Regulation And Mechanism Of Juvenile Hormone On Reproductive Diapause Of Harmonia Axyridis

Lady beetle Harmonia axyridis is an important biological control insect,which has been widely used in pest management.Generally,to be a commercial viable pest control method requires that the beetle can be mass-reared,and that can be flexible stored and transported to prolong their shelf-life.Although the mass-rearing method of this beetle is available,the shelf-life adjustment technology of this insect still needs to be exploited.Previous studies shows that diapause manipulation can be exploited for storing and shipping insects without lose of their fitness.Diapause is induced by environmental signals and regulated via hormone signals.Although previous studies have identified the environmental conditions for induction of reproductive diapause of H.axyridis,the physiological characteristics and endocrine regulation mechanism of reproductive diapause of this beetle remains unclear,which hinders the development of diapause manipulation technology.In this study,the diapause characteristics of H.axyridis were systematically studied from the physiological level.The function and regulation mechanism of juvenile hormone on the reproductive diapause of H.axyridis were clarified.The main results are as follows:1.Comparison of pre-reproduction and pre-diapause physiological states of H.axyridis.To fill the knowledge gap of the physiological states of pre-diapause H.axyridis,we monitored the ovarian development,daily feeding amount,and nutrients accumulation of female beetles under 20℃,reproductive-induction condition and diapause-induction condition.The results showed that compared with the females under reproductive-induction condition,the ovarian development of females under diapause-induction condition was obviously arrested,and the daily feeding amount was significantly decreased from the 14 days post eclosion and continually kept at lower amount.Females under diapause-induction condition significantly accumulated carbohydrate and lipid,and the differences of nutrients content between females under the two induction condition reached significance level after 14 day post eclosion.Taken together,females enter diapause after 14 days induction in the mentioned diapause-induction condition.Those results provide a series obvious and stable diapause indicator for follow-up studies,and make it clear that the first 14 days post eclosion is the key period to study the endocrine regulation mechanism of reproductive diapause in H.axyridis.2.Regulation of juvenile hormone on reproductive diapause of H.axyridisAlthough the environmental induction condition and basic diapause physiology of reproductive diapause in H.axyridis have been clarified,the endocrine regulation mechanism is still unclear.Juvenile hormone has been evidenced to be a key regulator of reproductive diapause in variety of insects.Therefore,in this study,we explored the role of juvenile hormone in regulating reproductive diapause of female H.axyridis.The results showed that the juvenile hormone titer of diapause females was significantly lower than that of reproductive females.The application of exogenous juvenile hormone analogue methoprene to females under diapause-induction condition significantly promoted the ovarian development and expression of Vg1 and Vg2,decreased the accumulation of carbohydrate and triglyceride,and increased the expression of lipogenesis genes ADH and FAS and stress-response genes HSP21 and HSP68.Those results indicated that juvenile hormone analogue promoted the reproductive development and inhibited diapause incidence.RNAi mediated gene silencing of juvenile hormone receptor gene Met in females under reproductive-induction condition can cut off the juvenile hormone signaling,which inhibited the ovarian development and vitellogenin deposition,decreased the expression of Vgl and Vg2,increased the accumulation of carbohydrate and triglyceride and the expression of lipogenesis genes and stress-response genes.Those results indicated that knocking down Met blocked the reproductive development and promoted the incidence of reproductive diapause.In addition,knocking down Met in females under diapauseinduction conditions can eliminated the inhibitory effect of methoprene on reproductive diapause.In conclusion,those results showed that the low level of juvenile hormone is the direct endocrine reason of reproductive diapause.3.Regulation mechanism of low level juvenile hormone during reproductive diapause in H.axyridisThe previous study have confirmed that the low level juvenile hormone is the key reason of reproductive diapause,however,the regulation mechanism of the low juvenile hormone level before diapause is still unclear.Previous studies have shown that the juvenile hormone titer homeostasis is regulated by both juvenile hormone biosynthesis and degradation pathway.In order to clarify the regulatory effect of both pathway on decrease the juvenile hormone level before reproductive diapause,we identified the key genes involved in both pathway.The genes was identified based on the gene different expression between females under reproductive-induction condition and diapause-induction condition by RNA-seq.The genes expression pattern analysis showed that genes involved in juvenile hormone biosynthesis were significantly down-regulated in females under diapauseinduction condition,and that of genes involved in juvenile degradation were significantly up-regulated.The RNAi mediated gene silencing indicated that knocking down the juvenile hormone biosynthesis genes FPPS1,FPPS2,FOLD1,and JHAMT1 in females under reproductive-induction condition decreased the expression of juvenile hormone-response gene Kr-hl,inhibited the ovarian development,promote the accumulation of carbohydrate and lipid,and up-regulated the expression of stress-resistance genes,such as HSPs.Knocking down the juvenile hormone degradation genes JHDK,JHEH1,JHEH2,and JHEH3 in females under diapause-induction condition increased the expression of Kr-h1,decreased the accumulation of carbohydrate and lipid and expression of stress-resistance genes,but had no effect on ovarian development.This results indicate that the attenuation of juvenile hormone biosynthesis and enhancement of juvenile hormone degradation jointly led to the low level of juvenile hormone before diapause.Attenuation of juvenile hormone biosynthesis is the main reason for the maintaining of low level of juvenile hormone.4.Up-stream regulator of juvenile hormone synthesis during reproductive diapause in H.axyridis.Insect diapause is jointly regulated by environmental signaling and endocrine signaling.It is an important scientific question in the field of diapause research to clarify how environmental signals act on the endocrine system and finally regulate diapause.Our previous studies have evidenced that low level of juvenile hormone,which determined by attenuation of juvenile hormone biosynthesis,is the main reason of reproductive diapause in H.axyridis.However,the factors which regulates juvenile hormone biosynthesis remains unknown.To uncover this question,we identified DMAP1 may be an upstream regulator involved in the regulation of juvenile hormone biosynthesis during the process of reproductive diapause of H.axyridis by literature analysis.We explored the role and regulation mechanism of DMAP1 in regulating juvenile hormone biosynthesis before reproductive diapause by using RNAi and DNA methylation inhibitors.The results showed that DMAP1 can respond to photoperiodic signaling at the transcriptional level.Knocking down DMAP1 in females under reproductive-induction condition inhibited the expression of juvenile hormone genes FOLD1、FPPS1、FPPS2、JHAMT1 and reduced the juvenile hormone titer,which finally promoted reproductive diapause.Juvenile hormone analogue can rescued the inhibited ovarian development,increased nutrients accumulation,and increased expression of lipogenesis genes ADH and FAS,and stress-response genes HSP21 and HSP68,which caused by DMAP1 Knocking down.Those results indicated that DMAP1 regulated reproductive diapause via regulating juvenile hormone biosynthesis.Although knock down DMAP1 can significantly reduced the DNA methylation level and decreased the expression of DNMT1,the key enzyme of DNA methylation,knocking down DNMT1 and injection of DNA methylation inhibitors,which is a activity inhibitor of DNMT1,had no significant effect on the ovarian development of females under reproductive-induction condition.Those results indicate that DMAP1 regulated reproductive diapause in H.axyridis independently of DNMT1.In this study,we confirmed that low level of juvenile hormone is the direct endocrine factor leading to reproductive diapause of H.axyridis.We clarified that the attenuation of juvenile hormone biosynthesis rather than the enhancement of degradation is the main reason for the low level of juvenile hormone before reproductive diapause in H.axyridis.Moreover,DMAP1 was found to be an upstream regulator of juvenile hormone biosynthesis in reproductive diapause of H.axyridis.Those results can not only help people to further understand the reproductive diapause regulation mechanism of biological control insect H.axyridis,but also provide theoretical support for the development of accurate diapause manipulation technology based on the reproductive diapause regulation target,so as to promote the commercial production of H.axyridis.