| The large black chafer Holotrichia parallela(Motschulsky)(Coleoptera Scarabaeidae)is one of the most harmful underground pests.In recent years,it is more urgent to prevent and control H.parallela because of its serious damage.As people pay more and more attention to their own health and food safety,pheromone lures and plant volatile lures,which are environmentally friendly,low toxic,safe and efficient control methods,have been paid more and more attention.Therefore,it is important to identify the active plant volatiles for the control of H.parallela Olfactory system and taste system are the main pathways for insects to recognize plant volatiles and sex pheromones in the surrounding environments.However,the molecular mechanism of chemoreception is still unclear.Therefore,study on the molecular mechanism of chemoreception,identification of olfactory related proteins will be helpful to discover more potential target genes for pest control.Our results are as follows(1)In order to compare the sensilla of antennae between sexes,the ultrastructure of sensilla was observed and analyzed.H.parallela antennae were a typical lamellate type,including a basal scape,a pedicel,a flagellum,and a lamellar club composed of the three terminal flagellomeres.Twenty-two subtypes of sensilla were identified on the outer and inner surfaces of the lamellae in both sexes:three sensilla trichodea(TRS),three sensilla chaetica(CHS),four sensilla placodea(PLAS),two sensilla coeloconica(COS),six sensilla auricilica(AUS),one Bohm’s bristle(BB),and three sensilla basiconica(BAS).TRS,CHS,and BB were distributed primarily on the outer surfaces of the antennae as mechanoreceptor sensilla,while PLAS,COS,AUS,and BAS were concentrated on the inner surfaces of the lamellae as chemoreceptors.The number of PLAS is about the sum of COS and AUS,suggesting PLAS plays an important role in the recognition of various odorant molecules.PLAS and BAS were more abundant in males than in females,but females had more COS and AUS.PLAS were distributed along the lamellar periphery,while COS,BAS,and AUS were distributed in the center of the lamellae.It is suggested that the antennae of males and females perform different functions using different sensilla.(2)Behavioral and electrophysiological experiments were performed to analyze the preference of H.parallela to plant volatiles.EAG experiments were examined to determine the antennal responses of H.parallela to 11 plant volatiles.The phenylacetaldehyde and cis-3-Hexen-l-ol could elicit the strongest EAG responses in male and female antennae,respectively.In addition,the sensitivity of antennae to plant volatiles was different between male and female.Female antennae were sensitive to 2-phenylethyl propionate and geraniol,while male antennae were more sensitive to phenylacetaldehyde,trans-2-hexen-l-ol and eugenol.The dose response curves showed that:for females,cis-3-hexen-ol at 10μg/μl,trans-2-Hexen-l-al at 0.1μg/μl and cis-3-Hexenyl acetate at 1μg/μl could elicite the highest EAG response.For males,the strongest EAG response was found at a low concentration of 0.1 μg/μl for trans-2-Hexen-l-ol and 1μg/μl for phenylacetaldehyde.What’s more,the behavioral responses of H.parallela to 11 volatiles were tested by"Y" olfactometer assays.It was found that male preferred phenylacetaldehyde and female preferred cis-3-Hexenyl acetate.Finally,we tested the synergistic effect of phenylacetaldehyde on sex pheromone by cage experiment.The number of male H.parallela captured increased obviously with the increasing of phenylacetaldehyde concentration,and the best concentration ratio of phenylacetaldehyde:sex pheromone was 1:3.(3)Based on RNA-seq,the antennal transcriptome of female and male were systematically analyzed.43,624 unigenes and 38,785 unigenes were obtained from female antennae and male antennae,respectively.Finally,34,706 distinct unigenes were assembled.A total of 19,925 unigenes were annotated by aligning to Nr,NT,Swiss-Prot,KEGG,COG and GO databases.The main physiological processes were analyzed by GO and COG functional annotations.A total of 4172 KEGG Orthology(KO),378 KEGG pathways and 248 KEGG modules were obtained through KEGG annotations.“Signal transduction" is the most important metabolic pathway in antennae.1870 differentially expressed genes were obtained by comparing male and female antennal transcriptome data,including 158 male up-regulated genes and 1712 female up-regulated genes.Furthermore,53 male up-regulated genes and 432 female up-regulated genes were annotated by GO functional annotation.393 female up-regulated and 40 male up-regulated genes were annotated to KEGG database,and then 180 female up-regulated and 38 male up-regulated genes were annotated into KEGG pathway layer.20 female up-regulated and 9 male up-regulated genes were further assigned into KEGG module layer.It was found that differentially expressed genes were mainly concentrated in pathways related to substance regulation,metabolism and signal transduction.(4)A total of 145 chemosenory related genes were identified based on antennal transcriptomes,including 7 OBPs,3 CSPs,2 SNMPs,47 ORs,15 GRs,27 IRs 43 P450s and 1 CPRs.Two differentially expressed cytochrome P450 genes were significantly up-regulated in female antennae by comparative transcriptome analysis.(5)Bioinformatics analysis and tissue expression analysis of chemosensory genes were carried out to identify the characteristics and expression profiles of these olfactory genes and predict their possible olfactory functions.Four or six cysteine conserved residues and six alpha helix sites were obtained by aligning seven OBPs and three CSPs.Eight conserved motif regions in Coleoptera OBPs and CSPs were obtained by motif analysis.These motifs constitute 11 and 2 common motif patterns for OBP and CSP,respectively,inferring different functional characteristics of OBP and CSP.The phylogenetic analysis predicted the relationship of OBP and CSP with those of other species.FPKM values and qRT-PCR were used to compare the expression levels of OBP and CSP between male and female antennae and to predict the functional differences of olfactory proteins between sexes.Five OBPs(HparOBP33,34,35,36 and 37)and one CSP(HparCSP17)showed higher expression in female antennae.The expression profiles of OBP and CSP between olfactory and non-olfactory tissues were compared by qRT-PCR,and explored olfactory-specific or biased expressed genes(HparOBP36,HparOBP 37 and HparCSP 17),which may perform important olfactory functions in antennae.Other non-olfactory biased genes(HparOBP35)may be involved in other physiological processes.(6)Candidate chemoreceptor genes were identified,including 47 ORs,15 GRs and 27 IRs,and 2 sensory neuron membrane proteins(SNMPs).26 ORs,4 GRs,16 IRs and 2 SNMPs have complete open reading frames(ORFs).Seven transmembrane domains of ORs and two transmembrane domains of SNMPs are predicted.Phylogenetic analysis revealed the relationships of chemoreceptor genes between H.parallela and other species,including OR co-expression receptors(HparOR40),IR co-expression receptors(HparIRl and HparIR27),carbon dioxide receptors(HparGR5 and HparGR14)and sugar receptors(HparGR8,HparGRll and HparGR13).The expression of 13 receptor genes in female antennae was higher than that in male antennae,while the expression of other 4 receptor genes in male antennae was higher than that in female antennae.Twelve receptor genes were mainly expressed in the antennae(olfactory tissues),but at a low level in the non-olfactory tissues.Two SNMPs showed both female biased and olfactory(antennae)biased expression profiles.(7)43 cytochrome P450s and 1 NADPH-cytochrome P450 reductase were identified.The conserved substrate recognition sites(SRS1-6),EXXR and WXXXR motifs,PERF and heme binding domains of P450 genes were successfully predicted by bioinformatics analysis.Three conserved regions of CPR,FMN,FAD and NADPH binding regions and several active catalytic residues(serine 472,cysteine 644,aspartate 687 and tryptophan 689)were also identified.Phylogenetic analysis revealed that 22 P450s clusterd into CYP3 family(16 P450s in CYP6 subfamily,4 in CYP345 and 2 in CYP9),20 P450s clusterd to CYP4 family(16 in CYP4 subfamily and 4 in CYP313)and 1 P450s clustered into CYP2 family(CYP303 subfamily).Comparative transcriptome analysis showed that HparCYP8 and HparCYP33 were significantly up-regulated in female antennae.qRT-PCR showed that 23 and 10 P450 genes were highly expressed in male and female antennae,respectively.In addition,four antenna-specific P450s(HparCYP7,9,15 and 36)and four antenna-biased P450s(HparCYP3,18,34 and 35)were also identified. |
