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.

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.

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.

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.

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.

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 β-glycosyltransferase chitin synthase is known to catalyze the key polymerization step of N-acetylglucosamine.

Structural basis for directional chitin biosynthesis.

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units. The key reactions of chitin biosynthesis are catalysed by chitin synthase, 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.

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.

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.

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.

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).

V-ATPase subunit a is required for survival and midgut development of Locusta migratoria.

The vacuolar-type H -ATPase (V-ATPase) is an ATP-dependent proton pump, which regulates various cellular processes. To date, most functional studies on V-ATPases of insects have focused on subunits of the V1 complex, and there is little information on the VO genes. In this study, two cDNA sequences of LmV-ATPase a were identified in Locusta migratoria.

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.

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.

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.

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.

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.

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.

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.

Rapid determination of binding parameters of chitin binding domains using chitin-coated quartz crystal microbalance sensor chips.

Chitin present in fungal cell walls has been considered as a diagnostic polymer for the detection of fungal infections. Chitin staining can be achieved with different dyes such as Calcofluor white or Congo red, but these methods have not entered into clinical routine diagnosis due to problems with sensitivity and specificity. More accurate detection can be achieved using chitin binding domains (CBDs) from a large variety of naturally occurring proteins that specifically interact with chitin.

Septin-associated protein kinase Gin4 affects localization and phosphorylation of Chs4, the regulatory subunit of the Baker's yeast chitin synthase III complex.

Chitin is mainly formed by the chitin synthase III complex (CSIII) in yeast cells. This complex is considered to be composed of the catalytic subunit Chs3 and the regulatory subunit Chs4, both of which are phosphoproteins and transported to the plasma membrane by different trafficking routes. During cytokinesis, Chs3 associates with Chs4 and other proteins at the septin ring, which results in an active CSIII complex.

Functional analysis of TcCTLP-5C, a chymotrypsin-like serine protease needed for molting in Tribolium castaneum.

In a previous study, we have characterized a gene family encoding chymotrypsin-like proteases from the red flour beetle, Tribolium castaneum (TcCTLPs). We identified 14 TcCTLP genes that were predominantly expressed in the midgut, where they presumably function in digestion. Two genes (TcCTLP-6C and TcCTLP-5C), however, additionally showed considerable expression in the carcass, and RNAi studies demonstrated that they are required for molting (Broehan et al.

In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae.

Chitin biosynthesis in yeast is accomplished by three chitin synthases (Chs) termed Chs1, Chs2 and Chs3, of which the latter accounts for most of the chitin deposited within the cell wall. While the overall structures of Chs1 and Chs2 are similar to those of other chitin synthases from fungi and arthropods, Chs3 lacks some of the C-terminal transmembrane helices raising questions regarding its structure and topology. To fill this gap of knowledge, we performed bioinformatic analyses and protease protection assays that revealed significant information about the catalytic domain, the chitin-translocating channel and the interfacial helices in between.