DOI: 10.5061/dryad.73n5tb2x0
title: Intergenerations effects of starvation on Athalia

description: Intergenerational effects, also known as parental effects in which the
 offspring phenotype is influenced by the parental phenotype, can occur in
 response to factors that occur not only in early but also in late parental
 life. However, little is known about how these parental life
 stage-specific environments interact with each other and with the
 offspring environment to influence offspring phenotypes, particularly in
 organisms that realize distinct niches across ontogeny. We examined the
 effects of parental larval starvation and adult reproductive environment
 on offspring traits under matching or mismatching offspring larval
 starvation conditions using the holometabolous, haplodiploid insect
 Athalia rosae (turnip sawfly). We show that parental larval starvation had
 trait-dependent intergenerational effects on both life-history and
 consumption traits of offspring larvae, partly in interaction with
 offspring conditions, while there was no significant effect of parental
 adult reproductive environment. In addition, while offspring larval
 starvation led to numerous gene- and pathway-level expression differences,
 parental larval starvation impacted far fewer genes and only the ribosomal
 pathway. Our findings reveal that parental starvation evokes complex
 intergenerational effects on offspring life-history traits, consumption
 patterns as well as gene expression, although the effects are less
 pronounced than those of offspring starvation.
description: <i>Set-Up of Insect Rearing and Plant
 Cultivation</i> Adults of <i>A.
 rosae</i> (F0) were collected in May 2019 at two locations
 (population A: 52°02'48.0"N 8°29'17.7"E, population B:
 52°03'54.9"N 8°32'22.2"E). These individuals were
 reared for further two generations for details of breeding design see S1).
 White mustard (<i>Sinapis alba</i>) was provided for
 oviposition and Chinese cabbage (<i>Brassica rapa</i> var.
 <i>pekinensis</i>) as food plant. Adults of the F2 generation
 were kept individually in Petri dishes and provided with a honey:water
 mixture (1:50). <i>Athalia rosae</i> is haplodiploid, i.e.
 virgin females produce male offspring (Naito and Suzuki 1991). Thus, mated
 as well as virgin females were placed individually into boxes (25 x 15 x
 10 cm) to increase the likelihood of gaining similar numbers of females
 and males. Females were supplied with middle-aged leaves of non-flowering
 cabbage plants for oviposition and a honey:water mixture, which were
 replenished daily. Females were removed from the boxes after one week and
 their offspring used to set up the experimental generations, which were
 kept in a climate chamber (20 °C:16 °C,
 16 h: 8 h light:dark, 70% r.h.). Plants of
 <i>S. alba</i> and <i>B. rapa</i> were grown from
 seeds (Kiepenkerl, Bruno Nebelung GmbH, Konken, Germany) in a greenhouse
 (20 °C, 16 h: 8 h light:dark, 70% r.h.) and a climate chamber (20 °C, 16 h:
 8 h light:dark, 70% r.h.). Plants of <i>Ajuga reptans</i> used
 for clerodanoid supply were grown from seeds (RHS
 Enterprise Ltd, London, UK) in the greenhouse and transferred
 outside in late spring when about 2 months old. Middle-aged leaves of 8
 months old plants were offered to adults.
 <i>Experimental Overview and Measurements of Life-History
 Traits</i> We conducted a fully factorial
 experiment where we manipulated the parental larval environment (parental
 larval starvation), the parental reproductive environment (exposure to
 clerodanoids) and the offspring environment (offspring larval starvation)
 (Fig. 1). We thus tested the effects of parental and offspring larval
 starvation, and parental adult reproductive environment on the offspring
 phenotype. On the day of hatching, larvae of the parental generation were
 individually placed into Petri dishes (5.5 cm diameter) with moistened
 filter paper and <i>B. rapa</i> leaf discs cut from 7-10 week
 old plants, which were replaced daily. Per maternal line, larvae were
 split equally between one of two larval starvation treatments, no
 starvation (N) or starvation (S). For starvation, individuals were starved
 twice for 24 h, the day after moulting into 2<sup>nd</sup>
 instar and the day of moulting into 4<sup>th</sup> instar,
 thereby mimicking the food deprivation larvae may experience when
 occurring in high densities (Riggert 1939). Larval instars were tracked by
 checking daily for the presence of exuviae. Eonymphs
 were placed in soil for pupation. Adults were kept individually in Petri
 dishes and provided with honey:water mixture. From the parental
 generation, pairs of non-sib females and males reared under the same
 larval starvation treatments were assigned to one of two reproductive
 environment treatments, where both parents either had clerodanoids (C+) or
 did not (C-). C+ individuals were exposed to a leaf section (1
 cm<sup>2</sup>) of <i>A. reptans</i> for 48 h
 prior to mating, giving individuals time to take up clerodanoids (preprint
 Paul et al. 2021). Mated females (2-9 days old) from each of these four
 treatments (NC-, NC+, SC-, SC+) and C- virgin females (NC-, SC-) were then
 placed in individual breeding boxes. Their offspring were distributed to
 offspring starvation treatments (N or S) that either matched the parental
 starvation treatment or differed from it (mismatch) (Fig. 1). In both
 generations, larval, pupal, and total development time (from larva to
 adult) as well as the adult body mass at the day of emergence (Sartorius
 AZ64, M-POWER Series Analytical Balance, Göttingen, Germany) were recorded
 for each individual. In total 358 larvae of the parental generation (177
 females, 181 males) and 484 larvae of the offspring generation (282
 females, 202 males) reached adulthood, out of the 607 and 688 larvae,
 respectively, that were initially reared in each generation.
 <i>Consumption Assays</i> To test
 effects of parental and offspring larval starvation experience on
 offspring consumption, assays were performed with larvae of the offspring
 generation at the start of the 3<sup>rd</sup> instar (directly
 after the first starvation event), measuring the relative growth rate,
 relative consumption rate, and efficiency of conversion of ingested food.
 Each larva was weighed at the beginning of the consumption assays (=
 initial body mass) (ME36S, accuracy 0.001 mg; Sartorius, Göttingen,
 Germany) and provided with four discs of middle-aged cabbage leaves
 (surface area of 230.87 mm<sup>2 </sup>per disc) on moistened
 filter paper. After 24 hours, larvae were weighed again (= final body
 mass) and the leaf disc remains scanned (Samsung SAMS M3375FD, resolution
 640 x 480). In that period, leaf discs showed no signs of wilting. The
 total area of leaf consumed (mm<sup>2</sup>) was then
 calculated as the difference between the average leaf area and the
 remaining leaf area. <i>Statistical
 Analyses</i> All data were analyzed using R 4.0.2
 (2020-06-22). We set <i>α</i> = 0.05 for all tests and checked
 model residuals for normality and variance homogeneity: All All linear
 mixed effects models (LMMs) and generalized linear mixed models (GLMM)
 were run in lme4 using maximum likelihood. Stepwise backwards deletion
 using <i>X<sup>2</sup></i> ratio tests
 (package:MASS; version 7.3-53.1) for the life history traits and, due to
 the much smaller sample sizes (Luke 2017), conditional
 <i>F</i>-tests with <i>df</i> correction using
 Satterthwaite method (package lmerTest; version 3.1-3;
 Kuznetsova et al. 2017) for the consumption analyses were employed to
 reach the minimum adequate models (Crawley 2012). Posthoc analyses were
 carried out using the package ‘multcomp’ (version 1.4-13; Hothorn et al.
 2008). Post data entry, raw data were visually inspected thrice, all
 variables plotted and outliers and possible anomalies in the data (e.g.
 strings of similar values) interrogated (package:pointblank; version
 0.6.0). Intragenerational effects of starvation on life-history traits of
 individuals of the parental generation were tested as described in
 S1. In <i>A. rosae</i>, usually 6 instars
 for female and 5 instars for males are found (Sawa et al. 1989). Due to
 observations made during the experiment (no <i>a priori</i>
 hypothesis), we tested in the offspring generation whether the likelihood
 of an additional larval instar (7 for females and 6 for males) differed
 based on offspring larval starvation treatment (independent of parental
 treatments) using a binomial GLMM (package: lme4), where the predictor was
 offspring starvation treatment and parental pair was included as a random
 effect. The effects of the parental larval starvation treatment, parental
 clerodanoid exposure, offspring starvation treatment and their interaction
 on larval, pupal and total developmental time as well as adult mass of
 offspring individuals were assessed in separate LMMs, with parental pair
 included as a random effect (controlling for non-independence of sibling
 larvae). Female and male data were analyzed separately to enable model
 convergence. Relative growth rate, relative consumption
 rate, and food conversion efficiency of larvae of the offspring generation
 were analyzed using LMMs. We excluded parental clerodanoid exposure as a
 predictor variable from the consumption assay analysis due to the low
 number of individuals in certain treatments (Fig. 1) and analyzed male and
 female data separately. To assess variation in relative growth rate, the
 change in larval body mass [final mass — initial mass] was used as the
 response variable and initial larval body mass, parental starvation
 treatment, offspring starvation treatment, and their interactions were
 used as predictors. To assess relative consumption rate, we used the total
 area of consumed leaf material as the response variable and initial larval
 body mass, parental starvation treatment, offspring starvation treatment,
 and their interactions as the predictors. Finally, for food conversion
 efficiency the change in larval body mass was taken as the response
 variable and the predictor variables were total area of consumed leaf
 material, parental starvation treatment, offspring starvation treatment,
 and their interactions. Parental pair was included in all three models as
 a random effect. <i>Sample Collection and
 Sequencing</i> A
 total of 24 male larvae (4<sup>th</sup> instar, 9 d old),
 comprising six biological replicates per treatment level (parent/offspring
 treatment: N/N, N/S, S/N, S/S, all C-, Fig. 2), were collected to
 investigate the effects of parental and offspring starvation treatment on
 gene expression in offspring larvae. Individuals were chosen in a way that
 maximized the equal spread of siblings across treatments and frozen at -80
 °C prior to extraction. RNA was extracted with an Invitrogen PureLink™ RNA
 Mini Kit (ThermoFisher Scientific, Germany), including a DNase step
 (innuPREP DNase I Kit, analyticJena, Jena, Germany). RNA quality was
 assessed on a bioanalyzer 2100 (Agilent, CA, United States) and Xpose (PLT
 SCIENTIFIC SDN. BHD, Malaysia). Library preparation (Ribo-Zero for rRNA
 removal) and sequencing (NovaSeq6000 and S4 Flowcells, Illumina, CA,
 United States) were provided by Novogene (Cambridge, UK). Sequence quality
 before and after trimming was assessed using FastQC (v. 0.11.9; Andrews
 2010). After short (&lt; 75 bp), low quality (Q &lt; 4, 25 bp
 sliding window), and adapter sequences were removed using Trimmomatic
 (Bolger et al. 2014), more than 98% of reads remained. <i>Differential Expression
 Analysis</i> Cleaned reads were mapped to the
 annotated genome of<i> A. rosae</i>, version AROS v.2.0
 (GCA_000344095.2) with RSEM v1.3.1 (Li and Dewey 2011), implementing
 mapping with STAR v2.7.1a (Dobin et al. 2013). Analysis of differential
 gene expression was conducted with DESeq2 (version 1.28.1; Love et al.
 2014). The results of mapping with RSEM were passed to DESeq2 for
 gene-level analyses using Tximport (version 1.16.1; Soneson et al. 2015).
 Prior to analysis, genes with zero counts in all samples and those with
 low counts (&lt; 10) in less than a quarter of samples were excluded.
 Model fitting was assessed by plotting dispersion estimates of individual
 gene models and outlier samples were inspected using principle component
 analysis of all expressed genes and pairwise-distance matrices between
 samples. Expression was modelled based on the entire dataset, with the
 four levels representing the combination of parental.offspring starvation
 treatments (N.N, N.S, S.N, and S.S). Differential expression was assessed
 in four pairwise comparisons between the treatments: 1) N.N vs N.S and 2)
 S.N vs S.S were used to assess the effects of offspring starvation
 treatment for individuals whose parents experienced the same starvation
 treatment, whereas 3) N.N vs S.N and 4) N.S vs S.S were used to assess the
 effects of differing parental starvation treatment on individuals that
 experienced the same offspring starvation treatment. Significance was
 based on a Wald Test and shrunken log fold-change values with apeglm
 (version 1.10.1; Zhu et al. 2019). <i>P</i>-values were
 adjusted for multiple testing using a Benjamini-Hochberg procedure
 (Benjamini and Hochberg 1995); we retained significantly differentially
 expressed genes (relatively up- or downregulated) if this adjusted
 <i>P</i>-value was &lt; 0.05. A Venn diagram
 (Venn.Diagram, version 1.6.20) was used to depict the overlap of
 significantly differentially expressed genes for each pairwise comparison.
 The expression of the significantly differentially expressed genes of each
 comparison was visualized in a heatmap using normalized counts scaled per
 gene (scale=“row”) (pheatmap, version 1.0.12). To examine differential expression
 of genes with known roles in stress and/or starvation response, we
 searched specifically for target significantly differentially expressed
 genes, based on their identity (from the genome annotations), using the
 keywords “heat shock protein” (and “hsp”), “cytochrome P450”, “octopamine”
 and “tyramine” in the putative gene names. <i>Pathway-Level Analysis of
 Differential Expression</i> We used the KEGG (Kyoto Encyclopedia of
 Genes and Genomes) database to assign the predicted genes in the
 <i>A. rosae</i> genome to gene pathways with the KEGG
 Automatic Annotation Server (KAAS; Moriya et al. 2007) (S3). Of the
 annotated genes for which we had read counts, 65% were assigned to at
 least one KEGG pathway. A gene set enrichment analysis was then performed
 on the normalized counts using GAGE (Luo et al. 2009) and pathview (Luo
 and Brouwer 2013) in R, applying a false discovery rate-adjusted
 <i>P</i>-value cut-off of &lt; 0.05 to identify
 differentially expressed pathways. We derived the non-redundant
 significant gene set lists, meaning those that did not overlap heavily in
 their core genes, using esset.grp and a <i>P</i>-value cut-off
 for the overlap between gene sets of
 10e<sup>-10</sup>. Unmapped reads were inspected to check
 for the differential expression of genes not present in the reference
 genome. These were extracted from the RSEM-produced BAM file using
 samtools view (Li et al. 2009) and converted to fastq (using bamtools
 bamtofastq; Barnett et al. 2011), and <i>de novo</i> assembled
 using Trinity (default settings) (Grabherr et al. 2011). This
 assembly was done jointly with reads from additional samples (preprint
 Paul et al. 2021) to optimize coverage.
 The unmapped reads were mapped back to this reference and
 expected read counts extracted using eXpress (Roberts et al. 2011).
 Transcripts with &gt;10,000 mapped reads were identified using BLASTN
 (default settings, limited to one match per gene and
 1e<sup>-1</sup>) against the NCBI <i>nt</i>
 nucleotide database. Differential expression of
 unmapped reads in the larval samples was carried out as for mapped reads
 (see above).