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Research On The Olfactory Mechanism Of Parasitic Behaviour In Anastatus Japonicus

Posted on:2019-06-11Degree:MasterType:Thesis
Country:ChinaCandidate:J LiFull Text:PDF
GTID:2333330563953630Subject:Zoology
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Anastatus japonicus is a parasitic wasp used for biological control,that lays the progeny eggs in the host eggs and uses the nutrients in them for growth,eventually destroying the host eggs and achieving the purpose of prevention and control.A.japonicus as a natural enemy of the litchi and longan pest Tessaratoma papillosa has been widely released into the field to parasitize T.papillosa eggs,because it is harmless to crops,does not cause environmental pollution,is conducive to maintaining ecological balance,and achieve long-lasting pest control effects.The key to the research is to understand host-seeking mechanism of A.japonicus,to improve the efficiency of biological control in the field,and to increase field release efficiency.In this study,the odorant receptors and ionotropic receptors of A.japonicus were identified by transcriptome sequencing,and their expression characteristics in various tissues and organs of the wasp were studied,preliminaryly selected that the chemical cues played a significant attractant or repellant role during the host-seeking process;The use of molecular biology,behavior and other methods,proved that the host-seeking behavior of Anastatus japonicus is related to peripheral olfactory receptors,and mainly related to the olfactory co-receptor Orco,and not related to ionotropic co-receptors IR8 a and IR25a;this study for the first time locates the olfactory receptors of the functional semiochemicals during the host-seeking process.The main results are as follows:(1)Identification of chemical pheromones related to host-seeking process of Anastatus japonicusFirstly the experiment monitored the developmental duration of the wasps,including 2 days of egg period,4 days of larval period,4-5 days of pre-pupa period,and 9-10 days of pupa period.From the end of the pupa period to adults,it took less than 24 hours.At this time,the olfactory system of the wasp has not matured,therefore,RNA interference is performed immediately after adult hatched,and the success rate of the interference is ensured.Our previous GC-MS data derived twenty-nine compounds from host plants and host insects.We performed electrophysiological and oviposition assays on A.japonicus,and eight odorants were found to elicit a significant electroantennogram(EAG)response.Among these odorants,?-Caryophyllene,Undecane,(E)-?-Farnesene(+)-Aromadendrene and Cis-3-Hexen-ol had strong attractant effects on oviposition,whereas 2-Ethyl-1-Hexan-ol,Ethyl Acetate and ?-Caryophyllene had a strong repellant effects.Thus,these chemicals might influence the host-seeking and positioning,the also can be used in subsequent experiments to identify the functional receptors that play a role in the host searching process of A.japonicus.(2)Identification and selection the ORs and IRs based on Anastatus japonicus antennal transcriptome analysisPrevious studies have shown that the odorant recognition of insects in the host-seeking process is mainly related to the odorant receptors(ORs)and ionotropic receptors(IRs).In order to further determine which olfactory pathways play a role in the host-seeking behavior of A.japonicus,firstly,we need get the relative gene sequences.Through transcriptome sequencing,we obtained 92,499 Transcripts and 70,473 Unigenes,from which 165 odorant receptors were screened,of which 48 were full-length sequences,including the olfactory co-receptor Orco sequence and the common odorant receptor AjapOr1-AjapOr47 sequences;13 ionotropic receptors,of which 9 were full-length sequences,including the ionotropic co-receptor IR8 a and IR25 a sequences and AjapIR68 a,AjapIR3,Ajap64a1,Ajap64a2,AjapIR6,Ajap76 b,and Ajap93 a.Phylogenetic trees were constructed with the corresponding amino acids sequences of other species of Hymenoptera and their homology were analyzed.Previous studies have suggested that odorant identification in host-seeking behavior may be related to the ORs-based pathways.RT-PCR was used to identify the specific expression of AjapORs and AjapIRs genes in different tissue parts of adults.Combined with the FPKM values in the transcriptome databases,AjapORs genes with both specific expression and high expression levels were screened out,including AjapOR10,AjapOR11,AjapOR20,AjapOR27,AjapOR29,AjapOR33,AjapOR34 and AjapOR35.These genes may be directly involved in insect olfactory perception and play an important role in the recognition of external odorant molecules,therefore,the screening of these eight genes provides the possibility of further olfactory receptor identification of functional chemical cues.(3)Determination of related olfactory pathways in the host-seeking behavior in Anastatus japonicusThe known sequences of the three co-receptor genes Orco,IR8 a and IR25 a obtained by transcriptome sequencing were used to synthesize dsRNA for RNA interference.The results of qPCR validation showed that the expression levels of olfactory genes in the experimental groups injected with dsOrco,dsIR8 a,and dsIR25 a were significantly decreased after 5 days of injection compared with the untreated group and the dsGFP-injected control group,indicating that with the increasing of time,dsRNA in adult worms significantly inhibited mRNA expression of these three genes in olfactory tissues,indicating that RNA interference was successful.Then,A.japonicas after the gene silencing make parasitic reaction and host-seeking time assays.The results showed that the AjapOrco injection group had a lower parasitism rate at all tested egg densities,and differences were not observed between the GFP control and the AjapIR8a/25 a group.Interestingly,the length of time required before the first eggs were found in the AjapOrco-silencing group was longer than the time required for the other groups,and our observations indicated that certain individuals in the AjapOrco-silencing group took an extremely long time to search for suitable eggs.Even when they finally found an egg,these individuals were more likely to locate the oviposition sites by contact sensations rather than olfaction.These results indicate that the chemical cues for sensing during the host-seeking and oviposition processes of A.japonicus are primarily reliant on OR-relevant pathways rather than IR-relevant pathways.Compared with the two control groups,the searching efficiency of the dsOrco,dsIR8 a,and dsIR25a-injected groups did not change significantly,indicating that the influence of A.japonicas own density to seeking efficiency had nothing to do with the olfactory co-receptor Orco and ionotropic co-receptors IR8 a,IR25a.(4)The olfactory receptor localization of Anastatus japonicusIn the above experiments,we found that chemical sensing during the host-seeking process and oviposition is known to be dependent on the OR-relevant pathways.In order to further determine which specific odorant receptors the search-related chemical cues are recognized by,the 8 candidate ORs described above were highly abundant or expressed for olfactory-specific purposes to identify the olfactory receptors of functional semiochemicals.The AjapOR35-silencing group showed a significant decrease in the EAG response to ?-Caryophyllene and(E)-?-Farnesene,indicating that AjapOR35 is tuned to these two oviposition attractants ?-Caryophyllene and(E)-?-Farnesene.Further binary-choice oviposition assays showed that the oviposition attractant effect of ?-Caryophyllene and(E)-?-Farnesene vanished after AjapOR35 was silenced,indicating that the emmision of these attractants from host plants can guide A.japonicus to locate eggs for ovipositioning and indicated that AjapOR35 is correlated with the olfactory detection oviposition behavior of of this species.In summary,this study was conducted on the expression characteristics of odorant receptors and ionotropic receptors in A.japonicas,and it was found that the olfactory co-receptor Orco may play an odor-recognition role in its host-seeking behavior,to lay a foundation for improving the olfactory mechanism of insects;At the same time,it is initially found that in the process of host searching,the recognition of(E)-?-Famesene and ?-Carophyllene may be related to the OR35 common odorant receptor.This study provides a better understanding of the molecular basis and functional chemicals underlying the oviposition behavior of A.japonicus,and the results may help improve biocontrol approaches.
Keywords/Search Tags:Anastatus japonicas, odorant receptors, biocontrol, host-seeking, oviposition
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