Insect cuticular extracellular matrices (ECM) including the eggshell and exoskeleton play vital roles in protecting them from natural environmental stresses. However, these chitinous ECMs must be degraded at least in part during embryonic and post-embyonic molting periods to accommodate continuous growth all the way to the adult stage. In this study we investigated the functions of groups I and II chitinases, TcCHT5 and TcCHT10, in turnover of the eggshell and cuticle in Tribolium castaneum. RNAi and TEM analyses revealed that TcCHT10 is required for digestion of chitin in the serosal cuticle for embryo hatching as well as in the old cuticle during post-embryonic molts including larval-pupal and pupal-adult metamorphosis. However, although TcCHT5 is apparently involved in these vital physiological events, TcCHT10 could substitute for TcCHT5 except during the pupal-adult molting when both enzymes are indispensable to degrade chitin in the old pupal cuticle.

Insect eggshell and cuticle/exoskeleton play vital roles in protecting them from natural environmental stresses. However, these chitinous cuticular extracellular matrices must be degraded at least in part during embryo hatching and molting/ecdysis periods to accommodate continuous growth all the way to the adult stage. In this study, we investigated the functional importance of groups I and II chitinases, TcCHT5 and TcCHT10, in the turnover of chitinous cuticle during both embryonic and post-embryonic development in Tribolium castaneum. RNAi and TEM analyses revealed that TcCHT10 is required for digestion of chitin in the serosal cuticle for embryo hatching as well as in the old cuticle during post-embryonic molts including larval-pupal and pupal-adult metamorphosis. TcCHT10 appears to be able to substitute for TcCHT5 in all these vital physiological events except for the pupal-adult molting in which TcCHT5 is indispensable for complete digestion of chitin in the old pupal cuticle.

Chitin deacetylases (CDAs) are extracellular-modifying enzymes that deacetylate chitin to produce chitosan. In insects, this modification may contribute to the affinity and/or cross-linking of chitin/chitosan-like polysaccharides for a variety of structural proteins, which may lead to diverse mechanical properties of the cuticle. We previously reported the functional importance of Group I CDAs, TcCDA1 and TcCDA2, as well as the two alternative spliced isoforms of the latter, TcCDA2a and TcCDA2b from the red flour beetle, Tribolium castaneum in molting, morphology of cuticle and movement of legs. In this study, we further analyzed protein localization, ultrastructural defects of the cuticles and leg joints after RNAi of those genes. Both proteins are mainly present in the innermost procuticle region called the “assembly zone”. Loss of function of either TcCDA1 or TcCDA2 caused disorganized chitinous horizontal laminae and vertical pore canals in both the rigid and soft cuticles. RNAi of TcCDA2b affects cuticle integrity similar to that seen in RNAi of the two alternatively spliced forms of TcCDA2. In contrast, TcCDA2a-deficient adult, like that seen in the hypomorphic phenotype produced by RNAi of TcCDA1, exhibited ruptured tendons between femur and tibia, resulting in loss of locomotion ability. These results suggest that Group I CDAs play critical roles in molting, morphology, ultrastructure and mobility in T. castaneum. This work was supported by NRFs (NRF-2015R1A6A3A04060323 and NRF-2018R1A2B6005106).