Introduction
Social interactions among conspecific individuals create a scenario in which interacting phenotypes influence trait variation (Farine, Montiglio, & Spiegel, 2015; Moore, Brodie, & Wolf, 1997). When the phenotype of an individual is influenced by environmental variation attributable to the genotypes of its social partners, this is termed an indirect genetic effect (Moore et al., 1997). Because individuals rely on social care to develop into adults in eusocial species, indirect genetic effects can be a heritable, evolutionarily-responsive, source of trait variation (Linksvayer, 2015; Linksvayer & Wade, 2005; Wolf, Brodie, Cheverud, Moore, & Wade, 1998). Variation in aggression (Avalos et al., 2020), size, and reproductive allocation (Linksvayer, 2006; Vojvodic et al., 2015) have each been attributed to indirect genetic effects in studies of eusocial insects.
Recent studies have uncovered many instances of supergenes (i.e., tight clusters of two or more loci each affecting a different developmental or behavioral characteristic), which provide integrated control of complex adaptive phenotypes segregating within species (Schwander, Libbrecht, & Keller, 2014; Thompson & Jiggins, 2014 ; Wellenreuther & Bernatchez, 2018). In eusocial insects, supergenes are frequently responsible for intraspecific variation in social organization (Kay et al., 2022b). Understanding the direct and indirect genetic effects of supergenes is of particular interest in social organisms (Kay et al., 2022b) or in situations where supergenes induce variation in mating systems (Mank, 2022).
In the fire ant Solenopsis invicta , indirect genetic effects of a supergene underpin the behavioral regulation of colony queen number by workers (Ross & Keller, 2002; Wang et al., 2013). In monogyne colonies, a single reproductive queen resides with her worker offspring, while in polygyne colonies multiple reproductive queens reside with workers composed of multiple family groups (Keller, 1993). This variation in queen number is associated with a suite of important individual- and colony-level phenotypic differences (DeHeer, 2002; DeHeer, Goodisman, & Ross, 1999; Gotzek & Ross, 2007; Ross & Keller, 1995; Ross & Keller, 1998). The supergene controlling this fundamental social polymorphism is formed by three overlapping chromosomal inversions that result in high levels of linkage disequilibrium between >500 protein coding genes (Helleu, Roux, Ross, & Keller, 2022; Huang, Dang, Chang, & Wang, 2018; Wang et al., 2013; Yan et al., 2020). The fire ant supergene thus provides a system with prominent roles for direct and indirect genetic effects in shaping variation in social organization.
Reproductive queens in monogyne colonies of S. invicta are all homozygous for the Social B (SB ) supergene haplotype, which has largely retained ancestral synteny, whereas reproductive queens in polygyne colonies in the U.S. are all heterozygous forSB and the inverted supergene haplotype Social b(Sb ) (Ross, 1997; Wang et al., 2013; Yan et al., 2020). Importantly, the presence of workers with the Sb haplotype affects the behavior of SB/SB workers. When the queen is removed from colonies containing only SB/SB workers, the workers accept only a single replacement queen with the genotype SB/SB(SB/Sb and supernumerary queens are executed), but SB/SBworkers that coinhabit colonies with Sb -bearing workers will accept multiple SB/Sb replacement queens (Gotzek & Ross, 2008; Ross & Keller, 2002). Supergenes mediating polymorphic social organization have now been discovered in at least four additional ant lineages and it is likely that they play an important role underlying variation in social organization in many other eusocial insects (Kay et al., 2022b).
So far only a few studies have investigated the consequences of indirect genetic effects on gene expression in social insects. In the honey beeApis mellifera , the mixing of workers with different genotypes has been shown to have indirect effects on individual worker behavior and transcription of genes in the brain (Gempe, Stach, Bienefeld, & Beye, 2012). By contrast, no detectable indirect genetic effects were detected in the clonal raider ant Ooceraea biroi when gene expression was compared between workers exposed to social partners from different clonal lineages (in this species thousands of genes are differentially expressed between individuals of the different lineages) (Kay et al., 2022a). In S. invicta , indirect genetic effects of the social supergene have previously been investigated on several occasions (Arsenault et al., 2020; Dang, Cohanim, Fontana, Privman, & Wang, 2019; Wang, Ross, & Keller, 2008). In a first study using microarrays of pooled whole bodies of workers (Wang et al., 2008), a greater number of genes were influenced by indirect effects associated with the Gp-9 genotypic composition of their colony than by the direct effect of their own Gp-9 genotype (Gp-9 is a an odorant binding gene located in the social supergene (Wang et al., 2013)). In another study comparing gene expression of pools of antennae from multiple workers there were similar numbers of genes influenced by direct and indirect genetic effects (Dang et al., 2019). Finally, a comparison of gene expression of two tissues of individual pre-reproductive queens revealed substantially fewer genes differentially expressed because of indirect genetic effects than direct genetic effects within each tissue type (Arsenault et al., 2020). The contribution of direct and indirect effects of the supergene on transcriptional variation seems to vary according to biological context and scale in S. invicta . Thus, an assessment of general trends across castes and tissues is warranted to test for general properties of direct versus indirect genetic effects in the system.
Here we present the first report of direct and indirect effects of the fire ant supergene in brains and gasters of the worker caste of S. invicta . We integrated the analyses of our data with previously reported data for pre-reproductive queens (Arsenault et al., 2020) to provide a comprehensive view of the direct and indirect genetic effects of the supergene across castes (Figure 1). We examined direct genetic effects using transcriptomes from brains and abdominal tissues ofSB/SB and SB/Sb workers and unmated winged queens (gynes) from polygyne colonies. We examined indirect genetic effects of the supergene using transcriptomes from adult SB/SB workers and gynes that developed in a monogyne social environment (where all nestmate workers have the genotype SB/SB ) and a polygyne social environment (where nestmate workers are mix of genotypes SB/SBand SB/Sb ; Figure 1). Our results suggest that direct genetic effects of the fire ant supergene result in larger gene expression differences than indirect genetic effects and that direct genetic effects are more consistent than indirect genetic effects across tissue types and castes. We also conducted cross-fostering experiments of late-stage pupae to investigate how the social environment affects patterns of gene expression in adults. Previous studies revealed thatSB/SB workers shift from rejecting all SB/Sb queens to accepting multiple SB/Sb queens when their colony contains more than 10-15% Sb- bearing adult workers (Gotzek & Ross, 2008; Ross & Keller, 2002). We raised these SB/Sb individuals until two weeks post-eclosion to investigate the role of social environment during the first weeks of adulthood. These cross-fostering experiments revealed no significant indirect effects of the supergene on transcription when the type of social environment experienced by sampled individuals differed only during adulthood.