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Functional transcriptome analyses of Drosophila suzukii midgut reveal mating-dependent reproductive plasticity in females

BMC Genomics volume 23, Article number: 726 (2022) Cite this article

Abstract

Background

Insect females undergo a huge transition in energy homeostasis after mating to compensate for nutrient investment during reproduction. To manage with this shift in metabolism, mated females experience extensive morphological, behavioral and physiological changes, including increased food intake and altered digestive processes. However, the mechanisms by which the digestive system responds to mating in females remain barely characterized. Here we performed transcriptomic analysis of the main digestive organ, the midgut, to investigate how gene expression varies with female mating status in Drosophila suzukii, a destructive and invasive soft fruit pest.

Results

We sequenced 15,275 unique genes with an average length of 1,467 bp. In total, 652 differentially expressed genes (DEGs) were detected between virgin and mated D. suzukii female midgut libraries. The DEGs were functionally annotated utilizing the GO and KEGG pathway annotation methods. Our results showed that the major GO terms associated with the DEGs from the virgin versus mated female midgut were largely appointed to the metabolic process, response to stimulus and immune system process. We obtained a mass of protein and lipid metabolism genes which were up-regulated and carbohydrate metabolism and immune-related genes which were down-regulated at different time points after mating in female midgut by qRT-PCR. These changes in metabolism and immunity may help supply the female with the nutrients and energy required to sustain egg production.

Conclusion

Our study characterizes the transcriptional mechanisms driven by mating in the D. suzukii female midgut. Identification and characterization of the DEGs between virgin and mated females midgut will not only be crucial to better understand molecular research related to intestine plasticity during reproduction, but may also provide abundant target genes for the development of effective and ecofriendly pest control strategies against this economically important species.

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Introduction

Reproduction is an energetically costly process which induces numerous physiological and functional adaptations in the gastrointestinal tract during pregnancy and lactation in multifarious species [1, 2]. Female individuals invest more energy and resources than male individuals into reproduction, and restructure energy balance to maximise their reproductive success. These shifts fulfill sufficient nutrient intake to the increasing energy demands in females [2]. In insects, such as the fruit fly Drosophila melanogaster, dietary protein is required for yolk protein synthesis, and changing female’ s protein intake can affect her fecundity [3,4,5,6,7,8,9]. Moreover, mated females enhance feeding and consume energy and protein rich diets preferentially to support the metabolic needs of oviposition [10,11,12,13,14,15]. As the major place of digestion and nutrient absorption, the female midgut is a crucial regulator of alterations in post-mating energy balance, and signals between midgut and ovary are critical for raising egg production after mating [16,17,18,19]. The adult D. melanogaster intestine is a plastic organ, and the female midgut undergoes striking remodeling in size and physiology by stimulating intestinal stem cell (ISC) driven epithelial expansion in responding to mating [17, 20,21,22].

The Drosophila midgut is sexually dimorphic which is reflected in the differences of physiology and gene expression [17, 20, 23, 24]. Cell division-related processes genes and carbohydrate metabolism genes are abundantly expressed in females and males respectively [23]. Compared to the males, ISC proliferation is higher in virgin females and this sex difference is further improved by mating, since ISC proliferation is significantly higher in mated females than in virgin females [17, 23, 25]. Furthermore, the midgut in virgin and mated females are morphologically and physiologically diverse [17, 20]. Mated females change defecation frequency, fecal pH and water content of the gut, and this mating responsiveness is crucial for regulating female post-mating nutrient absorption and egg production [17, 19, 20]. As food passes through the guts of mated females more slowly, this allows more time for nutrient absorption and thus more concentrated excreta [20]. The mated D. melanogaster female midgut also can accelerate gametogenesis by releasing enteroendocrine cells (EECs)-derived Neuropeptide F [17, 18]. Besides, genes involved in fatty acid metabolism are up-regulated in enterocytes (ECs) after mating in D. melanogaster, which may facilitate fecundity in females [17]. Post-mating transcriptomes in Anopheles coluzzii female midguts also showed that sugar transport, metabolism, and innate immune response genes were expressed inductively [26].

During mating, males transfer seminal fluid proteins that trigger the switch between virgin and mated female states. The transition involves a series of molecular, morphological, behavioral and physiological changes, and occurs in rapid and sustained phases [27,28,29,30,31]. Short-term post-mating responses occur during the first 24 h, while long-term post-mating responses can last up to two weeks after mating [27,28,29,30, 32]. The female post-mating response includes increased egg production and food intake, changes in food preference, decreased receptivity to remating, and diminished immune response [10,11,12,13, 28, 29, 33,34,35,36,37,38]. Male-derived Sex Peptide (SP), actings through Sex Peptide receptor (SPR) neurons in the female reproductive tract, has been connected to increases in intestinal transit time and stimulation of Neuropeptide F release from EECs in D. melanogaster midgut, and thus enhanced nutrition and fecundity [18, 20, 39, 40]. Mating significantly facilitates intestine growth specifically in females and enhances reproductive output as a result of juvenile hormone and ecdysone promoted ISC proliferation [17, 19, 22]. Despite the important connection between nutrition, gut physiology, and mating, little is known about the integrative and coordinated process involving numerous transcriptional changes triggered by mating in the female midgut and which processes are modulated to adjust midgut size and digestion to the demands of egg production.

The spotted wing Drosophila, Drosophila suzukii (Matsumura), is a global devastating and invasive agricultural pest that invaded Europe and the Americas. It causes severe economic loss due to damage to a wide variety of fruit crops such as waxberry, blueberries, strawberries, peaches, cherries, persimmon, and grapes. D. suzukii poses a huge threat to commercial soft fruit production and security due to its polyphagy, adaptability and robust fecundity [41,42,43,44,45,46,47]. Unlike the majority of Drosophila species, such as D. melanogaster, who oviposit on overripe fruit, D. suzukii lays eggs in healthy and undamaged ripening fruit, destroying crops through the rot and abscission of fruits. The infestation of D. suzukii female to ripe fruits is facilitated by the presence of a sclerotized and serrated ovipositor that enables piercing intact fruit skin and deposition of eggs into ripe fruits [44, 48, 49]. Chemical insecticides are currently considered as the most effective tool to control D. suzukii. However, the increase in resistance to commonly applied chemical insecticides necessitates the development of environment friendly pest management strategies [41, 50, 51]. Population replacement control strategies that utilize genetically modified pests show promise for integrated pest control, and these approaches are dependent on successful mating and reproduction [52,53,54]. Establishment of a positive energy homeostasis may be particularly important to D. suzukii that involves rapid production of large numbers of progeny, and the intestine plasticity may play significant role in this process during reproduction. Thus, identification of genes driven by mating in the D. suzukii female midgut will provide important insights for the development of novel approaches to control this pest by targeting the reproduction.

In the present study, the transcriptome of virgin and mated female D. suzukii midguts were sequenced using the Illumina HiSeq 2500 system. Furthermore, analysis of the differentially expressed genes between virgin and mated female midgut was carried out to identify the potential genes that respond to mating. The expression levels of the genes involved in the post-mating response at different time points were analyzed by qRT-PCR.

Results

Sequencing and assembly of virgin and mated female midgut transcriptomes

First, we examined the changes in midgut lengths at 1 d, 2 d and 3 d after mating and the midgut length was significantly longer than that of virgin controls at each time point in female D. suzukii (P < 0.001) (Fig. 1A and 1B and Figure S1). The average midgut length was 5614.25, 6102.75, 5529 um in the mated female midgut compared to the the average midgut length of 4406.5, 5048, 4612.25 um from the virgin controls at 1 d, 2 d and 3 d after mating respectively (Fig. 1A and Figure S1). The visibly longer and larger midgut phenotypes were observed under brightfield conditions at each time point after mating (Fig. 1B and Figure S1). This midgut enlargement in female D. suzukii is in accordance with the time frame of SP-mediated post-mating responses, such as increased egg production and reduced receptivity to remating, which persists for ten days [28, 33]. We also found that mating increases the number of intestinal cells significantly and leads to a visibly larger midgut diameter as revealed by DAPI stainings in female D. suzukii (Fig. 1C and Figure S1 and S2). To ascertain the impact of mating on digestive physiology, we characterized the transcriptome of whole midguts of virgin females, and females mated to males at 2 d post-mating. Transcriptome libraries of the virgin and mated female midguts were constructed and sequenced in the Illumina platform using paired-end sequencing. This generated a total of 262.92 million reads with high sequence quality (BioProject accession number: PRJNA827258). After removing low-quality reads, virgin female midgut libraries generated 43.13, 42.96 and 43.09 million clean reads while mated female midgut libraries generated 43.08, 43.18 and 43.00 million clean reads. Among these clean reads, 22.47–23.40 million (52.16%-54.20%), were mapped to genes in the whole genome sequence (WGS) of D. suzukii (Supplementary Table S1). The percentage of clean reads ranged from 98.03% to 98.43% in virgin female midgut libraries and 98.13% to 98.53% in mated female midgut libraries (Figure S3). The filtered sequence reads from all samples were assembled and produced 17,452 unigenes, which the total length, mean length, N50 and GC contents were 25,611,966 bp, 1,467 bp, 2,448 bp and 50.27%, respectively (Table 1). Gene sequences were annotated by searching the nonredundant NCBI protein database using BLASTX. A total of 15,275 unigenes (87.53%) were matched to known genes, among which 12,609 unigenes had the complete coding sequence (CDS) and 1,730 unigenes encoding transcription factor were predicted (Supplementary Table S2). Most of these sequences (84.03%) showed strong similarity to those of Drosophila species. Amongst them, 65.68% of these sequences best matched sequences from D. suzukii, followed by D. biarmipes (11.33%), D. takahashii (3.19%), D. melanogaster (2.45%), D. elegans (1.37%), and other insect species (15.97%) (Fig. 2). The average unigene size was 1,467 bp with lengths ranging from 200 to 25,224 bp (Table 1). There were 8,415, 4,299 and 2,232 genes whose length was larger than 1,000, 2,000, and 3,000 bp, respectively (Table 1 and Figure S4). The total analysis of transcriptome sequences suggested that the assembly quality was high and the data accuracy was reliable in D. suzukii.

Fig. 1
figure 1

Post-mating change in midgut two days after mating in female D. suzukii. A Midgut length quantifications. The unit of length is um. Error bars indicate the SEM of three independent biological replicates and asterisks (***) indicate the statistically significant differences (P < 0.001) between virgin and mated female midgut based on Student’ s t-test. B Representative images of virgin and mated female midgut phenotypes. The scale bar is 500 um. c Changes in midgut cell proliferation revealed by DAPI staining. The scale bar is 20 um. The region of the midgut is anterior midgut

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Table 1 Summary of the virgin and mated D. suzukii female midgut transcriptomes
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Fig. 2
figure 2

Species distribution of unigenes in the virgin and mated female midgut transcriptomes of D. suzukii

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Comparison of gene expression profiles in virgin and mated female midgut

To assess the relative expression level of genes in the D. suzukii virgin and mated female midgut transcriptomes, we normalized the gene read counts by transforming them into Fragments Per Kilobase of transcript per Million mapped reads (FPKM). A broad extent of gene expression levels from less than 1 FPKM to 62,092 FPKM were obtained (Supplementary Table S2). 13.85% to 16.45% of the genes had a low expression level (FPKM <  = 1), 56.15% to 58.50% of the genes had a moderate expression level (FPKM 1–10), and 27.37% to 27.65% exhibited a high expression level (FPKM >  = 10) in the virgin female midgut libraries. While 16.32% to 31.58% of the genes had a low expression level (FPKM <  = 1), 46.46% to 57.34% of the genes had a moderate expression level (FPKM 1–10), and 21.96% to 26.34% exhibited a high expression level (FPKM >  = 10) in the mated female midgut libraries (Fig. 3). Principal components analysis of all six samples showed that both virgin and mated female midgut samples clustered together with their respective replicates (Fig. 4).

Fig. 3
figure 3

Fragments per kilobase of transcript per million mapped reads (FPKM) distribution of the D. suzukii virgin and mated female midgut transcriptomes. 1, 2, 3 represent the virgin female midgut samples and 4, 5, 6 represent the mated female midgut samples

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Fig. 4
figure 4

Principal component analysis of the six transcriptome samples. Samples are virgin female midgut (red) and mated female midgut (blue)

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We further identified differentially expressed genes (DEGs) between virgin and mated female midgut. At a setting of p < 0.05, 652 DEGs were identified. 400 DEGs exhibited relatively higher expression levels in virgin female midgut than mated female midgut, and 252 DEGs showed relatively higher expression levels in mated female midgut than virgin female midgut (Fig. 5 and Supplementary Table S3). We then analyzed the functions of DEGs based on gene ontology (GO) classification. 652 DEGs were characterized into three groups: 236, 263 and 299 DEGs categorised into biological processes, cellular components, and molecular function, respectively (Fig. 6 and Supplementary Table S4). The largest representations were in cellular processes and metabolic processes (biological processes), cellular anatomical entity and intracellular (cellular component) and binding and catalytic activity (molecular function). In addition, enrichment comparisons showed that the cellular process, metabolic process, biological regulation, response to stimulus, developmental process and immune system process were included during mating in the biological processes (Fig. 6). Furthermore, enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for the DEGs was also performed (Fig. 7 and Supplementary Table S5). The results showed that metabolism pathways were the main groups, including carbohydrate metabolism, lipid metabolism, amino acid metabolism, metabolism of cofactors and vitamins, glycan biosynthesis and metabolism, and energy metabolism (Fig. 7).

Fig. 5
figure 5

Volcano plot showing differentially expressed unigenes (|log2(FoldChange)|> 0 and padj < 0.05) in virgin and mated female midgut transcriptomes. Genes shown on top right corner are up-regulated in virgin female midgut and genes shown on top left corner are up-regulated in mated female midgut. The orange, dark and green dots indicate the genes involved in carbohydrate metabolism, lipid metabolism and amino acid metabolism, respectively. The pink dots indicate the genes involved in carbohydrate metabolism and lipid metabolism simultaneously. The violet dots indicate the genes involved in carbohydrate metabolism and amino acid metabolism simultaneously. The brown dots indicate the genes involved in carbohydrate metabolism, lipid metabolism and amino acid metabolism simultaneously

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Fig. 6
figure 6

Gene Ontology (GO) significant enrichment analysis for differentially expressed genes (DEGs) between virgin and mated female midgut transcriptomes of D. suzukii

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Fig. 7
figure 7

Kyoto Encyclopedia of Genes and Genomes (KEGG) significant enrichment analysis for differentially expressed genes (DEGs) between virgin and mated female midgut transcriptomes of D. suzukii

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Transcriptional metabolism and immune changes of post-mating response in female midgut

To identify the influence of mating on the dynamic responses in female midgut, we sequenced the transcriptome of virgin female midguts and 2 d post-mating female midguts. Differential expression analysis disclosed 652 genes were differentially expressed in the midgut between virgin females and 2 d post-mating females (Supplementary Table S3), among which 252 and 400 genes were up-regulated and down-regulated respectively. We identified the up-regulated protein and lipid metabolism genes and down-regulated carbohydrate metabolism genes upon mating (Supplementary Table S3). To further validate numerous metabolism-related genes with respect to expression at different time points after mating, they were analyzed by quantitative Real-Time PCR (qRT-PCR). The results showed that the Jonah family of proteases, Jonah 66Cii (Jon66Cii) gene, the trypsin family protease Trypsin (Try) gene, the SLC family of transporters Vesicular glutamate transporter (VGlut) gene and the amino acid transporters Nutrient Amino Acid Transporter 1 (NAAT1) gene were highly expressed in 24 and 48 h mated female midgut compared to the virgin control female midgut (Fig. 8). We also found up-regulation of genes involved in fatty acid and lipid metabolism (Fig. 8). For instance, Sterol regulatory element binding protein (SREBP), Lipin, lipase 1 (lip1), lipase 3 (lip3) were highly expressed in 24 and 48 h mated female midgut. Consistent with this up-regulation of lipid genes, female flies accumulated neutral lipid content revealed by Bodipy staining after 48 h mating in the midgut of D. suzukii (Fig. 9 and Figure S5). Meanwhile, we examined the down-regulated genes upon mating to males and found an enrichment of genes related to carbohydrate metabolism (Fig. 8). Carbohydrate metabolism genes such as maltase A1 (Mal-A1), Phosphoglucose isomerase (Pgi), glucose dehydrogenase (GDH), Trehalase (Treh) were significantly down-regulated in 24 and 48 h mated female midgut compared to the virgin control. Altogether, these data suggest that mating induces a shift in midgut gene expression from carbohydrate metabolism to protein and lipid metabolism in D. suzukii.

Fig. 8
figure 8

The effect of mating on the expression levels of metabolism-related genes was detected by qRT-PCR in the midgut of D. suzukii females. RNA was extracted from the midgut of mature vigin females as well as mated females at 24, 48 h after mating. Error bars indicate the SEM of three independent biological replicates and various letters represent statistically significant differences of the expression level of genes (p < 0.05, Student’ s t-test)

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Fig. 9
figure 9

Mating increases neutral lipid content revealed by Bodipy staining in the virgin and mated female midgut of D. suzukii. The scale bar is 20 um. The region of the midgut is anterior midgut

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To analyze the mating-induced changes in immunity in D. suzukii virgin and mated female midgut transcriptomes, we identified multiple immune-related genes that were differentially expressed between virgin and mated female midgut (Supplementary Table S3). In order to explore the complicated post-mating response in immunity, the expression profiles of numerous immune-related genes in the female midgut at 24 and 48 h after mating were checked by qRT-PCR. The results showed that the positive regulators of the immune deficiency (IMD) pathway genes Imd, peptidoglycan-recognition protein LE (PGRP-LE), the antimicrobial peptides (AMPs) genes defensin (def), and the Toll signaling pathway genes Toll, Tube, Dorsal were down-regulated in the 24 and 48 h mated female midgut compared to the virgin control female midgut (Fig. 10). While the negative regulators of the IMD pathway genes peptidoglycan-recognition protein SC1a (PGRP-SC1a), peptidoglycan-recognition protein LB (PGRP-LB), peptidoglycan-recognition protein LF (PGRP-LF) were up-regulated in the mated female midgut (Fig. 10). All the qRT-PCR results of metabolism-related and immune-related genes were consistent with our deep sequencing data, which indicated that the current analysis is accurate. For functional evidence of immune suppression induced by mating in D. suzukii, we evaluated the differences in survivorship between virgin females and mated females that were all infected with the bacterium, Providencia rettgeri, 48 h after mating. The results showed that mated females had significantly lower survival rate than virgin females during the experimental process when infected with P. rettgeri in D. suzukii, while the virgin and mated females suffered similar low mortality rates when infected with PBS (Fig. 11). Higher levels of infection-induced mortality in mated females may indicated that there was a trade-off between reproduction and immunity in D. suzukii.

Fig. 10
figure 10

The effect of mating on the expression levels of immune-related genes was detected by qRT-PCR in the midgut of D. suzukii females. RNA was extracted from the midgut of mature vigin females as well as mated females at 24 h and 48 h after mating. Error bars indicate the SEM of three independent biological replicates and various letters represent statistically significant differences of the expression level of genes (p < 0.05, Student’ s t-test)

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Fig. 11
figure 11

The effect of mating on immune defense after mating in D. suzukii. Survival over time of mated females was significantly lower than that of virgin controls when infected with P. rettgeri. Mortality was recorded each day for six days after infection, and survival curves were estimated using the Kaplan–Meier method. Control flies had negligible mortality over the course of the experiment regardless of mating treatment. ***p < 0.001

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Discussion

In this study, we performed a genome-wide transcriptomic analysis of the main digestive organ, the midgut, to investigate how mating status trigger shifts of gene expression in female midgut of D. suzukii. A total of 652 DEGs were identified, among which the expression pattern of 252 DEGs were up-regulated in mated female midgut and 400 DEGs were up-regulated in virgin female midgut. Differential expression profiles of transcripts involved in the metabolism and immune processes at different time points after mating were validated by qRT-PCR. As the first analysis of transcriptional mechanisms driven by mating in D. suzukii female midgut, this dataset not only provides a valuable resource necessary for a better understanding of the genetic basis of intestine plasticity during reproduction, but also provides abundant target genes for effective control of this agricultural pest.

Production of offspring requires significant energy investments and involves dramatic metabolic adaptations to enhanced energy demands in females. D. melanogaster increases egg production tenfold after mating and triggers multiple metabolic and behavioural adaptations such as food intake, digestion, and nutrient preference [10, 11, 17, 20, 28, 30, 55]. These shifts are largely induced by signals delivered by the male during copulation, in particular the seminal fluid molecule SP [28, 30]. In our study, we found that the midgut enlargement is detected at 1 d, 2 d and 3 d post-mating females of D. suzukii which is similar to the situation in D. melanogaster [17, 22]. This midgut enlargement is consistent with the time frame of SP-mediated post-mating responses, for instance increased egg production and reduced receptivity to remating which persists for 10 d [28, 33]. Besides, we have also identified SPR in the midgut with low expression, indicating the possibility that SP could act directly on the SPR in gut to stimulate post-mating gut growth. Across diverse studies of mating-regulation of gene expression in whole females or different female tissues such as midgut, spermathecae, head/brain, and reproductive tract, metabolic processes are the main shifts induced by mating in multiple insect species, for example, Aedes aegypti, Anastatus disparis, Anopheles gambiae, Apis mellifera, Bactrocera dorsalis, Callosobruchus maculatus, D. melanogaster [26, 56,57,58,59,60,61,62,63,64,65,66,67,68]. We observed down-regulation of genes involved in carbohydrate metabolism and up-regulation of genes involved in protein digestion and lipid metabolism in mated female midgut compared to virgin female midgut in D. suzukii, which show the similar mating-induced gene expression changes to D. melanogaster reported previously [22, 67,68,69]. Pgi, Mal-A1, GDH and Treh were down-regulation of genes involved in carbohydrate metabolism in mated D. suzukii female midgut, which are consistent with the observations of down-regulation of maltase genes in the whole-organism transcriptome of 3 to 5 d-old mated D. melanogaster females [69] and down-regulation of carbohydrate metabolic genes in D. melanogaster female abdomens at 3 h post-mating and in D. melanogaster female midgut at 2 days after mating [22, 70]. Jon66Cii, Try, VGlut, NAAT1 were up-regulated genes involved in protein digestion and SREBP, Lipin, lipase were up-regulated genes involved in lipid metabolism in mated D. suzukii female midgut, which are consistent with the observations of post-mating up-regulation of Jonah family serine-type endopeptidase genes in D. melanogaster [68, 69] and up-regulation of several proteases in female and proteolysis-related genes in female abdomens 3 h after mating [67, 70]. The accumulation of neutral lipid content revealed by Bodipy staining after 48 h mating confirmed the up-regulation of lipid genes in mated female midgut of D. suzukii. We detected up-regulation of SREBP in the midgut after mating by qRT-PCR, which is consistent with the observation that the expression of fatty acid metabolic genes is induced upon mating [17, 22]. The lipid biosynthesis pathway is significantly up-regulated by mating in A. ludens and genes involved in fatty acid synthesis are up-regulated by mating in both A. ludens and Anastatus disparis [71, 72]. We also found down-regulation of GstS1 which is involved in detoxification, and this mating induced down-regulation could affect the capacity of the female to cope with toxic dietary foods and oxidative stress [22, 73]. All these results demonstrated that mating could induce an increase in protein and lipid digestion in female insects, which is consistent with increased protein and lipid food intake after mating, which is necessary for yolk protein production and female fecundity [9, 74]. The up-regulation of protein and lipid metabolic genes and the coincident down-regulation of carbohydrate metabolic genes in mated D. suzukii female midgut may reflect the fact that females alter digestive parameters to adapt to new nutritional demands.

Reproduction and immunity are physiologically and energetically fastidious courses and the trade-off between these two crucial processes exists in numerous insects [38]. The trade-off between reproduction and immunity are driven by resource-allocation. The more biological resources insects invest in reproductive capacity, the lower the immune function, and vice versa. Indeed, many studies have documented trade-offs between reproduction and immunity in a diversity of insects. In the ground cricket, Allonemobius socius, reduced hemocyte number, encapsulation ability and lytic activity with increasing mating effort, results in an increased mortality rate of both sexes [75]. Increasing mating success led to a reduction in phenoloxidase activity and consequent immune suppression in mealworm beetles, Tenebrio molitor, and wood ants, Formica paralugubris [76, 77]. Besides, sperm viability is negatively correlated with immunity such as encapsulation ability and lysozyme in leaf-cutting ant queens and honey bee queens [78, 79], and this negative relationship between sperm viability and immune function is widespread in various male insects [80,81,82,83,84,85]. In our study, the positive regulators of the IMD pathway genes Imd, PGRP-LE, the AMPs genes def, and the Toll signaling pathway genes Toll, Tube, Dorsal were down-regulated in mated D. suzukii female midgut. While the negative regulators of the IMD pathway genes PGRP-SC1a, PGRP-LB, PGRP-LF were up-regulated in mated female midgut at different time points. All these results showed that mating induces a reduction of immunity in D. suzukii female midgut at 24 h and 48 h post-mating. The expression level of AMP gene def is up-regulated after mating in Ae. aegypti, Atta colombica, B. dorsalis, Ceratitis capitata, D. melanogaster, and Lasius niger [56, 57, 67, 86,87,88], and is down-regulated after mating in A. mellifera [62]. However, this is different from the results of the previous report on C. capitata, which revealed a large reduction in def expression after mating in the female abdomen [87]. Mating also reduces the survival rate of female D. melanogaster under all kinds of pathogenic infections, and mated females have higher pathogen loads and reduced expression level of AMP genes after pathogenic infection [86, 89, 90]. The expression level of def and other AMP genes were down-regulated and up-regulated in mated females compared with virgin females at 12 h and 24 h post-infection in D. melanogaster, respectively [86]. The expression differences among these genes indicated that mated females were vulnerable to adequate defence against bacterial infection than virgin females in D. melanogaster [90]. More studies are needed to elucidate the complex relationship between the expression level of AMP genes and female immune response at different post-infection time points after mating. We have discovered that mating reduces survivorship after infection in D. suzukii similar to the studies described previously [38, 86, 89]. Interestingly, some studies reported that the expression level of AMP genes were induced by mating in different tissues [67, 91,92,93,94], which conflict with results showing that mating reduces female resistance to infection in D. melanogaster. However, this up-regulation of AMP genes were mainly confined to the reproductive tract [93, 94], and this tissue-specific up-regulation may be a result of a regional defence against sexually transfered infection [95, 96] that may barely affect the immune process in defence of systemic infection. A previous study has revealed that juvenile hormone (JH) prevented autoimmunity in D. melanogaster reproductive tissues by suppressing immune signaling to support reproductive output [97]. JH also increased reproductive output via raised lipid metabolism [17], and sterile D. melanogaster females were resistant to the impact of mating on immunity [86]. Besides, ecdysone signalling promoted intestinal growth, particularly in mated D. melanogaster females, and facilitated fecundity [19]. Thus, more work should address the exploration of the complicated relationships among hormone signalling, immunity and reproduction in mated female insects in the future.

Conclusion

This study demonstrates post-mating modulation at the transcriptional level of genes involved in the midgut of D. suzukii, a destructive and invasive soft fruit pest. Mating causes a shift in the transcriptome of midgut, and the post-mating midgut increased transcription of genes involved in lipid and protein metabolism, while decreasing mRNA levels of carbohydrate metabolism genes and immune-related genes. All these shifts may help the female meet the energetic demands of egg production. Thus, the identification of genes between virgin and mated females midgut will not only be crucial to a better understanding of molecular research related to intestine plasticity during reproduction, but also provide abundant target genes for the development of effective and ecofriendly pest control strategies.

Materials and methods

Insect rearing

D. suzukii were fed on an artificial diet consisting of cornmeal, yeast, soy flour, maltose syrup and agar at Hunan Normal University (Changsha, China) and cultured at 25 Â°C under 12 h light: 12 h dark photoperiod [98]. Methyl 4-hydroxybenzoate was used as a preservative to prevent the overgrowth of fungi and bacteria. The diet has been optimized and has no negative fitness on D. suzukii development, survival and fecundity which is described in a previous study [99]. Newly emerged virgin females and males were collected and sorted separately. Mature females and males were kept in standard Drosophila vials together for mating (ten females and ten males per vial). Midguts were dissected from two days after female mating and at the same age as virgin females.

Midgut length measurements and stainings

Virgin females and males were collected and single-pair matings were conducted three days after eclosion. Matings were monitored within 5–30 min and females who mated for less than 15 min were discarded. After mating, females were aged in groups of 10 in new food vials. Age-matched virgin female control groups were also maintained. Guts were dissected in phosphate-buffered saline (PBS) at the stated time points (1 d, 2 d,3 d after mating) and fixed with 4% paraformaldehyde solution in PBS for 60 min. Samples were stained with DAPI and Bodipy in PBS-0.01% Triton X-100 (1:50,000; Sigma Aldrich) and mounted on slides. All staining images were obtained using a Zeiss AxioImager M2 fluorescence inverted microscope. The whole midgut images were obtained under brightfield and analysed quantitatively using ImageJ. Brightfield images and staining images were loaded into ImageJ and the line tool used to outline the midguts. The region of interest (ROI) was analyzed using the polygon tool of ImageJ. Maximum projections were adjusted for levels and offsets and filtered to remove noise. The same parameters for scans were used within one experiment. The integrated density of fluorescence was quantified by multiplying their relative intensity and the area in the relevant channel. Quantification of cell numbers in the midgut of virgin and mated females was carried out by counting individual nuclei marked by the DAPI through a 40 × objective. The length of spline curve drawn down the midline was regarded as the midgut length.

RNA extraction and transcriptome sequencing

Midguts of the same age virgin and mated females were collected with three independently biological replicates, and each replicate contained 60 midguts. Total RNA was extracted from midguts of D. suzukii using Trizol (Invitrogen, Carlsbad, CA, USA) according to manual instruction. The degradation and contamination of all RNA samples was checked on a 1.0% agarose gel, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). The library construction for Illumina sequencing was conducted with a total amount of 1 Î¼g RNA from midguts of virgin and mated females by using NEBNext® Ultraâ„¢ RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s instructions. Sample sequencing was conducted on the HiSeq4000 platform using paired-end (PE) technology.

Transcript sequence analysis

Raw reads of fastq format was filtered with SOAPnuke (v1.5.2). Clean reads were generated by removing reads containing the sequencing adapter, reads containing ploy-N and low-quality reads from raw datasets. The reads whose low-quality base ratio (base quality less than or equal to 5) was more than 20% were removed. Meanwhile, Q20, Q30 and GC content of the clean data were calculated. All the subsequent analyses were based on the clean reads with high quality. The clean reads were mapped to the whole genome sequence (WGS) of D. suzukii using HISAT2 (v2.0.4) (https://www.ncbi.nlm.nih.gov/genome/?term=txid28584) [100, 101]. The discordant or unpaired alignments were discarded. The mapped reads were assembled by StringTie (v1.3.3b) in a reference-based method. The read numbers mapped to each gene were counted using HTSeq v0.9.1 and each gene expression level was further calculated by FPKM (Fragment Per Kilobase of exon model per million mapped reads) based on the gene length and the gene read counts. Due to the effect of sequencing depth and gene length for the read counts, both were taken into consideration, and the FPKM is currently the most generally utilized approach for evaluating gene expression levels [102, 103].

Differential expression and GO, KEGG enrichment analysis

Differential expression analysis of virgin and mated female midgut libraries was accomplished utilizing the DESeq2 R package (1.16.1). The resulting P-values were corrected using the Benjamini and Hochberg’s method for managing the false discovery rate (FDR). Genes with an corrected P-value < 0.05 found by DESeq2 were appointed as differentially expressed genes (DEGs) [98]. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs was performed by Phyper based on Hypergeometric test [104]. The significant levels of terms and pathways were corrected by P-value with a rigorous threshold (P-value < 0.05) by Bonferroni [98].

Quantitative real-time PCR

The expression profiles of metabolism and immune-related genes were surveyed using quantitative Real-Time PCR (qRT-PCR). Total RNA was extracted using RNAiso Plus reagent (TaKaRa, Dalian, China) from 60 midguts per replicate, with 200 ng for each sample subjected to reverse transcription utilizing the PrimeScriptâ„¢ RT Master Mix (TaKaRa, Dalian, China). The reverse transcription products were subsequent utilized for qRT-PCR using primers listed in Supplementary Table S6. qRT-PCR was implemented using the SYBR Green qRT-PCR mix following the manufacturer’s instructions in a real-time thermal cycler (Bio-Rad, Hercules, CA, USA) utilizing the cycling conditions: 95 Â°C for 10 min, 40 cycles of 95 Â°C for 15 s, 60 Â°C for 30 s and 72 Â°C for 30 s. Three biological and three technical replicates were accomplished with expression data analyzed by the 2−△△Ct approach [105]. Dissociation curves were determined for each gene to confirm unique amplification. The expression of ribosomal protein 49 (Rp49) was utilized as an internal control to normalize gene expression.

Survival assays

Virgin females and males were collected and housed in groups of 10 individuals and 30 individuals, respectively. Three days after eclosion, females and males were transferred to new food vials for the copulation and this process was monitored within 5–30 min to ensure all females were typically paired with males. The Gram-negative bacterium, Providencia rettgeri, was used for the infection experiments. CO2-anesthetized females at 48 h after mating and the same age virgin females were injected with P. rettgeri cultures (OD600 = 7) in the thorax using a pulled capillary needle mounted on a Nanoject II apparatus (Drummond Scientific). Females were placed into new food vials in groups of 10 immediately after infection. Females that did not recover from the injection within 8 h were removed as their death was due to experimental handling rather than infection. Survival was recorded daily for 6 days and the PBS-injected females rarely resulted in mortality (< 1%).

Statistical analysis

All experiments were repeated in triplicate and analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) or Microsoft Excel (Microsoft, Redmond, WA, USA) with results expressed as the mean ± SEM. Data was compared with either a two-way ANOVA, with subsequent t tests using Bonferroni post-tests for multiple comparisons, or with the Student’s t test. For all tests, differences were considered significant when P < 0.05.

Availability of data and materials

All the RNA-sequencing reads have been deposited in the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA827258) with the accession codes (BioProject accession number: PRJNA827258).

Abbreviations

DEGs:

Differentially expressed genes

RNA-seq:

RNA sequencing

WGS:

Whole genome sequence

FPKM:

Fragments Per Kilobase of transcript per Million mapped reads

ISC:

Intestinal stem cell

GO:

Gene ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

EEC:

Enteroendocrine cell

EC:

Enterocytes

SP:

Sex Peptide

SPR:

Sex Peptide receptor

CDS:

Complete coding sequence

PGRPs:

Peptidoglycan recognition proteins

Imd:

Immune deficiency

AMPs:

Antimicrobial peptides

PBS:

Phosphate-buffered saline

FDR:

False discovery rate

Rp49:

Ribosomal protein 49

References

  1. Hammond KA. Adaptation of the maternal intestine duringlactation. J Mammary Gland Biol Neoplasia. 1997;2(3):243–52.

    Article  CAS  PubMed  Google Scholar 

  2. Roa J, Tena-Sempere MJM, Endocrinology C. Connecting metabolism and reproduction: roles of central energy sensors and key molecular mediators. Mol Cell Endocrinol. 2014;397(1–2):4.

    Article  CAS  PubMed  Google Scholar 

  3. Bownes M, Scott A, Shirras AJD. Dietary components modulate yolk protein gene transcription in Drosophila melanogaster. Development. 1988;103(1):119.

    Article  CAS  PubMed  Google Scholar 

  4. Drummond-Barbosa D, Spradling AC. Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Dev Biol. 2001;231(1):265–78.

    Article  CAS  PubMed  Google Scholar 

  5. Terashima J, Bownes M. Translating available food into the number of eggs laid by Drosophila melanogaster. Genes. 2004;167(4):1711–9.

    CAS  Google Scholar 

  6. Bradley TJ, Simmons FH. An analysis of resource allocation in response to dietary yeast in Drosophila melanogaster. J Insect Physiol. 1997;43(8):779–88.

    Article  PubMed  Google Scholar 

  7. Schultzhaus JN, Carney GE. Dietary protein content alters both male and female contributions to Drosophila melanogaster female post-mating response traits. J Insect Physiol. 2017;99:101–6.

    Article  CAS  PubMed  Google Scholar 

  8. Piper MD, Blanc E, Leitão-Gonçalves R, Yang M, He X, Linford NJ, Hoddinott MP, Hopfen C, Soultoukis GA, Niemeyer C, et al. A holidic medium for Drosophila melanogaster. Nat methods. 2014;11(1):100–5.

    Article  CAS  PubMed  Google Scholar 

  9. Bownes, Entomology MJARo. Expression of the genes coding for vitellogenin (Yolk Protein). Annu Rev Entomol. 2003;31(1):507–531.

  10. Carvalho GB, Kapahi P, Anderson DJ, Benzer S. Allocrine modulation of feeding behavior by the sex peptide of drosophila. Curr Biol. 2006;16(7):692–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ribeiro C, Dickson BJ. Sex peptide receptor and neuronal TOR/S6K signaling modulate nutrient balancing in Drosophila. Curr Biol. 2010;20(11):1000–5.

    Article  CAS  PubMed  Google Scholar 

  12. Vargas MA, Luo N, Yamaguchi A, Kapahi P. A role for S6 kinase and serotonin in postmating dietary switch and balance of nutrients in D. melanogaster. Curr Biol. 2010;20(11):1006–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Barnes AI, Wigby S, Boone JM, Partridge L, Chapman T. Feeding, fecundity and lifespan in female Drosophila melanogaster. Proc Biol Sci. 2008;275(1643):1675–83.

    PubMed  PubMed Central  Google Scholar 

  14. Corrales-Carvajal VM, Faisal AA, Ribeiro C. Internal states drive nutrient homeostasis by modulating exploration-exploitation trade-off  Elife. 2016;5:e19920.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Camus MF, Huang CC, Reuter M, Fowler K. Dietary choices are influenced by genotype, mating status, and sex in Drosophila melanogaster. Ecol Evol. 2018;8(11):5385–93.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Lemaitre B, Miguel-Aliaga I. The digestive tract of Drosophila melanogaster. Annu Rev Genet. 2013;47:377–404.

    Article  CAS  PubMed  Google Scholar 

  17. Reiff T, Jacobson J, Cognigni P, Antonello Z, Ballesta E, Tan KJ, Yew JY, Dominguez M, Miguel-Aliaga I. Endocrine remodelling of the adult intestine sustains reproduction in Drosophila  ELife. 2015;4:e06930.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Ameku T, Yoshinari Y, Texada MJ, Kondo S, Amezawa K, Yoshizaki G, Shimada-Niwa Y, Niwa R. Midgut-derived neuropeptide F controls germline stem cell proliferation in a mating-dependent manner. PLoS Biol. 2018;16(9): e2005004.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Ahmed SMH, Maldera JA, Krunic D, Paiva-Silva GO, Pénalva C, Teleman AA, Edgar BA. Fitness trade-offs incurred by ovary-to-gut steroid signalling in Drosophila. Nature. 2020;584(7821):415–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cognigni P, Bailey AP, Miguel-Aliaga I. Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. Cell Metab. 2011;13(1):92–104.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Klepsatel P, Gáliková M, De Maio N, Ricci S, Schlötterer C, Flatt T. Reproductive and post-reproductive life history of wild-caught Drosophila melanogaster under laboratory conditions. J Evolution Biol. 2013;26(7):1508–20.

    Article  CAS  Google Scholar 

  22. White MA, Bonfini A, Wolfner MF, Buchon N. Drosophila melanogaster sex peptide regulates mated female midgut morphology and physiology. Proc Natl Acad Sci U S A. 2021;118(1):e2018112118.

    Article  CAS  PubMed  Google Scholar 

  23. Hudry B, Khadayate S, Miguel-Aliaga I. The sexual identity of adult intestinal stem cells controls organ size and plasticity. Nature. 2016;530(7590):344–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hudry B, de Goeij E, Mineo A, Gaspar P, Hadjieconomou D, Studd C, Mokochinski JB, Kramer HB, Plaçais PY, Preat T, et al. Sex differences in intestinal carbohydrate metabolism promote food intake and sperm maturation. Cell. 2019;178(4):901–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Regan JC, Khericha M, Dobson AJ, Bolukbasi E, Rattanavirotkul N, Partridge L. Sex difference in pathology of the ageing gut mediates the greater response of female lifespan to dietary restriction. ELife. 2016;5:e10956.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Dahalan FA, Churcher TS, Windbichler N, Lawniczak MKN. The male mosquito contribution towards malaria transmission: mating influences the Anopheles female midgut transcriptome and increases female susceptibility to human malaria parasites. PLoS Pathog. 2019;15(11): e1008063.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Kalb JM, DiBenedetto AJ, Wolfner MF. Probing the function of Drosophila melanogaster accessory glands by directed cell ablation. Proc Natl Acad Sci U S A. 1993;90(17):8093–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Liu H, Kubli E. Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2003;100(17):9929–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Peng J, Chen S, Büsser S, Liu H, Honegger T, Kubli E. Gradual release of sperm bound sex-peptide controls female postmating behavior in Drosophila. Curr Biol. 2005;15(3):207–13.

    Article  CAS  PubMed  Google Scholar 

  30. Avila FW, Sirot LK, LaFlamme BA, Rubinstein CD, Wolfner MF. Insect seminal fluid proteins: identification and function. Annu Rev Entomol. 2011;56:21–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Peng D, Kakani EG, Mameli E, Vidoudez C, Mitchell SN, Merrihew GE, MacCoss MJ, Adams K, Rinvee TA, Shaw WR et al. A male steroid controls female sexual behaviour in the malaria mosquito. Nature. 2022.

  32. Manning A. A sperm factor affecting the receptivity of Drosophila Melanogaster females. Nature. 1962;194(4825):252–3.

    Article  Google Scholar 

  33. Chapman T, Bangham J, Vinti G, Seifried B, Lung O, Wolfner MF, Smith HK, Partridge L. The sex peptide of Drosophila melanogaster: female post-mating responses analyzed by using RNA interference. Proc Natl Acad Sci U S A. 2003;100(17):9923–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yapici N, Kim YJ, Ribeiro C, Dickson BJ. A receptor that mediates the post-mating switch in Drosophila reproductive behaviour. Nature. 2008;451(7174):33–7.

    Article  PubMed  Google Scholar 

  35. Soller M, Bownes M, Kubli E. Control of oocyte maturation in sexually mature Drosophila females. Dev Biol. 1999;208(2):337–51.

    Article  CAS  PubMed  Google Scholar 

  36. Soller M, Bownes M, Kubli E. Mating and sex peptide stimulate the accumulation of yolk in oocytes of Drosophila melanogaster. Eur J Biochem. 1997;243(3):732–8.

    Article  CAS  PubMed  Google Scholar 

  37. Walker SJ, Corrales-Carvajal VM, Ribeiro C. Postmating circuitry modulates salt taste processing to increase reproductive output in Drosophila. Curr Biol. 2015;25(20):2621–30.

    Article  CAS  PubMed  Google Scholar 

  38. Schwenke RA, Lazzaro BP, Wolfner MF. Reproduction-immunity trade-offs in insects. Annu Rev Entomol. 2016;61:239–56.

    Article  CAS  PubMed  Google Scholar 

  39. Apger-McGlaughon J, Wolfner MF. Post-mating change in excretion by mated Drosophila melanogaster females is a long-term response that depends on sex peptide and sperm. J Insect Physiol. 2013;59(10):1024–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ameku T, Niwa R. Mating-induced increase in germline stem cells via the neuroendocrine system in female Drosophila. PLoS Genet. 2016;12(6): e1006123.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Lee JC, Bruck DJ, Curry H, Edwards D, Haviland DR, Van Steenwyk RA, Yorgey BM. The susceptibility of small fruits and cherries to the spotted-wing drosophila, Drosophila suzukii. Pest Manag Sci. 2011;67(11):1358–67.

    Article  CAS  PubMed  Google Scholar 

  42. Cini A, Ioriatti C, Anfora G. A review of the invasion of Drosophila suzukii in Europe and a draft research agenda for integrated pest management. Bull Insectol. 2012;65(1):149–60.

    Google Scholar 

  43. Asplen MK, Anfora G, Biondi A, Choi DS, Chu D, et al. Invasion biology of spotted wing Drosophila (Drosophila suzukii): a global perspective and future priorities. J Pest Sci. 2015;88:469–94.

    Article  Google Scholar 

  44. Karageorgi M, Bräcker LB, Lebreton S, Minervino C, Cavey M, Siju KP, Grunwald Kadow IC, Gompel N, Prud’homme B. Evolution of multiple sensory systems drives novel egg-laying behavior in the fruit pest Drosophila suzukii. Curr Biol. 2017;27(6):847–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Dweck HK, Talross GJ, Wang W, Carlson JR. Evolutionary shifts in taste coding in the fruit pest Drosophila suzukii. eLife. 2021;10:e64317.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Walsh DB, Bolda MP, Goodhue RE, Dreves AJ, Lee J, et al. Drosophila suzukii (Diptera: Drosophilidae): invasive pest of ripening soft fruit expanding its geographic range and damage potential. J Integr Pest Manag. 2011;2:1–7.

    Article  Google Scholar 

  47. Rota-Stabelli O, Blaxter M, Anfora G. Drosophila suzukii. Curr Biol. 2013;23:R8–9.

    Article  CAS  PubMed  Google Scholar 

  48. Atallah J, Teixeira L, Salazar R, Zaragoza G, Kopp A. The making of a pest: the evolution of a fruit-penetrating ovipositor in Drosophila suzukii and related species. Proc Royal Soc B-Biol Sci. 2014;281(1781):20132840.

    Article  Google Scholar 

  49. Green JE, Cavey M, Médina Caturegli E, Aigouy B, Gompel N, Prud’homme B. Evolution of ovipositor length in Drosophila suzukii is driven by enhanced cell size expansion and anisotropic tissue reorganization. Curr Biol. 2019;29(12):2075–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Timmeren SV, Isaacs R. Control of spotted wing drosophila, Drosophila suzukii, by specific insecticides and by conventional and organic crop protection programs. Crop Prot. 2013;54:126–33.

    Article  Google Scholar 

  51. Smirle MJ, Zurowski CL, Ayyanath MM, Scott IM, MacKenzie KE. Laboratory studies of insecticide efficacy and resistance in Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) populations from British Columbia, Canada. Pest Manag Sci. 2017;73(1):130–7.

    Article  CAS  PubMed  Google Scholar 

  52. Esvelt KM, Smidler AL, Catteruccia F, et al. Concerning RNA-guided gene drives for the alteration of wild populations. eLife. 2014;3:3277–88.

    Article  Google Scholar 

  53. Champer J, Buchman A, Akbari OS. Cheating evolution: engineering gene drives to manipulate the fate of wild populations. Nat Rev Genet. 2016;17(3):146–59.

    Article  CAS  PubMed  Google Scholar 

  54. Hammond A, Galizi R, Kyrou K, Simoni A, Siniscalchi C, Katsanos D, Gribble M, Baker D, Marois E. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol. 2016;34(1):78–83.

    Article  CAS  PubMed  Google Scholar 

  55. Rogina B, Wolverton T, Bross TG, Chen K, Müller H, Carey JR. Distinct biological epochs in the reproductive life of female Drosophila melanogaster. Mech Ageing Dev. 2007;128(9):477–85.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Zheng W, Luo D, Wu F, Wang J, Zhang H. RNA sequencing to characterize transcriptional changes of sexual maturation and mating in the female oriental fruit fly Bactrocera dorsalis. BMC Genomics. 2016;17:194.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Alfonso-Parra C, Ahmed-Braimah YH, Degner EC, Avila FW, Villarreal SM, Pleiss JA, Wolfner MF, Harrington LC. Mating-induced transcriptome changes in the reproductive tract of female Aedes aegypti. PLoS Negl Trop Dis. 2016;10(2): e0004451.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Alonso DP, Campos M, Troca H, Kunii R, Tripet F, Ribolla PEM. Gene expression profile of Aedes aegypti females in courtship and mating. Sci Rep. 2019;9(1):15492.

    Article  PubMed  PubMed Central  Google Scholar 

  59. Dalton JE, Kacheria TS, Knott SR, Lebo MS, Nishitani A, Sanders LE, Stirling EJ, Winbush A, Arbeitman MN. Dynamic, mating-induced gene expression changes in female head and brain tissues of Drosophila melanogaster. BMC Genomics. 2010;11:541.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Immonen E, Sayadi A, Bayram H, Arnqvist G. Mating changes sexually dimorphic gene expression in the seed beetle callosobruchus maculatus. Biol Evol. 2017;9(3):677–99.

    CAS  Google Scholar 

  61. Kocher SD, Richard FJ, Tarpy DR, Grozinger CM. Genomic analysis of post-mating changes in the honey bee queen (Apis mellifera). BMC Genomics. 2008;9:232.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Manfredini F, Brown MJ, Vergoz V, Oldroyd BP. RNA-sequencing elucidates the regulation of behavioural transitions associated with the mating process in honey bee queens. BMC Genomics. 2015;16:563.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Newell NR, Ray S, Dalton JE, Fortier JC, Kao JY, Chang PL, Nuzhdin SV, Arbeitman MN. The Drosophila post-mating response: gene expression and behavioral changes reveal perdurance and variation in cross-tissue interactions. G3 Genes Genom Genet. 2020;10(3):967–83.

    CAS  Google Scholar 

  64. Rangel J, Shepherd TF, Gonzalez AN, Hillhouse A, Konganti K, Ing NH. Transcriptomic analysis of the honey bee (Apis mellifera) queen spermathecae reveals genes that may be involved in sperm storage after mating. PLoS ONE. 2021;16(1): e0244648.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Al-Wathiqui N, Dopman EB, Lewis SM. Postmating transcriptional changes in the female reproductive tract of the European corn borer moth. Insect Mol Biol. 2016;25(5):629–45.

    Article  CAS  PubMed  Google Scholar 

  66. Huo Z, Liu Y, Yang J, Xie W, Wang S, Wu Q, Zhou X, Pang B, Zhang Y. Transcriptomic analysis of mating responses in Bemisia tabaci MED females. Insects. 2020;11(5):308.

    Article  PubMed Central  Google Scholar 

  67. McGraw LA, Gibson G, Clark AG, Wolfner MF. Genes regulated by mating, sperm, or seminal proteins in mated female Drosophila melanogaster. Curr Biology. 2004;14(16):1509–14.

    Article  CAS  Google Scholar 

  68. Gioti A, Wigby S, Wertheim B, Schuster E, Martinez P, Pennington CJ, Partridge L, Chapman T. Sex peptide of Drosophila melanogaster males is a global regulator of reproductive processes in females. Proc Biol Sci. 2012;279(1746):4423–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Zhou S, Mackay T, Anholt RR. Transcriptional and epigenetic responses to mating and aging in Drosophila melanogaster. BMC Genomics. 2014;15(1):927.

    Article  PubMed  PubMed Central  Google Scholar 

  70. Fowler EK, Bradley T, Moxon S, Chapman T. Divergence in transcriptional and regulatory responses to mating in male and female fruitflies. Sci Rep. 2019;9(1):16100.

    Article  PubMed  PubMed Central  Google Scholar 

  71. Sirot L, Bansal R, Esquivel CJ, Arteaga-Vázquez M, Herrera-Cruz M, Pavinato VAC, Abraham S, Medina-Jiménez K, Reyes-Hernández M, Dorantes-Acosta A, et al. Post-mating gene expression of Mexican fruit fly females: disentangling the effects of the male accessory glands. Insect Mol Biol. 2021;30(5):480–96.

    Article  CAS  PubMed  Google Scholar 

  72. Liu PC, Hao DJ. Behavioural and transcriptional changes in post-mating females of an egg parasitoid wasp species. R Soc Open Sci. 2019;6(1): 181453.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Tu CP, Akgül B. Drosophila glutathione S-transferases. Methods Enzymol. 2005;401:204–26.

    Article  CAS  PubMed  Google Scholar 

  74. Parra-Peralbo E, Culi J. Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism. PLoS Genet. 2011;7(2): e1001297.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Fedorka KM, Zuk M, Mousseau TA. Immune suppression and the cost of reproduction in the ground cricket. Allonemobius socius. Evolution. 2004;58(11):2478–85.

    Article  PubMed  Google Scholar 

  76. Castella G, Christe P, Chapuisat M. Mating triggers dynamic immune regulations in wood ant queens. J Evol Biol. 2009;22(3):564–70.

    Article  CAS  PubMed  Google Scholar 

  77. Rolff J, Siva-Jothy MT. Copulation corrupts immunity: a mechanism for a cost of mating in insects. Proc Natl Acad Sci U S A. 2002;99(15):9916–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Baer B, Armitage SA, Boomsma JJ. Sperm storage induces an immunity cost in ants. Nature. 2006;441(7095):872–5.

    Article  CAS  PubMed  Google Scholar 

  79. McAfee A, Chapman A, Pettis JS, Foster LJ, Tarpy DR. Trade-offs between sperm viability and immune protein expression in honey bee queens (Apis mellifera). Commun Biol. 2021;4(1):48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. McKean KA, Nunney L. Increased sexual activity reduces male immune function in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2001;98(14):7904–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Simmons LW, Roberts B. Bacterial immunity traded for sperm viability in male crickets. Science. 2005;309(5743):2031.

    Article  CAS  PubMed  Google Scholar 

  82. Simmons LW, Tinghitella RM, Zuk M. Quantitative genetic variation in courtship song and its covariation with immune function and sperm quality in the field cricket Teleogryllus oceanicus. Behav Ecol. 2010;21:1330–6.

    Article  Google Scholar 

  83. Simmons LW. Resource allocation trade-off between sperm quality and immunity in the field cricket. Teleogryllus oceanicus Behav Ecol. 2011;23:168–73.

    Article  Google Scholar 

  84. Stürup M, Baer-Imhoof B, Nash DR, Boomsma JJ, Baer B. When every sperm counts: factors affecting male fertility in the honeybee Apis mellifera. Behav Ecol. 2013;24:1192–8.

    Article  Google Scholar 

  85. Wigby S, Suarez SS, Lazzaro BP, Pizzari T, Wolfner MF. Sperm success and immunity. Curr Top Dev Biol. 2019;135:287–313.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Short SM, Wolfner MF, Lazzaro BP. Female Drosophila melanogaster suffer reduced defense against infection due to seminal fluid components. J Insect Physiol. 2012;58(9):1192–201.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Gomulski LM, Dimopoulos G, Xi Z, Scolari F, Gabrieli P, Siciliano P, Clarke AR, Malacrida AR, Gasperi G. Transcriptome profiling of sexual maturation and mating in the Mediterranean fruit fly, Ceratitis capitata. PLoS ONE. 2012;7(1): e30857.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Chérasse S, Baer B, Schiøtt M, Boomsma JJ. Differential immune gene expression in sperm storage organs of leaf-cutting ants. J Exp Biol. 2018;221(Pt 6):jeb173435.

    Article  PubMed  Google Scholar 

  89. Fedorka KM, Linder JE, Winterhalter W, Promislow D. Post-mating disparity between potential and realized immune response in Drosophila melanogaster. Proc Biol Sci. 2007;274(1614):1211–7.

    PubMed  PubMed Central  Google Scholar 

  90. Short SM, Lazzaro BP. Female and male genetic contributions to post-mating immune defence in female Drosophila melanogaster. Proc Biol Sci. 2010;277(1700):3649–57.

    PubMed  PubMed Central  Google Scholar 

  91. Innocenti P, Morrow EH. Immunogenic males: a genome-wide analysis of reproduction and the cost of mating in Drosophila melanogaster females. J Evol Biol. 2009;22(5):964–73.

    Article  CAS  PubMed  Google Scholar 

  92. Lawniczak MK, Begun DJ. A genome-wide analysis of courting and mating responses in Drosophila melanogaster females. Genome. 2004;47(5):900–10.

    Article  CAS  PubMed  Google Scholar 

  93. Mack PD, Kapelnikov A, Heifetz Y, Bender M. Mating-responsive genes in reproductive tissues of female Drosophila melanogaster. Proc Natl Acad Sci U S A. 2006;103(27):10358–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Domanitskaya EV, Liu H, Chen S, Kubli E. The hydroxyproline motif of male sex peptide elicits the innate immune response in Drosophila females. FEBS J. 2007;274(21):5659–68.

    Article  CAS  PubMed  Google Scholar 

  95. Knell RJ, Webberley KM. Sexually transmitted diseases of insects: distribution, evolution, ecology and host behaviour. Biol Rev Camb Philos Soc. 2004;79(3):557–81.

    Article  PubMed  Google Scholar 

  96. Miest TS, Bloch-Qazi MC. Sick of mating: sexual transfer of a pathological bacterium in Drosophila melanogaster. Fly. 2008;2:215–9.

    Article  PubMed  Google Scholar 

  97. Schwenke RA, Lazzaro BP. Juvenile hormone suppresses resistance to infection in mated female Drosophila melanogaster. Curr Biol. 2017;27(4):596–601.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Deng D, Xing S, Liu X, Ji Q, Zhai Z, Peng W. Transcriptome analysis of sex-biased gene expression in the spotted-wing drosophila, Drosophila suzukii (Matsumura). G3 (Bethesda). 2022;12(8):127.

    Article  Google Scholar 

  99. Bing XL, Winkler J, Gerlach J, Loeb G, Buchon N. Identification of natural pathogens from wild Drosophila suzukii. Pest Manag Sci. 2021;77(4):1594–606.

    Article  CAS  PubMed  Google Scholar 

  100. Chiu JC, Jiang X, Zhao L, Hamm CA, Cridland JM, Saelao P, Hamby KA, Lee EK, Kwok RS,Zhang G, et al. Genome of Drosophila suzukii, the spotted wing drosophila  G3 Genes Genom Genet. 2013;3(12):2257–71.

    Google Scholar 

  101. Paris M, Boyer R, Jaenichen R, Wolf J, Karageorgi M, Green J, Cagnon M, Parinello H, Estoup A, Gautier M, et al. Near-chromosome level genome assembly of the fruit pest Drosophila suzukii using long-read sequencing. Sci Rep. 2020;10(1):11227.

    Article  PubMed  PubMed Central  Google Scholar 

  102. Arlt MF, Brogley MA, Stark-Dykema ER, Hu YC, Mueller JL. Genomic structure, evolutionary origins, and reproductive function of a large amplified intrinsically disordered protein-coding gene on the X chromosome (Laidx) in mice.  G3 Genes Genom Genet. 2020;10(6):1997–2005.

    CAS  Google Scholar 

  103. Ma C, Wang S, Cao Y, Tang W, Wuniqiemu T, Teng F, Zhu X, Wei Y, Dong J. Screening and verification of differentially expressed long non-coding RNAs in the peripheral blood of patients with asthma. Front Pharmacol. 2022;13: 834009.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Alfred M. Handler of U.S. Department of Agriculture, Agricultural Research Service, Center for Medical, Agricultural, and Veterinary Entomology for the revision of the manuscript.

Funding

This research was supported by International Atomic Energy Agency’s Coordinated Research Project (No. D44003), the Natural Science Foundation of Hunan Province, China (No. 2022JJ40269), the Natural Science Foundation of Zhejiang Province, China (No. LQ21C140007), the Natural Science Foundation of Changsha City, China (No. kq2202244), the Key Project of Education Department of Hunan Province (No. 21A0042).

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Authors and Affiliations

  1. Hunan Provincial Key Laboratory of Animal Intestinal Function and Regulation, State Key Laboratory of Developmental Biology of Freshwater Fish, HunanInternational Joint Laboratory of Animal Intestinal Ecology and Health, Hunan Normal University, Changsha, 410081, China

    Shisi Xing, Dan Deng, Wen wen & Wei Peng

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  1. Shisi Xing
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  2. Dan Deng
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  3. Wen wen
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  4. Wei Peng
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Contributions

WP conceived and designed the study. SX performed the experiments; DD and WW assisted with the preparation of samples. WP analyzed the data; SX and WP wrote the manuscript, the final version of which all authors have read and approved.

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Correspondence to Wei Peng.

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Supplementary Information

Additional file 1: Supplementary Table S1.

Alignment statistics of the virginand mated D. suzukii female midgut RNA-Seq analysis. Supplementary Table S2. All genes description and FPKM value in D.suzukii virgin and mated female midguttranscriptomes. Supplementary Table S3. Differentially expressed genes in pairwise comparison in D. suzukii virgin and mated female midguttranscriptomes. Supplementary Table S4. GO classification of the differentially expressed genes in pairwise comparisonin D. suzukii virgin and mated femalemidguttranscriptomes. Three main categories, namely biological process (BP), cellularcomponent (CC), and molecular function (MF) were assigned to DEGs. Supplementary Table S5. KEGG pathwayenrichment analysis for differentially expressed genes in pairwise comparisonin D. suzukii virgin and mated femalemidguttranscriptomes. Supplementary Table S6. Primersused in our study. Supplementary FigureS1. Post-mating change in midgut length 1 and 3 days after mating in D.suzukii. Midgut length quantifications (A) and (D), representative imagesof virgin and mated female midgut phenotypes (B) and (E), changes in midgutrevealed by DAPI staining (C) and (F). The scale label is 500 um in picture Band E, and 20 um in picture C and F. SupplementaryFigure S2. Mating increases cell proliferation in female D. suzukii midgut.Error bars indicate the SEM of three independent biological replicates andasterisks (**) indicate the statistically significant differences (P < 0.01)between virgin and mated female midgut based on Student’ s t-test.SupplementaryFigure S3. Evaluation of sequence quality for the D. suzukii virgin and mated female midguttranscriptomes. SupplementaryFigure S4. Distribution of protein coding genes lengths in D.suzukii virgin and mated female midguttranscriptomes. The sizes of all protein coding genes were calculated. SupplementaryFigure S5. Mating increases neutrallipidcontent revealed by Bodipy staining in the whole virgin and mated female midgutof D. suzukii. The quantification is showed in the right. The scale bar is 500 um.

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Xing, S., Deng, D., wen, W. et al. Functional transcriptome analyses of Drosophila suzukii midgut reveal mating-dependent reproductive plasticity in females. BMC Genomics 23, 726 (2022). https://doi.org/10.1186/s12864-022-08962-2

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BMC Genomics

ISSN: 1471-2164

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