To identify viruses and compare their abundance levels in the venom glands of hymenopteran species, we conducted venom gland-specific transcriptome assemblies and analyses of 22 Aculeate bees and wasps and identified the RNA genomes of picornaviruses. Additionally, we investigated the expression patterns of viruses in the venom glands over time following capture. Honeybee-infecting viruses, including black queen cell virus (BQCV), deformed wing virus (DWV), and Israeli acute paralysis virus (IAPV), were highly expressed in the venom glands of Apis mellifera and social wasps. This finding suggests that the venoms of bees and wasps likely contain these viruses, which can be transmitted horizontally between species through their stinger usage. A. mellifera exhibited an increasing pattern of abundance levels for BQCV, DWV, IAPV, and Triatovirus, while the social wasp Vespa crabro showed increasing abundance levels of IAPV and Triatovirus over different capture periods. This suggests that the venom glands of honeybees and wasps may provide suitable conditions for active viral replication and may be an organ for virus accumulation and transmission. Some viral sequences clearly reflected the phylogeny of Aculeate species, implying host-specific virus evolution. On the other hand, other viruses exhibited unique evolutionary patterns of phylogeny, possibly caused by specific ecological interactions. Our study provides insights into the composition and evolutionary properties of viral genes in the venom glands of certain Aculeate bees and wasps, as well as the potential horizontal transmission of these viruses among bee and wasp species.

One of the endoparasitoid wasp, Cotesia plutellae (Braconidae), parasitizes young larvae of the diamondback moth, Plutella xylostella. For the successful parasitization, C. plutellae required suppression of immune response in P. xylostella. Maternal (polydnavirus, venom proteins and ovary proteins) and embryonic (teratocytes) factors have been involved in immune-suppression. In this study, we performed transcriptome analysis of venom of C. plutellae and identify neprilysin-1 (Cp-NEP1) as a potential immunosuppressive protein. Cp-NEP1 encoded 451 amino acids and largely belongs to the hymeopteran neprilysin family via phylogenetic analysis. It is of interest that Cp-NEP1 has no conserved motifs such as zinc-binding domain (HExxH), substate binding domain (NAYY/F) and protein folding and maturation domain (CxxW) generally identified in other neprilysin family. In order to examine the biochemical function of Cp-NEP1, the recombinant Cp-NEP1 tagged with N-terminally 6X His was constructed and expressed in Escherichia coli. Expression of Cp-NEP1 was confirmed with SDS-PAGE and peptide sequencing. Recombinant Cp-NEP1 significantly suppressed nodule formation when the co-injection with E. coli. These results suggest that Cp-NEP1 contributes to suppression of immune response in P. xylostella and that the conserved motifs reported from other neprilysin do not involve immunosupperssion.

Vespa crabro is a cosmopolitan social wasp species whereas Vespa analis is commonly found in Asia. Both species are widely distributed in Korea and known to be aggressive when disturbed, resulting in frequent sting accidents. Although major venom components of well known Vespa wasps have been reported, no comparative transcriptomic analysis of venom gland between V. crabro and V. analis has been conducted to date. To investigate the differences in venom properties between these two wasps, total RNA was extracted from each venom gland and used for RNA-sequencing. A total of 31 venom-specific genes were identified in both venom gland transcriptomes but their expression profiles were different between V. crabro and V. analis. Venom allergen 5, premastoparan A and phospholipase A were the top three genes that were most prevalently transcribed in the venom gland of V. crabro, and their transcription rates were 902-, 112- and 4164-fold higher compared with V. analis, respectively, as judged by FPKM values. Their differential transcription profiles were confirmed by quantitative real-time PCR. In the venom gland of V. analis, however, premastoparan A was most abundantly transcribed gene, followed by calponin and tropomysin. In general, most venom-specific genes were more abundantly expressed in V. crabro but some genes exhibited higher transcription rates in V. analis, including muscle LIM protein, troponin, paramyosin, calponin, etc. Our findings suggest that V. crabro produce venom with much more enriched venom components, thereby with higher toxicity compared with V. analis.

Cotesia plutellae known as an endoparasitoid parasitizes larvae of the diamondback moth, Plutella xylostella which is a major pest in cruciferous crops. For the successful parasitization, maternal and embryonic factors of C. plutellae such as polydnavirus, ovarian proteins, teratocytes and venom are required. In this study, we identified calreticulin (Cp-CRT) gene from transcriptome data of the venom gland in C. plutellae. cDNA of CRT was cloned from total RNA of the venom gland via PCR and encodes 403 amino acids harboring several structural motifs such as a signal peptide sequence, a repetitive sequence, a putative coiled-coil sequence encompassing, and endoplasmic reticulum-recognizing domain (-KDEL). Phylogenetic analysis showed that the Cp-CRT gene formed a unique cluster with other hymenopteran CRT genes, indicating that the Cp-CRT belongs to the CRT family. To examine the physiological function of Cp-CRT, recombinant Cp-CRT, fused with 6X-His at N-terminal was constructed and expressed in E. coli. Recombinant Cp-CRT was successfully expressed via Western blot analysis and suppressed significant nodule formation when co-injected with E. coli as immune response inducer. These results suggest that the Cp-CRT involves in suppression of cellular immune response in the host

Cotesia plutellae as an endoparasitoid wasp is the natural enemy against the diamondback moth, Plutella xylostella. For successful parasitism of the offspring, the female parasitoid always injects venom with other parasitic factors such as polydnavirus, teratocytes and ovary proteins. These venom products prevent their eggs from host immune system as well as modulate their host. To identify of constituents of venom in relation of host-parasitoid interaction, total RNA of the venom gland were extracted and analyzed with RNA-seq. RNA-seq reveals that 5.5 Gbp were read and approximately 3322 transcripts were identified. The transcripts related to the biological process, cellular components and molecular functions occupied about 20%, 15% and 18%, respectively. Among these transcripts, 301 genes were related to the immune activities such as calreticulin, defensing, phosphatases and serpin etc. These results suggest that some proteins exhibiting biological function related to the immune response can be contribute to the development of wasp in the host.

To investigate genes differentially expressed in the venom of social and solitary wasps, a comparative transcriptome analysis was conducted. Subtractive expressed sequence tag (EST) libraries specific to the venom gland and sac (gland/sac) of a social wasp species, Vespa tropica and a solitary hunting wasp species, Rhynchium brunneum, was constructed by suppression subtractive hybridization. In BLASTx analysis, 41% and 56% of the total ESTs showed statistically best-matched hits (E ≤ 10-4) in the libraries of V. tropica and R. brunneum, respectively. Although the functional category analysis did not show remarkable differences in the distribution of functional categories between the two venom gland/sac cDNA libraries, perhaps due to the lack of functional information on many of the venom components, there were groups of genes that are specific to either V. tropica or R. brunneum. Venom allergen 5 and serine protease were found to be social wasp-specific venom transcripts. In contrast, venom peptides, metalloendopeptidases, arginine kinase and dendrotoxin were observed in solitary wasp at much higher frequencies.

Rhynchium brunneum is a widely distributed wasp species in South Eastern Asia. R. brunneum females were collected from rural provinces of Cambodia, and their total RNA and venom were extracted on site. To search for novel substances in venom, a subtracted cDNA library specific to the venom gland and sac was constructed. A total of 1118 expressed sequenced sequence tags (ESTs) were sequenced and assembled into 349 contigs (107 multiple sequences and 242 singletons). In this result, we found the putative neurotoxin (DTX protein precursor), antimicrobial peptides (teratocyte-specific caboxylesterase) together with typical major components of wasp venom (venom hyaluronidase, arginine kinase, phospholipase A2, serine/theonine protein phosphatase). Additional in-depth annotation would be required for further characterization of many unidentified genes found in the EST library.

Vespa tropica is a tropical species of Vespa found in Southeast Asia. V. tropica wasps were collected from rural provinces of Cambodia, and their total RNA and venom were extracted on site. To search for novel substances in venom, a subtracted cDNA library specific to the venom gland and sac was constructed and venom protein was analyzed by nano-LC-MS/MS. A total of 1127 expressed sequenced sequence tags (ESTs) were sequenced and assembled into 572 contigs (152 multiple sequences and 420 singletons). The short venom peptides were identified to be encoded from 5 contigs (43 ESTs) by proteomic analysis. In addition, putative antimicrobial peptides together with typical major components of wasp venom (venom allergen 5, mastoparan-like peptide, serine protease, and hyaluronidase) were identified in the EST Library. Additional in-depth annotation would be required for further characterization of many unidentified genes found in the EST library.

To determine differential gene expression profiles in the venom gland and sac (gland/sac) of a solitary hunting wasp species, Eumenes pomiformis Fabricius (1781), a subtractive cDNA library was constructed by suppression subtractive hybridization. A total of 541 expressed sequence tags (EST) were clustered and assembled into 102 contigs. In total, 37 genes were found from the library by BLASTx search and manual analysis. A eumenitin-like venom peptide, EpVP1, occupied ca. 26% of the library. A novel venom peptide, EpDTX, shared sequence similarity with trypsin inhibitors and dendrotoxin-like venom peptides known as K+ channel blockers, implying it could be responsible for the paralysis of prey. As well as phospholipase A2 and hyaluronidase known to be main components of wasp venoms, several contigs encoding enzymes, including metalloendopeptidases and a decarboxylase likely involved in the processing and activation of venomous proteins, peptides, and neurotransmitters, were also isolated from the library. The presence of a gene encoding insulin-like growth factor binding protein suggests that solitary hunting wasps might control the prey to stay in larval stage by their venom. The abundance of these venom components in the venom gland/sac and alimentary canal was confirmed by quantitative real-time PCR.