Behavioural and transcriptional changes in post-mating females of an egg parasitoid wasp species

In many animals, mating is essential for the production of offspring by females; however, mating seems to not be necessary in Hymenoptera insects. Virgin females can produce offspring, although the sex of the offspring is all male. Usually, behavioural and physiological changes are induced by mating in female insects, including parasitoid wasps. However, very little is known about the resulting changes in gene expression that contribute to the post-mating response in females; thus, we studied this aspect in the egg parasitoid wasp species Anastatus disparis (Hymenoptera: Eupelmidae) by transcriptional analysis. A total of 55 differentially expressed genes were identified in post-mating females, and most of the genes (90.9%) were downregulated. Upregulated genes encoded products that were mainly involved in fatty acid synthesis and pyrimidine metabolism, while the downregulated genes were mainly involved in substance transport and metabolism. In addition, post-mating A. disparis females exhibited a tendency to accelerate egg maturation and became unreceptive to further mating. Based on the transcriptional data, we discuss how specific genes mediate these behavioural and physiological changes. Overall, our study provided new and comprehensive insights into post-mating changes in females and provided a basis for future mechanistic studies.

P-CL, 0000-0002-4092-9709; D-JH, 0000-0001-7743-9968 In many animals, mating is essential for the production of offspring by females; however, mating seems to not be necessary in Hymenoptera insects. Virgin females can produce offspring, although the sex of the offspring is all male. Usually, behavioural and physiological changes are induced by mating in female insects, including parasitoid wasps. However, very little is known about the resulting changes in gene expression that contribute to the post-mating response in females; thus, we studied this aspect in the egg parasitoid wasp species Anastatus disparis (Hymenoptera: Eupelmidae) by transcriptional analysis. A total of 55 differentially expressed genes were identified in post-mating females, and most of the genes (90.9%) were downregulated. Upregulated genes encoded products that were mainly involved in fatty acid synthesis and pyrimidine metabolism, while the downregulated genes were mainly involved in substance transport and metabolism. In addition, post-mating A. disparis females exhibited a tendency to accelerate egg maturation and became unreceptive to further mating. Based on the transcriptional data, we discuss how specific genes mediate these behavioural and physiological changes. Overall, our study provided new and comprehensive insights into post-mating changes in females and provided a basis for future mechanistic studies.

Background
In many animals, mating is essential for the production of offspring by females [1,2]; however, mating seems to not be necessary in Hymenoptera, in which sex determination is haplodiploid. Usually, males develop from unfertilized eggs and are haploid, while females develop from fertilized eggs and are diploid. Thus, virgin females can produce offspring, but the sex of all these offspring is male [3,4]. Mating often induces behavioural and physiological changes in female insects [5], including parasitoid wasps. Most commonly, these changes include increased oviposition behaviour and repression of subsequent sexual activity as a result of the transfer of male accessory gland substances [6]. In addition, in Hymenoptera parasitoids, virgin and mated female wasps may behave differently because of differences in the benefits and costs of mating [1]. Compared to mated females, unmated females are usually likely to exhibit reduced fitness, especially in highly structured populations, where brothers compete for mates and the reproductive return through sons is low, requiring females to minimize the number of male offspring [7][8][9]. Unmated females can increase their fitness by producing only a few small sons, sufficient for mother -son mating, to produce female offspring [10,11]. In many species, mated females produce more offspring than virgin females [12][13][14][15]; however, there have been a few reports of virgin females producing more offspring than mated females [16 -18], and many species exhibit no significant difference [1,[19][20][21][22][23].
In addition, there have been several studies on the effect of female mating status on other aspects including superparasitism [24,25], host discrimination capacity [26], longevity [1,17,27], foraging [28] and offspring fitness [9]. Hypothetically, virgin females need to trade-off between either (1) searching for hosts and producing sons immediately or (2) searching for mates and perhaps producing both sons and daughters later in life [13,16,28,29]. Generally, studies on the difference between virgin and mated female parasitoid wasps have focused on behaviour and demographic parameters. However, very little is known about the resulting changes in gene expression in females that contribute to the post-mating response. Thus, we attempted to study the changes in gene expression in post-mating females in the egg parasitoid wasp species Anastatus disparis (Hymenoptera: Eupelmidae) by transcriptomic analysis.
Anastatus disparis is an egg parasitoid of several harmful species of Lepidoptera that are primarily considered forest pests in China [30]. In previous studies, A. disparis is considered a potential biological control agent for Lymantria dispar which is an important defoliator of broad-leaved and coniferous trees [30][31][32]. As with other parasitoids, the sex determination of A. disparis is haplodiploid; both virgin and mated females can produce several hundred offspring in a lifetime and live for more than a month in the wild [23,30]. In terms of oviposition and longevity, there are no significant differences between virgin and mated A. disparis females [23,30]. Here, we also focused on other reproductive aspects of changes induced by mating in A. disparis females (e.g. mating ability and egg load). Besides identifying changes in gene expression prompted by mating, our study attempted to provide new and comprehensive insights into post-mating changes in females and provide a basis for future mechanistic studies.

Insect cultures
Anastatus disparis colonies were first established from a population reared on an L. dispar egg mass collected in Longhua County, Hebei Province (41831 0 N, 117874 0 E) in March 2012, and the colony was subsequently maintained on Antheraea pernyi eggs. Antheraea pernyi is of commercial interest due to the use of its pupae in silk production. Pupae of A. pernyi were purchased from Qinhuangdao, Hebei Province, China. Adult A. pernyi emerged from the pupae at 25-308C. Eggs of A. pernyi were obtained by dissecting adult female abdomens and maintained at 08C [25,33]. Approximately 20 hosts were offered to a female for oviposition lasting 24 h at 26-288C. Then, we isolated the hosts individually in polyethylene tubes (height: 7.5 cm; diameter: 1 cm) whose openings were covered with cotton balls to prevent any mating behaviour before the start of the experiment. The parasitized hosts were incubated at a temperature of 28 + 0.58C, a relative humidity of 70 + 5% and a photoperiod of 14 L : 10 D. After approximately 18 days [23], females and males started to emerge and were collected daily. Before the experiment began, the adult wasps were fed honey water (honey : water ¼ 4 : 6) on cotton balls [30,33].

Transcriptomic analyses
For the transcriptomic experiment, 2-day-old virgin and 2-day-old mated female adults were selected. Each treatment included three replicates, and each replicate included 15 adults (virgin or mated females). Similar to Anastatus sp. [30], most A. disparis adults emerge daily in the morning, especially royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181453 from 9.00 to 10.00. These virgin females were collected during this period, then divided into two groups. One group of females was maintained in a virgin condition, while each female from the other group was offered one new emerged male, and the mated females who mated within 2 h were selected. At 12.00 on day 2, the whole bodies of adults in the same treatment were pooled into a plastic tube (1.5 ml), snap frozen in liquid nitrogen, and transferred to a 2808C freezer for long-term storage. RNA from each sample group was extracted with TRIzol reagent (Invitrogen, USA). A total of 3 mg of total RNA from each sample was converted into cDNA using the NEBNext w Ultra TM RNA Library Prep Kit for Illumina w (NEB, USA). In total, six cDNA libraries were constructed and subsequently sequenced with the Illumina HiSeq 2000 platform by Beijing Biomarker Technologies Co. Ltd, yielding raw reads. Raw sequence data generated were deposited into Sequence Read Archive database of NCBI with the accession no. PRJNA505044. Clean reads were obtained by removing reads containing the adapter sequence, poly-N reads and low-quality reads from the raw data using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/), and these clean reads were used for further analysis. Then, all the high-quality reads from the six samples were pooled and assembled using Trinity software (v. 2.5.1) with the default parameters [34]. A tool of TransRate was used in our study to evaluate the transcriptome assembly [35]. We chose the longest isoform of each gene to construct the unigene set. After the isoforms were selected, these assembled transcripts were predicted to be the unigenes produced. Bowtie was used to align reads to unigenes [36], then identified putatively expressed genes by RSEM [37] using the reads per kb per million reads (RPKM) method. For functional annotation, the pooled assembled unigenes were searched using BLASTX (v. 2.2.31) against five public databases, namely, Swiss-Prot, euKaryotic Orthologous Groups (KOG), NCBI nonredundant protein sequences (nr), KEGG Ortholog database (KO) and Gene Ontology (GO), with an E-value cut-off of 10 25 . Differentially expressed genes (DEGs) were indentified using DESeq2 package (v. 1.6.3) in R, and incorporate RSEM reads into DESeq2 using tximport [38]. Genes with at least a two-fold change (FC) (i.e. log 2 jFCj greater than or equal to 1) and a false discovery rate (FDR) less than 0.01 were considered to be differentially expressed. The GOseq R package [39] was used to implement the statistical enrichment of DEGs in the GO database, and an adjusted p-value , 0.05 was chosen as the significance cut-off.

Mating
All adults were collected from 9.00 to 11.00 every day. Then, a virgin female (1-day-old) was supplied with a newly emerged virgin male in a Petri dish (height: 1.5 cm; diameter: 8 cm) at an environmental temperature of 26 + 18C. We recorded whether and when the female exhibited mating behaviour over a period of 60 min. If multiple matings occurred in this period, we also recorded the mating times. Then, the mated females were selected and removed and then offered another newly emerged male for 60 min, and the condition of mating (e.g. whether and when mating behaviour was exhibited, and mating times) was examined. Additionally, females that mated on the first day were fed honey water (honey : water ¼ 4 : 6) on cotton balls. On days 2, 3, 4 and 5 at 11.00, these females were supplied with newly emerged males to examine mating. As a control, 2-, 3-, 4-and 5-day-old virgins were also tested.

Quantitative real-time polymerase chain reaction
Mating generally causes changes in attractiveness, that is correlated with pheromone levels [40,41]. Many studies of lepidopteran species [42] and other insect orders [43,44] have shown that pheromone biosynthesis in females is stimulated by a brain factor known as pheromone biosynthesis-activating neuropeptide (PBAN). Therefore, we aimed to test whether a change in pheromone may result in mated females becoming less attractive by evaluating the mRNA expression of PBAN between mated female and virgin through quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Total RNA was extracted from the whole bodies of mated female and virgin female adults using TRIzol (Invitrogen, USA) according to the manufacturer's protocols, and then resuspended in nuclease-free water; finally, the RNA concentration was measured using a Nanodrop (Thermo Scientific Nanodrop 2000; USA). Approximately 0.5 mg of total RNA was used as a template to synthesize the first-strand cDNA using a PrimeScript RT Reagent Kit (TaKaRa; Japan) following the manufacturer's protocols. The resultant cDNA was diluted to 0.1 mg ml 21

Egg load determination
Unmated A. disparis females and females mated with conspecific males were dissected at ages ranging from 1 to 5 days for determination of egg loads. Egg loads were measured in terms of the number of mature eggs in the ovaries [45]. Unmated individuals that emerged each day from 9.00 to 11.00. were collected. To obtain mated females, newly emerged males were supplied to a virgin female (1-dayold) for mating, and mating behaviour was observed. All female adults were fed honey water (honey : water ¼ 4 : 6) until dissection. The selected adults were subjected to sudden death at 2808C, and then the abdomens were placed into a Petri dish with a saline solution. We counted the number of mature eggs by dissecting the abdomens using forceps under a microscope (Leica M205A, Germany). In total, 15 replicates were performed for each treatment.

Statistical analysis
All analyses were performed using R software, version 2.14.1. The chi-square test was used to determine the effects of female age on the rate of mating. Prior to analysis, the raw data were tested for normality and homogeneity of variance with Kolmogorov-Smirnov and Levene's tests, respectively, and the data were transformed if necessary. The q-PCR data comparing gene expression in mated females and virgin were analysed with an independent t-test. In addition, a generalized linear mixed model (GLMM) was applied to test for the effects of mating status and female age on egg loads. For the analysis of GLMM, we used the lme4 package [46]. Egg loads were measured as response variables, with mating status and female age as fixed effects. Interactions are presented only where significant at a level of p , 0.01; this criterion for significance is recommended when testing interactions [47]. The positive/negative relationship between maternal age and egg load numbers was tested by correlation analysis.

Transcriptomic analyses
We constructed six cDNA libraries derived from three A. disparis mated female and virgin adult samples. Approximately 8.57 Gb of paired-end reads were produced for each RNAseq sample. After removing reads containing adapter sequences, poly-N reads and low-quality reads from the raw data, approximately 7.17 Gb of clean reads were obtained from each sample. The percentages of Q30 were higher than 93.62% in each sample, which showed that sequencing of each sample was of high quality.
All high-quality reads from the six samples were pooled and assembled using Trinity with the default parameters, and the TransRate score of our assembly was 0.19 (optimized score of 0.23). A total of 132 543 transcripts with lengths longer than 300 bp were generated. More than half of the transcripts (73,211, 55.23%) were longer than 1 kb in length, whereas 44.76% (13 951) were between 300 and 1000 bp in length, and the N50 size was 5020 bp. Then, these assembled transcripts were predicted to be produced from a total of 57 152 unigenes. The N50 size of the unigenes was approximately 1935 bp, Table 1. Primer pairs used for expression analysis using qRT-PCR. and their mean length was 1044.11 bp. 49.61% unigenes were between 300 and 500 bp in length, and half were longer than 500 bp (electronic supplementary material, table S1). For annotation, the pooled assembled unigenes were searched using BLASTX against five public databases with an E-value cut-off of 10 25 . A total of 28 174 unigenes were successfully annotated (table 2). Using our assembled transcriptome as a reference, we identified putatively expressed genes using the RPKM method, and genes with at least a two-FC and FDR less than 0.01 were defined as DEGs. Consequently, 55 DEGs were identified, including 5 upregulated and 50 downregulated genes in mated females (table 3). As shown in table 2, 12 genes were found in the GO database, 19 in KOG, 44 in nr, 25 in Swiss-Prot and 7 in KEGG. The upregulated genes after mating included those that encoded products that were mainly involved in fatty acid synthesis and pyrimidine metabolism. Downregulated genes were mainly involved in substance transport and metabolism (e.g. amino acids, carbohydrates and lipids).

Mating
When males were offered to virgins ranging from 1 to 5 days in age, approximately 80.42% of the virgins exhibited successful mating, which was not significantly affected by age ( Most of the mating behaviour occurred 10 min after a male was offered. After a virgin female mated with a male, she was not observed to mate again with the same male or another male. With increasing age, the mated females also ceased to exhibit mating behaviour ( figure 1). Additionally, we observed that males still fan and run towards mated females as they do virgin females.
The expression of the PBAN gene determined through qRT-PCR and RNASeq was calculated by the 2 2DDCt and RPKM methods, respectively. Results showed that the expression of the PBAN gene was not significantly different between virgin and mated females (figure 2a: qRT-PCR, t ¼ 20.71, d.f. 1

Discussion
Mating often induces behavioural and physiological changes in female insects [5]. In the egg parasitoid wasp A. disparis, post-mating females become unattractive and exhibit accelerated egg maturation (figures 1 and 3), which is accompanied by substantial changes in gene expression (table 3). A total of 55 DEGs were identified in post-mating females, and most (90.9%) of the DEGs were downregulated. Overall, the changes in gene expression prompted by mating observed in our study provide insight and useful information to improve comprehension of behavioural and physiological changes, which are discussed below. With respect to the mode of egg production, parasitoids can be classified as pro-ovigenic or synovigenic. Pro-ovigenic species mature all or most of their lifetime complement of eggs prior to emergence from hosts, whereas synovigenic species emerge with very few or no mature eggs and have to mature the eggs as they age [48,49]. Therefore, A. disparis is thought to be a synovigenic species in which the number of eggs per day (figure 3, max. ¼ 15) is significantly less than the lifetime fecundity of hundreds [23], and the number of mature eggs increases with female age. In addition, we found that as female age increased, the egg loads in mated females increased more rapidly than those in virgin females (figure 3). Gillott & Friedel [50] and Wheeler [51] reviewed 'fecundity-enhancing substances' in addition to sperm that are transferred by male insects during mating and that stimulate oogenesis, egg maturation and oviposition. Our transcriptional data indicate that increased egg loads in mated females are associated with high expression of the fatty acid synthase (FASN) gene, which encodes the enzyme catalysing fatty acid synthesis [52][53][54] and is upregulated in mated females (table 3). FAS expression has been demonstrated to be related to fecundity in insects; in Nilaparvata lugens, when FAS expression decreased, female weights, ovarian total lipids and the number of oviposited eggs also significantly decreased [55]. A similar finding showed that FAS silencing suppressed fatty acid biosynthesis and decreased fecundity in the mosquito Aedes aegypti [56]. In addition, increased egg production in mated females might require that females allocate resources royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181453 away from somatic maintenance and invest resources in reproductive processes [57], which may suggest that many genes related to metabolism exhibit changes after female mating according to our transcriptional data. For example, there were two upregulated genes involved in pyrimidine metabolism (c40539.graph_c0, c40539.graph_c1). While most of the genes were involved in the metabolism of lipids, carbohydrates and amino acids (e.g. c47166.graph_c0; c43794.graph_c0; c49861.graph_c0; c21488.graph_c0; c46393.graph_c7; c44788.graph_c0; table 3), hexamerin (c45559.graph_c0) was also downregulated, which may reflect a trade-off between reproductive and nonreproductive processes [58], likely because egg production is energetically costly and females shift from nutrient storage to utilization as their stores are depleted [59].
Polyandrous females can gain direct and indirect benefits [60][61][62][63][64]. Similar to many parasitoid wasp species (reviewed by Ridley [65]), the A. disparis females in this study also exhibited the characteristics of monandry, in which post-mating females rejected subsequent mating (figure 1). As male A. disparis mate only once, they can supply females with sufficient sperm for subsequent reproduction [23]; thus, females may refuse to mate multiple times to avoid wasting time and energy. Several studies have shown that females of some parasitoid species may re-mate if they have mated with sperm-depleted males [16,66], which will be studied further. Furthermore, during copulation, males can transfer certain chemicals with the spermatozoa [67,68], which may include toxic compounds, such as those found in Drosophila fruit flies [69], the bruchid Acanthoscelides obtectus [70] and the nematode Caenorhabditis elegans [71]. Other negative effects of multiple mating include concomitant increased vulnerability to predation, sexual diseases, parasites and pathogens [72,73]. A cytochrome P450 gene (c47989.graph_c4) was found to be upregulated by mating in females, which may be involved in detoxification [74]. In addition, the post-mating expression levels of four protease genes change, among which predicted serine protease genes (c43486.graph_c0) and a chymotrypsin gene (c48890.graph_c0) were downregulated after mating. Induced proteases in virgin female could protect females from harmful proteins introduced during mating [58]. Females receive sperm from their mates, then maintain the sperm in storage organs to await opportunities for fertilization. A serine protease inhibitor (c49077.graph_c0) was downregulated after mating, which may play a role in protecting sperm from degradation or expose sperm surface proteins needed for storage or fertilization [58].
Females may cease to attract males after mating resulting in mating only once [75]. It has been shown that mating generally causes changes in attractiveness in many species of moths and parasitoids, which are correlated with pheromone levels [41,77,76]. However, as shown in Spalangia endius [78], we observed that males fan and run towards mated females, and our q-PCR results (figure 2a) and transcriptional analyses (figure 2b) showed that expression of the PBAN gene was not significantly different between virgin and mated females. This finding suggested that because the production of attractants may not cease or decrease after mating, mating might not cause changes in the attractiveness of females to males, and the mating of A. disparis females only once may therefore be unlikely to be caused by lower attractiveness of mated females (also see Cotesia flavipes [79]). Besides, odorant-binding proteins (OBPs) are a class of olfactory proteins and are thought to aid in the capture and transport of odorants and pheromones to receptors [80]. In fruit flies, OBP expression levels in females changed significantly after mating [81], and ectopic expression of Obp99b in female fat body tissue led to reduced receptivity and mating success [82]. Our transcriptome data showed that a total of three royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181453 annotated genes associated with OBPs were downregulated in A. disparis females after mating (table 3; c41154.graph_c0; c29056.graph_c0; c21285.graph_c0), which may explain why mated females become unreceptive to further mating. In addition, our transcriptional data also showed that four genes (c40480.graph_c0; c41422.graph_c0; c44319.graph_c1; c32591.graph_c0) that are expressed in the adult eye or are known to function in visual transduction (including opsin, rhodopsin and carcinine transporter [59,83]; table 3) were downregulated after mating (see also the honeybee and Apis florae [84,85]). Altered expression of vision genes could impact a female's response to other females or males [83]. Therefore, the downregulation of vision-related genes after mating in our species may also influence the re-mating behaviour of females. Rather than a change in female pheromone related attractive, our results suggested that decreased visual and odorant-binding abilities also resulted in mated females becoming unreceptive and refusing to mate again.
In addition, as shown in other studies [58,59], other genes in our study, for example, involved in chitin metabolism (c43794.graph_c0; c21488.graph_c0), signal transduction (c41137.graph_c0; c42015.graph_c0), that exhibit ectopic expression after mating involved in post-mating behavioural and physiological responses, while those with unknown or unclear function require further study. By identifying changes in gene expression prompted by mating, our study provided new insights into changes in behavioural and physiological aspects. Simultaneously, this dataset provides a basis for future mechanistic studies examining how specific genes mediate behavioural and physiological changes in females post-mating. Additionally, understanding how these changes in gene expression orchestrate the post-mating response in this species may provide insight into the reproductive behaviour of more complex animals.
Ethics. There was no requirement to seek ethical approval to carry out the work described above. However, the use of insects in the above experiments was kept to a minimum.
Data accessibility. The datasets supporting this article have been uploaded as part of the electronic supplementary material. All raw sequence data files are available from the Sequence Read Archive database (accession no. PRJNA505044).