| Insects can accurately locate host plants and seek mates from sophisticated environments through their sensitive sense of olfactory system.The meadow moth Loxostege sticticalis(Lepidoptera: Pyralidae)is an important omnivorous agricultural pest which feeds on the leaves of various economic crops such as beet,soybean,potato,etc.,and seriously threaten the economic development of agriculture in China.The larvae of L.sticticalis have the characteristics of migrating and foraging in clusters,and the collective foraging behavior of the larvae is thought to be involved in the search for new food sources and substantially contributes to the expansion of the infested area,however,the olfactory molecular mechanisms that regulate the collective foraging behavior of are still unknown.In the whole life cycle of lepidoptera,besides larval foraging behavior,another important behavior is the mating behavior of adults.The adult moth is a typical insect that uses sex pheromones to mediate mating behavior,however,the mechanism underlying the selectivity of its pheromone-binding proteins(PBPs)remains unknown.In this study,we analyzed the mechanisms of chemical signals and sex pheromones recognition of L.sticticalis at the peripheral olfactory level by using molecular biology,electrophysiology,insect behavior,RNAi,molecular docking,site-directed mutagenesis techniques,etc.This study would provide molecular targets and theoretical basis for the green prevention and control of the meadow moth.The main results are as follows:1.The 8 ORs expressed in the antennae of larvae were most sensitive to herbivore-induced plant volatiles(HIPVs).In this section,8 ORs expressed in larval antennae were successfully cloned and deophanized in vitro using heterologous expression system of Xenopus laevis oocytes combined with two-electrode voltage clamp records.The results showed that 8 ORs showed different degree of binding sensitivity to 42 common host plant volatiles,LstiOR5,LstiOR6 and LstiOR39,which were narrowly tuned to methyl salicylate,3-octanone and ?-caryophyllene,respectively.In addition,LstiOR14,LstiOR16,LstiOR24,LstiOR26 and LstiOR40 were broad-tuned ORs,the ligands with the largest responds were nerolidol,acetophenone,linalool,eucalyptol and hexyl acetate,respectively.Among these ligands,methyl salicylate,?-caryophyllene,nerolidol,acetophenone are all important HIPVs.2.The HIPV of soybean leaves,methyl salicylate acts as a chemical signal to induce the collective foraging behavior of L.sticticalis,and LstiOR5 is the main receptor for the larvae to recognize methyl salicylate.In this study,GC-MS was used to detect volatiles of soybean leaves with different treatments.The results showed that 8 HIPVs were produced in soybean leaves damaged by larvae,which were toluene,1-decen-3-one,2-hexene,3,5,5-trimethyl,3,5-octadien-2-one,(E,E),2,4-octadienal,(E,E),2-nonen-1-ol,(E),6-undecanol and methyl salicylate;Among these 8compounds,the recognition receptor of methyl salicylate was identified as LstiOR5 by ORs functional expression in vitro.The electrophysiological experiments showed that methyl salicylate induced significant electrophysiological response(p<0.05).The results of Y tube olfactometer assay showed that the methyl salicylate exerted attractive effects on the L.sticticalis larvae at all tested concentrations(p<0.01).Further foraging choice assays showed that the L.sticticalis larvae preferred foraged soybean leaves over unforaged leaves,the mean preference is 67.96%,when methyl salicylate was artificially added to unforaged leaves,the larvae preferred to choose the side of soybean leaf with methyl salicylate volatilization,the mean preference is66.22%.These all results suggest that methyl salicylate is the HIPV of soybean leaf,which could be recognized by the larvae as a chemical signal that trigger the collective foraging behavior of L.sticticalis.In addition,in situ hybridization showed that LstiOR5 was highly expressed in larval antenna neurons;In order to further explore the function of LstiOR5,we successfully reduced the expression level of LstiOR5 in the antennae of larva by RNA interference.After being injected with ds LstiOR5,both the EAG response of larval antennae to methyl salicylate and the attractive effect of methyl salicylate was significantly decreased(p<0.05),and the binary choice foraging assay showed that the average attractiveness of LstiOR5-silenced larvae to foraged soybean leaves and methyl salicylate volatilized side significantly decreased to 48.34%(p =0.001)and 45.57%(p <0.05),respectively.These results suggest that LstiOR5 is the main receptor which involved in in the collective foraging behavior of L.sticticalis.3.LstiPBP1 selectively binds to the sex pheromone component,E11-14:OH,with significantly higher affinity than plant volatiles.In this study,LstiPBP1 and LstiPBP3 were successfully cloned,expressed,and purified,finally,the final proteins which could be used for fluorescence binding assay was obtained.The binding characteristics of LstiPBP1 and LstiPBP3 to a total of 71 compounds including 68 plant volatiles and 3 sex pheromone components were analyzed by fluorescence binding assay,the results showed that LstiPBP1 could selectively bind to E11-14:OH,and its binding ability was significantly stronger than that of plant volatiles,the Ki value is 4.97 ± 0.44,LstiPBP1 also showed lower binding affinity to the other 9 plant volatiles,with Ki values ranging from 10.89 ± 0.93 to 88.91 ± 16.76.LstiPBP3 only bound to six plant volatiles,and the Ki values ranged from 9.20 ± 0.81 to 75.93 ±15.52.4.Hydrogen bond interaction played key role in the binding of LstiPBP1 to E11-14:OH,and the hydrogen bonding site was Leu37.In this study,the binding mode of LstiPBP1 and ligand was studied by homology modeling,molecular docking,site-directed mutagenesis.According to the sequence similarity principle,the 3D model of LstiPBP1 was reasonably constructed based on the structure of Bmor PBP,the 3D structure of LstiPBP1 included six ?-helices,six conserved cysteine residues(Cys),Cys23-Cys58,Cys54-Cys112 and Cys101-Cys121,formed three disulfide bonds in pair,forming a stable protein structure.98.3% of amino acids in the predicted model were in the allowable region,exceeding 90% of the stability threshold,proving that the constructed structure was stable and reliable.The reliable model was used for molecular docking of protein and ligand,the results showed that there was a hydrogen bonding force between LstiPBP1 and E11-14:OH,and the binding site was Leu37.After Leu37 was mutated to Ala37,the E11-14:OH-binding affinity decreased drastically(Ki value changed from 4.97 ± 0.44 to 35.69 ± 2.12).5.Interference with the expression of LstiPBP1 in the antennae of males significantly affected the perception of E11-14:OH in both electrophysiological and behavioral levels.In this study,RNAi was successfully used to reduce the expression level of LstiPBP1 in male antennae to further study the function of LstiPBP1.After LstiPBP1 was silenced,both the antennal response and attractiveness of E11-14:OH decreased significantly(p<0.05).Interference with the expression of LstiPBP1 in the antennae of males significantly affected the perception of E11-14:OH in both electrophysiological and behavioral levels,suggesting that LstiPBP1 is the main binding protein to recognize E11-14:OH.In conclusion,this study provides more evidence to elucidate the molecular mechanism of collective foraging behavior and sex pheromone communication of L.sticticalis,and provides a theoretical basis for further research on the control strategy of L.sticticalis. |
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