The Mechanism Of The Highly Effective Attraction Of A Sex Pheromone Analogue To Male Athetis Lepigone

Olfactory system is an important measure for insects to perceive the external environment.Insects have developed a highly sensitive olfactory system during the long-term evolutionary process.Through this system,insects can complete various behaviors such as foraging,searching for mates,locating hosts,defending against enemies and selecting oviposition sites.The harm of pests has seriously threatened the sustainable development of agriculture and food security in China.In recent years,pest olfactory behavior control technology for the development of sensitive olfactory system of pests has been paid attention to.Up to now,the sex pheromone components of more than 1,600 moth species have been identified.Sex pheromones of many important agricultural pests of Lepidoptera have been identified and commercialized for use in forecasting,mating disturbance and trapping control.With the deepening of research on sex pheromones,problems such as fast release rate of sex pheromones in the field,poor stability,and high synthesis cost have become increasingly prominent.Insect sex pheromone analogues,as the derivant of natural insect sex pheromones,have better physical and chemical properties and can effectively make up for the shortage of natural sex pheromones than natural sex pheromones,and therefore can regulate chemical communication and behavioral activities of insect more effectively.Based on the above advantages of sex pheromone analogues,our team selected a sex pheromone analogue E3 and found that it was highly attractive to male Athetis lepigone in the field.In this study,Athetis lepigone(Lepidoptera: Noctuidae)was selected as the research object.Firstly,laboratory behavioral experiment was used to verify the efficient attraction of the sex pheromone analogues.And then relevant odorant receptors were analyzed by antennae transcriptome sequencing,and related odor receptors were cloned.In addition,the in vitro function of odor receptors were verified by using Xenopus oocytes and two-electrode voltage clamp.Investigating the mechanism of the sex pheromone analogue E3 sensed by Athetis lepigone provides a scientific basis for developing more efficient sex attractants and also provides new ideas for green control of pests.The main results of this study are as follows:1.Behavioral assay and antennal responses of sex pheromone analogue to Athetis lepigoneThe Y-type olfactometer was used to design a selective behavioral experiment in order to verify the attraction effect of E3 on A.lepigone.The results showed that the sex pheromone analogue E3 could specifically attract males,but had no effect on females.The sex pheromone analogue E3 was more attractive to males than the sex pheromone.Of the different concentrations of sex pheromone analogue E3,the best attraction effect was at 10μg.The electrophysiological response of antennae to E3 was verified by EAG,and it was proved that E3 could induce antennae electrical response of Athetis lepigone.The response of male antennaes to E3 increased with the increasing concentration of E3.2.Antennae Transcriptome sequencing and analysis of Athetis lepigoneTo identify the receptors for the sex pheromone analogue E3,we sequenced the antennae transcriptome of Athetis lepigone using the Illumina Hiseq.35.97 Mb and 38.44 Mb cleanreads were generated.Assemblies led to the generation of 44337 and 44806 unigenes,respectively.After merging and clustering,53388 all-unigenes were obtained.The total length,average length,N50 and GC content were 76013235 bp,1423 bp,2932 bp and41.18%,respectively.After annotation and identification of the transcriptome,a total of 60 ORs were obtained.Two new PRs,AlepOR47 and AlepOR62,were obtained in addition to four known sex pheromone receptor PRs.In addition,an OR located in a new PR branch of Lepidoptera,AlepOR9,was also identified.3.Function of sex pheromone receptors in vitro of Athetis lepigoneThe full-length sequences of six classical PRs(AlepOR3,AlepOR4,AlepOR5,AlepOR6,AlepOR47,AlepOR62)and one novel PR(AlepOR9)were obtained by cloning.These PRs were expressed in vitro using Xenopus oocytes,and their responses to the sex pheromone analogue E3,the sex pheromone components of Athetis lepigone and related species were tested using a two-electrode voltage clamp technique.Among them,AlepOR6 and AlepOR62 showed significant electrophysiological responses to the analogue E3.4.Function of male-biased expression odorant receptors in vitro of Athetis lepigoneThe full-length sequences of the six common ORs reported in the literature to be highly expressed in male moths of Athetis lepigone were cloned and expressed in vitro using Xenopus oocytes(AlepOR8,AlepOR14,AlepOR36,AlepOR37,AlepOR51,AlepOR54).The responses to the sex pheromone components of Athetis lepigone,the sex pheromone analogue E3 and plant volatiles were tested using a two-electrode voltage clamp technique.The results showed that none of the above six ORs reacted to E3,but they reacted to some important host plant volatiles.AlepOR8 had specific electrophysiological responses to benzaldehyde,AlepOR14 had robust electrophysiological responses to phenylethylaldehyde,and AlepOR54 had specific electrophysiological responses to eugenol.