Integration of transcriptomic and proteomic data uncovers the nutritional requirement of the immature development of an endoparasitoid wasp

Abstract

Endoparasitic wasps produce several factors, such as polydnavirus, venom and a specialized group of wasp embryonic cells named teratocytes, to condition their hosts and support the development of parasitoid offspring. However, the role of the developing parasitoid larvae in host adaption and regulation remains largely unknown. By analyzing RNA-seq data of Cotesia vestalis, an endolarval parasitoid of the diamondback moth Plutella xylostella, we discovered that most of the larval stage highly expressed genes are associated with nutrient metabolism, including carbohydrate, protein, and lipid metabolism. We also identified hundreds of C. vestalis larvae-secreted proteins. A large proportion of the secreted proteins (126/404) are encoded by larval highly expressed genes. Besides several immune-related proteins, most of the secreted proteins are closely related to carbohydrate, protein, and amino acid metabolism. We further demonstrated that eleven genes linked to amino acid metabolism that encode secretory proteins were upregulated in 2nd instar larvae. These results suggest a relatively positive correlation between gene expression and secreted proteins. In summary, our study presents the secretory proteome of an endolarval parasitoid, highlighting its potential function in nutritional and immune regulation within the host, which would benefit the growth and development of parasitoid progeny.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-02290-7.

Introduction

Parasitoids occur in several insect orders, including Coleoptera, Diptera, Hymenoptera, and Lepidoptera¹. It is predicted that parasitoid wasps, referring to parasitic Hymenoptera, account for approximately 10–20% of all insects, which makes them one of the most important resources of insect species on Earth¹. Typically, a female parasitoid deposits its egg either inside or outside the body of another arthropod, upon which its offspring rely to complete their development^2,3. The parasitism will eventually result in the death of the host. As most of the hosts are important agricultural pests, parasitoid wasps are widely used as natural or augmented biological control agents in agricultural and forest ecosystems^4,5. During parasitization, a number of the parasitic factors are produced by the parasitoid wasp, i.e., venom, calyx fluid, which includes a symbiotic polydnavirus (PDVs) and ovarian proteins, and teratocytes^6–10, contributing to host physiological and metabolic modulation^11–13. For example, in some parasitoid species, venom plays important roles in regulating the concentrations of nutrient components, such as lipids, free amino acids, proteins, acylglycerols and sugars in the host^12,14–16. PDVs are a group of large double-stranded DNA viruses that integrate their segmented genome into the host but cannot reproduce in the caterpillar host¹⁷. The infection of Cotesia vestalis bracovirus (CvBV) reduces lipid levels in the hemolymph of the host Plutella xylostella¹⁸. Teratocytes are dissociated from the embryonic membrane of the parasitoid egg during its hatching and may provide nutrition for the parasitoid larvae⁸. Recently, several studies have revealed that the developing parasitoid larva itself releases proteins into its hosts, which might also play a role in altering the host physiology. For instance, larval salivary secretions from the ectoparasitic wasp Eulophus pennicornis and the endoparasitic wasp Pteromalus puparum suppress the immune response of their hosts^19,20. Larval secretions of Leptopolina boulardi impair host immune defenses²¹; Anal secretions from the larvae of Chelonus near curvimaculatus cause host developmental arrest²²; The larvae of Chelonus inanitus produce factors that induce the onset of precocious metamorphosis in its host Spodoptera litoralis²³. In addition, trypsin isolated from the salivary secretion of Eupelmus orientalis can liquefy the host tissue and provide a suitable food source for the developing parasitoid larvae²⁴. Thus, parasitoid larvae might also play an important role in host adaptation and regulation. However, the involvement of parasitoid larval secretions in host modulation is largely underestimated.

The endoparasitic wasp C. vestalis (Hymenoptera; Braconidae) is one of the most efficient natural enemies of the diamondback moth (DMB), P. xylostella (Lepidoptera: Plutellidae), the most destructive pest of cruciferous plants worldwide^25,26. Adult female parasitoid deposits an egg into the early or middle 3rd instar host larva. The embryo hatches 48 h post oviposition, and the larval stages last approximately 6 days²⁵. It has been largely known that three major parasitic factors, i.e., venom, PDV, and teractocytes^6–9, facilitate the survival of C. vestalis offspring, while the significance of parasitoid larvae remains unclear. In this study, we analyzed C. vestalis larval secretory proteins and their relationship to transcriptome profiling to uncover their potential function.

Materials and methods

Insects rearing

The parasitoid wasp, C. vestalis, and its host DMB were originally obtained from cabbage (Brassica spp.) fields in the suburbs of Hangzhou (30.3009 N, 120.0870E), Zhejiang Province, China. The DMB and C. vestalis used in this study were reared as previously described²⁷. Adult parasitoids were fed with 20% (w/v) honey-water solution and propagated using DMB larvae as hosts. To obtain parasitized larvae, the third instar DMB larva was individually exposed to a single C. vestalis female within a 10 mm × 80 mm plastic tube until oviposition was observed, then cultured on the cabbage at 25 °C.

Transcriptome sequencing and analysis of parasitoid larvae

Larval development of the parasitoid in the host lasts approximately six days: the first instar larva lasts approximately three days, while the second instar larva lasts about three days²⁵. Ten first instar and ten second instar C. vestalis larvae were collected by dissecting parasitic DMB three days (P3d) and six days (P6d) post parasitization for transcriptome sequencing, respectively. To avoid sample contamination, the parasitoid larvae were dissected from the hosts and washed in a new Petri dish with 200 µL of TNM-FH medium (HyClone, Logan, UT, USA) at least 10 times.

Total RNA was isolated using the whole body of C. vestalis larvae with TRIzol reagent (Ambion, Foster City, CA, USA). RNA library construction, sequencing and raw sequencing data quality-filtering were performed by Shanghai NewCore Biotechnology Co., Ltd. The C. vestalis genome index was built using Bowtie 2 (v2.1.0)²⁸, and the paired-end clean reads were aligned to the C. vestalis genome (GCA_001675545.1) using TopHat (v2.1.1)^29,30. The expression level of each gene was measured and normalized as FPKM (fragments per kilobase of transcript) by StringTie (v2.1.1)³¹. All the read counts of each sample were normalized to the mean total number of reads across all samples. After obtaining the normalized gene expression level, the differential expression analysis was performed using the DEGSeq R package (v1.10.1) mode to screen the differentially expressed genes (DEGs) between the two comparative groups. To correct for multiple testing, the false discovery rate (FDR) was calculated to adjust the threshold of P value. The genes with FDR ≤ 0.05 and the absolute value of | log₂ (fold change) | ≥ 1 and a q value < 0.05 were set as the criteria for DEGs. In addition, the pheatmap package (version 1.0.8, https://cran.r-project.org/web/packages/pheatmap/index.html) in R was utilized to perform the hierarchical cluster analysis of the DEGs between these two comparative groups, with FPKMs standardized by natural logarithm.

Collection of secretory proteins of C. vestalis larvae

The DMB larvae were collected four days post parasitization (2nd instar), surface-sterilized with 75% ethanol, and then dissected with fine forceps by gently tearing the epidermis, without disrupting the gut, in TNM-FH medium (HyClone, Logan, UT, USA) containing antibiotics (ampicillin and kanamycin, each at a final concentration of 0.1 g/L). The parasitoid larvae were removed from the host larvae and repeatedly put into a new Petri dish containing 200 µl culture medium for washing, at least ten times, with gentle shaking. The washed parasitoid larvae were incubated first in a culture medium with 10% fetal bovine serum (FBS, Gibco, Hudson, NH, USA) at 27 °C for 24 h, in which the parasitoid larvae can survive for at least seven days, and then transferred to the culture medium, devoid of FBS, and incubated for an additional 24 h. Thus, the main components of the culture medium were secretory proteins from parasitoid larvae. The parasitoid larvae were removed, and the culture medium was collected for protein concentration. The secretory proteins from 200 C. vestalis larvae were set as one biological replication. A total of 600 C. vestalis larvae were collected. To rule out the contamination by teratocytes, the existence of C. vestalis nucleotides (both DNA and RNA) was detected by Qubit (Thermo Fisher Scientific, Hudson, NH, USA). The amount of the collected proteins was determined by the Bradford method using the Pierce Coomassie Plus Bradford Assay Kit (Thermo Fisher Scientific, Hudson, NH, USA)³² and the proteins were kept in liquid nitrogen for further use. The culture medium without larvae was used as a blank control.

Single reverse-phase (RP) liquid chromatography and mass spectrometry

Protein pellets were dissolved in 8 M urea, 100 mM Tris-HCl, pH 8.5. Tris (2-carboxyethyl) phosphine hydrochloride TCEP (Sigma-Aldrich, St. Louis, MO, USA) was added (final concentration of 5 mM) to the solution and incubated for 20 min at room temperature. The residue was alkylated by iodoacetamide (final concentration of 10 mM) for 15 min at room temperature. The protein mixture was diluted four times and digested with trypsin at 1:100 (w/w) (Sigma-Aldrich, St. Louis, MO, USA).

The digested peptides were desalted by spin column (Pierce) and then analyzed by an in-house packed reversed-phase C18 column (360 μm OD x 75 μm ID) connected to an Easy-nLC 1000 HPLC system by a 3 h-gradient at a flow rate of 300 nL/min. The eluted peptides were ionized and introduced into a Q Exactive mass spectrometer (Thermo Orbitrap, Hudson, NH, USA) using a nanospray source. Full MS spectra (from m/z 300–1800) were acquired by the precursor ion scan using the Orbitrap analyzer with resolution r = 70,000 at m/z 200, followed by 20 MS/MS events in Orbitrap analysis with resolution r = 17,500 at m/z 200. The twenty most intense ions were sequentially isolated and fragmented with a normalized energy of 27% of HCD.

The database searching was performed against the OGS1.0 of C. vestalis²⁹ using Integrated Proteomics Pipeline version 3 (http://www.integratedproteomics.com). Mass tolerances for precursor ions and LC-MS/MS were set at 20 ppm. Trypsin was defined as a cleavage enzyme with the three most missed cleavages, the mass of the amino acid cysteine was statistically modified by + 57.02146 Dalton, and the protein FDR was set at 0.01.

GO and KEGG enrichment analyses

Gene functions were assigned based on the best match derived from the annotation by Blastp against the SwissProt and TrEMBL databases³³. We annotated motifs and domains using InterPro³⁴ by searching against publicly available databases, including Pfam, PRINTS, PROSITE, ProDom, and SMART. Gene Ontology³⁵ information was retrieved from InterPro. We used Blast2GO to annotate the sequence and make it correspond to the corresponding GO entry. The Kyoto Encyclopedia of Genes and Genomes database (KEGG) enrichment analysis was performed to predict the role of genes in cell activity³⁶.

Real-time RT-PCR (RT-qPCR) validation and expression analysis

Total RNAs of C. vestalis larvae of P3d and P6d were extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and incubated with DNase to remove any DNA contamination. The resulting RNA (1 µg) was used as a template to synthesize first-strand cDNA with reverse-transcriptase using ReverTra Ace qPCR RT Master Mix (Code FSQ-301, TOYOBO, Dojima Hama, Osaka, Japan) according to the manufacturer’s instructions. RT-qPCR was carried out with a CFX96 real-time PCR detection system (BioRad) using SYBR^® Green Realtime PCR Master Mix (Code QPK-201, TOYOBO, Dojima Hama, Osaka, Japan). The Cv-18 S rRNA and Cv-β-tubulin of C. vestalis were used as internal reference genes. All the primers and the GenBank accession number of the corresponding selected genes used for qPCR in this study are displayed in Table S1. The data from three independent experiments were analyzed by the 2^−△△CT method³⁷.

Statistic analysis

Statistical analyses were performed in GraphPad Prism version 9.5 (https://www.graphpad.com/). Statistical significance was determined using a two-tailed unpaired Student’s t-test when comparing two different kinds of samples. Data are shown as the mean ± SD. Significance values are indicated as * P < 0.05; ** P < 0.01; *** P < 0.001.

Results

Transcriptome data analysis of parasitoid larvae

The transcriptomes of 1st instar (P3d) and 2nd instar (P6d) (Fig. 1A) parasitoid larvae were sequenced, respectively. A total of six cDNA libraries were constructed through the Illumina HiSeq 2500 sequencing platform. We obtained approximately 250 million raw reads from all samples (Table S2). After qualify-filtering, the remaining 247 million clean reads, with an average Q30 of 92.29%, were mapped to the C. vestalis genome assembly CvesOGS1.0²⁹. The proportion of total clean reads mapped to the genome ranges from 88.64 to 95.741% (Table S2). The highest proportion of clean reads were mapped to known exons, with a mapping rate of 66.42-78.42% (Table S3).

Fig. 1.

Sampling and transcriptome analysis of C. vestalis larvae. (A) Developmental and sampling time (red arrow) of C. vestalis larvae. 2d–7d: days post parasitization; 1st, 1st instar larvae; 2nd, 2nd instar larvae. (B) Numbers of DEGs between of 1st and 2nd instar C. vestalis larvae. P3d, 3 days post parasitization (1st instar); P6d, 6 days post parasitization (2nd instar); Up represents the number of up-regulated genes; Down represents the number of down-regulated genes. (C) The heatmap and a hierarchical cluster of all the DEGs between P3d and P6d parasitoid larvae.

There was a total of 3845 DEGs between the two sample groups, of which 2179 genes were upregulated and 1666 genes were downregulated in the P6d parasitoid larvae (Fig. 1B and Table S4). To observe the gene expression patterns, we performed hierarchical clustering of all the DEGs based on the log₁₀ (FPKM + 1) (Fig. 1C), which showed that the expression of identified DEGs could correctly distinguish the two different kinds of samples.

All DEGs were used for the enrichment analysis of GO and KEGG. GO analysis showed that the up-regulated DEGs in the second instar larvae were mainly involved in metabolic and catalytic processes: (i) lipid-related biological processes, such as lipid metabolism, lipid transport and location, lipid droplet organization, and cellular lipid metabolic process. (ii) carbohydrate-related processes, such as carbohydrate metabolic process and carbohydrate derivative metabolic process (Fig. 2A and Table S4). Similarly, KEGG analysis revealed that the upregulated DEGs were significantly enriched and categorized into 48 pathways, of which mainly belong to metabolism branches. The top 30 pathways with the most abundant upregulated DEGs showed that seven amino acid metabolisms, seven carbohydrate metabolisms, and three lipid metabolisms were enriched (Fig. 2B and Table S4). According to GO enrichment analysis, the downregulated genes in the biological process assessment were involved in rRNA processing, rRNA metabolic processes, and so on. Downregulated DEGs linked to molecular function include RNA binding, DNA binding, and nucleic acid binding. Downregulated genes were enriched in the nucleolus, nuclear lumen, and other structures in the cellular component (Fig. S1A and Table S4). KEGG enrichment analysis of the downregulated genes showed that the top five pathways were Ribosome biogenesis in eukaryotes, DNA replication, Mismatch repair, Nucleotide excision repair, and Cell cycle (Fig. S1B and Table S4).

Fig. 2.

Functional enrichment analysis of up-regulated genes in 2nd instar larvae. (A) The GO functional classification of the up-regulated genes. The distributions are summarized in three main categories: biological process (BP), molecular function (MF), and cellular component (CC). The x-axis indicates the gene ratio, which means the ratio of the differentially expressed gene number to the total gene number in a certain GO term and the y-axis indicates the different GO terms. (B) Classified KEGG pathways of the up-regulated genes. The pathways were further classified into five major groups: cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems.

To identify whether 3845 larval DEGs are specifically expressed in the larval stage, we re-analyzed the transcriptome dynamics of C. vestalis throughout its lifetime, combining our previously reported transcriptomic profile³⁸. Overall, we found that about half of the DEGs (2005/3845) were enriched in the larval stage (C3, C4, C7 and C8), of which 738 and 599 DEGs were specific-highly expressed at 1st instar (P3d) and 2nd instar (P6d) larval stage, respectively (Fig. 3). Genes in specificcally expressed in 1st instar (C7) were related to ribosome biogenesis, spliceosome and nucleocytoplasmic transport, suggesting parasitoid larva is undergoing mRNA translation and modification, and protein synthesis at this stage; while genes enriched in 2nd instar (C4) were linked to lipid, amino acid metabolism, and protein digestion and absorption, implying nutritional requirements at this stage. The other enriched DEGs were also highly expressed at the pupal (C8) or adult (C4) stage, which were involved in aminoacyl-tRNA biosynthesis, cell cycle and meiosis, and DNA replication and RNA polymerase, suggesting increasing cell proliferation at these stages.

Fig. 3.

Heatmap and hierarchical clustering for DEGs in the developmental stage of C. vestalis. The colors represent the relative gene expression values after normalization adjustments. The red and blue color refers to high- and low-expression levels, respectively. KEGG enrichment was shown on the right panel.

Parasitoid larvae secreted proteins into the host Hemocoel

So far, several parasitism-specific proteins have been discovered, which are of parasitoid larvae origin based on the SDS-PAGE analysis. Here, we conducted a proteomic study to characterize the diversity of proteins secreted by endoparasitoid larvae. When C. vestalis larvae were incubated in culture medium in vitro, they released a variety of proteins, with molecular weight estimates ranging from 10 to 210 kDa. When wasp larvae on days 3 and 6 of parasitism were incubated in a culture medium, it was clear that the electrophoretic profile of the proteins released by the parasitoid larvae differed from each other (Fig. 4A and Fig. S2). Three biological repeats were performed, and we detected 993, 809 and 1174 secretory proteins, respectively (Fig. 4B). Finally, 404 shared secretory proteins were identified (Table S5). Among all the identified proteins, 33 proteins (8.17%) contain a predicted signal peptide at the N-terminal (Table S5). Some of the proteins were enriched in protein export, protein processing in the endoplasmic reticulum and ribosome with possible function in protein synthesis, translation, transport, and degradation (Fig. 4C and Table S5); The other secreted proteins were predominantly enriched in metabolism, with more than 100 secreted proteins were involved in nutrient-related metabolic pathways, such as carbohydrate metabolism (glycolysis/gluconeogenesis, pyruvate metabolism, citrate cycle (TCA cycle), pentose phosphate pathway), amino acid metabolism (glycine, serine and threonine metabolism, cysteine and methionine metabolism, valine, leucine and isoleucine degradation, arginine and proline metabolism, phenylalanine, tyrosine and tryptophan biosynthesis), and lipid metabolism, suggesting possible roles of these 404 larval secretory proteins in parasitoid-host nutritional interactions.

Fig. 4.

Proteomic analysis of C. vestalis larval secretions. (A) SDS-PAGE analysis of larval secreted proteins of endoparasitoid C. vestalis. Larval wasp secretions were collected from the larval-conditional medium from P3d and P6d. P3d: 3 days post parasitization (1st instar larvae); P6d: 6 days post parasitization (2nd instar larvae). Equal protein amounts of 80 µg per lane were loaded. The gel was cropped from a full-length gel displayed in Fig. S2. (B) Venn diagram of the shared secreted proteins of three biological repeats. (C) KEGG pathway enrichment analysis for the conserved larval secretory proteins.

While venom and PDVs play roles in suppressing host immune defenses^6,39,40, we found several secreted proteins that might be associated with immunity, which have been demonstrated in other parasitoid wasps. For example, serine proteases and serine protease homologs, Cu/Zn superoxide dismutase, metalloprotease, serpinB4, and calreticulin⁴¹; lysozyme⁴², c-type lectin⁴³, and laccase-4⁴⁴. Taken together, larval-secreted proteins of C. vestalis are of great potential to modulate the host nutritional status and suppress host immune defenses.

Correlation between larval transcriptome and secreted proteome

To figure out the relationship between larval transcriptome and secreted proteome, the expression levels of the 404 secretory protein-coding genes were analyzed. Some of these genes were highly expressed during the C. vestalis larval stage, and 31 and 126 secretory proteins are specifically expressed in the 1st and the 2nd instar larvae, respectively (Fig. 5A-B and Table S5). Notably, the 126 secreted proteins, which were upregulated in the 2nd instar parasitoid larvae (C4), were mainly enriched in amino acid metabolism (Figs. 3 and 4C), such as valine, leucine and isoleucine degradation, cysteine and methionine metabolism, and arginine and proline metabolism. Furthermore, qPCR analysis showed that 11 amino acid metabolism related genes, which are also secretory proteins, were upregulated in 2nd instar parasitoid larvae (Fig. 5C). These results suggest a relative positive correlation between gene expression and secreted proteins.

Fig. 5.

Correlation between transcripts and proteins. (A) The heatmap and a hierarchical cluster of 404 secretory protein-coding genes in the developmental stage of C. vestalis. (B) Venn diagram presentation of the DEGs of transcriptomes of parasitoid larvae whose corresponding proteins were detected in larval wasp secretions. (C) Relative expression levels of 15 genes involved in amino acid metabolism by qRT-PCR analysis. The x-axis represents different genes: ALDH: Aldehyde dehydrogenase, SMOX: Spermine Oxidase, ACAT: Acetyl-CoA acetyltransferase A, ASNS: Asparagine synthetase, SSADH: Succinate-semialdehyde dehydrogenase, ALT2: Alanine aminotransferase 2, GS2: Glutamine synthetase 2, PSAT: Probable phosphoserine aminotransferase, AST: Probable aspartate aminotransferase, PGDH: D-3-phosphoglycerate dehydrogenase, PRMT1: Arginine N-methyltransferase 1. Cv-18 S rRNA and Cv-β-Tubulin were used as the reference genes, and data are shown as mean ± SD with three biological repeats. (Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001).

Discussion

As an endoparasitoid, C. vestalis offspring develop for about six days (egg and larval stages) inside the body of the DMB larvae and then cocoon outside. The ability of the developing larvae to produce and release proteins into their host hemolymph and their possible functions are largely unknown. Here, the present study revealed the gene transcriptional profiles during the larval stage of C. vestalis using RNA-seq, and the secretory proteins of C. vestalis larvae through proteomics.

We obtained 993, 809, and 1174 secreted proteins from three different samples of larval wasp secretions, respectively. Among them, we identified 404 shared larval secreted proteins, with the number of proteins accounted for less than 50% of those obtained from three biological repeats, which suggests the process of parasitoid larvae secreting proteins into their hosts during the growth stage may be ongoing and dynamic. In addition, based on the SDS-PAGE analysis of changes in secreted protein between different instars, there were more proteins secreted in the 2nd instar larvae than in the 1st instar larvae. This result corresponds to that found in other parasitoid wasps. For example, for Chelonus inanitus, the mid-1st instar larvae onwards release an approximately 212 kD protein and the 2nd and 3rd instar larvae mainly release additional smaller proteins into the host⁴⁵; A 27 kD secretory protein started to be synthesized and released by the E. pennicornis larva on three days after parasitization and increased its level subsequently⁴⁶. Combined with our results, the secretion of proteins by parasitoid larvae is probably stage-dependent and environment-dependent.

Functional annotation showed that both the larval-stage-specific expressed genes and the secreted proteins were mainly enriched in metabolism, including carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Genes involved in the nutrient metabolism pathway were differentially expressed in the parasitized hosts, and parasitism also induces nutritional alteration in the host hemolymph, including but not limited to increasing trehalose level, altering specific amino acid concentration and lowering TAG content, etc^47–49. Based on the traditional nutrient removal method, in vitro assays have demonstrated the requirements for amino acids, carbohydrates and lipids of several parasitoid wasps^50,51. Taking the amino acid for example, it has been reported that parasitoid wasps lack the de novo synthesis pathway of several amino acids²⁹. It is reasonable to find that a relatively large number of the upregulated genes (40/599) and secreted proteins (79/404) in 2nd instar parasitoid larvae were enriched in the amino acid metabolism pathway. Thus, we infer that the dramatic growth of C. vestalis larvae from the early 1st instar to the late 2nd instar requires rapid absorption or transformation of metabolic intermediates into a suitable form and larva-secreted proteins may contribute greatly to this transition.

We also discovered several larval proteins that are involved in the regulation of host immunity. Serpins play an important role in regulating melanization^52,53, and Toll pathway signaling⁵⁴. There are 25 serpin genes in the C. vestalis genome and serpins secreted by venom and teratocytes of C. vestalis have been reported to regulate host immune response^29,55,56, of which C. vestalis teratocyte-expressed serpins CvT-serpin1, CvT-serpin18, and CvT-serpin2 inhibit the PPO activation of host P. xylostella. Here, a larval-secreted protein serpin B4 (Cv-serpin7) shares a high similarity (86%) with Cc-serpinB4 from Cotesia chilonis venom, which shows phenoloxidase (PO) inhibitory activity⁵⁷. In insects, C-type lectins (CTLs) play important roles in innate immunity. They are involved in opsonization, nodule formation, agglutination, encapsulation, melanization, and prophenoloxidase activation⁵⁸. There are twelve CTLs in the C. vestalis genome. A CTL coded by CvBV serves as an immune resistance component to protect the developing parasitoid larvae by repressing the capsulation process of host hemocytes and cleaning a classical bacterium, which is synthesized in the hemocytes of the host, and secreted into the hemolymph⁵⁹. One of the two CTLs (CVE08066) that was detected in the larval secretions shows an 87% sequence similarity with Bombyx mori CTL-S3, which functions in bacterial clearance in the larval hemolymph⁶⁰. Therefore, we speculate that the two CTLs identified in larval secretion may function together with other CTLs from different sources (PDV or venom) to protect the developing parasitoid larvae in the host. Taken together, larval-secreted proteins may also be involved in the regulation of host immune defenses.

While the primary pathway for protein delivery is unknown, larval secretions may come from several sources^19,20,61,62. The so-called anal vesicle, which is formed in the larval hindgut of many braconid wasps (Hymenoptera: Braconidae), facilitates close physiological communication with the host and aids in the absorption of amino acids, glucose, and trehalose^62,63. The anatomical analysis of Pimpla turionellae hindgut and its secretory system has demonstrated that the parasitoid anal vesicle has a specific function for the production, temporary storage, and periodic expulsion of anal secretions⁶¹. According to the proteomic study of salivary secretions from Diaphorina citri⁶⁴ and Aphis craccivora⁶⁵, salivary proteins may function in nutrient metabolism (malate dehydrogenase, glucose dehydrogenase, succinate dehydrogenase, peroxidase, inosine monophosphate dehydrogenase). Besides, research on salivary secretions from parasitoids are mainly enriched in suppressing host immunity^19,20. Being two important parts of larvae, we assume that anal and salivary secretions may be the two major sources of larval secretion. However, we could not exclude the possibility that another protein secreted pathway was involved, and further study is required to validate the approach for wasp larval protein delivery.

It is known that parasitoid wasps can manipulate hosts to produce a nutritionally favorable environment for the growth and development of their offspring through injecting venoms, PDV and teratocytes^6,8,15,55,66. Our study suggests larval wasp secretions may also play an important role in host regulation. There is a positive correlation between secreted proteins and stage-specific expressed genes. Functional analysis also reveals that larval-secreted proteins were mainly involved in nutrient metabolism and immune response. However, further investigation is required to explore the detailed molecular mechanisms of secreted proteins involved in the regulation of nutrition and immunity in the host.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

L.Z W. performed experiments for Figs. 4 and 5 and drafted the manuscript; Z.H. W., and S. X. collected samples; P. Z. W., X. Q.Y., analyzed the data; J. H.H., Z. Z.W. and X. X. C. conceived the research idea and outlined the methods. All authors reviewed the manuscript.

Data availability

Sequence data supporting the findings of this study have been deposited in the National Center for Biotechnology Information Short Read Archive, BioProject PRJNA1193885 and PRJNA1193824. The transcriptome data for the pupal and adult stages of C. vestalis samples were previously submitted under PRJNA701718.

Declarations

Competing interests

The authors declare no competing interests.