Host Location And Functional Analysis Of Odorant Binding Protein In Cnaphalocrocis Medinalis

Feeding, courtship and mating are fundamental for insects, and most insects rely on their sensitive antennae that express specific olfactory proteins to detect survival and reproductive-related chemical cues from the environment. The rice leaffolder, Cnaphalocrocis medinalis is widely pest. However, the molecular mechanisms of how pheromones are perceived by male C. medinalis moths are still rudimentary. The identification and functional characterization of antennal olfactory proteins in C. medinalis will enhance our knowledge of the molecular basis of insect chemoreception. We have taken systematic functional studies of the two key olfactory proteins in the C. medinalis, we have provided detailed evidences of OBPs in C. medinalis at transcriptional level and proteins level. The main results are as follows:1. Electrophysiological responses of C. medinalis to rice plant volatiles Electrophysiological responses of this species to 38 synthetic volatiles known to be released from rice plants(Poaceae: Oryza spp.) were studied using the electroantennogram(EAG) method. Compounds that elicited the strongest EAG responses for each physiological condition were selected. It is nonadecanoic,(Z)-3-hexenol,(E)-2-hexenol,(Z)-2-hexenol, Heptanol, cyclohexanol, linalool, hexanal and Methyl salicylate. Dose responses suggested that C. medinalis may rely on various EAG-active compounds in particular ratios to find the plant for feeding or for oviposition. The EAG responses to lower doses of the extract may well correspond to attraction, whereas the EAG responses to higher doses may correspond to repellency or behavioral inhibition. Statistical analyses revealed that both volatile compound chemical structure and C. medinalis physiological condition(age, sex, and mating condition) had an effect on EAG response.2. The oviposition of C. medinalis to non-Bt and Bt rice The impact of Bt rice and non- Bt rice to C. medinalis was investgated. The results of electronic nose analysis showed that the radar map and PCA analysis of Bt rice cultivars was analogous to the non- Bt rice cultivars at seedling, booting and tillering stages. There were no significant differences between the Bt rice and non-Bt rice for volatiles produced or in its physical characteristics and there were no negative impacts on C. medinalis oviposition behavior. These results add to the mounting evidence that Bt rice has no negative impact on the target insect oviposition behavior.3. Ultrastructural observations on antennal sensilla of C.medinalis Eight morphological types of sensillum were recorded in both sexes: sensilla trichodea, sensilla basiconc, sensilla coeloconica, sensilla styloconica, sensilla squamous, sensilla auricillica, B?hm bristles and sensilla cavity. S. trichodea might play a major role in sensing mechanical stimuli and in detecting chemical stimuli in insects. S. basiconc might be considered to detect sex pheromone. TEM of S. squamous reveal non-porous walls suggesting a non-olfactory function. S. coeloconica, S. styloconica and S. cavity may be involved in the perception of humidity, temperature, heat, and CO2. The results obtained provide direct morphological evidence that antennae possess structures that can play a role in finding mates and locating host plants.4. Analysis of a c DNA library from the antenna of C. medinalis and the expression pattern of olfactory genes In this study, preliminary characterization and isolation of c DNA clones from C.medinalis antennal libraries identified two general odorant-binding proteins(Cmed OBP2 and Cmed OBP3). QPCR showed that Cmed OBPs in C. medinalis seemed to be antenna-specific, but differentially expressed in male and female antennae. And also, the transcription levels of olfactory genes depended on the age, sex, and mating status of the adults. These findings support the hypothesis that OBPs and CSPs play dynamic roles during development of C. medinalis and are likely to be involved in broader physiological functions.5. Functional analysis of odorant binding protein in C. medinalis Ligand-binding characteristics, which were studied under single and binary OBP mixture conditions at different p H levels, showed that the binding affinity of Cmed OBP2 and Cmed OBP3 exhibited a wide flexibility depending on the nature of the ligand, the p H and the synergistic effect of Cmed OBPs. Also the binding sites of Cmed OBP2 and Cmed OBP3 were predicted by three-dimensional structure modelling and molecular docking, and several residues which may play an important role in binding ligands were selected. Molecular docking revealed that some residues of Cmed OBPs might play a key role in binding with ligands. It is the first time to provide an evidence of olfactory compensation, when the expression of olfactory gene downregulated, a significant upregulation could be observed for the similar gene to compensate the olfactory function.The essential cooperative interaction of Cmed OBP2 and Cmed OBP3 in the perception of rice plant volatiles was ultimately demonstrated by ds RNA-Cmed OBP2 & 3 injections that induced a higher reduction of the EAG responses than that induced by single ds RNA.The identification and functional characterization of OBPs in C. medinalis will enhance our knowledge of the molecular and cellular basis of insect chemoreception. More importantly, can provide us new methods to control this pest through interring their olfaction perception.