Functional specialization among insect chitinase family genes revealed by RNA interference

Abstract

The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum (Tc), were examined by using gene-specific RNAi. One chitinase, TcCHT5, was found to be required for pupal-adult molting only. A lethal phenotype was observed when the transcript level of TcCHT5 was down-regulated by injection of TcCHT5-specific dsRNA into larvae. The larvae had metamorphosed into pupae and then to pharate adults but did not complete adult eclosion. Specific knockdown of transcripts for another chitinase, TcCHT10, which has multiple catalytic domains, prevented embryo hatch, larval molting, pupation, and adult metamorphosis, indicating a vital role for TcCHT10 during each of these processes. A third chitinase-like protein, TcCHT7, was required for abdominal contraction and wing/elytra extension immediately after pupation but was dispensable for larval-larval molting, pupation, and adult eclosion. The wing/elytra abnormalities found in TcCHT7-silenced pupae were also manifest in the ensuing adults. A fourth chitinaselike protein, TcIDGF4, exhibited no chitinolytic activity but contributed to adult eclosion. No phenotypic effects were observed after knockdown of transcripts for several other chitinase-like proteins, including imaginal disk growth factor IDGF2. These data indicate functional specialization among insect chitinase family genes, primarily during the molting process, and provide a biological rationale for the presence of a large assortment of chitinase-like proteins.functional genomics ͉ gene family ͉ molting defects ͉ wing abnormalities C hitinases (␤-1,4-poly-N-acetylglucosaminidase; EC 3.2.1.14)are members of the O-glycoside hydrolase superfamily and are found in many species, including microbes, plants, insects, and mammals (1). Insect chitinases, which belong to family 18 glycosylhydrolases, have been detected in molting fluid and gut tissues and are predicted to mediate the digestion of chitin present in the exoskeleton and peritrophic membrane (PM) in the gut to chitooligosaccharides (2, 3). Genes and cDNAs encoding insect chitinases have been identified and characterized from several lepidopteran, dipteran, and coleopteran insects (4-6). Even though only one (or occasionally two) chitinase gene had been previously identified in studies involving many insect species, database searches of fully sequenced genomes from Drosophila, Anopheles, and, more recently, Tribolium, have revealed that each of these insects has a rather large family of genes encoding chitinase and chitinase-like proteins with 16-23 members, depending on the species (refs. 7 and 8 and unpublished data). Based on amino acid sequence similarity and phylogenetic analysis, insect chitinase family proteins have been classified into five or possibly more groups (7,8). From currently available data on a large number of biochemically characterized insect chitinases, we have hypothesized that many and perhaps all group I insect chitinases are involved in chitinolysis associated wit...

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

“…3B), we suspected that TcKnk might have a previously unrecognized function in protection of nascent cuticle from molting fluid, a role previously attributed exclusively to the envelope. We performed TcKnk RNAi and assessed whether the resulting chitin depletion could be rescued by simultaneous down-regulation of transcripts for either or both of the two chitinase-encoding genes TcCht-5 and TcCht-10 because these two genes have been shown to be critical for molting and turnover of chitin in the old cuticle (8). Remarkably, in agreement with the above hypothesis, confocal microscopy and quantitative chitin content analysis revealed that chitin levels could be restored to normal when both TcCht-5 and TcCht-10 were down-regulated along with TcKnk ( Fig.…”

“…A finding that chitinolytic or proteolytic enzymes in molting fluid are excluded from the nascent procuticle would lend some support to the view that the envelope forms a barrier for protection of newly secreted layers of chitin or protein. Zheng et al (6) immunolocalized a chitinase in spruce budworm [orthologous to chitinase 5 of Tribolium castaneum (TcCht-5) (7,8)] in the molting fluid and the integument but did not determine whether this chitinase was colocalized with chitin in the newly synthesized cuticle. In this study, we refute the venerable notion that access of molting enzymes to nascent cuticle is restricted by the presence of a protective envelope and provide evidence for an entirely unexpected alternative mechanism.…”

RNA Interference with Special Reference to Combating Viruses of Crustacea

“…The phenomenon of RNAi has subsequently been described in a wide range of eukaryotic organisms including arthropods and mammals [40,45,48,77,100,121,147]. To date, RNAi has been used as a strategy to investigate gene function [5,15,213] (for example, it has been used to analysis the function of close to 17,000 of the 19,000 (approximate figure) genes in Caenorhabditis elegans [76,186]) and as an antiviral mechanism to combat viral infections in plants [182,211], invertebrates [29,185,209] and vertebrates (in particular, influenza, cancer and human immunodeficiency virus (HIV) [53,54,122,170,195].…”

RNAi in Agriculturally-Important Arthropods

“…Prior to RNAi analysis, the spatiotemporal pattern of gene expression is determined via RT-PCR to ensure RNAi is performed at appropriate time points. Chitin/cuticle pathway genes, including those required for chitin synthesis (Arakane et al, 2005b;, molting, survival and fecundity (Arakane et al, 2010b;Arakane et al, 2009;Broehan et al, 2010;Hogenkamp et al, 2007;Zhu et al, 2008) and tanning of the epidermal cuticle (Arakane et al, 2010a;Arakane et al, 2009;Arakane et al, 2005a) have been functionally characterized, revealing a wealth of potential biotargets for arthropod-specific pest control. While the candidate gene approach has been of enormous value in T. castaneum, it imposes limitations due to reliance on sequence conservation.…”