Abstract
In insect societies, the queen monopolizes reproduction while workers
perform tasks such as brood care or foraging. Queen loss leads to ovary
development and lifespan extension in workers from many ants. However,
the underlying molecular mechanisms of this phenotypic plasticity remain
unclear. Recent studies highlight the importance of epigenetics in
regulating plastic traits in social insects. We investigated the role of
histone acetylation in the regulation of worker reproduction in the antTemnothorax rugatulus . We removed queens from their colonies to
induce worker fecundity, and either fed workers with chemical inhibitors
of histone acetylation (C646), deacetylation (Trichostatin A), or the
solvent (DMSO) as control. We monitored worker number for six weeks
after which we assessed ovary development and sequenced fat body mRNA.
Workers survived better in queenless colonies and developed their
ovaries after queen removal in control colonies as expected, but not in
colonies treated with chemical inhibitors. Both inhibitors affected gene
expression, although the inhibition of histone acetylation using C646
influenced the expression of more genes with immunity, fecundity, and
longevity functionalities. Interestingly, these C646-treated workers
shared many upregulated genes with infertile workers from queenright
colonies. We also identified one gene with antioxidant properties
commonly downregulated in infertile workers from queenright colonies and
both C646 and TSA-treated workers from queenless colonies. Our results
indicate that histone acetylation is involved in the molecular
regulation of worker reproduction and lifespan, and thus point to an
important role of histone modifications in modulating phenotypic
plasticity of life history traits in social insects.
Keywords: gene regulation, epigenetics, fecundity, longevity,
ants
Introduction
The societies of social insects exhibit a reproductive division of labor
where the queen reproduces, whereas workers perform all other tasks
including brood care, nest defense, and foraging (Hölldobler & Wilson,
1990). Workers thus sacrifice their own reproduction. This evolutionary
incongruity is commonly explained by Hamilton’s inclusive fitness
theory, which states that genes of sterile altruists can be transmitted
indirectly to the next generation by helping closely related
reproductives (Hamilton, 1964). The proximate mechanisms underlying the
maintenance of worker sterility in social insect colonies have been
extensively investigated as well. Worker reproduction is regulated via
chemical signals emitted by the queen or her brood (Endler et al., 2004;
Matsuura et al., 2010; Van Oystaeyen et al., 2014), or through social
control meditated by the queen or workers themselves (Foster &
Ratnieks, 2000; Oldroyd et al., 2001; Ruhland, Moulin, Choppin, Meunier,
& Lucas, 2020). However, in rare cases, social insect workers
circumvent those restraints even in queenright colonies and successfully
gain direct fitness benefits by laying haploid eggs developing into
males (Beekman & Oldroyd, 2008; Giehr, Wallner, et al., 2020).
Conversely, worker reproduction is frequently found in queenless
colonies. Queen loss first induces fights among workers, who establish a
new reproductive hierarchy, develop their ovaries, and start laying
haploid eggs (Giehr, Senninger, Ruhland, & Heinze, 2020; Heinze, 2008;
Monnin & Peeters, 1999). Reproduction has strong effects on the
physiology and immunity of workers, who can become more resistant to
oxidative stress and often live longer (dos Santos Conceição Lopes,
Campbell, & Contrera, 2020; Kohlmeier et al., 2017; Majoe, Libbrecht,
Foitzik, & Nehring, 2021; Negroni, Segers, Vogelweith, & Foitzik,
2020). These positive effects of reproduction have been linked to the
activation of signaling pathways such as insulin/insulin-like growth
factor (IIS), the target of rapamycin (mTOR), and the
alpha-ketoglutarate (alpha-KG) (Negroni, Macit, Stoldt, Feldmeyer, &
Foitzik, 2021). Indeed, gene expression changes profoundly in workers
after queen loss in social wasps (Taylor, Cini, Sumner, Wyatt, &
Reuter, 2021), honeybees (Cardoen et al., 2011), and ants (Negroni et
al., 2021; Wurm, Wang, & Keller, 2010) and in various tissues from the
brain to the fat body. Similarly, gene expression differs between
reproductive and sterile bumblebee workers (Marshall, Lonsdale, &
Mallon, 2019). Worker fecundity thus appears to be a highly plastic
trait positively linked to lifespan (Heinze & Schrempf, 2008). This
opens up exciting new avenues to study the molecular regulation of
plasticity in fecundity and longevity in social insect workers (Monroy
Kuhn & Korb, 2016). Indeed, the transcriptomic changes linked to worker
reproduction have been well characterized, while the underlying gene
regulatory mechanisms remain largely unexplored.
Epigenetic mechanisms including DNA methylation and histone
modifications have been proposed to play a major role in the
extraordinary phenotypic plasticity exhibited by social insects
(Bonasio, 2012, 2014; Herb, 2014; Maleszka, 2016; Vaiserman, 2015;
Vaiserman, Lushchak, & Koliada, 2018; Yan et al., 2015). In Carpenter
ants, histone modifications have been associated with behavioral
differences between major and minor ant workers (Glastad et al., 2019;
Simola et al., 2016, 2013) and worker polymorphism (Alvarado, Rajakumar,
Abouheif, & Szyf, 2015). Histone acetylation has also been associated
with the ability of workers to adjust to new daily rhythms (Libbrecht,
Nadrau, & Foitzik, 2020). Besides, there is growing evidence for the
role of histone modifications in caste differentiation. In honey bees,
queen development is largely controlled by royal jelly, a secretion that
has histone deacetylase inhibitor (HDACi) activity (Spannhoff et al.,
2011). Moreover, caste-determined female larvae exhibit genome-wide
differences in histone acetylation and methylation patterns, which are
linked to caste-specific gene expression (Wojciechowski et al., 2018).
Besides, the transition from workers to reproductive gamergates has been
associated with changes in gene expression related to epigenetic
pathways in the ant Harpegnathos saltator (Bonasio et al., 2010).
In this study, we use the ant Temnothorax rugatulus to
investigate the role of histone acetylation in the regulation of
reproduction and lifespan in queenless workers. This common Myrmicine
ant builds small nests of 50 to 2000 workers with one to several queens
and evolved two queen morphs, the large macrogynes, and the small
microgynes, associated with alternative reproductive strategies (Choppin
et al., 2021; Rüppell, Heinze, & Hölldobler, 1998, 2001). Queens can
live over ten years and their gene expression in the brain and fat body
changes with age (Negroni, Foitzik, & Feldmeyer, 2019). Following queen
loss, T. rugatulus workers are known to develop their ovaries,
start laying haploid eggs, live longer, and show transcriptomic changes
in the fat body, a physiologically active tissue (Negroni et al., 2021,
2020). Here, we asked whether histone acetylation is required for
workers to plastically respond to queen loss by altering their ovary
development and associated gene expression. We used queen removal to
induce fecundity in workers while feeding them with chemical inhibitors
of histone acetylation (C646) or deacetylation (TSA). Based on previous
studies, we predicted that workers would develop ovaries, survive
better, and show transcriptomic changes following queen removal. If
histone acetylation does play a role in the regulation of worker
reproduction and lifespan, we expected the chemical inhibitors C646 and
TSA to negatively impact worker ovary development following queen
removal, and to alter the regulation of fecundity and longevity genes,
preventing workers in queenless colonies to reproduce and live longer.
Materials and Methods
Ant collection and maintenance
Temnothorax rugatulus ants are distributed throughout the western
part of North America and reside in high elevation coniferous forests,
under stones or in rock crevices. In August 2018, we collected colonies
from nine different locations in the Chiricahua Mountains (Arizona, USA,
Table S1). In the laboratory, each colony was kept in a three-chambered
box (9.7 x 9.7 x 2.9 cm) covered with a lid and containing an artificial
nest made of a plastic insert between two glass slides covered by a red
foil to block the light. The colonies were maintained at 21°C and 70%
humidity with a 12:12 light:dark cycle. They were fed weekly with half a
cricket and a drop of honey and were provided with water ad libitum.
Colony monitoring
We selected 90 monogynous colonies with 54 to 100 workers and reduced
worker number to 50 per colony. Colonies were then moved to a climate
chamber at 25°C and 70% humidity with a 12:12 light:dark cycle for two
weeks. Before starting the experiment, colonies were randomly assigned
to one of five experimental groups with a total of 18 colonies per group
(Table 1).
On the first day of the experiment, we removed all eggs, pupae, and
males and adjusted the number of larvae to five per colony. Queens from
the queen removal groups were removed and returned to their natal
colonies. Colonies were fed with either the solvent dimethyl sulfoxide
(DMSO; Carl Roth) only, the inhibitor of histone acetylation C646 which
targets the histone acetyltransferase p300 (50 µM in DMSO;
Sigma-Aldrich) (Bowers et al., 2010), the inhibitor of histone
deacetylation Trichostatin A (TSA) known as class I and class II histone
deacetylase inhibitor (50 µM in DMSO; Sigma-Aldrich) (Yoshidas, 1990),
or a combination of C646 and TSA (both 50 µM in DMSO). All preparations
were diluted in 0.102 g/mL sucrose solution. The ants were fed for six
weeks every other day with 15 µL of fresh solution per colony (Figure
S1). Additionally, each colony received half a cricket every other day
and water ad libitum. Once a week, we anesthetized all colonies with
CO2. We removed and counted the eggs in queenless
colonies to get precise numbers of worker-laid eggs. Once every two
weeks, we counted all colony members (queens if applicable, workers,
eggs, larvae, and pupae) in colonies from all groups.
We tested the effect of queen removal on worker survival by comparing
worker number over time between the groups “queenright” and
“queenless” using a linear mixed-effects model (LMM) with the package
“lme4” (Bates, Mächler, Bolker, & Walker, 2015). In queenless
colonies, we investigated the effect of treatment (DMSO, DMSO+C646,
DMSO+TSA, and DMSO+C646/TSA) on worker number in interaction with time
using a similar model. Colony identification (ID) was used as a random
factor in both models to account for inter-colony variability. We
assessed the fit of our LMMs using visual inspections of the residual
distributions. Treatment effect on egg production at week six was
analyzed using a generalized linear mixed-effects model (GLMM, binomial
family) with egg production as a binary variable. Our GLMM was tested
for overdispersion using the package “DHARMa” (Hartig, 2020). All
statistical analyses were conducted in R v3.5.1 (R Core Team, 2020).
Dissections, RNA extraction, and fecundity measures
After six weeks, we selected a subset of colonies for dissections (SI
Methods). For each colony, we isolated all the workers close to the
brood in a Petri dish and dissected them on ice in a drop of a sterile
saline solution until two workers with developed ovaries were found (2
to 14 workers dissected per colony). From these two workers, we cleaned
the ovaries and took pictures for fecundity measurements using a
stereomicroscope (details below). The fat bodies, including the first
cuticle plate of the gaster, were collected from the two workers and
pooled in the same Eppendorf tube containing 50 µl of TRIzol
(Thermofisher) for further RNA-sequencing. Tissue collection took less
than 10 minutes. The samples were flash-frozen in liquid nitrogen and
preserved at -80°C. Before the RNA extraction of each sample, we crushed
the fat bodies with a pestle, added 50 µl of
phenol:chloroform:isoamyl alcohol
(25:24:1) (Carl Roth), mixed manually, and centrifuged at 1200 xg for 15
minutes. Afterwards, the upper phase was transferred to a new tube and
mixed with 25 µl of ethanol 100% (Carl Roth). RNA was then extracted
using a NucleoSpin RNA XS kit (Macherey-Nagel). After quantity and
quality control, 20 samples were sent for sequencing (Table 1). Library
preparation was conducted following the standard protocol of BGI
(Hongkong), which sequenced 150 bp paired-end reads on an Illumina Hiseq
X ten.
We measured ovariole length and counted the number of white eggs (i.e.,
eggs in development) and yellow bodies in the ovaries using the Leica
software LAS v4.5. Yellow bodies are an indication of recent egg laying
in ants (Cini, 2014; Heinze, Hölldobler, & Trenkle, 1995; Peeters &
Tinaut, 2014). We analyzed the effects of queen removal and treatment
(DMSO, DMSO+C646, DMSO+TSA, DMSO+C646/TSA) on worker ovariole length
using LMMs. We used GLMMs (binomial family) to test for effects of queen
removal and treatment on the presence of white eggs and yellow bodies in
the ovaries. Colony ID was used as a random factor to account for
inter-colony variability. The models’ fit was assessed as described
above.
Gene expression analysis
Raw reads were trimmed with Trimmomatic v0.39 (Bolger, Lohse, & Usadel,
2014) (Table S2) and quality checked using FastQC v0.11.7 (Andrew,
2010). The paired reads were then mapped against the Temnothorax
rugatulus draft genome (Jongepier et al. unpub.) using HISAT2 v2.1.0
(Kim, Langmead, & Salzberg, 2015) (Table S2). We converted and sorted
the output files using SAMtools v1.7 (Li et al., 2009) and obtained a
quality report from Qualimap v2.2.1 (Okonechnikov, Conesa, &
García-Alcalde, 2016). A genome guided transcriptome assembly was
created using StringTie v2.1.3 (Pertea et al., 2015) and transcript
sequences were extracted using GffRead v0.11.8 on the merged GTF file.
Transcriptome quality was assessed using TransRate v1.0.3 (Smith-Unna,
Boursnell, Patro, Hibberd, & Kelly, 2016). Transcripts with an Open
Reading Frame (ORF) < 100 bp were removed and the Python
script “prepDE.py” from the online StringTie Manual was used to
generate the gene count matrix (Table S3).
We assessed the effect of queen removal on worker gene expression by
comparing the groups “queenless” and “queenright”. Then, we tested
the effects of the chemical inhibition of histone acetylation and
deacetylation by first comparing the groups “queenless” to
“queenless+C646”, and then “queenless” to “queenless+TSA”. To
avoid factitious DESeq2 results and for each comparison, we first
filtered the gene count matrix so at least 70% of samples had a read
count of ten or more reads per gene in at least one experimental group.
We additionally plotted the maximum cook distance against the average
gene expression per sample to identify and remove putative outliers. We
used the filtered count matrix (Table S4) to perform the differential
gene expression analysis using DESeq2 (Love, Huber, & Anders, 2014)
setting an adjusted p-value < 0.05 as significance threshold.
We plotted principal component analyses (PCAs) with all genes using the
package “ggplot2” (Wickham, 2010) to assess the group-based clustering
of our samples (Figure S2, S3, and S4). We created heatmaps with the
package “pheatmap” (Kolder, 2012) to visualize expression differences
and clustering between samples.
To annotate transcripts we conducted a BlastX homology search with BLAST
v2.10.1+ (Altschul, Gish, Miller, Myers, & Lipman, 1990) using the
non-redundant invertebrate protein database from NCBI (May 2020) and
only considered hits with an E-value < 10-5(SI Methods). We combined the blast annotations with gene information
from UniProt (www.uniprot.org) . We
used TransDecoder v5.5.0 (Haas et al., 2013) to translate nucleotide
sequences into amino-acid sequences and then ran InterProScan v5.45-80.0
(Quevillon et al., 2005) to obtain Gene Ontology (GO) term annotations.
Then, we performed a GO term enrichment analysis using the R package
“topGO” (Alexa & Rahnenführer, 2020) with the algorithm “weight01”
(SI Methods). We conducted the GO enrichment analysis separately for
upregulated and downregulated genes in the groups compared to the
queenless control. Statistical significance was given using Fischer
exact tests. We extracted the overlap of upregulated and downregulated
genes between the groups “queenright”, “queenless+C646” and
“queenless+TSA” and assessed whether the overlap size between two
groups was larger than expected by chance by resampling random gene
lists (500 iterations). Finally, we plotted expression levels (i.e.,
normalized read counts) of genes of interest using “plotCounts” from
DESeq2.
Results
Effects of queen removal
Worker number and fecundity
Worker number decreased over time
(X² = 441.663, df = 2, p < 0.001), although less strongly in
queenless colonies (X² = 9.723, df = 2, p = 0.008; Figure S5). Workers
from queenless colonies had longer ovarioles (X² = 30.578, df = 1, p
< 0.001; Figure 1A), were more likely to have yellow bodies
(X² = 9.588, df = 1, p = 0.002; Figure 1B), and also tentatively more
likely to have white eggs in their ovaries
(X² = 2.828, df = 1, p = 0.093;
Figure S6).
Gene expression and functional enrichment
We found 346 differentially expressed genes (DEGs) between workers from
the groups “queenless” and “queenright” (Table S5A-B), among which
206 were upregulated and 140 downregulated in the queenright group. The
samples clearly clustered according to treatment in the heatmap based on
all DEGs (Figure 1C). The enrichment analysis revealed that workers in
presence of their queen downregulated genes related to five functions
including “innate immune response” (Figure 1D, Table S6B). This is
reflected by the presence of multiple immune genes in our top 15
downregulated genes in the queenright group including “FK506-binding
protein 2 isoform X1” or “chymotrypsin-2-like”. Also, the gene coding
for “vitellogenin-1-like” (Figure 2) was found to be downregulated in
queenless workers. Queen presence affected the expression of many
regulatory genes as well, such as transcription factors including “zinc
finger protein 454-like”.
Effects of chemical inhibitors
Worker number, egg production, and fecundity
Here we focus on queenless colonies that were either fed with DMSO only
(control) or additionally treated with C646, TSA or both inhibitors.
Again, worker number generally decreased over time (X² = 719.732, df =
2, p < 0.001), but irrespective of treatment (X² = 7.493, df =
6, p = 0.278; Figure S7). After six weeks, 23% of colonies had eggs and
the presence of eggs was unaffected by treatment (X² = 2.521, df = 3, p
= 0.472; Figure S8). However, workers treated with the epigenetic
inhibitors had shorter ovarioles (X² = 11.569, df = 3, p = 0.009; Figure
3A) and a smaller proportion of treated workers had yellow bodies in
their ovaries (X² = 9.721, df = 3, p = 0.021; Figure 3B) compared to
control workers. More precisely, C646-treated workers exhibited shorter
ovarioles (tC646 = -3.290, pC646 =
0.002) and were less likely to have yellow bodies in the ovaries
(zC646 = -2.366, pC646 = 0.018) compared
to workers fed with DMSO only. In the TSA treatment, fewer workers had
yellow bodies in their ovaries compared to control workers
(zTSA = -2.949, pTSA = 0.003), although
ovariole length was unaffected by TSA treatment (tTSA =
-0.891, pTSA = 0.379). None of the inhibitors affected
the proportion of workers with white eggs in the ovaries (X² = 5.817, df
= 3, p = 0.121; Figure S9).
Gene expression and functional enrichment
We found 306 differentially expressed genes between workers from the
control group and workers treated with the inhibitor of histone
acetylation C646 (Table S5C-D), among which 247 were upregulated and 59
were downregulated in the C646-treated workers. The heatmap revealed
that C646 samples clustered well together (Figure 3C). C646-treated
workers downregulated genes with longevity functionalities such as
“protein I’m not dead yet” also called “Indy” (Rogina & Helfand,
2013) (Figure 2), or genes with an immune function like “proclotting
enzyme-like” (Villanueva-Segura et al., 2020) (Figure 2). Workers fed
with C646 also downregulated seven genes related to the synthesis of
fatty acids, versus one only in the control. Our enrichment analysis
revealed the overrepresentation of five functions associated with
downregulated genes of C646-treated workers including
“oxidation-reduction process” (Figure 3D, Table S6D).
Between workers from the control group and workers treated with the
inhibitor of histone deacetylation TSA, we found 32 differentially
expressed genes (Table S5E-F). Only a single gene was upregulated and 31
were downregulated in the TSA-treated workers. The heatmaps created
using the 32 DEGs revealed a looser clustering of our samples by group
(Figure S10), which is consistent with the weaker effect of TSA
treatment on the number of DEGs. Based on the low number of DEGs between
the two groups we only found the functions “transposition,
DNA-mediated” and “autophagy” significantly overrepresented in the
DEGs of the TSA-treated workers (Table S6E-F). Although the TSA
treatment also had weaker effects on worker fecundity, we did find the
aging and fecundity-associated gene “juvenile hormone acid
O-methyltransferase-like isoform X1” (Hartfelder, 2000; Yamamoto, Bai,
Dolezal, Amdam, & Tatar, 2013) downregulated in TSA-treated workers
(Figure 2).
Overlapping genes between groups
As indicated above, the chemical inhibitors of histone acetylation and
deacetylation impaired worker ovary development following queen removal.
Thus, we asked whether the transcriptomes of inhibitor-treated workers
were similar to the ones of infertile workers from queenright colonies.
Indeed, between the groups “queenright” and “queenless+C646” we
found 82 genes commonly upregulated (Figure 4A, Table S5G) and five
genes commonly downregulated (Figure 4B, Table S5H) in workers. We
additionally found five genes commonly downregulated between the groups
“queenright” and “queenless+TSA” (Figure 4B, Table S5I). These three
numbers of overlapping genes were higher than expected by chance as
evidenced by resampling random gene lists (Figure S11). Among the
commonly upregulated genes between workers with a queen and C646-treated
workers, we found genes associated with circadian rhythm like
“circadian clock-controlled protein” (Figure 2). We additionally found
many genes associated with digestion like “mucin-5AC-like”, “probable
salivary secreted peptide” or “silk gland factor 1”. Finally, the
gene “transketolase-like protein 2 isoform X2” with antioxidant
properties (Kovarova et al., 2018; Schenk, Duggleby, & Nixon, 1998; Xu
et al., 2016) was commonly downregulated in all three groups in
comparison to the control (Figure 2).
Discussion
In this study, we investigated the role of histone acetylation in the
regulation of genes associated with worker reproduction and lifespan in
the ant Temnothorax rugatulus . We removed queens from their
colonies while feeding the workers with chemical inhibitors of histone
acetylation (C646) or deacetylation (TSA). Our phenotypic and
transcriptomic results confirmed that worker fecundity and survival
increased after queen removal, as shown before in this species (Negroni
et al., 2021, 2020). However, the ovary development of queenless workers
was impaired when treated with the chemical inhibitors. On a molecular
level, the inhibition of histone acetylation using C646 had a stronger
effect than the inhibition of histone deacetylation using TSA, as shown
by the difference in the number of differentially expressed genes (306
vs 32). Yet, workers from both treatments downregulated many genes
related to fecundity, immunity, and longevity, compared to the queenless
control. More importantly, a large number of genes were commonly
upregulated between workers from queenright colonies (i.e., infertile
workers) and C646-treated workers from queenless colonies, pointing to
similar transcriptomes. We additionally found one gene with antioxidant
properties commonly downregulated between infertile workers and both
C646 and TSA-treated workers, in comparison to fertile ants in the DMSO
control, which might be linked to the extended survival of these fertile
ants.
Ant workers start to reproduce and live longer after the loss of their
queen in many ant species (Heinze, 2008; Kohlmeier et al., 2017; Konrad,
Pamminger, & Foitzik, 2012) including our model Temnothorax
rugatulus (Negroni et al., 2021, 2020). We took advantage of
this plasticity by inducing fecundity in workers using queen removal and
confirmed that workers in queenless colonies developed their ovaries,
started laying eggs, survived better, and shifted their gene expression
in the fat body. Our transcriptomic analysis revealed avitellogenin (vg ) gene to be upregulated in workers in the
absence of their queen. During social insect evolution, vg genes
underwent several duplications followed by diversification and
sub-functionalization. Various vg orthologues now take over
different functions in behavior (Kohlmeier, Feldmeyer, & Foitzik, 2018)
and physiology, including fecundity (Amdam, Norberg, Hagen, & Omholt,
2003) and aging (Parker, 2010) in social insects. For example, the
expression of conventional vitellogenins is linked to oxidative
stress resistance in honeybee workers (Seehuus, Norberg, Gimsa,
Krekling, & Amdam, 2006). We investigated where our
“vitellogenin-1-like” copy falls within the vitellogeninphylogeny (Kohlmeier et al., 2018, data not shown) and found that it
clusters close to the conventional vitellogenins , indicating an
ancestral fecundity function as an egg yolk precursor (Amdam et al.,
2003) rather than a derived role, as in worker caste differentiation
(Feldmeyer, Elsner, & Foitzik, 2014; Morandin et al., 2014).
Interestingly, a previous study on the effect of queen removal on worker
gene expression in T. rugatulus detected a vitellogeninreceptor to be upregulated in fertile, queenless workers (Negroni et
al., 2020), pointing to the importance of vg -associated pathways
for worker reproduction.
Longevity is traded-off with immunity in many organisms due to the cost
of an active immune system and the production of reactive oxygen species
during immune reactions (DeVeale, Brummel, & Seroude, 2004; Garschall
& Flatt, 2018). Nevertheless, our results show that the longer-lived,
fertile workers from queenless colonies activate genes with an innate
immune response functionality, which is consistent with previous
findings in Temnothorax rugatulus showing that fertile workers
express more immunity genes following an immune challenge, compared to
infertile workers (Negroni et al., 2020). Similarly, the highly fertile
young queens of T. rugatulus upregulate immune genes in the
Toll-pathway (Negroni et al., 2019), which plays a crucial role in
insect immunity (Valanne, Wang, & Rämet, 2011). These findings provide
evidence that fecund ants invest in a longer lifespan and immunity
concurrently, supporting other studies on social insects indicating that
life history trade-offs have shifted during their social evolution
(Korb, 2016; Schrempf, Giehr, Röhrl, Steigleder, & Heinze, 2017).
Interestingly, ant workers treated with chemical inhibitors following
queen removal had a lower ovary development and were less likely to
exhibit yellow bodies, which provide evidence for egg-laying (Cini,
2014; Heinze et al., 1995; Peeters & Tinaut, 2014) compared to control
workers from queenless nests. This indicates that dynamic changes in
histone acetylation might be required for workers to shift to
longer-lived, fecund phenotypes. In social insects, histone acetylation
has previously been linked to various processes including the regulation
of foraging behavior and caste determination (Simola et al., 2016, 2013;
Spannhoff et al., 2011; Wojciechowski et al., 2018), but evidence for
the regulation of life history traits such as fecundity and longevity
have been lacking so far. In contrast, in solitary insects such as the
pea aphid, the inhibition of histone acetylation and deacetylation
affects development, fertility, and longevity (Kirfel, Kessel, Skaljac,
Seip, & Vilcinskas, 2020). In fact, fertility appears to be regulated
by this epigenetic mark in various taxonomic groups, which includes
other insects such as the planthopper Nilaparvata lugens (Zhang
et al., 2018), but also mammals including mice (Ma, Pan, Montgomery,
Olson, & Schultz, 2012) and men (Wang, Gao, Li, & Liu, 2019).
The inhibition of histone acetylation using C646 had more severe
consequences on gene expression in worker fat bodies compared to the
inhibition of histone deacetylation using TSA, shifting the expression
of nearly 10 times as many genes. Among the most strongly downregulated
genes in ants treated with the chemical inhibitors, we found interesting
candidates such as the Indy protein, which has been linked to longevity
in Drosophila (Rogina & Helfand, 2013), the enzyme proclotting,
which plays a role in the innate immune response (Villanueva-Segura et
al., 2020), or a gene associated with juvenile hormone, which is
involved in many physiological processes including aging (Yamamoto et
al., 2013). We also detected seven downregulated genes associated with
the fatty-acid synthesis in the queenless, C646-treated group compared
to only one in the queenless control. Fatty acids are involved in the
synthesis of cuticular hydrocarbons (Blomquist & Bagnères, 2010) and
reproductive and non-reproductive individuals exhibit different odors in
social insects (Heinze, Stengl, & Sledge, 2002; Monnin, 2006),
including Temnothorax ants (Kleeberg, Menzel, & Foitzik, 2017).
Besides, many histone-related genes were upregulated in the group where
histone acetylation was inhibited compared to our control, attesting to
the efficiency of our treatment on a molecular level. Nevertheless,
Chromatin Immunoprecipitation sequencing (ChIP-sequencing) would be our
next logical step to both confirm changes in acetylation on the histone
level and to associate histone acetylation patterns with the expression
of genes of interest.
Between 33% and 40% of all differentially expressed genes were
commonly upregulated in the queenright control and the queenless group
treated with the inhibitor of histone acetylation C646, both groups
containing workers with less developed ovaries. Among those overlapping
genes, many were involved in the regulation of the circadian rhythm. On
one side, this is in line with a previous study in anotherTemnothorax species where the use of C646 led to the loss of the
ability to adjust to novel daily rhythms (Libbrecht et al., 2020). On
the other side, queen presence has been found to affect worker and
colony activity in the honeybee (Grodzicki, Piechowicz, & Caputa, 2020;
Moritz & Sakofsky, 1991), which could explain the upregulation of
circadian rhythm-related genes in workers from colonies where the queen
remained. We could alternatively speculate that worker sterility is
maintained by the upregulation of genes, which are then downregulated
when workers become fecund following queen removal, explaining the large
overlap of genes between our two groups.
The candidate gene “transketolase-like protein 2 isoform X2”was commonly downregulated in infertile workers and workers treated with
the two chemical inhibitors, despite the relatively small number of
genes (i.e., five) in the two lists of commonly downregulated genes.
Transketolases are enzymes involved in the non-oxidative part of the
pentose phosphate pathway (PPP) in all living organisms (Schenk et al.,
1998). They are known to maintain low levels of reactive oxygen species
(ROS) and are thus used in cancer treatment (Xu et al., 2016) and
parasitic disease control (Kovarova et al., 2018). More broadly,
antioxidant production has been positively associated with lifespan in
the fruit fly (Orr & Sohal, 1994) and the nematode Caenorhabditis
elegans (Sampayo, Olsen, & Lithgow, 2003). In our study, this gene was
always upregulated in fertile ants, providing first mechanistic insights
into how fecund ant workers may be able to live longer, and reinforcing
findings showing that long-lived ant queens invest more in antioxidant
production with age (Negroni et al., 2019).
Conclusions
The experimental use of chemical inhibitors of histone acetylation and
deacetylation allowed us to show that this epigenetic mark might be
required for workers to dynamically shift their physiology following
queen removal. Our manipulation did not only affect life history traits
such as fecundity, but also shifted the expression of genes with
fecundity, immunity, and longevity functionalities. Our results thus
provide insights into the molecular regulation of fecundity and
longevity in social insects, which are prime examples of phenotypic
plasticity.