Characterization of the pheromone receptors in Mythimna loreyi reveals the differentiation of sex pheromone recognition in Mythimna species

Pheromone receptors(PRs)are key proteins in the molecular mechanism of pheromone recognition,and exploring the functional differentiation of PRs between closely related species helps to understand the evolution of moth mating systems.Pheromone components of the agricultural pest Mythimna loreyi have turned into(Z)-9-tetradecen-1-yl acetate(Z9-14:OAc),(Z)-7-dodecen-1-yl acetate(Z7-12:OAc),and(Z)-11-hexadecen-1-yl acetate,while the composition differs from that of M.separata in the genus Mythimna.To understand the molecular mechanism of pheromone recognition,we sequenced and analyzed antennal transcriptomes to identify 62 odorant receptor(OR)genes.The expression levels of all putative ORs were analyzed using differentially expressed gene analysis.Six candidate PRs were quantified and functionally characterized in the Xenopus oocytes system.MlorPR6 and MlorPR3 were determined to be the receptors of major and minor components Z9-14:OAc and Z7-12:OAc.MlorPR1 and female antennae(FA)-biased MlorPR5 both possessed the ability to detect pheromones of sympatric species,including(Z,E)-9,12-tetradecadien-1-ol,(Z)-9-tetradecen-1-ol,and(Z)-9-tetradecenal.Based on the comparison of PR functions between M.loreyi and M.separata,we analyzed the differentiation of pheromone recognition mechanisms during the evolution of the mating systems of 2 Mythimna species.

Functional characterization of sex pheromone receptors in Spodoptera frugiperda, S. exigua, and S. litura moths

Moths possess an extremely sensitive and diverse sex pheromone processing system,in which pheromone receptors(PRs)are essential to ensure communication between mating partners.Functional properties of some PRs are conserved among species,which is important for reproduction.However,functional differentiation has occurred in some homologous PR genes,which may drive species divergence.Here,using genome analysis,17 PR genes were identified from Spodoptera frugiperda,S.exigua,and S.litura,which belong to 6 homologous groups(odorant receptor[OR]6,11,13,16,56,and 62);of which 6 PR genes(OR6,OR11,OR13,OR16,OR56,and OR62)were identified in S.frugiperda and S.exigua,and 5 PR genes were identified in S.litura,excluding OR62.Using heterologous expression in Xenopus oocytes,we characterized the functions of PR orthologs including OR6,OR56,and OR62,which have not been clarified in previous studies.OR6 orthologs were specifically tuned to(Z,E)-9,12-tetradecadienyl acetate(Z9,E12-14:OAc),and OR62 orthologs were robustly tuned to Z7-12:OAc in S.frugiperda and S.exigua.The optimal ligand for OR56 was Z7-12:OAc in S.frugiperda,but responses were minimal in S.exigua and S.litura.In addition,SfruOR6 was male antennae-specific,whereas SfruOR56 and SfruOR62 were male antennae-biased.Our study further clarified the functional properties of PRs in 3 Spodoptera moth species,providing a comprehensive understanding of the mechanisms of intraspecific communication and interspecific isolation in Spodoptera.

Identification of an odorant receptor responding to sex pheromones in Spodoptera frugiperda extends the novel type-I PR lineage in moths

moths,pheromone receptors(PRs)are crucial for intraspecific sexual communication between males and females.Moth PRs are considered as an ideal model for studying the evolution of insect PRs,and a large number of PRs have been identified and functionally characterized in different moth species.Moth PRs were initially thought to fall into a single monophyletic clade in the odorant receptor(OR)family,but recent studies have shown that ORs in another lineage also bind type-I sex pheromones,which indicates that type-I PRs have multiple independent origins in the Lepidoptera.In this study,we investigated whether ORs of the pest moth Spodoptera frugiperda belonging to clades closely related to this novel PR lineage may also have the capacity to bind type-I pheromones and serve as male PRs.Among the 7 ORs tested,only 1(SfruOR23)exhibited a male-biased expression pattern.Importantly,in vitro functional characterization showed that SfruOR23 could bind several type-I sex pheromone compounds with Z-9-tetradecenal(Z9-14:Ald),a minor component found in female sex pheromone glands,as the optimal ligand.In addition,SfruOR23 also showed weak responses to plant volatile organic compounds.Altogether,we characterized an S.frugiperda PR positioned in a lineage closely related to the novel PR clade,indicating that the type-I PR lineage can be extended in moths.

Moth sex pheromones affect interspecific competition among sympatric species and possibly population distribution by modulating pre-mating behavior

Premating behaviors mediated by pheromones play pivotal roles in animal mating choices.In natural populations of the striped stem borer Chilo suppressalis and the rice leaf roller Cnaphalocrocis medinalis in the rice field habitat,we discovered that Z11-16:Ald,a major component of the C.suppressalis pheromone,modulated the premating behavior of C.medinalis.Z11-16:Ald evoked a strong olfactory response in male antennae and strongly inhibited the sex pheromone trapping of male C.medinalis in the field.The functions of three C.medinalis sex pheromone receptor genes(CmedPR1–3)were verified through heterologous expression in Xenopus oocytes.CmedPR1 responded to Z11-18:OH and Z11-18:Ald,as well as the interspecific pheromone compound Z11-16:Ac of sympatric species;CmedPR2 responded to Z13-18:OH and Z13-18:Ald,as well as the sex pheromone compounds Z11-16:Ald and Z9-16:Ald of sympatric species;and CmedPR3 responded to Z11-18:OH and Z13-18:OH,as well as the interspecific pheromones Z11-16:OH,Z9-16:Ald,Z11-16:Ac,and Z11-16:Ald of sympatric species.Thus,CmedPR2 and CmedPR3 share the ligand Z11-16:Ald,which is not a component of the C.medinalis sex pheromone.Therefore,the sex pheromones of interspecific species affected the input of neural signals by stimulating the sex pheromone receptors on the antennae of male C.medinalis moths,thereby inhibiting the olfactory responses of the male moths to the sex pheromones.Our results demonstrate chemical communication among sympatric species in the rice field habitat,the recognition of intra-and interspecific sex pheromones by olfactory receptors,and how insect premating behaviors are modulated to possibly affect resource partitioning.

Functional characterization of pheromone receptor candidates in codling moth Cydia pomonella(Lepidoptera:Tortricidae)

Sex pheromones serve a critical role in Lepidopterans finding mates.Male moths perceive and react to sex pheromones emitted by conspecific females through a delicate pheromone communication system.Pheromone receptors(PRs)are the key sensory elements at the beginning of that process.The codling moth(Cydia ponwonella)is an important pome fruit pest globally and a serious invasive species in China.Pheromonebased techniques have been used successfully in monitoring and controlling this species.We conducted ribonucleic acid sequencing analysis of the codling moth antennal transcriptome and identified 66 odorant receptors(ORs)in a population from Xinjiang province,China,of which 14 were PRs,including two novel PRs(CpomOR2e and CpomOR73).Four PRs that contain full-length open reading frames(CpomORl,OR2a,OR5,OR7)and four PRs with ligands that have not been reported previously(CpomORl,OR2a,OR5,OR7)were selected to deorphanize in the heterologous Xenopus oocyte expression system.Specifically,we found that CpomOR2a and CpomOR5 responded to(£,£)-8,10-dodecadien-l-yl acetate(codlemone acetate).Furthermore,CpomOR5(EC50=1.379 x 10-8 mol/L)was much more sensitive to codlemone acetate than CpomOR2a(EC50=1.663×10^(-6) mol/L).Since codlemone acetate is an important component of C.pomonella sex pheromone,our results improve the current understanding of pheromone communication in codling moths and will be helpful for the development of pest management strategies.

Identification and initial characterization of the 3＇ end of gene transcripts encoding putative members of the pheromone receptor subfamily in Lepidoptera

Semiochemicals, including pheromones and kairomones, used in pest man- agement programs reduce the need for chemical insecticides, and understanding their interactions with their membrane receptors may help make them more effective in the field. Identification of odorant receptors in the Lepidoptera has mainly been achieved us- ing bioinformatics to search DNA sequences generated by genome or expressed sequence tag （EST） sequencing projects. This study reports a rapid method to identify members of the pheromone receptor subfamily in Lepidoptera. Degenerate oligonucleotide primers were designed against a conserved amino acid sequence in the carboxyl terminus of known lepidopteran pheromone receptors, and the primers were used in a 3＇ rapid amplifica- tion of complementary DNA （cDNA） ends procedure. Polymerase chain reaction products generated from seven different lepidopteran species were TA cloned and sequenced. The cDNA sequences of 25 transcripts were determined to encode potential members of the pheromone receptor subfamily. These cDNAs ranged from 238 to 642 bp and encoded 49-54 amino acids of the carboxyl terminus. Analysis of the 3＇ untranslated region reveals that most of the transcripts contain multiple polyadenylation signal sequences, and in the case ofManduca sexta, an alternate polyadenylation signal appears to be used in transcript processing. The 3＇ untranslated region was also useful in determining unique receptors en- coded by transcripts having highly similar nucleotide and amino acid sequences. Overall, this technique provides a complementary method of pheromone receptor identification in EST sequencing projects, or can be used as a stand-alone method in conjunction with 5＇ rapid amplification of cDNA ends procedures.