Discussion
Our study suggests that direct genetic effects (i.e., SB/SBversus SB/Sb genotype) have a greater influence on patterns of
gene expression than indirect genetic effects (i.e., being raised in a
monogyne social environment containing only SB/SB workers or a
polygyne social environment containing both SB/SB and SB/Sbworkers). This contrasts with two earlier studies, which concluded (i)
that fire ant worker gene expression profiles (whole body) are more
strongly influenced by indirect genetic effects associated with
differences in the genetic composition of monogyne and polygyne colonies
than by the direct genetic effects of their own supergene genotype (Wang
et al., 2008) and (ii) that, in worker antennae, the number of genes
differentially expressed in response to direct genetic effects is
similar to the number differentially expressed in response to indirect
genetic effects (Dang et al., 2019). These contrasting results may stem
from differences in experimental design and statistical approach. In our
experiments, we used body parts of single individuals (6-8 replicates
per tissue/caste/genotype/social environment) (Arsenault et al., 2020).
By contrast, Wang et al. (2008) used pools of 7-10 individuals for each
of the 20 replicates and Dang et al. (2019) pools of 36-55 individuals
for each of the 4 replicates. Because our analyses showed that indirect
genetic effects have a lower effect on the magnitude of changes in gene
expression than direct genetic effects, it is possible that using pools
of individuals (i.e., because of lower variance) or more replicates
increased the likelihood of detecting genes influenced by indirect
genetic effects compared to direct genetic effects. The possibility that
the variance in the indirect genetic effects experienced by different
individuals is higher than the variance in direct genetic effects
experienced by different individuals is an intriguing hypothesis that
warrants further study.
Whether direct or indirect genetic effects more greatly impact
transcription is a multidimensional question: the degree of difference
in expression and the number of affected loci are both relevant. By some
metrics we do observe a greater number of genes undergoing expression
changes in response to indirect than direct genetic effects (worker
gaster and queen ovaries and combined sample GLM, each at FDR
< 0.1 but not more stringent cutoffs), offering apparent
agreement with prior findings (Wang et al., 2008). However, our study
suggests general agreement between tissue types and castes on the
following: gene expression ratios are generally larger for direct than
indirect genetic effects and indirect genetic effects on transcription
appear more tissue- and caste-specific than direct effects. Moreover, we
detected no significantly differentially expressed genes in the brains
of workers in response to indirect genetic effects of the supergene,
despite the presence of many differentially expressed genes arising from
direct genetic effects with the same statistical power. This stands in
contrast to the prediction of pervasive indirect genetic effects
stemming from prior results from whole workers in S. invicta(Wang et al., 2008) and is more in line with results from a recent study
of indirect genetic effects on brains in the clonal raider ant (Kay et
al., 2022a).
The overrepresentation in the supergene region of genes differentially
expressed due to direct genetic effects, but not indirect genetic
effects, is consistent with cis -regulatory effects of supergene
evolution and/or differences in chromatin accessibility between theSB and Sb haplotypes. Prior analysis of the gyne data
alone also pointed to such effects (Arsenault et al., 2020).
Evolutionary divergence of supergene-encoded proteins (Martinez-Ruiz et
al., 2020; Pracana et al., 2017a; Pracana, Priyam, Levantis, Nichols, &
Wurm, 2017b) likely also make trans- regulatory contributions to
the observed direct genetic effects, but these are not expected to
disproportionately impact expression within the supergene (Signor &
Nuzhdin, 2018). Moreover, a large-scale analysis of multiple tissue
types in humans revealed that cis- regulatory sequence variants
often influence transcription in a constitutive fashion across tissues
(Ardlie et al., 2015). We found that direct genetic effects on gene
expression patterns were often consistent across tissues, offering
apparent support to the hypothesized cis -regulatory impact of
evolution of the fire ant social supergene.
It has previously been noted that differentially expressed genes arising
from direct genetic effects (Arsenault et al., 2020) and combined direct
and indirect genetic effects (Martinez-Ruiz et al., 2020) of the fire
ant supergene are overwhelmingly more highly expressed in SB/Sbthan SB/SB individuals. Remarkably, this holds true for genes
residing outside the supergene region, suggesting thattrans- mediated gene upregulation is caused by the
inversion-derived Sb haplotype (Arsenault et al., 2020;
Martinez-Ruiz et al., 2020). The genetic change(s) responsible for
widespread gene upregulation in response to the Sb haplotype
remains unknown, but effects of cis- regulatory evolution and gene
duplication (Fontana et al., 2020) on trans -regulatory factors
could be of relevance. Remarkably, we also observed more gene
upregulation than expected by chance due to indirect genetic effects of
the Sb haplotype – that is, higher expression of differentially
expressed genes in SB/SB individuals cohabitating withSB/Sb individuals than those cohabitating with only SB/SBindividuals. This suggests the Sb haplotype may lead to gene
upregulation through both direct and indirect genetic effects, despite a
largely non-overlapping sets of genes being influenced.
The lack of significant gene expression changes associated with
cross-fostering SB/SB worker pupae from a monogyne to a polygyne
social environment until two weeks post-eclosion suggests thatSB/SB worker tolerance of multiple SB/Sb queens may not
require a transcriptional response (at least not at a scale detectable
with our methods). A possible explanation for this finding is that the
presence of SB/Sb workers may induce passive habituation ofSB/SB workers to the Sb chemical greenbeard and thereby
induce them to accept SB/Sb queens. Such a behavioral
change without a detected change in brain gene expression is consistent
with findings from a study of variation in aggressive behavioral
response to social cues in honey bees (Rittschof, 2017) and a study of
indirect genetic effects on brain gene expression in the clonal raider
ant (Kay et al., 2022a). Thus, a growing body of evidence suggests that
context-specific behavioral variation can arise from variation in the
social environment without requiring fundamental changes to gene
regulation at a detectable level in whole brains. Nevertheless, our
results do suggest that indirect genetic effects accrue through the full
course of development in S. invicta (when the social environment
is consistent from egg to two-weeks as adults), as detected in abdominal
tissues. Establishing a more fine-scale time course of the social
supergene’s effects on development, both direct and indirect, remains an
outstanding problem.
Queens of S. invicta face a unique selective pressure during
reproductive development in polygyne colonies, which likely explains why
their ovarian tissues exhibit the greatest observed indirect genetic
effects. As they mature in polygyne colonies, SB/SB gynes become
targets of spiteful greenbeard-mediated elimination by Sb -bearing
workers (Gotzek & Ross, 2008; Keller & Ross, 1998; Trible & Ross,
2016). The SB/SB gynes that survive in polygyne colonies until
mating flights have been found to be slightly, but significantly,
lighter than SB/SB gynes embarking on flights from monogyne
colonies (DeHeer et al., 1999; Keller & Ross, 1995), reducing their
independent colony-founding potential (DeHeer, 2002). The differential
expression between the SB/SB gynes of monogyne and polygyne
colonies could be associated with morphological and/or physiological
differences associated with these weight differences (Arsenault et al.,
2020).
Some of the conclusions we reached are similar to those of a genome wide
association study (GWAS) of direct and indirect genetic effects
conducted in laboratory mice (Baud et al., 2021) despite the very
different approach of mapping trait variation as opposed to
transcriptional variation. Effect sizes of loci associated with direct
genetic effects on trait variation were found to be higher than effect
sizes of loci associated with indirect genetic effects on trait
variation (Baud et al., 2021), mirroring the larger gene expression
ratios we observed for direct genetic effects than for indirect genetic
effects. In contrast to this GWAS study (Baud et al., 2021), we observed
a modest but significant overlap in the genes with significant indirect
and direct genetic effects on transcription in S. invicta .
Among the genes that exhibit a significant transcriptional response to
both direct and indirect genetic effects are a zinc-finger
domain-containing transcription factor, which represents a candidate
trans-regulatory protein that may contribute to the widespread direct
genetic effects observed outside the supergene region in S.
invicta (Arsenault et al., 2020; Martinez-Ruiz et al., 2020). Also
responsive to direct and indirect effects is limulus clotting
factor C , which encodes an enzyme linked to antibacterial hemolymph
clotting (Iwanaga, 1993; Iwanaga, Miyata, Tokunaga, & Muta, 1992) and,
notably, three odorant receptor genes. Odorant receptors play an
important role in chemical communication and therefore in social
organization. There are also many documented amino acid substitutions
between odorant receptor proteins encoded by the SB and Sbhaplotypes of the fire ant supergene (Cohanim, Amsalem, Saad, Shoemaker,
& Privman, 2018).
As in other social animals (Avalos et al., 2020; Baud et al., 2021;
Gempe et al., 2012; Linksvayer, 2006; Vojvodic et al., 2015), direct and
indirect genetic effects are pronounced in S. invicta . By
examining direct and indirect genetic effects of the fire ant supergene
on gene regulatory dynamics in samples from distinct tissues and castes
we show that direct genetic effects on transcription are generally more
detectable than indirect genetic effects and that direct genetic effects
are more consistent across biological contexts than indirect genetic
effects. Our results also add evidence to the notion that behavioral
plasticity in response to the social environment need not be accompanied
by detectable differences in brain gene expression (Kay et al., 2022a).
Comparative study of other supergene systems (Kay et al., 2022b) will
help to test the generality of our findings across taxa.