Experimental increase in fecundity causes upregulation of fecundity and body maintenance genes in the fat body of ant queens

Abstract

In most organisms, fecundity and longevity are negatively associated and the molecular regulation of these two life-history traits is highly interconnected. In addition, nutrient intake often has opposing effects on lifespan and reproduction. In contrast to solitary insects, the main reproductive individual of social hymenopterans, the queen, is also the most long-lived. During development, queen larvae are well-nourished, but we are only beginning to understand the impact of nutrition on the queens' adult life and the molecular regulation and connectivity of fecundity and longevity. Here, we used two experimental manipulations to alter queen fecundity in the ant Temnothorax rugatulus and investigated associated changes in fat body gene expression. Egg removal triggered a fecundity increase, leading to expression changes in genes with functions in fecundity such as oogenesis and body maintenance. Dietary restriction lowered the egg production of queens and altered the expression of genes linked to autophagy, Toll signalling, cellular homeostasis and immunity. Our study reveals that an experimental increase in fecundity causes the co-activation of reproduction and body maintenance mechanisms, shedding light on the molecular regulation of the link between longevity and fecundity in social insects.

1. Introduction

The interplay between fecundity and longevity and their molecular regulation are long-standing questions in evolutionary biology [1–3]. The negative association between reproduction and lifespan observed in most solitary organisms is thought to result from antagonistic pleiotropy or resource allocation trade-offs [2,4,5]. Yet, in social insects, reproduction and lifespan are positively linked: the most fertile individuals in insect societies, the queens, live the longest. Moreover, fecundity and longevity are highly plastic traits in social insects, as the phenotypic differences between the queen and worker castes arise from the same genetic background [6]. These characteristics make social insects ideal for investigating the molecular regulation of fecundity and its link to body maintenance and lifespan [7].

Nutrition is one of the best-studied factors in the regulation of reproduction and lifespan. In solitary species, food intake positively affects reproduction, but often concurrently shortens the lifespan via the activation of the TOR signalling and insulin/IGF-1 signalling (IIS) pathways [5,8,9]. However, which food components are influencing lifespan is still under debate [10]. For example, nutrients rather than calories were identified to affect lifespan in fruit flies in some studies [11,12], but not in others [13]. In the reproductives of the social insects, the opposing effects of food intake on fecundity and lifespan seem to be absent, suggesting a reshaping of molecular pathways regulating these life-history traits in solitary organisms [7]. However, the effect of diet on lifespan and fecundity in social insects largely depends on developmental stage and caste. For example, a protein-rich diet results in a shorter lifespan in workers, while being beneficial during queen development [14,15].

Here, we investigate the molecular regulation of fecundity in queens of the ant Temnothorax rugatulus. We conducted two experiments aimed to alter queen fecundity. In the first experiment, we manipulated food availability to test whether queen fecundity would decline under dietary restriction. In a second manipulation, we removed eggs laid by the queen, as this can increase egg production in ant queens [16]. As Temnothorax queens can live for up to two decades [17], we did not expect queen survival to differ between treatments, although our treatments might have affected lifespan eventually. Rather, we analysed whether genes involved in body maintenance changed their expression, which would indirectly indicate an interconnection in the regulation of fecundity and longevity. For these transcriptomic analyses focused on the fat body, a physiologically active tissue [18]. We were particularly interested whether changes in fecundity led to an up- or down-regulation of genes involved in fecundity and body maintenance pathways, shedding light on the molecular basis of the positive association between fecundity and longevity in social insects.

2. Material and methods

Temnothorax rugatulus is a small ant with colonies of a few hundred workers and one to several queens. Two queen morphs can occur and we only used colonies of the common larger queen morph [19]. These ants reside in rock crevices in forests throughout Western North America. We collected 105 T. rugatulus colonies in the Chiricahua Mountains, Arizona in August 2015 (electronic supplementary material, table S1). In our laboratory, colonies were transferred to artificial nest boxes and kept at 22°C and 12 h light/12 h dark in a climate chamber.

For the dietary-restriction experiment, we limited the queens’ access to workers, as these might buffer food restrictions imposed on the queen. The queen was isolated with five workers to ensure food provisioning in the upper part of an artificial nestsite (queenright part, QR), while the reminder of the colony inhabited the lower section (queenless, QL). The two parts were separated by a metal grid, allowing the exchange of volatiles, but not food (electronic supplementary material, figure S1). The QR-parts of the dietary-restriction treatment were provided with two cricket legs and a droplet of honey every second week (N = 32 colonies, electronic supplementary material, figure S1), while the QR-parts of the control (N = 30 colonies) and the QL-parts of both treatments received the same amount of food twice weekly. At each feeding session, all nests were opened and any remaining food was removed. Food was replaced with fresh food at every session for the QL-parts and the QR-part control, but only at every fourth time for the QR-part of dietary-restriction treatment. Thus, these ants had food available only a quarter of the time, whereas all others had continuous access to food. All colonies had continuous access to water. Worker survival was monitored weekly. Queens reduced egg-laying over time in both treatments, as eggs laid at the beginning developed into larvae. In order to increase the likelihood to detect a treatment effect, all eggs and young larvae were removed from the QR parts at week eight (electronic supplementary material, figure S2). The experiment ended after 13 weeks. All eggs were counted. All queens were dissected.

For the egg-removal experiment, 44 polygynous colonies were used to create 58 experimental colonies. Fourteen colonies were split and colony fragments were allocated to different treatments. We standardized the number of queens, workers, and larvae to 2, 50 and 12, respectively, and removed all eggs. In the egg-removal treatment (N = 29 colonies) all eggs were removed once per week, while in the control treatment (N = 39 colonies) eggs were just moved around with forceps. Colonies were anesthetized with CO₂ to remove or simulate egg removal. Queen survival was recorded weekly. The experiment was performed over six weeks. All queens were dissected two weeks after the experiments' end.

Ovaries and fat bodies of eight queens per treatment were dissected on ice (N = 32). The fat body was individually homogenized in 50 µl TRIZOL (Invitrogen) and stored at −20°C. RNA was extracted using the RNeasy mini kit (Qiagen) with a preceding chloroform step. Library preparation and sequencing of 100 bp paired-end reads on an Illumina HiSeq 2000/2500 was conducted at BGI Hong Kong. The ovaries of all remaining queens were dissected and photographed for fertility measurements (Leica DFC425 20x; LAS v. 4.5). Ovary length in the dietary-restriction experiment was analysed by using a Wilcoxon test. We used generalized-linear models with a Poisson distribution (link function = log) to investigate the effect of treatment on the number of white eggs in the ovaries and in the colony as a dependent variables. For the egg-removal experiment, fecundity differences were analysed with a linear-mixed model with ovary length (in mm) as dependant variable, and a generalized linear-mixed model with a Poisson distribution (link function = log) with the number of white eggs in the ovaries as dependent variable. Experimental fragment ID and colony ID were added as random factors. For both experiments, we separately analysed queen survival by running survival models, with treatment as an explanatory variable. As all queens were independent in the dietary-restriction experiment, we used the R package survival, while we used the package coxme for the analysis of the egg removal data by adding colony ID as a random factor. The statistical analyses were conducted in R v. 3.0.2 (R Development Core Team 2008).

For the transcriptome analyses, raw reads from all 32 samples were trimmed with Trimmomatic v. 0.36 [20], quality checked using FastQC v. 0.11.5 [21]. Paired reads were de-novo assembled using Trinity v. 2.4.8 [22], resulting in 328 731 transcripts. For annotation, we conducted a BlastX homology search [23] against the non-redundant invertebrate protein database ( June 2018) with an E-value cut-off of E-05. Read count estimates per transcript and sample were obtained using RSEM v. 1.3.0 [24] with Bowtie2 aligner for each experiment separately. To eliminate low read counts likely representing noise, we removed transcripts with fewer than 10 reads in fewer than four samples [25]. The differential expression analyses were performed with R package Deseq2 v. 1.2.10 [26] (contrast function) by comparing treatment to control for each experiment. We added colonyID as a random factor in the egg-removal treatment as some samples were dependent. Nucleotide sequences were translated into amino acid sequences with Transdecoder v. 5.5.0 [22], before conducting a gene ontology (GO) annotation using InterProScan v. 5.34–73.0 [27]. We performed GO term enrichment analyses based on subsets of differentially expressed genes (DEGs) using the R package TopGo v. 3.6 [28], with the ‘weight01’ algorithm. For each DEG, we extracted the geneID from the BlastX results to retrieve additional GO and biological functions from the Uniprot database (www.uniprot.org) with Homo sapiens, Mus musculus and Drosophila melanogaster as query organisms using an in-house python script (Supplement: maintenance_test.R). Thereafter, we searched for terms associated with fecundity (fecund, fertile, meiosis, meiotic, zygote, reproductive, reproduction, embryo, pregnancy, mating, fetal, sexual, brood, egg, ovule, ovary and ovarian), body maintenance (Toll, response to oxidative stress, apoptosis, TOR, tumour repressor, transposable element, response to UV damages, DNA repair, stress response, ageing, autophagy and cellular homeostasis), epigenetics (chromatin, histone), fatty acid metabolism (fatty) and immunity (immune). χ²-tests were used to contrast the frequency of DEGs with these functions between treatment and controls. We conducted these additional analyses to obtain insights into putative functions of DEGs in fecundity, longevity (body maintenance, immunity), food processing (fatty acid metabolism) and gene regulation (epigenetics).

3. Results

Dietary restriction had a negative effect on fecundity as it reduced the number of eggs produced ( χ² = 252.6, df = 1, p < 0.001; electronic supplementary material, figure S2), the ovariole length (W = 522, p = 0.015; figure 1a), as well as number of eggs in development in the ovaries ( χ² = 47.58, df = 1, p < 0.001; figure 1b). Queen survival was unaffected by dietary restriction ( χ² = 0.6, df = 1, p = 0.42) as only two queens in control and four in the dietary restriction treatment died. Seventy-two genes were differentially expressed in the fat body of queens from the dietary restriction and control treatment, 29 and 43 being up- or downregulated, respectively (electronic supplementary material, table S2). The gene sequestosome-1, a gene involved in autophagy [29,30], was downregulated under dietary restriction. The enrichment analyses revealed the function protein dephosphorylation (GO:0006470, p = 0.021) to be overrepresented in the dietary restriction DEGs and phenylalanyl-tRNA aminoacylation (GO:0006432, p = 0.011) enriched among the control DEGs. Biological functions of candidate genes revealed a negative effect of dietary restriction on the expression of genes with autophagy, Toll signalling, cellular homeostasis and immunity functionality (e.g. tetraspanin-1-like; table 1 and figure 1c). No genes with apparent fecundity functionalities were differently expressed (electronic supplementary material, table S2).

Figure 1.

Effects of dietary restriction and egg removal on queen fecundity and fat body gene expression. Dietary restriction negatively affected queen fecundity reducing (a) ovariole length and (b) the number of eggs in development. (c) Number of genes with function in body maintenance, epigenetics, fatty acid metabolism and immunity among the DEGs of the dietary restriction experiment. Egg removal (d) did not alter the length of queen ovarioles, but led to (e) an increase in the number of eggs in development. (f) More genes with fecundity and body maintenance functionality were found among the upregulated genes of queens of the egg removal treatment.

Table 1.

List of longevity, immunity and/or fecundity candidate genes upregulated in either control or treatment groups. We report the effect of treatment on fecundity, transcript ID, log-fold change, p-value, as well as the Blast and Uniprot annotation of the transcripts.

treatment	fecundity	transcript ID	log-fold change	p	annotation	species	Uniprot function	species
no dietary restriction	high	TRINITY_DN91813_c0_g1_i6	10.7	0.001	sequestosome-1	Monomorium pharaonis	autophagy	Homo sapiens
		TRINITY_DN98272_c1_g1_i3	9.1	0.013	mitochondrial Rho GTPase	Wasmannia auropunctata	cellular homeostasis	Drosophila melanogaster
		TRINITY_DN82958_c0_g1_i1	9.0	0.001	proteasome subunit alpha type-7-1	Monomorium pharaonis	Toll signalling pathway	Drosophila melanogaster
		TRINITY_DN83891_c0_g1_i3	3.6	0.001	tetraspanin-1	Vollenhovia emeryi	innate immune response	Caenorhabditis elegans
no egg removal	low	TRINITY_DN93193_c0_g2_i1	1.6	0.001	protein Toll-like	Wasmannia auropunctata	innate immune response	Drosophila melanogaster
egg removal	high	TRINITY_DN97419_c1_g2_i1	11.6	0.006	DNA ligase 1	Vollenhovia emeryi	DNA repair	Drosophila melanogaster
		TRINITY_DN93193_c0_g1_i2	11.6	0.045	protein Toll-like	Wasmannia auropunctata	innate immune response	Drosophila melanogaster
		TRINITY_DN98272_c1_g1_i3	9.2	0.032	mitochondrial Rho GTPase	Wasmannia auropunctata	cellular homeostasis	Drosophila melanogaster
		TRINITY_DN97049_c0_g1_i11	5.2	0.029	breast cancer type 2 susceptibility protein	Solenopsis invicta	DNA repair	Drosophila melanogaster

Egg removal did not affect ovariole length of queens ( χ² = 1.15, df = 1, p = 0.28; figure 1d), but caused an increase in the number of eggs present in their ovaries ( χ² = 7.12, df = 1, p = 0.007; figure 1e). Survival was unaffected by treatment as only two queens died during the experiment ( χ² = 0.7, df = 1, p = 0.41). A total of 372 genes were differentially expressed, with 194 upregulated in the control and 178 upregulated in the egg-removal treatment (electronic supplementary material, table S2). DEGs were not enriched for any function. Candidate genes important for fecundity, DNA repair and immunity were upregulated in response to egg removal (table 1). One gene, mitochondrial Rho GTPase involved in cellular homeostasis, was differentially expressed in fat bodies of highly fecund queens from both experiments, i.e. upregulated in response to egg removal and no dietary restriction (table 1). Significantly more fecundity ( χ² = 6.9, df = 1, p = 0.0088) and body maintenance ( χ² = 4.8, df = 1, p = 0.029, figure 1f) genes were upregulated in the egg-removal treatment, i.e. under high fecundity, while there was no enrichment of fecundity or body maintenance genes among the DEGs of the dietary restriction experiment.

4. Discussion

Nutrient intake in solitary species increases reproduction often at the price of reducing lifespan via an increase in oxidative stress through reduced investment in body maintenance [31]. Cross-talk between molecular pathways is thought to contribute to the commonly observed longevity–fecundity trade-off [32]. As social insect queens seem to be an exception to this pattern, they also might not experience these opposing effects of nutrients on reproduction and lifespan [33]. We experimentally manipulated queen fecundity by restricting food availability and by removing eggs, and demonstrate clear consequences on the expression of genes associated with fecundity and somatic maintenance. Indeed, mitochondrial Rho GTPase was commonly upregulated across both manipulations in the more fecund treatment group, a gene that plays a role in body maintenance via cellular homeostasis and innate immune response [29,30].

Dietary restriction caused a decrease in queen egg-laying rate with no impact on queen survival. Queens under food limitation showed a lower expression of sequestosome-1, a gene involved in autophagy [29,30], which plays a role in the regulation of lifespan via the inactivation of TOR signalling [34,35]. Our findings may thus suggest a reshaping of pathways associated with autophagy in social species, rendering sequestosome-1 an interesting candidate potentially involved in the positive link between lifespan and reproduction in social insects. However, while the lack of survival differences was expected and can be explained by the long lifespan of Temnothorax queens [17], we provide no evidence that our manipulations altered longevity in ant queens and thus our inferences on the longevity–fecundity trade-off remain indirect.

As already shown for Cardiocondyla ants [16], egg removal caused a stimulation in ovarian activity of T. rugatulus ant queens, which triggered the expression of numerous fecundity and body maintenance genes in the fat body. For example, we found interesting candidates with functions in oogenesis (e.g. protein sarah, actin-binding protein anillin isoform X2, γ-tubulin complex component 4 [36]) and DNA repair (e.g. DNA ligase 1, breast cancer type 2 susceptibility protein) to be upregulated in queens of the egg-removal treatment. Indeed, a significant enrichment of genes with fecundity and maintenance functionalities suggests that fecundity in ant queens goes in concert with increased activity of body repair genes, which was also found when comparing highly fecund queens with sterile ant workers [37,38]. The extent to which the latter may also be an indication for associated lifespan changes remains to be elucidated.

Our experimental manipulations revealed that fecundity stimulation in ant queens triggers an upregulation of genes involved in biological processes important for fecundity and longevity such as egg production and somatic repair. Dietary restriction caused a decrease in queen fecundity and lowered the expression of genes with roles in immunity and autophagy. Our study provides first insights into how the molecular regulation of these life-history traits might have shifted during social evolution, leading to a rewiring of the link between fecundity and longevity in social insects [6,39].

Ethics

Ant collection permits were obtained from Coronado National Forest via the Southwestern Research station. Import and export licences are not required for the transport of our study species. We followed the guidelines of the Study of Animal Behaviour and the legal and institutional rules.

Data accessibility

All raw data are provided in the Dryad Digital Repository: https://doi.org/10.5061/dryad.sxksn0322. RNAseq samples from the control treatment of the egg removal experiment were also used for another study (Negroni et al., unpublished ms) and are accessible under the BioProject accession nos. PRJNA658854, SAMN15891875-SAMN15891898 (https://www.ncbi.nlm.nih.gov/bioproject/).

Authors' contributions

M.A.N., B.F. and S.F. collected the ant colonies and designed the experiments. M.A.N. conducted the experiments, dissected the ants, extracted the RNA and analysed the gene expression data supported by B.F. and S.F. All authors contributed to writing and revising the manuscript. All authors agree to be held accountable for the content therein and approve the final version of the manuscript.

Competing interests

S.F. is a member of the Biology Letters Editorial Board. The authors declare no competing interests.

Funding

We thank the Deutsche Forschungsgemeinschaft that funded this project grant to B.F. and S.F. (nos. DFG FE 1333/6-1 and FO 298/19-1) within the research unit FOR-2281.