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.

Insulin/insulin-like peptide-binding protein (IBP) is abundantly found in venom of the solitary hunting wasp, Eumenes pomiformis (Hymenoptera: Eumenidae). E. pomiformis IBP (EpIBP) is most similar to insect IBP-like proteins that are known to inhibit insect growth and insulin signaling. To investigate the toxicity and target protein, EpIBP was in vivo expressed by Escherichia coli. Spodoptera exigua (Lepidoptera: Noctuidae) larvae injected with EpIBP showed a 20% lower pupation rate than the control larvae, although their body weight was not significantly different from the control when the larvae were provided artificial diet after the injection. EpIBP extended the larval stage without inducing paralysis of S. exigua larvae. To investigate the effects of EpIBP on caterpillar under a starvation condition, survivorship and body weight of the EpIBP-injected were evaluated without providing artificial diet until all the larvae died. The survivorship of the EpIBP-injected larvae was 24-36% higher than the control larvae at 4-5 d post-injection. The body weight of the control larvae reduced to 59% that is approximately 10% lower than the body weight of the EpIBP-injected larvae. These results suggest that EpIBP might inhibit the metabolism of the caterpillars, which is likely related with insulin-like peptide signaling pathway, suppress the loss of body weight and eventually extend the larval stage. An EpIBP-binding protein (EpIBPBP) isolated by immunoprecipitation was matched with a coiled-coil domain-containing protein of the fruit fly. The full-length sequence analysis of EpIBPBP is in progress.

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.

To determine differential gene expression profiles in the salivary gland of a predatory flower bug species, Orius laevigatus Fieber, a subtractive cDNA library was constructed by suppression subtractive hybridization. Digestion-related genes, including trypsin, hemolysin, lipase, cathepsin, and peritrophin, occupied 35% of the EST library. The major transcripts encoded trypsin-like serine proteases (223 ESTs, 28.8% of the total ESTs). A hemolysin gene occupied ca. 8% (42 ESTs) of the library. Hemolysin in saliva may be cytotoxic against the prey cells, thereby allowing O. laevigatus to facilitate feeding. An anticoagulation factor, lumbrokinase, also appears to act as a feeding-facilitating factor. Carbonic anhydrase, glutation peroxidase, alkaline phosphatase, and glutation S-tranfterase are expected to function as antioxidants, pH regulation- or homeostasis-related reagents in the saliva. Bactericidal permeability-increasing protein and peritrophin might protect insects from microbial infection. Interestingly, a neuroendocrinein and a huntingtin-interacting protein that could disturb the neuronal system were found in the salivary gland-specific 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 salivary gland of a water stick-insect, Ranatra chinensis Mayrt, a subtractive cDNA library was constructed by suppression subtractive hybridization. The salivary gland was determined among three salivary gland-like tissues by investigating transcription levels of five trypsin genes isolated from R. chinensis. The major transcripts encoding trypsins (64.4% of the total ESTs) were eliminated from the library and then remaining salivary gland-specific genes were searched. A total of 643 expressed sequence tags (ESTs) were clustered and assembled into 148 contigs (49 multiple sequences and 99 singletons), among which 35 contigs had matched BLASTx hits (E ≤ 1.00E-4). Salivary apyrase occupied 5.6% (36 ESTs) of the library. Apyrase is known to be released by female mosquitoes or blood-sucking assassin bugs to prevent blood clots during blood sucking. Therefore, apyrase in the salivary of R. chinensis might allow R. chinensis to facilitate feeding. Several contigs encoding acid phosphatase, hyaluronidase, prophenoloxidase, and dipeptidylpeptidase IV, commonly found in venoms of Hymenoptera, were also identified from the salivary gland-specific library. Discovery of salivary glandspecific genes should promote further studies on biologically active components in the saliva of R. chinensis.

To determine differential gene expression profiles in the salivary gland of a predatory flower bug species, Orius laevigatus Fieber, a subtractive cDNA library was constructed by suppression subtractive hybridization. The major transcripts encoding trypsins (28.6% of the total ESTs) were eliminated from the library and then remaining salivary gland-specific genes were searched. A total of 513 expressed sequence tags (ESTs) were clustered and assembled into 129 contigs (64 multiple sequences and 65 singletons). About 58% were matched with insect genes. In total, 38 genes (179 ESTs) were found from the library by BLASTx search. A hemolysin-like protein occupied ca. 8% (41 ESTs) of the library. Hemolysin is known to destruct cells including blood cells by forming pores on the cell membrane. A hemolysin-like salivary protein of O. laevigatus might be hemolytic against the prey cells, thereby allowing O. laevigatus to facilitate feeding. Several contigs encoding lipase was also identified from the salivary gland-specific library. Discovery of salivary gland-specific genes should promote further studies on biologically active components in the saliva of O. laevigatus.

To search for novel biologically active venom components, secretory venom proteins of two solitary hunting wasps, Orancistrocerus drewseni Saussure (1857) and Eumenes pomiformis Fabricus (1781), were identified by SDS-PAGE in conjunction with mass analysis with the aid of venom gland and sac-specific EST libraries constructed by suppression subtractive hybridization. Arginine kinase was the most predominant protein in both wasp venoms. Along with the full-length arginine kinase, a truncated form, which was known to have paralytic activity on a spider, was a common predominant protein in the two wasp venoms. Insulin/insulin-like peptide-binding protein was abundantly found only in E. pomiformis venom and the EST library, which might be due to its unique behaviors of oviposition and provision. It seemed that some venom proteins are secreted into venom fluid from venom gland cells via exosomes, not by signal sequence-mediated transport processes. Amphipathic α-helical peptides (10-15 amino acids) were predominantly transcribed in the venom gland/sac than protein components, and showed cell lytic activities against insect cells, mammalian cells, bacteria, and fungi. Phospholipase A2 and hyaluronidase, which are known to be the main components of wasp venoms, were found in both wasp venoms. In addition, a dendrotoxin-like peptide known to be a K+ channel blocker was also found in the venom of E. pomiformis.

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.

Three venom peptides, OdVP1, OdVP2, and OdVP3 were isolated from the venom of the solitary wasp Orancistrocerus drewseni (Hymenoptera: Eumenidae). The venom peptide amino acid sequences were determined by Q-TOF/MS/MS. The OdVP1, 2, and 3 with amidated C-terminals showed similar peptide sequences to the mastoparan from Vespula lewisii or the protonectin from Protonectarina sylveirae, suggesting that they adopt an amphipathic α-helix secondary structure. The amidation of C-terminal Leu of the venom peptides have been known to be required for their biological activities. The full-length open reading frame (ORF) sequences of the OdVP1, 2, and 3 were analyzed by 5’- and 3’-rapid amplification of cDNA ends (RACE). The overall gene structure of OdVPs showed a high homology to that of mastoparan B from Vespa basalis by containing signal sequence, prosequence, mature peptide and C-terminal glycine, but the mature peptide sequences were distinct from each other. The toxicological property and antimicrobial activity of OdVPs were characterized using synthetic peptides. This study on the venom peptides from O. drewseni should promote further studies on bioactive ingredients in the venom of solitary hunting wasps.

Cytochrome P450s (P450s) are known to oxidize a variety of insecticides including pyrethroids, thereby conferring metabolic resistance in diamondback moth (DBM), Plutella xylostella. Synergism assay with piperonyl butoxide indicated that the enhanced activity of P450 is associated with pyrethroid resistance in a cypermethrin-resistant (CR) strain. However, there were little differences in the basal transcription levels of all the P450s examined between susceptible (Sus) and CR strains, suggesting that constitutive overexpression of P450 is not likely involved in the cypermethrin resistance but induction of P450 by cypermethrin is rather associated with metabolic resistance. To determine the conditions resulting in maximum levels of P450 induction, several factors including the way of adminstration (topical application vs. leaf dipping), exposure dose and exposure duration were examined. In general, leaf dipping method resulted in greater levels of induction in a wider array of P450s. The conditions of ‘low dose (sublethal dose or concentration) and short exposure (less than 3 hr)' to cypermethrin were more efficient in P450 induction than those of ‘high dose (around LD50 or LC50) and long exposure (more than overnight)’, which have been employed in many other studies to date. Cross-strain comparison revealed that 9 of 11 P450s were induced 1.4-2.2 fold in CR whereas only 3 P450s in Sus under the optimal induction conditions, demonstrating that metabolic resistance in CR strain is actually conferred by the mechanism of selective P450 induction when exposed to cypermethrin.

To determine differential gene expression profiles in a cypermethrin-resistant strain (CR) of diamondback moth, Plutella xylostella Linnaeus (1758), a subtractive cDNA library was constructed by suppression subtractive hybridization. A total of 1196 expressed sequence tags (EST) were clustered and assembled into 579 contigs (100 multiplets and 479 singletons). About 46% (267) of 579 contigs had the matched BLASTx hits (E ≤ 10-5). Among these, 143 contigs had similarity to proteins with assigned molecular function in the Functional Catalogue database, and most of them (86%) were homologous to the genes from insects, particularly to Lepidoptera (56%). The contigs encoding carboxylesterase and cytochrome P450 known to be involved in the insecticide resistance were found in the library. They were identified as pxest3, pxest4, and CYP9G2 gene by 5' and 3' RACE. Among these, pxest4 was determined to be 2-fold over-transcribed in the CR strain by qrtPCR. Several contigs encoding enzymes including cytochrome oxidase subunit I that are likely involved in the insecticide resistance were also identified from the library. Discovery of the genes specific to cypermethrin resistance should promote further studies on the molecular mechanisms of insecticide resistance in P. xylostella.