Results
Direct genetic effects. Seventy-one genes were differentially expressed between brains of SB/SB and SB/Sbworkers and twenty-three between brains of SB/SB and SB/Sbgynes (False Discovery Rate [FDR] < 0.1; Figure 2A). Eighty-six genes were differentially expressed between gasters (the abdominal segments following the post-petiole) of workers and 215 genes between ovaries of gynes with different supergene genotypes (FDR < 0.1; Figure 2A). In all tissues and castes where the gene expression of SB/SB and SB/Sb was compared, the differentially expressed genes tended to be upregulated in SB/Sbindividuals (Figure 2E) and more of the differentially expressed genes were located within the supergene region than expected by chance (Figure 2I).
Genes that were differentially expressed in one tissue or caste because of direct genetic effects were frequently differentially expressed in the other tissue or caste (Figure 3A). A majority of differentially expressed genes in both worker and gyne brains were differentially expressed in other sample types, and eight of these genes were differentially expressed in all four direct genetic effect comparisons (Figure 3A). Four of these eight genes had functionally annotated homologs: growth arrest specific 8 (gas-8 ), nose resistant to fluoxetine-6 (nrf-6 ), and two Harbinger Transposase derived-1 (harbi1 ) genes (Supplementary Data C). To further characterize the direct genetic effects on transcription, we used a generalized linear model to identify genes that were significantly differentially expressed by genotype when including samples of both castes and tissues and modeling the effects of genotype, social environment, caste, and tissue (McCarthy, Chen, & Smyth, 2012). This analysis revealed 145 significant genes that were differentially expressed by the direct genetic effects of genotype (FDR < 0.1; 115 differentially expressed genes at FDR < 0.05, 77 at FDR < 0.01; Figure 3C). Gene Ontology functional enrichment tests (Fisher’s Exact Test, P < 0.01) for these direct genetic effect genes (FDR < 0.1) revealed that locomotion ,reproduction , and numerous metabolism terms were overrepresented (Supplementary Data A).
Indirect genetic effects . The number of genes expressed differentially by indirect genetic effects (comparison of SB/SBadults reared in different social environments from egg through two weeks of adulthood) was lower than the number of genes differentially expressed by direct genetic effects (comparison of SB/SB andSB/Sb adults from the same social environment) in all comparisons (worker brain, worker gaster, gyne brain, and gyne ovaries), when using an FDR of 0.05 or 0.01 as a cutoff for significance. At the least stringent statistical cutoff FDR of 0.1, this was also true for brains of workers and gynes but not for abdominal tissues (Figure 2A,B). Genes differentially expressed by indirect genetic effects tended to be upregulated in individuals from polygyne colonies as compared with individuals from monogyne colonies in the gasters and ovaries of workers and gynes respectively (Figure 2F, positive log2expression ratios). In contrast to direct genetic effects, genes differentially expressed by indirect genetic effects were not located within the supergene region more frequently than expected by chance (Figure 2J). The transcriptional signature of indirect genetic effects was also much less pronounced than that of direct genetic effects across sample types; on average, the fold-change was significantly lower for genes differentially expressed by indirect genetic effects than for genes differentially expressed by direct genetic effects for 3 of 4 caste and tissue combinations (worker brains, worker gasters, and gyne ovaries at FDR < 0.1; Welch Two-Sample t-tests, P < 0.01; Figure 2E,F). These findings each mirror the previous findings using solely gyne data (Arsenault et al., 2020). Our analysis of age- and size-matched worker samples helps to rule out the possibility that differences in gene expression could result from age-induced differences (Lucas, Romiguier, & Keller, 2017) because the age of gynes can vary at the time of nuptial flight (Arsenault et al., 2020; Nipitwattanaphon, Wang, Dijkstra, & Keller, 2013). While there are well-documented behavioral differences between workers in monogyne and polygyne colonies (Gotzek & Ross, 2008; Keller & Ross, 1998; Ross & Keller, 1998, 2002; Trible & Ross, 2016; Zeng, Millar, Chen, Keller, & Ross, 2022), we observed only few genes that were differentially expressed in brains of individuals exposed to these different social environments, with none observed in workers and only five in gynes (FDR < 0.1; Figure 2B). Thus, pronounced indirect genetic effects of the fire ant supergene were primarily detectable in abdominal tissues, in contrast to the direct genetic effects, which were prevalent in abdominal tissues and in the brain.
To quantify the indirect genetic effects that occurred from eclosion until the pupal stage, we compared SB/SB workers that always experienced a polygyne social environment and SB/SB workers that experienced a monogyne social environment until the pupal stage and then a polygyne social environment through the first 14 days as adults. This analysis revealed no significant differences in gene expression (FDR > 0.1; Figure 2C). To quantify the indirect genetic effects that occurred during the first 14 days as adults, we comparedSB/SB workers that always experienced a monogyne social environment and SB/SB workers that experienced a monogyne social environment until the pupal stage and then a polygyne social environment during the first 14 days as adults. This analysis also revealed no significant differences in gene expression (FDR > 0.1; Figure 2C).
The proportion of differentially expressed genes (FDR < 0.1) that overlapped between castes or tissues was 22 times higher for the set of genes influenced by direct genetic effects (Figure 3A) than those influenced by indirect genetic effects (Figure 3B). To further characterize the indirect genetic effects of monogyne versus polygyne social environments, we utilized a generalized linear model to identify genes that were significantly differentially expressed when including samples of both castes and tissues in a model that considered the effects of genotype, social environment, caste, and tissue (McCarthy et al., 2012). This analysis revealed 700 significant genes that were differentially expressed by the indirect genetic effects of the social environment at a lenient statistical cutoff (FDR < 0.1; 7 differentially expressed genes at FDR < 0.05, 0 at FDR < 0.01; Figure 3D).
The genes differentially expressed in the direct genetic effects generalized linear model (FDR < 0.1) exhibited higher absolute log2-transformed expression ratios than genes differentially expressed in the indirect genetic effects generalized linear model (Welch Two-Sample t-test; P < 0.01; Figure 3C,D). Gene Ontology functional enrichment tests (Fisher’s Exact Test, P < 0.01) revealed an overrepresentation of genes differentially expressed in response to indirect genetic effects (FDR < 0.1) that were annotated with the terms response to stimulus ,oogenesis , and various neural development terms (Supplementary Data A).
Direct + indirect genetic effects. Given that the behavior of workers depends both on their own genotype and social environment (due to the presence of SB/Sb workers only in polygyne colonies), we wanted to test for a combinatorial effect of both colony social environment and supergene genotype on transcription. Analyses of these combined direct and indirect genetic effects were performed by comparing gene expression of SB/SB individuals from a monogyne social environment to SB/Sb individuals from a polygyne social environment (Figure 2D). The numbers of the differentially expressed genes (FDR < 0.1) in brains due to these combined effects in workers (n = 78) and gynes (n = 22) were similar to the sum of differentially expressed gene tallies from separate analyses of direct and indirect genetic effects in each caste (workers: n = 71 + 0 = 71; gynes: n = 23 + 5 = 28). In worker gasters, many fewer genes were differentially expressed (FDR < 0.1) by combined indirect and direct effects (n = 117) than the sum of tallies from separate analyses of these effects (n = 86 + 139 = 225). This could be explained by overlap between direct and indirect effect gene sets (examined below), opposing directionality of direct and indirect effects on expression, and/or noise in the data. Gyne ovaries offer a stark contrast to the other sample types, in that there were over 2000 more differentially expressed genes (FDR < 0.1) observed in response to the combination of direct and indirect genetic effects (n = 3451) than in separate tallies of indirect and direct genetic effects (n = 215 and 966, respectively).
A generalized linear model including samples of both castes and tissues revealed that the overlap between direct genetic effects and indirect genetic effects on differential gene expression was higher than expected by chance (Fisher’s Exact Test; p < 0.05; Figure 4A). However, when considering tissues and castes separately, the overlap between direct and indirect genetic effects on gene expression was significantly greater than expected by chance in only one of the four pairwise comparisons (gyne brains), where it was small (Fisher’s Exact Test; p < 0.05; Figure 4B-E). In gyne ovaries, the overlap was significantly less than expected by chance (Fisher’s Exact Test; p < 0.05; Figure 4C). The genes differentially expressed by both direct and indirect genetic effects, considered separately, included a zinc-finger containing transcription factor, limulus clotting factor C , two transposase-derived HARBI1 proteins, and three odorant receptor genes (Supplemental Data D).