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Molecular And Functional Characterization Of Olfactory Genes In Helicoverpa Armigera,Chrysopa Pallens Andchrysopaperla Sinica

Posted on:2016-04-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z Q LiFull Text:PDF
GTID:1313330512472646Subject:Agricultural Entomology and Pest Control
Abstract/Summary:PDF Full Text Request
A sophisticated olfactory system is a key physiological trait for insect survival and reproduction.Olfaction is crucial to many behaviors,including feeding,mating,toxin avoidance,and negative taxis.Previous studies have shown that the major olfactory-related proteins in insects are odorant-binding proteins(OBPs),odorant receptors(ORs),ionotropic receptors(IRs),sensory neuron membrane proteins(SNMPs)and odorant-degrading enzyme(ODEs).The process of.insect olfaction can be generalized as having several main steps.First,odorants reaching the pore tubules enter a sensillum and are bound and solubilized by OBPs;then,the OBPs are transported through the sensillar lymph that fills the cavity around the dendrites,where they finally activate membrane-bound ORs.After OR activation,the odorant will be rapidly removed by ODEs to restore the sensitivity of the sensory neuron.In addition,some other proteins are also thought to be involved in insect olfaction.Similar to OBPs,CSPs are thought to affect insect chemoreception by enhancing the solubility of semiochemicals and delivering them to the chemosensory receptors.Compared with the many available reports on functional and structural studies of olfactory genes in pests,such data from natural emeny are scarce.Most Neuroptera insects are natural enemies of agroecosystem pests.The lacewing Chrysopa pallensRamburand Chrysoperla sinicaTjederare the most important natural predators of various pests such as aphids,coccids,thrips,and and the eggs and young larvae of lepidopteran insects.This two lacewing speices are voracious predators that adapt well to different agroecosystems,so they are very useful auxiliaries in biological control.Butstudies on olfactory system of Neuroptera insects were blank.The cotton bollworm,Helicoverpa armigeraHubner,is a polyphagous species posing a major threat to cotton,maize and some vegetable crops.Although rapid adaptation of Bt cotton has greatly suppressed H.armigera damage,pest resistance to Bt toxin is a potential problem for all Bt crops.An olfaction based behavioral interference strategy has been used for a number of moth pests and could provide a valid alternative for sustainable control of H.armigera.To enhance the effectiveness of C.pallens and C.sinica in biological control via tools such as the "push-pull" strategy,in which combinations of repellent and attractive stimuli are used to alter the populations of insect pests and natural enemies,research about olfactory behavior,including hunting,mating,and oviposition,is essential.Here,we reported the molecular and functional characterization of olfaction related genes from C.pallens,C.sinica and H.armigera.The main results are as follows:1.Transcriptome and digital gene expression analysis in Neuroptera with an emphasis on chemoreception genes in Chrysopa pallens.To facilitate identifying sets of genes involved in olfaction,the transcriptome of C.pallens were sequenced.A large number of candidate chemosensory genes were identified,including 16CpalOBPs,17CpalCSPs,39CpalORs,and genes potentially involved in olfactory modulation.To better understand the CpalOBPs,CpalCSPs and CpalORs,The expression pattern of 16CpalOBPs,17CpalCSPs and 39CpalORs were determined by qPCR,and demonstrated antennae dominantly expression of most CpalOBP and CpalOR genes.On the other hand,10 digital gene expression libraries of different tissues were constructed and gene expression profiles were compared among different tissues in males and females.The DGE results were consistent with the major functions of different tissues,most of the olfaction genes were up-regulated in male and female antennae,the center of olfaction.Most of the differentially-expressed genes were up-regulated in FB.The majority of up-regulated genes were involved in metabolic processes,suggesting that the thorax and abdomen have enhanced metabolic activities,perhaps because these tagmataare the most important sites of digestion,circulation,and reproduction.This large number of insect chemosensory will provide the basis for further functional studies.2.Identification and expression pattern of candidate olfactory genes in Chrysoperla sinica by antennal transcriptome analysis.C.sinica is one of the most prominent natural enemies of many agricultural pests.We sequenced female-and male-antennae transcriptome of C.sinica.Many candidate chemosensory genes were identified,including 12 CsinOBPs,19CsinCSPs,and 37CsinORs from C.sinica.Tissue distribution profiles of all CsinCSP,CsinOBP,and CsinOR genes were investigated in our study.The expression patterns of candidate olfaction genes in C.sinica might help characterize the function of these proteins in future research.Our study showed that most of OBPs were highest expression in antennae than the other tissues,suggesting an olfactory role for these genes in.Insects CSPs serve varied functions,including chemosensationand development,as well as other processes.The broad and diverse expression patterns suggest that different CSPs play different roles during the adult stage.Those genes highly expressed in antennae,CsinCSP4,6,14,and 16,may be involved in insect chemoreception.All of 37 ORs displayed antennae extremely or highly expression pattern,indicating they may play crucial roles in olfaction of C.sinica.Among the CsinORs,CsinORco gene the most abundant in antennae of C.sinica,which is similar with AgoORcoA.gossypii.Because ORco is a co-receptor in insect,which serve as chaperones with the other ORs,so more ORco transcripts were needed to active different ORs.3.Comparison of olfaction genes expressed in the antennae of two lacewing species reveal different feeding specializations in adults.C.pallens and C.sinica have different adult feeding specializations.We compared the 17 CpalCSPs,10 CpalOBPs,and 39 CpalORs from female-and male-antennae transcriptomes ofC.pallens with 19 CsinCSPs,12 CsinOBPs,and 36 CsinORs from female-and male-antennae transcriptomes ofC.sinica.Tissue expression patterns and phylogenetic and abundance analyses suggested that olfaction genes might have played an important role in the speciation of the two species.Comparing orthologous groups of CSPs,OBPs,and ORs,we found that many gain and loss events had happened during evolution.Additionally,CpalOBP and CpalOR transcripts were more abundant in the antennae of C.sinica(33662 and 444)than C.pallens(20718 and 334),suggesting different olfactory system between these two lacewing species.4.Odorant-binding proteins display high affinities for behavioral attractants and repellents in the natural predator Chrysopa pallens.In this study,we purified nine OBP genes from C.pallens(CpalOBPs).Ligand-binding assays showed that there were 10 ligands that showed relatively higher binding affinities(Ki<20 ?M)for CpalOBPs,suggesting that CpalOBPs might play important roles in searching habitat,oviposition sites and recognizing and usingthe herbivory insect induced plant volatiles to improve its hunting efficiency.These differences in the binding properties are probably caused by different roles in chemoreception among the CpalOBPs.Behavioral trials using the 30 compounds exhibiting high binding affinities for the CpalOBPs revealed that 18 were able to elicit significant behavioral responses from C.pallens.Among them,farnesene and its corresponding alcohol,farnesol,elicited remarkable repellent behavioral responses from C.pallens.Thus,C.pallens might recognize and use the aphid alarm pheromone to improve its hunting efficiency for aphids.Our study provides several compounds that could be selected to develop slow-release agents that attract/repel C.pallens and to improve the search for strategies to eliminate insect pests.The compounds that elicited significant behavioral responses could be selected to develop slow-release agents to attract/repel C.pallens and to improve the research for the "push-pull" strategy,in which combinations of repellent and attractive stimuli are used to alter the populations of insect pests and natural enemies to eliminate insect pests.5.Binding assays in the odorant binding protein genes of Chrysoperla sinica.In this study,we purified six OBP genes from C.sinica(CsinOBPs).A fluorescence competitive binding assay was used to determine the binding activities of the.six CsinOBPs for 85 volatile compounds.The results showed that CsinOBPs have different binding profles.CsinOBP4 does not show any binding affinities with any of the 85 tested compounds.CsinOBP2,6 and 9 exhibited high binding affinities for one ligands(Ki<20?M).There were 8 ligands that showed relatively higher binding affinities(Ki<20 pM)for CsinOBP1 and 21 ligands for CsinOBP10.CsinOBP1 exhibited high binding affinities for dodecane,2-hexyl-1-decanol,caryophyllene,(-)-trans-caryophyllene,and farnesene,suggesting that CsinOBP1 might play important roles in searching habitat,oviposition sites and hunting.Additionally,CsinOBP10 had high binding capacities for phenylacetaldehyde and hexyl butyrate which are herbivory insect induced plant volatiles.These data indicate that CsinOBP10 is involved in detecting and recognizing the herbivory insect induced plant volatiles to improve the hunting efficiency.6.Two Minus-C odorant binding proteins from Helicoverpa armigera display higher ligand binding affinity at acidic pH than neutral pH.In this study,two novel Minus-C OBP genes(HarmOBP17 and 18)were characterized from the cotton bollworm,H.armigera,one of the most important pests in cotton and other crops.qPCR results indicate that that HarmOBP17 was expressed highly in the head of female,and the antennae of both sexes,whereas HarmOBP18 had a dominant expression only in female wing tissue.Binding properties of the two OBPs with plant volatiles were investigated at different pH levels using a competitive binding assay.Both OBPs displayed the highest binding affinities(Ki<13?M)with Beta-ionone among the 85 tested compounds.Surprisingly,the two OBPs(HarmOBP17 in particular)displayed higher binding at pH 5.0 than at pH 7.4 and 10.0.This is the first reported Lepidopteran OBPs showing a contrary pH-dependent ligand binding behavior to other OBPs,implying a different mechanism of ligand binding and release.In addition,binding assays of the mutant OBPs(with the C-terminus eliminated)showed that the C-terminus was important for the ligand binding,but this importance varied with different ligands and OBPs,suggesting that their binding properties depend on the specific interactions between OBP and ligand.7.Expression analysis and binding assays in the chemosensory protein gene family indicate multiple roles in Helicoverpa armigera.We identified and cloned 24 CSP genes(HarmCSP)to better understand the physiological function of CSPs in H.armigera.qPCR assays indicate that CSP genes are ubiquitously expressed in adult H.armigera tissues.Broad expression patterns in adult tissues suggest that CSPs are involved in diverse cellular processes.HarmCSP6,9,18,and 19,all highly expressed in sensory organs or pheromone glands,were expressed in bacteria to explore CSP function.Fluorescence competitive binding assays were used to measure the binding affinities of HarmCSP6,9,18,and 19 for 85 plant volatiles and 4 pheromone components.Results reveal that only HarmCSP6 exhibited higher binding affinity(Ki<1.5?M)for pheromone components.The other three proteins do not show any binding affinities with any of the 89 tested compounds.HarmCSP6 was found to be expressed in numerous cells located in or very close vicinity to long and short sensilla trichodea on both female and male antennae.These results suggest that HarmCSP6 may be involved in the transportation of female sex pheromones in H.armigera.In conclusion,the female-and male-antennal transcriptomes of C.pallens and C.sinica were sequenced using next-generation sequencing technology.We identified large number of olfactory gene from C.pallens,C.sinica and H.armigera.This large number of insect chemosensory genes will provide the basis for functional studies.To better understand the physiological function of the olfactory gene,phylogenetic,mRNA abundance analyses,Semi-quantitative RT-PCR,qPCR,ligand-binding assays,behavioral trials and in situ hybridization were used.Our study has yielded large-scale sequence information for further studies and can be used to identify potential targets for the bio-control.And,the compounds that elicited significant behavioral responses could be selected to develop slow-release agents to attract/repel C.pallens and to improve the research for strategies to eliminate insect pests.
Keywords/Search Tags:Chrysopa pallens, Chrysoperla sinica, Helicoverpa armigera, chemosensory protein, odorant-binding protein, odorant receptor
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