Studies On The Expression And Function Of Insulin Signaling Pathway Genes From Bactrocera Dorsalis

The oriental fruit fly, Bactrocera dorsalis(Hendel)(Dipttera: Tephritidae), is a world-wide devastating agricultural pest, which widely distributed in tropical and subtropical areas. Due to the wide host ranges, high adaptive capacity, fecundity and dispersal ability, B. dorsalis is treated as an export barrier in the international trade, and documented on the quarantine target lists in many countries. Females typically oviposit in fruit, and the larvae develop tunnel through the fleshy mesocarp on which they feed, causing fruit rotting and drop, directly leading to a great damage to the edible of fruit and vegetables and economical loss. The control of B. dorsalis relyed on the method of traping during the early period, but the pest management is mostly depended on the insecticide spraying nowadays. As a result, many populations of B. dorsalis have developed high-level resistance to some classes of insecticides. Therefore, it’s an urgent task for seeking a more reasonable method to control of B. dorsalis. Insulin signal transduction plays major roles in many important physiological processes, including growth and development, metabolism, reproduction and aging. Therefore, it is urgent to clarify the molecular mechanism of insulin signaling pathway in regulating metamorphosis, and lay the foundation for searching new potential target for novel insecticide development.This dissertation focused on B. dorsalis. The RT-qPCR was employed to analyze the expression profiles of the seven genes in different developmental stages and different tissues. The RT-qPCR was also used to evaluate the effects of starvation and exogenous 20 E treatment in molecular responses under stress. Additionally, the functional analysis of these genes in development of B. dorsalis was performed by RNAi. Finally, by HPLC technology, we detected the effect on the titer of ecdysone in B. dorsalis of BdFoxO gene. This result further clarified the closely relation between insulin signaling pathway and ecdysone. This study could deepen the understanding of function and regulation mechanism of the genes, providing potentional tagrets and theoretical basis in transgenic food applying and new insectcides screening in pest control. The main results are as follows: 1 Expression patterns of three insulin signaling pathway genes of B. dorsalis 1.1 Developmental expression patterns of three insulin signaling pathway genesThe RT-qPCR method was used to characterize the expression patterns of the three insulin signaling pathway genes(BdFoxO, BdFBP and BdPEPCK) during different developmental stages. The results showed that BdFoxO gene was primarily expressed during the larval-pupal transition and in the late pupal stage, and the highest expression level appeared in day-1 larvae. BdFBP gene was mainly expressed during the larval stage,the rest of the stages almost had no significant changes. BdPEPCK gene was mainly expressed during the pupal stage,but the highest expression level appeared in day-1 adult. The results showed that most insulin signaling pathway genes had higher expression during the larval and pupal stage. They had participated in the molting process of B. dorsalis. 1.2 Tissue-specific expression patterns of three insulin signaling pathway genesRT-qPCR was performed to characterize the expression patterns of the three insulin signaling pathway genes(BdFoxO, BdFBP and BdPEPCK) in different tissues, including, head, thorax, abdomen, ovary, midgut, Malpighian tubules and fat body, which were dissected from the day-4 adult. The results showed that the highest mRNA expression level of BdFoxO was observed in the midgut, followed by the fat body. The highest mRNA expression level of BdFBP or BdPEPCK was observed in the fat body. Interestingly, there had a very significant different between the expression in the fat body and ovary of BdPEPCK. Fat body can integrate a variety of nutritions and hormone signal transduction to control the individual and cell size of insect. Therefore, insulin signaling pathway genes play a decisive role to control the individual and cell size of B. dorsalis, and affecting its normal growth and development. 2 The effects of 20 E and starvation treatment on expressions of insulin signaling pathway genes 2.1 The effect of 20 E on expressions of insulin signaling pathway genesThis study has been carried out to investigate the effects of 20 E on the day-5 larvae of B. dorsalis by injecting different doses of 20 E. And RT-qPCR has been used to detect the relative expressions of seven insulin signaling pathway genes(Akt, PDK, PI3K21 B, InR, BdFoxO, BdFBP and BdPEPCK). The results showed that 20 E caused these genes increased significant except Akt. It is suggested that increased of some insulin signaling pathway gene expression to achieve dynamic balance of different hormones in response to exogenous 20 E. 2.2 The effect of starvation on expressions of insulin signaling pathway genesThe study showed that mRNA levels of the insulin signaling pathway genes were remarkably changed after starvation treatment using RT-qPCR analyses. The results showed that most genes increased significant except Akt and BdFBP. We speculated that the mRNA transcription levels changes of insulin signaling pathway genes coordinated and strengthened the signals in B. dorsalis to avoid the advers environmental. 3 The functional study of seven insulin signaling pathway genes in B.dorsalisHigh quality of dsRNA was synthesized by in vitro transcription, and delivered into B. dorsalis by microinjection. Quantitative PCR showed that, the expression level of insulin signaling pathway genes was reduced significantly compared to PBS treatement after injected. The expressiones was depressed to a very low level, down-regulated over 50% compared to the control. To research the interaction of these seven insulin signaling pathway genes, we respectively to detected the remaining six genes after an insulin signaling pathway gene RNAi. We found that these seven insulin signaling pathway genes existed different influences. There had a close relationship between this result and the mechanisms of feedback and negative feedback regulation. Insect started the stress mechanism in special situations, which leading to other signaling pathway participated in the signal transduction of insulin. It can be inferred that a compensation mechanism might exist in the system to enhance the expression levels of other genes, which would ensured the nomal insulin signal transduction in insect body. Through to abserve the changes after injection, we found most insulin signaling pathway genes after silence caused big impacts on its own pupation. It is possible that this result is associated with physiological changes, such as nutrient sensing and hormone changes(ecdysone or juvenile hormone), resulting from gene silencing. The further observation found that the RNAi-mediated silencing of BdFoxO had larger body sizes relative to control-injected individuals. In short, insulin signaling pathway genes play an import role in B. dorsalis development. 4 The effects of BdFoxO gene on the title of ecdysone in B. dorsalisUsing HPLC technology to test the changes of ecdysone title after BdFoxO gene silencing in B. dorsalis. The results showed that the title of ecdysone significantly decreased after Bd FoxO gene silencing in B. dorsalis. It’s not only further clarified the reason that the pupation delayed after BdFoxO gene RNAi, at the same time, clarified the close relationship between insulin signaling pathway and ecdysone.