Functional Analysis Of Odorant-binding Proteins Involved In Sex Pheromone Induced Feeding Of Spodoptera Litura Larvae

Spodoptera litura（Lepidoptera: Noctuidae）is an important gluttonic agricultural pest in the world,which can feed on hundreds of host plants and cause huge losses for many important cash crops such as vegetables,tobacco,corn,beans and cotton in many countries.A variety of chemical agents have been used to control S.litura for a long time.However,due to the frequent abuse of chemical agents,it has developed resistance to a variety of insecticides,and has also seriously harmed the ecological environment.Insect sex pheromones play a role in monitoring population,interference mating and trapping adults.Using insect sex pheromones,as green prevention and control agents,not only has the advantages of low consumption,high biological activity,strong specificity,no harm to natural enemies,and no drug resistance,in addition,it can be combined with other biological pesticides such as natural enemies,bacteria,fungi or their metabolites for integrated insect control.Although sex pheromones have been successfully used in the prediction and control of insect pests in some countries or regions,they have been used in prediction and control of adult insects’ population,and there is little research on the control of larvae,which directly play a harmful role.Based on the moth larvae feeding behavior induced by the sex pheromone,the specific functional characteristics of OBPs in the moth larvae would be analyzed in vivo and in vitro.The research results can provide important scientific basis for understanding the mechanism of larval communication behavior mediated by insect sex pheromones and for developing behavioral disruptors targeting larvae.The analysis results are as follows:（1）The results of determination of feeding attraction behavior of Spodoptera litura larvae showed that when the content of sex pheromone was 3 ng,only Mix2 had significant attraction to larval feeding behavior;under the condition of 10 ng of sex pheromone content,Z9,E12-14:Ac,Z9-14:Ac,Mix2 had significant effect on larval feeding behavior and could attract larval feeding;furthermore,the six components had no effect on larval feeding behavior at 30 ng sex pheromone content.Excluding the influence of food factors,components lost their attraction to larvae.These results indicated that Z9,E12-14:Ac and Z9-14:Ac had significant effects on the feeding of s.litura larvae,and these two sex pheromones only acted in the food environment.It also suggests that the same chemical that is a sexual attractant for adults is a foraging indicator for larvae.（2）In this study,we identified 158 putative olfactory genes based on transcriptomic and genomic data for S.litura,including 45 Odorant-binding proteins（OBPs,9 were new）,23 Chemosensory proteins（CSPs）,60 Odorant receptors（ORs,3 were new）,and 30 Gustatory receptors（GRs,3 were new）,Based on FPKM results,39 OBPs,22 CSPs,22 ORs and 13 GRs were identified in the larval head（LH）.Phylogenetic analysis showed that 36 Slit OBPs and 9 new Slit OBPs were widely distributed in all branches.Slit PBP1-3 and Slit GOBP1-2 cluster into the PBP/GOBP subfamily,suggesting that Slit OBPs have similar functions to other moth OBPs.（3）In order to further identify OBPs recognizing Z9,E12-14:Ac and Z9-14:Ac in the antennae of S.litura larvae.At first,we conducted an induction analysis of pheromone exposure on the fourth instar larvae after 24 hours of starvation.After 0.5h of exposure to each component,the antennae of larvae were collected to analyze by quantitative PCR.The results showed that after Z9,E12-14:Ac induction,the GOBP2,OBP7,OBP15,OBP18,OBP22 and OBP30 were up-regulated;After Z9-14:Ac induction,only GOBP2 was found to be up-regulated.Subsequently,these 6 OBPs were expressed and purified by Escherichia coli expression system,and the binding-abilities of the protein and sex pheromone were analyzed by fluorescence competitive binding assay in vitro.The results showed that only GOBP2 and OBP7 has different binding abilities with six sex pheromone components.These data suggest that GOBP2 and OBP7 might be involved in the recognition of Z9,E12-14:Ac and Z9-14:Ac by larvae,and regulate feeding behavior of larvae.（4）According to the results of fluorescence competition binding experiment in vitro,we used RNAi method to silence GOBP2 and OBP7 in the antennae of S.litura larvae.q RT-PCR analysis showed that the expression levels of target OBP in the two treatment groups were significantly decreased compared with the control group.GOBP2 decreased by 70.37% in si GOBP2 group and OBP7 decreased by 63.27% in si OBP7 group.Feeding behavior experiment showed that the si RNA treated larvae showed no sex pheromone-induced feeding behavior,suggesting that GOBP2 and OBP7 should be able to recognize Z9,E12-14:Ac,Z9-14:Ac and Mix2,and regulate the feeding behavior induced by sex pheromone.