Revista Brasileira de Entomologia Revista Brasileira de Entomologia
Rev Bras Entomol 2017;61:224-31 - Vol. 61 Núm.3 DOI: 10.1016/j.rbe.2017.05.001
Systematics, Morphology and Biogeography
Variation of cuticular chemical compounds in three species of Mischocyttarus (Hymenoptera: Vespidae) eusocial wasps
Eva Ramona Pereira Soaresa,b,, , Nathan Rodrigues Batistab, Rafael da Silva Souzac, Viviana de Oliveira Torresb, Claudia Andrea Lima Cardosoc, Fabio Santos Nascimentod, William Fernando Antonialli-Juniora,b
a Universidade Federal da Grande Dourados, Programa de Pós-graduação em Entomologia e Conservação da Biodiversidade, Dourados, MS, Brazil
b Universidade Estadual de Mato Grosso do Sul, Laboratório de Ecologia Comportamental, Dourados, MS, Brazil
c Universidade Estadual de Mato Grosso do Sul, Centro de pesquisa em biodiversidade, Dourados, MS, Brazil
d Universidade de São Paulo, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, SP, Brazil
Recebido 12 Janeiro 2017, Aceitaram 08 Maio 2017
Abstract

The social wasps have a remarkable system of organization in which chemical communication mediate different behavioral interactions. Among the compounds involved in this process, cuticular hydrocarbons are considered the main signals for nestmate recognition, caste differentiation, and fertility communication. The aims of this study were to describe the cuticular chemical compounds of the species Mischocyttarus consimilis, Mischocyttarus bertonii, and Mischocyttarus latior, and to test whether these chemical compounds could be used to evaluate differences and similarities between Mischocyttarus species, using gas chromatography coupled to mass spectrometry (GC–MS). Workers from these three species presented a variety of hydrocarbons ranging from C17 to C37, and among the compounds identified, the most representative were branched alkanes, linear alkanes and alkenes. The results revealed quantitative and qualitative differences among the hydrocarbon profiles, as confirmed by discriminant analysis. This study supports the hypothesis that cuticular chemical profiles can be used as parameters to identify interspecific and intercolony differences in Mischocyttarus, highlighting the importance of these compounds for differentiation of species and populations.

Keywords
Chemical profile, Cuticular hydrocarbons, Mischocyttarus, Polistinae, Surface pheromones
Introduction

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.

Materials and methodsCollection

Twenty colonies were collected in different areas of the municipalities, two in Ivinhema (22°21′22.5″ S; 53°45′26.1″ W), 14 in Mundo Novo (23°56′23″ S; 54°17′25″ W), and three in Dourados (22°13′16″ S; 54°48′20″ W) in Mato Grosso do Sul State; and one in the municipality of Palotina (24°16′23″ S; 53°52′38″ W) in Paraná State, Brazil. Colonies of three species were collected: Mischocyttarus (Kappa) bertonii, Mischocyttarus (Kappa) latior, and Mischocyttarus (Monocyttarus) consimilis. All the adults were killed by freezing and stored until the moment of extraction of the compounds. Only workers were used, because differences between castes could influence the individual chemical composition. Castes were distinguished by behavioral observation prior to collection using the behavioral repertoire described by Torres et al. (2012) in M. consimilis. In addition, all colonies collected were in the post-emergence phase, according to the classification of Jeanne (1972).

Extraction of cuticular compounds for chemical analysis

The cuticular compounds were extracted by washing each individual for 2min in 2mL of hexane (Vetec, HPLC grade). The extracts were dried in an exhaustion chapel and stored until the day of the analysis, when the samples were solubilized in 200μL of hexane. The CHCs were extracted from each female and the variation depended on the number of individuals in the colony, the colonies collected, and the species. The analyses were performed using a gas chromatograph coupled to a mass spectrometer (Model 2010 GCMS-QP, Shimadzu). A DB5-MS column was used (30.0m length×0.25mm internal diameter, 0.25μm film thickness), with heating from an initial temperature of 150°C to 280°C, at a rate of 3°C/min, and maintaining the final temperature for 25min. Helium (99.99%) was used as carrier gas, at a flow rate of 1mL/min, and 1μL sample volumes were injected in splitless mode. The temperatures of the injector, detector, and transfer line were 250°C, 250°C, and 290°C, respectively. The mass spectrometer parameters included electron impact ionization voltage of 70eV, mass range of 40–600m/z, and scan time of 0.3s.

The cuticular compounds were identified using the retention indexes calculated using a series of linear alkanes (Van den Dool and Kratz, 1963), the library of the equipment (NIST21 and WILEY229), and analysis of the mass spectra. In the case of the linear alkanes (C8–C40), standards of the compounds were also used.

The peak area for each compound was determined by manual integration of the total ion chromatogram (TIC). All the areas were then transformed into relative percentage areas.

Statistical analysis

We used a discriminant analysis to separate the groups defined previously according to their chemical profiles of inter- and intraspecific differences. Wilks’ Lambda was employed as a measure of the contribution of each variable. In these multivariate analyses, the percentage values were calculated from the chromatogram peak areas used as the independent variables.

ResultsInterspecific variation among the three species of Polistinae wasps

The chemical compounds identified in the analyses of samples from the three species ranged from C17 to C37 (Table 1), corresponding to over 47% of the compounds detected and representing a relative proportion exceeding 76%. The three species presented 10 common compounds: 3-methyloctadecane, pentacosane, heptacosane, 3-methylheptacosane, octacosane, X-methyloctasane, 3-methyloctacosane, nonacosane, 13-methylnonacosane, and 3-methyltriacontane (Fig. 1). However, the use of relative proportions of these compounds in the chromatograms permitted the identification of quantitative differences between the species. For example, the relative percentage of 3-methyloctacosane was significantly higher in M. latior than in the two other species, and there were important contributions of 3-methylheptacosane and nonacosane in M. bertonii and M. consimilis, respectively (Fig. 1).

Table 1.

Average relative proportions and their standard deviations for the cuticular hydrocarbons identified in three species of Mischocyttarus wasps.

Compound  Index calculated  M. consimilis
Mean±SD (%) 
M. bertonii
Mean±SD (%) 
M. latior
Mean±SD (%) 
5-Methylheptadecane  1752  0.01±0.10  0.03±0.04  – 
Octadecane  1800  0.13±0.63  0.03±0.02  – 
3-Methyloctadecane  1872  0.15±0.68  0.19±0.08  0.09±0.03 
Nonadecane  1900  0.01±0.70  –  0.06±0.01 
2-Methylnonadecane  1964  1.78±0.70  0.59±0.94  0.09±0.12 
Eicosane  2000  0.79±0.27  0.55±0.13  0.07±0.04 
Heneicosane  2100  0.07±2.63  0.09±0.13  0.03±0.03 
9-Methylheneicosane  2140  2.62±2.58  3.23±4.53  0.40±0.72 
2-Methylheneicosane  2170  6.04±2.07  7.71±3.45  – 
Docosane  2200  0.35±0.20  0.24±0.15  – 
2-Methyldocosane  2269  0.01±0.16  –  0.12±0.06 
Tricosene  2272  0.02±0.16  0.03±0.07  – 
Tricosane  2300  0.34±0.16  0.89±1.35  0.02±0.01 
X-methyltricosane  2329  0.05±0.03  0.01±0.01  – 
7-Methyltricosane  2341  0.03±0.03  0.01±0.01  – 
3-Methyltricosane  2373  0.03±0.05  0.10±0.12  – 
Tetracosane  2400  0.03±0.04  0.05±0.09  0.01±0.01 
X,Y-dimethyltricosane  2407  0.01±0.08  –  – 
8-Methyltetracosane  2430  0.04±0.16  0.17±0.09  0.06±0.03 
10-Methyltetracosane  2431  0.03±0.16  –  – 
7-Pentacosene  2477  0.19±0.17  0.19±0.21  – 
Pentacosane  2500  0.32±0.17  2.27±1.36  0.34±0.12 
X-methylpentacosane  2534  0.03±0.09  0.01±0.01  – 
6-Methylpentacosane  2542  –  0.01±0.01  – 
5-Methylpentacosane  2549  0.22±0.09  0.48±0.23  0.38±0.46 
3-Methylpentacosane  2573  0.14±0.06  0.67±0.23  0.02±0.02 
Hexacosane  2600  0.06±0.50  1.16±1.82  0.18±0.03 
2-Methylhexacosane  2656  0.02±0.50  –  – 
3-Methylhexacosane  2672  0.06±1.49  –  0.48±0.08 
1-Heptacosene  2676  –  27.95±9.09  – 
4,8-Dimethylhexacosane  2690  0.01±1.47  –  – 
Heptacosane  2700  1.61±1.47  9.75±1.33  11.48±1.42 
X,Y-dimethylheptacosane  2714  0.11±1.43  1.35±2.29  0.02±0.05 
X-methylheptacosane  2731  0.04±1.43  0.53±0.87  0.17±0.10 
7-Methylheptacosane  2741  0.01±1.43  0.05±0.06  – 
5-Methylheptacosane  2750  0.11±1.43  0.34±0.55  0.06±0.02 
3-Methylheptacosane  2774  4.80±1.44  7.80±2.02  7.76±0.67 
Octacosane  2800  0.27±0.27  0.61±0.38  0.82±0.17 
3,11-Dimethylheptacosane  2810  0.04±0.27  –  – 
X-methyloctacosane  2828  0.55±1.32  0.77±0.83  0.55±0.26 
2-Methyloctacosane  2859  0.59±1.33  0.58±1.15  8.23±16.32 
3-Methyloctacosane  2874  0.22±1.34  6.48±4.57  34.5±18.64 
Nonacosene  2885  –  0.72±0.79  – 
4,8-Dimethyloctacosane  2890  0.11±1.34  –  – 
Nonacosane  2900  7.47±1.32  3.88±0.75  4.16±2.34 
13-Methylnonacosane  2934  1.26±2.07  0.32±0.23  2.92±1.55 
7-Methylnonacosane  2939  0.29±2.14  –  – 
5-Methylnonacosane  2952  0.15±2.13  –  0.05±0.04 
X,Y-dimethylnonacosane  2956  0.29±2.14  –  – 
11,19-Dimethylnonacosane  2963  0.08±2.13  –  – 
7,17-Dimethylnonacosane  2970  0.81±2.13  –  – 
5,9-Dimethylnonacosane  2977  6.33±2.09  2.29±0.54  8.08±0.69 
Triacontane  3000  2.84±1.40  0.32±0.29  0.03±0.03 
14-Methyltriacontane  3034  1.56±1.47  0.07±0.08  0.02±0.01 
2-Methyltriacontane  3060  0.90±2.64  –  0.04±0.03 
3-Methyltriacontane  3076  0.18±2.66  –  2.12±0.50 
13,17-Dimethyltriacontane  3078  –  0.69±0.43  – 
Hentriacontane  3100  2.42±2.58  3.25±1.29  1.15±0.40 
X-methylhentriacontane  3135  4.33±2.51  2.03±1.07  0.25±0.11 
13,17-Dimethylhentriacontane  3157  7.27±2.33  –  – 
3-Methylhentriacontane  3178  1.44±1.15  0.65±0.67  – 
Dotriacontane  3200  2.95±1.12  0.22±0.37  0.51±0.11 
11-Methyldotriacontane  3233  2.21±0.82  –  – 
12,16-Dimethyldotriacontane  3254  0.47±0.34  0.18±0.32  0.01±0.01 
2-Methyldotriacontane  3260  0.86±0.44  –  – 
Tritriacontane  3300  0.38±0.68  0.38±0.26  0.03±0.06 
13-Methyltritriacontane  3333  1.59±1.06  1.46±1.44  – 
11,21-Dimethyltritriacontane  3339  0.29±1.10  –  0.01±0.01 
7-Methyltritriacontane  3343  0.17±1.09  –  – 
13,17-Dimethyltritriacontane  3355  2.08±1.09  0.24±0.16  – 
3-Methyltritriacontane  3379  0.56±0.98  –  – 
5,17-Dimethyltritriacontane  3387  0.66±0.85  0.28±0.39  0.05±0.11 
Tetratriacontane  3400  0.72±0.68  0.01±0.01  – 
X-methyltetratriacontane  3421  0.05±0.67  –  – 
Pentatriacontane  3500  0.16±0.46  0.01±0.02  – 
X-methylpentatriacontane  3529  0.65±0.33  –  – 
X,Y-dimethylpentatriacontane  3556  0.06±0.28  0.04±0.06  – 
11,21-Dimethylpentatriacontane  3563  0.48±0.29  –  – 
X,Y-dimethylpentatriacontane  3556  0.05±0.28  –  – 
7,11-Dimethylpentatriacontane  3568  0.19±0.28  –  – 
Hexatriacontane  3600  0.34±0.27  –  – 
X-methylheptatriacontane  3721  0.06±0.07  –  – 
11,21-Dimethylheptatriacontane  3767  0.75±0.56  –  – 

SD, standard deviation.

Fig. 1.
(0.13MB).

Representative chromatograms for three species of social wasps of the genus Mischocyttarus, indicating the 10 compounds common to all of them. 1=3-methyloctadecane; 2=pentacosane; 3=heptacosane; 4=3-methylheptacosane; 5=octacosane; 6=X-methyloctacosane; 7=3-methyloctacosane; 8=nonacosane; 9=13-methylnonacosane; 10=3-methyltriacontane.

The compounds 6-methylpentacosane, 1 heptacosane, nonacosene, and 13,17-dimethyltriacontane only occurred in workers of the M. bertonii species. Workers of M. latior did not show any unique compounds (Table 1).

M. consimilis showed the highest number of detected (163) and identified (60) compounds, of which 13 were exclusive to this species: 4,8-dimethyloctacosane, 13,17-dimethylhentriacontane, 11-methyldotriacontane, 11,21-dimethyltritriacontane, 7-methyltritriacontane, X-methyltetratriacontane, X-methylpentatriacontane, 11,21-dimethylpentatriacontane, X,Y-dimethylpentatriacontane, 7,11-dimethylpentatriacontane, hexatriacontane, X-methylheptatriacontane and 11,21-dimethylheptatriacontane.

In M. consimilis, the identified compounds represent a relative proportion of 76.72±4.44%. In M. bertonii, 93 compounds were detected and 61 (64.5%) were identified, representing a relative proportion of 90.26±4.71%. Finally, 81 compounds were detected in M. latior and from these 43 (53%) were identified, representing a relative proportion of 88.56±3.07%. These findings confirmed qualitative differences among the different species, regarding cuticular compounds. For all the species, the compounds most frequently identified were the branched alkanes, linear alkanes, and alkenes (Fig. 2A and B). Notably, the presence of the alkene 1-heptacosene was only detected in M. bertonii, in higher relative proportion compared to other alkenes as well as other identified compounds (Fig. 2A and B).

Fig. 2.
(0.11MB).

Relative proportions (A) and numbers in percentage terms (B) of the compounds identified in the three social wasp species of the genus Mischocyttarus: Mischocyttarus consimilis, Mischocyttarus bertonii, and Mischocyttarus latior.

Discriminant analysis showed significant quantitative differences in the relative proportions of the compounds common to the different species (Wilks’ Lambda=0.019, F=59.136, p<0.001; Fig. 3), and among the 11 compounds used in the analysis, 8 were important for separation of the species. The first and second canonical roots explained 61.2 and 27.4% of the results, respectively (total of 88.6%). The scatter plot (Fig. 3) shows clear separation among the species, indicating the existence of a typical cuticular chemical profile for each species.

Fig. 3.
(0.14MB).

Scatter diagram of the discriminant analysis results, showing the two canonical roots for differentiation of the three social wasp species of the genus Michocyttarus, based on the relative areas (percentages) of the cuticular compounds of females, obtained by GC–MS.

Intraspecific variation of cuticular chemical compounds among colonies of the three species of Mischocyttarus

In the case of M. consimilis, 44 of the 60 compounds identified (73.3%) were common to all colonies. Discriminant analysis revealed significant differences among the colonies (Wilks’ Lambda=0.001, F=2.426, p<0.001; Fig. 4), with 53 compounds being important for separation of the groups. The first and second canonical roots explained 80 and 8% of the results (total of 88%), as shown in Fig. 4.

Fig. 4.
(0.16MB).

Scatter diagram of the discriminant analysis results, showing the two canonical roots for differentiation of 11 colonies of Mischocyttarus consimilis, based on the relative areas (percentages) of the cuticular compounds of females, obtained by GC–MS.

For M. bertonii, only 21 of the 61 compounds identified (35%) were common to all colonies. The discriminant analysis also showed significant differences among colonies (Wilks’ lambda=0.001, F=6.647, p<0.001; Fig. 5), and out of the 19 compounds used, 13 were important for separation of the groups. The first and second canonical roots explained 83 and 9% of the results, respectively (Fig. 5).

Fig. 5.
(0.14MB).

Scatter diagram of the discriminant analysis results, showing the two canonical roots for differentiation of 5 colonies of Mischocyttarus bertonii, based on the relative areas (percentages) of the cuticular compounds of females, obtained by GC–MS.

Finally, of the 43 compounds identified in M. latior, 26 (60.4%) were common to all the colonies. Discriminant analysis showed significant differences among the colonies (Wilks’ Lambda=0.002, F=7.356, p<0.001; Fig. 6), with 9 of the compounds being important for separation of the groups. The first canonical root explained 63.3% of the results, and the second explained 34.4% (Fig. 6).

Fig. 6.
(0.13MB).

Scatter diagram of the discriminant analysis results, showing the two canonical roots for differentiation of 4 colonies of Mischocyttarus latior, based on the relative areas (percentages) of the cuticular compounds of females, obtained by GC–MS.

Discussion

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.

Conflicts of interest

The authors declare no conflicts of interest

Acknowledgments

The authors are grateful to Dr. Orlando T. Silveira (Museu Paraense Emílio Goeldi) for species identification. Financial support was provided by the Brazilian agency Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (VOT grant number 23/200.285/2014). The authors also thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (WFAJ grant number 307998/2014-2), (CALC grant number 307998/2014-2), Fapesp for productivity grant to FSN (2015/25301-9) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the first author's scholarship.

References
Antonialli-Junior et al., 2007
W.F. Antonialli-Junior,S.M. Lima,L.H.C. Andrade,Y.R. Súarez
Comparative study of the cuticular hydrocarbon in queens, workers and males of Ectatomma vizzotoi (Hymenoptera: Formicidae) by Fourier transform-infrared photoacoustic spectroscopy
Genet. Mol. Res., 6 (2007), pp. 492-499
Arnold et al., 2000
G. Arnold,B. Quenet,C. Masson
Influence of social environment on genetically based subfamily signature in the honeybee
J. Chem. Ecol., 26. (2000), pp. 2331-2333 http://dx.doi.org/10.1007/s10886-016-0798-4
Arnold et al., 1996
G. Arnold,B. Quenet,J.M. Cornuet,C. Masson,B. Deschepper,A. Estoup,P. Gasquil
Kin recognition in honeybees
Nature, 379 (1996), pp. 498
Bagneres and Blomquist, 2010
A.G. Bagneres,G.J. Blomquist
Site of synthesis, mechanism of transport and selective deposition of hydrocarbons
Insect Hydrocarbons: Biology, Biochemistry and Chemical Ecology, pp. 75-99
Bagneres and Wicker-Thomas, 2010
A.G. Bagneres,C. Wicker-Thomas
Site of synthesis, mechanism of transport and selective deposition of hydrocarbons
Insect Hydrocarbons: Biology, Biochemistry, and Chemical Ecology, pp. 75-99
Baracchi et al., 2010
D. Baracchi,L. Dapporto,S. Tesseo,R. Hashim,S. Turillazzi
Medium molecular weight polar substance of the cuticle as tools in the study of the taxonomy, systematics and chemical ecology of tropical hover wasps (Hymenoptera: Stenogastrinae)
J. Zool. Syst. Evol., 48 (2010), pp. 109-114
Billen, 2006
J. Billen
Signal variety and communication in social insects
Proc. Neth. Entomol. Soc. Meet., 17 (2006), pp. 9
Blomquist, 2010
G.J. Blomquist
Structure and analysis of insect hydrocarbons
Insect Hydrocarbons: Biology, Biochemistry, and Chemical Ecology, pp. 19-34
Blomquist and Bagneres, 2010
G.J. Blomquist,A.G. Bagneres
Insect Hydrocarbons: Biology, Biochemistry, and Chemical Ecology
Cambridge University Press, (2010)
Bonavita-Cougourdan et al., 1987
A. Bonavita-Cougourdan,J.L. Clément,C. Lange
Nestmate recognition: the role of cuticular hydrocarbons in the ant Camponotus vagus
Scop. J. Entomol. Sci., 22 (1987), pp. 1-10
Bonavita-Cougourdan et al., 1991
A. Bonavita-Cougourdan,G. Theraulaz,A.G. Bagneres,M. Roux,M. Pratte,E. Provost,J.L. Clement
Cuticular hydrocarbons, social organization and ovarian development in a polistine wasp: Polistes dominulus Christ
Comp. Biochem. Physiol., 100 (1991), pp. 667-680
Brown et al., 1991
W.V. Brown,J.P. Spradbery,M.J. Lacey
Changes in the cuticular hydrocarbon composition during development of the social wasp, Vespula germanica (F.) (Hymenoptera: Vespidae)
Comp. Biochem. Physiol., 99 (1991), pp. 553-562
Bruschini et al., 2010
C. Bruschini,R. Cervo,S. Turillazzi
Pheromones in social wasps
Vitam. Horm., 83 (2010), pp. 447-492 http://dx.doi.org/10.1016/S0083-6729(10)83019-5
Bruschini et al., 2011
C. Bruschini,R. Cervo,A. Cini,G. Pieraccini,L. Pontieri,L. Signorotti,S. Turil- lazzi
Cuticular hydrocarbons rather than peptides are responsible for nestmate recognition in Polistes dominulus
Chem. Sens., 36 (2011), pp. 715-723
Buckner, 1993
J.S. Buckner
Cuticular polar lipids of insects
Insect Lipids: Chemistry, Biochemistry and Biology, pp. 227-270
Calderón-Fernández et al., 2005
G. Calderón-Fernández,M.P. Juárez,M.C. Monroy,M. Menes,D.M. Bustamante,S. Mijailovsky
Intraspecific variability in Triatoma dimidiata (Hemiptera: Reduviidae) populations from Guatemala based on chemical and morphometric analyses
J. Med. Entomol., 42 (2005), pp. 29-35
Carpenter, 1993
J.M. Carpenter
Biogeographic patterns in the Vespidae (Hymenoptera): two views of Africa and South America
Biological Relationships between Africa and South America, pp. 139-155
Carpenter and Wenzel, 1988
J.M. Carpenter,J.W. Wenzel
A new species and nest type of Mischocyttarus from Costa Rica (Hymenoptera: Vespidae; Polistinae), with descriptions of nests of three related species
Psyche, 95 (1988), pp. 89-99
Cervo et al., 2008
R. Cervo,L. Dapporto,L. Beani,J.E. Strassmann,S. Turillazzi
On status badges and quality signals in the paper wasp Polistes dominulus: body size, facial colour patterns and hierarchical rank
Proc. R. Soc. Lond. B, 275 (2008), pp. 1189-1196
Cotoneschi et al., 2009
C. Cotoneschi,F.R. Dani,R. Cervo,C. Scala,J.E. Strassmann,D.C. Queller,S. Turillazzi
Polistes dominulus (Hymenoptera, Vespidae) larvae show different cuticular patterns according to their sex: workers seem not use this chemical information
Chem. Sens., 34 (2009), pp. 195-202
Cotoneschi et al., 2007
C. Cotoneschi,F.R. Dani,R. Cervo,M.F. Sledge,S. Turillazzi
Polistes dominulus (Hymenoptera: Vespidae) larvae possess their own chemical signatures
J. Insect Physiol., 53 (2007), pp. 954-963 http://dx.doi.org/10.1016/j.jinsphys.2006.12.016
Dahbi et al., 1996
A. Dahbi,A. Lenoir,A. Tinaud,T. Taghizadeh,W. Francke,A. Hefetz
Chemistry of the postpharyngeal gland secretion and its implication for the phylogeny of the Iberian Cataglyphis species (Hymenoptera: Formicidae)
Chemoecology, 7 (1996), pp. 163-171
Dani et al., 1996
F.R. Dani,S. Turillazzi,E.D. Morgan
Dufour gland secretion of Polistes wasp: chemical composition and possible involvement in nestmate recognition (Hymenoptera: Vespidae)
J. Insect Physiol., 42 (1996), pp. 541-548
Dapporto et al., 2004
L. Dapporto,E. Palagi,S. Turillazzi
Cuticular hydrocarbons of Polistes dominulus as a biogeographic tool: a study of populations from the Tuscan archipelagoand surrounding areas
J. Chem. Ecol., 30 (2004), pp. 2139-2151
Dapporto et al., 2005
L. Dapporto,F.M. Sledge,S. Turillazzi
Dynamics of cuticular chemical profiles of Polistes dominulus workers in orphaned nests (Hymenoptera, Vespidae)
J. Insect Physiol., 51 (2005), pp. 969-973 http://dx.doi.org/10.1016/j.jinsphys.2005.04.011
Espelie et al., 1994
K.E. Espelie,R.F. Chapman,G.A. Sword
Variation in the surface lipids of the grasshopper, Schistocerca americana (Drury)
Biochem. Syst. Ecol., 22 (1994), pp. 563-575
Espelie and Hermann, 1990
K.E. Espelie,H.R. Hermann
Surface lipids of the social wasp Polistes annularis (L.) and its nest and nest pedicel
J. Chem. Ecol., 16 (1990), pp. 1841-1852 http://dx.doi.org/10.1007/BF01020498
Ferreira et al., 2012
A.C. Ferreira,C.A.L. Cardoso,E.F. Neves,Y.R. Súarez,W.F. Antonialli-Junior
Distinct linear hydrocarbon profiles and chemical strategy of facultative parasitism among Mischocyttarus wasps
Genet. Mol. Res., 11 (2012), pp. 4351-4359 http://dx.doi.org/10.4238/2012.September.25.3
Gadagkar, 1991
R. Gadagkar
Belonogaster, Mischocyttarus, Parapolybia, and independent founding Ropalidia
The Social Biology of Wasps, pp. 149-190
Gamboa et al., 1996
G.J. Gamboa,T.A. Grudzien,K.E. Espelie,E.A. Bura
Kin recognition pheromones in social wasps: combining chemical and behavioural evidence
Anim. Behav., 51 (1996), pp. 625-629
Hermann and Chao, 1983
H.R. Hermann,J. Chao
Nesting biology and defensive behavior of Mischocyttarus (Monocyttarus) mexicanus cubicola (Vespidae: Polistinae)
Psyche, 91 (1983), pp. 51-65
Howard, 1993
R.W. Howard
Cuticular hydrocarbon and chemical communication
Insect Lipids: Chemistry, Biochemistry and Biology, pp. 179-226
Howard and Blomquist, 1982
R.W. Howard,G.J. Blomquist
Chemical ecology and biochemistry of insect hydrocarbons
Annu. Rev. Entomol., 27 (1982), pp. 149-172
Howard and Blomquist, 2005
R.W. Howard,G.J. Blomquist
Ecological, behavioral, and biochemical aspects of insect hydrocarbons
Annu. Rev. Entomol., 50 (2005), pp. 371-393 http://dx.doi.org/10.1146/annurev.ento.50.071803.130359
Izzo et al., 2010
A. Izzo,M. Wells,Z. Huang,E. Tibbetts
Cuticular hydrocarbons correlate with fertility, not dominance, in a paper wasp, Polistes dominulus
Behav. Ecol. Sociobiol., 64 (2010), pp. 857-874
Jeanne, 1970
R.L. Jeanne
Chemical defense of brood by a social wasp
Science, 168 (1970), pp. 1465-1466
Jeanne, 1972
R.L. Jeanne
The Social biology of the Neotropical wasp Mischocyttarus drewseni
Bull. Mus. Comp. Zool. Harvard, 144 (1972), pp. 63-150
Jeanne, 1980
R.L. Jeanne
Evolution of social behavior in the Vespidae
Annu. Rev. Entomol., 25 (1980), pp. 371-396
Kaib et al., 1991
M. Kaib,R. Brandl,R.K.N. Bagine
Cuticular hydrocarbons profiles: a valuable tool in termite taxonomy
Naturwissenschaften, 78 (1991), pp. 176-179
Karlson and Luscher, 1959
P. Karlson,M. Luscher
Pheromones: a new term for a class of biologically active substances
Nature, 183 (1959), pp. 55-56
Kather and Martin, 2012
R. Kather,S.J. Martin
Cuticular hydrocarbon profiles as a taxonomic tool: advantages, limitations and technical aspects
Physiol. Entomol., 37 (2012), pp. 25-32
Layton et al., 1994
J.M. Layton,M.A. Camann,K.E. Espelie
Cuticular lipids profiles of queens, workers and males of social wasp Polistes metricus say are colony-specific
J. Chem. Ecol., 20 (1994), pp. 2307-2321 http://dx.doi.org/10.1007/BF02033205
Lockey, 1988
K.H. Lockey
Lipids of the insect cuticle: origin, composition and function
Comp. Biochem. Physiol. B, 89 (1988), pp. 595-645
Lorenzi et al., 1997
M.C. Lorenzi,A.G. Bagneres,J.L. Clement,S. Turillazzi
Polistes biglumisbimaculatus epicuticular hydrocarbons and nestmate recognition (Hymenoptera, Vespidae)
Insect. Soc., 44 (1997), pp. 123-138
Lorenzi et al., 2004
M.C. Lorenzi,R. Cervo,F. Zacchi,S. Turillazzi,A.G. Bagnères
Dynamics of chemical mimicry in the social parasite wasp Polistes semenowi (Hymenoptera: Vespidae)
Parasitology, 129 (2004), pp. 643-651
Martin et al., 2008
S.J. Martin,H. Helantera,F.P. Drijfhout
Evolution of species-specific cuticular hydrocarbon patterns in Formica ants
Biol. J. Linn. Soc., 95 (2008), pp. 131-140
Monnin, 2006
T. Monnin
Chemical recognition of reproductive status in social insects
Ann. Zool. Fenn., 43 (2006), pp. 515-530
Neves et al., 2012
E.F. Neves,L.H.C. Andrade,Y.R. Súarez,S.M. Lima,W.F. Antonialli-Junior
Age-related changes in the surface pheromones of the wasp Mischocyttarus consimilis (Hymenoptera: Vespidae)
Genet. Mol. Res., 11 (2012), pp. 1891-1898 http://dx.doi.org/10.4238/2012.July.19.8
O’Donnell, 1999
S. O’Donnell
The function of male dominance in the eusocial wasp Mischocyttarus mastigophorus (Hymenoptera: Vespidae)
Ethology, 105 (1999), pp. 273-282
Page et al., 1991
R.E. Page Jr.,R.A. Metcalf,R.L. Metcalf,E.H. Erickson Jr.,R.L. Lampman
Extractable hydrocarbons and kin recognition in honeybee (Apis mellifera L.)
J. Chem. Ecol., 17 (1991), pp. 745-756 http://dx.doi.org/10.1007/BF00994197
Provost et al., 2008
E. Provost,O. Blight,A. Tirard,M. Renucci
Hydrocarbons and insects’ social physiology
Insect Physiology: New Research, pp. 19-72
Silveira, 1998
O.T. Silveira
Mischocyttarus (Mischocyttarus) aripuanaensis. A new social wasp from western-central Brazil, and redescription of Mischocyttarus lindigi Richards (Hym., Vespidae, Polistinae)
Pap. Avulsos Zool., 40 (1998), pp. 359-367
Silveira, 2008
O.T. Silveira
Phylogeny of wasps of the genus Mischocyttarus de Saussure (Hymenoptera, Vespidae, Polistinae)
Rev. Bras. Entomol., 52 (2008), pp. 510-549
Singer et al., 1998
T.L. Singer,E.K. Espelie,G.J. Gamboa
Nest and nestmate discrimination in independent-founding wasps
Pheromone Communication in Social Insects, pp. 104-125
Sledge et al., 2001
M.F. Sledge,F. Boscaro,S. Turillazzi
Cuticular hydrocarbons and reproductive status in the social wasp Polistes dominulus
Behav. Ecol. Sociobiol., 49 (2001), pp. 401-409
Strassmann et al., 1995
J.E. Strassmann,D.C. Queller,C.R. Solis
Genetic relatedness and population structure in the social wasp Mischocyttarus mexicanus (Hymenoptera: Vespidae)
Insect. Soc., 42 (1995), pp. 379-383
Tannure-Nascimento et al., 2007
I.C. Tannure-Nascimento,F.S. Nascimento,I.C. Turatti,N.P. Lopes,J.R. Trigo,R. Zucchi
Colony membership is reflected by variations in cuticular hydrocarbon profile in a Neotropical paper wasp, Polistes satan (Hymenoptera, Vespidae)
Genet. Mol. Res., 6 (2007), pp. 390-396
Tannure-Nascimento et al., 2008
I.C. Tannure-Nascimento,F.S. Nascimento,R. Zucchi
The look of royalty: visual and odour signals of reproductive status in a paper wasp
Proc. R. Soc. B, 275 (2008), pp. 2555-2561 http://dx.doi.org/10.1098/rspb.2008.0589
Torres et al., 2012
V.O. Torres,T.S. Montagna,J. Raizer,W.F. Antonialli-Junior
Division of labor in colonies of the eusocial wasp, Mischocyttarus consimilis
J. Insect. Sci., 12 (2012), pp. 21 http://dx.doi.org/10.1673/031.012.2101
Van den Dool and Kratz, 1963
H. Van den Dool,P.D. Kratz
A generalization of the retention index system including linear temperature programmed gas–liquid partition chromatography
J. Chromatogr., 11 (1963), pp. 463-471
Vander Meer and Morel, 1998
R.K. Vander Meer,L. Morel
Nestmate recognition in ants
Pheromone Communication in Social Insects, pp. 79-103
Von Ihering, 1896
R. Von Ihering
The state of social wasps in Brazil
Bull. Soc. Zool. Fr., 21 (1896), pp. 159-162
(in French)
Wenzel, 1991
J.W. Wenzel
Evolution of nest architecture
The Social Biology of Wasps, pp. 480-519
Wilson, 1965
E.O. Wilson
Chemical communication in the social insects: insect societies are organized principally by complex systems of chemical signals
Science, (1965), pp. 1064-1071
Wilson, 1971
E.O. Wilson
The Insect Societies
Belknap Press, Harvard University Press, (1971)pp. 548
Corresponding author. (Eva Ramona Pereira Soares evapereirasoares@gmail.com)
Copyright © 2017. Sociedade Brasileira de Entomologia
Rev Bras Entomol 2017;61:224-31 - Vol. 61 Núm.3 DOI: 10.1016/j.rbe.2017.05.001