Functional Characterization Of Niemann-Pick Type C2 Proteins In The Parasitoid Wasp Microplitis Mediator(Hymenoptera:Braconidae)

Microplitis mediator is an important parasitic natural enemy of cotton bollworm and other Lepidoptera insect pests.In general,M.mediator mainly depend on it's olfactory system to identify semiochemicals from external environment and locate the hosts.The odor carrier proteins are involved in the first stage of odor perception,therefore,odor carrier proteins may play important roles in olfactory recongnition of wasps.Niemann–Pick type C2(NPC2)is a type of small soluble protein involved in lipid metabolism in vertebrates.Recent studies showed that NPC2 s also participated in chemical communication of arthropods.In this study,we investigated the potential function of NPC2 proteins in M.mediator at RNA level,protein level and electrophysiology level.The main results were as follows:1.From the previous transcriptome data,we obtained the full length of two NPC2 genes,MmedNPC2 a and MmedNPC2 b with coding 148 and 158 amino acids respectively,in M.mediator by RACE,and MmedNPC2 a and MmedNPC2 b shared sequence identity of 26.09%.Results of phylogenetic tree indicated that MmedNPC2 a and Mmed NPC2 b belonged to NPC2 gene family,but different branches.Moreover,Mmed NPC2 a and NPC2 of Camponotus Japonicus also belonged to the same clade.Real-time quantitative PCR(qPCR)analysis revealed that the MmedNPC2 a was expressed significantly higher in the antennae than in other tissues of adult wasps,compared with MmedNPC2 b.Therefore,we speculated that MmedNPC2 a might be involved in the olfactory communication of M.mediator.2.The recombinant MmedNPC2 a and MmedNPC2 b were obtained to make specific polyclonal antibodies after being cloned,expressed and purified.The immuno-gold localization results demonstrated that MmedNPC2 a was located in the lymph cavities of sensilla placodea,while MmedNPC2 b was located in the lymph of the center of antenna.The distribution differentiation of two proteins suggested that they may play different roles,and MmedNPC2 a was more likely to be involved in the olfactory communication of M.mediator.3.The fluorescence binding assay results showed that MmedNPC2 a had strong binding ability to seven candicated cotton volatiles,such as 4-ethylbenzaldehyde,3,4-dimethylbenzaldehyde,?-ionone,linalool,m-xylene,benzaldehyde,and trans-2-hexen-1-al,which preliminarily proved that MmedNPC2 a was involved in the olfactory recognition of host plants of M.mediator.The 3D model of MmedNPC2 a was built by SWISS-MODEL.Compared with the structure CjapNPC2 by using Swiss-PdbViewer,we found that MmedNPC2 a was composed of seven ?-sheets and three pairs of disulfide bridges.In this model,the key binding residues for oleic acid in CjapNPC2,Lue68,Lys69,Lys70,Phe97,Thr103,and Phe127,were replaced with Phe85,Ser86,His87,Leu113,Tyr119,and Ile143 in MmedNPC2 a,respectively..4.The antennal electrophysiological responses of M.mediator to seven volatiles which showed high binding abilities to recombinant MmedNPC2 a were evaluated by using EAG.The results showed that linalool,4-ethyl benzaldehyde,and trans-2-hexen-1-al could stimulate strong EAG responses of antanae of wasps.In short,MmedNPC2 a may play crucial roles in olfactory perception of M.mediator.These findings provide a theoretical basis for the next step of the research for the olfactory system of M.mediator and maybe helpful for designing the modulator of the olfactory behavior of M.mediators and the strategy of preserving natural enemies to control insect pests.