Cloning And Expression Profiles Of Olfactory Receptor Genes In Microplitis Mediator(Hymenoptera:Braconidae)

Olfaction plays important roles in insects’ behaviors, such as host-seeking, enemy-defending, mating and immigration. Olfactory receptors (ORs) are believed to fulfill an indispensable role in the connecting environment odorant and the olfactory receptor neurons (ORNs).The parasitic wasp, Microplitis mediator (Hymenoptera:Braconidae). is a polyphagous solitary larval endo-parasitoid of approximately40lepidopteran insect species, such as the cotton bollworm Helicoverpa armigera (Hiibner), the oriental armyworm Leucania separata (Walker) and the cabbage moth Barathra brassicae (Linnaeus). For the control of these lepidopteran insect species, M. mediator acts as an important component in the Integrated Pest Mangement (IPM). Using molecular biology approaches to study the olfactory mechanisms has become a hot spot, which can offer new strategies and approaches in the insect pest control and natural enemy use.1.The construction of antennal cDNA library of Microplitis mediatorIn present study, we constructed a high-quality antennal cDNA library of the parasitic wasp M. mediator. Through high-throughput sequencing of the expressed sequencing tags (EST),4785high-quality ESTs were acquired. Using BLASTx and BLASTn programs, a number of olfactory-related genes were identified from this antennal cDNA library. including25olfactory receptor genes.2. Cloning and analysis of olfactory receptor genes in Microplitis mediatorBased on the fragments from the cDNA library,13full-length OR genes (MmedOrl-MmedOrl3) were obtained using rapid amplification of cDNA ends (RACE) method and these sequences were analyzed by bioinfornatics. Although the similarities between these ORs and with ORs from other insect species were low, some conserved amino aicd residues were found (G-W-K-Y-H-F-C-W). By prediction of ORs structures, it was found that all ORs had7transmembrane regions, the intracellular N-terminal and extracelluar C-terminal.3. Tissue distribution of olfactory receptors in the parasitoid Microplitis mediatorThe expression profiles of these OR genes and one atypical odorant receptor gene (MmedOrco) were evaluated by Real-time PCR. The results indicated that these OR genes had different expression levels among tissues and genders. However, all OR genes are highly expressed in antenna. Three OR genes (MmedOr2, MmedOr12and MmedOr13) are highly expressed in female antennae, while eight ORs (MmedOrco, MmedOr3, MmedOr4, MmedOr5, MmedOr7, MmedOr8, MmedOr9and MmedOr11) are highly expressed in male antennae. The expression levels of three ORs (MmedOr1, MmedOr6and MmedOr10) showed no differences between male and female antennae. Furthermore, MmedOr6and MmedOr8.appeared relatively high expression in legs, while MmedOr10and MmedOr11seemed to have relatively high expression in heads.The quantitative changes of MmedOr2, an antenna-specific olfactory receptor highly expressed in female antennae, at different developmental stages were detected using real-time PCR. Its expression levels reached the maximum amount on the3rd emergence day of female while the male reached the maximum amount at the4th day after emergence. After the the maximum amount, the expression level of MmedOr2in male and female adult drops gradually. Moreover, the expression level in female decreased after mating.