Abstract

Simple Summary

   Research on the composition of egg parasitoid venom is very limited. In
   this study, we used a combination of transcriptomic and proteomic
   approaches to identify the protein fractions of the venom in both
   eupelmid egg parasitoids, Anastatus japonicus and Mesocomys trabalae.
   We identified 3422 up-regulated venom gland genes in M. trabalae and
   3709 in A. japonicus and analyzed their functions comparatively. By
   proteome sequencing, we identified 956 potential venom proteins in the
   venom pouch of M. trabalae, of which 186 were contained in up-regulated
   genes simultaneously. A total of 766 proteins were detected in the
   venom of A. japonicus, of which 128 venom proteins were highly
   expressed in the venom glands. The results of functional analysis for
   these identified venom proteins indicated that the venom proteins in M.
   trabalae are well known but not in A. japonicus, which may be related
   to the host range. These results will provide a theoretical basis for
   studying the function of egg parasitoid venom and its parasitic
   mechanism.

Abstract

   Parasitic wasps are abundant and diverse Hymenoptera insects that lay
   their eggs inside or on the external surface of the host and inject
   venom into the host to create a more favorable environment for the
   larvae to survive and regulate the host’s immunity, metabolism, and
   development. But research on the composition of egg parasitoid venom is
   very limited. In this study, we used a combination of transcriptomic
   and proteomic approaches to identify the protein fractions of the venom
   in both eupelmid egg parasitoids, Anastatus japonicus and Mesocomys
   trabalae. We identified 3422 up-regulated venom gland genes (UVGs) in
   M. trabalae and 3709 in A. japonicus and analyzed their functions
   comparatively. By proteome sequencing, we identified 956 potential
   venom proteins in the venom pouch of M. trabalae, of which 186 were
   contained in UVGs simultaneously. A total of 766 proteins were detected
   in the venom of A. japonicus, of which 128 venom proteins were highly
   expressed in the venom glands. At the same time, the functional
   analysis of these identified venom proteins was carried out separately.
   We found the venom proteins in M. trabalae are well known but not in A.
   japonicus, which may be related to the host range. In conclusion,
   identifying venom proteins in both egg parasitoid species provides a
   database for studying the function of egg parasitoid venom and its
   parasitic mechanism.

   Keywords: parasitic wasp, Eupelmidae, Caligula japonica, venom proteins

1. Introduction

   Parasitic wasps, the most abundant and diverse insect species in
   Hymenoptera, are critical natural control factors for insects and
   effective biocontrol agents [[32]1]. Parasitic wasps usually carry
   parasitic factors released into the host during egg laying or embryo
   and larval development to regulate critical physiological processes in
   the host to ensure successful parasitism of the host pest and normal
   development of its offspring [[33]2,[34]3,[35]4]. These parasitic
   factors include polydnavirus (PDV), venom, virus-like particles (VLP),
   female-carrying factors such as ovarian proteins, and parasitic factors
   carried or released by embryos or larvae such as teratocytes
   [[36]5,[37]6,[38]7,[39]8,[40]9,[41]10,[42]11]. And the venom of
   parasitic wasps is a crucial factor affecting the success of their
   parasitism [[43]12]. The venom of parasitoids is a complex mixture of
   active substances with unknown biological functions that can be used to
   modulate host behavior, immunity, development, and metabolism [[44]13].
   The presence of venom is conservative among parasitic wasps. Venom
   proteins are produced in special glands of Hymenoptera parasitoids
   associated with the female reproductive system [[45]14].

   In parasitoids, various components are introduced into the host when
   they oviposit and facilitate the development of their progeny,
   including venom [[46]15]. Traditionally, venom has been defined as a
   toxic fluid that inflicts sudden death or paralysis on the host or
   prey. They have been recognized as a rich source of biologically active
   compounds. In particular, the venom of snakes, scorpions, spiders, and
   bees has received significant attention from the biological community,
   and these species have been intensively studied. Venom proteins of
   medical value have been identified and characterized by combining
   transcriptomic, proteomic, and peptide-led techniques
   [[47]16,[48]17,[49]18,[50]19,[51]20,[52]21]. Some studies have been
   conducted on several parasitoid venom components
   [[53]13,[54]22,[55]23,[56]24,[57]25,[58]26,[59]27,[60]28,[61]29,[62]30,
   [63]31,[64]32]. Still, these studies on the function of parasitoid
   venom have mainly focused on larval and pupal parasitoids, and our
   understanding of the venom protein fractions and function of egg
   parasitoids is still limited.

   Anastatus japonicus and Mesocomys trabalae are two egg parasitoids in
   Eupelmidae, Hymenoptera. They can be used as natural enemy insects to
   control a vital forestry pest, the Japanese Giant Silkworm, Caligula
   japonica (Lepidoptera: Saturniidae). A. japonicus has a wide host range
   and can parasitize Hemiptera insects, such as Erthesina fullo and
   Halyomorpha picu. M. trabalae does not parasitize Hemiptera insects,
   but it is the dominant species parasitizing C. japonica eggs in the
   natural field [[65]33]. It has been reported that A. japonicus has no
   parasitic factors other than the venom received in the reproductive
   organs [[66]34]. Its venom gland is similar to that of the
   endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae)
   [[67]35], and the venom of P. puparum has been reported to cause
   significant changes in the total number of host blood cells, disrupt
   the cytoskeleton, alter the behavior of blood cells such as stretching,
   adsorption, and aggregation, and affect the cystic host response to
   parasitoid eggs, thereby suppressing host cellular immunity [[68]36].
   Similarly, the venom of the parasitic wasp Leptopolina boulardi
   actively suppresses the host’s immune response [[69]37].

   As one of the weapons of successful parasitism in parasitic wasps,
   venom plays an essential role in the evolution of the host range of
   both parasitoids, and it has been shown that venom proteins in
   parasitic wasps are rapidly evolving protein taxa, with both conserved
   proteins and a large number of specific proteins between different
   parasitic wasps [[70]38]. Humoral immunity mainly includes hemolymph
   nigrosis, synthesis of antimicrobial peptides, and protease and
   association reactions [[71]39,[72]40].

   This study used a combined transcriptomic and proteomic approach to
   identify and sequence the venom glands and venom of two egg
   parasitoids, A. japonicus and M. trabalae ([73]Figure 1), and annotate
   and analyze their functions. Identifying these two egg parasitoids’
   venom proteins will provide a database for future study of the function
   of egg parasitoids’ venom and its parasitic mechanism.

Figure 1.

   [74]Figure 1
   [75]Open in a new tab

   Schematic representation of combined proteomic and transcriptomic
   analyses to identify putative venom proteins in Anastatus japonicus and
   Mesocomys trabalae. (a): female adult of A. japonicus; (b): female
   adult of M. trabalae; (c): venom organs of A. japonicus; (d) venom
   organs of M. trabalae; Vr: venom reservoir; Vg: venom gland; Ds:
   duodenal gland.

2. Materials and Methods

2.1. Insects Rearing

   The egg parasitoids, A. japonicus and M. trabalae, were collected from
   the field in Kang County, Gansu Province (105–106° E, 32.9–33.7° N) in
   August 2018 [[76]41] and bred in the Key Laboratory of Green Pesticide
   and Agricultural Bioengineering of the Ministry of Education, Guizhou
   University. The parasitoids used the eggs of Antheraea pernyi as
   alternative hosts. They were incubated in a light and constant
   temperature incubator at 25 ± 5 °C, photoperiod 14 L:10D, and 70 ± 5%
   humidity for 26–28 days and then fed with 20% honey water to ensure
   their continued growth and development after emergence.

2.2. Venom Glands Collection

   Two hundred females of A. japonicus and M. trabalae emerged over 6–7
   days, were fully mated, and were used to set up three replicates,
   respectively. The venom glands and the parasitoid bodies without venom
   glands were placed in Trizol (Ambion, Foster City, CA, USA), frozen in
   liquid nitrogen, and stored at −80 °C in a refrigerator.

2.3. RNA Extraction and cDNA Library Preparation

   Total RNA was extracted from the venom glands and the residues with
   Trizol reagent, and the total amount and quality of RNA were measured
   by NanoDrop One/Onec2000 (Thermo Fisher Scientific Inc., Waltham, MA,
   USA). The integrity of the RNA was then checked precisely with a
   bioanalyzer (Thermo Fisher Scientific Inc., USA).

   The cDNA library construction and sequencing were performed by Beijing
   Nuohe Zhiyuan Technology Co., Ltd. (Beijing, China). The cDNA libraries
   were constructed for sequencing using the NEBNext^® Ultra™ RNA Library
   Prep Kit for Illumina^® (NEB, Ipswich, MA, USA) kit. The extracted RNA
   was enriched with mRNA with polyA tails from 20 μg of total RNA by
   poly-T oligo-attached magnetic beads to remove ribosomal RNA, resulting
   in mRNA. The mRNA was fragmented at high temperatures using divalent
   cations in Illumina’s uniqueNEB Fragmentation Buffer. The first strand
   of cDNA was synthesized in the M-MuLV Reverse Transcriptase System,
   followed by degradation of the RNA strand with RNaseH and synthesis of
   the second strand of cDNA with dNTPs under the DNA polymerase I system.
   The purified double-stranded cDNA was end-repaired, A-tailed, and
   ligated with sequencing connectors. cDNAs of about 250–300 bp were
   screened with AMPure XP beads, PCR amplified, and the products were
   purified again using AMPure XP beads to finally obtain libraries. The
   libraries were initially quantified using a Qubit2.0 fluorometer,
   diluted to 1.5 ng/uL, and then the insert size of the libraries was
   checked using an Agilent 2100 bioanalyzer. After the insert size met
   expectations, the effective concentration of the libraries was
   accurately quantified by qRT-PCR. After the insert size met
   expectations, qRT-PCR was performed to accurately quantify the
   effective concentration of the library (the effective concentration of
   the zasasp library was higher than 2 nM) to ensure the quality of the
   library.

2.4. RNA-Seq and Data Analysis

   After passing the library inspection, the transcriptomes of the venom
   glands and residues of the two wasps were sequenced using the Illumina
   NovaSeq 6000 (Illumina, San Diego, CA, USA) sequencing platform. We
   filtered the raw data for adapter sequences, ploy-N, and low-quality
   reads and then checked the sequencing error rate and GC% content
   distribution to obtain clean reads for subsequent analysis.

   Clean reads were quickly and accurately compared to the reference
   genome (unpublished data) using HISAT2 software to obtain information
   on the reading positioning on the reference genome [[77]42]. The number
   of reads covered by each gene (including newly predicted genes) from
   the start to the end range is thus counted based on the position
   information of the genes’ alignment on the reference genome. This
   analysis was performed using the featureCounts tool in the subread
   software. We performed a quantitative analysis of gene expression
   levels for each venom gland and residual sample separately and then
   combined them to obtain the expression matrix for all samples.
   Differential gene expression signature analysis was performed using
   DESeq2 software [[78]43], and the screening criteria for differential
   genes were |log2(FoldChange)| ≧ 1 and padj ≦ 0.05. We used
   clusterProfiler software to perform GO functional enrichment analysis
   of the differential gene sets between venom glands and remnants and
   KEGG pathway enrichment analysis. We also compared the functional
   annotations of genes significantly up-regulated in the venom glands
   (UVGs) of the parasitoids A. japonicus and M. trabalae.

2.5. Venom Protein Collection

   After the venom gland synthesizes, venom proteins will be stored in its
   venom sacs. So, we dissected venom sacs to obtain the venom. Three
   replicates of 400 females of both parasitoid species were taken,
   respectively. The venom sacs were dissected and separated from the end
   of the abdomen and washed three times in sterile 1× Pringle’s
   phosphate-buffered saline buffer (1× PBS) (Biosharp, Hefei, China). The
   venom sacs were punctured with the tip of dissecting forceps and
   dissolved in 1× PBS buffer and protease inhibitor (TransGen Biotech,
   Beijing, China), centrifuged at 12,000 g for 15 min at 4 °C. The
   supernatant was collected, frozen in liquid nitrogen, and stored at −80
   °C for proteome sequencing.

2.6. SDS-PAGE Electrophoresis

   We used the Bradford Protein Quantification Kit (Bio-Rad, Hercules, CA,
   USA) to prepare the BSA standard protein solution according to the
   instructions. The absorbance of the standard protein solution was used
   to draw a standard curve and calculate the protein concentration of the
   sample to be tested. After the electrophoresis, the samples were
   stained with Thomas Brilliant Blue R-250 (Amresco, Solon, OH, USA) and
   decolorized until the bands were clear.

2.7. Enzymatic Digestion of Venom Proteins and Identification by LC-MS/MS
Mass Spectrometry

   One hundred and twenty microgram venom protein samples of two
   parasitoids were taken, respectively, and the volume of protein
   solution was added to make up to 100 μL and mixed with three μL 1
   μg/μL-trypsin and 500 μL 100 mM-TEAB buffer, mixed well, and digested
   overnight at 37 °C. An equal volume of 1% formic acid was added to the
   mixture, mixed well, and centrifuged at 12,000 g for 5 min at room
   temperature. Then, the supernatant was slowly passed through the C18
   desalting column.

   Mobile phases A (100% water, 0.1% formic acid) and B (80% acetonitrile,
   0.1% formic acid) were prepared in advance. The lyophilized powder was
   dissolved in 10 µL of liquid A, centrifuged at 15,000 rpm for 20 min at
   4 °C, and 1 µg of the supernatant was sampled for liquid
   chromatography. The EASY-nLCTM 1200 nano UHPLC system (Thermo Fisher
   Scientific Inc., USA) with a homemade pre-column (2 cm × 75 μm, 3 μm)
   and a homemade analytical column (15 cm × 150 μm, 1.9 μm) was used in
   this study. The Q ExactiveTM series mass spectrometer (Thermo Fisher
   Scientific Inc., USA) with a Nanospray Flex™ (ESI) ion source was used.
   The ion spray voltage was set at 2.3 kV. The ion transfer tube
   temperature was set at 320 °C. The mass spectrum was acquired in a
   data-dependent mode. The full scan range of the mass spectrum was
   350–1500 m/z. The resolution of the primary mass spectrum was set at
   60,000 (200 m/z). The parent ion with ion intensity TOP 20 (40) in the
   full scan was selected and fragmented by the high-energy collisional
   cleavage (HCD) method for secondary mass spectrometry with a resolution
   of 15,000 (200 m/z) and a maximum C-trap capacity of 5 × 10^4. The
   maximum injection time of the C-trap was 45 ms. The collision energy of
   peptide fragmentation was set to 27%. The threshold intensity was 2.2 ×
   10^4 with a 20s dynamic exclusion range to generate the raw data for
   mass spectrometry detection. The raw data were directly imported into
   Proteome Discoverer v3.0 software (Thermo Fisher Scientific Inc., USA)
   for searching the database and quantifying spectral peptides and
   proteins.

2.8. Transcriptomic and Proteomic Data Analysis

   We combined transcriptome and proteome methods to identify the venom of
   both parasitoid species. The proteins significantly up-regulated in the
   venom glands and detected in the venom proteome were used as candidate
   venom proteins. The insect venom and online analysis database, iVenomDB
   [[79]44], were used to identify venom proteins, annotate them, and
   classify protein family functions. GraphPad Prism v9 uses statistical
   analysis and the production of pictures.

3. Results

3.1. Transcriptome Analysis

   The venom gland serves as the organ of venom protein synthesis, so
   identifying proteins requires ensuring that the venom protein gene is
   highly expressed in the venom gland. Through transcriptome sequencing,
   we obtained a total of 12 transcripts ([80]Table 1). Briefly, 84.84 Gb
   of raw data were obtained from M. trabalae, and the proportion of bases
   with quality values greater than 30 to the total bases (Q30) ranged
   from 91.22–92.28%. The clean data obtained from sequencing were
   compared to the reference genome to obtain information on the
   positioning of reads on the reference genome. An average of 94% of the
   reads of the M. trabalae transcriptome could be compared to the
   reference genome, and 22,691 genes were compared in the annotation
   information of the genome. According to the differential gene screening
   criteria of |log2(FoldChange)| ≧ 1, and padj ≦ 0.05, the total number
   of differentially expressed genes between bodies without venom glands
   and venom glands of M. trabalae was 4237, among which 3422 highly
   expressed genes were identified in the venom gland of M. trabalae
   ([81]Figure 2a). The transcriptome sequencing of A. japonicus resulted
   in 86.63 Gb of data and Q30 ranging from 90.98–93.68%. On average, 93%
   of the reads of the A. japonicus transcriptome could be compared to the
   reference genome, and 20,860 genes were compared in the annotation
   information of the genome. Furthermore, 4879 differentially expressed
   genes were found between the wasp bodies without venom glands and venom
   glands, of which 3709 were UVGs ([82]Figure 2b). UVGs as potential
   candidates for venom proteins need to be further identified in
   combination with venom proteomic data.

Table 1.

   Summary of the transcriptomes of Mesocomys trabalae and Anastatus
   japonicus.
   Species Samples Total Number of Reads Total Number of Bases Q30 (%) GC
   (%) Total Number of Genes Percentage Compared to Reference Genome (%)
   M. trabalae Mt_vg_1 ^1 49,726,064 7.46 Gb 92.02 43.93 22,691 94
   Mt_vg_2 47,288,482 7.09 Gb 92.3 49.46
   Mt_vg_3 49,488,022 7.42 Gb 92.28 44.24
   Mt_body_1 ^2 45,211,074 6.78 Gb 92.24 41.87
   Mt_body_2 44,573,442 6.69 Gb 91.22 42.32
   Mt_body_3 44,435,666 6.67 Gb 92.12 45.34
   A. japonicus Aj_vg_1 ^3 45,558,606 6.83 Gb 92.11 41.65 20,860 93
   Aj_vg_2 45,633,086 6.84 Gb 92.26 35.98
   Aj_vg_3 47,038,826 7.06 Gb 93.68 36.9
   Aj_body_1 ^4 48,425,812 7.26 Gb 91.88 35.21
   Aj_body_2 54,616,318 8.19 Gb 90.98 36.43
   Aj_body_3 45,866,146 6.88 Gb 91.72 36.22
   [83]Open in a new tab

   Note: ^1: Mt_vg_1-3: M. trabalae venom glands; ^2: Mt_body_1-3: M.
   trabalae bodies without venom gland; ^3: Aj_vg_1-3: A. japonicus venom
   glands; ^4: Aj_body_1-3: A. japonicus bodies without venom gland.

Figure 2.

   [84]Figure 2
   [85]Open in a new tab

   Volcano plot of differentially expressed genes of Mesocomys trabalae
   (a) and Anastatus japonicus (b).

3.2. Functional Annotation of UVGs

   To investigate the functions of the UVGs in M. trabalae and A.
   japonicus, they were subjected to GO functional enrichment analysis
   ([86]Figure 3). The results showed that the functional distribution of
   UVGs between the two wasps was divergent. The UVGs of M. trabalae were
   enriched for 79 functions, mostly focused on molecular functions, with
   2717 genes. And the number of genes with transporter activity functions
   was the highest, with 141, followed by genes related to enzyme
   activity. A total of 1176 genes were annotated in the classification of
   biological processes, of which the most significant number of genes
   related to protein hydrolase activity was 134. This was followed by
   stimulus response-related genes with 133. Accordingly, it is speculated
   that these UVGs may be involved in regulating the immune and
   physiological metabolism of the host. Among both parasitoid species,
   the cellular components were annotated rare genes, with only M.
   trabalae annotated 30 genes and UVGs of A. japonicus not annotated to
   this functional classification. In addition, 543 genes were enriched to
   26 pathways in the KEGG pathway enrichment analysis for UVGs in M.
   trabalae. Among the top 20 pathways, the highest percentage was
   neuroactive ligand-receptor interaction (60), followed by fatty acid
   metabolism (42) and lysosome (38), respectively ([87]Figure 4a).

Figure 3.

   [88]Figure 3
   [89]Open in a new tab

   The most enriched GO terms of up-regulated genes (UVGs) in Mesocomys
   trabalae and Anastatus japonicus. MF: molecular function; CC: cellular
   component; BP: biological process.

Figure 4.

   [90]Figure 4
   [91]Open in a new tab

   The most enriched KEGG pathways of UVGs of Mesocomys trabalae (a) and
   Anastatus japonicus (b).

   The UVGs of A. japonicus were annotated in the GO database with 1916
   genes in 38 functional classifications ([92]Figure 3). As with M.
   trabalae, most of these genes are distributed in molecular functional
   classifications (27 in total), for 1488 genes. The most significant
   number of genes with transmembrane transporter activity function was
   194, followed by oxidoreductase activity (173). In the biological
   process classification, 428 genes were annotated into ten pathways, and
   the functional classification with the highest number of genes was
   transmembrane transport, which contained 171 genes. In the KEGG
   database, there are 555 genes enriched to a total of 31 pathways. The
   top three most numerous were the hedgehog signaling pathway,
   biosynthesis of cofactors, and lysosome, with 37, 36, and 35 genes,
   respectively ([93]Figure 4b). The UVGs may include genes encoding venom
   proteins and other metabolic pathways related to their synthesis
   pathways. Thus, UVGs contain a larger number of genes and are enriched
   to a larger number of pathways. However, the results of this section
   indicate that the UVGs of M. trabalae are not only more numerous than
   those of A. japonicus but also more functionally abundant.

3.3. SDS-PAGE and LC-MS/MS Analysis

   The M. trabalae and A. japonicus venom proteins were separated by
   SDS-PAGE and found to be distributed in molecular sizes from less than
   15 KD to greater than 130 KD ([94]Figure 5). The venom protein of M.
   trabalae had a broader band at 20 KD, indicating that the protein size
   was more abundant at 20 KD. The bands of A. japonicus had three more
   distinct bands at less than 20 KD, indicating a higher protein content
   at the corresponding sizes of these three bands. LC-MS/MS detection of
   venom proteome raw files and database search using Proteome Discoverer
   software showed that the number of candidate venom proteins identified
   in the M. trabalae venom samples (average of 956) was significantly
   higher than that of A. japonicus (average of 766) (p < 0.01)
   ([95]Figure 6).

Figure 5.

   [96]Figure 5
   [97]Open in a new tab

   The SDS-PAGE analysis of venom protein in Mesocomys trabalae (1–3) and
   Anastatus japonicus (4–6). Marker: molecular weight marker.

Figure 6.

   [98]Figure 6
   [99]Open in a new tab

   Total number of proteins in venom reservoir of Mesocomys trabalae and
   Anastatus japonicus identified by LC-MS/MS. Asterisks (****) above the
   standard error bars indicate significant differences (t-test, p <
   0.01).

3.4. Venom Protein Identification and Functional Analysis

   Venom proteins of M. trabalae and A. japonicus were identified by a
   combined venom gland transcriptome and venom proteome approach. The
   3422 UVGs of M. trabalae had 186 proteins identified in the proteome
   ([100]Figure 7a). The functions of these proteins are listed, and 179
   potential venom proteins are known, such as lipid transport and
   metabolism, amino acid transport and metabolism, and signal
   transduction mechanisms. Meanwhile, there are 7 proteins with unknown
   functions ([101]Table S1). The 3709 UVGs of A. japonicus had 128
   proteins identified in the proteome ([102]Figure 7b); the functions of
   these proteins were annotated, and 98 of them were known, such as lipid
   transport and metabolism, carbohydrate transport and metabolism, and
   amino acid transport and metabolism. Meanwhile, there were 30 proteins
   with unknown functions ([103]Table S2).

Figure 7.

   [104]Figure 7
   [105]Open in a new tab

   Venom diagram of putative venom proteins of two parasitoids combining
   transcriptomic and proteomic analysis. (a) The intersection of UVGs and
   those matched to the proteomic databases from Mesocomys trabalae. (b)
   The intersection of UVGs and those matched to the proteomic databases
   from Anastatus japonicus.

4. Discussion

   Parasitoids are essential species in natural ecosystems and can also be
   used as valuable and environmentally friendly biocontrol agents to
   control various pests [[106]45]. Their successful parasitism depends on
   parasitic factors, and venom is one of the important parasitic factors
   [[107]11,[108]39]. Therefore, it is imperative to understand the venom
   protein to clarify the parasitic mechanism. Research on the protein
   composition of egg parasitoid venom is relatively lacking. The existing
   research has identified more than 10 enzymes from parasitoids venom
   through enzyme activity determination, gene sequence comparison
   analysis, and proteome methods, including metalloproteinase, peptidase,
   serine proteinase, phenol oxidase, trehalase, chitinase, and
   acid/alkaline phosphatase [[109]46]. These enzymes have different
   functions in the host [[110]47]. In this study, the residues of M.
   trabalae and A. japonicus and the transcriptome of the venom gland were
   sequenced by high-throughput transcriptome sequencing technology, and
   the genes highly expressed in the venom gland were screened out.
   Because the venom protein was synthesized in the venom gland, secreted,
   and stored in the venom sac, these UVGs may be transcripts of the genes
   encoding the venom protein or may participate in the expression of the
   venom proteins; other auxiliary factors may also be translated and
   assembled. Therefore, we sequenced the venom proteome to identify only
   the components of the venom protein. The potential venom protein exists
   in both the transcriptome and the proteome.

   With the development of sequencing technology, the identification of
   parasitoid venom proteins has also made significant progress
   [[111]29,[112]30,[113]31,[114]45,[115]48,[116]49,[117]50,[118]51].
   Despite strict filtration, it is still found that their venom
   composition is very complex, and the number and species are higher than
   those of other Hymenoptera insects with venom [[119]19], which may be
   related to their need to adapt to a variety of hosts [[120]2,[121]52].
   The venom of social insects such as ants, bees, spiders, and
   centipedes, is mainly composed of small molecular peptides
   [[122]53,[123]54]. The venom proteins of parasitoids are mostly
   macromolecular proteins [[124]55]. However, parasitic wasps may release
   different factors when parasitizing different hosts. In this study, the
   venom proteins of both parasitoid species were also more than 45 kD
   ([125]Figure 5). The venom proteins of parasitoids vary greatly among
   distant species [[126]27,[127]31]. Ye found that the genes encoding
   venom proteins belong to a rapidly evolving group in the genome with
   rich diversity [[128]38]. In this study, M. trabalae and A. japonicus
   are parasitoids of two genera, and the latter has more host species
   than the former. We assumed that the number of venom proteins would
   also be more abundant. However, the results were entirely unexpected.
   Although the number of UVGs (3422) in the M. trabalae venom gland was
   less than that of A. japonicus (3709), not only did the proteome get
   more protein ([129]Figure 6), but the number of potential venom
   proteins screened by combining the two methods was more than that of A.
   japonicus ([130]Figure 7). But of the 186 venom proteins of M.
   trabalae, 179 are known in function, and only 7 are unknown. However,
   30 venom proteins in A. japonicus are unknown, and 98 have known
   functions. It can be speculated that A. japonicus may evolve a unique
   venom protein due to its adaptation to the need to find a host, which
   is not found in many other parasitoids. Although the quantity of venom
   protein of M. trabalae is dominant, most of it can be identified, so it
   may be relatively conservative.

   Parasitic wasp venom mainly targets immunity, development, and
   metabolism [[131]56], and sometimes even the host nervous system, to
   ensure successful parasitism [[132]19,[133]20]. This is very different
   from the venom function of social Hymenoptera insects, which are mainly
   used for predation and defense [[134]46]. However, research on its
   function in egg parasitoids is very scarce, which may be due to the
   fact that the egg is a relatively fragile period of an insect and its
   physiological functions are not fully developed, so it is difficult to
   carry out functional verification, which is also a technical problem to
   be overcome in the future. When commenting on the GO function of venom
   proteins in both parasitoid species, we found that the venom proteins
   mostly belong to the classification of molecular function and
   biological process and rarely involve the cellular component. The
   results also conform to the characteristics of venom proteins, most of
   which have clear biological functions, such as lipid transport and
   metabolism, amino acid transport and metabolism, and lyase and
   hydrolase activities ([135]Table S2). We also found that M. trabalae
   venom proteins, relative to those of A. japonicus, have a large
   distribution of functions, especially in molecular function.

   The venom proteins of M. trabalae and A. japonicus have been
   preliminarily identified. Due to the lack of functional research on the
   venom protein of egg parasitism, future research will focus on some
   proteins with special functions. Therefore, the data sets of both
   parasitoids obtained in this study will provide a theoretical basis for
   exploring the parasitic mechanism of egg parasitoids and improving the
   parasitic ability of parasitic wasps to improve the biological control
   effect in the future.

5. Conclusions

   In this study, we used a combination of transcriptomic and proteomic
   approaches to identify the protein fractions of the venom in both
   eupelmid egg parasitoids, A. japonicus and M. trabalae. We identified
   3422 up-regulated venom gland genes in M. trabalae and 3709 in A.
   japonicus and analyzed their functions comparatively. By proteome
   sequencing, we identified 956 potential venom proteins in the venom
   pouch of M. trabalae, of which 186 were contained in up-regulated genes
   simultaneously. A total of 766 proteins were detected in the venom of
   A. japonicus, of which 128 venom proteins were highly expressed in the
   venom glands. The results of functional analysis for these identified
   venom proteins indicated that the venom proteins in M. trabalae are
   well known, but not in A. japonicus, which may be related to the host
   range.

Supplementary Materials

   The following supporting information can be downloaded at:
   [136]https://www.mdpi.com/article/10.3390/biology12050700/s1, Table S1:
   Function Description of Venom proteins of Mesocomys trabalae; Table S2:
   Function Description of Venom proteins of Anastatus japonicus.
   [137]Click here for additional data file.^ (25KB, zip)

Author Contributions

   Conceptualization, L.-S.Z. and Y.-M.C.; methodology, X.C. and L.-S.Z.;
   software, X.C.; validation, Q.-Y.Z. and L.-S.Z.; formal analysis,
   Q.-Y.Z. and R.-Z.W.; resources, L.-S.Z. and Y.-M.C.; data curation,
   Q.-Y.Z., X.C. and R.-Z.W.; writing—original draft preparation, Q.-Y.Z.
   and X.C.; writing—review and editing, X.C. and L.-S.Z.; visualization,
   X.C. and L.-S.Z.; supervision, L.-S.Z.; project administration, L.-S.Z.
   All authors have read and agreed to the published version of the
   manuscript.

Institutional Review Board Statement

   This study did not involve humans or animals.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The raw transcriptome data of M. trabalae and A. japonicus have been
   uploaded to the NCBI Sequence Read Archive (SRA) database with
   accession numbers SRR23851092, SRR23851091, SRR23851090, SRR23851089,
   SRR23851088, SRR23851087, SRR23864786, SRR23864787, SRR23864788,
   SRR23864789, SRR23864790, SRR23864791 (Bioproject: PRJNA943600). The
   venom protein data have been uploaded to Integrated Proteome Resources
   (iProX) in Project ID: IPX0006159000.

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This research was funded by the National Natural Science Foundation of
   China, grant number: 32172469, the Natural Science Research Program of
   Guizhou University, grant number: 202202, and the Foundation of
   Postgraduate of Guizhou Province, grant number: YJSKYJJ [2021]041.

Footnotes

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References