points -- trail bifurcations5
. As it is
volatile, it provides advance warning -- like
human road signs situated before junctions.
Across a trail network, the pheromone could
help direct foragers to food by closing off
unrewarding sections. Exactly how negative
pheromones enhance foraging efficiency in
trail networks is not known, but they might
complement attractive trail pheromones6,7
­20
­10
­20
­30
­10
0
10
20
0 S
S Fb
Fe
Nb
Ne
P<0.01
P<0.001
P<0.05
P<0.001
P<0.001
(NS)
Fb
P<0.05
Fe
P<0.05
Nb
P<0.01
Ne
P<0.05
Percentagezigzagorstraight-
walkingantsrelativetocontrols
NumberofantsU-turning
relativetocontrols
10
30
20
40
a
b
­15
­5
5
15
Figure 1 | Identifying the location of the negative
pheromone. The ants' response is monitored by
their walking behaviour, with U-turning or
zigzagging on unbranched trails indicating
detection. Test sections: S, 1 mm before
bifurcation; Fb and Nb, 3 mm after bifurcation
on feeder and non-feeder branches, respectively;
Fe and Ne, 60 mm from bifurcation at the ends
of feeder and non-feeder branches, respectively.
(For details and chi-squared tests, see
supplementary information.) a, Number of ants
that U-turned while walking on different test
sections, relative to controls. b, Percentage of
straight-walking (left bars) or zigzagging (right
bars) ants, relative to controls.
INSECT COMMUNICATION
`No entry' signal in ant foraging
NaturePublishing Group2005