In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system. (19th October 2012)

• Record Type: Journal Article
• Authors: Toprak, Umut; Baldwin, Doug; Erlandson, Martin; Gillott, Cedric; Harris, Stephanie; Hegedus, Dwayne D.
• Is Part Of: Insect science. Volume 20:Number 1(2013:Feb.)
• Page Start: 92
• Page End: 100
• Publication Date: 2012-10-19
• Subjects: Insects -- Periodicals; Entomology -- Periodicals; 595.705
• DOI: 10.1111/j.1744-7917.2012.01562.x
• Languages: English
• ISSNs: 1672-9609

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p> <bold>Abstract </bold> 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, <italic>Mamestra configurata</italic>, was examined <italic>in vitro</italic> and <italic>in vivo</italic>. <italic>In vitro</italic> 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. <italic>In vivo</italic> experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent <italic>in vitro</italic>. 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 <italic>per os</italic> feeding in lepidopterans.</p> </abstract>