Ultrastructural Studies Of Olfactory Sensilla Of Two Parasitoids And Functional Characterization Of Sex Pheromone Receptors Of Three Dendrolimus Spp.

Olfactory plays an important role in the survival and population reproduction of insects,the research on the olfactory perception mechanism of pests will provide a theoretical basis and technical reserve for the development of new control technologies.This article takes two types of insects as research objects,to study the structural and molecular basis of insect olfactory perception.The first part was the ultrastructural studies of olfactory sensilla of two parasitoids via the scanning electron microscope,and compared the antennae sensilla types between pine caterpillar parasitoids we have studied and other related parasitoids.The second part was the functional characterization of sex pheromone receptors of Dendrolimus spp..First,six candidate PR genes of D.punctatus were expressed using the Xenopus oocyte expression system in vitro.We successfully identified two PR genes of D.punctatus by two electrode voltage clamps.After screen the PR genes,we silenced the PR genes of D.punctatus by RNAi technology,wanted to verified the function of PRs in vivo.Then,the PR genes from D.houi and D.kikuchii were screened in the same way.The main findings are as follows:(1)Through scanning electron microscope observation,eight olfactory sensilla were observed on the adults bodies of two parasitoid(Aphidius gifuensis and Pachyneuron aphidis),including: trichoid sensilla(TS),basiconic sensilla(BS),placodea sensilla(PS),B(?)hm bristle(BB),chaetica sensilla(Ch S),squamous sensillum(SS),coeleoconica sensilla(Co S)and basiconic capitate peg sensilla(BCPS).The antennae,as the most important olfactory organs,had the most sensilla types.The sensilla on the antennae of the two parasitoids are basically the same.Some of the rare sensilla are different and may perform special functions.Finally,we compared the antennae sensilla types between pine caterpillar parasitoids we have studied and other related parasitoids.(2)Using the Xenopus oocyte expression system,six candidate PR genes of D.punctatus were expressed in vitro,combined with two-electrode voltage clamps,we identified two PR genes from D.punctatus(Dpun PR45/46).In previous studies,the PR genes of Lepidoptera insects that recognized Type I sex pheromone always clustered together in phylogenetic tree,but our phylogenetic analysis showed that the PR genes of the D.punctatus were not gathered to the traditional PR clade.The two-electrode voltage clamp results showed that Dpun PR45 response to five components: Z5,E7-12: OH;E5,Z7-12: OH;Z5,E7-12: OAc;E5,Z7-12:OAc;Z5,E7-12: OPr,and have a modest response to Z5-12: OH.Dpun PR46 shows a strong response to Z5,E7-12: OPr,and has a weak response to Z5,E7-12: OAc.Dose-response curves showed that Dpun PR45 is more sensitive to minor sex pheromone components than to major components.In addition,because the efficiency of RNAi is highly variable in Lepidoptera,the interference test effect for Dpun PR45/46 and Dpun Orco is not obvious.(3)D.houi and D.kikuchii are two closely related species distributed in the same domain.Using the Xenopus oocyte expression system,we expressed eight,five candidate PR genes in vitro,respectively.And finally identified one PR gene,respectively(Dhou PR26,Dkik PR21).Dhou PR26 response to six components: Z5,E7-12: OH,E5,Z7-12: OH,Z5,E7-12: OAc,E5,Z7-12: OAc,Z5,E7-12: OPr,Z5-12: OH.Dkik PR21 response to seven components: Z5,E7-12 :OH,E5,Z7-12: OH,Z5,E7-12: OAc,E5,Z7-12: OAc,Z5,E7-12: OPr,Z5-12: OH,Z5-12:OAc.Both of them showed broad-spectrum response characteristics to sex pheromones.In this paper,the structural and molecular basis of insect olfactory perception were studied via the scanning electron microscope,Xenopus oocyte expression system,two-electrode voltage clamp,RNAi and so on.The antenna ultrastructure of two parasitoids was clarified,functional characterization of PRs illuminated the intraspecific sex pheromone recognition mechanism of D.punctatus,and sex pheromone recognition mechanism between two closely related Dendrolimus spp.in the same domain.Antennae are the most important olfactory organs of insects,their structural and molecular basis will provide help for the future ecological control of pests.