Functional Characterization Of Sex Pheromone Receptors And Neurons In The Armyworms,Mythimna Separata(walker)

Insects' behaviors were closely related to its olfactory sensory,which detects several volatile compounds relying on the sensilla located on the antenna.The various odorant receptors expressed on it could recognize external chemical signal,convert into electrical signal and further input to the central nervous system.In Lepidoptera,males can recognize sex pheromones released by females from long-distance and then mating and reproduction.Studying on the neural and molecular basis of peripheral encoding of sex pheromone in insect has important scientific significance in screening and developing more effective insect-behavior regulators.Here,Mythimna separata were taken as our experimental subject,and we used Electroantennogram recordings to test the responses of male and female antennae to its sex pheromone components and analogues;single sensillum recording technology was used to detect different functional types of sensilla trichodea and identify the main ORNs on the antennae of male M.separata to main sex pheromone component.We identified and cloned 6 pheromone receptors(PRs)based on the antennae transcriptome.All the PRs were functionally analyzed by Xenopus oocytes system together with two-electrode voltage clamp in vitro.The main results are as follows:1.This study selected 7 sex pheromone components and analogues of M.separata,tested the EAG responses of both male and female antennae.Results demonstrated that Z11-16:Ald could elicit strong physiological responses of male antennae,followed by analogue Z9-14:Ald,while Z9-16:Ald only elicit a small response.Female antennae showed small response to all tested components.The responses of male antennae to Z11-16:Ald and Z9-14:Ald were significantly higher than female.This result show that male antennae could recognize main sex pheromones released by females,and plays a crucial role in the mating behavior of M.separata.2.The single sensillum recording technology is used to test the function of ORNs on different types of sensilla trichodea of the male antennae of M.separata.Results showed four functional types of sensilla trichodea were found on male antennae,Type A,Type B,Type C and Type D.Type A occupied the major amount of sensilla trichodea,and the neuron b housed in it could be activated by two odors and showed strong response to the main sex pheromone component Z11-16:Ald.Three ORNs are housed in Type B.Neuron a could be activated by analogue Z11-16: OAc and Z9-14:Ald.Among the results,the response to Z9-14: Ald is sensitive and strong.Neuron b could be activated by minor sex pheromone component Z9-16:Ald and Z11-16:OH.There are three ORNs housed in Type C,and neuron b could be activated specifically by analogue Z11-16: OAc.Two ORNs are housed in Type D,and neuron b could be activated by minor sex pheromone component 16:Ald.The dose responses of ORNs to these sex pheromone components and analogues were also tested.3.Based on the 6 PRs genes reported from the transcriptome analysis of M.separata,we designed specific primers and cloned the full lengths of the cDNA of MsepPR1,MsepPR2,MsepPR3,MsepPR4,MsepPR5 and MsepPR6 by Rapid Amplification of cDNA Ends(RACE)from the antennae of M.separata.Then we aligned the pheromone receptors with closely related species Helicoverpa armigera,Heliothis assulta,Heliothis virescens and Spodoptera mauritia in Lepidoptera.Results showed MsepPR1 gathered with OR16,MsepPR2 gathered with OR11,MsepPR3 gathered with OR13,MsepPR4 gathered with OR15,and MsepPR5 gathered with MsepPR6 alone.The sequence similarity of homologous genes is between 81.34% and 91.92%,which indicated sex pheromone receptors of M.separata are highly conserved with closely related species.4.The cloned PR genes were co-expressed with Orco in the Xenopus oocytes,and functional analyzed by the two-electrode voltage clamp.The results showed that MsepPR1/Orco turned to analogue Z9-14:Ald and minor component Z11-16:OH with strong responses.MsepPR5/Orco showed responses to main sex pheromone component Z11-16:Ald,analogue Z9-14:Ald and minor sex pheromone component Z11-16:OH.MsepPR6/Orco showed broad responses to Z11-16:Ald,Z11-16:OAc,Z9-14:Ald and Z9-16:Ald.MsepPR2/Orco,MsepPR3/Orco and MsepPR4/Orco did not respond to any chemical tested in this study.