In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system
- 20 August 2012
- journal article
- research article
- Published by Wiley in Insect Science
- Vol. 20 (1), 92-100
- https://doi.org/10.1111/j.1744-7917.2012.01562.x
Abstract
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.Keywords
This publication has 40 references indexed in Scilit:
- Chitin synthases are required for survival, fecundity and egg hatch in the red flour beetle, Tribolium castaneumInsect Biochemistry and Molecular Biology, 2008
- Control of coleopteran insect pests through RNA interferenceNature Biotechnology, 2007
- Digestive enzyme compartmentalization and recycling and sites of absorption and secretion along the midgut of Dermestes maculatus (Coleoptera) larvaeArchives of Insect Biochemistry and Physiology, 2006
- The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrixInsect Molecular Biology, 2005
- Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNABMC Biotechnology, 2003
- Specific developmental gene silencing in the honey bee using a homeobox motifInsect Molecular Biology, 2002
- Hemolin gene silencing by ds‐RNA injected into Cecropia pupae is lethal to next generation embryosInsect Molecular Biology, 2002
- Parental RNAi in Tribolium (Coleoptera)Current Biology, 2002
- The peritrophic membrane of Spodoptera frugiperda: Secretion of peritrophins and role in immobilization and recycling digestive enzymesArchives of Insect Biochemistry and Physiology, 2001
- Using RNAi to investigate orthologous homeotic gene function during development of distantly related insectsEvolution & Development, 1999