Insect cuticle or exoskeleton is a complex extracellular matrix formed primarily from structural polysaccharide chitin and protein, and it plays a critical role in protecting them from various environmental stresses and pathogenic infection. Despite of limited composition, insect cuticle has remarkably diverse mechanical properties, ranging from soft and flexible to hard and rigid. My research has been focusing on functional importance of the genes involved in chitin metabolism and cuticle tanning (sclerotization and pigmentation) to comprehensively understand the genetic, enzymatic as well as molecular mechanism underlying differentiation, development and formation of insect cuticular extracellular matrices.

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)

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)

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).

Insect cuticle tanning (pigmentation and sclerotization) is a complex and vital process, which includes hydroxylation of initial amino acid, tyrosine, to DOPA and decarboxylation of DOPA to dopamine. In the pigmentation process, dopamine further undergoes two N-acylation reactions to yield N-acetyldopamine (NADA) and N-β-alanyldopamine (NBAD). In the former reaction, arylalkylamine N-acetyltransferase (AANAT1) converts dopamine to NADA, and in the later reaction, aspartate 1-decarboxylase (ADC) provides β-alanine, which is conjugated with dopamine catalyzed by NBAD synthase (Ebony) for production of NBAD. In this study, we performed functional genomics of TmAANAT1, TmADC and Tmebony to determine whether they are required for cuticle pigmentation in Tenebrio molitor adults. Loss of function of these genes by RNAi caused the significantly darker body color than that of control animals. Note that, although all phenotypes exhibited dark cuticle pigmentation, RNAi of either TmADC or Tmebony only altered brownish outer region of the cuticle to dark/black. In contrast, RNAi of TmAANAT1 had no effect on the brown hue of the outer cuticle layer, but less or no pigmented inner region of the cuticle became significantly darker than those of control adults. These results suggest that, like that seen in TcAANAT1- or TcADC-deficient Tribolium castaneum adults, NADA produced by a reaction by TmAANAT1 contributes the lighter inner cuticle layer(s), whereas NBAD appears to do the highly pigmented outer cuticle layer(s) of the cuticle of T. molitor adults. This work was supported by NRFs (NRF-2015R1A6A3A04060323 and NRF-2018R1A2B6005106).

Insect structural cuticular proteins (CPs) play a major role in determining the diverse physical properties of the cuticle as a result of interactions/cross-linking among themselves and with chitin. CP genes compose a large gene family and have been classified more than ten distinct families based on the presence of unique amino acid sequence motifs. In this study, we performed RNAi-based functional analysis of eleven genes (TcCPLCP1-11) in Tribolium castaneum, which belong to CPLCP (Cuticular Proteins of Low Complexity, Proline rich) cuticular protein family. RNAi for TcCPLCP7-11 caused lethal pupal-adult molting defects and/or abnormal cuticle morphology in the resulting adults. Ultrastructural defects of the cuticles from TcCPLCP7-11-deficient insects by TEM are also discussed.

Eggs from the mosquito genus Aedes exhibit high desiccation resistance that likely facilitate spreading some of them as a vector of human disease throughout the world. However, molecular mechanism underlying the embryonic resistant to desiccation has not well understood. In this study, we performed functional study of two ovary-specific yellow genes, AalY-g and AalY-g2, in the Asian tiger mosquito, Aedes albopictus. The eggs obtained from AalY-g or AalY-g2 RNAi females showed poor desiccation resistance. TEM analysis revealed that, unlike that seen in the dsEGFP-control eggs, no high electron-dense outer-endochorion was evident in the eggs from AalY-g- or AalY-g2-deficient females. These results suggest that both yellow genes at least play roles in the chorion formation, which appears to be critical for integrity and desiccation resistance in Ae. albopictus eggs.

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.

The eggs of Asian tiger mosquito, Aedes albopictus, possess high desiccation resistance, which could lead rapidly spreading this mosquito across the world. Melanization and/or hardening of mosquito eggshell are critical for desiccation resistance to protect embryo from adverse environment. Dopachrome-conversion enzymes (Yellows) catalyze melanin synthesis in insects. Here we report functional importance of two ovary-specific yellow genes, AalY-g and AalY-g2, in desiccation resistance of Ae. albopictus egg. Loss of function of these genes by RNAi causes the fragile exochorion and delay initial melanization of the endochorion. Furthermore, eggs from control females exhibit high desiccation resistance under the air-dry condition, whereas those from AalY-g- or AalY-g2-deficient females are collapsed. TEM analysis reveals ultrastructural defects in the endochorion and serosal cuticle in these eggs. The results indicate that AalY-g and AalY-g2 are critical for integrity and desiccation resistance of the Ae. albopictus egg.

Tyrosine-mediated cuticle tanning (pigmentation and sclerotization) is a vital process for insect growth and development. In this metabolism, dopamine and N-β-alanyldopamine (NBAD) are major precursors to black melanin-like and yellowish quinonoid pigments, respectively. NBAD synthase (Ebony) catalyzes the synthesis NBAD by conjugation of dopamine and β-alanine, while NBAD hydrolase (Tan) dose a reverse reaction of NBAD synthesis catalyzed by Ebony. In this work, we cloned cDNAs of ebony and tan from two beetle species, Tribolium castaneum and Tenebrio molitor. Loss of function phenotypes produced by RNAi for these genes indicate that Ebony, but not Tan, is required for cuticle pigmentation in both beetles. This work was supported by NRFs (NRF-2015R1A2A2A01006614).

Insect chitinases (CHTs), an extracellular enzyme, belong to family 18 glycosyl hydrolases that hydrolyze chitin by an endo-type manner. In insect genomes, there are a large number of genes encoding CHT-like proteins, and they have been classified into eleven groups based on phylogenetic analysis. In this study, we have investigated functions of a group III chitinase (TcCHT7) in Tribolium castaneum. Although, unlike most insect CHTs, TcCHT7 contains a predicted transmembrane segment in N-terminal, immunohistochemical analysis reveals that it is localized in the newly forming procuticle, suggesting that TcCHT7 is released from the plasma membrane of underlying epidermal cells. RNAi for TcCHT7 does not affect on any types of molting. However the resulting pupae and adults fail to undergo wing-expansion and abdominal contraction. In addition, TcCHT7-deficient insects exhibit ultrastructural defects in both rigid (e.g. elytron) and soft (e.g. hindwing) cuticles. These results demonstrate that functional importance of TcCHT7 in the formation of the rigid and soft cuticles of the beetle.

Chitin deacetylases (CDAs) are extracellular-modifying enzymes that deacetylate chitin to produce chitosan. Insect CDAs have been divided into five groups based on phylogenetic analysis. We previously reported the functional importance of group I CDAs, TcCDA1 and TcCDA2, from Tribolium castaneum in molting, morphology of cuticle as well as in movement of legs. However, ultrastructure in the cuticle after RNAi for these genes have not been investigated. In this study, we further analyzed precise localization of these proteins and ultrastructural changes/defects of the cuticles in TcCDA1- and TcCDA2-deficient insects. Loss of function of TcCDA1 and TcCDA2 causes disorganized horizontal laminae and vertical pore canals in both rigid (e.g. elytron and ventral body wall) and soft (e.g. hindwing and dorsal body wall) adult cuticles. These results indicate that TcCDA1 and TcCDA2 are critical for development and formation of the beetle cuticles

Insect cuticle/exoskeleton covering the entire external surface of the body is essential for protecting insects from various environmental stresses. Tyrosine metabolism plays a major role in not only the darkening of cuticle but also its hardening. In this work, we have focused on the functional analysis of nine genes involved in tyrosine-mediated cuticle tanning (pigmentation and sclerotization) pathway in Tenebrio molitor, which has a unique adult cuticle coloration, dark/black dorsal thorax and elytron, and reddish ventral thorax and abdomen. The temporal and spatial expression patterns of the genes were analyzed by real-time PCR, and RNA interference (RNAi) was performed to study the functional importance of these genes in cuticle coloration and/or hardening in T. molitor. This work was supported by NRFs (NRF-2015R1A2A2A01006614 and NRF-2015R1A6A3A04060323).

Insect chitinases (CHTs) belong to family 18 glycosylhydrolases and hydrolyze chitin by an endo-type manner. One of the functions of CHTs is in the turnover of chitin-containing extracellular matrices such as the cuticle and peritrophic matrix of the midgut. There are a large number of genes encoding CHT-like proteins in insects, and they have been classified into eleven groups based on phylogenetic analysis. We have investigated functions of a group III chitinase in Tribolium castaneum (TcCHT7) containing a predicted transmembrane segment in N-terminal region. Recombinant TcCHT7 exhibits chitinolytic activity against CM-Chitin-RBV. Immunohistochemical analysis shows that TcCHT7 is localized in newly formed procuticle in elytral cuticles, suggesting that TcCHT7 is released from the plasma membrane of underlying epidermal cells. TcCHT7-deficient pupae and adults fail to undergo wing-expansion and abdominal contraction. In addition, cuticular chitin accumulates in the inner region of the procuticle where disorganized horizontal laminae and pore canals are evident. These results demonstrate that TcCHT7 plays a critical role in the formation of the rigid and soft cuticles of the beetle. This work was supported by NRFs (NRF-2015 R1A2A2A01006614).