Molecular Mechanism Of Kr-h1 Regulating Reproduction Of Female Adult In Bactrocera Dorsalis (Hendel)

The oriental fruit fly,Bactrocera dorsalis(Hendel)(Dipttera: Tephritidae),widely distributed in tropical and subtropical areas of the world,is a dangerous fruit and vegetable pest.Its host range is wide,causing serious economic loss.It is one of most important pests,mainly because of its extreme ability to damage numerous fruits and vegetables,as well as the feasible adaption,dispersion and highly fecundity.B.dorsalis is classified as a quarantine pest by many countries and regions,which has caused many restrictions on international trade.Therefore,some entomologists and biologists consider that B.dorsalis is one of the most important agricultural pests in the world.Currently,the field control of B.dorsalis is dominated by insecticides,but due to the widespread and irrational use of insecticides,its resistance to insecticides has developed rapidly.Reproductive maturation is an important physiological process of Bactrocera dorsalis,which has important research significance.In insects,ecdysone interacts with juvenile hormones to participate in the regulation of insect metamorphosis and reproductive maturation.Therefore,to study the regulation mechanism of key genes involved in the reproduction development of B.dorsalis will help find novel insecticidal targets and may provide new ideas and ways for pest control.Based on the genome and transcriptome database of B.dorsalis,the Met and Kr-h1 open reading frames were firstly cloned by using RT-PCR technique.The phylogenetic analysis of Met and Kr-h1 were implemented,respectively.Subsequently,the spatiotemporal expression patterns of Met and Kr-h1 genes in adults of B.dorsalis were determined using qPCR.At the same time,the effects of exogenous hormone(methoprene)treatment on transcript levels of JH signaling pathway and Vg genes were investigated.Furthermore,the functional analysis of Met and Kr-h1 in reproduction development of adult B.dorsalis were performed by RNA interference(RNAi).Besides,the effects of methoprene on expression levels of JH signaling pathway and Vg genes and related phenotypes were explored.The main research results are as follows: 1 Cloning and sequence analysis of Met and Kr-h1 in B.dorsalisThe ORFs of Met(GenBank accession No.MG763072)and Kr-h1(MG763073)of JH signaling pathway genes were cloned from B.dorsalis.Subsequently,the encoded amino acid sequences of BdMet and BdKr-h1 were deduced using DNAMAN v.6.03 software.Furthermore,the amino acid sequence characteristics of BdMet and BdKr-h1 were analyzed using bioinformatics tools such as TMHMM,SignalP 4.1,ClustalW and ProtParam.The phylogenetic trees were finally constructed.2 Spatiotemporal expression patterns of Met and Kr-h1 genes in B.dorsalis 2.1 Expression patterns of BdMet and BdKr-h1 in different ages of female adultsThe qRT-PCR was used to analyze the expression patterns of BdMet and BdKr-h1 genes in different ages of female adults.The results showed that the transcript levels of BdKr-h1 fluctuated a lot compared to that of BdMet.The relative expression levels of BdKr-h1 and BdMet gradually increased and peaked in 7-day-old adults,and then gradually decreased.It suggests that BdKr-h1 and BdMet exhibit important functions in the reproduction development of B.dorsalis.2.2 Expression patterns of BdMet and BdKr-h1 in different tissues of female adultsThe qRT-PCR was used to analyze the expression patterns of BdMet and BdKr-h1 genes in different tissues(fatbody,midgut,hindgut,Malpighian tubules,ovary and testis)of 7-day-old female adults.The results showed that BdKr-h1 and BdMet were both highly expressed in the fat body,midgut,and Malpighian tubules.In contrast,low transcript levels were recorded in ovary and testis.Fatbody is an important tissue for the formation of yolk,which plays an important role in promoting the formation of vitellogenin.The high expression levels of BdMet and BdKr-h1 in fatbody suggests that they may be involved in the development and reproductive maturation of B.dorsalis.3 Effects of methoprene on expression profiles of JH signaling pathway and Vg genes in female adults of B.dorsalisTo clarify the effects of JH exogenous analogue(methoprene)on the expression levels of JH signaling pathway genes in B.dorsalis,the relative expression levels of BdMet,BdKr-h1,BdVg1 and BdVg2 genes were detected using qRT-PCR after 1-day-old female adults were treated by methoprene.The results showed that after treatment of newly enclosed adults with methoprene,the transcript levels of JH signaling pathway and Vg genes were significantly up-regulated relative to those in the control.Meanwhile,compared with that in the control treated with acetone,female ovary development was slightly accelerated by methoprene treatment on 3-to 7-day-old adults.4 Physiological function of Met and Kr-h1 in reproduction of female B.dorsalis 4.1 Effects of BdMet or BdKr-h1 silencing on ovary development and egg productionBased on previous results of spatiotemporal expression patterns in B.dorsalis,the 4-day-old female adults were chosen for RNAi silence of BdMet and BdKr-h1 genes using injection method.The results showed that the relative expression levels of BdMet,BdKr-h1,BdVg1,and BdVg2 were significantly down-regulated after injection with dsMet.Similarly,silencing of BdKr-h1 significantly down-regulated the expression of BdVg1 and BdVg2.Compared with the control group injected with dsGFP,silencing of BdMet or BdKr-h1 delayed ovary development and knockdown of BdMet or BdKr-h1 inhibited the egg production of female adults.4.2 Effects of methoprene in combination with RNAi on ovary developmentBased on the RNAi results above,methoprene was applied onto 4-day-old female adult two hours after RNAi with BdMet or BdKr-h1.Expression levels of JH signaling pathway and Vg genes were measured 24 and 72 h later.The results showed that the application of methoprene after RNAi significantly stimulated the transcript levels of JH signaling pathway and Vg genes,and time-dependent effect existed for methoprene application.Although there were still phenotypic differences between the ovaries of rescued and control(pre-RNAi)groups,ovary redevelopment with a significantly larger surface area occurred following methoprene rescue after RNAi with BdMet or BdKr-h1.This result is consistent with the methoprene-induced changes in spatiotemporal expression patterns and phenotypes described earlier.