Advances in understanding insect chitin biosynthesis.

Chitin, a natural polymer of N-acetylglucosamine chains, is a principal component of the apical extracellular matrix in arthropods. Chitin microfibrils serve as structural components of natural biocomposites present in the extracellular matrix of a variety of invertebrates including sponges, molluscs, nematodes, fungi and arthropods. In this review, we summarize the frontier advances of insect chitin synthesis. More specifically, we focus on the chitin synthase (CHS), which catalyzes the key biosynthesis step. CHS is also known as an attractive insecticidal target in that this enzyme is absent in mammals, birds or plants. As no insect chitin synthase structure have been reported so far, we review recent studies on glycosyltransferase domain structures derived from fungi and oomycetes, which are conserved in CHS from all species containing chitin. Auxiliary proteins, which coordinate with CHS in chitin biosynthesis and assembly, are also discussed.

Dextran sulfate sodium and uracil induce inflammatory effects and disrupt the chitinous peritrophic matrix in the midgut of Tribolium castaneum.

Dextran sulfate sodium is used in inflammatory bowel disease (IBD) mice models to trigger chronic intestinal inflammation. In this study, we have analyzed DSS effects in the genetic model and pest beetle, Tribolium castaneum, which can be easily and cost-effectively cultivated and examined in very large quantities compensating for individual variations. We fed the larvae with DSS and uracil, which is known to induce the production of reactive oxygen species by activating DUOX, a member of the NADPH oxidase family. Both chemicals induced IBD-like phenotypes, including impaired growth and development, midgut thickening, epithelial swelling, and a loss of epithelial barrier function. RNAi mediated knockdown of DUOX expression enhanced the effects of DSS and uracil on mortality. Finally, we showed that both treatments result in an altered activity of the intestinal microbiome, similar as observed in IBD patients. Our findings suggest that both chemicals impair the epithelial barrier by increasing the permeability of the peritrophic matrix. The loss of the barrier function may facilitate the entry of midgut bacteria triggering innate immune responses that also affect the intestinal microbiome. As the observed effects resemble those induced by DSS treatment in mice, T. castaneum might be suitable high-throughput invertebrate model for IBD research and preclinical studies.

Analyses of ecdysteroid transporters in the fat body of Tribolium castaneum.

The control of insect moulting and metamorphosis involves ecdysteroids that orchestrate the execution of developmental genetic programs by binding to dimeric hormone receptors consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). In insects, the main ecdysteroids comprise ecdysone (E), which is synthesized in the prothoracic gland and secreted into the haemolymph, and 20-hydroxyecdysone (20E), which is considered the active form by binding to the nuclear receptor of the target cell. While biosynthesis of ecdysteroids has been studied in detail in different insects, the transport systems involved in guiding these steroid hormones across cellular membranes have just recently begun to be studied. By analysing RNAi phenotypes in the red flour beetle, Tribolium castaneum, we have identified three transporter genes, TcABCG-8A, TcABCG-4D and TcOATP4-C1, whose silencing results in phenotypes similar to that observed when the ecdysone receptor gene TcEcRA is silenced, that is, abortive moulting and abnormal development of adult compound eyes during the larval stage. The genes of all three transporters are expressed at higher levels in the larval fat body of T. castaneum. We analysed potential functions of these transporters by combining RNAi and mass spectrometry. However, the analysis of gene functions is challenged by mutual RNAi effects indicating interdependent gene regulation. Based on our findings, we propose that TcABCG-8A, TcABCG-4D and TcOATP4-C1 participate in the ecdysteroid transport in fat body cells, which are involved in E → 20E conversion catalysed by the P450 enzyme TcShade.

Both LmDicer-1 and two LmDicer-2s participate in siRNA-mediated RNAi pathway and contribute to high gene silencing efficiency in Locusta migratoria.

Dicers belong to a class of large RNase III multidomain ribonucleases and are central components of the RNA interference (RNAi) pathways. In insects, Dicer-2 has been known to cleave long double-stranded RNA (dsRNA) in small interfering RNA (siRNA)-mediated-RNAi pathway. However, Dicer-1 is responsible for cleaving precursor microRNAs (pre28 miRNAs) in miRNA-mediated RNAi pathway. In this study, we identified one LmDicer-1 and two LmDicer-2 (LmDicer-2a and LmDicer-2b) genes in Locusta migratoria. The RNAi of RNAi assay showed that knockdown of each of the Dicer genes reduced RNAi efficiency against a target gene (Lmß-Tubulin), suggesting that all these genes participated in the siRNA-mediated RNAi pathway. Sequence analyses of the siRNAs generated from dsLmß-Tubulin after silencing each LmDicer gene showed no significant difference in the pattern of siRNAs mapped to dsLmß-Tubulin. This result indicated that all the three LmDicers are capable of generating siRNAs from the dsRNA. We then generated recombinant proteins consisting of different domains using Escherichia coli expression system and incubated each recombinant protein with dsLmß-Tubulin. We found that the recombinant Dicer proteins successfully cleaved dsLmß-Tubulin. However, LmDicer-2a-R lacking dsRBD domain lost activity, suggesting that dsRBD domain is critical for Dicer function. Furthermore, overexpression of these proteins in Drosophila S2 cells improved RNAi efficiency. Our siRNA affinity chromatography and LC-MS/MS analysis identified LmDicer-2a, LmDicer-2b, LmR2D2, LmAgo2a, LmAgo1, LmStaufen and LmTARBP2 as constituents of RNA-induced silencing complex. Taken together, these data show that both LmDicer-1 and two LmDicer-2s all participate in siRNA-mediated RNAi pathway and likely contribute to high RNAi efficiency in L. migratoria.

High-throughput screening of caterpillars as a platform to study host-microbe interactions and enteric immunity.

Mammalian models of human disease are expensive and subject to ethical restrictions. Here, we present an independent platform for high-throughput screening, using larvae of the tobacco hornworm Manduca sexta, combining diagnostic imaging modalities for a comprehensive characterization of aberrant phenotypes. For validation, we use bacterial/chemical-induced gut inflammation to generate a colitis-like phenotype and identify significant alterations in morphology, tissue properties, and intermediary metabolism, which aggravate with disease progression and can be rescued by antimicrobial treatment. In independent experiments, activation of the highly conserved NADPH oxidase DUOX, a key mediator of gut inflammation, leads to similar, dose-dependent alterations, which can be attenuated by pharmacological interventions. Furthermore, the developed platform could differentiate pathogens from mutualistic gastrointestinal bacteria broadening the scope of applications also to microbiomics and host-pathogen interactions. Overall, larvae-based screening can complement mammals in preclinical studies to explore innate immunity and host-pathogen interactions, thus representing a substantial contribution to improve mammalian welfare.

Fatty acid binding protein is required for chitin biosynthesis in the wing of Drosophila melanogaster.

Chitin, the major structural polysaccharide in arthropods such as insects and mites, is a linear polymer of N-acetylglucosamine units. The growth and development of insects are intimately coupled with chitin biosynthesis. The membrane-bound ß-glycosyltransferase chitin synthase is known to catalyze the key polymerization step of N-acetylglucosamine. However, the additional proteins that might assist chitin synthase during chitin biosynthesis are not well understood. Recently, fatty acid binding protein (Fabp) has been suggested as a candidate that interacts with the chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster. Here, using split-ubiquitin membrane yeast two-hybrid and pull-down assays, we have demonstrated that the Fabp-B splice variant physically interacts with Kkv in vitro. The global knockdown of Fabp in D. melanogaster using RNA interference (RNAi) induced lethality at the larval stage. Moreover, in tissue-specific RNAi experiments, silenced Fabp expression in the epidermis and tracheal system caused a lethal larval phenotype. Fabp knockdown in the wings resulted in an abnormal wing development and uneven cuticular surface. In addition to reducing the chitin content in the first longitudinal vein of wings, Fabp silencing also caused the loss of procuticle laminate structures. This study revealed that Fabp plays an important role in chitin synthesis and contributes to a comprehensive understanding of the complex insect chitin biosynthesis.

Structural basis for directional chitin biosynthesis.

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2-4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a 'gate lock' that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.

SERCA interacts with chitin synthase and participates in cuticular chitin biogenesis in Drosophila.

The biogenesis of chitin, a major structural polysaccharide found in the cuticle and peritrophic matrix, is crucial for insect growth and development. Chitin synthase, a membrane-integral ß-glycosyltransferase, has been identified as the core of the chitin biogenesis machinery. However, a yet unknown number of auxiliary proteins appear to assist in chitin biosynthesis, whose precise function remains elusive. Here, we identified a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), in the fruit fly Drosophila melanogaster, as a chitin biogenesis-associated protein. The physical interaction between DmSERCA and epidermal chitin synthase (Krotzkopf verkehrt, Kkv) was demonstrated and analyzed using split-ubiquitin membrane yeast two-hybrid, bimolecular fluorescent complementation, pull-down, and immunoprecipitation assays. The interaction involves N-terminal regions (aa 48-81 and aa 247-33) and C-terminal regions (aa 743-783 and aa 824-859) of DmSERCA and two N-terminal regions (aa 121-179 and aa 369-539) of Kkv, all of which are predicted be transmembrane helices. While tissue-specific knock-down of DmSERCA in the epidermis caused larval and pupal lethality, the knock-down of DmSERCA in wings resulted in smaller and crinkled wings, a significant decrease in chitin deposition, and the loss of chitin lamellar structure. Although DmSERCA is well-known for its role in muscular contraction, this study reveals a novel role in chitin synthesis, contributing to our knowledge on the machinery of chitin biogenesis.

Clathrin heavy chain is essential for the development and reproduction of Locusta migratoria.

Clathrin heavy chain (Chc) is a constituent of clathrin-coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but appears to have physiological roles also at the organismal level. Most of what we know about Chc functions originates from studies performed in fungal or vertebrate cells. However, the physiological functions of Chc in insects remain poorly understood. Here, we identified a Chc ortholog from a Locusta migratoria transcriptome database. RT-qPCR revealed that LmChc was constitutively expressed in fifth-instar nymphs. In this developmental stage, LmChc showed the highest expression in the ovary followed by hemolymph, testis, hindgut, midgut, and foregut. In isolated hemocytes, we detected the Chc protein in patches at the plasma membrane. To examine the role of LmChc in L. migratoria during development, RNA interference was performed by injecting dsRNA into the early fifth-instar nymphs. Silencing of LmChc caused a lethal phenotype with molting defect from nymph to adult. In addition, silencing of LmChc resulted in abnormal development of the ovaries, the size of which was significantly smaller than that in controls. Taken together, our results suggest that LmChc is a vital gene in L. migratoria that plays an important role in growth, development, and reproduction. LmChc may be used as an efficient RNAi target gene for developing dsRNA-based biological insecticides to manage insect pests.

Vacuolar (H+ )-ATPase subunit c is essential for the survival and systemic RNA interference response in Locusta migratoria.

BACKGROUND: Vacuolar (H+ )-ATPase (V-ATPase) is a multi-subunit enzyme that hydrolyzes adenosine triphosphate (ATP) to transport protons across a cellular membrane, and it plays an important role in numerous biological processes, including in growth, development and immune responses. The c subunit of V-ATPase is a highly conserved subunit of the rotatory proteolipid ring that is required for binding and transporting protons. To date, there are only a few published reports on V-ATPase-c functions in insects. RESULTS: We identified and characterized the V-ATPase-c gene in Locusta migratoria, one of the most destructive agricultural insect pests in the world. LmV-ATPase-c was predominately expressed in Malpighian tubules of nymphs, followed by the hindgut and ovary, while the other tissues showed relatively low expression levels. Silencing of LmV-ATPase-c caused severe molting defects in nymphs and a high mortality rate of > 90%. Histological staining and microscopic examination of sections from the abdominal cuticle revealed the absence of newly formed cuticle in nymphs that were injected with dsLmV-ATPase-c. In addition, silencing of LmV-ATPase-c transcript levels significantly impaired RNA interference (RNAi) efficiency of a reporter gene. By quantifying double-stranded RNA (dsRNA) amounts by quantitative polymerase chain reaction (PCR), we found that RNAi against LmV-ATPase-c provoked a dramatic accumulation of dsRNA in the endosomes of epidermal and midgut cells of Locusta migratoria. CONCLUSION: Our results indicate that LmV-ATPase-c is indispensable for the formation of new cuticle during the molting process and has pivotal functions in dsRNA escape from endosomes. LmV-ATPase-c might be a valuable target for developing new strategies for insect pest management. © 2022 Society of Chemical Industry.

Choline transporter-like protein 2 interacts with chitin synthase 1 and is involved in insect cuticle development.

Chitin is an aminopolysaccharide present in insects as a major structural component of the cuticle. However, current knowledge on the chitin biosynthetic machinery, especially its constituents and mechanism, is limited. Using three independent binding assays, including co-immunoprecipitation, split-ubiquitin membrane yeast two-hybrid assay, and pull-down assay, we demonstrate that choline transporter-like protein 2 (Ctl2) interacts with krotzkopf verkehrt (kkv) in Drosophila melanogaster. The global knockdown of Ctl2 by RNA interference (RNAi) induced lethality at the larval stage. Tissue-specific RNAi to silence Ctl2 in the tracheal system and in the epidermis of the flies resulted in lethality at the first larval instar. The knockdown of Ctl2 in wings led to shrunken wings containing accumulated fluid. Calcofluor White staining demonstrated reduced chitin content in the first longitudinal vein of Ctl2 knockdown wings. The pro-cuticle, which was thinner compared to wildtype, exhibited a reduced number of chitin laminar layers. Phylogenetic analyses revealed orthologues of Ctl2 in different insect orders with highly conserved domains. Our findings provide new insights into cuticle formation, wherein Ctl2 plays an important role as a chitin-synthase interacting protein.

Chitin metabolism as a potential target for RNAi-based control of the forestry pest Hyblaea puera Cramer (Lepidoptera: Hyblaeidae).

BACKGROUND: Hyblaea puera, commonly known as the teak defoliator, is a serious pest in teak plantations. Despite the availability of control measures, this pest causes losses in yield and quality of timber through voracious feeding. RNA interference (RNAi) is a promising strategy for the control of this pest. Chitin metabolism, which is vital for the growth and development of arthropods, is a potential target for developing RNAi-based insecticides. RESULTS: To assess the effects of chitin metabolism inhibition, H. puera larvae were treated with a chitin synthesis inhibitor, diflubenzuron (DFB). DFB treatment caused pupal deformities and disrupted eclosion. Partial gene sequences for three key genes of H. puera chitin metabolism were cloned and sequenced: chitin synthase 1 (HpCHS1), chitinase-h (HpChi-h) and ecdysone receptor (HpEcR). Feeding dsRNA cognate for these three target genes to the first instar of H. puera resulted in mortality and reduction in the corresponding transcript levels as assessed through qRT-PCR. This is the first report of RNAi in this forestry pest. The highest mortality was 45.9%, in response to dsHpEcR treatment; HpChi-h transcripts were the most down-regulated in response to dsHpEcR feeding. DsHpEcR RNAi resulted in growth inhibition and molting arrest. The mortalities were 29.7% and 32.4% for dsHpCHS1 and dsHpChi-h feeding, respectively. CONCLUSION: Chitin metabolism could be a potential target for RNAi-based control of H. puera, and HpCHS1, HpChi-h and HpEcR could be suitable target genes. However, the RNAi efficacy needs to be improved through formulations that improve stability and uptake, and employing better delivery strategies. © 2021 Society of Chemical Industry.

Identification of two ABCC transporters involved in malathion detoxification in the red flour beetle, Tribolium castaneum.

ABC transporters have been suggested to be involved in insecticide detoxification in different insect species mainly based on the indirect observation of transcriptional upregulation of ABC gene expression in response to insecticide exposure. Previous studies performed by us and others in the red flour beetle, Tribolium castaneum, have analyzed the function of TcABCA-C and TcABCG-H genes using RNA interference (RNAi) and demonstrated that specific TcABCA and TcABCC genes are involved in the elimination of the pyrethroid tefluthrin and the benzoylurea diflubenzuron, because gene silencing increased the beetle's susceptibility to the insecticides. In this study, we focused on the potential functions of TcABCA-C genes in detoxification of the pyrethroid cyfluthrin (CF), the organophosphate malathion (MAL) and the diacylhdyazine tebufenozide (TBF). Analysis of transcript levels of selected TcABCA-C genes in response to treatment with these three chemically unrelated insecticides revealed that some genes were particularly upregulated after insecticide treatment. In addition, the ABC inhibitor verapamil synergized significantly the toxicity of MAL but only negligibly CF and TBF toxicities. Finally, silencing of two TcABCC genes by RNAi revealed a significant increase in susceptibility to MAL. In contrast, we did not observe a significant increase in insecticide-induced mortalities when knocking down TcABC genes in larvae treated with CF or TBF, although they were upregulated in response to insecticide treatment. Our results suggest that two pleiotropic ABCC transporters expressed in metabolic and excretory tissues contribute to the elimination of MAL.

Functional analysis of ABCG and ABCH transporters from the red flour beetle, Tribolium castaneum.

BACKGROUND: ATP-binding cassette transporter (ABC transporter) subfamilies ABCA-C and ABCG-H have been implicated in insecticide detoxification, mostly based on findings of elevated gene expression in response to insecticide treatment. We previously characterized TcABCA-C genes from the model beetle and pest Tribolium castaneum and demonstrated that TcABCA and TcABCC genes are involved in the elimination of diflubenzuron, because RNA interference (RNAi)-mediated gene silencing increased susceptibility. In this study, we focused on the potential functions of TcABCG and TcABCH genes in insecticide detoxification. RESULTS: When we silenced the expression of TcABCG-H genes using RNAi, we noticed a previously unreported developmental RNAi phenotype for TcABCG-4F, which is characterized by 50% mortality and ecdysial arrest during adult moult. When we knocked down the Drosophila brown orthologue TcABCG-XC, we did not obtain apparent eye colour phenotypes but did observe a loss of riboflavin uptake by Malpighian tubules. Next, we determined the expression profiles of all TcABCG-H genes in different tissues and developmental stages and analysed transcript levels in response to treatment with four chemically unrelated insecticides. We found that some genes were specifically upregulated after insecticide treatment. However, when we determined insecticide-induced mortalities in larvae that were treated by double-stranded RNA injection to silence those TcABCG-H genes that were upregulated, we did not observe a significant increase in susceptibility to insecticides. CONCLUSION: Our findings suggest that the observed insecticide-dependent induction of TcABCG-H gene expression reflects an unspecific stress response, and hence underlines the significance of functional studies on insecticide detoxification. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Tribolium castaneum: A Model for Investigating the Mode of Action of Insecticides and Mechanisms of Resistance.

The red flour beetle, Tribolium castaneum, is a worldwide insect pest of stored products, particularly food grains, and a powerful model organism for developmental, physiological and applied entomological research on coleopteran species. Among coleopterans, T. castaneum has the most fully sequenced and annotated genome and consequently provides the most advanced genetic model of a coleopteran pest. The beetle is also easy to culture and has a short generation time. Research on this beetle is further assisted by the availability of expressed sequence tags and transcriptomic data. Most importantly, it exhibits a very robust response to systemic RNA interference (RNAi), and a database of RNAi phenotypes (iBeetle) is available. Finally, classical transposonbased techniques together with CRISPR/Cas-mediated gene knockout and genome editing allow the creation of transgenic lines. As T. castaneum develops resistance rapidly to many classes of insecticides including organophosphates, methyl carbamates, pyrethroids, neonicotinoids and insect growth regulators such as chitin synthesis inhibitors, it is further a suitable test system for studying resistance mechanisms. In this review, we will summarize recent advances in research focusing on the mode of action of insecticides and mechanisms of resistance identified using T. castaneum as a pest model.

Transcriptional plasticity of different ABC transporter genes from Tribolium castaneum contributes to diflubenzuron resistance.

The development of insecticide resistance challenges the sustainability of pest control and several studies have shown that ABC transporters contribute to this process. ABC transporters are known to transport a large range of chemically diverse molecules across cellular membranes, and therefore the identification of ABC transporters involved in insecticide resistance is difficult. Here, we describe a comprehensive strategy for the identification of whole sets of ABC transporters involved in insecticide resistance using the pest beetle, Tribolium castaneum (Tc) as a model. We analyzed the expression of ABCA to ABCC genes in different tissues and developmental stages using larvae that were sensitive or resistant to diflubenzuron (DFB). The mRNA levels of several ABC genes expressed in excretory or metabolic tissues such as midgut, Malpighian tubules or fat body were markedly upregulated in response to DFB. Next, we monitored mortality in the presence of the ABC inhibitor verapamil, and found that it causes sensitization to DFB. We furthermore established a competitive assay for the elimination of DFB, based on Texas Red (TR) fluorescence. We monitored TR elimination in larvae that were treated with DFB or different ABC inhibitors, and combinations of them. TR elimination was decreased significantly in the presence of DFB, verapamil and the ABCC inhibitor MK-571. The effect was synergized when DFB and verapamil were both present suggesting that the transport of TR and DFB involves overlapping sets of ABC transporters. Finally, we silenced the expression of DFB-responding ABC genes by RNA interference and then followed the survival rates after DFB exposure. Mortality increased particularly when specific ABCA and ABCC genes were silenced. Taken together, we were able to show that different ABC transporters expressed in metabolic and excretory tissues contribute to the elimination of DFB. Up- or down-regulation of gene expression occurs within a few days already at very low DFB concentrations. These results suggests that transcriptional plasticity of several ABC genes allows adaptation of the efflux capacity in different tissues to eliminate insecticides and/or their metabolites.

Chitin Prevalence and Function in Bacteria, Fungi and Protists.

Chitin is an important structural polysaccharide, which supports and organizes extracellular matrices in a variety of taxonomic groups including bacteria, fungi, protists, and animals. Additionally, chitin has been recognized as a molecule that is required for Rhizobia-legume symbiosis and involved in arbuscular mycorrhizal signaling in the symbiotic interaction between terrestrial plants and fungi. Moreover, it serves as a unique molecular pattern in the plant defense system against pathogenic fungi and parasites, and in the innate and adaptive immune response of mammals and humans. In this review, we will focus on the prevalence and structural function of chitin in bacteria, fungi, and protists, with a particular focus on the evolution of chitin synthases and the function of chitin oligosaccharides as a signaling molecule in symbiosis and immunity.

Immune Responses of Mammals and Plants to Chitin-Containing Pathogens.

Chitin-containing organisms, such as fungi and arthropods, use chitin as a structural component to protect themselves from harsh environmental conditions. Hosts such as mammals and plants, however, sense chitin to initiate innate and adaptive immunity and exclude chitin-containing organisms. A number of protein factors are then expressed, and several signaling pathways are triggered. In this chapter, we focus on the responses and signal transduction pathways that are activated in mammals and plants upon invasion by chitin-containing organisms. As host chitinases play important roles in the glycolytic processing of chitin, which is then recognized by pattern-recognition receptors, we also pay special attention to the chitinases that are involved in immune recognition.

Chitin Organizing and Modifying Enzymes and Proteins Involved In Remodeling of the Insect Cuticle.

Chitin, the extracellular matrix polysaccharide of insects and arthropods is widely distributed in nature in all kingdoms of life and serves a variety of functions. After synthesis by membrane-bound chitin synthases, it is extensively remodeled before incorporation into divergent matrices with wide-ranging physical and biological properties. This chapter discusses the properties of a variety of insect enzymes and proteins involved in this process. Chitin remodeling involves chitin synthases, which make the nascent chitin chains, and chitin deacetylases that partially deacetylate some of the N-acetylglucosamine residues either randomly or sequentially to yield local chitosan-like regions. Other proteins secreted into the procuticle or the midgut help in the assembly of single chitin chains into larger crystalline aggregates that measure in a few 100 nanometers. They are further embedded in a complex matrix of cuticular proteins or become associated with proteins containing chitin-binding domains to constitute the laminar procuticle or the lattice-like peritrophic matrix. During molting, previously formed laminar cuticle or PM are decrystallized/depolymerized to unmask the chitin chains, which then are degraded by a mixture of chitinolytic enzymes consisting of chitinases and N-acetylglucosaminidases present in molting fluid or in gut secretions. Some of the degradation products may be recycled for the synthesis of new matrices. We present a model of chitin synthesis, assembly, and degradation and the roles of these chitin-remodeling enzymes in this overall process.