Function Of A Male Accessory Gland Protein,Jhbp-Like,Involved In The Reproduction Of Bactrocera Dorsalis (Hendel)

The oriental fruit fly,Bactrocera dorsalis?Hendel??Diptera,Trypetidae?is a worldwide pest that harms more than 430 important fruits and vegetables.Because of its powerful ability of destruction,feasible adaption,fecundity,B.dorsalis has been classified as one of the quarantine pests by many countries and regions,causing restrictions on international trades.Mating is the basic step in which an insect chooses the best spouse and produces offspring.Males transfer seminal fluid to the female during mating to complete the generation.In addition to the sperm used for insemination,the main component of seminal fluid are male accessory gland proteins?Acps?which were synthesized and secreted by male accessory glands.Acps act as the main functional active molecules in the seminal fluid,and interact with other factors such as juvenile hormone?JH?to affect female reproductive physiology.In previous study,we identified a new Acp in the male accessory gland of B.dorsalis using transcriptome-proteome analysis,which is predicted to be a JHBP family member according to the conserved functional domain.In this study,the open reading frame?ORF?of BdJHBP-like were cloned using RT-PCR.Furthermore,its expression profiles in different developmental stages and different tissues,as well as the expression in response to biological rhythm,exogenous Methoprene and 20E,were determined.Furthermore,its physiological functions in the reproductive behavior was observed by using RNAi.In addition,we tried to knock out the BdJHBP-like using the CRISPR/Cas9 technology system.The results of this project are crucial to gain insight for the physiological function of Acps in reproduction,and also will pave the way to study the regulation mechanism of other Acps in reproduction in the future.On the other hand,this will also provide a new idea and new pesticide target for ecological control of pests.The main findings are as follows:1.Identification of BdJHBP-like and analysis of it's expression patternBased on the B.dorsalis genome and transcriptome,the ORF of BdJHBP-like was cloned from B.dorsalis.The encoded amino acid sequence was deduced using DNAMAN.Furthermore,the potential physiological functions such as conserved sequence of BdJHBP-like deduced protein were analyzed by bioinformatics softwares such as ClustalW,ProtParam,SignalP 4.1 and TMHMM.The expression patterns of BdJHBP-like in different developmental stages were analyzed by qRT-PCR.The results showed that BdJHBP-like was lowly expressed in eggs,larvae,pupa and female adults,but highly expressed in the sexual matured male adults.The qRT-PCR method was used to analyze the expression patterns of BdJHBP-like among different tissues of male adults,The results showed that BdJHBP-like was highly expressed in the male accessory gland,and lowerly expressed in the head,thorax,midgut,fat body,malpighian tube and testis.While male accessory gland is an important reproductive organ of insects,it was speculated that BdJHBP-like was associated with the reproduction of B.dorsalis.2 Effects of different inductions on the expression of BdJHBP-like in male adultsIn order to investigate the effects of circadian rhythms and exogenous hormone?Methoprene?and 20E on the expression of BdJHBP-like in male adult.The relative expression levels of BdJHBP-like was detected using qRT-PCR method within a day and night,as well as treated by methoprene and 20E.The results showed that the relative expression levels of BdJHBP-like was regular changed as the circadian rhythms changed;At the same time,after treated with methoprene,the relative expression levels of BdJHBP-like was found to be significantly down-regulated at 1 h,then gradually increased to a normal level and then up-regulated at 24 h time-point.While the expression of BdJHBP-like was significantly down-regulated at 1 h,3 h,6 h,12 h and24 h after 20E treatment of male adults.3.Physiological function of BdJHBP-like in male adultBased on the BdJHBP-like spatiotemporal expression pattern,RNAi was performed on 7-days-old male adults by microinjection.The results showed that BdJHBP-like can be significantly down-regulated after injected with dsRNA.Continuous injection of dsRNA into 5th and 7th days of male adults,The results show that the mating rate and duration of injected males with dsRNA were significantly decreased compared with control.The egg production and hatching rate of female adults which mated with suppressed males also significantly decreased compared with control.4.Knocking out BdJHBP-like by CRISPR/Cas9 systemAccording to the genomic DNA sequence of BdJHBP-like and the selection of target sites for gene editing,three target sites were selected on the first and second exon.Then sgRNAs were synthetized in vitro and mixed with Cas9 enzyme at a certain concentration ratio for embryo injection.In this experiment,a total of 573 embryos were injected,128 embryos successfully hatched into larvae,30 of them edveloped into pupa.Finally,13 pupa developed to adults and 5 of them were malformed with wings or legs.Extracted the genomic DNA of pupas that fail to emerge and malformed adults,the target fragment was amplified by PCR,and then sequenced.PCR products which appear overlapping peaks was thereafter cloned into E.coli cell for sequencing.A total of 15 positive clones were detected,three of them were positive knocked out at a position of 113 bp from the target 2.Nine mutations were detected at a position of 32 bp from the target 3.Subsequently,the survived 8 G₀ generation were reared to the sexual matured stage and allowed to mated with the wild type adults.The genomic DNA from G₀ generation were extracted after offspring producing,however the PCR amplification ndd sequencing of BdJHBP-like showed no mutations occurred.