Group phenotypic composition drives task performances in ants.

Differences in individual behaviour within a group can give rise to functional dissimilarities between groups, particularly in social animals. However, how individual behavioural phenotypes translate into the group phenotype remains unclear. Here, we investigate whether individual behavioural type affects group performance in a eusocial species, the ant Aphaenogaster senilis. We measured individual behavioural traits and created groups of workers with similar behavioural type, either high-exploratory or low-exploratory workers. We tested these groups in four different, ecologically relevant, tasks: reaction to an intruder, prey retrieval from a maze, nest relocation and tool use. We show that, compared to groups of low-exploratory workers, groups of high-exploratory workers were more aggressive towards intruders, more efficient in collecting prey, faster in nest relocation and more likely to perform tool use. Our results demonstrate a strong link between individual and collective behaviour in ants. This supports the 'behavioural type hypothesis' for group dynamics, which suggests that an individual's behaviour in a social environment reflects its own behavioural type. The average behavioural phenotype of a group can therefore be predicted from the behavioural types of individual group members.

Queen fecundity, worker entourage and cuticular chemistry in the ant Formica fusca.

Cooperative breeding entails conflicts over reproductive shares that may be settled in different ways. In ants, where several queens simultaneously reproduce in a colony, both queens and workers may influence the reproductive apportionment and offspring quality. Queens may vary in their intrinsic fecundity, which may influence the size of the worker entourage attending individual queens, and this may eventually dictate the reproductive output of a queen. We tested whether the reproductive success of queens is affected by the size of their worker entourage, their fecundity at the onset of the reproductive season, and whether the queen cuticular hydrocarbon profile carries information on fecundity. We show that in the ant Formica fusca both queen fecundity and egg hatching success increase with the size of their entourage, and that newly hatched larvae produced by initially highly fecund queens are smaller. Furthermore, higher relatedness among workers increased queen fecundity. Finally, the queens that received a large worker entourage differed in the cuticular chemistry from those that received a small worker entourage. Our results thus show that workers play a pivotal role in determining queen fitness, that high intracolony relatedness among workers enhances the overall reproductive output in the colony, and that queen fecundity is reflected in their cuticular hydrocarbon profile.

Ants act as olfactory bio-detectors of tumours in patient-derived xenograft mice.

Early detection of cancer is critical in medical sciences, as the sooner a cancer is diagnosed, the higher are the chances of recovery. Tumour cells are characterized by specific volatile organic compounds (VOCs) that can be used as cancer biomarkers. Through olfactory associative learning, animals can be trained to detect these VOCs. Insects such as ants have a refined sense of smell, and can be easily and rapidly trained with olfactory conditioning. Using urine from patient-derived xenograft mice as stimulus, we demonstrate that individual ants can learn to discriminate the odour of healthy mice from that of tumour-bearing mice and do so after only three conditioning trials. After training, they spend approximately 20% more time in the vicinity of the learned odour than beside the other stimulus. Chemical analyses confirmed that the presence of the tumour changed the urine odour, supporting the behavioural results. Our study demonstrates that ants reliably detect tumour cues in mice urine and have the potential to act as efficient and inexpensive cancer bio-detectors.

Formic acid modulates latency and accuracy of nestmate recognition in carpenter ants.

Decision-making processes face the dilemma of being accurate or faster, a phenomenon that has been described as speed-accuracy trade-off in numerous studies on animal behaviour. In social insects, discriminating between colony members and aliens is subject to this trade-off as rapid and accurate rejection of enemies is of primary importance for the maintenance and ecological success of insect societies. Recognition cues distinguishing aliens from nestmates are embedded in the cuticular hydrocarbon (CHC) layer and vary among colonies. In walking carpenter ants, exposure to formic acid (FA), an alarm pheromone, improves the accuracy of nestmate recognition by decreasing both alien acceptance and nestmate rejection. Here, we studied the effect of FA exposure on the spontaneous aggressive mandible opening response (MOR) of harnessed Camponotus aethiops ants presented with either nestmate or alien CHCs. FA modulated both MOR accuracy and the latency to respond to odours of conspecifics. In particular, FA decreased the MOR towards nestmates but increased it towards aliens. Furthermore, FA decreased MOR latency towards aliens but not towards nestmates. As response latency can be used as a proxy of response speed, we conclude that contrary to the prediction of the speed-accuracy trade-off theory, ants did not trade off speed against accuracy in the process of nestmate recognition.

The Scent of Ant Brood: Caste Differences in Surface Hydrocarbons of Formica exsecta Pupae.

Chemical communication is common across all organisms. Insects in particular use predominantly chemical stimuli in assessing their environment and recognizing their social counterparts. One of the chemical stimuli used for recognition in social insects, such as ants, is the suite of long-chain, cuticular hydrocarbons. In addition to providing waterproofing, these surface hydrocarbons serve as a signature mixture, which ants can perceive, and use to distinguish between strangers and colony mates, and to determine caste, sex, and reproductive status of another individual. They can be both environmentally and endogenously acquired. The surface chemistry of adult workers has been studied extensively in ants, yet the pupal stage has rarely been considered. Here we characterized the surface chemistry of pupae of Formica exsecta, and examine differences among sexes, castes (reproductive vs. worker), and types of sample (developing individual vs. cocoon envelope). We found quantitative and qualitative differences among both castes and types of sample, but male and female reproductives did not differ in their surface chemistry. We also found that the pupal surface chemistry was more complex than that of adult workers in this species. These results improve our understanding of the information on which ants base recognition, and highlights the diversity of surface chemistry in social insects across developmental stages.

Ant cuticular hydrocarbons are heritable and associated with variation in colony productivity.

In social insects, cuticular hydrocarbons function in nest-mate recognition and also provide a waxy barrier against desiccation, but basic evolutionary features, including the heritability of hydrocarbon profiles and how they are shaped by natural selection are largely unknown. We used a new pharaoh ant (Monomorium pharaonis) laboratory mapping population to estimate the heritability of individual cuticular hydrocarbons, genetic correlations between hydrocarbons, and fitness consequences of phenotypic variation in the hydrocarbons. Individual hydrocarbons had low to moderate estimated heritability, indicating that some compounds provide more information about genetic relatedness and can also better respond to natural selection. Strong genetic correlations between compounds are likely to constrain independent evolutionary trajectories, which is expected, given that many hydrocarbons share biosynthetic pathways. Variation in cuticular hydrocarbons was associated with variation in colony productivity, with some hydrocarbons experiencing strong directional selection. Altogether, this study builds on our knowledge of the genetic architecture of the social insect hydrocarbon profile and indicates that hydrocarbon variation is shaped by natural selection.

Trail pheromone modulates subjective reward evaluation in Argentine ants.

The Argentine ant, Linepithema humile, is native to South America but has become one of the most invasive species in the world. These ants heavily rely on trail pheromones for foraging, and previous studies have focused on such signals to develop a strategy for chemical control. Here, we studied the effects of pre-exposure to the trail pheromone on sugar acceptance and olfactory learning in Argentine ants. We used the synthetic trail pheromone component (Z)-9-hexadecenal, which triggers the same attraction and trail-following behavior as the natural trail pheromone. We found that pre-exposure to (Z)-9-hexadecenal increases the acceptance of sucrose solutions of different concentrations, thus changing the ants' subjective evaluation of a food reward. However, although ants learned to associate an odor with a sucrose reward, pheromone pre-exposure affected neither the learning nor the mid-term memory of the odor-reward association. Taking into account the importance of the Argentine ant as a pest and invasive organism, our results highlight the importance of pheromonal cues in resource evaluation, a fact that could be useful in control strategies implemented for this species.

Pheromone-Induced Accuracy of Nestmate Recognition in Carpenter Ants: Simultaneous Decrease in Type I and Type II Errors.

The ecological and evolutionary success of social insects relies on their ability to efficiently discriminate between group members and aliens. Nestmate recognition occurs by phenotype matching, the comparison of the referent (colony) phenotype to the one of an encountered individual. Based on the level of dissimilarity between the two, the discriminator accepts or rejects the target. The tolerated degree of mismatch is predicted by the acceptance threshold model, which assumes adaptive threshold shifts depending on the costs of discrimination errors. Inherent in the model is that rejection (type I) and acceptance (type II) errors are reciprocally related: if one type decreases, the other increases. We studied whether alarm pheromones modulate the acceptance threshold. We exposed Camponotus aethiops ants to formic acid and subsequently measured aggression toward nestmates and nonnestmates. Formic acid induced both more nonnestmate rejection and more nestmate acceptance than a control treatment, thus uncovering an unexpected effect of an alarm pheromone on responses to nestmates. Nestmate discrimination accuracy was improved via a decrease in both types of errors, a result that cannot be explained by a shift in the acceptance threshold. We propose that formic acid increases the amount of information available to the ants, thus decreasing the perceived phenotypic overlap between nestmate and nonnestmate recognition cues. This mechanism for improved discrimination reveals a novel function of alarm pheromones in recognition processes and may have far-reaching implications in our understanding of the modus operandi of recognition systems in general.

Necessary Conditions for Social Transmission of Food Preference Through Feces in the House Mouse, Mus musculus domesticus.

Mice can obtain information about a new food source through olfactory cues of conspecifics and consequently develop an attraction for this diet. The social transmission of food preference (STFP) takes place directly, during an encounter with a conspecific or indirectly, via feces. In indirect STFP, the digestive process can degrade odorant compounds characterizing the food, impairing the matching between feces and food. In a previous study, indirect STFP was efficient when the information support was a composite odorant. We, thus, hypothesized that the acquisition of indirect STFP depends on the multiplicity of the odorant compounds present in diets. Tested in female house mice (Mus musculus domesticus) our results showed that a single odorant compound as information support was not sufficient to induce an indirect STFP. Chemical analysis did not reveal the presence of the compounds in feces suggesting that the degradation of diet cues during the digestive process prevented the pairing between feces and food. By using a process that limits the degradation of molecules, we performed indirect STFP when the pertinent information was represented by a single odorant compound and multiple odorant compounds. Unlike with multiple odorant compounds, our results did not show a clear indirect STFP with single odorant compound, despite their presence in feces confirmed by chemical analysis. We conclude that constraints associated to indirect STFP can be removed by the multiplicity of information characterizing the diet both by reducing the degradation risk during the digestive process and by allowing an accurate assessment of diet consumed by the conspecific.

Seasonal Variations in Femoral Gland Secretions Reveals some Unexpected Correlations Between Protein and Lipid Components in a Lacertid Lizard.

Animals modulate intraspecific signal shape and intensity, notably during reproductive periods. Signal variability typically follows a seasonal scheme, traceable through the expression of visual, acoustic, chemical and behavioral patterns. The chemical channel is particularly important in lizards, as demonstrated by well-developed epidermal glands in the cloacal region that secrete lipids and proteins recognized by conspecifics. In males, the seasonal pattern of gland activity is underpinned by variation of circulating androgens. Changes in the composition of lipid secretions convey information about the signaler's quality (e.g., size, immunity). Presumably, individual identity is associated with a protein signature present in the femoral secretions, but this has been poorly investigated. For the first time, we assessed the seasonal variability of the protein signal in relation to plasma testosterone level (T), glandular activity and the concentration of provitamin D3 in the lipid fraction. We sampled 174 male common wall lizards (Podarcis muralis) over the entire activity season. An elevation of T was observed one to two months before the secretion peak of lipids during the mating season; such expected delay between hormonal fluctuation and maximal physiological response fits well with the assumption that provitamin D3 indicates individual quality. One-dimensional electrophoretic analysis of proteins showed that gel bands were preserved over the season with an invariant region; a result in agreement with the hypothesis that proteins are stable identity signals. However, the relative intensity of bands varied markedly, synchronously with that of lipid secretion pattern. These variations of protein secretion suggest additional roles of proteins, an issue that requires further studies.

Cooperative defence operates by social modulation of biogenic amine levels in the honey bee brain.

The defence of a society often requires that some specialized members coordinate to repel a threat at personal risk. This is especially true for honey bee guards, which defend the hive and may sacrifice their lives upon stinging. Central to this cooperative defensive response is the sting alarm pheromone, which has isoamyl acetate (IAA) as its main component. Although this defensive behaviour has been well described, the neural mechanisms triggered by IAA to coordinate stinging have long remained unknown. Here we show that IAA upregulates brain levels of serotonin and dopamine, thereby increasing the likelihood of an individual bee to attack and sting. Pharmacological enhancement of the levels of both amines induces higher defensive responsiveness, while decreasing them via antagonists decreases stinging. Our results thus uncover the neural mechanism by which an alarm pheromone recruits individuals to attack and repel a threat, and suggest that the alarm pheromone of honey bees acts on their response threshold rather than as a direct trigger.

Pheromones modulate responsiveness to a noxious stimulus in honey bees.

Pheromones are chemical substances released into the environment by an individual, which trigger stereotyped behaviors and/or physiological processes in individuals of the same species. Yet, a novel hypothesis has suggested that pheromones not only elicit innate responses but also contribute to behavioral plasticity by affecting the subjective evaluation of appetitive or aversive stimuli. To test this hypothesis, we exposed bees to three pheromonal components whose valence was either negative (i.e. associated with aversive events: isopentyl acetate and 2-heptanone) or positive (i.e. associated with appetitive events: geraniol). We then determined the effect of this exposure on the subjective evaluation of aversive stimuli by quantifying responsiveness to a series of increasing electric shock voltages before and after exposure. Two experiments were conducted varying the time lapse between shock series (15 min in experiment 1, and 24 h in experiment 2). In experiment 1, we observed a general decrease of shock responsiveness caused by fatigue, due to the short lapse of time between the two series of shocks. This decrease could only be counteracted by isopentyl acetate. The enhancing effect of isopentyl acetate on shock responsiveness was also found in experiment 2. Conversely, geraniol decreased aversive responsiveness in this experiment; 2-heptanone did not affect aversive responsiveness in any experiment. Overall, our results demonstrate that certain pheromones modulate the salience of aversive stimuli according to their valence. In this way, they would affect the motivation to engage in aversive responses, thus acting as modulators of behavioral plasticity.

Chemical Characterization of Young Virgin Queens and Mated Egg-Laying Queens in the Ant Cataglyphis cursor: Random Forest Classification Analysis for Multivariate Datasets.

Social insects are well known for their extremely rich chemical communication, yet their sex pheromones remain poorly studied. In the thermophilic and thelytokous ant, Cataglyphis cursor, we analyzed the cuticular hydrocarbon profiles and Dufour's gland contents of queens of different age and reproductive status (sexually immature gynes, sexually mature gynes, mated and egg-laying queens) and of workers. Random forest classification analyses showed that the four groups of individuals were well separated for both chemical sources, except mature gynes that clustered with queens for cuticular hydrocarbons and with immature gynes for Dufour's gland secretions. Analyses carried out with two groups of females only allowed identification of candidate chemicals for queen signal and for sexual attractant. In particular, gynes produced more undecane in the Dufour's gland. This chemical is both the sex pheromone and the alarm pheromone of the ant Formica lugubris. It may therefore act as sex pheromone in C. cursor, and/or be involved in the restoration of monogyny that occurs rapidly following colony fission. Indeed, new colonies often start with several gynes and all but one are rapidly culled by workers, and this process likely involves chemical signals between gynes and workers. These findings open novel opportunities for experimental studies of inclusive mate choice and queen choice in C. cursor.

Aversive learning of odor-heat associations in ants.

Ants have recently emerged as useful models for the study of olfactory learning. In this framework, the development of a protocol for the appetitive conditioning of the maxilla-labium extension response (MaLER) provided the possibility of studying Pavlovian odor-food learning in a controlled environment. Here we extend these studies by introducing the first Pavlovian aversive learning protocol for harnessed ants in the laboratory. We worked with carpenter ants Camponotus aethiops and first determined the capacity of different temperatures applied to the body surface to elicit the typical aversive mandible opening response (MOR). We determined that 75°C is the optimal temperature to induce MOR and chose the hind legs as the stimulated body region because of their high sensitivity. We then studied the ability of ants to learn and remember odor-heat associations using 75°C as the unconditioned stimulus. We studied learning and short-term retention after absolute (one odor paired with heat) and differential conditioning (a punished odor versus an unpunished odor). Our results show that ants successfully learn the odor-heat association under a differential-conditioning regime and thus exhibit a conditioned MOR to the punished odor. Yet, their performance under an absolute-conditioning regime is poor. These results demonstrate that ants are capable of aversive learning and confirm previous findings about the different attentional resources solicited by differential and absolute conditioning in general.

Olfactory experience shapes the evaluation of odour similarity in ants: a behavioural and computational analysis.

Perceptual similarity between stimuli is often assessed via generalization, the response to stimuli that are similar to the one which was previously conditioned. Although conditioning procedures are variable, studies on how this variation may affect perceptual similarity remain scarce. Here, we use a combination of behavioural and computational analyses to investigate the influence of olfactory conditioning procedures on odour generalization in ants. Insects were trained following either absolute conditioning, in which a single odour (an aldehyde) was rewarded with sucrose, or differential conditioning, in which one odour (the same aldehyde) was similarly rewarded and another odour (an aldehyde differing in carbon-chain length) was punished with quinine. The response to the trained odours and generalization to other aldehydes were assessed. We show that olfactory similarity, rather than being immutable, varies with the conditioning procedure. Compared with absolute conditioning, differential conditioning enhances olfactory discrimination. This improvement is best described by a multiplicative interaction between two independent processes, the excitatory and inhibitory generalization gradients induced by the rewarded and the punished odour, respectively. We show that olfactory similarity is dramatically shaped by an individual's perceptual experience and suggest a new hypothesis for the nature of stimulus interactions underlying experience-dependent changes in perceptual similarity.

Biological activity of the enantiomers of 3-methylhentriacontane, a queen pheromone of the ant Lasius niger.

Queen pheromones are essential for regulation of the reproductive division of labor in eusocial insect species. Although only the queen is able to lay fertilized eggs and produce females, in some cases workers may develop their ovaries and lay male-destined eggs, thus reducing the overall colony efficiency. As long as the queen is healthy, it is usually in the workers' collective interest to work for the colony and remain sterile. Queens signal their fertility via pheromones, which may have a primer effect, affecting the physiology of workers, or a releaser effect, influencing worker behavior. The queen pheromone of the ant Lasius niger was among the first queen pheromones of social insects to be identified. Its major component is 3-methylhentriacontane (3-MeC31), which is present in relatively large amounts on the queen's cuticle and on her eggs. 3-MeC31 regulates worker reproduction by inhibiting ovarian development. Most monomethyl-branched hydrocarbons can exist in two stereoisomeric forms. The correct stereochemistry is fundamental to the activity of most bioactive molecules, but this has rarely been investigated for methyl-branched hydrocarbons. Here, we tested the bioactivity of the (S)- and (R)-enantiomers of 3-MeC31, and found that whereas both enantiomers were effective in suppressing worker ovarian development, (S)-3-MeC31 appeared to be more effective at suppressing aggressive behavior by workers. This suggests that the natural pheromone may be a mixture of the two enantiomers. The enantiomeric ratio produced by queens remains unknown because of the small amounts of the compound available from each queen.

Chemical disguise of myrmecophilous cockroaches and its implications for understanding nestmate recognition mechanisms in leaf-cutting ants.

BACKGROUND: Cockroaches of the genus Attaphila regularly occur in leaf-cutting ant colonies. The ants farm a fungus that the cockroaches also appear to feed on. Cockroaches disperse between colonies horizontally (via foraging trails) and vertically (attached to queens on their mating flights). We analysed the chemical strategies used by the cockroaches to integrate into colonies of Atta colombica and Acromyrmex octospinosus. Analysing cockroaches from nests of two host species further allowed us to test the hypothesis that nestmate recognition is based on an asymmetric mechanism. Specifically, we test the U-present nestmate recognition model, which assumes that detection of undesirable cues (non-nestmate specific substances) leads to strong rejection of the cue-bearers, while absence of desirable cues (nestmate-specific substances) does not necessarily trigger aggression. RESULTS: We found that nests of Atta and Acromyrmex contained cockroaches of two different and not yet described Attaphila species. The cockroaches share the cuticular chemical substances of their specific host species and copy their host nest's colony-specific cuticular profile. Indeed, the cockroaches are accepted by nestmate but attacked by non-nestmate ant workers. Cockroaches from Acromyrmex colonies bear a lower concentration of cuticular substances and are less likely to be attacked by non-nestmate ants than cockroaches from Atta colonies. CONCLUSIONS: Nest-specific recognition of Attaphila cockroaches by host workers in combination with nest-specific cuticular chemical profiles suggest that the cockroaches mimic their host's recognition labels, either by synthesizing nest-specific substances or by substance transfer from ants. Our finding that the cockroach species with lower concentration of cuticular substances receives less aggression by both host species fully supports the U-present nestmate recognition model. Leaf-cutting ant nestmate recognition is thus asymmetric, responding more strongly to differences than to similarities.

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants.

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.