Social insects have a sophisticated colony organization system regulated mainly by chemical signals or pheromones, which are involved in all social activities (Wilson, 1965). Among these insects are the social wasps, which belong to the Vespidae family, divided in six subfamilies. Among these subfamilies is Polistinae, which englobes the genus Mischocyttarus Saussure (1853). This genus is the only representative of the tribe Mischocyttarini (Carpenter, 1993) and is the largest genus of social wasps, with more than 240 species distributed in nine subgenera (Carpenter and Wenzel, 1988; Silveira, 2008).

Their colonies are established by independent foundresses varying from one to a few queens (reproductive females) that start to build the nests (Jeanne, 1980; Von Ihering, 1896). A typical nest consists of a single comb attached to the substrate by a peduncle (Gadagkar, 1991; Jeanne, 1972; Wenzel, 1991). Mischocyttarus is an essentially Neotropical genus, with exception of a few species that occur in northern Mexico and Florida, USA (Hermann and Chao, 1983; Silveira, 1998, 2008), and has been considered of great importance in studies regarding the sociobiology (Jeanne, 1970, 1972; O’Donnell, 1999; Strassmann et al., 1995).

An important trait that plays a role in the cohesion of insect societies is the ability to distinguish between nestmates and non-nestmates (Singer et al., 1998). Chemical communication is very important for this purpose (Billen, 2006), as these insects use information provided by chemical compounds known as pheromones. Karlson and Luscher (1959) define pheromones as chemical signals produced by an organism that, even released in small quantities, may induce behavioral and/or physiological changes in another individual of the same species. These pheromones are generally divided into two types: light and volatile substances secreted by the exocrine glands and heavy hydrocarbon molecules found in the cuticle (Howard, 1993).

Cuticular hydrocarbons (CHCs) are compounds that essentially consist of carbon and hydrogen (Blomquist and Bagneres, 2010) and compose part of the lipid layer covering the cuticle of insects. They are essential to the survival of the individuals, because their primary function is to prevent dehydration (Lockey, 1988); at the same time, they form a protective barrier against microorganisms (Provost et al., 2008). The CHCs are synthesized by secreting cells derived from epidermal cells (Lockey, 1988) and are transported to the cuticle via hemolymph by lipophorin proteins (Bagneres and Blomquist, 2010).

Among the hundreds of CHCs identified in insects, there are three groups that stand out: n-alkanes, methyl branched alkanes, and unsaturated hydrocarbons (Blomquist, 2010). These compounds also act as signals during communication among insects, especially in social insects, enabling the recognition of conspecific individuals, nestmates, age, sex, and caste (Howard, 1993; Singer et al., 1998; Vander Meer and Morel, 1998). Variation can occur in the CHCs composition, depending on genetic and exogenous factors (Arnold et al., 1996; Blomquist and Bagneres, 2010; Dahbi et al., 1996; Gamboa et al., 1996; Kather and Martin, 2012; Page et al., 1991).

Another function of CHCs that has been previously explored in social wasps is their use as a complementary tool to assess variations among insect populations (Calderón-Fernández et al., 2005; Dapporto et al., 2004), as in the case of the species Polistes dominula (Christ, 1791) (Dapporto et al., 2004). Furthermore, previous studies have used the CHCs to differentiate species of termites (Kaib et al., 1991), ants (Martin et al., 2008), and Stenogastrinae wasps (Baracchi et al., 2010). Despite the recognized importance of these compounds as signaling or biochemical markers, there have been few studies with social wasps of the genus Mischocyttarus.

Mischocyttarus has been considered important for the study of sociobiology in wasps due to the incipient social organization and reproductive totipotence (Jeanne, 1970, 1972; Strassmann et al., 1995). However, the success of these studies depends on parallel efforts to rebuild relationships within this highly diverse genus (Silveira, 2008). Therefore, the aims of this study were 1) to describe the cuticular chemical compounds of the species Mischocyttarus consimilis (Zikan 1949), Mischocyttarus bertonii (Ducke, 1918), and Mischocyttarus latior (Fox, 1898), and 2) to test whether these chemical compounds could be used to evaluate differences and similarities between Mischocyttarus species.

The results indicate that the identified compounds comprised the greatest fraction (over of 76% of compounds) and are therefore likely to be the most important for the composition of the chemical signature. Among the identified compounds, the most abundant were branched alkanes, linear alkanes, and (to a lesser extent) alkenes (Fig. 2A and B). Branched alkanes were the most important compounds for interspecies separation (Fig. 3), evidencing the existence of a particular cuticular chemical profile for each species. Hence, the findings suggest that cuticular chemical profiles can be used as complementary tools to distinguish Mischocyttarus species.

Bagneres and Wicker-Thomas (2010) reported that CHCs could be used as chemotaxonomic parameters for species distinction. Studies using CHCs for distinction of social insect species were described by Kaib et al. (1991) for termites, Martin et al. (2008) for ants, and Baracchi et al. (2010) for hover wasps (Stenogastrinae), highlighting the importance of CHCs as a useful tool for species distinction.

Baracchi et al. (2010) suggested that the polar compounds present in the epicuticles of females of Stenogastrinae wasps could be used to identify different species, because the chemical parameters were consistent with the observed morphological characteristics. However, the authors point out that this tool is not always effective in distinguishing phylogenetically close species, as occurred in the species Liostenogaster campanulae (Turillazzi, 1990) and Liostenogaster flavolineata (Cameron, 1902). Kaib et al. (1991) analyzed the CHCs profiles of six species of Odontotermes (Holmgren, 1910) termites and concluded that intraspecific differences could be neglected when evaluating interspecific differences. Martin et al. (2008) investigated the chemical profiles of cuticular hydrocarbons in thirteen sympatric species of ants and concluded that the CHC profiles were stable, even considering ecological factors, with several species-specific hydrocarbons that could be used in taxonomic and evolutionary studies.

Ferreira et al. (2012), studying only the linear alkanes of three species of Mischocyttarus, identified 27 compounds of the same species. In the present study, in addition to the linear alkanes, 60 branched alkanes and 3 alkenes were also identified in Mischocyttarus species.

The use of these compounds as a taxonomic tool was previously proposed by Kather and Martin (2012), although it was suggested that chemotaxonomy might be more effective when used together with other traditional methods such as morphological, ecological, or molecular analysis. It was emphasized that the use of CHCs was reliable for this purpose, because these compounds are stable metabolic products that are hereditary and species-dependent.

Both qualitative and quantitative intracolonial differences were observed, and discriminant analysis confirmed significant differences between colonies of M. consimilis, M. bertonii, and M. latior. Therefore, for these three species of Mischocyttarus, each colony had a specific cuticular chemical profile.

Several studies have described the importance of CHCs for intraspecific distinction (Bonavita-Cougourdan et al., 1991; Bruschini et al., 2010; Dani et al., 1996; Dapporto et al., 2004; Espelie et al., 1994; Layton et al., 1994; Lorenzi et al., 1997; Sledge et al., 2001). Sledge et al. (2001) and Tannure-Nascimento et al. (2007) found that significant differences in CHCs enabled distinction between colonies of P. dominula and Polistes satan (Bequard, 1940), respectively. The distinctive chemical profile of an individual colony is related to the fact that social insects are usually able to discriminate nestmates (Arnold et al., 2000; Bonavita-Cougourdan et al., 1987; Howard and Blomquist, 1982; Wilson, 1971), with the main chemical signals involved in this process being the CHCs (Espelie and Hermann, 1990; Howard and Blomquist, 2005).

The existence of an individual chemical profile for each colony (Figs. 4–6) supports the role of CHCs as contact pheromones for conspecific identification, corroborating previous results obtained by Bruschini et al. (2011), Monnin (2006) and Provost et al. (2008). It therefore appears that these compounds can act as chemical signals not only at the individual level, but also at the colony level (Antonialli-Junior et al., 2007; Cotoneschi et al., 2009; Dapporto et al., 2004, 2005; Izzo et al., 2010; Layton et al., 1994; Monnin, 2006; Neves et al., 2012; Sledge et al., 2001; Tannure-Nascimento et al., 2008).

For both interspecific and intraspecific distinction, branched alkanes were most important for separation of the groups, together with some linear alkanes. Other studies also found that branched alkanes were the most relevant components for conspecific discrimination in colonies of P. dominula (Bonavita-Cougourdan et al., 1991; Dani et al., 1996), Polistes metricus (Say, 1831) (Layton et al., 1994), and Polistes biglumis bimaculatus (Geoffroy, 1785) (Lorenzi et al., 1997).

The higher content of branched alkanes can be explained by the fact that the results presented here and in the study of Tannure-Nascimento et al. (2007) were derived from the analysis of adults, rather than immature individuals. Cotoneschi et al. (2007) evaluated different developmental stages of P. dominula and found a greater abundance of linear alkanes in brood and a greater abundance of branched alkanes in adults. This emphasizes the importance of branched alkanes as recognition signals in adults.

According to Brown et al. (1991), Cervo et al. (2008), Espelie and Hermann (1990) and Lorenzi et al. (2004) during progression from the egg stage to the adult there is a decrease in the abundance of linear alkanes and an increase in the content of branched alkanes. On the other hand, Gamboa et al. (1996) suggested that the presence of branched alkanes might increase the susceptibility of the insect to desiccation. However, the wide variety of branched alkanes suggests that these compounds have additional functions whose benefit outweigh the disadvantage of decreased waterproofing (Buckner, 1993).

The results showed that the cuticular chemical compounds varied significantly among the colonies, confirming the existence of a colonial signature based on cuticular hydrocarbons. Independently of species and colony, the most relevant compounds (in decreasing order of importance) were methyl alkanes, linear alkanes, dimethyl alkanes, and alkenes. The results confirm that cuticular chemical profiles can be used as complementary parameters to evaluate interspecific and intercolony differences in wasps of the genus Mischocyttarus. However, it is clear that future studies that involve a larger number of species and phylogenetically closer are still necessary to validate the method, since a tool of great utility would help to solve difficult cases of differentiation between related species.

The authors declare no conflicts of interest