Molecularmechanisms Of Recognition Of Host Plant Volatiles In Grapholita Molesta Busck

The oriental fruit moth Grapholita molesta(Busck) is an important pest species that attacks stone and pome fruits throughout the world. The adults have a typical harmful habit of host-switch; the larva damage peach shoots in early and mid-season, and then the next generation of adults can switch to pear orchards in mid and late season causing substantial losses. This is an important cause that leads to frequent damage in companion planting area of peach and pear. Previous studies showed that the host-switch behavior was triggered by the change of host plant volatile compounds. The sensitive olfactory system of G. molesta plays a predominant role in seeking host plants, finding mates and selecting oviposition sites.However, the olfactory recognition mechanism of how host plant volatiles were perceived by the moths is still ambiguous. The identification and functional characterization of related olfactory proteins will enable a better understanding of the mechanisms of olfactory recognition at the molecular level of insect chemoreception. More importantly, the research can provide us the development of new methods to control the adults by hindering their olfactory perception. In this study, several kinds of putative olfactory genes in G. molesta antennae were identified by Illumina Mesiq transcriptome sequencing. We have taken systematic studies by tissue expression, binding characterization and key binding site of odorant binding proteins and chemosensory proteins. The main results are as follows:We generated transcriptome of female antennae of G.molestausing the next-generationsequencing technique, and assembled transcripts from RNA-seq reads using Trinity,SOAPdenovo-trans and Abyss-trans assemblers. Thede novotranscriptome assembly was further analyzed using DETONATE and Transrate.Using particular bioinformatics analysis, we identified 124 putative olfactorygenes, including 28 transcripts encoding for odorant binding proteins, 17 chemosensory proteins, 48 odorantreceptors, four gustatory receptors, 24 ionotropic receptors, two sensory neuron membraneproteins, and one odor degrading enzyme.Among the identified 28 OBP sequences, 21 had full ORFs with the length from 357 bpto 756 bp. Sequence alignment showed that almost all the intact OBPsshared the classic six-cysteinemotif, except Gmol OBP14, which was grouped into the“minus-C”subgroup with the secondand fifth cysteine residues missing. The RT-PCR results indicated that OBP genes were all expressed exclusively in antennae except for Gmol OBP1, Gmol OBP2,Gmol OBP6 and Gmol OBP18. Gmol OBP2 was expressed in both female antennae and theremaining body. In addition to the expression in both male and female antennae,Gmol OBP1 was also expressed in the female body.Gmol OBP6 and Gmol OBP18 were extensively expressed in antennae and bodies. The antennae-specific or enriched expressed OBPs may play key roles in host location and oviposition sites selection.Among the identified 17 CSP sequences, 15 unigenes had fulllength ORFs and signal peptide sequences formed 17 to 25 amino acids. The four conserved cysteine(with a pattern of C1-X6-8-C2-X18-19-C3-X2-C4) were found in all candidate CSPs. Compared with OBPs,almost all candidate CSPs were expressedin both the antennae and the body of both sexes,appearing no significant differences between malesand females.Only Gmol CSP6, Gmol CSP7 and Gmol CSP9 were expressed exclusively in male and femaleantennae, and these genes perhaps have special roles in detection and transduction of host plantodors molecules.We successfully expressed five Gmol OBP and five Gmol CSP proteins in prokaryotic expression system, and enriched via Ni ionaffinity chromatography. The binding properties of the r Gmol OBPs and r Gmol CSPs tosex pheromones and host-plant volatiles were investigated influorescence ligand-binding assays.(1) Five r Gmol OBPs were all exhibited strong binding affinities to at least one sex pheromones, whereas five r Gmol CSPs did not bind to any sex pheromones.(2) In addition to sex pheromones, r Gmol GOBP1 also showed broad binding properties towards 30 host plant-derived volatiles emitted from peach and pear.r Gmol GOBP1 had the highest binding affinity to decane with Ki vaule of 5.19 μM, and r Gmol GOBP1 also had a strong binding abilities with many alcohols and esters. The binding properties showed that r Gmol GOBP1 has dualfunctions in recognition of host plant volatiles and sex pheromones.(3) r Gmol GOBP2 exhibited the similar function as PBPs.It could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone dodecanol,suggesting that Gmol GOBP2 may play an important role in courtship and mating of adults.(4)r Gmol OBP8 and r Gmol OBP11 seemed to have similar binding affinity characteristics.The two proteins showed outstanding bindingaffinity todecanal, butyl hexanoate, and α-ocimene,with Ki values of9.82, 10.03, 11.06μM and 12.69, 12.97, 16.79μM, respectively.(5)Gmol OBP15 had a strong binding affinity to hexanal andheptanal, with Ki values of 5.52 and14.81 μM, respectively. We speculated that Gmol OBP15 acts in perception and recognition ofhexanal and heptanal in males and females.(6) r Gmol CSP8 showed specific bindingaffinity with small molecular weight alcohols, such as 1-hexanol and 3-Methyl-1-butanol.r Gmol CSP2, r Gmol CSP3, r Gmol CSP9 and r Gmol CSP11 showed very weak binding activities to host plant volatiles. Compared with Gmol OBPs, Gmol CSPs showed more specific binding affinity with odorant molecules, and the function of olfactory recognition needs further investigation.The crystal structure of the Bombyx mori Bmor GOBP2(PDB code: 2wck.1) and Culex pipiens Cpip OBP(PDB code:3ogn.1) as the template to build the 3D structure of Gmol GOBP2 and Gmol OBP8, respectively. Based on the docking study, Thr9, Val111 and Val114 were binding sites predicted for Gmol GOBP2, and Thr70, Leu74 and Trp108 were binding sites predicted for Gmol OBP8.Using site-directed mutagenesis, six potential key amino acid residues were substituted with alanine, yielding three Gmol GOBP2 mutants(T9A,V111 A and V114A) and three Gmol OBP8 mutants(T70A, L74 A and W108A). Fluorescence ligand-binding assays showed that mutant T9 A with a five times decline in affinity to dodecanol, while the binding affinity of V111 A and V114 A had no obvious difference. It was verified that Thr9 of Gmol GOBP2, was one of the key binding sites of dodecanol.The mutant W108 A completely did not bind to(E)-8-dodecenyl acetate, and the binding abilities of T70 A and L74 A were also significant decrease. The resultssuggested that Trp108 in Gmol OBP8 was a key binding site;Thr70 and Leu74 were binding sites but not the key sites.The whole mount fluorescence immunohistochemistry experiments indicated that a subpopulation of the antennal sensilla basiconica was intensively labeled with Gmol GOBP2 and Gmol OBP8. The number of labeled s. basiconica was significantly higher for anti-Gmol GOBP2 compared to anti-Gmol OBP8.Different from the previousviewpoint, it was interesting that the two OBPs were generally distributed in s. basiconica labeled by anti-Gmol GOBP2 and anti-Gmol OBP8,but involved in the detection of sex pheromones. We speculated that the ORNs of s. basiconica containing Gmol GOBP2 and Gmol OP8 were sensitive to sex pheromone components.Three sex pheromone components, Z8-12:Ac, E8-12:Ac, and Z8-12:OH, gave strong EAGresponses for males, but not females. Host plant volatiles, 1-hexanol,(E)-2-hexenal,hexanal, heptanal, nonanal, decanal, benzaldehyde, butyl acetate, Cis-3-hexenyl acetate, hexyl acetate and butyl hexanoate, can elicit intensive electrophysiological responses of both males and females antennaeat 10 μg/μl. There was a gender difference in the sensitivity of the antennae of male and female to host plant volatiles. The EAG response of female wass more sensitive to hexanal, heptanal, nonanal, decanal, butyl hexanoate than male, while the male was more sensitive to Cis-3-hexenyl acetate and hexyl acetate than female. The dual-choice bioassays showed that the volatiles with an electrophysiological activity did not always elicitbehavioral responses. The insect to recognize the host was mainly based on the verificationof the chemical odor fingerprint of plants, but some key volatiles emitted from host plants played an important role in the localization of insects, for example,(E)-2-hexenal and butyl hexanoate hada distinct attractive effect for female adults, and α-Pinene showed strong attraction for male adults.The highly expressed Gmol GST1 belongs to delta subfamily genes ofinsect cytoplasmic GSTs.The optimum reaction condition of Gmol GST1 was 35 ℃, p H 7.0 and 100 m M Tris-HCl. The activity of Gmol GST1 was measured using GSH and CDNB as standard substrates with a multiskan spectrum microplate spectrophotometer. The Michaelis constant Km and Vmax vaule was 0.232 m M and 172.80μmol/mg/min, respectively. Pesticides exhibited some inhibitory effect on Gmol GST1, but thepercentage of inhibition was all less than 30%.Gmol GST1 had the strongest degradation ability to sex pheromone component dodecanol,with the degrading efficiency of 74.63%. Gmol GST1 also displayed high degradation reactivity to thehost plant volatile, butyl hexanoate, withthe degrading efficiency up to49.19%. The main function of Gmol GST1 was to maintain the sensitivity and effectiveness of the olfactory system by detoxifying toxic xenobiotics.