Gene duplication and subsequent functional diversification of maltase in fig wasp (Chalcidoidea, Hymenoptera).

Maltase can catalyze the hydrolysis of α-1,4-glucosidic linkages and release α-d-glucoses that are used as a source of energy by insects. Maltase has been extensively studied in Lepidoptera and Diptera, while the characterization and evolutionary history of maltase are largely unknown in Hymenoptera. Here, we undertook a bioinformatics study and identified 105 maltase genes in 12 fig wasp species. Together with the maltase genes of Nasonia vitripennis and Apis mellifera, phylogenetic analysis showed that all the maltase genes were clustered into three clades. Clade I and III included maltase genes from all the fig wasp species, while clade II contained the maltase genes from non-pollinating fig wasps (NPFWs) only. Interestingly, the maltase genes located in clade II were intronless. Fig pollinators and NPFWs had lineage-specific gene expansion in clade I and II respectively, which were mainly derived from tandem duplications. The three clades displayed distinct gene structures. Furthermore, maltase showed significant functional divergence among the three clades and the critical amino acid sites were detected. These sites could be responsible for the ligand-binding preference and hydrolytic specificity. Overall, our results demonstrated that maltase might contribute to the discrepancy of life histories and feeding regimes between fig pollinators and NPFWs.

A Role of Newly Found Auxiliary Site in Phospholipase A1 from Thai Banded Tiger Wasp (Vespa affinis) in Its Enzymatic Enhancement: In Silico Homology Modeling and Molecular Dynamics Insights.

Phospholipase A1 from Thai banded tiger wasp (Vespa affinis) venom also known as Ves a 1 plays an essential role in fatal vespid allergy. Ves a 1 becomes an important therapeutic target for toxin remedy. However, established Ves a 1 structure or a mechanism of Ves a 1 function were not well documented. This circumstance has prevented efficient design of a potential phospholipase A1 inhibitor. In our study, we successfully recruited homology modeling and molecular dynamic (MD) simulation to model Ves a 1 three-dimensional structure. The Ves a 1 structure along with dynamic behaviors were visualized and explained. In addition, we performed molecular docking of Ves a 1 with 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) lipid to assess a possible lipid binding site. Interestingly, molecular docking predicted another lipid binding region apart from its corresponding catalytic site, suggesting an auxiliary role of the alternative site at the Ves a 1 surface. The new molecular mechanism related to the surface lipid binding site (auxiliary site) provided better understanding of how phospholipase A1 structure facilitates its enzymatic function. This auxiliary site, conserved among Hymenoptera species as well as some mammalian lipases, could be a guide for interaction-based design of a novel phospholipase A1 inhibitor.

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT7 genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.

A digestive tract expressing α-amylase influences the adult lifespan of Pteromalus puparum revealed through RNAi and rescue analyses.

BACKGROUND: Midgut and salivary gland α-amylases are digestive enzymes required for the development of insects and have been investigated in some insect species. However, α-amylases in the endoparasitioid wasps have not been reported. Pteromalus puparum (Hymenoptera: Pteromalidae) is a dominant endoparasitioid wasp that parasitizes many butterfly species, including the Brassicaceae pest Pieris rapae (Lepidoptera: Pieridae). Here, we studied the characteristics and functions of three α-amylases in P. puparum. RESULTS: We cloned three genes encoding α-amylases in P. puparum, PpAmy1, PpAmy2 and PpAmy3. The full length of the PpAmy1 cDNA is 1872 bp, encoding 496 amino acids, the PpAmy2 cDNA is 1863 bp long, encoding 518 amino acids, and PpAmy3 cDNA consists of 1802 bp encoding 521 amino acids. PpAmys are highly similar in amino acid sequences, but they have separate tissue distributions. Phylogenetic results show that gene duplications may occur between PpAmy2 and PpAmy3. PpAmy1 and PpAmy3 are most highly expressed in the digestive tract and the venom apparatus, respectively, while PpAmy2 is broadly expressed in all tissues. We report that PpAmy1 acts in the digestive tract, where it influences lifespan as demonstrated using RNAi and α-amylase rescue analyses, and there is no significant difference in longevity when PpAmy2 and PpAmy3 are knocked down. CONCLUSION: PpAmys probably have roles in carbohydrate metabolism of P. puparum and its host/parasitoid relationships. The characterization and functional study of PpAmys lays the foundation for the protection and utilization of parasitoid resources, and the biological control of agricultural pests. © 2019 Society of Chemical Industry.

Mapping key amino acid residues for the epimerase efficiency and stereospecificity of the sex pheromone biosynthetic short-chain dehydrogenases/reductases of Nasonia.

Males of the parasitic wasp genus Nasonia use blends of chiral hydroxylactones as sex pheromones to attract conspecific females. Whereas all Nasonia species use a mixture of (4R,5S)-5-hydroxy-4-decanolide (RS) and 4-methylquinazoline (MQ) as sex pheromones, Nasonia vitripennis evolved (4R,5R)-5-hydroxy-4-decanolide (RR) as an extra sex pheromone component. We recently identified and functionally characterized three short-chain dehydrogenases/reductases (SDRs) NV10127, NV10128, and NV10129 that are capable of catalyzing the epimerization of RS to RR via (4R)-5-oxo-4-decanolide (ODL) as intermediate. Despite their very high sequence identities of 88-98%, these proteins differ drastically in their ability to epimerize RS to RR and in their stereoselectivity when reducing ODL to RR/RS. Here, in order to unravel the sequence differences underlying these varying functional properties of NV1027, NV10128 and NV10129, we created chimeras of the three enzymes and monitored their catalytic activities in vitro. The results show that a few amino acid changes at the C-termini and active sites of Nasonia vitripennis SDRs lead to substantially altered RS to RR epimerization and ODL-reduction activities. Thus, our study adds to the understanding of pheromone evolution by showing that subtle mutations in key biosynthetic enzymes can result in drastic effects on the composition of chemical signals.

Superoxide dismutase from venom of the ectoparasitoid Scleroderma guani inhibits melanization of hemolymph.

Superoxide dismutase (SOD) known as an important antioxidative stress protein has been recently found in venoms of several parasitoid wasps. However, its functions and characteristics as a virulent factor remain scarcely described. Here, we report the characterization of two venomous SOD genes (SguaSOD1 and SguaSOD3) from the ectoparasitoid, Scleroderma guani. The metal binding sites, cysteine amino acid positions and signature sequences of the SOD family were conserved within SguaSOD1 and SguaSOD3. Relatively high levels of their transcripts were observed in pupae followed a decrease in early adults, after which they had the highest transcriptions, indicating that their productions would be regulated in venom apparatus. Although the two genes showed lower expression in venom apparatus compared to head and thorax, the enzymatic assay revealed that SOD indeed had activity in venom. Further, we showed that recombinant SguaSOD3 suppressed melanization of host hemolymph, implying that this protein used as a virulent factor uniquely impacts the prophenoloxidase cascade.

Phospholipase A1-based cross-reactivity among venoms of clinically relevant Hymenoptera from Neotropical and temperate regions.

Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.

Biochemical characterization of a phospholipase A2 homologue from the venom of the social wasp Polybia occidentalis

Background: Wasp venoms constitute a molecular reservoir of new pharmacological substances such as peptides and proteins, biological property holders, many of which are yet to be identified. Exploring these sources may lead to the discovery of molecules hitherto unknown. This study describes, for the first time in hymenopteran venoms, the identification of an enzymatically inactive phospholipase A2 (PLA2) from the venom of the social wasp Polybia occidentalis. Methods: P. occidentalis venom was fractioned by molecular exclusion and reverse phase chromatography. For the biochemical characterization of the protein, 1D and 2D SDS-PAGE were performed, along with phospholipase activity assays on synthetic substrates, MALDI-TOF mass spectrometry and sequencing by Edman degradation. Results: The protein, called PocTX, was isolated using two chromatographic steps. Based on the phospholipase activity assay, electrophoresis and mass spectrometry, the protein presented a high degree of purity, with a mass of 13,896. 47 Da and a basic pI. After sequencing by the Edman degradation method, it was found that the protein showed a high identity with snake venom PLA2 homologues. Conclusion: This is the first report of an enzymatically inactive PLA2 isolated from wasp venom, similar to snake PLA2 homologues.

Biochemical characterization and comparison of aspartylglucosaminidases secreted in venom of the parasitoid wasps Asobara tabida and Leptopilina heterotoma.

Aspartylglucosaminidase (AGA) is a low-abundance intracellular enzyme that plays a key role in the last stage of glycoproteins degradation, and whose deficiency leads to human aspartylglucosaminuria, a lysosomal storage disease. Surprisingly, high amounts of AGA-like proteins are secreted in the venom of two phylogenetically distant hymenopteran parasitoid wasp species, Asobara tabida (Braconidae) and Leptopilina heterotoma (Cynipidae). These venom AGAs have a similar domain organization as mammalian AGAs. They share with them key residues for autocatalysis and activity, and the mature α- and ß-subunits also form an (αß)2 structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and ß for LhAGA) is glycosylated instead of the two subunits for lysosomal human AGA (hAGA), and these glycosylations are partially resistant to PGNase F treatment. The two venom AGAs are secreted as fully activated enzymes, they have a similar aspartylglucosaminidase activity and are both also efficient asparaginases. Once AGAs are injected into the larvae of the Drosophila melanogaster host, the asparaginase activity may play a role in modulating their physiology. Altogether, our data provide new elements for a better understanding of the secretion and the role of venom AGAs as virulence factors in the parasitoid wasps' success.

An insect with a delta-12 desaturase, the jewel wasp Nasonia vitripennis, benefits from nutritional supply with linoleic acid.

The availability of linoleic acid (LA; C18:2(∆9,12)) is pivotal for animals. While vertebrates depend on a nutritional supply, some invertebrates, including the parasitic wasp Nasonia vitripennis, are able to synthesize LA from oleic acid (OA; C18:1(∆9)). This raises the question as to whether these animals nevertheless benefit from the additional uptake of LA with the diet. LA plays an important role in the sexual communication of N. vitripennis because males use it as a precursor for the synthesis of an abdominal sex pheromone attracting virgin females. We reared hosts of N. vitripennis that were fed diets enriched in the availability of stearic acid (SA: C18:0), OA or LA. N. vitripennis males developing on the different host types clearly differed in both the fatty acid composition of their body fat and sex pheromone titres. Males from LA-enriched hosts had an almost fourfold higher proportion of LA and produced significantly more sex pheromone than males from SA (2.2-fold) and OA (1.4-fold) enriched hosts, respectively. Our study demonstrates that animals being able to synthesize important nutrients de novo may still benefit from an additional supply with their diet.

Adaptation of Habrobracon hebetor (Hymenoptera: Braconidae) to Rearing on Ephestia kuehniella (Lepidoptera: Pyralidae) and Helicoverpa armigera (Lepidoptera: Noctuidae).

Food characteristics strongly regulate digestive enzymatic activity of insects through direct influences on their midgut mechanisms. Insect performance is better on diets that contain nutrients in proportions that fit its digestive enzymes. Little is known about the influences of rearing history on parasitism success of Habrobracon hebetor Say. This research focused on the effect of nutrient regulation on survival, development, and parasitism of H. hebetor. Life history and digestive enzyme activity of fourth-stage larvae of H. hebetor were studied when reared on Ephestia kuehniella Zeller. This parasitoid was then introduced to Helicoverpa armigera (Hübner), and above-mentioned parameters were also studied in the first and fourth generations after transfer. In term of parasitism success, H. hebetor preferred E. kuehniella over He. armigera. When the first and fourth generations of He. armigera-reared H. hebetor were compared, the rearing history affected the life history and enzymatic activity of the parasitoid. A better performance of H. hebetor was achieved after it was reared on He. armigera for the four generations. Because, digestive α-amylase and general protease of the parasitoid were matched with the new host, it used reserve energy for a better performance. Thus, a better performance of H. hebetor could be obtained when the parasitoid was reared on its original host for at least four generations.

Diapause induction, color change, and cold tolerance physiology of the diapausing larvae of the Chouioia cunea (Hymenoptera: Eulophidae).

The chalcid wasp Chouioia cunea Yang (Hymenoptera: Eulophidae) is one of the most dominant pupal parasitoids of Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), an invasive pest of many forestry trees and agricultural crops. For mass rearing C. cunea for biological control purposes, the pupae of Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae) have been widely used as a substitute host in China. In this article, photoperiodic effect on diapause induction in C. cunea within the pupae of A. pernyi was investigated, and the differences in cold tolerance physiology including supercooling point, water content, and activities of three protective enzymes (Peroxidase [POD], Catalase [CAT], and Superoxide dismuase [SOD]) between diapause and nondiapause mature larvae were comparatively determined. Our results revealed that C. cunea possess a short-day induced larval diapause. The critical photoperiods for diapause induction in C. cunea were estimated to be between a photoperiod of 13:11 and 14:10 (L:D) h at 18°C, or between a photoperiod of 12:12 and 13:11 (L:D) h at 21°C or 24°C. We also found that the color of C. cunea diapausing larvae was taupe, while the normally developed (nondiapausing) individuals were light yellow. This body color change can be used as an indicator of diapause entry of C. cunea larave. The average supercooling point of diapausing mature larvae were lower than those of nondiapausing ones. There were significant differences in the activity of three protective enzymes (POD, CAT, and SOD) between diapausing and nondiapausing mature larvae.

Identification and characterization of ace2-type acetylcholinesterase in insecticide-resistant and -susceptible parasitoid wasp Oomyzus sokolowskii (Hymenoptera: Eulophidae).

A full-length acetylcholinesterase (AChE) cDNA sequence (Os-ace2.s) from insecticide-susceptible (S) parasitoid Oomyzus sokolowskii (Hymenoptera: Eulophidae) and a partial cDNA sequence (Os-ace2.r) from insecticide- resistant (R) O. sokolowskii were identified firstly. Both Os-ace2.s (encoding a protein of 639 amino acid residues) and Os-ace2.r (encoding a protein of 530 amino acid residues) contained the typical conserved motifs, including FGESAGdomains, catalytic triad, acyl pocket, three oxy-anino hole, choline binding site, peripheral anionic site, omega loop and conserved aromatic residues. The multiple alignment and Blast results indicated that Os-ace2.s were ace2 member of AChE gene. There were three replacements of the amino acid residues (Glu 115 Leu, Phe 394 Leu, and Lys 424 Arg) between Os-ace2.s and Os-ace2.r. The ace2 of O. sokolowskii was the AChE gene firstly isolated from hymenopteran parasitoid so far. R O. sokolowskii displayed about 15-20-folds resistance ratios to methamidophos and avermectin. The bimolecular rate constant (k i) value in S O. sokolowskii was 3.8-folds for methamidophos and 12.3 for dichlorvos, respectively higher than those in R O. sokolowskii. The results indicated that the insensitive AChE and replacements of the amino acid residues in Os-ace2 might be involved in the resistance to methamidophos in R O. sokolowskii.

Identification of the main venom protein components of Aphidius ervi, a parasitoid wasp of the aphid model Acyrthosiphon pisum.

BACKGROUND: Endoparasitoid wasps are important natural enemies of the widely distributed aphid pests and are mainly used as biological control agents. However, despite the increased interest on aphid interaction networks, only sparse information is available on the factors used by parasitoids to modulate the aphid physiology. Our aim was here to identify the major protein components of the venom injected at oviposition by Aphidius ervi to ensure successful development in its aphid host, Acyrthosiphon pisum. RESULTS: A combined large-scale transcriptomic and proteomic approach allowed us to identify 16 putative venom proteins among which three γ-glutamyl transpeptidases (γ-GTs) were by far the most abundant. Two of the γ-GTs most likely correspond to alleles of the same gene, with one of these alleles previously described as involved in host castration. The third γ-GT was only distantly related to the others and may not be functional owing to the presence of mutations in the active site. Among the other abundant proteins in the venom, several were unique to A. ervi such as the molecular chaperone endoplasmin possibly involved in protecting proteins during their secretion and transport in the host. Abundant transcripts encoding three secreted cystein-rich toxin-like peptides whose function remains to be explored were also identified. CONCLUSIONS: Our data further support the role of γ-GTs as key players in A. ervi success on aphid hosts. However, they also evidence that this wasp venom is a complex fluid that contains diverse, more or less specific, protein components. Their characterization will undoubtedly help deciphering parasitoid-aphid and parasitoid-aphid-symbiont interactions. Finally, this study also shed light on the quick evolution of venom components through processes such as duplication and convergent recruitment of virulence factors between unrelated organisms.

Early presence of an enolase in the oviposition injecta of the aphid parasitoid Aphidius ervi analyzed with chitosan beads as artificial hosts.

Maternal factors of female wasps that are injected into hosts with their eggs at oviposition play a major role in strategies used by insect parasitoids to overcome host immunity, and to regulate host physiology during early stages of parasitism. Here, we attempted to precisely determine and compare the protein patterns injected by the endoparasitoid Aphidius ervi into two different host systems. Chitosan beads of aphid size designed as artificial and physiologically inert hosts were used as oviposition medium, to be compared with the natural aphid host as young nymphs of Macrosiphum euphorbiae. Proteins that the A. ervi wasp injects into hosts at oviposition were separated by SDS-PAGE, complemented with proteomic techniques. Analyses confirm the identification of A. ervi γ-glutamyl transpeptidase (γ-GT) as a key component in the venom of the endoparasitoid. Using proteomic techniques, the quantity of γ-GT injected by the A. ervi wasp into aphids along with the egg was estimated as approximately 4ng per oviposition strike. We suggest that similar quantities suffice to explain natural parasitization success in A. ervi, which do not rely on polydnavirus to establish into hosts. Moreover, an enolase that showed a high level of sequence identity with teratocyte A. ervi enolase was detected both in chitosan beads extracts, and in extracts of mature eggs excised from the A. ervi ovaries, but not in its venom glands extracts. Detecting enolase shortly after oviposition in the artificial inert hosts at a stage of parasitism when the A. ervi egg is still in the primary chorionated undifferentiated stage suggests the enolase as a chorionic protein of the mature egg. The possible functions of this enolase enzyme for the establishment and early development of A. ervi in aphid hosts are discussed.

Eicosanoids mediate cellular immune response and phenoloxidase reaction to viral infection in adult Pimpla turionellae.

Nodulation is the predominant insect cellular immune response to microbial infections. We posed the hypothesis that parasitoid insects in their adulthood express melanotic nodulation reactions to viral challenge and that eicosanoids mediate nodulation reactions and phenoloxidase (PO) activation in response to viral challenge. To test this idea, we injected Pimpla turionellae adults with indomethacin, a nonsteroidal anti-inflammatory drug, immediately prior to intrahemocoelic injection of Bovine herpes simplex virus-1 (BHSV-1). Treating newly emerged adults of P. turionellae with BHSV-1 induced nodulation reactions, and decreased PO activity at high viral doses. Relative to vehicle-treated controls, indomethacin-treated adults produced significantly reduced numbers of nodules following viral infection (down from approximately 21 nodules per adult to less than six nodules per adult). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 0.1%) were associated with decreasing nodulation (by six-fold) and PO (by about three-fold) reactions to BHSV-1 injection. Wasp adults orally fed with the lowest dietary indomethacin concentration (0.001%) expressed significantly increased PO activity (1.45 unit/min/mg protein) while nodulation reaction was not affected in response to viral challenge compared to control adults. We infer from these findings that cyclooxygenase (COX) products, at least prostaglandins, mediate nodulation response and PO action to viral infection in adults of these highly specialized insects.

Proteomic characterization of the hyaluronidase (E.C. 3.2.1.35) from the venom of the social wasp Polybia paulista.

Polybia paulista wasp venom possesses three major allergens: phospholipase A1, hyaluronidase and antigen-5. To the best of our knowledge, no hyaluronidase from the venom of Neotropical social wasps was structurally characterized up to this moment, mainly due to its reduced amount in the venom of the tropical wasp species (about 0.5% of crude venom). Four different glycoproteic forms of this enzyme were detected in the venom of the wasp Polybia paulista. In the present investigation, an innovative experimental approach was developed combining 2-D SDS-PAGE with in-gel protein digestion by different proteolytic enzymes, followed by mass spectrometry analysis under collision-induced dissociation CID) conditions for the complete assignment of the protein sequencing. Thus, the most abundant form of this enzyme in P. paulista venom, the hyaluronidase-III, was sequenced, revealing that the first 47 amino acid residues from the N-terminal region, common to other Hymenoptera venom hyaluronidases, are missing. The molecular modeling revealed that hyaluronidase-III has a single polypeptide chain, folded into a tertiary structure, presenting a central (ß/α)5 core with alternation of ß-strands and α-helices; the tertiary structure stabilized by a single disulfide bridge between the residues Cys189 and Cys201. The structural pattern reported for P. paulista venom hyaluronidase-III is compatible with the classification of the enzyme as member of the family 56 of glycosidase hydrolases. Moreover, its structural characterization will encourage the use of this protein as a model for future development of "component-resolved diagnosis".

Salivary secretions from the ectoparasitic wasp, Eulophus pennicornis contain hydrolases, and kill host hemocytes by apoptosis.

The current work demonstrates that larvae of the ectoparasitic wasp, Eulophus pennicornis, produce salivary secretions (Ep S) that contain a variety of hydrolases. This includes medium to high levels of N-acetyl-ß-glucosaminidase activity, relatively low levels of esterase (C 4) and lipase (C 14) activity, and trace levels of esterase lipase (C 8), acid phosphatise, ß-galactosidase, ß-glucuronidase, and α-glucosidase activity. In addition, in vitro monolayer assays indicate that both a 4 h and an 18 h incubation of hemocytes from host Lacanobia oleracea larvae in Ep S significantly increases (P < 0.001) the percentage of cells dying by apoptosis compared to the controls. Moreover, an 18 h incubation of hemocytes in Ep S (but not a 4 h incubation), also significantly increased the percentage of dead cells detected using a trypan-blue exclusion assay. The role of ectoparasitoid salivary secretions in conditioning the host by disabling hemocyte-mediated wound healing responses, and providing food for the developing wasp larvae is discussed.

DNA methylation plays a crucial role during early Nasonia development.

Although the role of DNA methylation in insect development is still poorly understood, the number and role of DNA methyltransferases in insects vary strongly between species. DNA methylation appears to be widely present among the social hymenoptera and functional studies in Apis have suggested a crucial role for de novo methylation in a wide variety of developmental processes. The sequencing of three parasitoid Nasonia genomes revealed the presence of three Dnmt1 (Dnmt1a, Dnmt1b and Dnmt1c) genes and one Dnmt2 and Dnmt3 gene, suggesting a role of DNA methylation in Nasonia development. In the present study we show that in Nasonia vitripennis all Dnmt1 messenger RNAs (mRNAs) and Dnmt3 mRNA are maternally provided to the embryo and, of these, Dnmt1a is essential during early embryogenesis. Lowering of maternal Dnmt1a mRNA results in embryonic lethality during the onset of gastrulation. This dependence on maternal Dnmt1a during embryogenesis in an organismal group outside the vertebrates, suggests evolutionary conservation of the function of Dnmt1 during embryogenesis.

Functional expression and characterization of dipeptidyl peptidase IV from the black-bellied hornet Vespa basalis in Sf21 insect cells.

The maturation of mastoparan B, the major toxin peptide in the venom of Vespa basalis, requires enzymatic cleavage of its prosequence presumably via sequential liberation of dipeptides. The putative processing enzyme, dipeptidyl peptidase IV, was expressed as a glycosylated His-tag fusion protein (rDPP-IV) via the baculovirus expression system. rDPP-IV purified by one-step nickel-affinity chromatography was verified by Western blot and LC-MS/MS analysis. The k(cat)/K(m) of rDPP-IV was determined to be in the range of 10-500 mM(-1)·S(-1) for five synthetic substrates. The optimal temperature and pH for rDPP-IV were determined to be 50 °C and pH 9. Enzymatic activity of rDPP-IV was significantly reduced by 80 and 60% in the presence of sitagliptin and phenylmethylsulfonyl fluoride respectively.