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.

Insect cuticle consists of numerous structural proteins, which could interact with polysaccharide, chitin, and alter properly mechanical property of the cuticle. Cuticular Protein Analogous to Peritrophins (CPAPs) are characterized by presence of one (CPAP1s) or three (CPAP3s) chitin-binding domain belong to CBM14/ChtBD2 family. In this study, we investigated physiological functions of TcCPAP1-H and TcCPAP3-C in Tribolium castaneum. RNAi for either TcCPAP1-H or TcCPAP3-C at late instar larvae had no effect on larval-pupal molt nor pupal development. However, the resulting pharate adults failed to shed their old pupal cuticle and died entrapped in it without undergoing eclosion. TEM analysis, in addition, revealed disorganized chitinous horizontal laminae and/or vertical pore canals of rigid cuticle from TcCPAP1-H- and TcCPAP3-C-deficient adults. Desiccation-induced death produced by injection of dsTcCPAP1-H into young instar larvae is also discussed.

Insect cuticle tanning (pigmentation and sclerotization) is a complex and vital physiological process that begins with tyrosine and is responsible for production of both melanin- and quinoid-type pigments. In addition, these quinones undergo isomerization to quinone methides and cross-linking reactions with cuticular proteins for cuticle sclerotization. In this study, we studied the functions of TmDDC and TmY-y as well as TmNAT1, TmADC and Tmebony from Tenebrio molitor, which are involved in the tyrosine-derived melanin- and quinoid-type pigment productions, respectively. The temporal and spatial expression patterns of the genes were analyzed by real-time PCR. RNA interference was performed to understand the genetic regulation and molecular mechanism underlying the darkening and hardening of beetle cuticle.

The eggs of Asian tiger mosquito, Aedes albopictus, possess high desiccation resistance, which contribute the rapid spread of this mosquito across the world. Melanization of eggshell appear to play a role in the resistance to desiccation. Dopachrome-conversion enzyme (DCE, Yellow) significantly accelerates the melanization of the eggshell. In this study, we demonstrated functional importance of two yellow genes, AalY-g and AalY-g2, in the chorion formation. Both genes were highly induced in the ovary at 48 h after blood meal. Injection of dsRNA for AalY-g or AalY-g2 into adult females had no effect on fecundity. However, the outermost colorless exochorion of the eggs obtained from both dsRNA-treated females was fragile and peeled off in places, and melanization of the endochorion was obviously delayed by several hours. In addition, unlike eggs from control females which acquired high desiccation resistance between 18 and 24 h after oviposition (HAO), 60-70% 24 HAO eggs from either AalY-g- or AalY-g2-deficient females were collapsed when they were moved to an air-dry condition, and the desiccation resistance was not increased in later stages of embryonic development analyzed. TEM analysis revealed that abnormal morphology and ultrastructure of the endochorion, particularly outer-endochorion, in the 24 HAO and older eggs from either AalY-g-and AalY-g2-deficient females. These results indicate that AalY-g and AalY-g2 are required for morphology and formation of the endochorion (outer-endochorion), a structure that appears to be critical for desiccation resistance of the Ae. albopictus eggs. This work was supported by NRFs (NRF-2015R1A6A3A04060323 and NRF-2018R1A2B6005106)