Functional Characterization Of Three Neuropeptide Genes On Regulation Of Juvenile Hormone In Clostera Anastomosis (Lepidoptera:Notodontidae)

Neuropeptides are a class of peptide hormone secreted by the nerve cells released into the blood or hemolymph. They are involved in almost all physiological behavioral and development processes in insects. Study on the expression patterns and biological functions of neuropeptide contribute to a better understanding on activities and development of insect, and provide more theoretical basis based on the new pesticide of neuropeptides. Juvenile hormone (JH) play a role in both growth and reproduction in insect. Blocking the synthesis of juvenile hormone is thought to be a specific insecticide, which target on insect growth and development. It is a major challenge to investigated the mechanisms and the mode of action on JH regulated by neuropeptides at molecular level. In this study, Clostera anastomosis tianscriptorne of mixed instars samples was successfully constructed, and based on this, a new gene ASTCC which encoding allatostatin was obtained. In addition, we also studied on the expression patterns of neuropeptide genes and its biological function on regulation JH by RNAi approach in Clostera anastomosis. The main results are as follows:Transcriptome sequences are assembled and 46304 Unigene sequences were obtained, which 23259 Unigenes were annotated to Nr protein database,14140 were an notated to KEGG database,7091 were annotated to COG database,6387 were annotated to GO database. These data not only provide an effective way for researching the function of neuropeptide genes in this article, but also provide a reference for future research and studying other functional genes.By analyzing the transcriptome data, the coding sequence of C. anastomosis ASTCC was successfully obtained, the full-length 324bp. Multiple sequence alignment and phylogenetic analysis showed ASTCC has a similar functional domain, two disulfide bonds formed by 4 cysteine, to the known gene ASTC which encoding allatostatin.The results of neuropeptide gene expression patterns showed, ASTC, ASTCC and AT mainly expressed in the brain and midgut, only trace amounts expressed in the body wall. This pattern has proved the hypothesis that they act as CA regulator. While these neuropeptide genes expressed highly in midgut also indicating that they may have other functions. Expression pattern of genes in JH synthesis pathway showed that these five JH synthase genes ACTA, HMGS, HMGR, FPPS, JHAMT expressed differently in development of C. anastomosis, but they all mainly expressed in the brain, while the expression in the gut and body wall is very low, indicating that at different times, the gene in charge of regulation JH synthesis may be different, and the brain may be the highly specific organization which synthetic JH.The expression level ofASTC and AT in various stages reflected the regulation of JH synthesis. ASTCC and ASTC might function in the same bio-pathway in the larvae and pupae, but not in female adults. Expression of ASTCC was significantly higher than the expression of ASTC in the whole development stages, this may be because ASTCC play more important roles in C. anastomosis. In addition, in this paper, using RNAi method study the function of neuropeptide gene, successfully and specifically silenced the target gene in different instars of C. anastomosis.RNAi of ASTC, ASTCC and AT in larvae and pupae of C. anastomosis did not significantly affect weight, pupation rate, emergence rate, mortality and JH titers of larvae, in natural and after RNA interference with ASTC or ASTCC, no correlations between ASTCIASTCC and genes in JH synthesis pathway showed in larvae and pupae. This results further evidenced ASTCC and ASTC function in the same bio-pathway in the larvae and pupae, can neither regulate JH synthesis nor affect the growth o f C. anastomosis. However, in the adult stage, after RNAi with ASTCC the eggs hatchability decreased. At molecular level, either in nature or after RNAi, the expression of ASTCC was showed significant negative correlations to the genes which in JH synthesis pathway, especially the key genes-JHAMT. It suggests that the role of ASTCC regulation on JH was stage-specific, only plays a role in the adult female. On the contrary, whatever in natural or after interference with ASTC, no correlations between ASTC and genes in JH synthesis pathway in adults, this indicating that the allatostatin neuropeptide gene which regulate JH in female adult of C. anastomosis was ASTCC rather than ASTC. The correlation between ASTC and ASTCC was not significant after RNAi of ASTC or ASTCC suggested that ASTC and ASTCC do not regulate each other in C. anastomosis.After injection H2O and dsGFP, there was no difference of target genes expression level compared to untreated insects, it indicated that the effect of RNAi is specific in C. anastomosis, there is no off-target effects.In this paper, we successfully characterized the function of ASTC, ASTCC and AT, three neuropeptide genes by RNAi. The results showed that in the larval and pupal stages, these three genes neither affect the development nor the expression level of genes in JH biosynthesis pathway in C. anastomosis. While in the adult stage, the allatostatin gene which functioned is ASTCC rather than ASTC. AT gene may regulate JH biosynthesis act on the gens HMGS, HMGR, FPPS and JHAMT. To establish the basis for understanding the mechanism of neuropeptide genes in the regulation of JH biosynthesis in insects.