The Mechanism Studies Of Sex Pheromone Signals Recognition In Helicoverpa Armigera And Helicoverpa Assulta

Though long-time animal evolution process,insects need to form a complicated olfactory system to recognize chemical information closely related to their life demands in complex surroundings and further to accomplish a serious of significant behaviors such as defense predators,finding host,mating preference and oviposition site selection.Of these behaviors,sex pheromone communication between the sexes presented high specificity and sensitivity,which contributed to intraspecific reproduction isolation.In addition,pheromones can also be exploited new attractants to widely application in pest control and monitoring.But,such control methods have some inevitable defects,such as pheromone instability structure,easily volatilization,function only on male and resistance to the attraction effects with long term using.So,making clearly pheromone communication system also provided some significant theory suggestions for pest control.Recently years,numerous studies showed that male depended on ten thousand of sensilla trichoidea located on antenna to detected one or more pheromone components female released and several olfactory protein families inner sensilla trichoidea have been reported to involve in peripheral signal conversion,such as Pheromone binding proteins(PBPs),Pheromone receptors(PRs),Sensory neuron membrane proteins(SNMPs),Pheromone degrading enzymes(PDEs).With the advent of Next Generation Sequence(NGS),these olfactory genes were identified in most of Lepidoptera species.Undoubtedly,these facilitated in documenting their in vivo function.However,little novel information was foncused on peripheral olfactory system,especially the co-expression models of pheromone receptors under pheromone-sensitivity sensilla function in related species,the function of pheromone antagonist.Given these,our studies focus on Helicoverpa armigera and Helicoverpa assulta.With the help of molecular biology,insect behavior,electrophysiological recording and gene editing techniques,we have succeeded in documenting the mechanism of pheromone-drive behavior in these important agricultural pests.The main results are as follows.1.The sensillar expression models of receptor-neuron in H.armigera and H.assulta perception pheromones.In this study,by using single sensillum recordings to identify the functions types of sensilla trichodea,heterologous expression system Xenopus occytes to analysis receptor function and two-colour in situ hybridization technique to identify which ORs were expressed in each type of sensilla,we were succeed in establishing the receptor-neuron functional relationship under sensilla trichodea of H.armigera and H.assulta.The details results showed that In A sensilla,there were housed two neurons,one expressing OR13 tuned to Z11-16:Ald,the other expressing OR11,so far without response to any pheromone components.In B sensilla,although contained two neurons,expressing OR 14b and OR 15 respectively,However,OR14b orthologs have different function in these two speices.OR14b in H.armigera was tuned to Z9-14:Ald,but did not show any response in H.assulta.Similar results also observed two neurons co-localization in C sensilla.One neuron expressed OR6 tuned to Z9-14:Ald and Z9-16:Ald consistently.Other one expressed OR16 appeared function differentiation,OR16 in H.armigera was tuned to Z9-14:Ald,Z11-16:OH and Z11-16:Ac,while OR16 in H.assulta was tuned to Z9-14:Ald,Z9-14:OH and Z9-16:OH.Besides this,the spatial distribution of these functional types of sensilla showed that functional and non-functional trichoid sensilla were clustered in the proximal and distal regions of each annulus respectively.2.Transcriptome analysis of larval antenna and labial palp of H.armigera and H.assulta.By analyzing the transcriptome data of H.armigera larval antenna and labial palp,a total of 56 olfactory genes were obtained including 1 Orco,2SNMPs,3IRs,3GRs 260BPs and 21CSPs.Similar in H.assulta,we obtained 56 olfactory genes including 1 Orco,2SNMPs,4GRs,2IRs,270BPs and 20CSPs.Later,the phylogenetic tree analysis showed that 4 HarmOBPs,6 HassOBPs,6 HarmCSPs and 6 HarmOBPs were identified as novel genes.The further RT-PCR results showed that there were 60BPs and 6CSPs presented larval-tissue special in H.armigera.To our disappointment,we haven't detected any expression of PBP or PR genes in larval olfactory tissues,suggested that larvae of these two species didn't involve in pheromone related activities,otherwise there are other olfactory genes accounted for larvae perception pheromone.3.Pheromone antagonist Zll-16:OH meditated female sexual development copulation of H.armigera.In this study,we firstly investigated whether there was a variation in the composition of these antagonists produced by different age females.Here we showed that among these antagonists,only Z11-16:OH presented age-related changes.Combined with changes in female reproduction conditions and male mating choice,we suggested that the composition of Z11-16:OH in females reflected their statues(mature or immature),which in turn convenience for male distinguishing and mating with mature females.To further demonstrated this hypothesis,next we used the prevalent genome editing tool,CRISPR/Cas9 system to knock out its stimulation receptor OR16.We found the OR16 mutant neurons have defects in physiological response to Z11-16:OH and Z9-14:Ald.In the term of behavior response,we found that OR16 mutant males were disrupted in Z11-16:OH and 10%Z9-14:Ald triggering inhibition behavior,but failed in altering 0.3%Z9-14:Ald triggering attraction behavior.More importantly,the subsequent results demonstrated that OR16 mutant male can mate with immature females,which decreased female fecundity.In summary,all these available findings indicated that pheromone antagonist Z11-16:OH served as regulatory switching permitting mating partners encountered to mate in optimal times which gurateed female maximum fecundity.