| Insects possess highly developed olfactory system.With this system,insects are able to detect different chemical cues from the external environments and respond with various physiological and behavioral activities,such as finding mate partner,food sources and oviposition sites.Targeting on the olfactory system,insect sex pheromones and plant volatiles have been used to monitor and control insect pests.To develop more effective and fully utilize the olfaction-based pest control technique,it is important to gain insights into the molecular mechanism of insect olfaction.It has been shown that the general process of insect olfaction involves several classes of proteins,including odorant binding proteins(OBPs),chemosensory proteins(CSPs),odorant receptors(ORs),ionotropic receptors(IRs),sensory neuron membrane proteins(SNMPs)and odorant degradation enzymes(ODEs).Among these proteins,OBPs and CSPs function to bind and transport the hydrophobic odorants across the sensilla lymph onto the ORs situated on the dendrite membrane of olfactory sensillae.However,tens of OBP and CSP homologus genes are found in a single insect species,making it important to clarify those olfaction related OBPs and CSPs and their selectivity to different odorants.Both OBPs and CSPs are small and water-soluble proteins with the hallmark of 6 conserved cysteine residues(for typical OBPs)or 4 conserved cysteines(for CSPs).In Lepidoptera,on the basis of similarity of amino acid sequence and postulated odorant specificity,OBPs are divided into pheromone-binding proteins(PBPs),general odorant binding proteins(GOBPs)and other OBPs.The rice striped stem borer Chilo suppressalis Walker is an important polyphagous pest in rice and some other crops.Up to now,tens of olfaction related genes have been molecularly identified by analyzing the genome and transcriptome data,but their olfactory functions are mostly unknown.In the present study,the electrophysiological reponses of male and female adults to female sex pheromones and various rice plant volatiles were firstly determined;then tissue expression patterns of the OBP and CSP genes were measured,and putative olfactory genes were suggested based on the antennal specificity or dominance in gene expression;with these putative OBP and CSP genes,protein expression and fluorescence competitive binding assay were further conducted to functionally characterize the roles of thses gene in the perception of sex pheromones and plant volatiles;finally,the male attraction and synergistic effect on sex pheromones by cuticular components(CCs)were explored in C.suppressalis.The main results are as follows:1.Electrophysiological responses of C.suppressalis to sex pheromones and plant volatilesSex pheromones and plant volatiles play important roles in mate and host plant searching in this pest To determine the plant volatiles with biological activity,the electrophysiological responses of male and female C.Suppressalis to 30 rice plant volatiles as well as three sex pheromone components were studied using the electroantennogram(EAG)method.EAG results revealed that most of the plant volatiles and all three sex pheromones elicited significant EAG response in male and/or females.Males showed significantly higher responses to all the three sex pheromones and seven plant volatile compounds(2-pentadecanone,nerolidol,(+)-cedrol,farnesol,methyl salicylate,linoleic acid and 2-tridecanone).On the other hand,females displayed significantly high EAG responses to 10 plant volatiles including 2-heptanol,(+)-cedrol,nerolidol,2-pentadecanone,farnesene 2-tridecanone,farnesol,benzaldehyde,?-ionone,and laurinaldehyde.Taken two sexes together,five compounds were able to elicit significantly high response in both sexes,which were(+)-cedrol,farnesol,nerolidol,2-pentadecanone and 2-tridecanone.We proposed that these plant volatiles with high EAG responses might play roles in the finding host plants for feeding and reproduction.2.Expression pattern and functional characterization of two general odorant binding proteins in C suppressalisOdorant sensitivity and selectivity play a crucial role in insect olfaction.GOBPs are thought to be involved in the detection of general odorants released by foods and host plants.However,recent studies suggest GOBPs may also play some role in the detection of sex pheromones.In the present study with the C.suppressalis,real time quantitative PCR(qPCR)measurements showed that both GOBP genes were specifically expressed in the antennae of both sexes,proposing their important roles in olfaction.To examine their specific functions,binding affinities to three sex pheromones and 35 plant volatiles were measured using fluorescence competitive binding assay.As a result,both GOBPs showed very strong binding affinities(Ki=0.33-1.50 ?M)to all three sex pheromone components(Zll-16:Ald,Z9-16:Ald and Z13-18:Ald).In addition,GOBP1 and GOBP2 displayed high binding affinities(Ki<10.00 ?M)to two volatiles(farnesol and oleic acid)and five volatiles((+)-cedrol,farnesol,myrcene,?-ionone and linoleic acid),respectively;GOBP1 and GOBP2 also displayed moderate binding affinities(Ki=10.00-20.00 pM)to two and one compounds,respectively.The results suggest that two CsupGOBPs tune to different array of plant volatiles,and also play roles in the perception of the female sex pheromones.In particular,the study for the first time denmonstrated that GOBP1 has high binding affinity with the sex pheromones in C.suppressalis.3.Tissue distribution profiles and functional characterization of odorant binding proteins in C.suppressalisIn addition to two GOBPs and four pheromone bnding proteins(PBPs),34 other OBP genes have been molecularly identified from C.suppressalis.To functionally characterize these OBPs,we first determined the tissue expression profiles by qPCR,and found that 9 genes(OBP1,3,4,11,15,17,19,20 and 24)were specifically or predominantly expressed in antennae of both sexes,suggesting their roles in olfaction;while three genes(OBP29,30 and 32)were almost not expressed in antennae.Focusing on olfactory roles,the ligand specificities of six antenna specifically or predominantly expressed genes were further investigated for three sex pheromones and 35 plant volatiles,using the fluorescence competitive binding assays.The results revealed that 6 OBPs had different ligand binding spectrum.For sex pheromones,OBP 11 and 15 displayed only low binding affinity(Ki=6.11 and 8.77 ?M)for Z9-16:Ald,respectively;while other 4 OBPs(OBP3,17,19 and 31)did not show binding for all three pheromone components.For the plant volatiles,OBP3,11,17,19 and 31 in total showed moderate(Ki=10.21-19.85 ?M)or high(Ki<10.00 ?M)binding affinity for 11 and one plant volatiles,respectively.In particular,all five OBPs showed high or moderate binding toa plant volatile ?-ionone.It is unexpected that OBP15 did not show any binding affinity to all tested plant volatiles.Taken together,the five OBP genes may play important roles in the perception of different host plant volatiles in C.suppressalis.4.Expression patterns and functional characterization of chemosensory proteins in C.suppressalisSimilar to OBPs,some CSPs are also thought to play roles in insect olfaction by binding and carrying the hydrophobic odorants across the aqueous sensillar lymph.To identify the olfactory CSPs from a repertoire of 21 CSP members in C.suppressalis,tissue expression patterns were firstly examined by qPCR.It showed that CsupCSP2 was antenna specific and seven more CsupCSPs(CSP1,3,4,6,15,16 and 17)were antenna biased in expression,suggesting their olfactory roles;while other CSPs were multiple-tissue expressed and non-antenna biased,suggesting other functions of these genes.To further determine the ligand binding specificity,three putative olfactory CSP genes(CSP1-3)were expressed in Escherichia coli cells,and binding affinity of these three recombinant CSP proteins were measured for three sex pheromones and 35 plant volatiles by the fluorescence competitive binding assays.None of these CSPs showed high binding affinities for all three sex pheromones.However,CsupCSPl and CsupCSP2 exhibited high binding affinities(Ki?10.00 ?M)for four(2-tridecanone,benzaldehyde,laurinaldehyde and 2-pentadecanone)and two(2-heptanol and(+)-cedrol)host plant volatiles,respectively;three CsupCSPs also showed moderate binding affinity(Ki=10.1-20.0 ?M)for 16 plant volatiles.The resuls indicate that the three CsupCSPs play important roles in the perception of different plant volatiles,providing important information for the elucidation of olfactory mechanisms in the rice striped stem borer.5.Male attraction and synergistic effect on sex pheromones by cuticular components in C.suppressalisCuticular components(CCs)in insects play important roles in chemical communication and inner-water protection,however,the evidence is limited in moth species regarding male attraction and synergism to sex pheromones.To address this topic,CCs from male and female bodies of adult C.suppressalis were extracted and analyzed in lab conditions.Results of behavioural assays demonstrated that both female body extract(FBE)and male body extract(MBE)significant induced the male attraction,indicated by parameters of the male selection and staying time after selection.In comparison,FBE was much stronger than MBE in male attraction,suggesting that distinct CCs ratio and/or compounds might exist in males.As expected,gas chromatography(GC)profiling showed two peaks that were almost specific in FBE,suggesting important roles of these compounds in the male attraction.Y-tube assay was further conducted to examine the synergism of FBE to the sex pheromones.It showed that males stayed for significantly longer time at the lure source containing FBE and sex pheromones compared than the lure source containing sex pheromone alone,but FBE did not increase the moth number attracted to the source.Taken together,the present results provide evidence for the chemical communication function of CCs in moth species,and new means on developing synergist to the sex pheromones in pest control. |
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