Functional Characterization Of Specific Odorant Receptors In Plutella Xylostella And Athetis Dissimilis

Moths have highly developed and sophisticated olfactory system,which makes them extremely sensitive and accurate to odorants in the environments,so as to complete the important behavioral and physiological activities such as foraging,courtship,and host searching.Odorant receptors(ORs)function as the bridge between insects and the odorant environments,playing central roles in the transduction from chemical to electrical signals.Insect OR can be divided into two classes:one is the atypical OR,the so-called odorant receptor co-receptor(Orco).One species normally has only one Orco gene,but Orcos in different species are highly conserved in amino acid sequence.The second class is the traditional ORs(simply called ORs).Different species have different number of ORs,ranging from a dozen to more than three hundreds,however,these ORs share very low homology in and between insect species.In insects,OR has to be co-expressed with Orco to form heteromultimer to perform normal functions.According to the type of odor they detected,ORs are divided into general ORs for common plant odorants,and pheromone receptors(PRs)mainly for sex pheromones.Plutella xylostella is a notoriou insecticide resistant pest,which urgently needs effective integrated control measures.Athetis distimilis is a new outbreak pest,and there is a lack of simple monitoring and control measures.In this study,we therefore selected these two pests to characyterize their odorant receptor genes for host specfic odorants(isothiocyanates,ITCs)in P.xylostella and for sex pheromones in A.distimilis.ITCs are the characteristic odor of the female moths of P.xylostella and other crucifer specialist insects looking for the host to oviposition.To characterize the ORs for ITCs,59 full sequence OR genes were firstly confirmed by bioinformatic analysis and molecular cloning verification;secondly,tissue and sex expression profiles were determined by qPCR,showing 12 female biased(female/male?2)ORs that were regarded as candidate ORs for ITCs functional analyses;then,functional analysis using Xenopus oocyte-two electrode voltage clamp system revealed that OR35 and OR49 are sensitive specifically to isothiocyanates.Finally,using CRSPR/Cas9 technology in vivo confirmed that knockout of these two ORs resulted in the loss of the female moth's oviposition preference to isothiocyanates compounds and Arabidopsis thaliana living plants.In addition,the two PRs(OR1 and OR14)were identified from Athetis dissimilis,through analyses of OR phylogenetic tree and tissue-sex expression profiles,and messurments using the Xenopus oocyte expression system.The main results are as follows:1.Determination of active ITCs that attract female P.xylostella for ovipositionTo analysis the active components of ITCs volatiles in attracting female P.xylostella to locate the host plants and in stimulating oviposition,13 ITCs that aer naturally existant in cruciferous plants were selected to do the oviposition preference assay and EAG test.The results showed that phenethyl ITC,4-Pentenyl ITC and iberverin could significantly induce the oviposition preference,with the choice index of 0.22,0.33 and 0.41 respectively.Meanwhile,the same three active ITCs consistently elicit high EAG responses in females.Therefore,it was confirmed that 3 ITCs(phenethyl ITC,4-Pentenyl ITC and iberverin)have attractant activity for oviposition of female P.xylostella.2.Confirmation of ORs in P.xylostella and analysis of tissue expression profileTo identify the ITC sensitive ORs in P.xylostella,all reported OR sequences were cloned and verified.The 92 ORs reported by the genome analysis were cloned when take out the partial sequences,and only 53 full sequence ORs were obtained;at meanwhile,the antennae transcriptome was sequenced,and 6 new full sequence ORs were obtained after analysis.In total,59 ORs were confimed in P.xylostella.Considering that ITCs function mainly on female moths,the female antenna bised ORs were determined and were regarded as the candidate ORs for further study.The qPCR was used to analyze the tissue expression profiles of the 50 ORs(9 OR have been reported).The results showed that 12 OR(OR10,18,25,33,35,37,38,40,47,48,49 and 59)were female antenna biased in expression(female/male?2.00),while OR27 was male biased(male/female? 2.00).All ORs were of low expression in tissues other than anennae,while the expression of OR23 and OR39 were relatively highere in heads,legs and wings.According to the results,the 12 female antenna biased ORs were used for the follow-up functional studies.3.In vitro functional analysis of the P.xylostella ORs sensitive to ITCsAfter the 12 OR candidates were obtained,the heterologous expression system of Xenopus oocyte with two-electrode voltage-clamp physiological recordings was used to teste the OR responses to 13 ITCs.The results showed that 1)only two OR(OR35 and OR49)responded robustly to iberverin and 4-pentenyl ITC,moderately to phenylethyl ITC,propyl ITC,butyl ITC,sulforaphane and sulforaphene.2)The two ORs shared identical substrate spectra,but OR35 was significantly more sensitive than OR49,suggesting that OR35 may play a greater role in ITC perception.At ITC screening concentration,the response(current value)of OR35 to iberverin and 4-pentenyl ITC was 2.0 and 1.6 times of OR49,respectively.3)A further dose-response study showed that the EC50 values of OR35 to iberverin and 4pentenyl ITC were 6.01×10-5 M and 8.38×10-5 M,while the EC50 values of OR49 to two ITC were 1.47×10-4 M and 3.52 ×10-4 M,respectively.4)OR35 and OR49 did not respond to any of the tested 25 plant odors and 11 sex pheromones(or analogues),suggesting the ITC specificity of the two ORs.4.In vivo functional verification of the P.xylostella ORs sensitive to ITCsThe ITC-sensitive OR35 and OR49 were further verified in vivo using the CRISPR/Cas9 technology to knockout the two ORs,then using EAG and oviposition choice tests to determine the electro-physiological and behavioral resposnes.The results showed that 1)EAG responses of OR49 and OR35 single knockout females to three active ITCs(iberverin,4-pentenyl ITC and phenylethyl ITC)were significantly reduced by 46%?42%?40%and 68%?71%?73%,respectively;the OR49 and OR35 double knockout females did not respond anymore to three active ITCs;2)Oviposition preference of OR49 and OR35 single knockout females to three active ITCs(iberverin,4-pentenyl ITC and phenylethyl ITC)were significantly reduced by 43%,38%,37%and 72%,67%,65%,respectively;while OR49 and OR35 double knockout females did not show any preference to each of three active ITCs;3)Oviposition preference of OR49 and OR35 single knockout females to wide type A.thaliana plants,compaired to GS-knockout plants(no longer produce ITCs),were significantly reduced by 19%and 37%,respectively;while the OR49 and OR35 double knockout females did not show any preference to wild type A.thaliana plants.The results of electrophysiological and oviposition selective tests consistently showed that OR35 and OR49 were involved specifically in the perception of ITCs in P.xylostella.Knockout of these two genes resulted in almost complete lost of the ITC perception.In comparison,OR35 played a more important role than OR49.These in vivo results were fully consistent with that obtained by the in vitro Xenopus oocyte system.5.Functional characterization of PRs in Athetis dissimilisSex pheromones,as the chemical language between males and females,are crucial for the mating and reproduction in moth insects,and pheromone receptors(PRs)play a key role in peripheral coding of sex pheromones.In recent years,the damage to maize by Athetis dissimilis is increasing,and thus the PR genes from this pest was also characterized in this study.Focusing on the four PR candidates(OR1,6,11 and 14)suggested in a previous study,we first cloned the full-length cDNAs and determined the tissue expression profiles by qPCR.The result revealed that expression of OR1,OR6 and OR14 were male antennae-specific,while OR11 was similar in expression between male and female antennae.Furthermore,the expression level of OR1 was much higher than those of the other three genes,suggesting the importance of this OR in sex pheromone perception.Then,functional analysis was conducted using Xenopus oocyte system.The results showed that OR1 responded strongly to the sex pheromone component Z9-14:OH and the potential pheromone component Z9,E12-14:OH with the current values of 180 nA and 320 nA at the ligand concentration of 10-4 M;OR14 showed specificity for Z9,E12-14:OH(current value was 45 nA),suggesting Z9,E12-14:OH as a potential sex pheromone component;while OR6 and OR11 did not respond to any of the pheromone components and analogs tested.This study contributes to elucidate the molecular mechanism of sex pheromone reception and provides potential targets for development of OR based pest control techniques in A.dissimilis.