Identification Of Chemosensory Genes And Functional Characterization Of Olfactory Receptors In Spodoptera Litura And Helicoverpa Armigera

As the most successful animal on the earth,insects have evolved an extremely species specific and sensitive olfactory system during the long time of biological evolution.With this system,insects can precisely perceive the multiple environmental odorants and subsequently accomplish various physiological and behavioral responses,such as locating mating partners,food,oviposition sites and avoidance of predation.Therefore,the study of insect chemosensory mechanism has important scientific significance.From an application perspective,our long-standing use of chemical pesticides not only lead to the pests resistance to insecticides and the reduction of prevention efficiency,but also the affect of pesticides to the environment and human life safety,which causes great concern especially when the environment is not so good.Therefore,more environmentally friendly and safer pest control techniques are urgently needed.Based on the highly specific chemical communication between the two sexes,the sex attractant has been successfully applied to pest control.But in general,it can hardly reach the expection.The main reason is the lack of deep understanding of the perception to sex pheromones and plant volatiles.Several proteins are involved in chemo-reception,such as odorant binding proteins?OBPs?,chemosensory proteins?CSPs?,odorant receptors?ORs?,ionotropic receptors?IRs?,sensory neuron membrane proteins?SNMPs?and odorant degrading enzymes?ODEs?.Deep understanding the function of these proteins and their role in chemo-reception,will facilitate the development and application of more efficient pest control technology.Here,by combined use of molecular biology,bioinformatics and electrophysiological techniques,we identified a number of chemosensory genes and functionally analyzed the olfactory receptors involved in sex pheromone and plant volatile recognition of the important agricultural pest Spodoptera litura and Helicoverpa armigera.The main results are as follows:1.Identification,expression pattern and functional analysis of pheromone receptors from S.lituraBased on the 4 PRs genes reported from the transcriptome analysis of S.littoralis,we designed 4 specific primers and cloned 4 PRs fragments from the male antennae of S.litura.The full lengths of the cDNA were further obtained by Rapid Amplification of cDNA Ends?RACE?.The names and Genbank numbers of these PRs were SlituOR6?KC188666?,SlituOR11?KC188667?,SlituOR13?KC188668?and SlituOR16?KC188669?.Gene expression patterns showed that expression of the 4 PRs was restricted to antennae.In addition,SlituOR6 and SlituOR13 were specifically expressed in male antennae while SlituORll and SlituOR16 were male-biased.Functional analysis in heterologous expression system Xenopus oocytes demonstrated that SlituOR6 was specifically tuned to the second major pheromone component,Z9,E12-14:OAc(EC₅₀= 1.99×10^-6 M).SlituORl3 was equally tuned to Z9,E12-14:OAc(EC₅₀=6.109×10^-6 M)and Z9-14:OAc(EC₅₀=1.184x10^-6 M),with a small response to the major pheromone component Z9,E11-14:OAc.SlituOR16 significantly responded to the behavioral antagonist Z9-14:OH(EC₅₀=2.920×10^-6M),while SlituOR11 did not show response to any of the pheromone compounds tested in this study.Our results provide molecular data to better understand the mechanisms of sex pheromone detection in the moth S.litura2.Identification,expression pattern and functional analysis of odorantThe OR repertoire housed within the dendritic membrane of sensory neurons is one of the primary determinants of the odor recognition.ORs in moths could be classified into PRs and non-pheromone receptors?non-PR ORs?.Much research in the field of insect olfaction recently has been focused on PRs of the male moth.However,few Lepidoptera studies have been done on the functional study of non-PR ORs.Based on the 4 ORs genes reported from the transcriptome analysis of S.littoralis,we designed 4 specific primers and cloned 4 ORs fragments from the male antennae of S.litura.The full lengths of the cDNA were further obtained by RACE.The names and Genbank numbers of these ORs were SlituOR12?JX999588?,SlituOR19?JX999589?,SlituOR44?JX999587?and SlituOR51 ?JX999586?.In this study,we identified and characterized 4 non-PR ORs from Spodoptera litura?Lepidoptera:Noctuidae?antennae.Tissue expression pattern showed that the four ORs were mainly expressed in adult antennae and further In situ hybridization revealed SlituOR12 was expressed in both long and short sensilla trichodea and sensilla basiconica.Functional analysis of the four SlituORs was conducted in heterologous expression system Xenopus oocytes.SlituOR12 was exclusively and sensitively tuned to cis-3-Hexenyl acetate(EC₅₀=3.393×10-7M)and SlituOR19 slightly responded to 4'-Ethylacetophenone.However,SlituOR44 and SlituOR51 didn't respond to any chemicals tested in this study.Based on the previous study of cis-3-Hexenyl acetate,it is proposed that SlituOR12 might partially account for some key behaviors of female,such as detection of host location and oviposition site.3.Identification and localization of two sensory neuron membrane proteins from S.lituraWe cloned the full length of two SNMPs genes,SlituSNMP1 and SltiuSNMP2,from S.litura.The names and Genbank numbers were SlituSNMP1?KC571258?and SlituSNMP2?KC571259?.Sequence alignment and phylogenetic analysis showed that the two SNMPs belong to two separate SNMP subtypes.Further expression profile conducted by RT-PCR and qPCR showed that SlituSNMPl was mainly expressed in the antenna,while SlituSNMP2 was broadly expressed in various tissues.By in situ hybridization experiments,it was found that SlituSNMP1 was expressed in the olfactory sensory neurons?OSNs?under pheromone sensitive sensilla,and SlituSNMP2 was expressed in the supporting cells surrounding the OSNs,suggesting different functions of the two SNMPs in insect olfaction.4.Antennal transcriptome analysis and identification of chemosensory gene families in Helicoverpa armigeraTo better understand the olfactory mechanisms of Helicoverpa armigera,we conducted transcriptome analysis of the adult antennae using Illumina HiSeqTM 2000 sequencing technology and identified 36 new olfactory genes?15 ORs,7 IRs,8 OBPs and 6 CSPs??Combined with the chemosensory genes we had identified previously in H.armigera by 454 sequencing,we identified 133 putative chemosensory unigenes in H.armigera including 60 ORs,19 IRs?34 OBPs,18 CSPs,and 2 SNMPs.The comparison of olfactory genes with related species H.assulta will surely provide valuable information for further studies of olfactory perception mechanism and feeding differentiation in Lepidoptera insects.5.Identification,expression and functional analysis of odorant receptors from H.armigeraBased on the ORs identified in transcriptome of H.armigera,RT-PCR was used to verifiy the tissue expression profile of the ORs.The results showed that 28 ORs were expressed in both adults and larvae antennae,2 ORs were expressed in larvae antennae,and other ORs were only expressed in the adults.Later we cloned 26 full length cDNA of ORs,and 12 of them have been further analyzed in heterologous expression system Xenopus oocytes and tested with 84 chemicals.The tuning breadths of 7 ORs?OR7,8,23,26,27,34 and 43?were so narrow that they only responded to 2-4 odorants;4 ORs?OR31,35,40 and 42?could respond to more than 5 odorants;and one OR had no response to any of the odorants.It is indicate that these ORs with narrow tuning curves may play important role in the recognition of host plant.In conclusion,we identified 4 PRs,4 ORs and 2 SNMPs from S.litura,investigated the expression patterns of these genes and explored the functions of these ORs.We identified 36 new chemosensory unigenes in H.armigera compared with 454 sequencing,including 15 ORs,7 IRs,8 OBPs and 6 CSPs.We cloned 26 full length cDNA of ORs in H.armigera,and 12 of them have been functionally studied.The results will provide considerable basis for further elucidation of the molecular mechanisms of sex pheromone and plant volatile recognition in S.litura,H.armigera and other moths,and provide important reference to develop behavioral attractants and disruptants with high efficacy for pest control.