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

Introduction

The evolution of reproductive castes is important for the organization of social insects (Queller & Strassmann, 1998). Among the hymenopteran social insects, the division of reproductive and nonreproductive labour was a fundamental step for their ecological success in most environments. In this context, Vespidae wasps are a key group for understanding the evolution of social behaviour because of their degrees of sociality, from solitary to highly eusocial (West-Eberhard, 1978; West-Eberhard, 1996; Wilson, 1985; Spradbery, 1991). They are important to understand the evolution of social behaviour as they are considered monophyletic, and a double origin of eusociality was recently supported in this group (Piekarski et al., 2018).

Within the subfamily Polistinae, the Neotropical tribe Epiponini (hence after epiponine) has perennial colonies and several inseminated breeders with a variable number of workers start new colonies (Jeanne, 1991; Jeanne, 2020). Epiponine and Polistes paper wasps are sister groups sharing a common caste-based ancestor (Menezes, Lloyd & Brady, 2020). It is then expected a conserved function of cuticular hydrocarbons (CHCs) in caste signalling and similarities of physiology and behaviour (Kelstrup et al., 2014a). The phenotype of reproductive individuals in several species of epiponine wasps is expressed by ovary activation (and insemination), which suggests post-imaginal caste determination (West-Eberhard, 1996), and retention of reproductive totipotence (Strassmann, Sullender & Queller, 2002; Mateus, Noll & Zucchi, 2004). For example, in Parachartergus fraternus, females with activated ovarioles play typical worker roles in their colonies (Mateus, Noll & Zucchi, 2004). However, in other species, caste fate seems to be determined by differential nourishment during larval development (Hunt et al., 1996).

During the colony cycle of epiponine, there is a reduction in the number of breeders (cyclical olygogyny) and workers evaluate queens’ fertility through aggressive solicitation, namely queen dance. In the queen dance, the queen reacts against workers. Then, submissive females bend their abdomen to perform a ritualized set of aggressive movements (West-Eberhard, 1978; Nascimento, Tannure-Nascimento & Zucchi, 2004; Kelstrup et al., 2014b). Yet, it is unknown if reproductive dominance is also pheromone-based in epiponine wasps, but past studies have shown that dominance signalling is likely to occur in two distinct epiponine species, Polybia micans and Synoeca surinama (Kelstrup et al., 2014a, 2014b). CHCs have been shown to function as queen pheromones across several lineages of social Hymenoptera (Holman et al., 2010; Holman, Lanfear & D’Ettorre, 2013; Oi et al., 2015; Oliveira et al., 2015; Van Oystaeyen et al., 2014; Holman et al., 2017). Most studies investigating CHCs as fertility cues in social insects are focused on ants (Holman, Lanfear & D’Ettorre, 2013; Van Oystaeyen et al., 2014; Monnin & Peeters, 1998; Liebig et al., 1999; Cuvillier-Hot et al., 2002), bees (Oliveira et al., 2015; Van Oystaeyen et al., 2014; Ayasse et al., 1995; Bloch & Hefetz, 1999; Andrade-Silva & Nascimento, 2015) and Polistinae or Vespinae wasps (Oi et al., 2016; Van Oystaeyen et al., 2014; Antonialli-Junior et al., 2021; da Silva et al., 2021; Sledge, Boscaro & Turillazzi, 2001; Sledge et al., 2004; Tannure-Nascimento, Nascimento & Zucchi, 2008; Oi et al., 2019). These studies demonstrated a correlation between the reproductive status of breeders and the presence of specific CHCs (Oi et al., 2015; Sledge et al., 2004; Tannure-Nascimento, Nascimento & Zucchi, 2008; Oi et al., 2019; Blomquist & Bagnères, 2010), and some of them evaluated the bioactivity of some CHCs (Oi et al., 2016; Van Oystaeyen et al., 2014; Holman et al., 2010). The correlative and empirical evidence support the hypothesis that the signals produced by reproductive females are likely honest messages of fertility rather than active inhibitors of workers’ reproduction (Keller & Nonacs, 1993; Beekman, 2004). For instance, in the common wasp Vespula vulgaris, two linear alkanes (n-C27 and n-C29) and one branched alkane (3-MeC29), which are present in relative higher quantities over the cuticular surface of queens, were found to reduce ovary activation in workers acting as queen pheromones (Van Oystaeyen et al., 2014). In the Saxon wasp, Dolichovespula saxonica, daily applications for two weeks of a mix of queen-characteristic CHCs (compounds overexpressed over the cuticular surface of queens), represented by three linear and two branched alkanes (n-C29, 3-MeC29, n-C30, n-C31, and 3-MeC31) resulted in reducing the proportion of workers with activated ovaries when compared with the solvent treated ones, meaning that one or more of the CHCs present in the tested blend, act as queen pheromones (Oi et al., 2016). Among the Polistes paper wasps, odd linear alkanes (from n-C₂₇ to n-C₃₅), methyl-branched alkanes (such as 7-MeC₂₇; 7-MeC₃₃; 13, 15, 17-C₃₃), and some alkenes (C₂₇:1, 9-C₂₉:1, 9-C₃₁:1, C₃₅:2) seem to be candidates to signal reproductive status (Sledge, Boscaro & Turillazzi, 2001; Sledge et al., 2004; Tannure-Nascimento, Nascimento & Zucchi, 2008; Oi et al., 2019; Dapporto, Sledge & Turillazzi, 2005). Higher proportions of alkanes and dimethyl-alkanes were observed in breeders of hover wasps (Turillazzi et al., 2004). Among the few evidences available for Mischocyttarus wasp (Ferreira et al., 2022), two branched alkanes (8,12-diMeC32, and 6-MeC32) were found in higher proportions over the cuticular surface of queens (females engaged in reproductive tasks) and in females expressing ovary activation (but not necessarily involved in reproductive tasks), in Mischocyttarus parallelogrammus (da Silva et al., 2020a). In Mischocyttarus cerberus, 5-MeC29 and C33:1 were detected exclusively in samples of alpha females from pre-worker emergence nests, whereas in samples from post-worker emergent nests, a dimethyl and two methyl alkanes were found either exclusively or higher in proportion in alpha females (da Silva et al., 2020b). Finally, in the epiponine species, CHCs such as 3-MeC₂₅ and n-C₂₅ appeared to be queen-associated in Polybia micans (Kelstrup et al., 2014b), and the linear alkanes n-C18, n-C25, and n-C26 are more abundant in queens of Polybia sericea (Soares et al., 2021). In the warrior wasp, Synoeca ilheensis (previously called Synoeca septentrionalis) (Lopes & Menezes, 2017) three linear alkanes were also found in greatest abundances in queens of three unrelated colonies (n-C25, n-C29, and n-C31) (Santos et al., 2018), whereas older queens of its sister species, Synoeca surinama, a single alkene C₂₅:1 was spotted as queen-characteristic (Kelstrup et al., 2014a).

Even though epiponine wasps contains about 250 species—distributed in 19 genera—which are almost exclusive to the Neotropical region (Carpenter, Kojima & Wenzel, 2000; Noll, 2013; Menezes, Lloyd & Brady, 2020), this tribe remains poorly explored concerning their chemical ecology. In the present work, we aimed to study the CHC diversity in eleven species from different genera and explore caste-linked differences. For that, we asked whether CHCs distinguish reproductive (queens) and non-reproductive (workers) individuals of epiponine wasps—including species with and without morphological caste differences. We hypothesized that CHCs are expressed differently between queens and workers, despite their morphological caste differences, similar to what is seen for other hymenopteran species, whether CHCs act as badges of caste in epiponine wasps. Additionally, we expected to find a clear chemical variation between queens and workers in species where reproductive females and non-reproductive females also differ in morphology, and a chemical discrete variation in species where morphological features are similar between queens and workers.

Material and Methods

Ovarian activation for caste assignment

Females were dissected and assigned to two different groups (i) queens, or (ii) workers based on their ovarian status. For the ovarian activity status, most of the females were dissected under a stereomicroscope Leica MZ75, however due to the difficult to sample rare species, some of the samples were dissected in the field after the sampling, for that, a LCD Digital microscope inskam-307 was used. Considering that not all species were collected at the same time, pictures are not available for all the eleven species (due to fieldwork constraints), however, the same parameters were adopted during the female assignment (see example Fig. 1). Ovaries were classified into two different categories without apparent activation—threadlike or filamentous ovarioles—(for workers), and with apparent activation—ovarioles with full mature oocytes—(for queens) (Fig. 1—The number of queens and workers per species is provided in Table S1). Only two categories of ovary activation were adopted in order to avoid misclassification. We were not able to check the spermatheca status for all the females; however, when possible, this information was used to confirm the group assignment (queen or worker together with their ovary status). To check whether the females were inseminated or not, the spermatheca was removed and prepared on a glass slide. The presence of yellow lines indicated that the spermatheca was inseminated, whereas the absence represented not inseminated.

Results

The CHC analysis revealed 232 compounds in total, mostly hydrocarbons, which included linear alkanes, methyl-alkanes, dimethyl-alkanes, trimethyl-alkanes, alkenes and alkadienes (Table 3, Tables S1 and S2). These compounds ranged from 18 to 40 of carbon length. Only four compounds were common to all study species, n-C25, n-C27, n-C28 and n-C29. The species with the highest chemical diversity was Charterginus sp., which expressed 64 chemical compounds, while the species with the least chemical diversity was N. xavantinensis with 21 chemical compounds. The percentage of CHC classes expressed by queens and workers of each species are shown in Fig. 2, Table 3 and Table S2.

Furthermore, we performed the chemical analyses according to caste syndromes that are depicted below:

Discussion

Overall, we confirmed our initial hypothesis that females belonging to different castes (queens and workers) express CHCs differently in epiponine wasps, seven out of eleven analyzed species were statistically different based on their overall chemical composition. Queens and workers expressed some caste specific CHCs (qualitative variation) in addition to different proportions of the shared compounds (quantitative variation), whereas in some other species the variation between queens and workers was subtle, consisting solely of quantitative variation. Contrary to our expectation, in species where females lack differential morphological traits, CHCs were either highly divergent but also similar between queens and workers depending on the species. As expected, species where females are morphologically distinct in caste, CHCs were majority distinct between queens and workers (with exception of A. pallipes). In this sense, CHCs are potential cues linked with caste in epiponine wasps.

CHCs have been demonstrated to be tightly connected with female fertility, and thus they are the candidates to act as queen pheromones in several unrelated lineages of social insects (Holman et al., 2010; Oliveira et al., 2015; Van Oystaeyen et al., 2014; Holman, 2018; Funaro et al., 2018; Layton, Camann & Espelie, 1994; Oliveira et al., 2017; Oi et al., 2016). In the case of epiponine queens, we found that in some species queens expressed exclusively certain CHCs, whereas in the remaining species, queens at least upregulate some of the CHCs that they share with workers. Interestingly, we reported four linear alkanes (n-C25, n-C27, n-C28, and n-C29) shared by females of all studied species, and overall, they were more abundant in the cuticular surface of queens rather than in workers. Considering the known properties of such compounds in advanced species of social wasps (V. vulgaris and D. saxonica) (Oi, 2022; Oi et al., 2016; Van Oystaeyen et al., 2014), these CHCs could potentially work as queen pheromones in swarm-founding species. From the literature, studies in epiponine wasps have revealed some specific compounds to be associated to queens. The results reported for S. ilheensis revealed that the overall proportion of linear alkanes was higher in queens compared to workers and males (Santos et al., 2018). In addition to that, three linear alkanes n-C25, n-C29, and n-C31, were consistently overexpressed by queens (Santos et al., 2018). In the species P. sericea, qualitative and quantitative variation were found between queens and workers, including females with and without ovary activation (Soares et al., 2021). For instance, the CHCs 3-MeC18, 9-MeC21, 14-;12-;10-MeC28, and n-C29 were found only in queens and workers with activated ovaries, whereas n-C20, 3-MeC25, and 2-MeC26 were identified exclusively in females with inactivated ovaries (Soares et al., 2021). Similarly, in P. micans, the linear alkane n-C25 and the branched alkane 3-MeC25, were significantly higher in queens (Kelstrup et al., 2014b). For now, the indication of putative queen pheromones in epiponine wasps is merely speculative and bioassays using synthetic versions of these chemicals should be conducted to test this hypothesis that linear alkanes act as queen pheromones in the swarm-founding wasps.

In epiponine wasps, it is still unknown whether the juvenile hormone (JH) plays a role regulating both queen fertility and queen signal production as it was shown in Vespinae wasps (Oliveira et al., 2017; Oi et al., 2021a; Oi et al., 2020). JH acts as a gonadotropic hormone and modulates CHCs expression in S. surinama (Kelstrup et al., 2014a), however, in Polybia micans (Kelstrup et al., 2014b), although it seems to control ovary activation, it does not have any correlation with CHCs expression. In Polybia occidentalis, JH regulates multiple factors in workers, such as age polyethism, ovarian status and CHCs expression (Prato et al., 2021). It was recently demonstrated that JH may also control female fertility and subtle modifications in CHCs in the primitively eusocial wasps Polistes and Mischocyttarus genera (Oi et al., 2021b). For the most part of our study species, epiponine queens were chemically distinguished from workers, it is possible that JH also controls CHCs production in these swarm-founding wasps, however, empirical evidence is needed. A systematic effort including species from different social insect taxa would be important to establish whether JH has a conserved function across the epiponine wasps.

We found a high chemical diversity among the epiponine wasp species, and each of them expressed a higher or lower CHC diversity. Even though queens and workers share similar proportions of CHCs classes, a species-specific qualitative variation was evident when the species were compared in Fig. 2. The chemical profiles of three species belonging to the Group I were majority composed by linear alkanes (C. communis, M. docilis and N. xavantinensis), while for S. surinama linear alkanes and alkenes were represented at similar proportional levels between castes, with queens being expressing more alkenes and workers linear alkanes, which is in line with the results previously published for the species (Kelstrup et al., 2014a). For the species P. fraternus and C. sulcata, which also belong to Group I, chemical profiles were more represented by branched alkanes. Females from Group II represented the ones with the most diversity in compound classes (Charterginus sp. = 6; B. augusti = 5; P. paulista = 4). Queens of Charterginus sp. exhibit branched alkanes in higher proportions whether compared to workers, which express linear and branched alkanes in similar proportions, on the other hand, in B. augusti the opposite pattern was seen, with queens being more represented by linear and workers by branched alkanes. Queens and workers of P. paulista show similar proportions of all compound classes. Lastly, females from both species comprising Group III were mainly characterized by branched alkanes (mostly methyl alkanes). More than 50% of the identified compounds comprise branched alkanes (methyl and dimethyl) and in a specific case (S. surinama), a high diversity of alkenes was found. Future studies could use CHCs as chemotaxonomic markers in epiponine, as most of our study species show a unique chemical signature. The use of CHCs as chemotaxonomic markers is common in the Hymenoptera order (Kather & Martin, 2015; Martin & Drijfhout, 2009; Dos Santos & Do Nascimento, 2015; Pokorny et al., 2014), solitary insects from Diptera (Ye et al., 2007; Moore et al., 2021), Hemiptera (Gemeno et al., 2012), and Orthoptera (Chapman, Espelie & Sword, 1995). For example, the chemical traits could also have a similar phylogenetic value compared to other often used characters, such as morphological, genetic or behavioural to build relationships among epiponine wasps.

Conclusion

In conclusion, we demonstrated that queens and workers of epiponine wasps are chemically distinct based on their CHC profiles—and this dissimilarity exists regardless of their caste-syndromes—which includes qualitative and quantitative variation, but also only quantitative differences. To date, considering that Neotropical swarm-founding wasps remained less explored whether compared with other groups of social insects, the results presented here reinforce that CHCs may act as reliable cues of castes in different unrelated groups of social insects. To strengthen the understanding of their chemical variation, future studies need to study the CHCs dynamics of queens and workers on a broader scale and a higher diversity of species, controlling the age of the sampled females. Overall, our correlative results suggest that division of labour within epiponine societies may be mediated by CHCs, queens and workers may benefit from these chemical dissimilarities and use this information to maintain their colonies functioning cohesively.

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