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.     

Chitin and chitinase: A kinetic model

A model is described for the action of insect molting chitinase on chitin microfibrils in cuticle. The model reconciles the disparate structures proposed for chitin in the literature. It also accounts for the kinetic characteristics of molting fluid chitinase insofar as known from in vitro studies, viz. positive co-operativity of possibly three catalytic sites, complexity, and processivity. These have hitherto been difficult to account for in vivo, given the arrangement of chitin in anhydrous microfibrils in arthropod cuticle.     

Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum

Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.     

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.     

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.     

Development and ultrastructure of the rigid dorsal and flexible ventral cuticles of the elytron of the red flour beetle,Tribolium castaneum

Insect exoskeletons are composed of the cuticle, a biomaterial primarily formed from the linear and relatively rigid polysaccharide, chitin, and structural proteins. This extracellular material serves both as a skin and skeleton, protecting insects from environmental stresses and mechanical damage. Despite its rather limited compositional palette, cuticles in different anatomical regions or developmental stages exhibit remarkably diverse physicochemical and mechanical properties because of differences in chemical composition, molecular interactions and morphological architecture of the various layers and sublayers throughout the cuticle including the envelope, epicuticle and procuticle (exocuticle and endocuticle). Even though the ultrastructure of the arthropod cuticle has been studied rather extensively, its temporal developmental pattern, in particular, the synchronous development of the functional layers in different cuticles during a molt, is not well understood. The beetle elytron, which is a highly modified and sclerotized forewing, offers excellent advantages for such a study because it can be easily isolated at precise time points during development. In this study, we describe the morphogenesis of the dorsal and ventral cuticles of the elytron of the red flour beetle, Tribolium castaneum, during the period from the 0 d-old pupa to the 9 d-old adult. The deposition of exocuticle and mesocuticle is substantially different in the two cuticles. The dorsal cuticle is four-fold thicker than the ventral. Unlike the ventral cuticle, the dorsal contains a thicker exocuticle consisting of a large number of horizontal laminae and vertical pore canals with pore canal fibers and rib-like veins and bristles as well as a mesocuticle, lying right above the enodcuticle. The degree of sclerotization appears to be much greater in the dorsal cuticle. All of these differences result in a relatively thick and tanned rigid dorsal cuticle and a much thinner and less pigmented membrane-like ventral cuticle.     

Chitin in the Silk Gland Ducts of the Spider Nephila edulis and the Silkworm Bombyx mori

Here we report the detection and localisation of chitin in the cuticle of the spinning ducts of both the spider Nephila edulis and the silkworm Bombyx mori. Our observations demonstrate that the duct walls of both animals contain chitin notwithstanding totally independent evolutionary pathways of the systems. We conclude that chitin may well be an essential component for the construction of spinning ducts; we further conclude that in both species chitin may indicate the evolutionary origin of the spinning ducts.     

Different Functions of the Insect Soluble and Membrane-Bound Trehalase Genes in Chitin Biosynthesis Revealed by RNA Interference

Background

Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1) and membrane-bound (Tre-2) trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported.

Principal Findings

The membrane-bound trehalase of Spodoptera exigua (SeTre-2) was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1) and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi) of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA) and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB) expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%.

Conclusions

SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2 has an important role in CHSB expression and chitin synthesis in the midgut.     

Activation of old cuticle chitin as a substrate for chitinase in the molt of Manduca

During the molt, chitin in the old cuticle of $\text{Manduca}$ is digested by chitinase taken up from molting fluid, but the chitin in intact (= premolt) cuticle is not accessible to chitinase. As a prerequisite of digestion, old cuticle chitin is rendered competent to serve as chitinase substrate in a reaction attributable to trypsin-like proteolytic activity of molting fluid.     

Cytochemical demonstration of chitin incorporation in the cuticle of Trialeurodes vaporariorum (Westwood) (Homoptera : Aleyrodidae) after buprofezin treatment

Buprofezin is an insect growth regulator acting specifically against sucking insects. Nymphs of the greenhouse whitefly Trialeurodes vaporariorum (Westwood) (Homoptera : Aleyrodidae) were treated with different concentrations of this compound and the deposition of the chitin nn the integument examined. The lectin, wheat germ agglutinin (WGA), which binds specifically to N-acetylglucosamine-containing polymers, conjugated with colloidal gold, was used as an electron-dense marker for localizing chitin on ultrathin sections. In untreated insects, the epicuticle and epidermis cells were devoid of label, while the procuticle was heavily labelled. In buprofezin-treated nymphs, only the pre-apolysial cuticle remained heavily labelled, independent of the concentration used. The post-apolysial cuticle, however, was hardly labelled, the intensity of labelling dependn: g on the applied concentration, and its thickness severely reduced. These results suggest that buprofezin affects only post-apolysial cuticle in a concentration-dependent way.     

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.     

Biochemistry of insect differentiation: A system for studying the mechanism of chitinase activity in vitro

Results obtained with an in vitro system for the study of chitinase are described. The system involves soluble enzyme protein(s) and an insoluble substrate preparation. With insect molting fluid chitinase, it shows properties that parallel those observed during in vivo breakdown of cuticle during the molt. For example, molting fluid chitinase activity not previously exposed to chitin is stronly and specifically adsorbed to the substrate, in contrast to other enzymatic activities including hexosaminidase (chitobiase) present in molting fluid. This leads to partial purification of molting fluid chitinase activity reflected in increased specific activity of chitinase associated with the insoluble chitin substrate; we have previously reported increase of specific chitinase activity of (deproteinized) cuticle resulting from its incubation with molting fluid (M. L. Bade and A. Stinson, 1978, Biochem. Biophys. Res. Commun.84, 381–388). Soluble end product is generated rapidly and linearly with time by the in vitro system; the end product is assumed to be N-acetylglucosamine since the specific radioactivity of this compound is unchanged during the 10 min required for assay. Molting fluid chitinase activity may involve a number of polypeptides ranging in molecular weight from 145,000 to less than 20,000 daltons. The system described gives results consistent with a processive mechanism for molting fluid chitinase, i.e., data are given demonstrating that molting fluid chitinase continues to act on the same chitin particle(s) with which it initially associates rather than diffusing freely from substrate particle to substrate particle, and the product of its action appears to be a monosaccharide rather than a mixture of oligosaccharides. Processive behavior for chitinase would be predicted from the known structure, and the in vivo measured rate of breakdown, of cuticle chitin during the molt; the preliminary nature of this conclusion, based on what is so far known about the structure of the substrate used in the in vitro system, is briefly discussed.     

The effects of DOPA decarboxylase inhibitors on the permeability and ultrastructure of the larval cuticle of the Australian sheep blowfly, Lucilia cuprina

Larvae of Lucilia cuprina, fed toxic levels of α-methyl DOPA (or other DOPA decarboxylase inhibitors) during the first or second instar, die at the completion of the next moult, soon after exposing their new cuticles. In electron micrographs of newly synthesised cuticle from these treated larvae, the ultrastructure of the lipid-rich outer epicuticle layer appears to be abnormal. This newly formed cuticle of the treated larvae is apparently defective in its role as a water permeability barrier (compared with that of normal larvae), since it permits the free movement of water in both directions. Thus, treated larvae die most probably as a direct result of dehydration. Larvae fed toxic levels of α-methyl DOPA can be rescued from death by simultaneously adding N-acetyldopamine (the cuticular sclerotizing agent) to the food. The rescued larvae are apparently normal in all respects. This suggests that sclerotization is required for the formation of a normal outer epicuticle. Diflubenzuron, which is known to inhibit chitin deposition in the cuticles of a number of different species of insect, also apparently affects chitin deposition in the larval cuticle of L. cuprina. Thus, in electron micrographs of cuticle from larvae fed toxic levels of diflubenzuron the ultrastructure of the chitin-containing endocuticle layer appears to be abnormal.     

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 expanding alloscutal cuticle in adults of the argasid tick Argas (Persicargas) robertsi (Acari: Ixodoidea)

The extensible cuticle of Argas (P.) robertsi is tuberculate and deeply folded when the tick is unfed but expands rapidly during feeding. During this expansion the epicuticle becomes less convoluted and the underlying endocuticle stretches but there is no significant alteration in thickness. However, the stretched cuticle has taken on a more open structure. Increase in surface area is restricted to a blister-like expansion because of an inextensible lateral suture which separates the dorsal and ventral surfaces. The cuticle is very hydrophobic, contains 9.9% chitin in the female and 8.9% in the male and the cuticular proteins are largely basic. The cuticle has similar properties to that of the ixodid tick Boophilus microplus but differs from it in fine structure. These differences appear to be related to the time sequence of cuticle synthesis and deposition and to the cycle of expansion and contraction which takes place each time A. (P.) robertsi feeds.