The Chemical Communication Behavior Mechanism Of "Floral-adelphocoris Lineolatus-pardosa Pseudoannulata" Mediated By Chemical Information

The life activities of herbivores,such as host location,mating and oviposition,avoiding natural enemies,as well as the search and selection of prey by predatory natural enemies,can not be separated from the chemical communication between the triple nutritional relationships of "plant-herbivores-natural enemy",this chemical signal is the"language" of their unique communication.Insects,as the most abundant species and quantity of animals in the world,have evolved into a complex and sensitive olfactory recognition mechanism after a long period of evolution.Different chemical signals can be accurately identified in a complex natural environment to accurately find food,search for mating,find suitable spawning sites,and avoid predators and so on.Predators also rely on their developed and keen olfactory system to search for prey and identify food quality.Therefore,elucidating the olfactory recognition mechanism of insects is helpful for us to excavate potential pest target genes and facilitate the development of efficient insect behavior regulators to serve agricultural production.At the same time,to find out the mechanism of olfaction in the process of predatory natural enemies preying on prey is helpful to give full play to the control effect of natural enemies on agricultural pests,to achieve the development of green prevention and control technology,and to reduce the amount of chemical pesticides.With the commercialization of transgenic Bacillus thuringiensis(Bt)cotton,alfalfa bug has become an important pest in cotton fields in China.It was found that alfalfa bug showed obvious preference for flowering plants in the process of host transfer.Pardosa pseudoannulata is an important predatory natural enemy,which is widely distributed in rice fields,cotton fields,alfalfa fields,tea orchards,apple orchards and chili fields.In this study,Adelphocoris lineolatus and Pardosa pseudoannulata were studied by bioinformatics,molecular biology,electrophysiology and classical chemical ecology.The chemical communication behavior mechanism of"cotton-Adelphocoris lineolatus-Pardosa pseudoannulata" mediated by volatile was studied.The main results were as follows:1.Sequencing of antennae transcriptome and identification of olfactory related gene in A.lineolatus.We sequenced the male and female antennae of A.lineolatus by using Illumina HiSeqTM 2500 platform.Then,we identified a series of olfactory related genes from the antennal transcripts of A.lineolatus through bioinformatics analysis,including 34 Odorant binding proteins(OBPs),19 Chemosensory proteins(CSPs),4 Niemann-Pick type C2 protein NPC2s(NPC2s),88 Olfactory receptors(ORs),12 Ionotropic receptors(IRs),4 Gustatory receptors(GRs)and 4 Sensory neuron membrane proteins(SNMPs).Phylogenetic analysis showed that OBPs and CSPs in miridae species had a very close evolutionary relationship,and ORs were extremely differentiated and formed various branches in Drosophila melanogaster,Acyrthosiphon pisum,Rhodnius prolixus and A.lineolatus,it showed that they have different functions in responding to different host odors.Different from the highly differentiated general ORs,the ORco of different insects could easily form an obvious direct homologous family.IRs were divided into antennal IRs and divergent IRs subfamilies,seven AlinIRs(AlinIR8a,AlinIR25a,AlinIR76b,AIinIR93a,AlinIR75d,AlinIR75q and AlinIR21a)belonged to the conservative antennal IRs family,of which two AlinIRco(AlinIR8a and AlinIR25a)were grouped into IR8a/25a subfamily,and five AlinIRs(AlinIR41a.1,AlinIR41a.2,AlinIR41a.3,AlinIR41a.4 and AlinIR41a.5 belonged to the divergent IRs subfamily.Different tissue and age expression profile analysis showed that 21 of 34 OBP genes were highly expressed in antennae,12 OBP genes were highly expressed in male antennae,and 7 OBP genes were highly expressed in female antennae.There were 2 OBP genes with the same expression level between male and female.The expression levels of 10 newly discovered CSP genes in male and female antennae were significantly higher than those in body parts.2 NPC2 genes were mainly highly expressed in the antennae,and the expression of the other two NPC2 genes in the body part was significantly higher than that in the antennae.The expression of almost all OR genes increased with the increase of age,and reached the highest level at 3 days of age,and 88 OR genes were clearly expressed in the antennae of adults.Among them,the expression of 20 OR genes in female antennae was higher than that in male antennae,and the expression of 37 OR genes in male antennae was higher than that in female antennae.Most IR genes(except AlinIR75q)were significantly expressed in male and female antennae;4 GR genes had extensive expression profiles;and 3 SNMP genes were specifically expressed in antennae.The identification and analysis of a large number of olfactory genes lay an important foundation for further functional research.2.Expression and functional characteristics of AlinOR59 in A.lineolatus.Based on the analysis of antennal transcripts and tissue and age expression profiles of A.lineolatus,an olfactory receptor gene-AlinOR59 specifically expressed in male and female tentacles of adults was screened.The results of FISH showed that AlinOR59 and AlinORco were co-expressed in the sensory neurons ORN located in the long curved sensilla trichodea,suggesting that AlinOR59 may be related to the recognition of information compounds.Further using Xenopus oocyte expression system and two-electrode voltage clamp recording technique,it was found that AlinOR59/ORco could react to 15 odor ligands:hexyl acetate,ethyl phenylacetate,indole,butyl acrylate,naphthalene,6-methyl-5-hepten-2-one,ethyl butyrate,acetophenone,methyl salicylate,3-hexanone,cis-3-hexenyl acetate,4-ethylbenzaldehyde,3,4-dimethylbenzaldehyde,?-caryophyllene,2-heptanone.All the 15 ligand compounds could stimulate the EAG response of the antennae of male and female adults of A.lineolatus.Such a broad binding spectrum suggests that AlinOR59 may play a key role in the host location of alfalfa bug.In addition,methyl salicylate,?-caryophyllene and 6-methyl-5-heptene-2-one are considered to be plant floral odors,indicating that AlinOR59 may be involved in the floral behavior of alfalfa bug.3.The functional characteristics of ORs highly expressed in the female antennae in A.lineolatus and the interaction between OBP and OR.Four olfactory receptor genes AlinOR2,AlinOR27,AlinOR28 and AlinOR30,specifically expressed in female antennae were screened by analysis of antennal transcripts and tissue and age expression profiles in A.lineolatus.The functions of four ORs were analyzed by Xenopus oocyte expression system and two-electrode voltage clamp recording technique.AlinOR2/ORco had obvious responses to four cotton volatiles ethyl laurate,tridecanol,dodecanol and decanal.AlinOR27/AlinORco has weak binding ability to two ligands,dodecanol and dodecyl aldehyde,respectively,and AlinOR28/AlinORco can response to butyl propionate,methyl salicylate and hexyl hexanoate.AlinOR30/AlinORco can response to methyl salicylate,cyclohexyl acrylate and acetophenone.These results suggest that the ORs specifically expressed in the female antennae may be involved in the localization of host and oviposition sites.In addition,the effect of AlinOBP1 on the function of AlinOR2 was also studied.It was found that AlinOBP1 could increase the reaction intensity of AlinOR2 to low concentration decanal,but significantly weaken the reaction intensity of AlinOR2 to high concentration decanal.AlinOBP1 protein alone could also stimulate the response of co-expressed cells.We speculate that odor molecules need to form a complex with OBPs to better activate the receptor in the process of odor molecule activation of ORs,and insects themselves have the ability to actively regulate the transport efficiency of OBPs to adapt to the concentration of odor molecules in the surrounding environment.4.The olfactory recognition of P.pseudoannulata to the volatiles of alfalfa bug nymphs.The olfactory recognition of natural enemy spiders during predation was studied by using P.pseudoannulata and A.lineolatus.First of all,the behavior test showed that both male and female adult spiders had obvious tendency to alfalfa bug nymphs.The volatiles of alfalfa bug nymphs were analyzed by GC-EAD and GC-MS,two components trans-2-hexenal and trans-2-hexenyl acetate were identified that could cause electrophysiological activity of the spider.Five IRs genes were identified from the appendage transcripts of spider,all of which belong to the conservative antennal IRs family.According to the evolutionary relationship,they were named PpseIR25a,PpseIR93a-1,PpseIR93a-2,PpseIR93a-3 and Ppse1R49,respectively.Tissue expression profile analysis showed that three IRs were highly expressed in the pedipalps,the expression of PpseIR25a,PpseIR93a-3 in the male pedipalps was significantly higher than that other tissues,while PpseIR49 was highly expressed in the female pedipalps.In addition,PpseIR93a-1 was highly expressed in the female abdomen.We preliminarily found out the mechanism of olfaction in the process of spider locating prey,which has guiding significance for further giving full play to the control effect of natural enemy spider on pests.