DOI: 10.5061/dryad.q2bvq83h2
title: Data from: 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: <em>Temnothorax rugatulus</em> 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 <em>T. rugatulus</em>
 colonies in the Chiricahua Mountains, Arizona, USA 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 <em>dietary-restriction</em> 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 some food provisioning in the upper part of an
 artificial experimental 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 of
 food (Fig. S1). The QR-parts of the <em>dietary-restriction
 </em>treatment were provided with two legs of 1cm-sized cricket
 nymphs (<em>Acheta domestica</em>) and an droplet of honey
 every 2<sup>nd</sup> week (N = 32 colonies, Fig. S1), while
 the QR-parts of the <em>control</em> received the same amount
 of food twice weekly, so four times as often (N = 30 colonies). QL-parts
 of both treatments were fed with crickets and honey twice weekly. At each
 twice-weekly feeding session, all nests of both treatments 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 <em>control</em>, but
 only at every fourth session for the QR-parts of
 <em>dietary-restriction</em> treatment. Thus, ants in
 this<em> </em>treatment had food available only a quarter of
 the time, whereas all others had continuous access to food. All ants had
 continuous access to water. Worker survival was monitored weekly. Queens
 reduced egg production in both treatments over time, as eggs produced at
 the beginning developed into larvae. In order to increase the likelihood
 of detect an effect of treatment, 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 and all queens were dissected
 For the <em>egg-removal</em> 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 <em>egg-removal
 treatment</em> (sample size = 29 colonies) all eggs were removed
 once per week, while in the <em>control treatment</em> (sample
 size  = 39 colonies) eggs were just moved within the nest 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 end of the experiment. 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 (<em>Leica DFC425</em> 20x; Leica
 software <em>LAS version 4.5</em>). Ovary length in the
 <em>dietary-restriction</em> experiment was analysed by using
 a Wilcoxon test. We used generalized-linear models with a
 <em>poisson</em> distribution (link function = log) to
 investigate the effect of treatment on the number of white eggs in the
 ovaries and the number of eggs in the colony as a dependent variable. For
 the <em>egg-removal </em>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
 <em>poisson</em> 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<em> </em>as explanatory
 variable<em>. </em>As all queens were independent in the
 <em>dietary-restriction</em> experiment, we used the R package
 <em>survival ()</em>, while we used the package
 <em>coxme()</em> 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 <em>Trimmomatic-v0.36</em> (20), quality checked using
 <em>FastQC-v0.11.5 </em>(21). Paired reads were
 <em>de-novo </em>assembled using <em>Trinity</em>
 v.2.4.8 (22), resulting in 328,731 transcripts. For annotation, we
 conducted a <em>BlastX</em> 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
 <em>Deseq2-v1.2.10 </em>(26) (<em>contrast
 </em>function) by comparing treatment to control for each
 experiment. We added colonyID as control factor in the
 <em>egg-removal</em> treatment as some samples were dependent.
 Nucleotide sequences were translated into amino-acid sequences with
 <em>Transdecoder-v5.5.0 </em>(22), before conducting a gene
 ontology (GO) term annotation using <em>InterProScan-v5.34-73.0
 </em>(27). We performed GO term enrichment analyses based on subsets
 of DEGs using the R package <em>TopGo-v-3.6</em> (28), with
 the “weight01” algorithm. For each DEG, we extracted the geneID from the
 <em>BlastX</em> results to retrieve additional GO and
 biological functions from the <em>Uniprot</em> database (www.uniprot.ong) with
 <em>Homo sapiens</em>, <em>Mus musculus</em>, and
 <em>Drosophila melanogaster</em> as query organisms using an
 in-house python script (Supplement: maintenance_test.R). Thereafter, we
 searched our results for terms associated with fecundity (fecund, fertile,
 meiosis, meiotic, zygote, reproductive, reproduction, embryo, pregnancy,
 mating, foetal, sexual, brood, egg, ovule, ovary, ovarian), body
 maintenance <em>(Toll,</em> 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 this additional analysis to obtain further details on putative
 functions of the DEGs in fecundity, longevity (body maintenance,
 immunity), food processing (fatty acid metabolism) and gene regulation
 (epigenetics).
description: # Data from: Experimental increase in fecundity causes upregulation of
 body maintenance genes in ant queens \[Access these datasets on
 Dryad](doi:10.5061/dryad.sxksn0322) File name --&gt; Content
 DEGs_Dietary_Restrictions_Egg_Removal.xlsx --&gt;   The list of
 upregulated contigs with their blast annotation for each group and the two
 experiments, with sheet 1: the list of upregulated contigs in the Non
 dietary restriction treatment compared to the Dietary restriction one from
 the dietary restriction experiment;  sheet 2: the list of upregulated
 contigs in the Dietary restriction treatment compared to the Non dietary
 restriction one from the dietary restriction experiment; sheet 3: the list
 of upregulated contigs in the No egg removal treatment compared to the Egg
 removal one from the egg removal experiment;  sheet 4: the list of
 upregulated contigs in the Egg removal treatment compared to the No egg
 removal one from the egg removal experiment; BlastX is the blast
 annotation; lfcSE is the logFoldChange standard error; pads is the FDR
 p-value; the search query is the gene name that was used for the uniprot
 annotation; GO is for gene ontology; BP is for biological process; MF is
 for molecular function; In this table the NAs mean that either there was
 no annotation for the transcripts or that the sequence did not match to
 any known sequence  DGE_food_rest_egg_rem.R      --&gt; The R script of
 the differential gene expression analysis for the dietary restriction
 experiment and for the egg removal experiment Egg_Foodrest.csv --&gt;
 Dataset with the number of egg counted per week for each colony in the
 food restriction experiment, with colony identity for the first column;
 the treatment (non dietary restricted / control, dietary restricted /
 restricted) for the second column; the week for the third column; the
 number of egg for the fourth column; and information about the death of
 the queen in the fifth column; The NA for the number of eggs when the
 queen was still alive means that the exact number of eggs could not be
 precisely accessed during observation without disturbing the nest: either
 that the egg pile was not fully visible during observation (portion of the
 egg pile under the separating wall of the nest) or that the egg pile was
 too dense, too compact or too large that the exact number of eggs could
 not be counted precisely. Egg_removal.R          The R script for the
 analysis of the phenotypical data for the egg removal experiment
 Food-rest.R               The R script for the analysis of the
 phenotypical data for the dietary restriction experiment
 maintenance-test.R The R script for the maintenance tests
 Ovaries_Foodrest.csv   Dataset of ovary measurment for the dietary
 restriction experiment with in the first column: colony identity; second
 column: the treatment (ctr/ non dietary restricted or rst/ dietary
 restricted) ; third column: the total number of ovariol; fourth column:
 the mean  in mm of the ovariols; fifth to  thenth columns: the ovary
 length of each ovariol in mm; eleventh column: the number of transparent
 egg in the ovaries; 12th column: the number of white egg in the ovaries;
 13th column: the number of yellow body in the ovaries;"
 Ovary_egg_rem.csv "Dataset of ovary measurment for the egg removal
 experiment with in the first column: colony identity; second column:
 colony fragment identity; third column: the queen identity; fourth column:
 the queen morph; fifth column: the treatment (ref: no egg removal or egg/
 egg removal); sixth column: the total number of ovariol; seventh column:
 the mean ovary length in mm; eighth column: the number of transparent egg
 in the ovary; nineth column: the number of white egg in the ovaries; tenth
 column: the number of yellow body in the ovaries; eleventh column:
 information about the use of the sample for RNA seq"; NA means a fail
 in the dissection (ovaries damaged during the dissection process)
 sampleFile.txt.csv    File containing the path of each raw reads used for
 the trimming step Supplement_Food_uniprot.xlsx   Supplement with the
 uniprot annotation for the contigs differentially expressed for the
 dietary restriction experiment with in the firt sheet the contigs
 upregulated in the non dietary restricted treatment and in the sheet 2 the
 contigs upregulated in the dietary restriction treatment.
 Supplement_Egg_uniprot.xlsx Supplement with the uniprot annotation for the
 contigs differentially expressed for the queen fecundity experiment with
 in the first sheet the contigs upregulated in the control treatment (no
 egg removal) and in the sheet 2 the contigs upregulated in the egg removal
 treatment. Surv_Foodrest.csv   dataset of the survival data per week for
 each queen in the dietary restriction experiment; Treat = treatment; Mac =
 Macrogyne survival_egg_rem.csv    dataset of the queen survival data per
 week for each colony in the egg removal treatment. experiment; Treat =
 treatment; Mac = Macrogyne; the NAs mean that the queen died after winter
 but before the start of the experiment so was excluded from the survival
 analysis of the experiment Table_S1_Collection_Food_res_Egg_rem.xlsx 
  Information about colony collection with the coordinateds, the date of
 collection, the number of worker at the time of collection the number of
 queen, whether the colony was used for the experimental treatment (treat)
 or in the control treatment (control), and the type of experiment the
 colony was used for (egg_removal or food restriction) Workflow.txt   the
 workflow for the transcriptomic analysis without the path
 Workflow__with_path    the workflow for the transcriptomic analysis with
 the path Note: In the data files, if cells were found empty it's
 because of no occurence.