Molecular Mechanism Of Olfactory Behavior Of Bactrocera Dorsalis Mediated By Olfactory Co-Receptor BdorOrco And BdorIR8a

Fruit fly is an important fruit and vegetable pest in the world.Due to the boredom of the larva and the extremely strong flight ability of the adult,the control of the pest by chemical pesticides is limited.The behavior regulator is an important technology to prevent and control the fruit fly,which mainly interferes with the key behavior of the fruit fly’s survival and reproduction through the sense of smell.The improvement and development of olfactor-based behavioral regulators usually rely on key olfactory molecular targets,mainly odor receptors and ionic receptors in insects.Because the olfactory co-receptor is the key element for the functions of the two types of receptors,it can predict the range of olfactory receptors corresponding to the key behaviors of important compounds and fruit flies by establishing the relationship between the co-receptor and important information compounds and allelopathic behaviors,and assist in the screening of future behavioral regulators.Therefore,this study first built and optimized the gene editing technology of fruit fly based on CRISPR-Cas9.Further,mutants of odor receptor co-receptor Bdor Orco and ionic receptor co-receptor Bdor IR8a were constructed using this technology.Then,the relationship between two kinds of co-receptors and the important information compounds of the fruit fly was established by using electrophysiological techniques;Finally,using behavioral technology,the effect of odor co-receptor deletion on several key behaviors of fruit flies was verified.The specific research results are as follows:1.From the design of guide RNAs（gRNAs）,embryo collection,embryo injection,post-injection insect breeding and mutant screening,the process of mutant construction based on CRISPR-Cas9 was described and optimized in detail and systematically.Using this process,the hatching rate of injection can reach 39-41.5%with a single injection of 200 capsules.Taking Bdor Orco as an example,the positive rate of G0 offspring can reach 81%.2.Further use the CRISPR/Cas9 technology constructed above to mutate Bdor Orco and Bdor IR8a respectively.Using the exon 2 region of Bdor Orco gene as the target region,a homozygous line with 2 bp deletion in exon 2 was obtained through screening,and its amino acid translation terminated prematurely at 160.Taking the exon 5 and 6 of IR8a gene as the target region,a homozygous line with 393 bp deletion of the large segment of exon 5 and 6 was obtained through screening,and the amino acid translation at 259 was terminated prematurely.3.According to the reference,64 kinds of important information compounds related to fruit flies were selected,and the differences of electrophysiological responses of fruit flies olfactory co-receptor mutants adults and wild type adults to these compounds were measured and compared by using the antenna potential technique.The results showed that the EAG responses of Orco^-/-females and males to all the tested odors,such as ester,alcohol,aldehyde and alkene,were significantly reduced compared with the wild type of fruit fly.There was no significant difference between the IR8a^-/-male and the wild type in the electrophysiological response to the above compounds.However,the female IR8a^-/-showed a significant decrease in the response to isovaleric acid odor.The electrophysiological response to allo-ocimene andα-terpinene increased.4.Using behavioral technology,the effect of olfactory co-receptor deletion on the attraction of two key information compounds,methyleugenol and 2,3,5-trimethylpyrazine,to adults was studied.The results showed that Orco mutant could not be induced by these two compounds,while IR8a mutant was not affected by the trend of these two compounds.Further study on the courtship and mating behavior of the fruit fly,the results showed that the absence of two kinds of olfactory co-receptors reduced the mating rate of the adult and delayed the mating peak of the adult.At the same time,the flapping behavior of the male was also significantly reduced,and the mating duration became shorter.Finally,select two kinds of lure sources,γ-Octalactone and orange juice were used to study the effect of olfactory co-receptor deletion on female oviposition behavior.After Orco knockout,females lost their preference forγ-Octalactone to lay eggs,and the number of eggs laid by IR8a mutant females decreased significantly.Addγ-Octalactone in orange juice can not promote the egg production of Orco^-/-,but it can promote the egg production of wild type female fruit fly and IR8a^-/-female.To sum up,this study used the optimized CRISPR-Cas9 technology to construct mutants of fruit fly Bdor Orco and Bdor IR8a,and combined with electrophysiological and behavioral techniques to establish the relationship between two olfactory co-receptors and important information compounds and key behaviors of fruit fly.It is found that the odor co-receptor plays a dominant role in the olfactor-based allelopathic communication behavior of the fruit fly,and the ionctropic receptor co-receptor plays an important role in some information compounds and behaviors,providing a reference for the screening of key molecular targets of future behavioral regulators,and providing a scientific theoretical and technical basis for the improvement and development of behavioral regulators.