Identification And Functional Characterization Of Olfactory Related Genes In Macrocentrus Cingulum

Olfaction or the sense of smell is one of the major modes of communication in wasps, playing an integral part in the wasp’s ability to locate mates for reproduction, location of host, oviposition sites and avoid toxin substances. Some of the key players of the wasps olfactory system include chemosensory sensilla, olfactory related protein especially odorant binding proteins (OBP) and olfactory receptors (OR). The aim of this study was to investigate the molecular mechanisms of olfaction in the polyembronic endoparasitoids, Macrocentrus cingulum. Consequently, in an effort to expand our understanding of chemosensory pathways, we construct the cDNA library from the male and female antennae for identifying the olfaction-related genes and olfactory signal transduction mechanisms in M. cingulum. For that, we sequenced3160unigenes and annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG ontology (KO). Through the homology search, we identified9ORs,3IRs and1OBP genes from the cDNA library sequences. Additionally, the expression patterns of these ORs and IRs in different tissues (antennae, heads, thoraxes, abdomens, and legs) were demonstrated by RT-PCR. The qualitative gene expression analyses showed that most of the ORs genes were more highly expressed in female antennae than male; whereas, IRs, unlike ORs; were more expressed in various male tissues than that of females.The McinOrco shares high degree of sequence homology with known Oreo proteins in other insects. Real-time PCR experiments indicate that the McinOrco is expressed mainly in adult antennae, with expression levels differing by sex. The expressing cells were visualized by adapting a whole mount fluorescence in situ hybridization method. Orco expressing sensory neurons were shown to be abundant in all39segments of male antenna. The highest densities of Orco-expressing cells were identified in segment20, whereas segment7comprised in low level. In case of female antenna, levels of Orco-expressing cells were lower than male antenna. Almost expressed cells were distributed over40flagellomeres, with decreasing numbers towards the proximal end. These demonstrate the sexual dimorphism concerning the expression cells of Orco.The McinOBPl was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR experiments indicate that the McinOBPl is expressed mainly in adult antennae, with expression levels differing by sex. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the McinOBPl can bind green-leaf volatiles, including aldehydes and terpenoids, but also can bind aliphatic alcohols with good affinity, in the order trans-2-nonenal>cis-3-hexen-l-ol>trans-caryophelle, suggesting a role of McinOBPl in general odorant chemoreception. Those three odorants used in further homology modeling and ligand docking based on their binding affinity. The Va158, Leu62and Glu130are the key amino acids in the binding pockets that bind with these three odorants. The three mutants, Va158, Leu62and Glu130, where the valine, leucine and glutamic residues were replaced by alanine, proline and alanine, respectively; showed reduced affinity to these odorants. This information suggests Va158, Leu62and Glu130are involved in the binding of these compounds, possibly through the specific recognition of ligands that forms hydrogen bonds with the ligands functional groups.The external morphology and ultrastructure of the antennal sensilla and sense organs on the female ovipositor and sheath of this parasitoid were examined by using scanning and transmission electron microscopy. Antennae of male and female of the M. cingulum are filiform in shape,5.90-6.64mm in length and consist of scape, pedicel, and flagellum with39and40flagellomeres, respectively. Cuticular pore and nine types of morphologically distinct sensilla were identified in both sexes, including two types of sensilla chaetica (nonporous), s. trichodea (nonporous), s. basiconica I (nonporous blunt tip), s. basiconica Ⅱ (porous wall) and basiconica III (nonporous wall) with branched blunt tip, s. coeloconica with finger-like projections, protruded s. campaniform with central tip pore, and plate-like s. placodea (porous). The number, morphology, and distribution of sensilla were compared between sexes. No differences in antenna shape and basic structure in the males and females, but male antennae length and width were significantly greater than those of females. Furthermore, males had more placodea than females. Three types of sensilla trichodea were shown to be abundant on the outer sheath of the ovipositor, with types Ⅱ and Ⅲ being most distal, and the inner surface of the ovipositor covered with microtrichia, more densely near the apex. S. coeloconica are distributed on both ventral and dorsal valves, while campaniform sensilla and secretory pores are only located on the dorsal valve. The olistheter-like interlocking mechanism, as well as the morphology of the ventral and dorsal valve tips and the ventral valve seal may be important in stinging, oviposition and in the host selection process.In Y-tube olfactometer behavioral assays, corn odorants of tereponids, aldehydes and alcohols that individually or collectively attract the parasitoid, M. cingulum, to its host habitat.