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).

Chitin deacetylases (CDAs) are chitin-modifying enzymes that deacetylate chitin to form 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 that may lead to diverse mechanical properties of the cuticle. DmCDA1 (serpentine) and DmCDA2 (vermiform) from Drosophila melanogaster play roles in development and morphology of embryonic tracheal tubes. We previously reported the functional importance of TcCDA1 and TcCDA2 orthologs to DmCDA1 and DmCDA2 from Tribolium castaneum, in molting, morphology of cuticle, and movement of legs. In this study, we further analyze ultrastructural defects of the cuticles and leg joints in TcCDA1- and TcCDA2-deficient insects. Loss of function of TcCDA1 and TcCDA2 causes disorganized chitinous horizontal laminae and vertical pore canals of rigid adult cuticle (e.g. elytron). Both proteins are also required for laminal organization in soft cuticle (e.g. hindwing). Morphological analysis of TcCDA1- and TcCDA2A-deficient adult revealed that ruptured tendons between femur and tibia cause the defects in movement of the leg joint. This work was supported by NRF (NRF-2015R1A2A2A01006614).

Pigmentation or melanization is an important physiological event in insects and is involved in cuticle tanning, wound healing and encapsulation as a defensive response. Dopachrome-conversion enzyme (DCE, Yellow) significantly accelerates the melanization of the chorion in mosquito eggs. In this study, we demonstrated functional importance of two ovary-specific yellow genes, AalY-g and AalY-g2, in the Asian tiger mosquito, Aedes albopictus. The transcripts of both genes were detected in the ovary of adult females only 48-72 h after blood feeding. RNAi for AalY-g or AalY-g2 had no effect on fecundity. However, the outermost colorless exochorion of the eggs was fragile and partially peeled off, and initial melanization of the endochorion was significantly delayed. Eggs from control females exhibited high desiccation resistance, whereas those from dsAalY-g- or dsAalY-g2-treated females were collapsed (50-80%) under the air-dry condition. Ultrastructural analysis revealed abnormal morphology of the endochorion and vitelline membrane in the AalY-g and AalY-g2-deficient eggshell. These results indicate that AalY-g and AalY-g2 are critical for integrity and desiccation resistance of the Ae. albopictus eggs. This work was supported by NRFs (NRF-2015R1A6A3A04060323).

In insect exoskeleton/cuticle, structural cuticular proteins (CPs) and the polysaccharide chitin are the major components of the procuticle. CPs are cross-linked by quinones or quinone methides produced by the laccase2 (Lac2)- mediated oxidation of N-acylcatechols. We reported that two major CPs, TcCPR27 and TcCPR18, belong to the CPR family that contain the RR-2 consensus motif (Rebers & Riddiford), are essential for formation and stabilization of the rigid cuticle of Tribolium castaneum adults. In this study, we characterized and investigated functions of the third most abundant protein, TcCP30, in extracts of elytra. TcCP30 cDNA encodes a protein with 171 amino acid residues containing a putative signal peptide. Unlike TcCPR27 and TcCPR18, TcCP30 mature protein lacks an RR motif, with a very unique amino composition, 36% Glu, 21% His, 20% Arg and 16% Gly. TcCP30 gene is highly expressed right before and after eclosion (in 5 d-old pupae and 0 d-old adults). Immunohistochemical studies revealed that TcCP30 protein was present in rigid cuticle such as elytra and ventral abdomen but not soft cuticle such as hindwings and dorsal abdomen of adult T. castaneum. Injection of dsRNA for TcCP30 into late instar larvae had no affect on larval and pupal growth and development. However, the subsequent pupal-adult molt, more than 50% adults were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults exhibited wrinkled, warped and split elytra. TcCP30-deficient adults could not fold their hindwings properly because probably due to the malformed elytra. These results indicate that TcCP30 is critical for formation of rigid adult cuticle as well as development and growth of T. castaneum.

Insect cuticle is a first physical barrier to protect their body from multifarious environments. Cuticle tanning (sclerotization and pigmentation) is a complex process involves hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA), decarboxylation of DOPA to dopamine, N-acylation of dopamine to N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD), oxidation of NADA and NBAD to their corresponding quinones, and reactions between the quinones or quinone derivatives with cuticular proteins (CPs) resulting in protein cross-linking. N-acetyltransferase (NAT) catalyzes the conversion of dopamine to NADA whose covalent-linkage of CPs is correlated with colorless cuticle (β-sclerotization). In this study, we analyzed functions of TcNAT1 on cuticle tanning of adult Tribolium castaneum by RNAi. Injection of dsRNA for TcNAT1 (dsTcNAT1) had no affect on animal development and growth. However, some of the resulting adults (~70%) showed split elytra that could not cover their abdomen, resulting in improper folding of their hindwings. Interestingly, body color of the mature adults was darker than that of control dsTcVer-treated adults because probably due to the buildup of abnormally high levels of dopamine, which is used for dopamine eumelanin pigment synthesis (black pigment). On elytra and hindwings of these adults, darker pigments were observed around the sensory bristles located at the intervein regions, suggesting that NADA mediated β-sclerotization is occurred in these regions. Similarly, darker pigment was evident at veins of the hindwings of TcNAT1-deficient adults. These results suggest that TcNAT1 plays important roles in cuticle tanning of T. castaneum adult. To characterize enzymatic properties of TcNAT1, furthermore, recombinant TcNAT1 protein expressed in E. coli was purified by utilizing Ni-NTA affinity column chromatography. This work was supported by NRF (NRF-2012R1A2A1A01006467).

Insect cuticle is a complex biocomposite material consisting of three major morphologically distinct layers, the waterproofing envelope, the protein-rich epicuticle and the chitin/protein-rich procuticle. Structural cuticular proteins (CPs) and the polysaccharide chitin are the major components of the exo- and endocuticular layers that comprise the procuticle. During cuticle tanning (sclerotization and pigmentation), CPs are cross-linked by quinones derived from the oxidation of catechols, resulting in hardening of the exoskeleton. However, the factors that lead to synthesis and assembly of cuticular regions with differing mechanical properties are not well understood. To gain a better understanding of the development and differentiation of rigid cuticle, we performed transmission electron microscopic (TEM) analysis of elytral cuticle (highly sclerotized and pigmented forewing) from 2 d-old pupae to 9 d-old adults of the red flour beetle, Tribolium castaneum. In 2-3 d-old pupae, pupal cuticle separated from the underlining epidermal cells (apolysis), and outermost envelope and protein-rich epicuticle begun to form. A numerous horizontal chitinous laminae and vertical pore canals were evident in the procuticle of 4-5 d-old pupae. By one day after adult eclosion, less-compact horizontal chitinous laminae were deposited, followed by block-type cuticular layers with no horizontal laminae were formed by 9 days. These results will lead to a) a better understanding of insect cuticle formation, structure and mechanics, b) the potential for development of novel insect control agents that target cuticle physiology, and c) the production of biomimetic materials with physical properties like those of the insect exoskeleton for use in biomedical or other technological devices. This work was supported by NRF (NRF-2012R1A2A1A01006467).

To accommodate growth, insects must periodically replace their exoskeletons. The cuticle or exoskeleton consists of multiple functional layers including the waterproof envelope (cuticulin layer), the protein-rich epicuticle (exocuticle) and the chitinous procuticle (endocuticle). After shedding the old cuticle, the newly formed soft and transparent cuticle must harden and tan. During tanning, cross-links form between adjacent polypeptide chains, causing progressive hardening, dehydration, and close packing of the polymers. This cross-linking occurs as a result of oxidative and nucleophilic reactions between highly reactive tanning agents derived from catechols and nucleophilic side chain groups of cuticular proteins (CPs). The initial steps of tanning in most cuticles involve formation of quinones and quinone methides derived from N-acylcatecholamines, followed by their oxidative conjugation with CPs, leading to changes in mechanical properties and pigmentation. This vital physiological step occurs during each stage of development and is required to stabilize and harden the exoskeleton. The mechanism of the insect sclerotization, however, is poorly understood, and the factors that lead to synthesis of cuticular structures with differing physical properties that are unique to each type of cuticle (e.g. elytron, hindwing, pronotum, dorsal and ventral body wall) are not well defined. In this study, we investigated development and differentiation of rigid cuticle using the red flour beetle, Tribolium castaneum adult, as a model insect. Tribolium as a beetle is superior model for studying rigid cuticle formation because they have a highly modified (sclerotized and pigment) forewing (elytron) which can be separated from other tissues easily and cleanly. We analyzed ultrastructure of elytral cuticle during development (from 3 d-old pupae to 3 d-old adults) by transmission electron microscopy (TEM). In 3 d-old pupae, pupal cuticle separated from the epidermal cells (apolysis), and the outermost envelop of adult cuticle was being formed. Protein-rich epicuticle and procuticle composed numbers of horizontal laminae and vertical canals were formed at 4 and 5 d-old pupal stages. After adult eclosion, additional thick horizontal laminae were evident and apical membrane of the epidermal cells became undulae like-structure at 1 d-old adult, and then final three layers with no horizontal laminae were formed by 3days after adult molting. Furthermore, protein localization of several high abundant adult CPs is also discussed. These results will contribute understanding cuticle formation and differentiation in insect during post-embryonic development.

Most traditional genome sequencing projects involving infectious viruses include culturing and purification of the virus. This can present difficulties as an analysis of multiple populations from multiple locations may be required to acquire sufficient amount of high-quality DNA for sequence analysis. The electrophoretic method provides a strategy whereby the genomic DNA sequences of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) were analyzed by purifying it from host DNA by pulsed-field gel electrophoresis, thus simplifying sampling and labor time. The genomic DNA of infected P. rapae was embedded in agarose plugs, digested with a restriction nuclease and methylase, and pulsed-field gel electrophoresis (PFGE) was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the separated viral DNA. The double-stranded circular genome of PiraGV-K encodes 120 open reading frames (ORFs), covering 92% of the sequenced genome. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (~99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF11), involved in the liquefaction of the host, were found in the genome. The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the pulsed field electrophoretic method. The method appears to be applicable to the analysis of genomes of large viruses. The chitinase, identified by PiraGV-K genome sequence, was functionally characterized by quantitative PCR, Western blot analysis, immunohistochemistry and transmission electron microscopy.

Cuticular proteins (CPs) and the polysaccharide chitin are the major components of the exo- and endocuticular layers or procuticle. CPs contain a conserved sequence known as the Rebers & Riddiford (R&R) motif, which may function as a chitin-binding domain that helps to coordinate the interaction between chitin fibers and the protein network. We identified two highly abundant RR-2 CPs, TcCPR18 and TcCPR27, in protein samples extracted from elytra (rigid cuticle) of Tribolium castaneum adults and determined that these two CPs are required for rigid cuticle morphology. In this study, we identified the third most abundant protein (TcCP30) extracted from the elytra, and cloned a full-length cDNA. It encodes a very unusual 171 amino acid residue protein of which 36% of the residues of the mature protein are Glu, 21% are His, 19% are Arg, and 16% are Gly, organized in a regular pattern but not R&R consensus motif. TcCPR18 and TcCPR27 genes are expressed at 4 d-old pupae, while TcCP30 is highly expressed at 5 d-old pupae (last pupal stage) and 0 d-old adults. Immunohistochemical studies revealed the presence of TcCP30 in rigid adult cuticle (e.g. elytron, pronotum and ventral abdomen) but not soft cuticle (e.g. hindwing and dorsal abdomen). Injection of dsRNA for TcCP30 into late instar larvae had no affect on larval and pupal growth and development. The subsequent pupal-adult molt, however, more than 50% adults were unable to shed their exuvium and died. In addition, the resulting adults exhibited wrinkled, warped and split elytra. TcCP30-deficient adults could not fold their hindwings properly. These results indicate that TcCP30 may play critical roles in rigid adult cuticle formation, development and insect growth and survival. This work was supported by NRF (NRF-2012R1A2A1A01006467).

Insect chitinases (CHTs), which belong to family 18 glycosylhydrolases (GH-18), have been detected in molting fluid and gut tissues and are predicted to mediate the digestion of chitin present in the exoskeleton and peritrophic matrix (PM) in the gut. Based on amino acid sequence similarity and phylogenetic analysis, insect CHT family proteins have been classified into eight groups (group I to VIII). The CHTs belonging to different groups have distinctly different developmental patterns of expression and tissue specificity, suggestive of distinct biological functions. CHT7s belong to Group III chitinase contain two catalytic domains and one chitin binding domain (CBD). The catalytic domain 1 of this group of chitinases exhibits greater sequence similarity to one another than to the catalytic domain 2 in the same protein(s), suggesting distinct functions and/or evolutionary origins for each of these two catalytic domains. This group of chitinases, unlike most insect CHTs, possesses a predicted transmembrane segment at the N-terminal region. The recombinant T. castaneum CHT7 that was expressed in Hi-5 insect cells was bound to the cell membrane. Apparently, the catalytic domains of this CHT face the extracellular space as revealed by its ability to hydrolyze an artificial chitin substrate added to the medium. DsRNA-based functional studies (RNAi) for several CHT genes in Tribolium castaneum indicated that CHTs belong to groups I (TcCHT5) and II (TcCHT10) are critical for molting and turnover of chitin in the old cuticle. In other hand, RNAi for TcCHT7 did not affect any types of molting such as larval-larval, larval-pupal and pupal-adult. The resulting pupae or adults, however, failed to wing-expansion and abdominal contraction. Immunohistochemical analysis revealed that TcCHT7 protein is localized in newly synthesized procuticle, suggesting that TcCHT7 could be released form the plasma membrane of epidermal cells by proteolysis. Chitin seems to accumulate within the assembly zone of the elytral and body wall cuticle in dsTcCHT7-treated animals. Transmission electron microscopy revealed that down-regulation of TcCHT7 transcripts resulted in disorganization of chitin laminar and vertical canals in the procuticle. These results suggest that TcCHT7 may have critical roles in the laminar assembly and synthesis and/or deposition of cuticular chitin. This work was supported by NRF (NRF-2012R1A2A1A01006467).

Insect cuticle/exoskeleton is a first physical barrier to protect their body from multifarious environments such as desiccation, natural enemies and entomopathogenic microorganisms. Cuticle tanning (sclerotization and pigmentation) is a vital procedure for generating suitable cuticle depending on body region by sclerotization and pigmentation in insects. Insect cuticle tanning is a complex process involves hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA), decarboxylation of DOPA to dopamine, N-acylation of dopamine to N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD), oxidation of NADA and NBAD to their corresponding quinones, and reactions between the quinones or quinone derivatives with cuticle protein (CP) side chains resulting in protein cross-linking. One type of pigmentation (quinone tanning) is associated with the covalent linkage of CPs to the ring component of NBAD. In contrast, linkage of CPs to the side chain of NADA (b-sclerotization) is correlated with colorless cuticle. N-acetyltransferase (NAT) catalyzes the conversion of dopamine to N-acetyl dopamine (NADA) in cuticle tanning pathway. In this study, we studied function of TcNAT1 on adult cuticle tanning by double stranded-RNA (dsRNA) mediated gene silencing. Injection of dsTcNAT1 had no affect on animal development, growth and molting such as larva to larva, larva to pupa and pupa to adult. However, some of the resulting adults (~70%) showed split elytra that could not cover their abdomen, resulting in improper folding of their hindwings. Interestingly, body color of the mature adults (older than 3 days) was darker than that of control dsTcVer treated adults because probably due to the buildup of abnormally high levels of dopamine, which is used for dopamine eumelanin pigment synthesis (black pigment) and dopamine quinone-mediated protein crosslinking. On elytra and hindwings of these adults, darker pigments were observed around the sensory bristles that are located in the intervein regions, suggesting that NADA mediated b-sclerotization is occurred at these regions. Similarly, darker pigment was evident at veins of the hindwings of the dsTcNAT1-mature adults. These results suggest that TcNAT1 have important roles in sclerotization and pigmentation of adult body and wings (elytron and hindwing). This work was supported by NRF (NRF-2012R1A2A1A01006467).

Insects have a protective exoskeleton consisted with cuticle to adapt various environments and pathogens. Insect cuticle mainly composed of the polysaccharide chitin and numerous of cuticular proteins (CPs). CPs are important for insect cuticle formation, development, and growth because it produces proper combination of mechanical and physical properties of cuticle depend on the regions of an exoskeleton. The largest family of CPs contains a 28-residue motif known as the Rebers-Riddiford (R&R) consensus sequence. When sequences containing the R&R consensus are aligned, they fall into three groups based on sequence similarity, and these groups tend to correlate with the type of cuticle (soft or hard) from which the proteins are derived. Proteins with the RR-1 motif have been found primarily in soft cuticle, whereas many proteins from rigid cuticle have an extended region of similarity called RR-2. We recently reportedthat two major CPs, TcCPR18 and TcCPR27 belong to RR-2, are essential for formation of highly sclerotized modified-forewings (elytra) of a beetle. In this study, we performed functional genomics of TcCPR4, which encodes RR-1 motif. The transcript levels of TcCPR4 drastically increased in 3 d-old pupae at when adult cuticle synthesis appears to be begun. Immunohistochemical studies revealed that TcCPR4 protein was detected in the rigid cuticle of elyton and ventral abdomen but not in the flexible cuticle of hindwing and dorsal abdomen of T. castaneum adult. Furthermore, TcCPR4 protein was specifically present at basal side of the procuticle (near the epidermal cells) and vertical canals, whereas TcCPR27 protein was found entire procuticle. Injection of double-stranded RNA of TcCPR4 (dsTcCPR4) into late instar larvae had no effect on development and any types of molting such as larval-larval, larval-pupal or pupal-adult. Interestingly, depletion of both TcCPR4 and TcCPR27 transcripts could rescue the elytral cuticle defect and mortality produced by injection of dsTcCPR27 alone. Transmission electron microscopy analysis revealed that depletion of TcCPR4 had abnormal vertical canals in rigid adult cuticle while dsTcCPR27 injection showed less electron-dense-horizontal laminae and vertical canals. Surprisingly, co-injection of dsRNA for TcCPR4 and TcCPR27 exhibited more severe cuticle defect with thinner elytral cuticle and abnormal vertical canals and chtin laminae compared to those from insects treated with dsRNA for each gene. These results suggest that TcCPR4 as a RR-1 is essential structural component in the rigid cuticle of T. castaneum adult.

14-3-3 proteins are known to play a pivotal role in a diverse array of cellular events such as cell survival, apoptosis, and signal transduction. Numerous 14-3-3 ζ have been cloned and characterized from a host of eukaryotic organisms including human, plants, yeast, fruit fly and silkworm. However, no study on Spodoptera exigua 14-3-3ζ in conjunction with virus infection has so far been reported in insects. It appears that expression of Se14-3-3ζ was decreased starting 24 h post-SeNPV infection as SeNPV titers seemed to increase as evidenced by intense bands of SeNPV IAP3. Interestingly, confocal microscopic analysis revealed that Se14-3-3ζ is expressed at the apical side of the NPV-uninfected gut cells, whereas it was detected mainly in the nucleus of the NPV-infected cells. Thus, despite the biological significance of Se14-3-3ζ in S. exigua in conjunction with molecular interactions between SeNPV and S. exigua is unclear now, our data suggest that Se14-3-3 ζ protein plays a role to protect S. exigua from the infection or inhibit replication of SeNPV.

Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is known to play a pivotal role in various cellular events and antiviral responses in both vertebrates and insects. In an attempt to elucidate the potential involvement of STAT on S. exigua-SeNPV interactions, the full length cDNA of SeSTAT was cloned from S. exigua. Analysis of temporal expression patterns shows that SeSTAT is expressed in all stages of life cycle such as larvae, pupae, and adult. Spatial expression analysis shows that it is highly expressed in fat body and Malpighian tubule. Interestingly, SeSTAT is induced at 24 h in response to either laminarin or LTA injection in larvae. Electrophoretic mobility shift assay (EMSA) shows that the binding of nuclear extracts from fat body cells immune-challenged with LTA to STAT5 probe was observed. In addition, SeSTAT was nuclear-translocalized in both fat body and gut cells that were challenged with LTA and laminarin, respectively. Finally, gene silencing of SeSTAT shows that SeNPV number appears to be increased. It suggests that SeSTAT may act as a negative regulator against SeNPV in midgut.