Abstract: Pacifastin proteinase inhibitors (PPIs) play key roles in the immunity and development of insects. For exploring the function of PPIs in parasitoid wasps, two cDNA sequences, nvpp-1 and nvpp-2, encoding the open reading frames of two PPIs in Nasonia vitripennis, were successfully cloned in this study. The cDNA sequences of both nvpp-1 and nvpp-2 sequences are 723 and 888 bp in length, encoding 240 and 295 aa, respectively. The deduced amino acid sequences of nvpp-1 and nvpp-2 both possess a 17-aa signaling peptide at their N-terminus. Sequence and phylogenetic analyses results indicated that NVPP-1 and NVPP-2 contain 5 and 4 typical pacifastin conserved domains, respectively. NVPP-1 and NVPP-2 are clustered into one group with CVP4, a venom protein from Pimpla hypochondriaca. Real-time quantitative RT-PCR results revealed that the mRNAs of both nvpp-1 and nvpp-2 were expressed in all tissues of female adults tested, and the transcript levels of these two genes were significantly higher in thorax, abdomen carcass (post dissection) and venom apparatus than in the other tissues. In venom apparatus, the transcript levels of both nvpp-1 and nvpp-2 were high in the initial stage of eclosion (0 and 1 d), and then maintained at low levels. Western blot results showed that NVPP-1 and NVPP-2 were detected only in venom, not in any other tissues. The contents of NVPP-1 and NVPP-2 were low in the initial stage of eclosion (0 d). nvpp-1 and nvpp-2 were recombinantly expressed utilizing pET-28a (+) vector, and the recombinant expression products were sequentially purified. The inhibition of the recombinant NVPP-1 and NVPP-2 to four serine proteinase inhibitors, including trypsin, chymotrypsin, proteinase K and elastase were determined. The results showed that the recombinant NVPP-1 and NVPP-2 could inhibit the activities of trypsin and chymotrypsin, respectively. Meanwhile, the influences of these two recombinant proteins on phenoloxidase (PO) activity and pro phenoloxidase (PPO) activating reaction in the hemolymph obtained from the pupal stage of host Musca domestica were evaluated. The results showed that the activation of PPO in the hemolymph of the pupal stage of host was impaired by these two recombinant proteins. However, there was no remarkable influence on the PO activity in host hemolymph by these two recombinant proteins. In conclusion, NVPP-1 and NVPP-2 are present in the venom of N. vitripennis, and belong to the members of trypsin and chymotrypsin inhibitor families, respectively. They probably suppress the humoral immunity of the host via suppressing the PPO activation in its hemolymph. The results acquired from this research make a further insight into the comprehension of PPIs in insects, especially in parasitoid wasps.

Key words: Nasonia vitripennis, Musca domestica, serine proteinase inhibitors, pacifastin proteinase, humoral immunity, transcript level, enzyme activity