Considerable interest surrounds the genetic architectures underlying fundamental adaptive traits in wild populations [1]–[5]. In social organisms, such interest centers on the numbers and types of genes directly regulating expression of the individual behaviors that, in aggregate, create social organization, as well as genes in interactants that indirectly influence expression of socially relevant behaviors by altering the social environment [6]–[12]. This indirect influence is mediated by interactions of the genotype of a given individual with those of other group members who collectively comprise the social environment. Information on the genetic architecture of social organization is essential to constructing realistic models of social evolution that can answer questions about the numbers and types of genetic changes necessary to change a solitary to a social animal or to convert a simple society to a large and highly complex one [13].

A remarkable case of a fundamental social polymorphism that appears to be under simple genetic control (single Mendelian factor of large effect) is variation in colony social organization in the fire ant Solenopsis invicta. In this species a single genomic element marked by the protein-encoding gene Gp-9 is implicated in the production of two distinct types of queens that differ in physiology, fecundity and behavior [14]–[19]. This genetic factor also determines whether workers tolerate a single fertile queen (monogyne social form) or multiple queens (polygyne social form) in their colony. Colonies containing only homozygous Gp-9 BB workers accept only a single queen, whereas colonies containing both Gp-9 BB and Gp-9 Bb workers invariably accept multiple queens, but only those bearing a Gp-9 b haplotype [20]–[23]. The near complete absence of adult workers and queens with a bb genotype stems from the deleterious effects associated with the genomic region marked by the b allele, inducing homozygous females to die shortly after they eclose from the pupa [22],[24]. The monogyne and polygyne social forms also differ in a number of important reproductive, behavioral, and life history traits besides colony queen number [25],[26], differences that are also completely associated with differences at the genomic region marked by Gp-9. In contrast, there is a complete lack of differentiation at genes not tightly linked to Gp-9, presumably because frequent matings between sexuals from sympatric monogyne and polygyne colonies result in extensive gene flow between the forms [22], [27]–[29].

Colony queen number in S. invicta is regulated by the workers, which collectively decide which and how many queens from within or outside the colony are recruited as new egg-layers [21], largely on the basis of chemical signals emanating from the queens [20]. Workers in monogyne colonies (all of which possess the BB genotype) accept only a single replacement queen that must also bear genotype BB, whereas workers in polygyne colonies accept multiple queens, each of which must possess the b haplotype. Significantly, the presence of as few as 5–10% of workers with the b haplotype induces the entire colony worker force, including BB workers, to become tolerant of multiple Bb queens and thus display the polygyne social phenotype [23]. Thus the genomic region marked by Gp-9 exerts indirect genetic effects [9], in that the presence of the b variant in a colony induces changes in the social behavior of all colony members, even those lacking the b haplotype. Although the identity of the product of Gp-9 as an odorant binding protein (OBP) and other lines of evidence suggest that the gene may play a direct part in regulating social organization via a role in chemical communication, it remains an open question whether other genes tightly linked to Gp-9 (possibly locked up in an inversion with it) are also involved [22]. The first aim of this study was to investigate whether variation in the genomic region marked by Gp-9 is associated with differences in patterns of expression of genes other than Gp-9 in workers. The second aim was to study how the social environment (i.e., presence or absence of nestmate workers with the b allele) can alter individual gene expression patterns. To answer these questions and begin to address the issue of how variation at a single Mendelian factor can directly and indirectly affect gene expression to produce a complex colony-level phenotype, we employed a fire ant microarray platform representing some 10,000 genes [30].

To determine the effect of Gp-9 genotype on gene expression in focal individuals, we compared expression profiles between BB and Bb adult workers from 20 polygyne S. invicta colonies. This comparison revealed 39 genes consistently differentially expressed between workers of the two genotypes, of which about two-thirds were more highly expressed in Bb than BB workers (Figure 1; see also confirmation of microarray data by quantitative RT-PCR [qRT-PCR] in Text S1 and Table S1). Sixteen of these genes did not significantly match any sequence in the public databases (BLASTX, threshold E-value = 1e-5), ten matched predicted proteins of unknown function, and the remaining 13 matched genes with a known or inferred function (Table 1).

Three gene categories were overrepresented among the genes differently expressed between workers of alternate genotypes (Table 1 and Table S2; all P<0.05). The first two are the allergen and odorant binding protein categories, which collectively include five genes likely to contribute to chemical signaling and response, the essential components of regulation of colony queen number and social organization. In ants, venom allergens are proteins released from the venom sac, an organ that, in queens, appears to store and release chemical signals allowing recognition by workers [25]. Similarly, the two odorant binding genes that encode members of the insect OBP protein family (as does Gp-9) and the antennal chemosensory protein may be involved in pheromone transduction, thereby potentially influencing the abilities of workers of the two Gp-9 genotypes to recognize and discriminate among queens. Experiments from other systems are suggestive that changes in the expression levels of OBPs could influence discriminatory behavior by modulating the threshold for a particular response; differential regulation of OBPs has been observed in Drosophila following mating [31], exposure to starvation stress [32], and alcohol tolerance development after exposure to alcohol [33]. Additionally, genetic and biochemical evidence suggests that OBPs may interact combinatorially in odor discrimination [34],[35].

The third overrepresented category comprises two transposons, which are of special interest with respect to properties that may be shared between the genomic region including Gp-9 and regions containing the sex-determining genes in species with sex chromosomes [36]. The b haplotype is found only in the polygyne social form, just as the Y chromosome is found only in males in species with male heterogamety. By analogy with the Y chromosome, theoretical predictions and empirical observations suggest that the Gp-9 b region should (i) accumulate genes beneficial in the polygyne social environment (as the Y chromosome accumulates genes beneficial to male function [37]), (ii) evolve reduced recombination to preserve associations of genes advantageous for polygyny (as occurs for genes advantageous to males on the Y chromosome [38],[39]), and (iii) accumulate deleterious alleles and transposable elements (because of reduced recombination [38],[39]). Consistent with these expectations, the genomic region marked by Gp-9 is characterized by low recombination [40],[41], the b haplotype is a homozygous lethal [15],[24],[40], and the piggyBac-like transposon, which is differentially expressed between workers of alternate genotypes, appears to occur almost exclusively in individuals possessing haplotype b (data not shown). Thus the strong expression of at least this transposon in b-bearing workers, which constitutes the most extreme expression difference among the 13 genes with annotated matches (Table 1), likely reflects its unique insertion in b haplotypes. While this distribution could signify that the piggyBac-like transposon directly affects the differential expression of other candidate genes in BB and Bb workers, we note that, consistent with earlier protein electrophoresis data [40], no significant difference in the expression levels of Gp-9 was detected between workers of the two genotypes; therefore, whatever elements control the differential expression of genes in parallel with Gp-9 genotype appear not to regulate the expression of Gp-9 itself.

To determine the indirect effects of colony Gp-9 genotype composition as well as other aspects of the social environment on worker gene expression, while controlling for individual Gp-9 genotype, we compared profiles of adult workers bearing genotype BB between 20 polygyne and 20 monogyne colonies. This comparison revealed 91 genes consistently differentially expressed between workers of the alternate forms, of which over three-quarters were more highly expressed in polygyne than monogyne workers (Figure 2; see also confirmation of microarray data by qRT-PCR in Text S1 and Table S1). Forty-five of these genes did not significantly match any sequence in the public databases (BLASTX, threshold E-value = 1e-5), 13 matched predicted proteins of unknown function, and the remaining 33 matched previously annotated genes (Table 2).

Three gene categories (mitochondrial, prefoldin complex, and viral genes) were overrepresented among the genes that were differentially expressed between BB workers from monogyne and polygyne colonies (Table 2 and Table S2; all P<0.05). The 11 genes encoding mitochondrial and prefoldin complex (molecular chaperone) proteins were all up-regulated in polygyne compared to monogyne workers. Increased mitochondrial gene expression may reflect increased oxidative metabolism, while increased prefoldin expression may indicate higher protein synthesis rates, possibly in relation to the relatively smaller size and higher metabolic rates of polygyne workers [42].

The pattern of expression of the six genes in the viral gene category is consistent with the expectation that differences in social organization affect susceptibility to pathogens and parasites. In the monogyne form, there is intense selection against susceptible infected individuals during independent colony founding, a stage that colonies of the polygyne form never display [26]. Accordingly, we found that workers in the polygyne form express more sequences corresponding to viral genes than their counterparts in the monogyne form, presumably because of relaxed selection and generally greater susceptibility in the former (see also [43]). Based on sequence similarity and correlated expression across our experiments, we identified six gene products that likely represent three different viruses, a ssRNA negative-strand (−) virus and two ssRNA positive-strand (+) viruses, one of which is the SINV-2 virus [44]. All 20 polygyne study colonies showed evidence of infection with at least one virus (mean number of viral types per colony, 2.5±0.67), whereas only three of the 20 monogyne colonies showed evidence of infection, in all cases by a single viral type. Finally, the pattern of expression of another socially-regulated gene, this one encoding a defensin (a class of small protein antibiotics active against viruses, bacteria, and fungi [45]), also is consistent with greater selection for resistance in the monogyne form, as this gene was more highly expressed in monogyne than polygyne workers.

The numbers of genes differentially expressed in the genotype (39) and social form (91) comparisons are relatively low compared to the numbers expected based on other published microarray experiments. There are several possible explanations for this. First, the use of whole worker bodies as the source of RNA may decrease the probability of detecting genes whose level of expression varies among cells or tissues. Second, our comparisons were performed on groups of workers originating from different colonies, thus adding a colony-level effect to, and thus increasing the total variance in, gene expression. Finally, workers of alternate genotype or social form apparently exhibit fewer phenotypic differences than queens [20],[21],[46],[47], possibly reflecting the involvement of fewer differentially expressed genes in the former caste.

Remarkably, there was almost no overlap between genes whose level of expression was influenced by the focal workers' Gp-9 genotypes and genes whose expression was influenced by the social environment, with only one of the 129 differentially expressed genes appearing in both categories. This demonstrates an almost complete decoupling of the direct effects of the genomic region marked by Gp-9 and the indirect effects mediated by social interactions within colonies. Moreover, there is little indication of an interaction between these direct and indirect effects; genes whose levels of expression depend on the Gp-9 genotype of focal individuals generally are expressed at similar levels in the two social forms when genotype is held constant (Figure 1), whereas genes whose levels of expression depend on the indirect influence of the social environment almost always are expressed at similar levels in polygyne workers of different Gp-9 genotypes (Figure 2; see also Figure S1).

This study reveals that variation at the S. invicta genomic region marked by Gp-9 is associated with the differential expression in workers of a relatively small number of genes that, with the exception of the piggyBac-like transposon, presumably are unlinked to Gp-9. A high proportion of these differentially expressed genes have putative functions implying a role in chemical signaling and behavior relevant to the regulation of colony queen number and, therefore, these genes may have a primary function in determining social organization. The number of such genes is unexpectedly low given the profound behavioral, physiological, and life-history differences between the two social forms and the fact that widespread changes in gene expression patterns can be observed after just a few generations of selection [48],[49]. A perhaps more surprising finding is that worker gene expression profiles are significantly more strongly influenced by indirect effects associated with the Gp-9 genotypic composition within their colony than by the direct effect of their own Gp-9 genotype (chi-squared test, P<0.001), with the indirect-effect genes largely implicated in the secondary differences in colony social characteristics expected between the forms. While several studies have demonstrated that the social environment can modulate gene expression [50]–[53], and others have revealed indirect genetic effects on phenotypes or levels of gene expression [10], [54]–[60], this is the first example of a naturally occurring polymorphic Mendelian element that affects gene expression in other group members. The finding of a complex genetic architecture directly and indirectly influencing the individual behaviors that, in aggregate, generate a fundamental colony-level social phenotype represents an unusual example of an “extended phenotype” [61].