DOI: 10.5061/dryad.sxksn0322
title: Experimental increase in fecundity causes upregulation of fecundity and body maintenance genes in the fat body of ant queens

description: 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.
description: <i>Temnothorax
 rugatulus</i> 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
 <i>T. rugatulus</i> colonies in the Chiricahua Mountains,
 Arizona in August 2015 (Table S1). In our laboratory, colonies were
 transferred to artificial nest boxes and kept at 22°C and 12h light / 12h
 dark in a climate chamber. For the
 <i>dietary-restriction</i> 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). Both parts were separated by a metal grid, allowing the
 exchange of volatiles, but not food (Fig. S1). The QR-parts of the
 <i>dietary-restriction </i>treatment were provided with two
 cricket legs and a droplet of honey every 2<sup>nd</sup> week
 (N = 32 colonies, Fig. S1), while the QR-parts of the
 <i>control</i> (N = 30 colonies) and the QL-parts of both
 treatments the 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 <i>control</i>, but only at every
 4<sup>th</sup> time for the QR-part of
 <i>dietary-restriction</i> 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 an treatment effect, all
 eggs and young larvae were removed from the QR parts at week eight (Figure
 S2). The experiment ended after 13 weeks. All eggs were counted. All
 queens were dissected. For the
 <i>egg-removal</i> experiment, 44 polygynous colonies were
 used to create 58 experimental colonies. 14 colonies were split and colony
 fragments were allocated to different treatments. We standardized the
 number of queen, workers, and larvae to 2, 50 and 12 respectively, and
 removed all eggs. In the <i>egg-removal treatment</i> (N = 29
 colonies) all eggs were removed once per week, while in the
 <i>control treatment</i> (N = 39 colonies) eggs were just
 moved around with forceps. Colonies were anesthetized with
 CO<sub>2</sub> 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 50μL 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
 100bp 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 (<i>Leica DFC425</i>
 20x; <i>LAS version 4.5</i>). Ovary length in the
 <i>dietary-restriction</i> experiment was analysed by using a
 Wilcoxon test. We used generalized-linear models with a
 <i>poisson</i> distribution (link function = log) to
 investigate the effect of treatment on the number of white eggs in the
 ovaries and in the colony as dependent variables. For the
 <i>egg-removal </i>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
 <i>poisson</i> 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<i> </i>as explanatory variable<i>.
 </i>As all queens were independent in the
 <i>dietary-restriction</i> experiment, we used the R package
 <i>survival ()</i>, while we used the package
 <i>coxme()</i> for the analysis of the egg removal data by
 adding colony ID as 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
 <i>Trimmomatic-v0.36</i> (20), quality checked using
 <i>FastQC-v0.11.5 </i>(21). Paired reads were <i>de-novo
 </i>assembled using <i>Trinity</i> v.2.4.8 (22),
 resulting in 328,731 transcripts. For annotation, we conducted a
 <i>BlastX</i> 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-v1.3.0 (24) with Bowtie2 aligner for each experiment separately. To
 eliminate low read counts likely representing noise, we removed
 transcripts with less than 10 reads in less than four samples (25). The
 differential expression analyses were performed with R package
 <i>Deseq2-v1.2.10 </i>(26) (<i>contrast
 </i>function) by comparing treatment to control for each
 experiment. We added
 colonyID as random factor in the <i>egg-removal</i> treatment
 as some samples were dependent. Nucleotide sequences were
 translated into amino-acid sequences with <i>Transdecoder-v5.5.0
 </i>(22), before conducting a gene ontology (GO) annotation using
 <i>InterProScan-v5.34-73.0 </i>(27). We performed GO term
 enrichment analyses based on subsets of DEGs using the R package
 <i>TopGo-v-3.6</i> (28), with the “weight01” algorithm. For
 each DEG, we extracted the geneID from the <i>BlastX</i>
 results to retrieve additional GO and biological functions from the
 <i>Uniprot</i> database (www.uniprot.org) with
 <i>Homo sapiens</i>, <i>Mus musculus</i>, and
 <i>Drosophila melanogaster</i> 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,
 foetal, sexual, brood, egg, ovule, ovary, ovarian), body maintenance
 <i>(Toll,</i> response to oxidative stress, apoptosis, TOR,
 tumour repressor, transposable element, response to UV damages, DNA
 repair, stress response, aging, autophagy, 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).