| 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 pests, mainly because of its extreme ability to damage numerous fruits and vegetables, as well as the feasible adaption, dispersion and highly fecundity. As a typical holometabolic insect, there are four developmental stages of B. dorsalis: egg, larva, pupa and adult. Females oviposit in fruit, and the larvae develop tunnel through the fleshy mesocarp on which they feed, causing major economic loss. Thus, it is of great significance to study on the important physiological process—metamorphosis. Insect molting and metamorphosis are orchestrated by ecdysteroids and juvinile hormone(JH). The molecular mechanisms of insect ecdysteroids signaling pathway have long been clarified, while the molecular mechanism of juvenile hormone signaling pathway remains to be a mistery. Juvenile hormone is important for regulating both metamorphosis and reproduction in insects, therefore, it is urgent to clarify the molecular mechanism of JH 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, the cDNAs of Met, Kr-h1 involved in JH signaling pathway were cloned. They are further investigated by using RT-PCR and 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 hormonal treatment in molecular responses under stress. Additionally, a functional analysis of Met, Kr-h1 in metamorphosis development was performed by RNA interference(RNAi). The results are helpful to explore the functions and regulating mechanism of JH signaling pathway genes in B. dorsalis. Our results may provide new ideas and ways for controlling this insect. The main results are as follows: 1 Molecular cloning and characterization of Met, Kr-h1 genes of B. dorsalisBased on the results of high-throughput transcriptome sequencing of B. dorsalis, the full-length cDNAs encoding Met, Kr-h1 were cloned, the open reading frames(ORF) and deduced amino acid sequences were confirmed by sequence analysis. Scanprosite, NetNGlyc 1.0, SignalP 4.1, TMHMM v2.0 and 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. 2 Expression patterns of Met, Kr-h1 genes of B. dorsalis 2.1 Developmental expression patterns of Met, Kr-h1 genesThe qPCR method was used to characterize the expression patterns of Met, Kr-h1 genes during different developmental stages: Egg, larvae, pupae and the 1-d adult. The results showed that Met continued expression throughout the developmental stages, but the expression in larval stage was significantly higher than pupal stage. Met mRNA persisted without major fluctuations through all larval instars and peaked in the 9-d larvae, afterwards, kept low expression in pupal stage. The constitutive presence of Met suggested its function in metamorphosis of B. dorsalis.Temporal profile of Kr-h1 was more dynamic.Kr-h1 sustained high expression in egg and the 1 instar but expressed the lowest in 3-d and 4-d larvae, then picked up from the 5-d larvae to 9-d larvae. In addition, both of Met, Kr-h1 were up-regulated during the transformation of pupa--adult. Kr-h1 gene in response to juvenile hormone changes, played a great role in metamorphosis of B. dorsalis. 2.2 Tissue-specific expression patterns of Met, Kr-h1 genesTissue-specific expression patterns of Met, Kr-h1 genes were analyzed in five different tissues from the 3rd-instar larvae, including fatbody, midgut, Malpighian tubules, integument and trachea. The results showed that Met, Kr-h1 genes were highly expressed in larval Malpighian tubules and fatbody, while lowest in trachea and midgut, respectively. 3 Effects of exogenous hormones on expressions of Met, Kr-h1 genes 3.1 Effects of exogenous hormones on expressions of Met, Kr-h1 genes and phenotype of B. dorsalis larvae.Topical administration of exogenous natural juvenile hormone JH III and methoprene to B. dorsalis 4-day-old larvae showed that, Met, Kr-h1 gene expression have suffered different degrees of increase after both hormone treatment 6 h, 10 h, 24 h. Phenotypic records show that, although there’s no significant difference in pupation time between treatment and control, the ―middle form‖ between larvae and pupae did appear after hormone treatment. Exogenous natural juvenile hormone JH III and methoprene stimulated Met, Kr-h1 genes and thus involved in the regulation of metamorphosis. 3.2 Effects of exogenous hormones on expressions of Met, Kr-h1 genes and phenotype of B. dorsalis pupae.Topical administration of exogenous natural juvenile hormone JH III and methoprene to early B. dorsalis pupae showed that, the expression of Met was not stimulated by neither of the hormones, and there’s no significant up-regulation compared with control.While Kr-h1 gene sustained increase throughout the pupal stage after 1 d, 3 d, 5 d, 7 d treatment, and the expression level increased by 79.5 times after 7 d treatment. The stimulation of Kr-h1 gene resulted in death of B. dorsalis pupae since it could not complete the molt. 4 Functional study of Met, Kr-h1 genes in B.dorsalisHigh quality of dsRNA was synthesized by in vitro transcription and mixed with dye cochineal. The 4-day larvae were fed with the dsRNA and subsequently, qPCR was carried out to determine the RNAi efficiency 24 h and 48 h later.RNAi of Met, Kr-h1 resulted in their significantly decreased expression levels. The results showed that the expression level of Met, Kr-h1 was significantly decreased compared to the control after 24 h and 48 h treatment, which indicating that the method of feeding interference is feasible. However, there’s no significant difference in pupation time between RNAi treatment and control(precocious phenomenon), only the size of the larvae under RNAi treatment were a little smaller than control. |
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