Analyses Of Chitinolytic Enzyme Gene Families In Nilaparvata Lugens

Chitinolytic enzymes including chitinases,β-N-acetylhexosaminidases and chitin deacetylases, are key enzymes required for insect chitin metabolism. We focused on rice pest, the brown planthopper Nilaparvata lugens, as model insect for molecular characterization and biological function analyses of three chitinolytic enzymes’gene family members. Analyses of gene sequences, conserved domain structures, phylogenetic trees, gene expression patterns and RNA interference experiments were performed, and the main results are as follows:1. Bioinformatic investigations with genomic and transcriptomic databases identified twelve genes encoding chitinases (NIChts), eleven genes encoding β-N-acetylhexosaminidases (NIHexs) and four genes encoding chitin deacetylases (NICDAs) in N. lugens. Phylogenetic analyses of insects in six orders showed that Cht genes were clustered into ten major groups, including group I-VIII, ENGase and SI-CLP; Hex genes were clustered into five ambiguous groups, except NAGidase; CD A genes were clustered into five clarified groups.2. The developmental and tissue-specific expression patterns of three chitinolytic enzyme families genes shown that eight NIChts (NIChtl, NlCht3, NICht5, NlCht6, NICht7, NlCht8, NlCht9and NICht10) and three NICDAs (NlCDA1, NlCDA2and NICDA4) were highly expressed in the integument and peaked periodically during every molting, which suggests their roles in chitin turnover of the insect old cuticle. NlCht4was specifically expressed in the male reproductive system, especially in the accessory glands, and NlCht2, NIIDGF and NIENGase were found in female reproductive system with expression in all developmental stages. NICDA3was a gut-specific CDA. NIHexl, NlHex4, NlHex7and NIHexll were also expressed periodically, but diverge in different tissues.3. Five NIChts(NIChtl, NlCht5, NlCht7, NlCht9and NICht10), one NIHex (NlHex4) and three NICDAs (NICDA1, NICDA2and NICDA4) were found to be required for molting. Lethal phenotypes that cuticle shedding failure and high mortality were came out after the injection of dsRNAs for these genes. The old cuticle of notum was split open or even not split, the insect bodies became slender and extended, and finally the insects failed to shed their old cuticle and died. No observable morphological and internal structural abnormality was obtained in insects treated with dsRNA for other18genes. However, injection of a mixture of dsRNAs for NlCht2, NlCht3, NlCht6, NlCht7and NlCht8exhibited an interesting phenomenon that challenged insects appeared to loss their claws of six legs and finally died.Chitinolytic enzymes play important roles in insect development. Based on our results,"5+1+3" pattern of chitinolytic enzymes was proposed which shows five NlChts, one NIHex and three NICDAs were requied for molting of N. lugens. Systematic analyses of three chitinolytic enzyme families facilitate further understanding of chitin metabolism in hemimetabolous sap-sucking hemipteran. Meanwhile, chitinolytic enzyme family genes are the potential targets for new biopesticides and RNAi-based management of N. lugens.