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

Cite this dataset

Foitzik, Susanne; Feldmeyer, Barbara; Negroni, Matteo (2024). Data from: Experimental increase in fecundity causes upregulation of fecundity and body maintenance genes in the fat body of ant queens [Dataset]. Dryad. https://doi.org/10.5061/dryad.q2bvq83h2

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.

README: 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 --> Content

DEGs_Dietary_Restrictions_Egg_Removal.xlsx -->   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      --> The R script of the differential gene expression analysis for the dietary restriction experiment and for the egg removal experiment

Egg_Foodrest.csv --> 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.

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, 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 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 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 dietary-restriction treatment were provided with two legs of 1cm-sized cricket nymphs (Acheta domestica) and an droplet of honey every 2nd week (N = 32 colonies, Fig. S1), while the QR-parts of the control 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 control, but only at every fourth session for the QR-parts of dietary-restriction treatment. Thus, ants in this 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 egg-removal 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 egg-removal treatment (sample size = 29 colonies) all eggs were removed once per week, while in the control treatment (sample size  = 39 colonies) eggs were just moved within the nest 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 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 (Leica DFC425 20x; Leica software LAS version 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 the number of eggs in the colony as a dependent variable. 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 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 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-v0.36 (20), quality checked using FastQC-v0.11.5 (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-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 Deseq2-v1.2.10 (26) (contrast function) by comparing treatment to control for each experiment. We added colonyID as control factor in the egg-removal treatment as some samples were dependent. Nucleotide sequences were translated into amino-acid sequences with Transdecoder-v5.5.0 (22), before conducting a gene ontology (GO) term annotation using InterProScan-v5.34-73.0 (27). We performed GO term enrichment analyses based on subsets of 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.ong) with Homo sapiens, Mus musculus, and Drosophila melanogaster 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 (Toll, 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).

Funding

Deutsche Forschungsgemeinschaft, Award: DFG FE 1333/6-1

Deutsche Forschungsgemeinschaft, Award: FO 298/19-1