Expression Profiles and Functional Analysis of Genes Encoding Chitin Deacetylases, Extracellular Matrix‐Modifying Proteins in Tribolium castaneum

Abstract

In insects chitin, the β‐(1,4)‐linked N‐acetylglucosamine homopolymer, is one of the major components of the exoskeleton or cuticle. The extracellular matrix (ECM) of this exoskeleton is modified in different ways to make it either very rigid and thick or thin and flexible, or to generate specialized structures such as mandibles with sharp cutting edges. Chitin deacetylases (CDAs) are secreted proteins belonging to a family of extracellular chitin‐modifying enzymes that deacetylate chitin to form chitosan, a polymer of β‐(1,4)‐linked D‐glucosamine residues. This modification possibly contributes to the affinity of chitin for a variety of proteins distinct from those that bind uniquely to chitin and also to mechanical properties of the cuticle.Two genes encoding putative chitin deacetylases in Drosophila, serpentine (CG32209) and vermiform (CG8756), play critical roles in shaping of tracheal tubes as well as in regulating the structural properties of epidermal cuticle. By searching Beetlebase (http://bioinformatics.ksu.edu/BeetleBase/), we identified nine genes encoding CDA‐like proteins in Tribolium. A comparative analysis of CDA families in other insect species including Drosophila, Anopheles and Apis indicated that the number of CDA genes varies from species to species. Of these, T. castaneum has the greatest number, including some recently duplicated genes found clustered in its genome. The expression profiles of all CDA genes during development were studied by RT‐PCR. The Tribolium genes TcCDA1 and TcCDA2 are orthologs of DmSerp and DmVerm, respectively, and are expressed throughout all stages of insect development. In contrast TcCDA6 – 9 are expressed predominantly during the larval feeding stages. in situ hybridization revealed that TcCDA1 and TcCDA2 are expressed in epidermal cells, whereas TcCDA6 through TcCDA9 are expressed in cells lining the midgut lining.To study the function(s) of TcCDA genes, double stranded RNA (dsRNA) specific for each gene was injected into insects at different developmental stages and phenotypes were observed. No visible phenotypic changes were observed after injection of dsRNAs for TcCDA3 to 9, while injection of dsRNAs for TcCDA1 or TcCDA2 affected all molts, including the larval‐larval, larval‐pupal and pupal‐adult. Insects treated with these dsRNAs did not shed their old cuticles and were trapped in the exuviae. Interestingly, different adult phenotypes were observed after injection of dsRNAs specific for alternatively spliced transcripts of TcCDA2, namely TcCDA2A and TcCDA2B. Neither dsTcCDA2A nor dsTcCDA2B had any effect on molting. However, the adults that developed after injection of dsTcCDA2A into larvae were unable to bend or unbend their legs, at the femoral‐tibial joints. In contrast, adults that emerged after larvae were injected with dsCDA2B exhibited defects in epidermal cuticle morphology, including roughened elytra. When adult females were injected with dsTcCDA1 or dsTcCDA2A, 70‐90% of their progeny were unable to hatch from the egg, and those that did hatch could not molt to second larval instar. These results support the hypotheses that; 1) TcCDA1 and TcCDA2 are critical for egg hatch and also larval‐larval, larval‐pupal and pupal‐adult molting; 2) alternatively spliced variants of TcCDA2, TcCDA2A and TcCDA2B, have different roles in determining adult cuticle morphology; and 3) Proper ratios of chitin/chitosan levels at different stages and/or in different tissues are critical for insect development.