Functional Differentiation Of Pheromone-Binding Protein/General Odorant-Binding Protein In Spodoptera Exigua And Spodoptera Litura

Most of noctuid species are the destructive pests with larvae that feed on numerous crops and forests.In recent years,a large and continuous usage of chemical insecticides has led to the high levels of insecticide resistance in the noctuid species,and meanwhile causes a serial of problems such as environmental pollution and insecticide residues.Therefore,the alternative strategies are in urgent needed to control the pests.At present,the strategies of pest control based on insect olfaction have been applied to the fields on management of agricultural pests and vector insects.However,the related molecular mechanism for olfactory reception is still unclear.Differing from other insects,noctuid moths generally have the nature of nocturnal habit.Thus,they need a more sophisticated and sensitive sex pheromone communication system to finish mating between females and males as well as population reproduction.Pheromone-binding protein(PBP)and general odorant-binding protein(GOBP)are two important sub-families of lepidopteran-specific odorant-binding proteins(OBPs)in the process of chemosensory reception.The PBP was formerly thought to be responsible for the binding and transport of sex pheromone molecules and the GOBP was thought to be involved in the recognition of general odorant molecules.However,considering two GOBPs and three PBPs generally exist within a single noctuid species and moreover share a high conservation among inter-species.It raises the issues what is the relationship of between and within noctuid PBP/GOBPs as well as whether they have functionally differentiated.Addressing these issues will not only facilitate us to understand the olfactory mechanism and evolution in insects,but also provide evidence and potential molecular target candidates for the design and development of novel pest control strategies based on olfaction.In this study,using molecular biological techniques,bioinformatics and biochemical techniques we investigated evolutionary relationships and functional differentiation within and between Spodoptera exigua and Spodoptera litura PBP/GOBPs,two important noctuid agricultural pests.The main results are as follows:1.Identification and expression pattern analysis of chemosensory genes in two Spodoptera species Using a bioinformatics screen and molecular biological strategies,we identified and characterized six crucial chemosensory gene families from two public Spodoptera developmental transcriptomes.Results showed that a total of 79 transcripts in S.exigua transcriptome were found consisting of 34 OBPs,20 CSPs,10 ORs,6 GRs,6 IRs and 3SNMPs,and in S.litura transcriptome 80 chemosensory genes were identified including 38OBPs,21 CSPs,8 ORs,4 GRs,6 IRs and 3 SNMPs.In particular,a novel candidate SNMP gene was identified from the two Spodoptera species for the first time,respectively.Based on sequence characteristic and phylogenetic analysis,we named it as SNMP3.Further,three SNMP3 orthologs were also found from other moths including Chilo suppressalis,Plutella xylostella and Sesamia inferens.Sequence alignment results showed that most of the orthologous chemosensory genes shared high amino acid identities(>80%)between the two Spodoptera species.Results from the phylogenetic tree showed that each of two Spodoptera species has three Minus-C OBPs and three Plus-C OBPs,and the rest are members of Classic OBP family.In addition,reverse transcription(RT)-PCR results showed that studied 67 chemosensory genes of S.exigua were detected in at least one tissue tested,and most of the genes(-80%)were expressed in adult antennae or larval heads with the antennae suggesting olfactory involvements.2.Gene structure and phylogenetic analysis of PBP/GOBPs in two Spodoptera species Based on known genomic DNA sequences of lepidopteran PBP/GOBPs,we analyzed62 PBP/GOBP sequences from 25 lepidopteran species by a bioinformatics screen.Results showed that all lepidopteran PBP/GOBPs have two introns and three exons with conserved exon/intron splice sites.In the phylogenetic analysis of noctuid PBP/GOBPs,the PBP/GOBPs were classified into five distinguished groups:Group1,2,3,4 and 5.The orthologous genes of each group shared relatively high amino acid identities to each other(>55%),while these genes of different groups displayed low identities(<50%).Further,results of selective pressure analysis showed that each of the groups has a low dN/ds value(?<0.70)suggesting a strongly negative selection and thus possibly shares some conserved functions.In addition,three PBPs in S.litura,Helicoverpa armigera and Helicoverpa assulta are clustered on the same chromosomes,respectively,within a close proximity(the distance is from 2.3 to 4.0 Kb).This indicates that these genes were derived from a common ancestor by gene duplications.Notably,S.litura GOBP2 is also situated very closely to three SlitPBPs on the same chromosome suggesting gene duplication events among these genes.This relationship of two Spodoptera PBPs and GOBPs on the chromosome is in line with that of non-noctuid PBP/GOBPs.Therefore,we suggest that lepidopteran PBP/GOBPs have similar evolutionary mechanisms.3.Functional differentiation of PBPs in two Spodoptera species In noctuid species,the communication between females and males is primarily dependent on sex pheromone components released by female moths,i.e.male moths use the antennae,the principal olfactory organ,to long-distance perceive the sex pheromones and finish the orientation to females.Thus,it is of significance to study tissue and sex expression patterns of PBPs.In this study,we first used RT-PCR to study the tissue specific-expression patterns of SexiPBPs.As a result,all these PBPs were abundant in adult antennae and some were also detected in non-antennal tissues.Further,qPCR results showed that the two Spodoptera PBPs displayed obviously different sex-biased expression in antennae of both sexes,with Sexi/SlitPBP1 being highly male-biased(5.96-fold and2.70-fold difference in S.exigua and S.litura,respectively),while PBP2 and PBP3displayed sex-equivalent(the absolute value<2.50).Among three PBPs,the relative ratios of PBP1:PBP2:PBP3 is 16.5:1.5:1.0 in male S.exigua and 10.0:3.3:1.0 in male S.litura,while the relative ratios of PBP1:PBP2:PBP3 is 1.1:1.1:1.0 in female S.exigua and 2.5:1.6:1.0 in female S.litura.This indicates that two Spodoptera PBP1 plays the most important role in female sex pheromone reception while the PBP2 and PBP3 may play minor roles.Binding assays revealed that two Spodoptera PBPs could bind their respective sex pheromones,but PBP1 showed the strongest binding and PBP3 weakest,well consistent with their sex-biased expression and relative expression levels in male antennae.For tested plant odorants,each of the PBPs selectively bound some plant odorants with high affinities(Ki<10?M).Taken together,this study suggests that the two Spodoptera PBPs have differentiated in functions:among three PBPs,PBP1 plays a major role in female sex pheromone reception while other two PBPs play some minor roles;on the other hand,three PBPs could also carry some plant odorants.4.Functional differentiation of GOBPs in two Spodoptera species In Lepidoptera,GOBP contains two conserved members of GOBP1 and GOBP2.The GOBP is believed to be involved in the detection of plant odorants.To study the functional differences between GOBP1 and GOBP2 in Noctuidae,two GOBPs from S.exigua and S.litura were systematically and functionally characterized regarding to the expression patterns,ligand-binding properties and key amino acids involving in ligand specificity.qPCR results showed that two Spodoptera GOBPs were primarily expressed in antennae at similar levels between females and males(the absolute value<2.0).Binding assays revealed that two Spodoptera GOBP1 exhibited a relatively broad binding spectrum with moderate affinities(Ki = 10?50?M),while GOBP2 was narrowly tuned to some compounds with strong binding activities(Ki<5.0?M).Intriguingly,GOBP2 could strongly bind the sex pheromones and pheromone analogs(Ki<1.5?M),but GOBP1 could not.Finally,by molecular docking we found different key residues in the binding pockets between SlitGOBPl and SlitGOBP2,and showed different ligand-binding mechanisms between SexiGOBP2 and SexiPBP1.Together,this study suggests that two GOBPs in S.exigua and S.litura have functionally differentiated in odorant recognition:GOBP1 and GOBP2 have distinguished ligand-binding spectra and particularly GOBP2 may also carry the sex pheromone molecules.5.Study on polymeric formations of PBPs in two Spodoptera species Crystal studies on the "PBP-odorant" complex in Bombyx mori and Lymantria dispar showed that PBP could form homodimer or heterodimer to bind the odorant molecules,which improves the specificity and sensitivity of PBPs to odorants.To study polymeric formations of two Spodoptera PBPs,the interactions of purified SexiPBPl,SexiPBP2,SexiPBP3 or SlitPBPl with a chemical cross-linker glutaraldehyde were investigated,respectively.Results showed that the optimal conditions for glutaraldehyde concentration,reaction time and protein concentration were 0.5%,30 min and 1.0 mg/mL,respectively.In the absence of glutaraldehyde,each of three SexiPBPs was primarily present in the form of monomer(95%)and a little homodimer(5%),but no homotrimer.In the presence of glutaraldehyde,the ratio of homodimer of three SexiPBPs was increased(9%)and moreover yielded some homotrimers(1%).Similar results were obtained in SlitPBPl.In addition,results by pH profiles showed that each of SexiPBPs was present in a mixture of monomer,homodimer and homotrimer,but no obvious changes for their relative ratios were observed under the conditions of different pHs.Taken together,this study suggests that the two Spodoptera PBPs can form homomultimeric proteins to bind numerous odorant molecules.In summary,this study suggests that the members of each Spodoptera PBP/GOBPs are derived from a common ancestor due to gene duplications but have functionally differentiated within and between the two sub-families.In particular,PBPs may also recognize plant odorants and GOBP2 is involved in the detection of sex pheromones.This study greatly sheds light on evolution and functional differentiation of noctuid PBP/GOBPs and assists in designing and developing environmentally-friendly pest control strategies on the basis of olfaction.