Functional Analysis of Genes of Chitin Metabolism in Tribolium castaneum by RNA interference

Abstract

A bioinformatics search of the genome of the red flour beetle, Tribolium castaneum, has identified families of genes encoding chitin synthases (CHS), chitinases (CHT), N‐acetylglucosaminidases (NAG), chitin deacetylases (CDA) and other proteins such as Retroactive (RTV) and Knickkopf (KNK), which may be involved in chitin assembly. The predicted gene models were confirmed or corrected by direct cDNA cloning. Tissue‐specific and developmental stage‐specific expression patterns were also investigated by RT‐PCR analyses. The roles of these proteins in synthesis, turnover and/or organization of chitin in the cuticle have been investigated in this model beetle species by RNA interference (RNAi), confocal and transmission electron microscopy. Injections of 2‐200 ng of double‐stranded RNAs into larvae, pupae or adults resulted in specific down‐regulation of the targeted transcripts within 2‐3 days. RNAi for many of these genes resulted in distinct phenotypes, which included lethality, molting arrest, developmental and cuticular abnormalities and failure of egg‐hatch.These studies have revealed that many apparently redundant genes of chitin metabolism have distinct physiological functions and/or tissue specificities. For example, the two CHS genes specialize in making chitin that becomes part of either the cuticle or the peritrophic matrix (PM). One chitinase (CHT10) is critical for every molt, whereas another chitinase (CHT5) is essential only for the adult molt. RNAi for two other chitinase/chitinase‐like proteins results in developmental arrest or morphological abnormalities. Interestingly, two CDAs appear to be essential for molting as well. KNK and RTV appear to be involved in the protection of chitin from chitinases as well as assembly of chitin laminae in the procuticle. Some proteins with peritrophin‐A type chitin‐binding domains (ChtBD2 domains) also appear to be essential for molting and/or survival.