Chitin is a necessary component to maintain the barrier function of the peritrophic matrix in the insect midgut

In most insects, the peritrophic matrix (PM) partitions the midgut into different digestive compartments, and functions as a protective barrier against abrasive particles and microbial infections. In a previous study we demonstrated that certain PM proteins are essential in maintaining the PM's barrier function and establishing a gradient of PM permeability from the anterior to the posterior part of the midgut which facilitates digestion (Agrawal et al., 2014). In this study, we focused on the effects of a reduction in chitin content on PM permeability in larvae of the red flour beetle, Tribolium castaneum. Oral administration of the chitin synthesis inhibitor diflubenzuron (DFB) only partially reduced chitin content of the larval PM even at high concentrations. We observed no nutritional effects, as larval growth was unaffected and neutral lipids were not depleted from the fat body. However, the metamorphic molt was disrupted and the insects died at the pharate pupal stage, presumably due to DFB's effect on cuticle formation. RNAi to knock-down expression of the gene encoding chitin synthase 2 in T. castaneum (TcCHS-2) caused a complete loss of chitin in the PM. Larval growth was significantly reduced, and the fat body was depleted of neutral lipids. In situ PM permeability assays monitoring the distribution of FITC dextrans after DFB exposure or RNAi for TcCHS-2 revealed that PM permeability was increased in both cases. RNAi for TcCHS-2, however, led to a higher permeation of the PM by FITC dextrans than DFB treatment even at high doses. Similar effects were observed when the chitin content was reduced by feeding DFB to adult yellow fever mosquitos, Aedes aegypti. We demonstrate that the presence of chitin is necessary for maintaining the PM's barrier function in insects. It seems that the insecticidal effects of DFB are mediated by the disruption of cuticle synthesis during the metamorphic molt rather than by interfering with larval nutrition. However, as DFB clearly affects PM permeability, it may be suitable to increase the efficiency of pesticides targeting the midgut.     

Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species Tribolium castaneum

Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin) are in wide use to control various insect pests. Although this class of compounds is known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored product pest Tribolium castaneum (red flour beetle) utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects and reduced chitin amounts in the larval cuticle, the peritrophic matrix and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unexpectedly unaffected, but many genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification pathways were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle turned out to be a good model organism for investigating the global effects of bioactive materials such as insect growth regulators and other insecticides. The results of this study recapitulate all of the different DFB-induced symptoms in a single model insect, which have been previously found in several different insect species, and further illustrate that DFB treatment causes a wide range of effects at the molecular level.     

Analysis of the chitin recognition mechanism of cuticle proteins from the soft cuticle of the silkworm, Bombyx mori

Insect cuticle is composed mainly of chitin, a polymer of N-acetylglucosamine, and chitin-binding cuticle proteins. Four major cuticle proteins, BMCP30, 22, 18, and 17, have been previously identified and purified from the larval cuticle of silkworm, B. mori. We analyzed the chitin-binding activity of BMCP30 by use of chitin-affinity chromatography. The pH optimum for the binding of BMCP30 to chitin is 6.4, which corresponds to hemolymph pH. Competition experiments using chitooligosaccharides suggested that BMCP30 recognizes 4-6 mer of N-acetylglucosamine in chitin fiber as a unit for binding. The comparison of the binding properties of BMCP30 with those of BMCP18 showed that their binding activities to chitin are similar in a standard buffer but that BMCP30 binds to chitin more stably than BMCP18 in the presence of urea. BMCPs possess the RR-1 form of the R&R consensus, about 70 amino acids region conserved widely among cuticle proteins mainly from the soft cuticle of many insect and arthropod species. Analysis of the binding activity using deletion mutants of BMCPs revealed that this type of conserved region also functions as the chitin-binding domain, similarly to the RR-2 region previously shown to confer chitin binding. Thus, the extended R&R consensus is the general chitin-binding domain of cuticle proteins in Arthropoda.     

Autoradiographic and fine structural study of chitin deposition in the cuticle of a barnacle using [3H]-D-glucosamine incorporation

[³H)-D-Glucosamine was injected into the rostral sinus of Balanus eburneus (barnacle) and the distribution of labelled chitin in the cuticle was studied with autoradiography and electron microscopy. When the pattern of labelling was examined in different body regions of the same organism where thickness of fully formed cuticle varied, it was observed that the rate of chitin deposition varied, being greater in thick than in thin regions. The density of Ag grains overlying cuticle was also greater in the thick regions. When the pattern of labelling was examined in regions of cuticle, comparable in thickness, taken from a series of organisms sacrificed at different time points a comparable value for the rate of chitin deposition was obtained. In addition, asynchrony in deposition of cuticle in different body regions of the same organism as well as uptake of the label by substances other than chitin, i.e. glycogen and glycoprotcins were described.     

A structural model of the chitin-binding domain of cuticle proteins

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Consequently, we have also verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). In this work, based on sequence and secondary structure similarity of cuticle proteins, and especially that of the consensus motif, to that of bovine plasma retinol binding protein (RBP), we propose by homology modelling an antiparallel beta-sheet half-barrel structure as the basic folding motif of cuticle proteins. This folding motif may provide the template for elucidating cuticle protein-chitin interactions in detail and reveal the precise geometrical formation of cuticle's helicoidal architecture. This predicted motif is another example where nature utilizes an almost flat protein surface covered by aromatic side chains to interact with the polysaccharide chains of chitin.     

Effects of benzoylphenylurea on chitin synthesis and orientation in the cuticle of the Drosophila larva

Chitin is an essential constituent of the insect exoskeleton, the cuticle, which is an extracellular matrix (ECM) covering the animal. It is produced by the glycosyltransferase chitin synthase at the apical plasma membrane of epidermal and tracheal cells. To fulfil its role in cuticle elasticity and stiffness it associates with proteins, thereby adopting a stereotypic arrangement of helicoidally stacked sheets, which run parallel to the surface of the animal. One approach to understand the mechanisms of chitin synthesis and organisation is to dissect these processes genetically. However, since only a few genes coding for factors involved in chitin synthesis and organisation have been identified to date using the model arthropod Drosophila melanogaster insight arising from mutant analysis is rather limited. To collect new data on the role of chitin during insect cuticle differentiation, we have analysed the effects of chitin synthesis inhibitors on Drosophila embryogenesis. For this purpose, we have chosen the benzoylphenylurea diflubenzuron and lufenuron that are widely used as insect growth regulators. Our data allow mainly two important conclusions. First, correct organisation of chitin seems to directly depend on the amount of chitin synthesised. Second, chitin synthesis and organisation are cell-autonomous processes as insecticide-treated larvae display a mosaic of cuticle defects. As benzoylphenylurea are used not only as insecticides but also as anti-diabetic drugs, the study of their impact on Drosophila cuticle differentiation may be fruitful for understanding their mode of action on a cellular pathway that is seemingly conserved between vertebrates and invertebrates.     

A possible structural model of members of the CPF family of cuticular proteins implicating binding to components other than chitin

The physical properties of cuticle are determined by the structure of its two major components, cuticular proteins (CPs) and chitin, and, also, by their interactions.A common consensus region (extended R&R Consensus) found in the majority of cuticular proteins, the CPRs, binds to chitin. Previous work established that β-pleated sheet predominates in the Consensus region and we proposed that it is responsible for the formation of helicoidal cuticle. Remote sequence similarity between CPRs and a lipocalin, bovine plasma retinol binding protein (RBP), led us to suggest an antiparallel β-sheet half-barrel structure as the basic folding motif of the R&R Consensus. There are several other families of cuticular proteins. One of the best defined is CPF. Its four members in Anopheles gambiae are expressed during the early stages of either pharate pupal or pharate adult development, suggesting that the proteins contribute to the outer regions of the cuticle, the epi- and/or exo-cuticle. These proteins did not bind to chitin in the same assay used successfully for CPRs. Although CPFs are distinct in sequence from CPRs, the same lipocalin could also be used to derive homology models for one A. gambiae and one Drosophila melanogaster CPF. For the CPFs, the basic folding motif predicted is an eight-stranded, antiparallel β-sheet, full-barrel structure. Possible implications of this structure are discussed and docking experiments were carried out with one possible Drosophila ligand, 7(Z),11(Z)-heptacosadiene.     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

Assembly of the Drosophila larval exoskeleton requires controlled secretion and shaping of the apical plasma membrane.

The apical plasma membrane of epithelial cells plays a central role in producing and shaping the apical extracellular matrix (aECM) that eventually adopts a stereotypic architecture required for the physical and physiological needs of the epithelium. To assess the implication of the apical plasma membrane on aECM differentiation, we have studied the function of the apical plasma membrane t-SNARE Syntaxin 1A in the embryo of the fruit fly Drosophila melanogaster during differentiation of the stratified exoskeleton, the cuticle, which is composed of proteins and the polysaccharide chitin. The cuticle layers of syntaxin1A deficient larvae are rudimentary. Consistently, Syntaxin 1A is required for the secretion of O-glycosylated proteins and components involved in pigmentation and protein cross-linking. By contrast, localization of chitin synthesis and organising proteins to the apical plasma membrane or to the extracellular space does not depend on Syntaxin 1A activity. However, chitin microfibrils have a random orientation instead of being arranged in parallel. This correlates with the lack of corrugations at the apical plasma membrane of epidermal cells, the apical undulae that have been proposed to be crucial for chitin microfibril orientation. Hence, Syntaxin 1A contributes to cuticle differentiation by controlling correct apical plasma membrane topology as well as mediating secretion of a subset of extracellular proteins required for layer organisation. Our data also indicate that yet another unidentified t-SNARE is needed in parallel to Syntaxin 1A to deliver extracellular material for complete cuticle assembly. Evidently, coordination of apical membrane modelling and two secretion routes are essential for stereotypic aECM organisation.     

"Soft"-cuticle protein secondary structure as revealed by FT-Raman, ATR FT-IR and CD spectroscopy.

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of a beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). In this study, we examined experimentally the secondary structure of intact and guanidine hydrochloride extracted cuticle and the cuticular protein extract. The studied cuticle came from the larval dorsal abdomen of the lepidopteran Hyalophora cecropia, a classical example of "soft" cuticle. Analysis with FT-Raman, ATR FT-IR and CD spectroscopy indicates that antiparallel beta-pleated sheet is the predominant molecular conformation of "soft-cuticle" proteins both in situ in the cuticle and following extraction. It seems that this conformation dictates the modes of chitin-protein interaction in cuticle, in agreement with earlier proposals (Atkins, J. Biosci. 8 (1985) 375).     

Cuticular proteins from the shrimp, Pandalus borealis.

1. The percentages of mineral salts, chitin, and urea-extractable and non-extractable proteins were determined in pieces of cuticle from selected body regions of the shrimp, Pandalus borealis. 2. Two-dimensional gel-electrophoresis of the urea-extractable proteins shows that a large number of different proteins are present. Identical protein patterns are obtained from the various cuticular regions. 3. A fractionation scheme is presented, which is suitable for obtaining the major proteins in quantities sufficient for further characterization. The amino acid compositions are reported for several of the proteins.     

Diflubenzuron-Induced alterations during in vitro development of Tenebrio molitor pupal integument

The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20-hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants in vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture in the presence of 20-hydroxyecdysone, DFB at high dose (≥ 20 μg/ml) was able to inhibit cuticle secretion, but lower doses (? 10 μg/ml) resulted in epicuticle deposition. These observations confirm in vivo studies showing antagonistic effects of DFB and ecdysteroids at the level of epidermal cells. In another series of experiments, the DFB effects were analyzed without addition of exogenous molting hormone in vitro. Because it had been observed in previous studies that pupal epidermal explants of Tenebrio secrete low but significant amounts of ecdysteroids in the culture medium, this in vitro secretion was measured by radioimmunoassay after DFB treatment. It was observed that DFB, when applied either before or during culture, significantly reduced the hormonal secretion in vitro. This reduction, observed at the level of epidermal cells, could be homologous with the diminution of the endogenous ecdysteroid peak previously described after in vivo DFB treatment in Tenebrio pupae.     

Physiological and morphological aspects of Aedes aegypti developing larvae: effects of the chitin synthesis inhibitor novaluron

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes.     

Analysis of expression and chitin‐binding activity of the wing disc cuticle protein BmWCP4 in the silkworm,Bombyx mori

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin‐binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic “R&R” chitin‐binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc‐containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full‐length BmWCP4 protein, “R&R” CBD peptide (CBD), non‐CBD peptide (BmWCP4‐CBD^?), four single site‐directed mutated peptides (M₁, M₂, M₃ and M₄) and four‐sites‐mutated peptide (M_F) were generated and purified, respectively, for in vitro chitin‐binding assay. The results indicated that both the full‐length protein and the “R&R” CBD peptide could bind with chitin, whereas the BmWCP4‐CBD^? could not bind with chitin. The single residue mutants M₁, M₂, M₃ and M₄ reduced but did not completely abolish the chitin‐binding activity, while four‐sites‐mutated protein M_F completely lost the chitin‐binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva‐to‐pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.     

The formation of the pupal cuticle by Drosophila imaginal discs in vitro

Mass-isolated imaginal discs of Drosophila melanogaster form a chitin-containing pupal procuticle In vitro. Optimal procuticle deposition occurs when the discs are incubated for 4–6 hr with 0.5–1.0 μg/ml of 20-hydroxyecdysone and then with less than 0.05 μg/ml of 20-hydroxyecdysone. The formation of the chitin-containing procuticle is demonstrated using three independent assays: with fluorescene-conjugated cuticle proteins that bind to chitin; by electron microscopy; by incorporation of [³H]glucosamine into a chitin fraction. Synthesis and deposition of pupal cuticle proteins are also demonstrated. Incorporation of [³H]glucosamine into chitin is sensitive to inhibitors of protein, RNA and chitin synthesis, but has little sensitivity to inhibitors of DNA synthesis, and dolichol-dependent glycosylation.