Identification of the Mamestra configurata （Lepidoptera Noctuidae） peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism

The peritrophic matrix （PM） is essential for insect digestive system physiol- ogy as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata （Lepidoptera： Noctuidae）, a major pest of cruciferous oilseed crops in North America. Liquid chromatography-tandem mass spectrometry analyses of the PM identified 82 proteins classified as： （i） peritrophins, including a new class with a CBDIII domain; （ii） enzymes involved in chitin modification （chitin deacetylases）, di- gestion （serine proteases, aminopeptidases, carboxypeptidases, lipases and ^-amylase） or other reactions （/^-l,3-glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase）; （iii） a heterogenous group consisting of polycalin, REPATs, serpin, C-Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut, cDNAs encoding chitin synthase-2 （McCHS-2）, chitinase （McCHI）, and fl-N-acetylglucosaminidase （McNAG） enzymes, in- volved in PM chitin metabolism, were also identified. McCHS-2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS-2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix （PM）, composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double-stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins （insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1） and the chitin biosynthetic or modifying enzymes （chitin synthase-B and chitin deacetylase 1） in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing （by 24 h） for the gene encoding chitin deacetylase 1 and a slower rate of silencing （by 72 h） for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase-B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double-stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.