Studies On Binding Characteristics Of Odorant-binding Proteins And Mulberry Leaf Volatiles In Glyphodes Pyloalis And Its Parasitoid Wasps

Glyphodes pyloalis Walker is a major mulberry pest,which break out in a short time and seriously impede the development of sericulture industry in China.The long-time application of chemical insecticides to control G.pyloalis has led to the issues of a sharp increase in the resistance of G.pyloalis,environmental pollution of mulberry orchards,and phytotoxicity of Bombyx mori.Therefore,it is extremely urgent to develop green and high-efficiency pest management of G.pyloalis.In recent years,green pest management methods represented by lure technology and parasitoid wasps have been gradually promoted and applied.The core of them is based on the profoundly revealing the insect recognition and perception mechanism of odor compounds,and illustrating the target odor molecules responded behavior,ultimately successfully applying to the research and development the biocontrol products of attractants or parasitoid wasps.So far,a large number of olfactory genes have been identified and their functions have been deeply analyzed.However,the studies of olfactory system in G.pyloalis and its parasitoid wasps have not been systematically carried out,which greatly impede the development of new green pest management methods for G.pyloalis.In this study,the chemical components of mulberry leaf volatiles were determined by gas chromatography-mass spectrometry(GC-MS).Key olfactory genes were identified based on antennal transcriptome database.Then,we obtained odorant-binding proteins(OBPs)by prokaryotic expression and purification in vitro,and their binding characteristics to mulberry leaf volatiles were analyzed.The binding modes of OBPs and ligands and the key binding sites were analyzed and explored by molecular dynamics(MD)simulation.The behavioral selection response of G.pyloalis to odors was analyzed by insect olfactometer.These findings provide an important reference for fully revealing the olfactory molecular mechanism of the G.pyloalis and its parasitoid wasps,and also lays a theoretical foundation for developing novel pest control strategies based on insect olfaction.The main findings are as follows:1.The chemical components of mulberry leaf(Yu 71-1)volatiles were identified and the binding characteristics of OBPs proteins and mulberry leaf volatiles were analyzed.Moreover,one of mulberry leaf volatiles that could attract G.pyloalis male moths were identified.The chemical components of healthy mulberry leaves and mulberry leaves infested by G.pyloalis were identified for the first time by headspace solid phase microextraction(SPME)and GC-MS.The results showed that α-pinene and β-pinene were the main volatiles of healthy and infested mulberry leaf.β-ocimene,2-pentadecanone and hexadecanal were the main volatiles of mulberry leaves infested by G.pyloalis.Two general odorant-binding proteins(GOBPs)of G.pyloalis(Gpyl GOBP1 and Gpyl GOBP4)were expressed and purified in vitro using prokaryotic expression system.The binding affinities of they to 56 ligands were determined by fluorescence competitive binding assays.Gpyl GOBP1 could bind to three plant volatiles,and the binding affinity of Gpyl GOBP1 to volatile of mulberry leaves infested by G.pyloalis(hexadecanal)was the strongest(Ki = 2.29 ± 0.18 μM).Gpyl GOBP4 could bind to four plant volatiles,including mulberry leaf volatile4-tert-octylphenol,and the binding affinity of Gpyl GOBP4 to farnesol was the strongest(Ki = 2.12 ± 0.03 μM).Behavioral selection assays showed that G.pyloalis male moths were significantly attracted to hexadecanal,a volatile of mulberry leaves infested by G.pyloalis,which suggested that there is a relationship between the damage of G.pyloalis larvae to mulberry trees and the behavioral selection of G.pyloalis adults to host plants,and that Gpyl GOBP1 was a key target protein for the recognition of hexadecanal.2.The binding modes of Gpyl GOBP1 and Gpyl GOBP4 proteins to ligands were explored by MD simulations,and the effects of mutations of key amino acid residues on the binding affinity of proteins were analyzed by experiments,then,the key binding sites of Gpyl GOBP1 and Gpyl GOBP4 proteins to ligands were identified by theoretical simulation and specific experiments.Three-dimensional models of Gpyl GOBP1 and Gpyl GOBP4 proteins were constructed by homology modeling,and MD simulation was used to explore the binding modes of Gpyl GOBP1 and Gpyl GOBP4 proteins to ligands.The binding free energies of the complexes were calculated by molecular mechanics/Poisson-Boltzmann surface area(MM/PBSA),and values are all greater than-20 kcal/mol.Van der Waals forces provide the largest energy contribution,while electrostatic interaction and non-polar solvation energy provide a weak energy contribution,while polar solvation energy hinders the formation and stability of the complexes.The computational alanine scanning(CAS)revealed that Phe12 residues of Gpyl GOBP1 and Gpyl GOBP4 are important amino acid residues for their binding to ligands.The mutants Gpyl GOBP1-F12 A and Gpyl GOBP4-F12 A were expressed and purified by site-directed mutation.Fluorescence competitive binding assays showed that the binding affinity of Gpyl GOBP4-F12 A to β-ionone was significantly decreased,and the binding affinities of two mutants to other ligands was directly lost.It was suggested that Phe12 residues of Gpyl GOBP1 and Gpyl GOBP4 proteins were key sites for binding to ligands.3.The RNA-Seq technology and bioinformatics analysis were used to identify key olfactory genes of parasitoid wasp of G.pyloalis.A total of 84 and 122 olfactory genes were identified from the antennal transcriptome database of Aulacocentrum confusum and Iseropus(Gregopimpla)kuwanae,respectively.The genes included 11 Acon OBPs genes of A.confusum and 11 Ikuw OBPs genes of I.kuwanae,respectively.Tissue expression profiles showed that Acon OBPs(Acon OBP1-7,9,and 11)genes were highly expressed in the antennae of A.confusum,with relative expression levels up to 16,000 folds.Seven Ikuw OBPs(Ikuw OBP1-3,5,9-11)genes were highly expressed in the antennae,and the relative expression level was up to 350,000 folds.The Ikuw OBP6 protein,which was highly expressed in the antennae and ovipositor of I.kuwanae,was obtained by prokaryotic expression and purification.The binding characteristics of Ikuw OBP6 protein with 56 ligands were determined by fluorescence competitive binding assays.The results showed that Ikuw OBP6 could only bind to the mulberry leaves volatile 4-tert-octylphenol(Ki = 35.24 ± 1.18 μM).These results indicated that OBP genes has typical expression specificity in antennae,suggesting their potential olfactory function in the antennae of Mulberry parasitoids.The Ikuw OBP6 protein may be a key target protein in the recognition of I.kuwanae to mulberry leaf volatiles,and plays an important role in host location.In conclusion,we identified the chemical components of mulberry leaf volatiles for the first time.Then,the binding characteristics of OBPs and mulberry leaf volatiles in G.pyloalis and its parasitoid wasps were explored,respectively.Meanwhile,we identified the volatile components of mulberry leaves that can significantly attract G.pyloalis male moths.These findings could provide a theoretical support for further establishing a new strategy of using attractants or parasitoid wasp products to control G.pyloalis.