Functional Identification And Differentiation Of Pheromone Receptors In Adelphocoris Lineolatus

The alfalfa plant bug Adelphocoris lineolatus(Hemiptera: Miridae)is a polyphagous agricultural insect pest with a wide host range up to 245 species,which damages to filed crops like alfalfa,cotton and corn and causes great financial losses.Currently,the management of alfalfa plant bugs is mainly based on the chemical pesticides,leading to more prominent chemical resis tance,less control effect and more control cost.Field investigation have demonstrated that the chemotaxis in alfalfa alfalfa plant bugs is of great importance and olfaction plays crucial role in locating host plants and mates.Hence,the research on olfactory recognition of alfalfa plant bugs and the regulatory mechanisms of mating is beneficial to interfere and disrupt their mating behaviors,leading to effective sustainable field control.I here selected five odorant receptors(ORs)highly expressed in male antennae as candidate pheromone receptors according to the expression profile of odorant receptors,and then performed a series of research.The main results are as following:1.Function of ORs: we analyzed the function of five ORs by Xenopus oocyte expression system and two-electrode voltage clamp?The results shows that Alin OR13 respond to trans-2-Hexenyl butyrate,one of the main pheromone components of alfalfa plant bugs,and Alin OR33 respond to trans-2-Hexenyl butyrate and three secondary pheromone components butyl butyrate,hexyl butyrate and Hexyl hexanoate.2.Phylogenetic analysis : the phylogenetic tree has been constructed with odorant receptors from Apolygus lucorum,A.lineolatus,Acyrthosiphon pisum,Tessaratoma papillosa and Drosophila melanogaster.In the phylogenetic tree,Alin OR33,Alin OR13 as well as Alin OR64 are in the same cluster,indicating that the Alin OR64 is the potential pheromone receptor to be functionally identified.And the phylogenetic tree is divided into several parts by Dmel ORs and Apis ORs.3.Expression pattern and localization of Alin OR33 and Alin OR13: I determined the expression pattern of Alin OR33 and Alin OR13 in nymph and adult stages by q RT-PCR.The q RT-PCR results showed that the expression of Alin OR13 and ALin OR33 gradually increased in nymph and peaked at 3 days of emergence.The expression of Alin OR33 peaked in 3days male mated adults after eclosion,yet Alin OR13 peaked in 5 days male mated adults after eclosion.Fluorescent in situ hybridization showed that Alin OR13 and Alin OR33 expressed in sensilla trichoidea specifically,there is no signal dected in other sensilla.4.Physiological changes: the RNAi has been adopted to knock down Alin OR33,and detect the RNAi efficiency by q RT-PCR.The results showed that the RNAi efficiency is of 70%,and the EAG response of alfalfa plant bugs' antennae halved.There is significant difference detected between before and after RNAi.5.Behavioral changes: the RNAi has been adopted to knock down Alin OR33,and Y-tube has been used to perform the behavioral exper iments.The results showed that alfalfa plant bugs cannot distinguish the trans-2-Hexenyl butyrate and mineral oil,indicating that RNAi changed the behaviors of bugs significantly.