Insect cuticle or exoskeleton is an extracellular matrix consisting of three major morphologically distinct layers, the water-proofing envelope, the protein-rich epicuticle and the chitin/protein-rich procuticle. To accommodate growth, insects must periodically replace their cuticles in a process called “molting or ecdysis”. During each molt cycle a new cuticle is deposited simultaneously with degradation of the inner part of the chitinous procuticle of the old one by molting fluid enzymes including epidermal chitinases. In this study, we show a novel role for an epidermal endochitinase containing two catalytic domains, TcCHT7, from the red flour beetle, Tribolium castaneum, belonging to a subfamily (group III) of insect chitinases in organizing chitin in the newly forming cuticle rather than in degrading chitin present in the prior one. RNAi of TcCHT7 reveals that this enzyme is nonessential for any type of molt or degradation of the chitinous matrix in the old cuticle. In contrast, TcCHT7 is required for formation of properly oriented long chitin fibers inside pore canals that are vertically oriented columnar structures, which contribute to maintain the integrity and the mechanical strength of a light-weight, yet rigid, adult cuticle. Because group III chitinases are highly conserved among insect and other arthropod species, these enzymes have a critical role in the higher ordered organization of chitin fibers for development of the structural integrity of many invertebrate cuticular extracellular matrices. This work was supported by NRFs (NRF-2015R1A2A2A01006614 and NRF-2018R1A2B6005106)

To accommodate growth, insects must periodically replace their chitin/protein-rich cuticles in a process called “molting or ecdysis”. During each molt cycle, a new cuticle is deposited simultaneously with degradation of the chitinous procuticle of the old one by molting fluid enzymes including epidermal chitinases. Here, we demonstrated a novel role for an endochitinase, TcCHT7, from the red flour beetle, Tribolium castaneum, belonging to a subfamily (Group III) that contain two catalytic domains, in organizing chitin in the newly forming cuticle rather than in degrading chitin present in the prior one. The conservation of CHT7-like proteins among many insect and other arthropod species indicates a critical role for the Group III class of chitinases in the higher ordered organization of chitin fibers for development of the structural integrity of many invertebrate exoskeletons.

An entomopathogenic filamentous fungus, Paecilomyces lilacinus strain HY-4, has a great potential as a promising bio-pesticide due to its superior pathogenicity against Adoretus tenuimaculatus and Tetranychus urticae. When the fungal strain infects host cuticle, it secrets a combination of hydrolytic enzymes including chitinase to solubilize the cuticle. Thus, we investigated effects of different carbon and nitrogen sources on the production of a chitinase from P. lilacinus strain HY-4. The organism produced an extracellular chitinase at a relatively high level (45.4 mU/ml) when cultivated for 5 days on a medium supplemented with insect pupa (0.5%) and colloidal chitin (1%), which was prepared by treating chitin from crab shells (Sigma-Aldrich Co. Ltd.) with 12 N HCl solution. However, extracellular secretion of chitinase by strain HY-4 was found to be significantly repressed in the presence of glucose (1%).

Baculovirus chitinase gene (ChiA) is a late gene and is essential for liquefying host insect at the late stage of infection for its hydrolyzing chitin function. In previous report, baculovirus ChiA can offer many interseting new opportunities for pest control. Recently, a putative chitinase gene (ChiA) was identified in the Spodopter litura nucleopolyhedorvirus (SlMNPV-K1) genome. The open reading frame (ORF) contains 1,692 nucelotides (nt) and encodes a protein of 563 amino acids (aa) with a predicted molecular weight of 62.62 kDa. To conform the insecticidal activity of ChiA from SlMNPV-K1, we constructed a baculovirus transfer vector, pBac-SlChiA, and this transfer vector was co-transfected with the bApGOZA DNA into sf9 cell to generate corresponding recombinant viru which designed Ap-SlChiA. Western blot analysis indicate that SlMNPV-K1 ChiA was successfully expressed. We found the chitinase activity of recombinant virus was enhanced 53% than wide type AcMNPV by chitinase assay, and the recombinant virus showed higher evidently insecticidal activity against 3rd instar larvae of Spodotera exigua than wide type AcMNPV (4.5 time). These results suggested that the chitinase gene from SlMNPV-K1 could be successfully applied to improve pathogenicity of bauclovirus