BACKGROUND
in many animal species, social interactions with conspecifics can profoundly influence individual physiology and behavior, including reproduction  <cit> .
eusocial insect societies represent an extreme case in which colonies consist of one or a small number of reproductively active queens or kings and tens to millions of sterile workers.
in some species, direct physical aggression establishes reproductive dominance, while other species use chemical signaling via pheromones to establish dominance hierarchies  <cit> .
it has been hypothesized that such chemical communication systems evolved from an ancestral state in which aggressive dominance interactions inhibited reproduction  <cit> .
comparative studies of the genomic mechanisms mediating reproductive dominance through aggression or chemical signaling can determine if these modes of communication share common genetic underpinnings.
comparisons of the genomic mechanisms underlying social regulation of reproductive dominance in primitively eusocial polistes wasps and advanced eusocial apis honey bees provide an excellent framework with which to study the evolution of social inhibition of worker reproduction  <cit> .
eusocial behavior evolved separately in bee and wasp lineages  <cit> , and thus, any shared mechanisms for sociality and reproduction between honey bees and paper wasps may represent deeply conserved elements that could be employed in multiple insect lineages  <cit> .
on one extreme, polistes wasps use physical aggression to initially establish dominance hierarchies, and then transition to ritualized behaviors, possibly using chemically based recognition to maintain these hierarchies  <cit> .
on the other extreme, honey bee queens use pheromones to establish reproductive dominance, and physical aggression by queens towards workers is not observed.
the effects of honey bee queen pheromones on worker physiology, behavior, and gene expression patterns have been extensively characterized  <cit> .
conversely, gene expression associated with dominance status in polistes has not been previously studied, but such work is now possible with the development of microarrays to monitor genome wide expression patterns in polistes metricus wasps  <cit> .
the goal of this study was to compare dominance-related gene expression in polistes to aggression- and pheromone-related gene expression patterns in honey bees and other species, allowing us to test whether reproductive dominance in polistes is associated with genes with known links to aggression, pheromonal regulation, or both.
the presence of distinct groups of females on polistes colonies exhibiting a range of caste, dominance, and reproductive states  <cit>  provides an excellent opportunity for dissecting the mechanistic bases of reproductive dominance.
the fact that dominance hierarchies occur in different stages of colony development also allows us to examine the importance of the colony environment, which has proven to be very important in recent studies in other social insects  <cit> .
in temperate species of polistes, one or a few sister females found annual colonies in the spring “founding phase”  <cit> .
if multiple females are present, these foundresses form a dominance hierarchy with dominant foundresses  taking over egg-laying and subordinate foundresses  taking over foraging and provisioning of larvae  <cit> .
during the “worker phase”, foundress-reared brood emerge as adult females, which typically become workers  <cit> .
at this time, the dominant foundress increases egg-laying and is called the queen, and subordinate foundresses either die or are forced from the nest  <cit> .
linear dominance hierarchy among the females characterizes worker phase nests, with the queen as the alpha, or most dominant, individual  <cit> .
among workers, both dominant workers  and subordinate workers  are socially inhibited from reproducing, but subordinates even more so due to receiving more aggressive contacts and engaging in energetically demanding foraging behavior  <cit> .
the initial position of a polistes individual in a dominance hierarchy is established within a few minutes via intense aggressive interactions with other females, including biting, grappling, and attempted stinging  <cit> .
since behavioral dominance is established rapidly, it is not likely to involve large-scale changes in gene expression or physiology, although prior physiological and hormonal state influences performance in dominance contests  <cit> .
subsequently, repeated, more ritualized dominance interactions maintain physiological, or reproductive, dominance  <cit> .
dominance hierarchy maintenance requires chemical and/or visual individual recognition between wasps  <cit> .
large physiological changes in ovary activation, juvenile hormone, and ecdysteroid titers accompany this longer-term reproductive dominance  <cit> .
here, we focus on reproductive  dominance rather than behavioral dominance, as this form of dominance is longer-term and thus more likely to be manifest at the level of gene expression.
this study focuses on transcriptomic and chemical correlates of reproductive dominance, both within and between the different female castes in polistes metricus wasps.
first, we examined chemical profiles in subordinate and dominant polistes workers, nest-founding females , and queens, to determine if there were chemical correlates of caste and reproductive dominance, as suggested by previous studies with other species of polistes <cit> , which could potentially function as chemical signals or cues to establish or maintain reproductive dominance hierarchies.
next, we examined the gene expression profiles in the brains and ovaries of these five groups of wasps, to explore transcriptomic correlates of caste and reproductive dominance.
the goals of the transcriptomic study were two-fold: first, to provide new baseline data on patterns of gene expression associated with dominance to identify candidate genes for future studies, and second, to conduct a comparative genomic analysis by quantitatively comparing wasp dominance-associated gene expression patterns to gene expression data in other species.
we hypothesized that paper wasp brain gene expression patterns would be related to gene expression associated with caste, exposure to queen pheromone and/or aggression in honey bee workers.
in addition, we extended our comparative analysis to available data on aggression-related gene expression from two non-eusocial species, the fruit fly drosophila melanogaster and the mouse mus musculus.
the results of these studies suggest reproductive hierarchies in primitively social species are associated with gene networks related to aggression in solitary species rather than pheromonal regulation in advanced eusocial species.
RESULTS
chemical analyses
we examined chemical profiles from dominant and subordinate co-foundresses  as well as queens and dominant and subordinate workers .
we identified four body areas as candidate carriers of dominance-related chemicals: 1) cuticular hydrocarbons, previously associated with dominance status in p. dominula <cit> ; 2) the mandibular glands, where queen pheromone is produced in honey bees  <cit> , 3) dufour’s glands because of their role in egg-marking in several social insect species  <cit> , and 4) the sternal glands because of their potential importance to abdomen rubbing behavior which may accompany dominance interactions in polistes <cit> .
each of the four body regions examined had distinct chemical profiles, none of which were clearly related to dominance status.
data from the three glands are presented and discussed in the supplementary materials .
from the cuticle, we identified  <dig> distinct hydrocarbons, a large proportion of which  showed significant differences across the five groups of wasps .
linear discriminant analysis  revealed a separation between the foundresses and the queen/worker groups ; this could possibly reflect differences in season or social environment.
using gc-ms, we identified at least  <dig> different hydrocarbons of varying chain lengths from 25- <dig> .
thirteen of these compounds were previously identified as present on the cuticle of polistes metricus .
hierarchical clustering of the  <dig> compounds with significant differences among the treatment groups  revealed a correlated cluster of five compounds in the range of 33- <dig> carbon chain length .
all five compounds varied significantly among treatments , and showed a similar pattern that reflected differences in season and/or social environment, with the highest levels in worker phase individuals  and lowest levels in founding phase individuals .
polistes metricus
all identifications were high confidence  except for those indicated with .
compounds that differed in anova analysis across the five groups are highlighted in bold and patterns presented graphically in figure  <dig>  compounds with *were also identified as present in polistes metricus in  <cit> .
brain gene expression
using previously developed custom oligo microarrays for p. metricus <cit> , we examined brain gene expression patterns of eight individuals from each of the five groups of wasps .
of the  <dig> transcripts represented on the arrays,  <dig> were expressed above background levels in a sufficient number of arrays to be included in the analysis.
<dig> of these  were differentially regulated across the five behavioral groups .
differentially regulated transcripts showed multiple distinct expression patterns across the five groups .
as in the case of the cuticular hydrocarbon profiles, there are clear differences associated with the two colony developmental phases.
this effect of season/social environment on brain expression patterns was apparent in both hierarchical clustering based on a distance matrix of all possible contrasts  and principal components analysis , where season/social environment accounted for 23% of the overall expression variation.
post-hoc contrasts across the groups  revealed relatively few transcripts were associated with dominance status.
we focused on two contrasts  because these contrasts represented females that were interacting together on the same nest and were not confounded by comparisons across castes.
there were  <dig> differentially regulated transcripts between dominant and subordinate foundresses and  <dig> differentially regulated transcripts between dominant workers and subordinate workers.
there was no overlap across these two contrasts, suggesting again a potent influence of season or social environment in that different mechanisms appear to be associated with dominance in foundresses and workers .
the  <dig> transcripts showing differences between dominant and subordinate females  are heretofore referred to as “brain dominance-associated” transcripts .
similarly, to identify caste-associated genes, we examined overlapping sets of genes from queen vs worker post-hoc contrasts  and found a somewhat larger signal of differential expression.
there were  <dig> differentially regulated transcripts between queens and dominant workers and  <dig> between queens and subordinate workers,  <dig> of which overlapped between the two contrasts .
the  <dig> transcripts showing differences between queens and workers  are heretofore referred to as “brain caste-associated” transcripts .
we validated array expression data for one gene, vitellogenin, which was previously examined in queens and subordinate workers using quantitative real time pcr   <cit> .
expression patterns uncovered with the array in the current study showed strikingly similar patterns to previous qrt-pcr data, with approximately 2-fold higher expression in queens compared to subordinate workers in both studies .
ovary gene expression
next, we examined ovary gene expression patterns of eight individuals from each of the five groups of wasps .
out of  <dig> transcripts represented on the array,  <dig> were expressed above background levels in a sufficient number of arrays to be included in the analysis.
of those, we found a large proportion  were differentially regulated across the five groups .
again, there was a diversity of expression patterns across the five groups .
one of the most prevalent expression patterns  reflects the gross level of ovary activation; queens and dominant foundresses had very high levels of ovary activation  and were likely to be actively egg-laying, whereas the other three groups had very low ovary activation  with no mature oocytes and were thus not actively egg-laying.
this pattern was recovered both by hierarchical clustering by gene  and pca , in which ovary activation levels accounted for 41% of the variation.
post-hoc contrasts across the groups  revealed a moderately large number of transcripts were associated with dominance status.
there were  <dig> differentially regulated transcripts between dominant and subordinate foundresses and  <dig> differentially regulated transcripts between dominant workers and subordinate workers.
there was an overlap of  <dig> transcripts across these two contrasts , suggesting that there may be both shared and divergent mechanisms associated with ovary activation across the reproductive and worker castes.
the  <dig> transcripts showing differences between dominant and subordinate females  are heretofore referred to as “ovary dominance-associated” transcripts .
by examining overlapping sets of genes from queen vs worker post-hoc contrasts , we again found a larger signal of differential expression associated with caste differences.
there were  <dig> differentially regulated transcripts between queens and dominant workers and  <dig> between queens and subordinate workers,  <dig> of which overlapped between the two contrasts .
the  <dig> transcripts showing differences between queens and workers  heretofore referred to as “ovary caste-associated” transcripts .
gene ontology  analysis on dominance and caste-associated gene lists
using david  <cit> , we tested to see which, if any, gene ontology categories  of genes were under or over-represented in our gene lists compared the background array.
the  <dig> “brain dominance-associated” transcripts were represented by several small clusters of genes , none of which were significantly overrepresented in the gene lists relative to the background gene set on the array: eye development, reproduction, and cytoskeletal organization.
for the “brain caste-associated” transcripts, only one go category, oxidation reduction, was significantly overrepresented relative to the background on the array, and this was only significant with unadjusted p-values .
other processes associated with, but not significantly overrepresented, in brain caste-associated genes included aging, synaptic transmission, and rna processing.
drosophila
brain dominance-associated
brain caste-associated
ovary dominance-associated
ovary caste-associated
based on a go analysis using drosophila homologs, the top  <dig> or  <dig> clusters of go terms corresponding to “biological process” are shown.
each cluster listed is accompanied by a description of the go terms that make up the “biological process of cluster”, “# genes” represented in each cluster, “# enriched subcategories” which incidates go subcategories that were significant within each cluster , and “example drosophila homologs” in each cluster.
in general, both “ovary dominance-associated” and “ovary caste-associated” genes showed functions related to protein folding, mitotic spindle organization, proteolysis, and metabolism .
for the “ovary caste-associated” list, there were a number of genes related to reproduction and ovary activation, though none of these were significantly enriched relative to the background.
there was a cluster of  <dig> genes related to “reproductive process”, which included insulin receptor substrate, sex lethal, female sterile  ketel, ecdysone induced protein 75b.
another cluster of six genes was related to “oocyte fate determination”, and included capping protein alpha, armadillo, and notch.
comparative analysis
to begin to identify conserved pathways associated with caste and dominance, we tested for overlap between our complete lists of differentially regulated transcripts in brain  and lists of differentially regulated transcripts from several studies in other species .
p. metricus
,
apis
,
drosophila
, and
mus
apis mellifera
apis mellifera
apis mellifera
d. melanogaster
mus musculus
brain de refers to the complete list of transcripts differentially expressed in the brain, “brain caste” refers to the subset of genes that are “brain caste-associated”, described in the main text.
the number of transcripts overlapping , as well as non-overlapping transcripts  and two-tailed p-values from fisher’s exact tests are shown.
lists of genes with significant overlaps are highlighted in bold and a complete list of overlapping transcripts are provided in additional file  <dig>
we found no significant overlap between wasp brain differentially expressed transcripts and those differentially expressed in honey bees in association with caste differences  <cit>  or response to queen pheromone  <cit> .
we did find a significant overlap between wasp brain differentially expressed gene lists and those associated with differences in worker foraging behavior in honey bees in one study  <cit> , but this result was not confirmed in comparisons with a second study on the same behavior  <cit> .
we next investigated whether there was significant overlap with genes relating to aggressive behavior in honey bees.
alaux et al.
<cit>  examined honey bee aggressive behavior in several contexts—as it relates to genotype , age , and response to alarm pheromone .
we first focused on the subset of genes that were found to be differentially expressed in all three contexts in honey bees.
we found a small, but significant overlap between this list of genes and the complete set of brain differentially expressed genes in wasps.
this suggestive result, along with the small size of the gene lists being compared, led us to compile an expanded list of all genes related to honey bee aggressive behavior in any context .
here we found no significant overlap with the wasp brain differentially expressed list; however, when we compared this expanded honey bee aggression list to the wasp "brain caste-associated" genes, we again found significant overlap .
there was no significant overlap when we compared to the  <dig> wasp "brain dominance-associated genes” .
to further investigate the potential connection between genes related to dominance  and aggression, we compared our complete wasp brain differentially expressed gene list to microarray studies identifying brain-expressed genes associated with aggression in drosophila melanogaster fruit flies  <cit>  and maternal aggression in mice  <cit> .
in both cases, we found evidence of a relatively small, but statistically significant overlap .
as a control, we compared our study to another mouse study that used the same microarray to examine brain gene expression patterns associated with sleep  <cit> —no significant overlap was detected.
discussion
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  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 , 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  <cit> .
finally, our results indicate the brain expression patterns associated with reproductive dominance are  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  <cit> .
we identified  <dig> 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 , ovarian activation in social insects , and even dominance status in other species of polistes .
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  and/or social environment .
further studies on these  <dig> 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  <cit> .
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  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  <cit> .
in workers, jh has a dual function in that it affects both reproductive dominance  <cit>  and age-related onset of foraging behavior  <cit> , and jh action depends on the physiological condition  of the female  <cit> .
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  <cit> , lifespan  <cit> , and learning abilities  <cit>  between workers and queens.
previous studies in honey bees have also uncovered differences in the expression of genes related to aging  and oxidation reduction  <cit> .
we suggest candidate genes related to aging , eye development , and reproduction  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  <cit> , 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  <cit> , drosophila <cit> , and mice  <cit> .
there was also some overlap with genes related to foraging in one  <cit>  of two  <cit>  previous honey bee studies.
this overlap may reflect differences in aggressive behavior between honey bee foragers and non-foragers  <cit> , or perhaps be explained by the fact that lower dominance status in wasps is typically associated with increased foraging behavior  <cit> .
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  <cit> .
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 .
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  are undoubtedly produced by changes in the regulation of genes related to egg production and maturation.
CONCLUSIONS
in summary, experiments presented here provide a wealth of new data about the chemical and transcriptomic correlates of reproductive dominance in polistes paper wasps, an important model system for studying dominance behavior and the evolution of sociality  <cit> .
several specific compounds and genes are excellent candidate for future studies of their causal role in establishing and maintaining dominance.
our data also highlight the importance of the season and/or social environment in gene expression and cuticular hydrocarbon production, and suggest there are distinct mechanisms responsible for communicating and maintaining dominance among foundresses, between queens and workers, and among workers.
comparisons with honey bees suggest that largely different sets of genes are associated with social regulation of reproduction in honey bees and paper wasps.
this is not entirely surprising, considering bees and social vespids diverged between 100- <dig> million years ago  <cit> , and that the form of social control of reproduction  differs greatly between the two species.
the most notable finding from our cross-species comparisons is that genes that are differentially expressed in brains of dominant and subordinate wasps are likely to be associated with aggression in other species, from honey bees, to flies, to mice.
our data suggest that in primitively social wasps, social regulation of reproduction may be regulated by genes with deeply conserved functions associated with aggression in solitary insects and other taxa.
thus, our data have begun to unravel the evolution of the mechanistic underpinnings of reproductive inhibition in workers, and that in some cases this may be built on fundamental elements of solitary behavior, such as aggression.

 in many animal species, social interactions with conspecifics can profoundly influence individual physiology and behavior, including reproduction. eusocial insect societies represent an extreme case in which colonies consist of one or a small number of reproductively active queens or kings and tens to millions of sterile workers. in some species, direct physical aggression establishes reproductive dominance, while other species use chemical signaling via pheromones to establish dominance hierarchies. it has been hypothesized that such chemical communication systems evolved from an ancestral state in which aggressive dominance interactions inhibited reproduction. comparative studies of the genomic mechanisms mediating reproductive dominance through aggression or chemical signaling can determine if these modes of communication share common genetic underpinnings. comparisons of the genomic mechanisms underlying social regulation of reproductive dominance in primitively eusocial polistes wasps and advanced eusocial apis honey bees provide an excellent framework with which to study the evolution of social inhibition of worker reproduction. eusocial behavior evolved separately in bee and wasp lineages, and thus, any shared mechanisms for sociality and reproduction between honey bees and paper wasps may represent deeply conserved elements that could be employed in multiple insect lineages. on one extreme, polistes wasps use physical aggression to initially establish dominance hierarchies, and then transition to ritualized behaviors, possibly using chemically based recognition to maintain these hierarchies. on the other extreme, honey bee queens use pheromones to establish reproductive dominance, and physical aggression by queens towards workers is not observed. the effects of honey bee queen pheromones on worker physiology, behavior, and gene expression patterns have been extensively characterized. conversely, gene expression associated with dominance status in polistes has not been previously studied, but such work is now possible with the development of microarrays to monitor genome wide expression patterns in polistes metricus wasps. the goal of this study was to compare dominance-related gene expression in polistes to aggression- and pheromone-related gene expression patterns in honey bees and other species, allowing us to test whether reproductive dominance in polistes is associated with genes with known links to aggression, pheromonal regulation, or both. the presence of distinct groups of females on polistes colonies exhibiting a range of caste, dominance, and reproductive states provides an excellent opportunity for dissecting the mechanistic bases of reproductive dominance. the fact that dominance hierarchies occur in different stages of colony development also allows us to examine the importance of the colony environment, which has proven to be very important in recent studies in other social insects. in temperate species of polistes, one or a few sister females found annual colonies in the spring “founding phase”. if multiple females are present, these foundresses form a dominance hierarchy with dominant foundresses taking over egg-laying and subordinate foundresses taking over foraging and provisioning of larvae. during the “worker phase”, foundress-reared brood emerge as adult females, which typically become workers. at this time, the dominant foundress increases egg-laying and is called the queen, and subordinate foundresses either die or are forced from the nest. linear dominance hierarchy among the females characterizes worker phase nests, with the queen as the alpha, or most dominant, individual. among workers, both dominant workers and subordinate workers are socially inhibited from reproducing, but subordinates even more so due to receiving more aggressive contacts and engaging in energetically demanding foraging behavior. the initial position of a polistes individual in a dominance hierarchy is established within a few minutes via intense aggressive interactions with other females, including biting, grappling, and attempted stinging. since behavioral dominance is established rapidly, it is not likely to involve large-scale changes in gene expression or physiology, although prior physiological and hormonal state influences performance in dominance contests. subsequently, repeated, more ritualized dominance interactions maintain physiological, or reproductive, dominance. dominance hierarchy maintenance requires chemical and/or visual individual recognition between wasps. large physiological changes in ovary activation, juvenile hormone, and ecdysteroid titers accompany this longer-term reproductive dominance. here, we focus on reproductive dominance rather than behavioral dominance, as this form of dominance is longer-term and thus more likely to be manifest at the level of gene expression. this study focuses on transcriptomic and chemical correlates of reproductive dominance, both within and between the different female castes in polistes metricus wasps. first, we examined chemical profiles in subordinate and dominant polistes workers, nest-founding females, and queens, to determine if there were chemical correlates of caste and reproductive dominance, as suggested by previous studies with other species of polistes, which could potentially function as chemical signals or cues to establish or maintain reproductive dominance hierarchies. next, we examined the gene expression profiles in the brains and ovaries of these five groups of wasps, to explore transcriptomic correlates of caste and reproductive dominance. the goals of the transcriptomic study were two-fold: first, to provide new baseline data on patterns of gene expression associated with dominance to identify candidate genes for future studies, and second, to conduct a comparative genomic analysis by quantitatively comparing wasp dominance-associated gene expression patterns to gene expression data in other species. we hypothesized that paper wasp brain gene expression patterns would be related to gene expression associated with caste, exposure to queen pheromone and/or aggression in honey bee workers. in addition, we extended our comparative analysis to available data on aggression-related gene expression from two non-eusocial species, the fruit fly drosophila melanogaster and the mouse mus musculus. the results of these studies suggest reproductive hierarchies in primitively social species are associated with gene networks related to aggression in solitary species rather than pheromonal regulation in advanced eusocial species.  chemical analyses we examined chemical profiles from dominant and subordinate co-foundresses as well as queens and dominant and subordinate workers. we identified four body areas as candidate carriers of dominance-related chemicals: 1) cuticular hydrocarbons, previously associated with dominance status in p. dominula; 2) the mandibular glands, where queen pheromone is produced in honey bees, 3) dufour’s glands because of their role in egg-marking in several social insect species, and 4) the sternal glands because of their potential importance to abdomen rubbing behavior which may accompany dominance interactions in polistes. each of the four body regions examined had distinct chemical profiles, none of which were clearly related to dominance status. data from the three glands are presented and discussed in the supplementary materials. from the cuticle, we identified distinct hydrocarbons, a large proportion of which showed significant differences across the five groups of wasps. linear discriminant analysis revealed a separation between the foundresses and the queen/worker groups; this could possibly reflect differences in season or social environment. using gc-ms, we identified at least different hydrocarbons of varying chain lengths from 25-. thirteen of these compounds were previously identified as present on the cuticle of polistes metricus. hierarchical clustering of the compounds with significant differences among the treatment groups revealed a correlated cluster of five compounds in the range of 33- carbon chain length. all five compounds varied significantly among treatments, and showed a similar pattern that reflected differences in season and/or social environment, with the highest levels in worker phase individuals and lowest levels in founding phase individuals. polistes metricus all identifications were high confidence except for those indicated with. compounds that differed in anova analysis across the five groups are highlighted in bold and patterns presented graphically in figure  compounds with *were also identified as present in polistes metricus in. brain gene expression using previously developed custom oligo microarrays for p. metricus, we examined brain gene expression patterns of eight individuals from each of the five groups of wasps. of the transcripts represented on the arrays, were expressed above background levels in a sufficient number of arrays to be included in the analysis. of these were differentially regulated across the five behavioral groups. differentially regulated transcripts showed multiple distinct expression patterns across the five groups. as in the case of the cuticular hydrocarbon profiles, there are clear differences associated with the two colony developmental phases. this effect of season/social environment on brain expression patterns was apparent in both hierarchical clustering based on a distance matrix of all possible contrasts and principal components analysis, where season/social environment accounted for 23% of the overall expression variation. post-hoc contrasts across the groups revealed relatively few transcripts were associated with dominance status. we focused on two contrasts because these contrasts represented females that were interacting together on the same nest and were not confounded by comparisons across castes. there were differentially regulated transcripts between dominant and subordinate foundresses and differentially regulated transcripts between dominant workers and subordinate workers. there was no overlap across these two contrasts, suggesting again a potent influence of season or social environment in that different mechanisms appear to be associated with dominance in foundresses and workers. the transcripts showing differences between dominant and subordinate females are heretofore referred to as “brain dominance-associated” transcripts. similarly, to identify caste-associated genes, we examined overlapping sets of genes from queen vs worker post-hoc contrasts and found a somewhat larger signal of differential expression. there were differentially regulated transcripts between queens and dominant workers and between queens and subordinate workers, of which overlapped between the two contrasts. the transcripts showing differences between queens and workers are heretofore referred to as “brain caste-associated” transcripts. we validated array expression data for one gene, vitellogenin, which was previously examined in queens and subordinate workers using quantitative real time pcr. expression patterns uncovered with the array in the current study showed strikingly similar patterns to previous qrt-pcr data, with approximately 2-fold higher expression in queens compared to subordinate workers in both studies. ovary gene expression next, we examined ovary gene expression patterns of eight individuals from each of the five groups of wasps. out of transcripts represented on the array, were expressed above background levels in a sufficient number of arrays to be included in the analysis. of those, we found a large proportion were differentially regulated across the five groups. again, there was a diversity of expression patterns across the five groups. one of the most prevalent expression patterns reflects the gross level of ovary activation; queens and dominant foundresses had very high levels of ovary activation and were likely to be actively egg-laying, whereas the other three groups had very low ovary activation with no mature oocytes and were thus not actively egg-laying. this pattern was recovered both by hierarchical clustering by gene and pca, in which ovary activation levels accounted for 41% of the variation. post-hoc contrasts across the groups revealed a moderately large number of transcripts were associated with dominance status. there were differentially regulated transcripts between dominant and subordinate foundresses and differentially regulated transcripts between dominant workers and subordinate workers. there was an overlap of transcripts across these two contrasts, suggesting that there may be both shared and divergent mechanisms associated with ovary activation across the reproductive and worker castes. the transcripts showing differences between dominant and subordinate females are heretofore referred to as “ovary dominance-associated” transcripts. by examining overlapping sets of genes from queen vs worker post-hoc contrasts, we again found a larger signal of differential expression associated with caste differences. there were differentially regulated transcripts between queens and dominant workers and between queens and subordinate workers, of which overlapped between the two contrasts. the transcripts showing differences between queens and workers heretofore referred to as “ovary caste-associated” transcripts. gene ontology analysis on dominance and caste-associated gene lists using david, we tested to see which, if any, gene ontology categories of genes were under or over-represented in our gene lists compared the background array. the “brain dominance-associated” transcripts were represented by several small clusters of genes, none of which were significantly overrepresented in the gene lists relative to the background gene set on the array: eye development, reproduction, and cytoskeletal organization. for the “brain caste-associated” transcripts, only one go category, oxidation reduction, was significantly overrepresented relative to the background on the array, and this was only significant with unadjusted p-values. other processes associated with, but not significantly overrepresented, in brain caste-associated genes included aging, synaptic transmission, and rna processing. drosophila brain dominance-associated brain caste-associated ovary dominance-associated ovary caste-associated based on a go analysis using drosophila homologs, the top or clusters of go terms corresponding to “biological process” are shown. each cluster listed is accompanied by a description of the go terms that make up the “biological process of cluster”, “# genes” represented in each cluster, “# enriched subcategories” which incidates go subcategories that were significant within each cluster, and “example drosophila homologs” in each cluster. in general, both “ovary dominance-associated” and “ovary caste-associated” genes showed functions related to protein folding, mitotic spindle organization, proteolysis, and metabolism. for the “ovary caste-associated” list, there were a number of genes related to reproduction and ovary activation, though none of these were significantly enriched relative to the background. there was a cluster of genes related to “reproductive process”, which included insulin receptor substrate, sex lethal, female sterile ketel, ecdysone induced protein 75b. another cluster of six genes was related to “oocyte fate determination”, and included capping protein alpha, armadillo, and notch. comparative analysis to begin to identify conserved pathways associated with caste and dominance, we tested for overlap between our complete lists of differentially regulated transcripts in brain and lists of differentially regulated transcripts from several studies in other species. p. metricus, apis, drosophila, and mus apis mellifera apis mellifera apis mellifera d. melanogaster mus musculus brain de refers to the complete list of transcripts differentially expressed in the brain, “brain caste” refers to the subset of genes that are “brain caste-associated”, described in the main text. the number of transcripts overlapping, as well as non-overlapping transcripts and two-tailed p-values from fisher’s exact tests are shown. lists of genes with significant overlaps are highlighted in bold and a complete list of overlapping transcripts are provided in additional file we found no significant overlap between wasp brain differentially expressed transcripts and those differentially expressed in honey bees in association with caste differences or response to queen pheromone. we did find a significant overlap between wasp brain differentially expressed gene lists and those associated with differences in worker foraging behavior in honey bees in one study, but this result was not confirmed in comparisons with a second study on the same behavior. we next investigated whether there was significant overlap with genes relating to aggressive behavior in honey bees. alaux et al. examined honey bee aggressive behavior in several contexts—as it relates to genotype, age, and response to alarm pheromone. we first focused on the subset of genes that were found to be differentially expressed in all three contexts in honey bees. we found a small, but significant overlap between this list of genes and the complete set of brain differentially expressed genes in wasps. this suggestive result, along with the small size of the gene lists being compared, led us to compile an expanded list of all genes related to honey bee aggressive behavior in any context. here we found no significant overlap with the wasp brain differentially expressed list; however, when we compared this expanded honey bee aggression list to the wasp "brain caste-associated" genes, we again found significant overlap. there was no significant overlap when we compared to the wasp "brain dominance-associated genes”. to further investigate the potential connection between genes related to dominance and aggression, we compared our complete wasp brain differentially expressed gene list to microarray studies identifying brain-expressed genes associated with aggression in drosophila melanogaster fruit flies and maternal aggression in mice. in both cases, we found evidence of a relatively small, but statistically significant overlap. as a control, we compared our study to another mouse study that used the same microarray to examine brain gene expression patterns associated with sleep —no significant overlap was detected. discussion 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 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, 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 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. we identified 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, ovarian activation in social insects, and even dominance status in other species of polistes. 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 and/or social environment. further studies on these 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. 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 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. in workers, jh has a dual function in that it affects both reproductive dominance and age-related onset of foraging behavior, and jh action depends on the physiological condition of the female. 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 and oxidation reduction. we suggest candidate genes related to aging, eye development, and reproduction 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, 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. 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 are undoubtedly produced by changes in the regulation of genes related to egg production and maturation.  in summary, experiments presented here provide a wealth of new data about the chemical and transcriptomic correlates of reproductive dominance in polistes paper wasps, an important model system for studying dominance behavior and the evolution of sociality. several specific compounds and genes are excellent candidate for future studies of their causal role in establishing and maintaining dominance. our data also highlight the importance of the season and/or social environment in gene expression and cuticular hydrocarbon production, and suggest there are distinct mechanisms responsible for communicating and maintaining dominance among foundresses, between queens and workers, and among workers. comparisons with honey bees suggest that largely different sets of genes are associated with social regulation of reproduction in honey bees and paper wasps. this is not entirely surprising, considering bees and social vespids diverged between 100- million years ago, and that the form of social control of reproduction differs greatly between the two species. the most notable finding from our cross-species comparisons is that genes that are differentially expressed in brains of dominant and subordinate wasps are likely to be associated with aggression in other species, from honey bees, to flies, to mice. our data suggest that in primitively social wasps, social regulation of reproduction may be regulated by genes with deeply conserved functions associated with aggression in solitary insects and other taxa. thus, our data have begun to unravel the evolution of the mechanistic underpinnings of reproductive inhibition in workers, and that in some cases this may be built on fundamental elements of solitary behavior, such as aggression. 
furthermore, there was a substantial impact of season/social environment on gene expression patterns, indicating the important role of external cues in shaping the molecular processes regulating behavior. our goals were to first characterize transcriptomic and chemical correlates of reproductive dominance and second, to test whether dominance-associated mechanisms in paper wasps overlapped with aggression or pheromone-related gene expression patterns in other species. this study provides the first comprehensive chemical and molecular analysis of reproductive dominance in paper wasps. we found little evidence for a chemical basis for reproductive dominance in p. metricus, and our transcriptomic analyses suggest that different pathways regulate dominance in paper wasps and pheromone response in bees. by examining dominant and subordinate females from queen and worker castes in early and late season colonies, we found that cuticular hydrocarbon profiles and genome-wide patterns of brain gene expression were primarily associated with season/social environment rather than dominance status. interestingly, genes associated with dominance in wasps were also associated with aggressive behavior in bees, solitary insects and mammals. to explore whether conserved molecular pathways relate to dominance, we compared wasp transcriptomic data to previous studies of gene expression associated with pheromonal communication and queen-worker differences in honey bees, and aggressive behavior in bees, drosophila, and mice. thus, genes involved in social regulation of reproduction in polistes may have conserved functions associated with aggression in insects and other taxa. in contrast, gene expression patterns in the ovaries were associated primarily with caste and ovary activation. here, we use a comparative framework to investigate the chemical and genomic underpinnings of reproductive dominance in the primitively eusocial wasp polistes metricus.
 in social groups, dominant individuals may socially inhibit reproduction of subordinates using aggressive interactions or, in the case of highly eusocial insects, pheromonal communication. it has been hypothesized these two modes of reproductive inhibition utilize conserved pathways. here, we use a comparative framework to investigate the chemical and genomic underpinnings of reproductive dominance in the primitively eusocial wasp polistes metricus. our goals were to first characterize transcriptomic and chemical correlates of reproductive dominance and second, to test whether dominance-associated mechanisms in paper wasps overlapped with aggression or pheromone-related gene expression patterns in other species. to explore whether conserved molecular pathways relate to dominance, we compared wasp transcriptomic data to previous studies of gene expression associated with pheromonal communication and queen-worker differences in honey bees, and aggressive behavior in bees, drosophila, and mice.  by examining dominant and subordinate females from queen and worker castes in early and late season colonies, we found that cuticular hydrocarbon profiles and genome-wide patterns of brain gene expression were primarily associated with season/social environment rather than dominance status. in contrast, gene expression patterns in the ovaries were associated primarily with caste and ovary activation. comparative analyses suggest genes identified as differentially expressed in wasp brains are not related to queen pheromonal communication or caste in bees, but were significantly more likely to be associated with aggression in other insects, and even a mammal.  this study provides the first comprehensive chemical and molecular analysis of reproductive dominance in paper wasps. we found little evidence for a chemical basis for reproductive dominance in p. metricus, and our transcriptomic analyses suggest that different pathways regulate dominance in paper wasps and pheromone response in bees. furthermore, there was a substantial impact of season/social environment on gene expression patterns, indicating the important role of external cues in shaping the molecular processes regulating behavior. interestingly, genes associated with dominance in wasps were also associated with aggressive behavior in bees, solitary insects and mammals. thus, genes involved in social regulation of reproduction in polistes may have conserved functions associated with aggression in insects and other taxa. waspssocial behaviorgenomicsaggressionpheromoneschemical communication 