| The oriental fruit fly, Bactrocera dorsalis (Hendel)(Dipttera: Tephritidae), is widely distributed in tropical and subtropical areas of the world. It is one of most important insect pests, mainly because of its extreme ability to damage numerous fruits and vegetables, as well as the feasible adaption, dispersion and highly fecundity. 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 this pest relyed on the method of traping during the early period, while the pest management is mostly depended on the insecticide spraying nowadays. As a result, many populations of this pest have developed high-level resistance to several classes of insecticides. In recent years, many researchers have focused on controlling insect pestsvia disrupting their growth and development. 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.Based on the results of high-throughput transcriptome sequencing of B. dorsalis, six cDNAs involved in insulin signaling pathway were cloned. They are further investigated by using RT-PCR and RACE, as well asbioinformatic softwares. Furthermore, the expression profiles of these genes were analyzed in different developmental stages and different tissues. The qPCR was also employed to evaluate the effects of starvation and hormonal treatment in molecular responses under stress. Additionally, a functional analysis of IRS in ovary development was performed by RNA interference (RNAi). The results are helpful to explore the functions and regulating mechanism of insulin signaling pathway genes in B. dorsalis. Our results may provide new ideas and ways for controlling this insect pest.The main results are as follows:1Molecular cloning and characterization of insulin signaling pathway genes of B. dorsalisBased on the results of high-throughput transcriptome sequencing of B. dorsalis, four full-length cDNAs encoding IRS, PI3K21B, PDK, Akt/PKB, and two cDNA fragments encoding InR and PI3K92E were cloned, and deposited in GenBank and accession numbers as follow: IRS (KF305824)、PI3K21B (KJ028006)、PDK (KJ011127)、Akt/PKB (KJ011125)、InR (KJ028005)、PI3K92E (KJ028006). The open reading frames (ORF) and deduced amino acid sequences were confirmed by sequence analysis. Scanprosite, NetNGlyc1.0, SignalP4.1, TMHMM v2.0and other bioinformatics softwares were further utilized to analyze the physical and chemical properties of the proteins, such as conserved domain, signal peptides, transmembrane regions, and potential physiological functions.2Expression patterns of insulin signaling pathway genes of B. dorsalis2.1Developmental expression patterns of insulin signaling pathway genesThe qPCR method was used to characterize the expression patterns of the six insulin signaling pathway genes during different developmental stages:third-instar larvae, pupae and adults of different ages. The results showed that these genes were expressed during the larval-pupal transition and in the late pupal stage. Specifically, InR, Akt/PKB and PDK were mainly expressed during the larval-pupal transition, while the other three genes were lowly expressed in this period. During the late pupal stage, high levels of all transcripts of the six genes were detected, suggesting that theinsulinsignaling pathway isrelated tometamorphosis of B. dorsalis. Besides, the insulin signaling pathway genes have age-specific expression patterns. The relative expression level of IRS was significantly higher in males than females, while other genes showed higher expressions in females. Subsequently, the expressions of PI3K21B and InR at the10th day and Akt/PKB at the4th day females were significantly higher than males at the same stage.2.2Tissue-specific expression patterns of insulin signaling pathway genesTissue-specific expression patterns of the six insulin signaling pathway genes were analyzed in five different tissues from adults, including head, thorax, midgut, Malpighian tubules and fat body by using qPCR. RT-PCR was performed to characterize the expression patterns of insulin signaling pathway genes in different tissues, including brain, fat body, midgut, Malpighian tubules, integument and trachea, which were dissected from the day-4third-instar larvae. The results showed that all six genes were highly expressed in larval brain and adult head. Furthermore, IRS, PI3K21B, PI3K92E and PDK were also highly expressed in larval fat body and Malpighian tubules. It is suggested that those genes might be involved in different physiological processes, including not only presumably related to neurosecretory system in the brain, but also nutrients storage and energy metabolism in fat body.3Effects of exogenous hormones and starvation on expressions of insulin signaling pathway genes3.1Effects of exogenous hormones on expressions of the insulin signaling pathway genesOn the basis of the finding that fat body is the main organ where the insulin signaling transduction happens, we investigated the effects of exogenous hormone on the expression of the insulin signaling pathway genes. The results showed that IRS, PI3K21B, Akt/PKB and PDK were upregulated remarkably with JHA and20E treatment compared with control larvae at12h post-treatment, while PI3K92E was downregulated with JHA treatment. However, the expression levels of InR showed no significantly difference after JHA and20E treatment. It is suggested that increased of some insulin signaling pathway gene expression to achieve dynamic balance of different hormones in response to exogenous hormone stimulation.3.2Effects of starvation on expressions of the insulin signaling pathway genesThe results showed that mRNA levels of the insulin signaling pathway genes were remarkably changed after starvation treatment using qPCR analyses. The results showed that theexpressions of these genes were significantly increased after a48h starvation compared to the fed larvae, except PI3K21B. However, the expressions of these genes were decreased when the larvae were re-fed for24h after a24h starvation. It is unclear whether this is an insect stress mechanism to adjust its metabolic balance or not, further investigation is needed for the elucidation of the hypothesis.4Functional study of IRS gene in B. dorsalisHigh quality of dsRNA was synthesized by in vitro transcription and injected into B. dorsalis females. Subsequently, qPCR was carried out to determine the RNAi efficiency. RNAi of IRS resulted in their significantly decreased expression levels, and caused smaller ovaries of female than the control. The results showed that the expression level of IRS was significantly decreased compared to the control after24h and48h treatment. Surprisingly, the expression levels of other insulin signaling pathway genes were increased after72h treatment. It is possibility that the 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 ensure the nomal insulin signal transduction in insect body. |
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