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Significance of chemical recognition cues is context dependent in ants
Animal Behaviour 80 (2010) 839e844
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Significance of chemical recognition cues is context dependent in ants Nick Bos a, *, Fernando J. Guerrieri a, b, Patrizia d’Ettorre a, c a
Centre for Social Evolution, Department of Biology, University of Copenhagen Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Germany c Laboratoire d’Ethologie Expérimentale et Comparée (LEEC), Université Paris 13, France b
a r t i c l e i n f o Article history: Received 10 March 2010 Initial acceptance 7 May 2010 Final acceptance 2 August 2010 Available online 9 September 2010 Keywords: ant associative learning Camponotus aethiops conditioning context cuticular hydrocarbon perception recognition
Recognition of group members is of fundamental importance in social animals, allowing individuals to protect resources against intruders and parasites, as well as ensuring social cohesion within the group. In ants and other social insects, social recognition relies on multicomponent chemical signatures, composed primarily of long-chain cuticular hydrocarbons. These signatures are colony specific and allow discrimination between nestmates and non-nestmates. Nevertheless, the mechanisms underlying detection, perception and information processing of chemical signatures are poorly understood. It has been suggested that associative learning might play a role in nestmate recognition. We investigated whether Camponotus aethiops ants can associate a complete cuticular hydrocarbon profile, consisting of about 40 compounds, with a food reward and whether the new association, developed in an appetitive context, affects aggression against non-nestmates carrying the hydrocarbon profile associated with food. Individual ant workers were able to associate the non-nestmate chemical profile with food. However, conditioned ants were still aggressive when encountering a non-nestmate carrying the odour profile used as training odour in our experiments. This suggests that ants, like some, but not all other insects, show interactions between different modalities (i.e. olfactory and visual), and can treat complex chemical cues differently, according to the context in which they are perceived. This plasticity ensures that learning in an appetitive context does not interfere with the crucial task of colony defence. Ó 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Associative learning is a widespread phenomenon in the animal kingdom (Giurfa 2007), allowing individuals to extract important information from their environment by establishing predictive relationships between different stimuli (i.e. honeybees, Apis mellifera: Bitterman et al. 1983; cuttlefish, Sepia officinalis: Cole & Adamo 2005; house mice, Mus domesticus: Watkins et al. 1998). Although insects have been generally neglected in studies addressing higherorder cognitive processes, during the last three decades the honeybee has become a model organism for the study of learning and memory (Giurfa 2007; de Brito-Sánchez et al. 2008), since they live in organized complex societies and show an amazing capacity for learning. All ants are eusocial, having cooperative brood care, reproductive division of labour and overlapping generations. Ants often live in very complex societies, and can learn to solve a variety of problems, such as navigating in complex environments (Cataglyphis: Wehner 2009), visiting feeding places at specific times during the day
* Correspondence: N. Bos, Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, bygning 12, DK-2100 Copenhagen, Denmark. E-mail address: [email protected] (N. Bos).
(Paraponera: Harrison & Breed 1987) or learning to avoid plants that are detrimental for their fungus garden (Atta: Saverschek et al. 2010). Some ants can learn and remember individual recognition cues (d’Ettorre & Heinze 2005; Dreier et al. 2007) and selectively police or punish colony members (e.g. Monnin et al. 2002; Van Zweden et al. 2007). However, ants have been largely ignored in studies on learning, memory and cognition other than studies on spatial memories (reviewed in Collett et al. 2006). A pioneering work by Dupuy et al. (2006) showed that individual Camponotus ants can learn to associate volatile substances with either a positive stimulus (sucrose solution) or an aversive stimulus (quinine). In their study, foraging ants had to make a choice between an odour associated with sucrose solution (appetitive conditioned stimulus) and another odour associated with quinine (aversive conditioned stimulus) in a Y-maze. Individual Camponotus aethiops ants can be trained to associate single synthetic long-chain hydrocarbons with a sucrose reward (S. Dreier et al., unpublished data). These hydrocarbons have a low volatility, are present on the ant cuticle and thus not usually found in a foraging context. Instead, cuticular hydrocarbons (CHCs), especially some classes of hydrocarbons, such as methyl-branched alkanes, are important for nestmate recognition (e.g. Guerrieri et al. 2009; reviewed in d’Ettorre & Lenoir 2010). Ant colonies are good targets for predators and parasites, since they contain many worker
0003-3472/$38.00 Ó 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2010.08.001
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ants, eggs, larvae, pupae and stored food. To maintain these valuable resources, ants need to defend their colonies from potential invaders. Also, because ant workers usually do not reproduce, fitness benefits are gained only if cooperative acts are directed towards nestmates, which are usually related, and not towards alien individuals. This requires an effective recognition system, allowing reliable discrimination of nestmates from non-nestmates. Nestmate recognition is therefore essential for the stability and success of insect societies. Because ant nests are usually dark, a nonvisual recognition system is needed. Recognition cues are chemical in nature and, as mentioned above, CHCs appear to be important substances for chemical recognition in social insects (i.e. bees: Breed 1998; wasps: Dani 2006; termites: Dronnet et al. 2006; ants: Hefetz 2007; d’Ettorre & Lenoir 2010). One of the first studies showing the role of CHCs in ant recognition was performed by Bonavita-Cougourdan et al. (1987), who removed the chemical profile of freshly killed workers of Camponotus vagus and replaced it with a chemical cuticular extract of a different colony. Workers were significantly more aggressive against these treated nestmates than against nontreated ones. In a subsequent study, a single synthetic hydrocarbon was added to the cuticle of individual C. vagus ants, which were then attacked by their nestmates (Meskali et al. 1995); however, a recent study showed that this might not be the case for all classes of hydrocarbons, although it is the presence and not the absence of specific hydrocarbons on the cuticle that promotes aggression (Guerrieri et al. 2009). According to the currently accepted hypothesis, which has recently been questioned (Ozaki et al. 2005; Guerrieri et al. 2009), nestmate recognition occurs following a ‘labeletemplate matching model’: each worker carries a set of recognition cues (the label) and when an individual detects this label, it compares the label and its inner template (a neural representation of the colony odour, stored in the long-term memory). If the label is dissimilar to the template, the worker will reject the encountered individual (Vander Meer & Morel 1998; Lenoir et al. 1999). Previous studies specifically showed that ants behave aggressively against the CHC extracts from non-nestmates (Lahav et al. 1999; Lucas et al. 2005; Ozaki et al. 2005). Guerrieri & d’Ettorre (2008) recently introduced a controlled protocol for recording a clear binary response, named the mandible opening response (MOR): harnessed ants (genus Camponotus and Formica) will immediately open their mandibles, as a sign of aggression, when presented with a glass rod coated with the cuticular extracts of non-nestmates. Conversely, ants keep their mandibles closed when presented with the cuticular extract of nestmates. These studies demonstrate that the chemical stimulus itself, that is, the CHC extract of non-nestmates, is sufficient to elicit aggression in several ant species. Knowing that ants behave aggressively against CHC extracts of non-nestmates, and also that ants can be conditioned to associate a synthetic chemical compound, including long-chain hydrocarbons, with food (Dupuy et al. 2006; S. Dreier et al., unpublished data), we investigated whether individual focal ants were able to associate the chemical extract of the CHC profile of a non-nestmate with food, thus transforming this non-nestmate cuticular hydrocarbon profile from an aversive stimulus (see Guerrieri & d’Ettorre 2008) into an appetitive stimulus. The question then arises, how would an ant that previously associated a non-nestmate CHC profile with food react to a real non-nestmate individual bearing the same CHC profile? We predicted two mutually exclusive scenarios: (1) the non-nestmate CHC profile changes its meaning, now indicating the presence of food and not of a potential enemy, and thus the focal ant will not behave aggressively towards the non-nestmate individual; (2) even if in a foraging context the CHC profile indicated the presence of a food source, the focal ant will still behave aggressively against a non-nestmate bearing the food-associated CHC profile. Our
experimental design allowed us to distinguish between these two predictions, although it remains to be investigated whether the ants learn the entire cuticular hydrocarbon mixture or a subset of it. METHODS Study Organism Twelve queenright colonies of C. aethiops (Latr.) were collected in April 2008 in the Italian Apennines. Six colonies were collected in Castel del Rio (44 21062.830 N, 11520 30.920 E) and six in Moraduccio (44100 32.750 N, 11290 3.080 E). Each colony was housed in a plastic box (27 17 cm and 9.5 cm high) with a plaster floor, serving as a nest. This was connected to another plastic box of the same size, serving as a foraging arena. The ants were fed twice a week with diluted honey and mealworms, Tenebrio molitor; water was provided ad libitum. The nests were kept in a climate room, at 25 2 C, and a 12:12 h light:dark regime. Ants were deprived of honey at least 1 week before the experiment to increase motivation for foraging on sucrose food sources. Preparation of CHC Extracts To condition individual ants to the chemical profile of nonnestmates, we used cuticular extracts in pentane as a solvent (SigmaeAldrich); this was the ‘training odour’. For each colony source of training odour, we prepared five extracts in the following way: 11 foragers (six major and five media workers) were frozen and after 30 min their abdomen was cut off to prevent any possible extraction of pheromones produced by glands. The remaining parts of the bodies were inserted into a glass vial and covered with 1 ml of pentane. After 10 min, the extract was transferred to a new vial and the solvent allowed to evaporate; chemical extracts were then redissolved in 500 ml of pentane before use in the following experiments. Conditioning Set-up and Procedure The ant nests used in conditioning trials were provided with a vertical wooden stick in their foraging arena for the ants to climb on. For each ant to be tested, seven petri dishes (100 mm diameter 15 mm high) were prepared by coating their walls with Fluon and covering their floor with clean filter paper. Two microscope cover slips (18 18 mm) were placed on each filter paper. One cover slip was treated with 20 ml of non-nestmate extract (the training odour, as described above) deposited on its borders and the solvent was allowed to evaporate completely. In the centre of the cover slip, a droplet of sucrose solution (1 ml, 30% w/w) was deposited. On the other cover slip, we put 1 ml of water, so that the two slips looked exactly alike. The slips were positioned on opposite sides of the petri dish (see Fig. 1a). At the start of the first conditioning trial, a medium foraging worker climbing on the vertical stick was gently allowed to walk onto a small piece of paper, which was then transferred into the petri dish where the ant was allowed to get off the paper at approximately equal distance from both cover slips (see Fig. 1a). We then recorded the time the ant required to find the sucrose reward. After the ant finished drinking the sugar reward, it was picked up with soft forceps, and marked with a dot of enamel paint on its abdomen. The ant was then allowed to walk on the piece of paper again, and transferred back to the vertical stick in its colony of origin. Once back in the nest, the ants usually transferred the food to their nestmates by trophallaxis and returned to the stick. After a minimum of 1 min, the same ant was picked up again from the stick and transferred to a new petri dish for a subsequent conditioning trial. A total of six conditioning
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(a)
Sucrose Non-nestmate CHC profile
Water
Focal ant (b)
Non-nestmate CHC profile
Solvent
Focal ant Figure 1. (a) Set-up for conditioning. (b) Set-up for choice test. For (a) the time the focal ant spent finding the reward was recorded. For (b) the time the focal ant spent in each of the four quadrants was recorded for 2 min.
trials were performed per ant. The location of the slide with the sucrose reward was randomized in each trial, but the cover slip was never in the same quadrant for more than two consecutive trials to prevent the ant from associating a given spatial direction with the reward. Following conditioning, a choice test was conducted to see whether the ant had developed a positive association between non-nestmate CHC extract and the sucrose reward. This choice test consisted of presenting the focal ant, in a new petri dish, with one cover slip coated with the CHC extract used as training odour, and another one treated with solvent only (pentane, P). In this test neither sucrose nor water was provided. The cover slips represent the contextual stimulus in these tests. The petri dish was divided into four quadrants by drawing a light cross in the middle of the filter paper with a pencil, and the two slips were placed randomly in two opposite quadrants (see Fig. 1b). The ant was transferred from the stick to the petri dish, analogously to the conditioning procedure, and was allowed to search for the expected reward for 2 min (although only the chemical stimulus, and not the reward, was present during this time). The time spent by the focal ant in each quadrant was recorded using the software EthoLog (Ottoni 2000). Afterwards, 1 ml of sucrose solution was provided on the CHC-coated cover slip to avoid any possible extinction effect. The ant was then transferred back to the vertical stick in the foraging arena of the colony. As a control, naïve ants were subjected directly to the choice test, without conditioning. Ten naïve ants were tested from each colony used for conditioning (40 control ants in total). Aggression Tests Once the choice test was completed, the conditioned ant was left in its colony for 10 min and then underwent an aggression test. We measured aggression by placing the focal ant from the stick into a neutral arena (50 mm diameter 60 mm height), where she was allowed to habituate to the new environment for 1 min. After this, a dead ant (freshly killed by freezing), from its own colony (nestmate control), the colony carrying the CHC profile used as the training
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odour or a novel colony (non-nestmate control), was placed in the centre of the arena. In these tests, the dead ant represents the contextual stimulus. The small size of the arena allowed for rapid contact between the focal ant and the freshly killed ant (average delay for first contact SE ¼ 7.2 1.4 s). The behaviour of the focal ant towards the dead ant was recorded continuously for 3 min using the software EthoLog (Ottoni 2000). We evaluated any possible aggression elicited by the target (dead ant) by recording six categorical behaviours: (1) no contact between the focal ant and the target; (2) antennation; (3) grooming; (4) mandible opening; (5) biting; and (6) gaster flexing (attempting to spread formic acid). We established a baseline of aggression before starting the conditioning experiments. Ten unconditioned focal ants and 10 target ants were used for each set of dyadic encounters (with an ant from the focal ant’s own colony, the colony carrying the CHC profile used as the training odour or a novel colony), giving a total of 30 aggression tests. This was replicated four times, using three new colonies for each replicate, for a total of 12 colonies and 120 aggression tests. In summary, 15 ants were conditioned. The aggression tests involving these conditioned focal ants were repeated five times with target nestmate ants, five times with target ants originating from the colony whose cuticular extracts were used as the training odour and five times with target ants from a novel non-nestmate colony. This was replicated four times, involving a total of 12 colonies and 60 conditioned ants. As a control for the conditioning experiment, 40 additional naïve ants were used in choice tests, and to establish the baseline of aggression, 120 additional unconditioned ants were used.
Data Analysis There were no significant differences in searching time between colonies (KruskaleWallis ANOVA: c24 < 9.49, P > 0.05), so the data could be pooled. Searching time in the course of the six conditioning trials (1e6) were analysed using a Friedman ANOVA followed by multiple comparisons (Siegel & Castellan 1988). For the choice tests, a preference index (PI) was calculated using the following formula, where tTO and tP is the time spent in quadrant TO (training odour) and P (pentane), respectively.
PI ¼
tTO tP tTO þ tP
PI data were normally distributed (KolmogoroveSmirnov test: P > 0.2 in all cases). There was no colony effect on PI (ANOVA: conditioned ants: F3,56 ¼ 1.47, P ¼ 0.23; naïve ants: F3,36 ¼ 1.08, P ¼ 0.37); hence the data were pooled. If ants preferred one quadrant over the other, the PI would differ significantly from zero. This was analysed using a t test for single means. Preference for quadrants was also compared between conditioned and unconditioned (naïve) ants using a t test for independent samples. The different behaviours observed during aggression tests were scored so that the most aggressive behaviour was assigned the highest score: 0 ¼ antennation and grooming; 1 ¼ mandible opening; 2 ¼ biting; 3 ¼ gaster flexing. The maximum level of aggression was recorded, giving a value between 0 and 3 for every ant tested. Since there was no colony effect on overall aggression (KruskaleWallis ANOVA: P > 0.05), data could be pooled. The difference in baseline aggression of unconditioned workers towards nestmates, non-nestmate ants bearing the CHC profile used for conditioning and novel non-nestmate ants was analysed with a Wilcoxon signed-ranks tests. The data were not normally distributed (KolmogoroveSmirnov test: P < 0.05) and thus a generalized linear model (GLM; Poisson
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distribution, log link function) was used for analysing aggression levels among naïve ants (the baseline of aggression) and conditioned ants. All analyses were performed using Statistica 7.1 (StatSoft, Tulsa, OK, U.S.A.).
0.4 Conditioned Unconditioned
0.35 0.3
Conditioning The searching time decreased significantly in the course of the six conditioning trials (Friedman ANOVA: c25 ¼ 97.17, N ¼ 60, P < 0.05; Fig. 2). In particular, searching time decreased markedly from the second trial on, showing that ants directed their search more promptly towards the training odour. In the control group (naïve ants), the PI did not differ significantly from zero (one-sample t test: t39 ¼ 0.27, P ¼ 0.79), meaning that ants had no spontaneous preference for a particular quadrant during the choice test. However, for the conditioned ants, the PI differed significantly from zero (one-sample t test: t59 ¼ 19.59, P < 0.001). During the choice test, conditioned ants spent significantly more time in the quadrant containing the training odour than control (naïve) ants (t test: t98 ¼ 10.60, P < 0.001; Fig. 3). Aggression When the baseline of aggression was established, aggression of unconditioned ant workers was significantly higher towards nonnestmate ants bearing the CHC profile used for conditioning (b) or novel non-nestmate ants (c) than towards nestmates (a; Wilcoxon signed-ranks test: aaeab: T ¼ 0.00, P < 0.01; aaeac: T ¼ 8.00, P < 0.01; abeac: T ¼ 7.50, P > 0.05; Bonferroni correction, adjusted significance level a ¼ 0.025). The results of the aggression tests are shown in Fig. 4. Aggression levels of conditioned ants against either ants from their own colony, non-nestmate ants bearing the CHC profile used for conditioning or novel non-nestmate ants did not differ significantly from the baseline of aggression of unconditioned ants (GLM: Wald c22 ¼ 2.783, P ¼ 0.249), showing that conditioning did not interfere with recognition abilities and expression of aggressive behaviour.
Preference index
RESULTS
0.25 0.2 0.15 0.1 0.05 0
Figure 3. Preference index of conditioned and unconditioned (naïve) ants (mean SE).
developed in an appetitive context, might affect nestmate discrimination, which is usually expressed in an aggressive context. We conditioned freely walking individual C. aethiops workers to associate a cuticular extract of a non-nestmate with a sucrose reward, showing that stimuli that originally elicit aggression in ants can be associated with food. This suggests plasticity in the significance of cues/signals that are typically important in the modulation of social interactions. The fact that the conditioned ant kept coming back to the stick, and that during the trials more ants were present on the stick, shows clearly that our set-up was appropriate to simulate a foraging context. Ants formed the association between the chemical blend and reward very rapidly; right after the first training trial, the searching time needed by the ant to find the reward decreased significantly. A subsequent choice test showed that the association between the
3.5 Conditioned Unconditioned
DISCUSSION
3
We investigated whether ants are able to associate a cuticular hydrocarbon mixture with food, and whether this association,
Aggression index
Searching time (s)
2.5
280 260 240 220 200 180 160 140 120 100 80 60 40 20 0
2
1.5
1
0.5
0
1
2
3
4
5
6
Trial Figure 2. Average searching time for each conditioning trial (N ¼ 60) showing median, quartiles and range.
Nestmate
Familiar
Novel
Figure 4. Aggression index of conditioned and unconditioned ants versus nestmates, familiar non-nestmates and novel non-nestmates, respectively (mean SE). Note that the ‘familiar non-nestmates’ are novel non-nestmates for the unconditioned ants, since they have not been familiarized by conditioning.
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food and the non-nestmate CHC profile was robustly established, although we do not know whether the ants learned the entire CHC profile or a subset of it (see below). Individual olfactory learning in ants was shown for the first time only recently (Dupuy et al. 2006), by conditioning individual ants to pure volatile substances using a Y-maze. Although the substances tested were present in either flowers or honeybee pheromones (see Balderrama et al. 2002), they were not potential cues involved in nestmate recognition. In addition, (S. Dreier et al., unpublished data) showed that individual Camponotus ants can be conditioned to associate single synthetic long-chain hydrocarbons with a sugar reward. Unlike these two studies, in which only pure synthetic chemical substances were used as conditioned stimuli, we used a multicomponent blend, namely the cuticular extract of C. aethiops workers, consisting of about 40 different hydrocarbons (Van Zweden et al. 2009), as training odour. The extracted cuticular hydrocarbons have the same properties as the natural mix on the ant’s cuticle, as shown by Bonavita-Cougourdan et al. (1987), Morel et al. (1988) and Nowbahari et al. (1990). These authors washed ants in solvent to remove the cuticular hydrocarbons, and then applied on these washed ants the cuticular extract of a nestmate or non-nestmate. This restored the expected level of aggression, while washed ants did not elicit an aggressive response. In addition, Ozaki et al. (2005) and Guerrieri & d’Ettorre (2008) showed that a non-nestmate cuticular extract, when applied to a glass bead (or rod), elicits aggression, proving that the CHC extract alone is sufficient to promote aggression. It remains to be determined whether the ants indeed learned the entire CHC profile or only some specific compounds within the profile. If the ants learned the entire CHC mixture of the profile, or a large part of it, learning could have happened in various ways. According to the elemental theory of learning (i.e. Rescorla & Wagner 1972), a mixture is processed as the sum of its components (AB ¼ A þ B), while the configural theory (i.e. Pearce 1987) suggests that the properties of a mixture are different from the properties of the components (AB ¼ X s A þ B; Giurfa 2003). In honeybees, a new model has been suggested: the key odorant hypothesis, which encompasses features of both elemental and configural learning (Reinhard et al. 2010). Even though honeybees were able to learn all individual odorants used in the experiments; when conditioned to a complex mixture, only certain key compounds were learned, suggesting that some odorants are more representative of the mixture than others. In our experiment, we cannot distinguish between these models. However, recent evidence indicates that methyl-branched alkanes are more important in nestmate recognition than linear alkanes (i.e. Guerrieri et al. 2009), suggesting that only key compounds of the cuticular hydrocarbon blend present on the ant might be used in recognition. Future studies should focus on which classes of hydrocarbons are learned when a mixture is used as the training odour. In our experiments, the association of the CHC profile with food did not affect the social meaning of the non-nestmate cuticular profile. Indeed, after conditioning, the focal ants were as aggressive towards an individual bearing the familiar odour (the cuticular profile associated with food) as towards an individual bearing a novel non-nestmate cuticular hydrocarbon profile. Context-dependent learning in the visual modality has been shown in a variety of organisms (e.g. ants: Chameron et al. 1998; honeybees: Collett et al. 1993; humans: Smith & Vela 2001; for a review on insects, see Collett et al. 2003). However, contextdependent olfactory learning in insects has only been shown in the cricket Gryllus bimaculatus (Matsumoto & Mizunami 2004) and the cockroach Periplaneta americana (Sato et al. 2006). For instance, individual crickets were conditioned to select one odour and to avoid
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another odour in one context (darkness), and to do the opposite in another context (light). In this experiment, neither the light condition nor the odour could predict a reward (water) or non-reward (saline solution); only a combination of the two could. In contrast, Drosophila melanogaster is not capable of solving a similar task (Yarali et al. 2008). Groups of flies were conditioned in a ‘biconditional discrimination’ design. One odour was paired with an electric shock in darkness, but not in light; another odour was paired with a shock in light, but not darkness. Flies failed to show any preference for the right odour in the specific light conditions. Therefore, interactions between different modalities (i.e. visual and olfactory), which are necessary for context-dependent learning, are not a general feature of all insects. Two explanations of context-dependent learning have been proposed (Pearce & Bouton 2001). In the occasion-setting theory, the contextual stimulus (in our case, the cover slip or the target ant) ‘sets the occasion’ for responding to another conditioned stimulus (in our case, the training odour: the non-nestmate CHC profile), without forming an association with the unconditioned stimulus (the food reward) itself. In the configural theory, an individual perceives different CSecontext combinations as different stimuli. Although learning in our experimental design was clearly context dependent, further studies will be needed to distinguish between these two theories. Living in a complex and changing environment requires being aware that the same cues/signals might have different significance according to context and the role of individual experience is extremely important. This learning capability and flexibility is an adaptive trait for the ants, as it maximizes their fitness and survival: on the one hand, ants can eventually find food by following the smell of non-nestmates, but still avoid or attack enemies that they encounter. Our study shows that ants have complex cognitive abilities and are capable of distinguishing the meaning of identical cues according to the context in which they are perceived. Acknowledgments This work was supported by the Marie Curie Excellence grant CODICES (MEXT-CT-2004-014202) and a Freia grant from the Faculty of Science, University of Copenhagen, both assigned to P.d’E. We are grateful to all members of the Copenhagen Centre for Social Evolution for a stimulating work environment. We thank three anonymous referees for useful comments and suggestions. References Balderrama, J., Núñez, J., Guerrieri, F. J. & Giurfa, M. 2002. Different functions of two alarm substances in the honeybee. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, 188, 485e491. Bitterman, M. E., Menzel, R., Fietz, A. & Schafer, S. 1983. Classical conditioning of proboscis extension in honeybees (Apis mellifera). Journal of Comparative Psychology, 97, 107e119. Bonavita-Cougourdan, A., Clément, J. L. & Lange, C. 1987. Nestmate recognition: the role of cuticular hydrocarbons in the ant Camponotus vagus Scop. Journal of Entomological Science, 22, 1e10. Breed, M. D. 1998. Pheromones of the honey bee. BioScience, 48, 463e470. de Brito-Sánchez, M. G., Deisig, N., Sandoz, J. C. & Giurfa, M. 2008. Neurobiology of olfactory communication in a social insect: the honeybee. In: Sociobiology of Communication: an Interdisciplinary Perspective (Ed. by P. d’Ettorre & D. P. Hughes), pp. 119e138. Oxford: Oxford University Press. Chameron, S., Schatz, B., Pastergue Ruiz, I., Beugnon, G. & Collett, T. S. 1998. The learning of a sequence of visual patterns by the ant Cataglyphis cursor. Proceedings of the Royal Society B, 265, 2309e2313. Cole, P. & Adamo, S. A. 2005. Cuttlefish (Sepia officinalis: Cephalopoda) hunting behavior and associative learning. Animal Cognition, 8, 27e30. Collett, T. S., Fry, R. & Wehner, R. 1993. Sequence learning by honeybees. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, 172, 693e706. Collett, T. S., Fauria, K. & Dale, K. 2003. Contextual cues and insect navigation. In: The Neurobiology of Spatial Behaviour (Ed. by K. J. Jeffery), pp. 67e82. Oxford: Oxford University Press.
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Animal Behaviour journal homepage: www.elsevier.com/locate/anbehav
Significance of chemical recognition cues is context dependent in ants Nick Bos a, *, Fernando J. Guerrieri a, b, Patrizia d’Ettorre a, c a
Centre for Social Evolution, Department of Biology, University of Copenhagen Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Germany c Laboratoire d’Ethologie Expérimentale et Comparée (LEEC), Université Paris 13, France b
a r t i c l e i n f o Article history: Received 10 March 2010 Initial acceptance 7 May 2010 Final acceptance 2 August 2010 Available online 9 September 2010 Keywords: ant associative learning Camponotus aethiops conditioning context cuticular hydrocarbon perception recognition
Recognition of group members is of fundamental importance in social animals, allowing individuals to protect resources against intruders and parasites, as well as ensuring social cohesion within the group. In ants and other social insects, social recognition relies on multicomponent chemical signatures, composed primarily of long-chain cuticular hydrocarbons. These signatures are colony specific and allow discrimination between nestmates and non-nestmates. Nevertheless, the mechanisms underlying detection, perception and information processing of chemical signatures are poorly understood. It has been suggested that associative learning might play a role in nestmate recognition. We investigated whether Camponotus aethiops ants can associate a complete cuticular hydrocarbon profile, consisting of about 40 compounds, with a food reward and whether the new association, developed in an appetitive context, affects aggression against non-nestmates carrying the hydrocarbon profile associated with food. Individual ant workers were able to associate the non-nestmate chemical profile with food. However, conditioned ants were still aggressive when encountering a non-nestmate carrying the odour profile used as training odour in our experiments. This suggests that ants, like some, but not all other insects, show interactions between different modalities (i.e. olfactory and visual), and can treat complex chemical cues differently, according to the context in which they are perceived. This plasticity ensures that learning in an appetitive context does not interfere with the crucial task of colony defence. Ó 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Associative learning is a widespread phenomenon in the animal kingdom (Giurfa 2007), allowing individuals to extract important information from their environment by establishing predictive relationships between different stimuli (i.e. honeybees, Apis mellifera: Bitterman et al. 1983; cuttlefish, Sepia officinalis: Cole & Adamo 2005; house mice, Mus domesticus: Watkins et al. 1998). Although insects have been generally neglected in studies addressing higherorder cognitive processes, during the last three decades the honeybee has become a model organism for the study of learning and memory (Giurfa 2007; de Brito-Sánchez et al. 2008), since they live in organized complex societies and show an amazing capacity for learning. All ants are eusocial, having cooperative brood care, reproductive division of labour and overlapping generations. Ants often live in very complex societies, and can learn to solve a variety of problems, such as navigating in complex environments (Cataglyphis: Wehner 2009), visiting feeding places at specific times during the day
* Correspondence: N. Bos, Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, bygning 12, DK-2100 Copenhagen, Denmark. E-mail address: [email protected] (N. Bos).
(Paraponera: Harrison & Breed 1987) or learning to avoid plants that are detrimental for their fungus garden (Atta: Saverschek et al. 2010). Some ants can learn and remember individual recognition cues (d’Ettorre & Heinze 2005; Dreier et al. 2007) and selectively police or punish colony members (e.g. Monnin et al. 2002; Van Zweden et al. 2007). However, ants have been largely ignored in studies on learning, memory and cognition other than studies on spatial memories (reviewed in Collett et al. 2006). A pioneering work by Dupuy et al. (2006) showed that individual Camponotus ants can learn to associate volatile substances with either a positive stimulus (sucrose solution) or an aversive stimulus (quinine). In their study, foraging ants had to make a choice between an odour associated with sucrose solution (appetitive conditioned stimulus) and another odour associated with quinine (aversive conditioned stimulus) in a Y-maze. Individual Camponotus aethiops ants can be trained to associate single synthetic long-chain hydrocarbons with a sucrose reward (S. Dreier et al., unpublished data). These hydrocarbons have a low volatility, are present on the ant cuticle and thus not usually found in a foraging context. Instead, cuticular hydrocarbons (CHCs), especially some classes of hydrocarbons, such as methyl-branched alkanes, are important for nestmate recognition (e.g. Guerrieri et al. 2009; reviewed in d’Ettorre & Lenoir 2010). Ant colonies are good targets for predators and parasites, since they contain many worker
0003-3472/$38.00 Ó 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2010.08.001
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ants, eggs, larvae, pupae and stored food. To maintain these valuable resources, ants need to defend their colonies from potential invaders. Also, because ant workers usually do not reproduce, fitness benefits are gained only if cooperative acts are directed towards nestmates, which are usually related, and not towards alien individuals. This requires an effective recognition system, allowing reliable discrimination of nestmates from non-nestmates. Nestmate recognition is therefore essential for the stability and success of insect societies. Because ant nests are usually dark, a nonvisual recognition system is needed. Recognition cues are chemical in nature and, as mentioned above, CHCs appear to be important substances for chemical recognition in social insects (i.e. bees: Breed 1998; wasps: Dani 2006; termites: Dronnet et al. 2006; ants: Hefetz 2007; d’Ettorre & Lenoir 2010). One of the first studies showing the role of CHCs in ant recognition was performed by Bonavita-Cougourdan et al. (1987), who removed the chemical profile of freshly killed workers of Camponotus vagus and replaced it with a chemical cuticular extract of a different colony. Workers were significantly more aggressive against these treated nestmates than against nontreated ones. In a subsequent study, a single synthetic hydrocarbon was added to the cuticle of individual C. vagus ants, which were then attacked by their nestmates (Meskali et al. 1995); however, a recent study showed that this might not be the case for all classes of hydrocarbons, although it is the presence and not the absence of specific hydrocarbons on the cuticle that promotes aggression (Guerrieri et al. 2009). According to the currently accepted hypothesis, which has recently been questioned (Ozaki et al. 2005; Guerrieri et al. 2009), nestmate recognition occurs following a ‘labeletemplate matching model’: each worker carries a set of recognition cues (the label) and when an individual detects this label, it compares the label and its inner template (a neural representation of the colony odour, stored in the long-term memory). If the label is dissimilar to the template, the worker will reject the encountered individual (Vander Meer & Morel 1998; Lenoir et al. 1999). Previous studies specifically showed that ants behave aggressively against the CHC extracts from non-nestmates (Lahav et al. 1999; Lucas et al. 2005; Ozaki et al. 2005). Guerrieri & d’Ettorre (2008) recently introduced a controlled protocol for recording a clear binary response, named the mandible opening response (MOR): harnessed ants (genus Camponotus and Formica) will immediately open their mandibles, as a sign of aggression, when presented with a glass rod coated with the cuticular extracts of non-nestmates. Conversely, ants keep their mandibles closed when presented with the cuticular extract of nestmates. These studies demonstrate that the chemical stimulus itself, that is, the CHC extract of non-nestmates, is sufficient to elicit aggression in several ant species. Knowing that ants behave aggressively against CHC extracts of non-nestmates, and also that ants can be conditioned to associate a synthetic chemical compound, including long-chain hydrocarbons, with food (Dupuy et al. 2006; S. Dreier et al., unpublished data), we investigated whether individual focal ants were able to associate the chemical extract of the CHC profile of a non-nestmate with food, thus transforming this non-nestmate cuticular hydrocarbon profile from an aversive stimulus (see Guerrieri & d’Ettorre 2008) into an appetitive stimulus. The question then arises, how would an ant that previously associated a non-nestmate CHC profile with food react to a real non-nestmate individual bearing the same CHC profile? We predicted two mutually exclusive scenarios: (1) the non-nestmate CHC profile changes its meaning, now indicating the presence of food and not of a potential enemy, and thus the focal ant will not behave aggressively towards the non-nestmate individual; (2) even if in a foraging context the CHC profile indicated the presence of a food source, the focal ant will still behave aggressively against a non-nestmate bearing the food-associated CHC profile. Our
experimental design allowed us to distinguish between these two predictions, although it remains to be investigated whether the ants learn the entire cuticular hydrocarbon mixture or a subset of it. METHODS Study Organism Twelve queenright colonies of C. aethiops (Latr.) were collected in April 2008 in the Italian Apennines. Six colonies were collected in Castel del Rio (44 21062.830 N, 11520 30.920 E) and six in Moraduccio (44100 32.750 N, 11290 3.080 E). Each colony was housed in a plastic box (27 17 cm and 9.5 cm high) with a plaster floor, serving as a nest. This was connected to another plastic box of the same size, serving as a foraging arena. The ants were fed twice a week with diluted honey and mealworms, Tenebrio molitor; water was provided ad libitum. The nests were kept in a climate room, at 25 2 C, and a 12:12 h light:dark regime. Ants were deprived of honey at least 1 week before the experiment to increase motivation for foraging on sucrose food sources. Preparation of CHC Extracts To condition individual ants to the chemical profile of nonnestmates, we used cuticular extracts in pentane as a solvent (SigmaeAldrich); this was the ‘training odour’. For each colony source of training odour, we prepared five extracts in the following way: 11 foragers (six major and five media workers) were frozen and after 30 min their abdomen was cut off to prevent any possible extraction of pheromones produced by glands. The remaining parts of the bodies were inserted into a glass vial and covered with 1 ml of pentane. After 10 min, the extract was transferred to a new vial and the solvent allowed to evaporate; chemical extracts were then redissolved in 500 ml of pentane before use in the following experiments. Conditioning Set-up and Procedure The ant nests used in conditioning trials were provided with a vertical wooden stick in their foraging arena for the ants to climb on. For each ant to be tested, seven petri dishes (100 mm diameter 15 mm high) were prepared by coating their walls with Fluon and covering their floor with clean filter paper. Two microscope cover slips (18 18 mm) were placed on each filter paper. One cover slip was treated with 20 ml of non-nestmate extract (the training odour, as described above) deposited on its borders and the solvent was allowed to evaporate completely. In the centre of the cover slip, a droplet of sucrose solution (1 ml, 30% w/w) was deposited. On the other cover slip, we put 1 ml of water, so that the two slips looked exactly alike. The slips were positioned on opposite sides of the petri dish (see Fig. 1a). At the start of the first conditioning trial, a medium foraging worker climbing on the vertical stick was gently allowed to walk onto a small piece of paper, which was then transferred into the petri dish where the ant was allowed to get off the paper at approximately equal distance from both cover slips (see Fig. 1a). We then recorded the time the ant required to find the sucrose reward. After the ant finished drinking the sugar reward, it was picked up with soft forceps, and marked with a dot of enamel paint on its abdomen. The ant was then allowed to walk on the piece of paper again, and transferred back to the vertical stick in its colony of origin. Once back in the nest, the ants usually transferred the food to their nestmates by trophallaxis and returned to the stick. After a minimum of 1 min, the same ant was picked up again from the stick and transferred to a new petri dish for a subsequent conditioning trial. A total of six conditioning
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(a)
Sucrose Non-nestmate CHC profile
Water
Focal ant (b)
Non-nestmate CHC profile
Solvent
Focal ant Figure 1. (a) Set-up for conditioning. (b) Set-up for choice test. For (a) the time the focal ant spent finding the reward was recorded. For (b) the time the focal ant spent in each of the four quadrants was recorded for 2 min.
trials were performed per ant. The location of the slide with the sucrose reward was randomized in each trial, but the cover slip was never in the same quadrant for more than two consecutive trials to prevent the ant from associating a given spatial direction with the reward. Following conditioning, a choice test was conducted to see whether the ant had developed a positive association between non-nestmate CHC extract and the sucrose reward. This choice test consisted of presenting the focal ant, in a new petri dish, with one cover slip coated with the CHC extract used as training odour, and another one treated with solvent only (pentane, P). In this test neither sucrose nor water was provided. The cover slips represent the contextual stimulus in these tests. The petri dish was divided into four quadrants by drawing a light cross in the middle of the filter paper with a pencil, and the two slips were placed randomly in two opposite quadrants (see Fig. 1b). The ant was transferred from the stick to the petri dish, analogously to the conditioning procedure, and was allowed to search for the expected reward for 2 min (although only the chemical stimulus, and not the reward, was present during this time). The time spent by the focal ant in each quadrant was recorded using the software EthoLog (Ottoni 2000). Afterwards, 1 ml of sucrose solution was provided on the CHC-coated cover slip to avoid any possible extinction effect. The ant was then transferred back to the vertical stick in the foraging arena of the colony. As a control, naïve ants were subjected directly to the choice test, without conditioning. Ten naïve ants were tested from each colony used for conditioning (40 control ants in total). Aggression Tests Once the choice test was completed, the conditioned ant was left in its colony for 10 min and then underwent an aggression test. We measured aggression by placing the focal ant from the stick into a neutral arena (50 mm diameter 60 mm height), where she was allowed to habituate to the new environment for 1 min. After this, a dead ant (freshly killed by freezing), from its own colony (nestmate control), the colony carrying the CHC profile used as the training
841
odour or a novel colony (non-nestmate control), was placed in the centre of the arena. In these tests, the dead ant represents the contextual stimulus. The small size of the arena allowed for rapid contact between the focal ant and the freshly killed ant (average delay for first contact SE ¼ 7.2 1.4 s). The behaviour of the focal ant towards the dead ant was recorded continuously for 3 min using the software EthoLog (Ottoni 2000). We evaluated any possible aggression elicited by the target (dead ant) by recording six categorical behaviours: (1) no contact between the focal ant and the target; (2) antennation; (3) grooming; (4) mandible opening; (5) biting; and (6) gaster flexing (attempting to spread formic acid). We established a baseline of aggression before starting the conditioning experiments. Ten unconditioned focal ants and 10 target ants were used for each set of dyadic encounters (with an ant from the focal ant’s own colony, the colony carrying the CHC profile used as the training odour or a novel colony), giving a total of 30 aggression tests. This was replicated four times, using three new colonies for each replicate, for a total of 12 colonies and 120 aggression tests. In summary, 15 ants were conditioned. The aggression tests involving these conditioned focal ants were repeated five times with target nestmate ants, five times with target ants originating from the colony whose cuticular extracts were used as the training odour and five times with target ants from a novel non-nestmate colony. This was replicated four times, involving a total of 12 colonies and 60 conditioned ants. As a control for the conditioning experiment, 40 additional naïve ants were used in choice tests, and to establish the baseline of aggression, 120 additional unconditioned ants were used.
Data Analysis There were no significant differences in searching time between colonies (KruskaleWallis ANOVA: c24 < 9.49, P > 0.05), so the data could be pooled. Searching time in the course of the six conditioning trials (1e6) were analysed using a Friedman ANOVA followed by multiple comparisons (Siegel & Castellan 1988). For the choice tests, a preference index (PI) was calculated using the following formula, where tTO and tP is the time spent in quadrant TO (training odour) and P (pentane), respectively.
PI ¼
tTO tP tTO þ tP
PI data were normally distributed (KolmogoroveSmirnov test: P > 0.2 in all cases). There was no colony effect on PI (ANOVA: conditioned ants: F3,56 ¼ 1.47, P ¼ 0.23; naïve ants: F3,36 ¼ 1.08, P ¼ 0.37); hence the data were pooled. If ants preferred one quadrant over the other, the PI would differ significantly from zero. This was analysed using a t test for single means. Preference for quadrants was also compared between conditioned and unconditioned (naïve) ants using a t test for independent samples. The different behaviours observed during aggression tests were scored so that the most aggressive behaviour was assigned the highest score: 0 ¼ antennation and grooming; 1 ¼ mandible opening; 2 ¼ biting; 3 ¼ gaster flexing. The maximum level of aggression was recorded, giving a value between 0 and 3 for every ant tested. Since there was no colony effect on overall aggression (KruskaleWallis ANOVA: P > 0.05), data could be pooled. The difference in baseline aggression of unconditioned workers towards nestmates, non-nestmate ants bearing the CHC profile used for conditioning and novel non-nestmate ants was analysed with a Wilcoxon signed-ranks tests. The data were not normally distributed (KolmogoroveSmirnov test: P < 0.05) and thus a generalized linear model (GLM; Poisson
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distribution, log link function) was used for analysing aggression levels among naïve ants (the baseline of aggression) and conditioned ants. All analyses were performed using Statistica 7.1 (StatSoft, Tulsa, OK, U.S.A.).
0.4 Conditioned Unconditioned
0.35 0.3
Conditioning The searching time decreased significantly in the course of the six conditioning trials (Friedman ANOVA: c25 ¼ 97.17, N ¼ 60, P < 0.05; Fig. 2). In particular, searching time decreased markedly from the second trial on, showing that ants directed their search more promptly towards the training odour. In the control group (naïve ants), the PI did not differ significantly from zero (one-sample t test: t39 ¼ 0.27, P ¼ 0.79), meaning that ants had no spontaneous preference for a particular quadrant during the choice test. However, for the conditioned ants, the PI differed significantly from zero (one-sample t test: t59 ¼ 19.59, P < 0.001). During the choice test, conditioned ants spent significantly more time in the quadrant containing the training odour than control (naïve) ants (t test: t98 ¼ 10.60, P < 0.001; Fig. 3). Aggression When the baseline of aggression was established, aggression of unconditioned ant workers was significantly higher towards nonnestmate ants bearing the CHC profile used for conditioning (b) or novel non-nestmate ants (c) than towards nestmates (a; Wilcoxon signed-ranks test: aaeab: T ¼ 0.00, P < 0.01; aaeac: T ¼ 8.00, P < 0.01; abeac: T ¼ 7.50, P > 0.05; Bonferroni correction, adjusted significance level a ¼ 0.025). The results of the aggression tests are shown in Fig. 4. Aggression levels of conditioned ants against either ants from their own colony, non-nestmate ants bearing the CHC profile used for conditioning or novel non-nestmate ants did not differ significantly from the baseline of aggression of unconditioned ants (GLM: Wald c22 ¼ 2.783, P ¼ 0.249), showing that conditioning did not interfere with recognition abilities and expression of aggressive behaviour.
Preference index
RESULTS
0.25 0.2 0.15 0.1 0.05 0
Figure 3. Preference index of conditioned and unconditioned (naïve) ants (mean SE).
developed in an appetitive context, might affect nestmate discrimination, which is usually expressed in an aggressive context. We conditioned freely walking individual C. aethiops workers to associate a cuticular extract of a non-nestmate with a sucrose reward, showing that stimuli that originally elicit aggression in ants can be associated with food. This suggests plasticity in the significance of cues/signals that are typically important in the modulation of social interactions. The fact that the conditioned ant kept coming back to the stick, and that during the trials more ants were present on the stick, shows clearly that our set-up was appropriate to simulate a foraging context. Ants formed the association between the chemical blend and reward very rapidly; right after the first training trial, the searching time needed by the ant to find the reward decreased significantly. A subsequent choice test showed that the association between the
3.5 Conditioned Unconditioned
DISCUSSION
3
We investigated whether ants are able to associate a cuticular hydrocarbon mixture with food, and whether this association,
Aggression index
Searching time (s)
2.5
280 260 240 220 200 180 160 140 120 100 80 60 40 20 0
2
1.5
1
0.5
0
1
2
3
4
5
6
Trial Figure 2. Average searching time for each conditioning trial (N ¼ 60) showing median, quartiles and range.
Nestmate
Familiar
Novel
Figure 4. Aggression index of conditioned and unconditioned ants versus nestmates, familiar non-nestmates and novel non-nestmates, respectively (mean SE). Note that the ‘familiar non-nestmates’ are novel non-nestmates for the unconditioned ants, since they have not been familiarized by conditioning.
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food and the non-nestmate CHC profile was robustly established, although we do not know whether the ants learned the entire CHC profile or a subset of it (see below). Individual olfactory learning in ants was shown for the first time only recently (Dupuy et al. 2006), by conditioning individual ants to pure volatile substances using a Y-maze. Although the substances tested were present in either flowers or honeybee pheromones (see Balderrama et al. 2002), they were not potential cues involved in nestmate recognition. In addition, (S. Dreier et al., unpublished data) showed that individual Camponotus ants can be conditioned to associate single synthetic long-chain hydrocarbons with a sugar reward. Unlike these two studies, in which only pure synthetic chemical substances were used as conditioned stimuli, we used a multicomponent blend, namely the cuticular extract of C. aethiops workers, consisting of about 40 different hydrocarbons (Van Zweden et al. 2009), as training odour. The extracted cuticular hydrocarbons have the same properties as the natural mix on the ant’s cuticle, as shown by Bonavita-Cougourdan et al. (1987), Morel et al. (1988) and Nowbahari et al. (1990). These authors washed ants in solvent to remove the cuticular hydrocarbons, and then applied on these washed ants the cuticular extract of a nestmate or non-nestmate. This restored the expected level of aggression, while washed ants did not elicit an aggressive response. In addition, Ozaki et al. (2005) and Guerrieri & d’Ettorre (2008) showed that a non-nestmate cuticular extract, when applied to a glass bead (or rod), elicits aggression, proving that the CHC extract alone is sufficient to promote aggression. It remains to be determined whether the ants indeed learned the entire CHC profile or only some specific compounds within the profile. If the ants learned the entire CHC mixture of the profile, or a large part of it, learning could have happened in various ways. According to the elemental theory of learning (i.e. Rescorla & Wagner 1972), a mixture is processed as the sum of its components (AB ¼ A þ B), while the configural theory (i.e. Pearce 1987) suggests that the properties of a mixture are different from the properties of the components (AB ¼ X s A þ B; Giurfa 2003). In honeybees, a new model has been suggested: the key odorant hypothesis, which encompasses features of both elemental and configural learning (Reinhard et al. 2010). Even though honeybees were able to learn all individual odorants used in the experiments; when conditioned to a complex mixture, only certain key compounds were learned, suggesting that some odorants are more representative of the mixture than others. In our experiment, we cannot distinguish between these models. However, recent evidence indicates that methyl-branched alkanes are more important in nestmate recognition than linear alkanes (i.e. Guerrieri et al. 2009), suggesting that only key compounds of the cuticular hydrocarbon blend present on the ant might be used in recognition. Future studies should focus on which classes of hydrocarbons are learned when a mixture is used as the training odour. In our experiments, the association of the CHC profile with food did not affect the social meaning of the non-nestmate cuticular profile. Indeed, after conditioning, the focal ants were as aggressive towards an individual bearing the familiar odour (the cuticular profile associated with food) as towards an individual bearing a novel non-nestmate cuticular hydrocarbon profile. Context-dependent learning in the visual modality has been shown in a variety of organisms (e.g. ants: Chameron et al. 1998; honeybees: Collett et al. 1993; humans: Smith & Vela 2001; for a review on insects, see Collett et al. 2003). However, contextdependent olfactory learning in insects has only been shown in the cricket Gryllus bimaculatus (Matsumoto & Mizunami 2004) and the cockroach Periplaneta americana (Sato et al. 2006). For instance, individual crickets were conditioned to select one odour and to avoid
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another odour in one context (darkness), and to do the opposite in another context (light). In this experiment, neither the light condition nor the odour could predict a reward (water) or non-reward (saline solution); only a combination of the two could. In contrast, Drosophila melanogaster is not capable of solving a similar task (Yarali et al. 2008). Groups of flies were conditioned in a ‘biconditional discrimination’ design. One odour was paired with an electric shock in darkness, but not in light; another odour was paired with a shock in light, but not darkness. Flies failed to show any preference for the right odour in the specific light conditions. Therefore, interactions between different modalities (i.e. visual and olfactory), which are necessary for context-dependent learning, are not a general feature of all insects. Two explanations of context-dependent learning have been proposed (Pearce & Bouton 2001). In the occasion-setting theory, the contextual stimulus (in our case, the cover slip or the target ant) ‘sets the occasion’ for responding to another conditioned stimulus (in our case, the training odour: the non-nestmate CHC profile), without forming an association with the unconditioned stimulus (the food reward) itself. In the configural theory, an individual perceives different CSecontext combinations as different stimuli. Although learning in our experimental design was clearly context dependent, further studies will be needed to distinguish between these two theories. Living in a complex and changing environment requires being aware that the same cues/signals might have different significance according to context and the role of individual experience is extremely important. This learning capability and flexibility is an adaptive trait for the ants, as it maximizes their fitness and survival: on the one hand, ants can eventually find food by following the smell of non-nestmates, but still avoid or attack enemies that they encounter. Our study shows that ants have complex cognitive abilities and are capable of distinguishing the meaning of identical cues according to the context in which they are perceived. Acknowledgments This work was supported by the Marie Curie Excellence grant CODICES (MEXT-CT-2004-014202) and a Freia grant from the Faculty of Science, University of Copenhagen, both assigned to P.d’E. We are grateful to all members of the Copenhagen Centre for Social Evolution for a stimulating work environment. We thank three anonymous referees for useful comments and suggestions. References Balderrama, J., Núñez, J., Guerrieri, F. J. & Giurfa, M. 2002. Different functions of two alarm substances in the honeybee. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, 188, 485e491. Bitterman, M. E., Menzel, R., Fietz, A. & Schafer, S. 1983. Classical conditioning of proboscis extension in honeybees (Apis mellifera). Journal of Comparative Psychology, 97, 107e119. Bonavita-Cougourdan, A., Clément, J. L. & Lange, C. 1987. Nestmate recognition: the role of cuticular hydrocarbons in the ant Camponotus vagus Scop. Journal of Entomological Science, 22, 1e10. Breed, M. D. 1998. Pheromones of the honey bee. BioScience, 48, 463e470. de Brito-Sánchez, M. G., Deisig, N., Sandoz, J. C. & Giurfa, M. 2008. Neurobiology of olfactory communication in a social insect: the honeybee. In: Sociobiology of Communication: an Interdisciplinary Perspective (Ed. by P. d’Ettorre & D. P. Hughes), pp. 119e138. Oxford: Oxford University Press. Chameron, S., Schatz, B., Pastergue Ruiz, I., Beugnon, G. & Collett, T. S. 1998. The learning of a sequence of visual patterns by the ant Cataglyphis cursor. Proceedings of the Royal Society B, 265, 2309e2313. Cole, P. & Adamo, S. A. 2005. Cuttlefish (Sepia officinalis: Cephalopoda) hunting behavior and associative learning. Animal Cognition, 8, 27e30. Collett, T. S., Fry, R. & Wehner, R. 1993. Sequence learning by honeybees. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, 172, 693e706. Collett, T. S., Fauria, K. & Dale, K. 2003. Contextual cues and insect navigation. In: The Neurobiology of Spatial Behaviour (Ed. by K. J. Jeffery), pp. 67e82. Oxford: Oxford University Press.
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