A comparative analysis of precision rescue behaviour in sand-dwelling ants

Abstract

A sand-dwelling Mediterranean ant, Cataglyphis cursor, recently was discovered to engage in two new forms of rescue behaviour, behavioural patterns that require would-be rescuers to recognize what, exactly, holds nestmates in place. That is, when sand digging and limb pulling, two well-known forms of rescue in ants, did not result in release of victims ensnared with nylon thread and partially buried beneath the sand, rescuers next began to transport sand away from the snare and to direct their behaviour to the snare in particular, biting and tugging at the snare itself. To determine whether these new forms of precisely directed rescue behaviour, as well as their exclusive delivery to nestmates, as in C. cursor, were characteristic of other ants occupying similar ecological niches, we conducted experiments with five sand-dwelling Mediterranean ant species: Cataglyphis floricola, Lasius grandis, Aphaenogaster senilis, Messor barbarus and Messor marocanus. Our experiments revealed the full range of rescue behaviour, including snare biting and sand transport, in two species, C. floricola and L. grandis. Both species directed rescue exclusively towards nestmates, treating other individuals, even conspecifics, with aggression, thus highlighting the ants' discriminative capacities. Differences in the performance of rescue behaviour between these and the remaining species mirror differences in their ecology, including the threat of predatory antlions. Finally, because this precisely directed and exclusively delivered rescue behaviour in ants resembles behaviour that has been labelled empathy in rats, our results demonstrate that what can appear to be complex, cognitively motivated behaviour might come about through much simpler mechanisms.

Full text

A comparative analysis of precision rescue behaviour in sand-dwelling
ants
Karen L. Hollis
a
,
*
, Elise Nowbahari
b
,
1
a
Interdisciplinary Program in Neuroscience & Behavior, Mount Holyoke College, MA, U.S.A.
b
Laboratoire d'Éthologie Expérimentale et Comparée, Université Paris 13, Sorbonne Paris Cité, France
article info
Article history:
Received 4 June 2012
Initial acceptance 23 July 2012
Final acceptance 28 November 2012
Available online 12 January 2013
MS. number: A12-00426R
Keywords:
altruistic behaviour
ant
Aphaenogaster senilis
Cataglyphis floricola
empathy
eusocial insect
Lasius grandis
Messor barbarus
Messor marocanus
rescue behaviour
A sand-dwelling Mediterranean ant, Cataglyphis cursor, recently was discovered to engage in two new
forms of rescue behaviour, behavioural patterns that require would-be rescuers to recognize what,
exactly, holds nestmates in place. That is, when sand digging and limb pulling, two well-known forms of
rescue in ants, did not result in release of victims ensnared with nylon thread and partially buried
beneath the sand, rescuers next began to transport sand away from the snare and to direct their
behaviour to the snare in particular, biting and tugging at the snare itself. To determine whether these
new forms of precisely directed rescue behaviour, as well as their exclusive delivery to nestmates, as in
C. cursor, were characteristic of other ants occupying similar ecological niches, we conducted experi-
ments with five sand-dwelling Mediterranean ant species: Cataglyphis floricola,Lasius grandis,Aphae-
nogaster senilis,Messor barbarus and Messor marocanus. Our experiments revealed the full range of rescue
behaviour, including snare biting and sand transport, in two species, C. floricola and L. grandis. Both
species directed rescue exclusively towards nestmates, treating other individuals, even conspecifics, with
aggression, thus highlighting the ants’discriminative capacities. Differences in the performance of rescue
behaviour between these and the remaining species mirror differences in their ecology, including the
threat of predatory antlions. Finally, because this precisely directed and exclusively delivered rescue
behaviour in ants resembles behaviour that has been labelled empathy in rats, our results demonstrate
that what can appear to be complex, cognitively motivated behaviour might come about through much
simpler mechanisms.
Ó2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Recently, Nowbahari et al. (2009) demonstrated that Cataglyphis
cursor, a common sand-dwelling Mediterranean ant, is capable of
performing forms of rescue behaviour that had not been previously
reported, despite a literature that dates as far back as Belt’s(1874)
monograph on ant behaviour. That is, when C. cursor ants were
presented with experimentally ensnared nestmates held in place
with nylon thread, ants not only engaged in sand digging and limb
pulling, both familiar forms of rescue behaviour in ants (e.g. Lafleur
1940;Wilson 1958;Markl 1965;Blum & Warter 1966;Spangler
1968;Hangartner 1969), but also somehow were able to detect
what, exactly, held victims in place: when digging and pulling did
not result in the victim’s release, ant rescuers next transported
excavated sand away from the victim’s body, exposed the nylon
thread, and then immediately bit and tugged at the snare directly.
Carefully aimed, snare biting never was misplaced, even though the
snare was in direct contact with the victim’s body.
Nowbahari et al. (2009) argued that sand transporting and,
especially, snare biting might require slightly more complicated
mechanisms than simple sand digging and limb pulling. That is,
digging and pulling require only that ants are alerted by a nest-
mate’s alarm signal, follow the sensory gradient to the source, and
then commence digging and pulling once they make contact with
the victim, a series of behaviour patterns easily explained by simple
releasing mechanisms. However, it is difficult to see how these
same simple mechanisms could guide rescuers to the precise
location of whatever object is holding the victim in place while
ignoring other ‘non-ant’objects nearby, enable them to expose it
further by transporting sand away from this object, and then finally
target their bites to the object itself, transporting additional sand as
necessary. In addition, because C. cursor directed this rescue
behaviour only towards nestmates, never even approaching ants
belonging to nearby colonies of the same species, the ‘call-for-help’
signal, in this species at least, necessarily contains a component
unique to each colony. Such precisely directed and exclusively
*Correspondence: K. L. Hollis, Interdisciplinary Program in Neuroscience &
Behavior, Mount Holyoke College, 50 College Street, South Hadley, MA 01075-1462,
U.S.A.
E-mail address: khollis@mtholyoke.edu (K. L. Hollis).
1
E-mail address:Elise.Nowbahari@leec.univ-paris13.fr (E. Nowbahari).
Contents lists available at SciVerse ScienceDirect
Animal Behaviour
journal homepage: www.elsevier.com/locate/anbehav
0003-3472/$38.00 Ó2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.anbehav.2012.12.005
Animal Behaviour 85 (2013) 537e544

targeted rescue behaviour would be expected to evolve only when
members of a particular species faced risks of possible entrapment,
and the benefits of releasing conspecifics exceeded what appear to
be particularly high costs of engaging in rescue behaviour.
Although the risks of entrapment would not be limited to sand-
dwelling ants like C. cursor, sand-dwelling species are likely
candidates for several reasons. The experiments with C. cursor were
prompted by field observations in which forager ants often
attempted to rescue nestmates that had been partially buried under
collapsing sand and debris (Nowbahari et al. 2009). Yet another
potential opportunity for rescue occurs when ants fall into the traps
of pit-digging larval antlions (Neuroptera: Myrmeleontidae).
Antlions’pit traps are found in some of the same habitats as sand-
dwelling ants and, although antlions prey upon many different
arthropods, ants are one of the most common prey items (Griffiths
1980;Lucas & Brockmann 1981;Botz et al. 2003;Mencinger-Vra
cko
& Devetak 2008). Antlions’pit traps, dug in loose sandy soils
(Devetak et al. 2005), are constructed in such a way to funnel prey
to the centre of the pit, where the predator is waiting; however,
prey sometimes are able to cling to the sides of pits for short
periods of time, despite attempts by antlions to dislodge their prey
by sand tossing (Hollis & Guillette 2011;Hollis et al. 2011). Thus,
rescue behaviour also may be an important antipredator defence.
Indeed, in several field experiments with Formica workers reared as
slaves, Czechowski et al. (2002) demonstrated that enslaved ants
immediately rushed to the aid of heterospecific nestmates captured
by antlions and began to engage in sand-digging and limb-pulling
behaviour.
Despite its obvious benefits, rescue behaviour entails costs that,
for some ant species, may be too high to pay. Rescue behaviour not
only is an energetically costly behaviour that prevents individuals
from devoting time to other important tasks, but also poses large
risks to rescuers (Nowbahari & Hollis 2010). For example, rescuers
themselves may become buried under sand as they attempt to aid
an entrapped nestmate. In addition, because some predators,
including antlions, are capable of seizing multiple prey items in
rapid succession, rescuers risk capture. Because of its costs, then,
we would predict that not all ant species are able to engage in
rescue behaviour. Thus, one goal of this study was to determine
how common the phenomenon of precisely directed rescue
behaviour is in ants, as well as to establish whether, like C. cursor,
foragers rescue only nestmates.
A second, related goal was to determine what might be some of
the ecological variables that promote rescue in ants. To address
both objectives, we conducted a comparative analysis of rescue
behaviour in five species of sand-dwelling Mediterranean ants
representing two subfamilies: Lasius grandis and Cataglyphis flo-
ricola (bicolour morph) from the subfamily Formicinae; and,
Aphaenogaster senilis,Messor barbarus and Messor marocanus from
the subfamily Myrmicinae. Because C. floricola not only inhabits the
same loose sandy soils as C. cursor, but also lives in close proximity
to pit-digging antlions, we expected that C. floricola would be likely
to show the same kinds of rescue behaviour as its close relative,
C. cursor. At the other end of the spectrum, Messor species inhabit
more compact soils than do Cataglyphis, and thus are probably less
likely to become trapped by collapsing sand. In addition, Messor
foragers form ant trails, long lines of individuals following marked
trails leading to food (López et al. 1993;Hölldobler & Wilson 2009),
making accidental encounters with antlion pits, or any other
dangerous situations, far less likely.
Yet another goal of this study was to shed light on the
phenomenon of rescue behaviour more generally. Although anec-
dotes of rescue in nonhuman animals abound in the popular media,
the scientific literature contains only two experimental analyses of
rescue behaviour, one in ants (Nowbahari et al. 2009) and one in
rats (Bartal et al. 2011). Interestingly, the report of rescue behaviour
in rats describes behaviour analogous to what Nowbahari et al.
(2009) observed in ants. Although the authors themselves do not
use the word ‘rescue’to describe rats’behaviour, preferring the
term ‘empathy’instead, our research with ants raises questions
about the necessary and sufficient conditions for interpreting
behaviour as empathic. That is, if ants respond to conspecifics in
ways that appear nearly identical to those of a mammal, then future
experiments exploring empathy in animals will need to include
control groups that help scientists to distinguish between analo-
gous behaviour involving different mechanisms (Vasconcelos et al.
2012).
METHODS
Subjects and Materials
We observed the behaviour of five sand-dwelling Mediterra-
nean ant species in the field between April 2010 and June 2011 in
response to four kinds of test stimuli, namely a homocolonial
nestmate, a heterocolonial ant, or a heterospecific ant held in place
using a filter paper and thread snare, or an empty paper-and-thread
snare in a control test. Only colonies that showed high activity
levels and large numbers of foragers presenton the surface near the
nest entrance were chosen for study. All data were collected during
ants’active period, either in the morning, at least 1 h after foragers
had emerged from the nest but before ants retreated to the nest at
midday, or later in the afternoon, at least 1 h after foragers had re-
emerged but before ants retreated to the nest near the end of the
day. We observed one of the ant species, M. barbarus, in Argelès-
sur-mer (Languedoc-Roussillon) with permission from Institut
National des Sciences de l’Univers; we observed all four of the
remaining ant species, L. grandis,C. floricola,A. senilis, and
M. marocanus, in the Doñana Biological Reserve (Reserva Biológica
de Doñana, RBD) in Southwestern Spain. All fieldwork in Doñana
was formally approved by the ICTS-RBD program. Specimens of
each species were returned to the laboratory to ensure correct
identification (Bernard 1968;Tinaut 1991).
Filter paper snares were prepared in advance of all trials: using
a sewing needle, a small loop (0.5e0.75 cm, depending on the test
species) of two-ply cotton thread was inserted near one end of
a strip (2 cm long 1 cm wide) of clean filter paper; the ends of the
thread were tied loosely underneath the paper and left hanging
until needed in the field. Prepared snares were kept in a clean
plastic box to avoid contamination and a clean snare was used for
each test.
Procedure
The testing procedures were similar to Nowbahari et al. (2009)
but adapted to field conditions where noted below. For homo-
colonial (nestmate) tests, the ant victim was chosen from amongst
the foragers near the nest entrance, grasped with insect handling
tweezers, gently inserted under the thread loop on the filter paper
such that the thread encircled the victim’s pedicel (waist), and then
secured to the filter paper by pulling on the ends of the thread
underneath the paper. A single knot held the victim in place. Next,
the filter paper was trimmed to a length of approximately 1.0e
1.5 cm (depending on species size) and the ends of the thread
were clipped close to the knot such that no thread extended beyond
the edges of the paper. For heterocolonial tests, the victim was
chosen from a nearby colonyof the same species, at least 30 m from
the test colony, and tied to the filter paper as described above.
Heterospecific tests were conducted in the same manner as het-
erocolonial tests, except that ant victims were chosen from colonies
K. L. Hollis, E. Nowbahari / Animal Behaviour 85 (2013) 537e544538

representing different species whose nests were typically between
30 and 50 m from the test colony. In this way, the ant victim would
represent a species with which the test species could have had
contact, at least potentially. For A. senilis, heterospecific tests were
conducted with both M. marocanus and L. grandis (a larger and
a smaller ant species, respectively). For C. floricola, heterospecific
tests were conducted with L. grandis,A. senilis and M. marocanus
(one smaller and two larger ant species, respectively). For L. grandis,
heterospecific tests were conducted with A. senilis (a larger
species). And, finally, both Messor species were tested with a single
species during heterospecific trials; M. marocanus was tested with
snared A. senilis ants, and M. barbarus was tested with snared
C. cursor ants. All control tests were conducted with a clean filter
paper containing a thread snare as described above; just prior to
the test, the paper and thread were trimmed and clipped exactly as
was done for trials with ant victims.
To conduct a test, the filter paper, either containing a victim or
left empty, was placed within 7e10 cm of the nest entrance and
covered with a thin layer of sand, such that the head and thorax of
each victim, or the empty loop, but not the filter paper, was visible.
Unlike the procedure described in Nowbahari et al. (2009), no arena
was used to confine the victim and potential rescuers; instead, the
5 min test began when at least one ant from the test colony either
contacted an ant victim, or walked within one body length of the
empty snare. Following the 5 min test, nestmate victims were
sequestered in a plastic box until any further trials on that day had
been conducted. Heterocolonial and heterospecific victims that did
not receive aggressive treatment, and thus were mostly uninjured,
were returned to their respective colonies following the test;
otherwise, they were destroyed. Used filter paper snares were
placed in a separate plastic box for experimentally contaminated
materials and disposed of at the end of each day.
A stopwatch wasused to time each 5 min trial and all behavioural
recording was done using an interval recording procedure, which is
especially useful when behaviour may be performed at low and
medium rates (Martin & Bateson 1993). Each 5 min trial was divided
into 10 s intervals, and target behaviours were recorded for each
interval. For each target behaviour (see Table 1), the data were the
proportion of intervals in which that behaviour pattern was
observed. To ensure reliability, we collected multiple samples of each
test condition from atleast three different colonies of each species.
Statistical Analysis
Each of the behaviour patterns was subjected to a univariate
ANOVA with two between-subjects factors, namely species and test
type. In addition, because individual target behaviours cannot
capture overall levels of rescue or aggression, we computed
a‘combined’rescue score and a ‘combined’aggression score. For
the combined rescue score, we computed the proportion of inter-
vals in which at least one of the four rescue behaviours occurred
(digging, pulling, snare biting or sand transport). For the combined
aggression score, we computed the proportion of intervals inwhich
at least one of the three aggressive behaviours occurred (aggressive
biting, dismembering or aggressive projecting). These combined
scores also were subject to ANOVAs. All ANOVAs were computed
using SPSS v.19 (SPSS Inc., Chicago, IL, U.S.A.). Significant main
effects and interactions were explored with post hoc tests, namely
paired comparisons using Bonferroni adjustments for multiple
comparisons; all reported Pvalues represent two-tailed tests.
Finally, we repeated our data analyses using nonparametric
statistics, that is, KruskaleWallis tests to evaluate main effects,
followed by ManneWhitney Utests to explore pairwise compari-
sons, and in all cases obtained exactly the same patterns of results
as are reported below with parametric analyses.
RESULTS
Rescue Behaviour
Combined rescue score
The five ant species differed significantly in their rescue
response to the test stimuli (Fig. 1). An ANOVA revealed significant
main effects of species (F
4,285
¼44.12, P<0.0001) and test type
(F
3,285
¼188.07, P<0.0001), as well as a significant interaction
between species and test type (F
12,285
¼43.33, P<0.0001). Explo-
ration of that interaction, using Bonferroni post hoc tests adjusted
for multiple comparisons, revealed that in the presence of an
ensnared nestmate, C. floricola and L. grandis engaged in very high
levels of rescue behaviour that were nearly identical to one another
(P¼1.000), and which differed significantly from the three
remaining species (P<0.0001), all of which engaged in extremely
low levels of rescue (between 0.01 and 0.16 of the 5 min test).
Table 1
Operational definitions of target behaviour patterns in sand-dwelling ants
Behaviour Operational definition
Sand digging Ant positions itself within 2 cm of, and facing, the test stimulus, either the ensnared ant victim or the paper-and-snare control stimulus, and flicks
sand backward, away from the test stimulus, using its anterior legs; the gaster (abdomen) is not flexed, and the antennae are facing forward, in the
characteristic nonaggressive posture
Limb pulling Ant grabs limb of the ensnared ant victim with its mandibles and drags the limb backwards with frequent antennation; the gaster (abdomen) is not
flexed, and the antennae are facing forward, in the characteristic nonaggressive posture
Snare biting Ant bites and tugs at the thread snare using its mandibles; the gaster (abdomen) is not flexed, and the antennae are facing forward, in the
characteristic nonaggressive posture
Sand transport Ant picks up one or more sand particles, covering the filter paper or ensnared ant victim, with its mandibles and moves the sand at least one
body length from its original position; the gaster (abdomen) is not flexed, and the antennae are facing forward, in the characteristic nonaggressive
posture
Paper tugging Ant bites and tugs at the filter paper using its mandibles, moving the paper away from the nest; the gaster (abdomen) is not flexed, and the antennae
are facing forward, in the characteristic nonaggressive posture
Biting Ant bites the limbs, antennae or other body parts of the ensnared ant victim with its mandibles; the gaster (abdomen) is flexed, curved underneath
the body, and the antennae are facing backward, in the characteristic aggressive posture
Dismembering Ant grabs limb or antenna of ensnared ant victim with its mandibles and drags the body part backwards; the gaster (abdomen) is flexed, curved
underneath the body, and antennae are flexed backward, in the characteristic aggressive posture.
Projecting Ant flexes its gaster (abdomen), curving it underneath its body, and projects a liquid substance in the direction of the test stimulus; used in aggressive
encounters, this substance is formic acid in ants belonging to the subfamily Formicinae (C. floricola and L. grandis); in the remaining three species
(Aphaenogaster senilis,Messor barbarus and Messor marocanus), the substances are alkaloids from the poison gland and/or hydrocarbons from the
Dufour gland; the antennae are flexed backward, in the characteristic aggressive posture
Retreating Ant orients towards test stimulus and immediately darts to a position inside the nest; subsequent retreats were scored whenever an ant appeared
at the nest entrance, or partially emerged, but immediately jerked back inside
K. L. Hollis, E. Nowbahari / Animal Behaviour 85 (2013) 537e544 539

Individual rescue behaviours
The same pattern of findings was obtained when each of the
four individual rescue behaviours was subject to analysis, namely
significant main effects of species and test type, as well as signifi-
cant interactions between species and test type (see Table 2 for
results of these individual ANOVAs). Exploration of each significant
interaction between species and test type, using Bonferroni post
hoc tests adjusted for multiple comparisons, revealed that in the
presence of an ensnared nestmate, both C. floricola and L. grandis
engaged in significantly higher levels of digging and pulling (all
Ps¼0.001), as well as the two newly discovered rescue behaviours,
namely snare biting (all Ps¼0.001) and sand transport (P0.044),
compared to extremely low levels of these same four behaviours in
the remaining three species (Fig. 2). However, as Fig. 2 also illus-
trates, C. floricola and L. grandis differed in the levels of these
individual behaviours, with C. floricola engaging in significantly
more pulling (P¼0.001) and snare biting (P¼0.015) than
L. grandis, but L. grandis engaging in more digging (P¼0.002) and
sand transport (P¼0.001) than C. floricola.
Messor marocanus, which appears to have the highest levels of
rescue behaviour amongst the three essentially ‘nonrescuer’
species, did indeed differ significantly from M. barbarus (P¼0.015),
although not from A. senilis (P¼0.081). However, M. marocanus
engaged in nearly as much paper-tugging behaviour as it did in
rescue behaviour (Fig. 2). Paper tugging was not included in the
combined rescue score because it appeared to be primarily a way to
remove the victim from near the nest. That is, once M. marocanus
revealed part of the filter paper by digging or pulling, the paper was
carried several centimetres away from the nest and the nestmate
was ignored for the remainder of the trial. Analysis of paper tugging
revealed a significant species by test type interaction
(F
12,285
¼2.049, P¼0.02), with M. marocanus engaging in signifi-
cantly more paper tugging than any of the four remaining species
(all Ps0.014).
Messor barbarus engaged in virtually no rescue behaviour
whatsoever. In one test, a single instance of snare biting occurred
and, in another, a single instance of sand transport. However, just
Homocolonial
Heterocolonial
Heterospecific
Control
1
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0L. grandis A. senilis M. marocanus M. barbarus
C. floricola
0.9
Mean proportion of intervals
Figure 1. Rescue behaviour in sand-dwelling ants in response to different test stimuli. Mean þSE proportion of intervals in which at least one individual engaged in rescue
behaviour (i.e. combined rescue score) in response to live test stimuli (a homocolonial nestmate, a heterocolonial ant or a heterospecific ant held in place using a filter paper and
thread snare) or a control test stimulus (an empty paper-and-thread snare). Five species of sand-dwelling Mediterranean ants were tested separately: Cataglyphis floricola, Lasius
grandis, Aphaenogaster senilis, Messor marocanus and Messor barbarus.
Table 2
Statistical analyses of individual rescue and aggressive behaviour patterns in sand-
dwelling ants using univariate ANOVA
Behaviour FdfP Effect sizes
Digging
Species 18.29 4, 285 <0.0001 0.204
Test type 78.60 3, 285 <0.0001 0.453
Speciestest type 19.88 12, 285 <0.0001 0.456
Pulling
Species 41.58 4, 285 <0.0001 0.368
Test type 152.03 3, 285 <0.0001 0.615
Speciestest type 44.29 12, 285 <0.0001 0.651
Snare biting
Species 13.55 4, 285 <0.0001 0.160
Test type 42.34 3, 285 <0.0001 0.308
Speciestest type 14.31 12, 285 <0.0001 0.376
Sand transport
Species 25.96 4, 285 <0.0001 0.267
Test type 40.14 3, 285 <0.0001 0.297
Speciestest type 22.76 12, 285 <0.0001 0.489
Paper tugging
Species 1.88 4, 285 0.113 0.026
Test type 1.37 3, 285 0.252 0.014
Speciestest type 2.05 12, 285 0.02 0.079
Biting
Species 17.24 4, 285 <0.0001 0.195
Test type 144.75 3, 285 <0.0001 0.604
Speciestest type 30.90 12, 285 <0.0001 0.565
Dismembering
Species 63.31 4, 285 <0.0001 0.471
Test type 135.82 3, 285 <0.0001 0.588
Speciestest type 45.90 12, 285 <0.0001 0.659
Projecting
Species 16.82 4, 285 <0.0001 0.191
Test type 20.19 3, 285 <0.0001 0.175
Speciestest type 18.52 12, 285 <0.0001 0.438
Retreating
Species 6.51 4, 285 <0.0001 0.084
Test type 5.48 3, 285 <0.001 0.055
Speciestest type 6.69 12, 285 <0.0001 0.220
For each behavioural pattern, the entries under ‘species’,‘test type’and ‘spe-
cies test type’refer to the source of between-subjects effects. Effect sizes refer to
partial eta-squared.
K. L. Hollis, E. Nowbahari / Animal Behaviour 85 (2013) 537e544540

prior to the snare biting, a portion of the paper had been exposed
and ants were engaged in moving the paper away from the nest.
Once moved, the paper and nestmate were completely ignored.
Thus, like the behaviour of M. marocanus,‘rescue’in M. barbarus
appears primarily as a way to remove an object from near the nest
entrance.
Tests with either a heterocolonial or heterospecific ant elicited
extremely low levels of the four rescue behaviours, if any at all, in
all five species, which were not significantly different from one
another (all Ps¼1.000). Exactly the same pattern of results was
obtained when combined rescue scores were analysed instead (all
Ps¼1.000); moreover, no differences were found between the
different heterospecific victims in species tested with more than
one species of heterospecific ant. Finally, control tests elicited
virtually no rescue behaviour in any of the five species. None the
less, the failure to respond with rescue behaviour in control tests
cannot be attributed to the failure to detect the presence of the
filter paper, as ants from all species repeatedly walked over the
filter paper and antennated the empty thread loop.
Aggressive Behaviour
Combined aggressive score
Cataglyphis floricola and L. grandis, the two species that deliv-
ered the most rescue behaviour to nestmates,not only refused such
aid to conspecifics from a different colony, but, instead, responded
to them with especially high levels of aggression. Moreover,
M. barbarus, which barely responded to nestmates with any form of
behaviour, either rescue or paper tugging, vigorously attacked
heterocolonial conspecifics (Fig. 3).
An ANOVA of the combined aggressive score revealed significant
main effects of species (F
4,285
¼42.16, P<0.0001) and test type
(F
3,285
¼279.99, P<0.0001), as well as a significant interaction
between species and test type (F
12,285
¼54.86, P<0.0001).
Exploration of that interaction, using Bonferroni post hoc tests
adjusted for multiple comparisons, revealed that in the presence of
a heterocolonial ant, C. floricola,L. grandis and M. barbarus engaged
in levels of aggressive behaviour that were similar to one another
(Ps0.91), but which differed from the two remaining species (all
Ps<0.0001), both of which engaged in significantly lower levels of
aggressive behaviour.
Individual aggressive behaviours
This same pattern of results is reflected in the analysis of two
individual aggressive behaviours, biting and dismembering of heter-
ocolonial victims. Exploration of the significant interactions between
species and test type for biting and dismembering (see Table 2)
revealed that C. floricola,L. grandis and M. barbarus engaged in
significantly higher levels of heterocolonial biting and dismembering
than did M. marocanus and A. senilis (all Ps0.001; see Fig. 4a). No
significant differences between species appeared in the frequency of
projecting (all Ps¼1.000), which was rare in heterocolonial tests.
Compared to heterocolonial tests, a very different pattern of
aggressive behaviour across species was elicited by heterospecific
victims (see Fig. 4b). Although L. grandis responded aggressively to
both heterocolonial and heterospecific victims (P¼0.246), neither
M. barbarus nor C. floricola did so (all Ps0.001). Indeed, C. floricola
did not respond at all to heterospecifics, even the comparatively
much smaller L. grandis victims, except to retreat from them.
Retreating by C. floricola took exactly the same form on every
heterospecific trial: As soon as the victim was positioned near the
nest, all nearby foragers fled back inside; repeatedly thereafter, one
or more foragers would appear at the nest entrance and then
immediately jerk back and disappear. This pattern of appearing and
retreating continued until the victim was removed at the end of the
trial, whereupon multiple foragers emerged and resumed activity.
Statistical analysis confirmed that C. floricola showed significantly
less aggression in heterospecific tests than all other species
(Ps0.001) except, perhaps, for M. barbarus, which barely reached
a statistically significant difference (P¼0.048).
Figures 3 and 4also show that heterocolonial and heterospecific
victims produced dramatically different patterns of behaviour in
A. senilis. Whereas A. senilis responded with weak aggression to
conspecifics from another colony, ants belonging to a different
species elicited much higher levels of aggression (P0.001), which
were not significantly different from those of the highly aggressive
L. grandis (P¼0.247). None the less, the individual aggressive
behaviour patterns differed slightly between these two aggressive
species, with L. grandis engaging in significantly more dismem-
bering and defensive projecting than A. senilis (both Ps0.001).
Finally, aggression never was observed in the presence of
a homocolonial nestmate, except on very rare occasions in two of
the five species, A. senilis and M. marocanus, nor was it ever
Digging
Pulling
Snare biting
Sand transport
Paper tugging
1
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0L. grandis A. senilis M. marocanus M. barbarus
0.9
0.8
Mean proportion of intervals
C.
f
loricola
Figure 2. Rescue behaviour in sand-dwelling ants in response to homocolonial nestmates. Mean þSE proportion of intervals inwhich ants showed rescue behaviour (sand digging,
limb pulling, snare biting or sand transport) or nonrescue behaviour (paper tugging) in response to a homocolonial nestmate. Five species of sand-dwelling Mediterranean ants
were tested separately: Cataglyphis floricola, Lasius grandis, Aphaenogaster senilis, Messor marocanus and Messor barbarus.
K. L. Hollis, E. Nowbahari / Animal Behaviour 85 (2013) 537e544 541

observed in any of the control tests. Statistical analyses confirmed
that no differences existed between species on either homocolonial
or control tests (all Ps¼1.000).
DISCUSSION
We examined ants in nature to ensure that rescue behaviour was
neither confined to the single species previously studied (Nowbahari
et al. 2009), nor reflected a laboratory artefact, and we succeeded on
both counts. In the presence of trapped nestmates, ants of both
C. floricola and L. grandis immediately approached the victims and
began to perform multiple behaviours to release them, including the
newly discovered, more complex forms of rescue, snare biting and
sand transport. Also similar to the laboratory experiments with
C. cursor, andcritical to our understanding of rescue behaviour more
generally, C. floricola and L. grandis rarely if ever attempted to rescue
non-nestmates, even conspecifics from nearby colonies. Indeed, not
only were heterocolonial conspecifics attacked aggressively, but also
the patterns of aggressive behaviour towards heterocolonial and
heterospecific victims reveal ants’capacity to discriminate between
different types of victims, as was demonstrated in C. cursor
(Nowbahari et al. 2009). Both C. floricola and L. grandis, as well as
C. cursor, belong to the subfamily Formicinae.
In stark contrast to the behaviour of the two new ‘rescuer’
species (C. floricola and L. grandis), the three remaining species in
this study, namely M. marocanus,M. barbarus and A. senilis
(subfamily Myrmicinae), rarely engaged in rescue behaviour.
However, note that the same rescue behaviour that occurred so
much more frequently, and, one might say, frenetically, in
C. floricola and L. grandis, including snare biting and sand transport,
appear to be within the behavioural capacity of the three non-
rescuer species. That is, although these nonrescuer species
neglected nestmate victims nearly all of the time, they nevertheless
are capable of performing precision rescue behaviour, as evidenced
by those occasions in which they executed the same behaviour
patterns, and in exactly the same way, as the two rescuer species.
Although additional comparative work is required, our analysis
of sand-dwelling ants’behaviour reflects some aspects of these
species’behavioural ecology. Lasius grandis nests were located in
easily disturbed soils, which could result in ants becoming buried
under collapsing sand and debris. Evidence for such risks comes
from the frequent appearance of wild ungulates in the area (e.g. Sus
scrofa), which occasionally trampled nests. In addition, this
microhabitat was shared by many predatory pit-digging antlions.
Although C. floricola could sometimes be found in more compact
soils than L. grandis,C. floricola also located its nests in loose sandy
soils shared by antlions. Interestingly, both C. floricola ants and pit-
digging antlions are drawn to a common flowering shrub of these
sandy soils, Halimium halimifolium. Antlions dig pits under the
overhanging branches of this shrub, which provides shelter from
wind and rain (Griffiths 1986; see Hollis et al. 2011, for a review)
and C. floricola ants feed heavily on its petals (Cerdá et al. 1996).
Very different from the microhabitats of the two rescuer species,
the nests of M. marocanus and M. barbarus were found in much
more hardened soils, nowhere near the vicinity of antlions’pit
traps. In addition, both M. marocanus and M. barbarus workers
foraged in very large groups, forming ant trails that often extended
10e15 m or more from the nest entrance. These trails are estab-
lished only when foragers return to the nest after locating a source
of food (Cerdan 1989;Hölldobler & Wilson 1990) and thus could
effectively prevent them from encountering antlion pits and other
sources of danger. In addition, the steady stream of nearby nest-
mates might offer a kind of ‘self-rescue’in which trapped foragers
could grab onto the legs or bodies of passing nestmates. Although
L. grandis also makes trails, solitary workers often were observed
foraging at some distance from the nest.
In contrast to the four species just discussed, the behaviour of
A. senilis is puzzling. Although A. senilis almost always was found in
loose, sandy soils within a few metres of L. grandis,A. senilis rarely
engaged in rescue behaviour. However, A. senilis does not share the
same behavioural profile as the two Messor species either, even
though all three belong to the subfamily Myrmicinae. That is,
1
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
A. senilis M. marocanus M. barbarus
0.8
C. floricola
Homocolonial
Heterocolonial
Heterospecific
Control
L. grandis
0.9
Mean proportion of intervals
Figure 3. Aggressive behaviour in sand-dwelling ants in response to different test stimuli. Mean þSE proportion of intervals in which at least one individual responded aggressively
(i.e. combined aggressive score) to live test stimuli (a homocolonial nestmate, a heterocolonial ant or a heterospecific ant held in place using a filter paper and thread snare) or
a control test stimulus (empty paper-and-thread snare). Five species of sand-dwelling Mediterranean ants were tested separately: Cataglyphis floricola, Lasius grandis, Aphaenogaster
senilis, Messor marocanus and Messor barbarus.
K. L. Hollis, E. Nowbahari / Animal Behaviour 85 (2013) 537e544542

whenever A. senilis engaged in any form of rescue behaviour,
rescuers did so for extended periods rather than sporadically, as in
Messor. Moreover, A. senilis rescuers never engaged in paper
tugging, the behaviour used by Messor ants to move the victim
away from the nest. Thus, A. senilis does not completely fit either
the rescuer or the nonrescuer profile. A close analysis of variables
that might have affected our observations, including weather
conditions, time of day or month, and locations of nests, revealed
no consistent patterns of this species’tendency to rescue or not.
Afinal comment speaksto the generality of rescue across multiple
taxa and the care that researchers must exert when attempting to
explain how such complex behaviour as rescue comes about. In an
experiment very similar in design to that used by Nowbahari et al.
(2009) with ants, Bartal et al. (2011) showed that rats behaved
similarly when they encountered a restrained cagemate, pulling on
the victim’s tail and biting the restraining tube. Moreover, when
given the opportunity to free theircagemate by tipping therestrainer
door, rats eventually learned to do so. In our field study, too, and
despite efforts to prevent escape, ants occasionally released victims
by biting repeatedly through the thread. The authors of the rat study
made the claim that rescuers were able to understand their cage-
mates’affective state, and that the results provide evidence of
intentional empathy, a higher level of empathic behaviour (de Waal
2011). However, the behaviour of ants reminds us how exceedingly
complex behaviour can come about through relatively simple
mechanisms. As Vasconcelos et al. (2012)argued, future experiments
exploringintentional empathyin animals will need to includecontrol
groups that help scientists to distinguish between analogous
behaviour involving different mechanisms.
Acknowledgments
For helpful comments on our manuscript, we thank Alain Lenoir
and an anonymous referee. For access to Reserva Científica de
Doñana and funding of this project through two separate grant
applications, we thank the ICTS-RBD Program. Several individuals
at Doñana were particularly helpful: Xim Cerdá, Ph.D., was an
invaluable resource on all matters pertaining to ants and the
1(a)
(b)
0.6
0.5
0.4
0.3
0.2
0.1
0L. grandis A. senilis M. marocanus M. barbarus
0.9
Biting
Dismembering
Projecting
Retreating
Biting
Dismembering
Projecting
Retreating
Heterocolonial test
Mean proportion of intervals
0.8
Mean proportion of intervals
0.7
1
0.6
0.5
0.4
0.3
0.2
0.1
0
0.9
0.8
0.7
Heterospecific test
C. floricola
L. grandis A. senilis M. marocanus M. barbarusC.
f
loricola
Figure 4. Aggressive behaviour in sand-dwelling ants in response to heterocolonial and heterospecific test stimuli. Mean þSE proportion of intervals in which ants showed
aggressive behaviour (biting, dismembering or projecting) or retreated in response to a live (a) heterocolonial ant or (b) heterospecific ant. Five species of sand-dwelling Medi-
terranean ants were tested separately: Cataglyphis floricola, Lasius grandis, Aphaenogaster senilis, Messor marocanus and Messor barbarus.
K. L. Hollis, E. Nowbahari / Animal Behaviour 85 (2013) 537e544 543

Doñana reserve, as well as a generous and gracious host. Begoña
Arrizabalaga and Rosa Rodríguez provided enormous help, not only
throughout the application process, but also for the duration of our
stays at Doñana. Additional support of this project was obtained
through a Faculty Grant from Mount Holyoke College (K.L.H.), as
well as from Laboratoire d’Éthologie Expérimentale et Comparée,
Université Paris 13 (E.N. and K.L.H.). Finally, Janelle Gagnon
provided statistical support and Cheryl McGraw provided technical
assistance to meet figure specifications.
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