This study is the first comprehensive examination of chemical profiles and genome-wide expression patterns associated with reproductive dominance in a primitively eusocial species. Our analysis of cuticular hydrocarbons identified over a dozen compounds with potential links to the phase of the colony cycle (which encompasses season and social environment) in P. metricus. In addition, we provide new baseline data on transcriptomic correlates of reproductive dominance and caste in both brains and ovaries. Many genes showed expression patterns related to the social environment/season (founding phase vs worker phase, Figure 2), suggesting there could be major effects of social environment on brain gene expression in wasps. Thus, both the chemistry and brain transcriptome data show patterns strongly associated with the social environment, and highlight the fact that there are major differences in the social milieu between founding and worker phase colonies. These data agree with other recent studies suggesting the social environment as one of the most potent influences on gene expression patterns in ants . Finally, our results indicate the brain expression patterns associated with reproductive dominance are (surprisingly) not conserved across wasps and honey bees, but rather that some genes associated with aggressive behaviors may have been co-opted to establish or maintain dominance hierarchies in Polistes wasps.
Previous studies have clearly demonstrated cuticular hydrocarbons change with female fertility in insects including Polistes, with some evidence for cuticular differences related to dominance status in foundresses/workers of other species of Polistes wasps [7, 34, 35, 47, 48]. We identified 13 cuticular hydrocarbons in P. metricus with significant differences across females. Several of these compounds have been identified on the cuticles of other insects, for example, in association with age in mosquitoes (pentacosane ), ovarian activation in social insects (dimethylpentatriacontane and dimethylhentriacontane , pentacosane, nonacosane, methylnonacosane, triacontane, and methyltriacontane ), and even dominance status in other species of Polistes (pentacosane, nonacosane, and methylnonacosane , methylpentatriacontane , and dimethylpentatriacontane ). However, in our study, none of these compounds were closely related to dominance status or levels of ovarian activation in P. metricus, but several showed associations with the time of collection (early vs late season) and/or social environment (foundress association vs queenright mature colony, Figure 1B). Further studies on these 13 compounds could provide additional insights into the role of cuticular hydrocarbons in response to the abiotic and social environment in Polistes.
Our microarray results suggest a relatively small subset of genes in the brain show patterns related to reproductive dominance status. There were several differentially expressed genes related to vision and eye development, which is intriguing because of the importance of visual communication in the genus Polistes, although P. metricus is not known to use visual cues for individual recognition [52, 53]. We found no overlap between sets of differentially expressed genes between dominant and subordinate foundresses and dominant and subordinate workers, suggesting that distinct subsets of genes may be involved in the maintenance of dominance status in the founding and worker stages of colony development. This is consistent with known differences in the role of juvenile hormone (JH) in the development and maintenance of dominance status and ovarian activation in queens versus workers in Polistes. In foundresses, JH regulates behavioral and ovarian reproductive dominance [22, 24, 30]. In workers, JH has a dual function in that it affects both reproductive dominance  and age-related onset of foraging behavior [54, 55], and JH action depends on the physiological condition (i.e. nutritional state) of the female [56, 57].
A slightly larger subset of genes showed caste-associated expression differences in the brain. These genes had functions related to oxidation reduction, aging, and synaptic transmission, which could be linked to known differences in metabolism , lifespan , and learning abilities  between workers and queens. Previous studies in honey bees have also uncovered differences in the expression of genes related to aging (such as telomerase) and oxidation reduction . We suggest candidate genes related to aging (Autophagy-specific gene 7, Excitatory amino acid transporter 1), eye development (microtubule star, rasputin, scabrous), and reproduction (Glutamate dehydrogenase, quick-to-court) may play an important role in establishing and maintaining adult caste differences in Polistes.
Our cross-species comparative analyses showed no significant overlap in sets of genes associated with dominance status in wasps and pheromonal regulation in honey bees. Thus, they do not support the hypothesis that pheromonal regulation of reproduction relies on the same molecular mechanisms as physical dominance in these two species. Furthermore, according to the ovarian and reproductive groundplan hypotheses [60–62], genes involved in reproduction have been co-opted to play a role in queen-worker caste differentiation and worker division of labor. However, in contrast to this theory, we find distinct brain gene expression patterns are associated with reproductive dominance hierarchies between dominant and subordinate co-foundresses and between dominant and subordinate workers, and dominance-associated genes differ between wasps and honey bees. Thus, there does not appear to be a conserved suite of genes regulating these processes in the brain. Interestingly, however, genes associated with dominance in Polistes significantly overlap with sets of genes associated with aggressive phenotypes in honey bees , Drosophila, and mice . There was also some overlap with genes related to foraging in one  of two  previous honey bee studies. This overlap may reflect differences in aggressive behavior between honey bee foragers and non-foragers , or perhaps be explained by the fact that lower dominance status in wasps is typically associated with increased foraging behavior . Overall, these data suggest that there may be a small number of genes with recurrent roles in aggressive behavior across diverse taxa. It is important to note, however, that the microarray only examined a subset of the genes in the paper wasp genome and was limited to transcripts showing significant homology to honey bee or other insect proteins. The role of novel genes or rapidly evolving genes in the regulation of dominance status in Polistes remains to be explored and is definitely worthy of further attention .
We found large differences in ovary gene expression, both associated with dominance status and with caste differences. Overall, many transcripts showed expression differences associated with gross differences in ovary size (Figure 3A). This pattern is reflected in the types of genes that were differentially expressed--there were numerous genes with functions related to cell division and proliferation, as well as production of nucleic acids and proteins. Thus, the large differences in ovary size across the groups (Figure 3A) are undoubtedly produced by changes in the regulation of genes related to egg production and maturation.