Pheromone Biosynthesis And Molecular Regulation Mechanism Of Olfactory Recognition Of Fig Wasps

Insect chemosensory system mainly includes two aspects:smell and taste,and antennae,as an important olfactory organ of insects,can detect chemical signals in the environment with high sensitivity and regulate the corresponding physiological and ecological behaviors of insects,such as feeding,courtship,oviposition,host-seeking,clustering and avoidance.Fig-fig wasp mutualism has coevolved for more than 90 million years,and it is a classic material for the study of co-evolution among species.With a few exceptions,usually one kind of figs is only pollinated by one kind of pollinating wasp,and there are some non-pollinating wasps.In order to accurately locate the appropriate receptive period figs,fig wasps mainly identify the volatile compounds released from figs at the female floral stage through chemical receptors,indicating that olfactory recognition plays a key role in maintaining fig-fig wasp mutualism and their long-term coevolution.In this study,olfactory regulation and pheromone biosynthesis were carried out based on the symbiotic system of two kinds of figs(Ficus hispida,Ficus Semicordata)and fig wasps.The main experimental results are as follows:(1)Cloning,sequence comparison and tissue expression analysis of olfactory genes in fig waspsEight OBP genes and two CSP genes of fig wasps were cloned,the amino acid sequence alignment analysis showed that each olfactory protein had a conserved cysteine structure of typical insect olfactory protein.Evolutionary analysis showed that six species of fig wasps OBP 10 were clustered with AcerOBP10 and AmelOBP10 into one branch,while CgraOBP1,PpilOBP1,PdunOBP1,Asp.OBP1,StriOBP1 and AcerOBP1,AmelOBP1 were clustered into one branch,and there was a group of homologous sequences among CSPs:StriCSP4-PdunCSP4-Asp.CSP4-CsolCSP4-AcerCSP4-AmelCSP4.QRT-PCR detection showed that the olfactory genes(CsolOBP10,CsolOBP6,CsolCSP6,CsolCSP4,CsolORco,CsolOR1,CsolIR8a,CsolIR76b,CsolSNMPl and CsolSNMP2)of Ceratosolen solmsi martchali were highly expressed in adult heads,but low in thoraxes and abdomens.(2)Prokaryotic expression,purification and binding characteristic analysis of olfactory protein of fig waspsThe recombinant target protein OBPs and CSPs of fig wasps were obtained by prokaryotic expression and affinity chromatography purification,and fluorescence competitive binding characteristics with 14 kinds of figs volatiles showed that CsolOBP10 had better binding with 5 attractants,among which the binding ability with Indole was the strongest,and the binding effect of(E)β-Ocimene was the best in the repellents binding experiment.The CgraOBP1 protein of Ficus Semicordata pollinating wasp also showed good binding ability with 6 attractants,among which Indole had the best binding ability.The results of binding analysis of 7 repellents showed that it had good binding ability with 1,8-Cineole,(E)-β-Ocimene and β-Pinene.The above results show that the physiological and ecological behaviors such as pheromone synthesis and olfactory recognition in the two species of fig-fig wasp mutualism have not only coevolutionary convergence,but also species differences.In addition,the OBP proteins of four non-pollinating fig wasps also showed different degrees of binding effects with the fig volatile compouds,and CsolCSP4 and CsolCSP6 hardly bind to the fig volatile compouds.(3)Docking analysis of OBP protein and pheromone of fig waspsBased on the analysis of pheromone ligand binding characteristics of olfactory protein,ligand compounds with strong binding ability were selected for molecular docking.It was found that CgraOBP1,CsolOBP10,PpilOBP1,PdunOBP1,Asp.OBP1 and StriOBP2 mainly depended on the hydrophobic interactions or hydrogen bond between key amino acid sites in protein and pheromone ligand molecules,such as PHE65,PHE66,ILE69,PRO70,Ala71 and TYR72 sites of CsolOBP10,ASN89,VAL90,LYS117 and PHE142 of CgraOBP1.ILE107,VAL133,ASN137,LEU140,TRP141 and PHE142 of PpilOBPl and VAL110,GLN115,MET126,VAL130 and TRP138 of PdunOBP1 of 142PHE and 130VAL,VAL130,ARG134 and PHE139 of Asp.OBP1,PHE77,ILE89,SER114,ILE116 and PHE141 of StriOBP2.(4)Construction and preliminary verification y of pheromone induced and repelled by fig waspsBased on the comparative analysis of transcriptome sequencing of different tissues of figs,the pheromone biosynthesis pathway of attractants and repellents of fig wasps was successfully constructed,and its key regulatory enzymes were verified by qPCR analysis.The results showed that the two attractant synthase genes Linalool synthase1(LIS1)and(-)-alpha-Terpineol synthase 1(α-TES1)were mainly highly expressed in the receptive stage of figs.On the other hand,the repellents synthase gene 1,8-Cineole synthase 1(1,8-CIS1)was mainly highly expressed in the post-receptive stage of figs.The results showed that attractants and repellents of fig wasps may be synthesized in large quantities during the receptive stage and the post-receptive stage of figs,respectively,and thus play key olfactory physiological functions in different developmental stages of figs.To sum up,the OBP proteins of Ficus hispida and Ficus Semicordata pollinating wasps play an important role in olfactory recognition,host location and flower pollination of different fig attraction and repellent pheromones.The tissue expression analysis of attractant and repellent synthase genes further supports the important role of mevalonate pathways,shikimate pathways and Biosynthetic pathway of benzoxazinoid in attracting species-specific pollinators.To some extent,it reveals the olfactory molecular mechanism of fig wasps recognizing host fig volatiles,and also helps to clarify the chemical receptive molecular mechanism of fig-fig wasp mutualism.At the same time,it provides more theoretical basis for protecting this key species of tropical rain forest and the whole tropical rain forest ecosystem.