Influence of Host Plants and Tending Ants on the Cuticular Hydrocarbon Profile of a Generalist Myrmecophilous Caterpillar.

In myrmecophilous organisms, which live in symbiosis with ants, cuticular hydrocarbons (CHCs) play a pivotal role in interspecific communication and defense against chemical-oriented predators. Although these interactions form complex information webs, little is known about the influence of biotic environmental factors on the CHC profiles of myrmecophiles. Here, we analyzed the effect of different host plants and tending ants on the larval CHC profile of Synargis calyce (Lepidoptera: Riodinidae), a polyphagous species with facultative myrmecophily. Groups of caterpillars were fed individually with three host plant species (without tending ants), and with two tending ant species. Through gas chromatography analysis, we compared the cuticular profiles of treatments and found a high similarity between plants and caterpillars (65-82%), but a low similarity between caterpillars and their tending ants (30 - 25%). Cluster analysis showed that caterpillars, ants, and plants form distinct groups, indicating that S. calyce caterpillars have their own chemical profile. These results are similar to those observed for Lycaenidae caterpillars indicating that there is functional convergence in the chemical strategies used by myrmecophilous caterpillar species with similar ecology. Also, the results suggest that the cuticular compounds of S. calyce are primarily influenced by their host plants rather than their tending ants. Thus, we propose that these caterpillars present a trade-off between camouflage and directly informing their presence to ants, maintaining their unique chemical profile, though slightly affected by biotic environmental factors.

Chemical signatures of egg maternity and Dufour's gland in Vespine wasps.

Cuticular hydrocarbons (CHCs) are often used in the chemical communication among social insects. CHCs can be used in nestmate recognition and as queen pheromones, the latter allows the regulation of the reproductive division of labor. In the common wasp Vespula vulgaris, CHCs and egg-marking hydrocarbons are caste-specific, being hydrocarbon queen pheromones and egg maternity signals. Whether these compounds are conserved among other Vespinae wasps remains unknown. Queens, virgin queens, reproductive workers, and workers belonging to four different wasp species, Dolichovespula media, Dolichovespula saxonica, Vespa crabro, and Vespula germanica, were collected and studied. The cuticular hydrocarbons, egg surface, and Dufour's gland composition were characterized and it was found that chemical compounds are caste-specific in the four species. Quantitative and qualitative differences were detected in the cuticle, eggs, and Dufour's gland. Some specific hydrocarbons that were shown to be overproduced in the cuticle of queens were also present in higher quantities in queen-laid eggs and in their Dufour's gland. These hydrocarbons can be indicated as putative fertility signals that regulate the division of reproductive labor in these Vespine societies. Our results are in line with the literature for V. vulgaris and D. saxonica, in which hydrocarbons were shown to be conserved queen signals. This work presents correlative evidence that queen chemical compounds are found not only over the body surface of females but also in other sources, such as the Dufour's gland and eggs.

Close-range cues used by males of Polistes dominula in sex discrimination.

Sexual pheromones are chemical molecules responsible for mediating sex recognition and mating events. Long- and close-range sexual pheromones act differently. The first type is released to attract potential partners, whereas the second coordinates the interactions after potential mating partners encounter each other. Cuticular hydrocarbons (CHCs) have been suggested to be important cues in the mating systems of several Hymenoptera species, although empirical data are still lacking for many species. Here, we evaluated whether males of the model species Polistes dominula can differentiate the sex of individuals based on their CHC composition. In August 2019, several post-worker emergent nests (n = 19) were collected in the vicinity of Leuven (Belgium) and taken to the lab (KU Leuven), where newly emerged females and males were sampled, marked individually, and kept in plastic boxes for at least a week before being used in the mating trials. Focal males were paired with females and males from different nests and subjected to five different conditions: (I) alive, (II) dead, (III) CHCs washed, (IV) CHCs partially returned, and (V) CHCs from the opposite sex. We videotaped the interactions for 10 min and analysed the duration and different behavioural interactions of the focal male. Our results indicate that CHCs may be used by males as cues to recognise a potential mating partner in P. dominula, since the focal males displayed specific courtship behaviours exclusively toward females. Although we cannot exclude that visual cues could also be used in combination with the chemical ones, we empirically demonstrate that CHCs may be important to convey sexual information at close range in mating systems, allowing fast decisions toward potential sexual partners or rivals.

Dominance Hierarchy, Ovarian Activity and Cuticular Hydrocarbons in the Primitively Eusocial Wasp Mischocyttarus cerberus (Vespidae, Polistinae, Mischocyttarini).

The dominance hierarchy in primitively eusocial insect societies has been shown to be mainly regulated through aggressive interactions. Females that are generally more dominant stand out and occupy the queen position, meaning that they monopolize reproduction while others perform other tasks. Chemical communication is important for maintaining social cohesion. Cuticular hydrocarbons are recognized as the main molecules responsible for mediating social interactions, especially nestmate recognition and queen signalling. Many highly eusocial groups have been studied in recent years, but primitively eusocial groups, which are key to understanding the evolution of social behavior, remain unexplored. In this study, we investigated the connection between cuticular hydrocarbons in females expressed in different social contexts in the primitively eusocial wasp Mischocyttarus cerberus. Colonies in two different ontogenetic phases, pre- and post-worker emergence, were used. We observed and categorized behavioral interactions between individual females and collected all individuals in a nest to obtain information on size, ovary activation and chemical composition. Furthermore, we conducted experiments in which the alpha (dominant) females were removed from nests to produce a new dominance hierarchy. We found that females in different hierarchical positions had small chemical difference corresponding with ovary activity. Our results support the hypothesis that cuticular hydrocarbons are associated with social context in this primitively eusocial species, with some compounds being associated with hierarchical position and ovarian activity.

Chemical Resemblance of Egg Surface Compounds and Dufour's Gland in Two Neotropical Polistinae Wasps Polistes versicolor (Olivier) and Mischocyttarus metathoracicus (de Saussure, 1854).

Chemical communication plays a major role in regulating social dynamics in social insect colonies. The most studied class of chemical compounds are the cuticular hydrocarbons (CHCs), compounds with high molecular weight that cover the insect body. CHCs are used in nestmate recognition and to signal reproductive status. Brood, in the form of larvae and eggs, is known to participate in chemical communication and social dynamics by performing hunger behaviour and inducing interaction with adults and conferring nest and maternity identity. CHCs of adults and egg surface compounds are similar in composition in social insect species. The main source of egg compounds is proposed to be Dufour's gland, an accessory reproductive gland found in several Hymenoptera females. There is still a lack of information about the level of similarity among CHCs, compounds of egg surface and Dufour's gland for several wasp species, which could provide correlational evidence about the origins of egg-marking compounds. Thus, we investigated whether egg surface compounds were more similar to CHCs or Dufour's gland secretions in two Neotropical primitively eusocial wasp species, Polistes versicolor (Olivier) and Mischocyttarus metathoracicus (de Saussure, 1854). As expected, there was a higher chemical similarity between eggs and Dufour's gland secretions in both studied species, supporting the hypothesis that this gland is the source of chemical compounds found over the eggs in these two primitively eusocial species.

Sex and lifestyle dictate learning performance in a neotropical wasp.

In contrast to extensive investigations on bee cognition, the cognitive capacities of wasps remain largely unexplored despite their key role as pollinators and predators of insect pests. Here we studied learning and memory in the neotropical wasp Mischocyttarus cerberus using a Pavlovian conditioning in which harnessed wasps respond with conditioned movements of their mouthparts to a learned odorant. We focused on the different castes, sexes, and ages coexisting within a nest and found that adults of M. cerberus learned and memorized efficiently the odor-sugar associations. In contrast, newly emerged females, but not males, were unable to learn odorants. This difference concurs with their different lifestyle as young males perform regular excursions outside the nest while young females remain in it until older age. Our results thus highlight the importance of socio-ecological constraints on wasp cognition and set the basis for mechanistic studies on learning differences across ages and castes.

Cuticular hydrocarbons as caste-linked cues in Neotropical swarm-founding wasps.

Wasps (Vespidae) are important organisms to understand the evolution of social behaviour. Wasps show different levels of sociality, which includes solitary to highly eusocial organisms. In social insect species, queens and workers differ in physiology and morphology. The Neotropical swarm-founding wasps (Epiponini) show a variety of caste syndromes. In this clade, the caste-flexibility is a unique characteristic, in which workers can become queens and swarm to start a new nest. The investigation of the caste system comparing several Epiponini species show a clear-cut morphological distinction between queens and workers, with a morphological continuum between queens and workers. However, whether cuticular hydrocarbons (CHCs) are used as cues for caste recognition in swarm-founding wasps is still unknown. We studied whether CHCs may display caste-linked differences in eleven species of Epiponini wasps and if CHCs differences would follow morphological patterns. Our results suggest that queens and workers of Epiponini wasps are chemically different from each other at two levels, qualitatively and quantitatively, or merely quantitatively. This variation seems to exist regardless of their morphological traits and may be useful to help us understanding how chemical communication evolved differently in these species.

Hormonal modulation of reproduction and fertility signaling in polistine wasps.

In social insects, it has been suggested that reproduction and the production of particular fertility-linked cuticular hydrocarbons (CHC) may be under shared juvenile hormone (JH) control, and this could have been key in predisposing such cues to later evolve into full-fledged queen pheromone signals. However, to date, only few studies have experimentally tested this "hormonal pleiotropy" hypothesis. Here, we formally test this hypothesis using data from four species of Polistine wasps, Polistes dominula, Polistes satan, Mischocyttarus metathoracicus, and Mischocyttarus cassununga, and experimental treatments with JH using the JH analogue methoprene and the anti-JH precocene. In line with reproduction being under JH control, our results show that across these four species, precocene significantly decreased ovary development when compared with both the acetone solvent-only control and the methoprene treatment. Consistent with the hormonal pleiotropy hypothesis, these effects on reproduction were further matched by subtle shifts in the CHC profiles, with univariate analyses showing that in P. dominula and P. satan the abundance of particular linear alkanes and mono-methylated alkanes were affected by ovary development and our hormonal treatments. The results indicate that in primitively eusocial wasps, and particularly in Polistes, reproduction and the production of some CHC cues are under joint JH control. We suggest that pleiotropic links between reproduction and the production of such hydrocarbon cues have been key enablers for the origin of true fertility and queen signals in more derived, advanced eusocial insects.

Effects of juvenile hormone in fertility and fertility-signaling in workers of the common wasp Vespula vulgaris.

In the highly eusocial wasp, Vespula vulgaris, queens produce honest signals to alert their subordinate workers of their fertility status, and therefore they are reproductively suppressed and help in the colony. The honesty of the queen signals is likely maintained due to hormonal regulation, which affects fertility and fertility cue expression. Here, we tested if hormonal pleiotropy could support the hypothesis that juvenile hormone controls fertility and fertility signaling in workers. In addition, we aimed to check oocyte size as a proxy of fertility. To do that, we treated V. vulgaris workers with synthetic versions of juvenile hormone (JH) analogue and a JH inhibitor, methoprene and precocene, respectively. We dissected the treated females to check ovary activation and analyzed their chemical profile. Our results showed that juvenile hormone has an influence on the abundance of fertility linked compounds produced by workers, and it also showed to increase oocyte size in workers. Our results corroborate the hypothesis that juvenile hormone controls fertility and fertility signaling in workers, whereby workers are unable to reproduce without alerting other colony members of their fertility. This provides supports the hypothesis that hormonal pleiotropy contributes to keeping the queen fertility signals honest.

Reproduction and signals regulating worker policing under identical hormonal control in social wasps.

In social Hymenoptera, fertility and fertility signalling are often under identical hormonal control, and it has been suggested that such hormonal pleiotropies can help to maintain signal honesty. In the common wasp Vespula vulgaris, for example, fertile queens have much higher juvenile hormone (JH) titers than workers, and JH also controls the production of chemical fertility cues present on the females' cuticle. To regulate reproductive division of labour, queens use these fertility cues in two distinct ways: as queen pheromones that directly suppress the workers' reproduction as well as to mark queen eggs and enable the workers to recognize and police eggs laid by other workers. Here, we investigated the hormonal pleiotropy hypothesis by testing if experimental treatment with the JH analogue methoprene could enable the workers to lay eggs that evade policing. In support of this hypothesis, we find that methoprene-treated workers laid more eggs, and that the chemical profiles of their eggs were more queen-like, thereby causing fewer of their eggs to be policed compared to in the control. Overall, our results identify JH as a key regulator of both reproduction and the production of egg marking pheromones that mediate policing behaviour in eusocial wasps.