Lepidopteran peritrophic membranes and effects of dietary wheat germ agglutinin on their formation and structure

Peritrophic membrane (PM) structure and the effects of dietary wheat germ agglutinin (WGA) on PM formation were studied in larvae of the European corn borer (ECB), Ostrinia nubilalis, and the tobacco hornworm (THW), Manduca sexta. Growth of ECB was strongly inhibited by low amounts of WGA in the diet (0.05%), whereas THW was not affected by amounts of up to 2%. In ECB larvae, chitin microfibrils were secreted to form an orthogonal network within the apical region of the anterior midgut microvilli. The network then moved to the tips of the microvilli where proteinacious matrix was added prior to delamination of a single PM into the lumen to enclose the food bolus. Multiple PMs rapidly appeared as the food moved posteriorly and some of these became greatly thickened in the middle and posterior regions of the midgut. WGA in the diet caused hypersecretion of unorganized PM in the anterior midgut lumen, disintegration of microvilli, and cessation of feeding. It was also shown to bind to both the chitinous network and to several PM proteins, perhaps causing voids in the PM and sparse matrix material. This allowed the passage of food particles through a defective PM into the ectoperitrophic space and penetration into the microvillar brush border. Stimulation of PM secretion and cessation of feeding may have been a response to damage to the brush border. Unlike ECB, the chitinous network of THW is a randomly organized felt-like structure embedded in a proteinaceous matrix. This PM is secreted as a thin multilayered structure in the anterior region of the midgut, but multiple and thickened PMs occur in the middle and posterior lumens of the midgut. THW tolerated high amounts of WGA in its diet with no disruption of PM formation or inhibition of growth. WGA did accumulate as large masses embedded in the PM, but caused no voids that would allow the penetration of food particles and subsequent damage to the brush border. Therefore, differences in PM formation and structure between ECB and THW appeared to affect how WGA interacts with chitinous and proteinaceous components of the PM and subsequent effects on larval feeding and growth.     

Structure and formation of the peritrophic membrane in the larva of the southern corn rootworm, Diabrotica undecimpunctata

The peritrophic membrane (PM) in larvae of the southern corn rootworm Diabrotica undecimpunctata (Coleoptera:Chrysomelidae) forms along the full length of the midgut epithelium, defining D. undecimpunctata as a Type I insect with respect to PM formation. PM formation occurs in three phases: organization of a continuous lamella of matrix from material secreted into the interstices between the microvilli, maturation and apical movement of the lamella along the microvilli, and shedding of the lamella from the tips of the microvilli into the midgut lumen. Subsequent cycles of synthesis and shedding give rise to multiple, concentric lamellae which surround the food in the gut lumen. PM lamellae are 0.2 mum in profile width and consist of a core of bundles of 5 nm-diameter microfibers encased in a finely-granular homogeneous material. The microfiber bundles are arranged in an orthogonal grid-like array with dimensions consistent with formation around the microvilli. The homogeneous material separates from the PM lamellae to enclose food particles suggesting it may contain digestive enzymes. The PM, microvilli and intracellular vesicles in the midgut epithelium stain intensely with wheat germ agglutinin reflecting the presence and sites of secretion and synthesis of chitin.     

Effect of wheat germ agglutinin on formation and structure of the peritrophic membrane in European corn borer (Ostrinia nubilalis) larvae

European corn borer (ECB; Ostrinia nubilalis (Hubner)) larvae (third instar) fed 0.05% w/w wheat germ agglutinin (WGA) in their diet for 72 h showed very little increase in body weight, whereas weight of control larvae increased nearly fourfold. Light and transmission electron microscopy studies showed that the morphology of the peritrophic membrane (PM) changed within 24 h after ECB larvae fed on the WGA diet. Whereas the PM in the anterior region of the midgut was a thin membranous structure in control larvae, the WGA-fed larvae secreted a multiple-layered and unorganized PM that contained embedded food particles, bacteria, and pieces of disintegrated microvilli. Gold-labeled WGA was localized specifically in the PM and microvilli. The PM of WGA-fed larvae was inundated with dark-staining amorphous structures that, when incubated with anti-WGA, showed heavy WGA localization. The antibody label indicated that most of the ingested WGA was found in the PM, with lesser amounts on the microvillar surface and the least amount within the epithelium. After 72 h, the middle portion of the mesenteron revealed a thin, compact PM in the control larvae, whereas the PM of the WGA-fed larvae was multilayered and discontinuous, which allowed plant cell-wall fragments to penetrate into the microvilli of the epithelium. Scanning electron microscopy of PMs from fifth instar ECB larvae fed the WGA diet revealed a breakdown in the chitinous meshwork by 48 h after initiation of feeding. The endo-PM surface from control larvae was smooth and intact, whereas the PM of WGA-fed larvae showed disintegration of the meshwork and a reduced proteinaceous matrix. This allowed bacteria and food particles to penetrate through the PM into the ectoperitrophic space and directly contact the microvilli. Therefore, WGA, a protein inhibitor of larval growth, interferes with the formation and integrity of the PM, which exposes the brush border to ingested material. This, in turn, appears to stimulate secretion of additional PM layers, the concomitant disintegration of the microvilli, and cessation of feeding.     

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.     

Isolation and characterization of an endodermally derived, proteoglycan-like extracellular matrix molecule that may be involved in larval starfish digestive tract morphogenesis

A monoclonal antibody, anti-Pisaster matrix-1 (anti-PM1) has been developed against an extracellular matrix antigen, Pisaster matrix-1 (PM1) found in embryos and larvae of the starfish Pisaster ochraceus . Pisaster matrix-1 was first observed in endodermal cells of the early gastrula, and shortly thereafter it was secreted into the blastocoel where it accumulated steadily during gastrulation. During the late gastrula stage it also appeared in the extracellular matrix (ECM) of the gut lumen. Immunogold electron microscopy with anti-PM1 revealed that PM1 was found in condensations of ECM associated with blastocoel matrix fibers, in the trans Golgi network, in Golgi-associated vesicles in endoderm and mesenchyme cells and throughout the ECM lining the digestive tract of late gastrula and bipinnaria larvae. When blastula or early gastrula stage embryos were grown in the presence of the PM1 antibody, archenteron elongation, bending and mouth formation failed to occur. Pisaster matrix-1 stained with alcian blue and its assembly could be disrupted with the common inhibitor of O-linked glycosaminoglycan assembly, β-xyloside but not by tunicamycin. It was not sensitive to enzymes that degrade vertebrate proteoglycans. Pisaster matrix-1 is a large (600 kDa) proteoglycan-like glycosaminoglycan, secreted exclusively by endodermal and/or endodermally derived cells that may be necessary for morphogenesis of the mouth and digestive tract of Pisaster ochraceus embryos/larvae.     

The peritrophic matrix limits the rate of digestion in adult Anopheles stephensi and Aedes aegypti mosquitoes

The peritrophic matrix (PM) is a chitin-containing acellular sheath that surrounds the blood meal and separates the food bolus from the midgut epithelium. Intense molecular traffic through the PM occurs during digestion. Digestive enzymes secreted by the midgut epithelium must traverse the PM to reach their substrates in the food bolus, and digestion products must cross the PM in the opposite direction to be absorbed by the epithelial cells. Here we report that the PM limits the rate of digestion. PM disruption by two independent means (chitinase and anti-PM antibodies) consistently increases the rate of blood digestion. The significance of these results in relation to PM function is discussed.     

Identification of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism

The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography‐tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and α‐amylase) or other reactions (β‐1,3‐glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a heterogenous group consisting of polycalin, REPATs, serpin, C‐Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase‐2 (McCHS‐2), chitinase (McCHI), and β‐N‐acetylglucosaminidase (McNAG) enzymes, involved in PM chitin metabolism, were also identified. McCHS‐2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS‐2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.     

Calcofluor disrupts the midgut defense system in insects

The insect midgut is generally lined with a unique protective chitin/protein structure, the peritrophic membrane (PM). We demonstrated that in Trichoplusia ni larvae, the majority of PM proteins were assembled with chitin as a consequence of their chitin binding properties. These proteins could be dissociated from the PM in vitro by Calcofluor, a well-known chemical with chitin binding properties. The chitin binding characteristics of PM proteins were confirmed by their high affinity binding in vitro to regenerated chitin. In vivo assays demonstrated that Calcofluor could inhibit PM formation in five lepidopteran insects tested. The inhibition of T. ni PM formation by Calcofluor, was accompanied by increased larval susceptibility to baculovirus infection. Continuous inhibition of PM formation by Calcofluor resulted in retarded larval development and mortality. The destructive effect of Calcofluor on PM formation was demonstrated to be transient and reversible depending on the presence of Calcofluor within the midgut. In addition, degradation of the insect intestinal mucin was observed concurrently with the inhibition of PM formation by Calcofluor. Our studies revealed a potential novel approach to develop strategies for insect control by utilizing chitin binding molecules to specifically target PM formation in a broad range of insect pest species.     

A naturally occurring plant cysteine protease possesses remarkable toxicity against insect pests and synergizes Bacillus thuringiensis toxin

When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitating the movement of other insecticidal proteins in the diet to the midgut microvilli and thereby enhancing their toxicity. To directly determine the toxicity of Mir1-CP, the purified recombinant enzyme was directly tested against four economically significant Lepidopteran pests in bioassays. Mir1-CP LC(50) values were 1.8, 3.6, 0.6, and 8.0 ppm for corn earworm, tobacco budworm, fall armyworm and southwestern corn borer, respectively. These values were the same order of magnitude as those determined for the Bacillus thuringiensis toxin Bt-CryIIA. In addition to being directly toxic to the larvae, 60 ppb Mir1-CP synergized sublethal concentrations of Bt-CryIIA in all four species. Permeabilization of the PM by Mir1-CP probably provides ready access to Bt-binding sites on the midgut microvilli and increases its activity. Consequently, Mir1-CP could be used for controlling caterpillar pests in maize using non-transgenic approaches and potentially could be used in other crops either singly or in combination with Bt-toxins.     

The role of peritrophic membrane in the resistance of Anticarsia gemmatalis larvae (Lepidoptera: Noctuidae) during the infection by its nucleopolyhedrovirus (AgMNPV)

The aim of this study was to analyze morphologically the peritrophic membrane (PM) of Anticarsia gemmatalis larvae resistant (RL) and non-resistant (susceptible) (SL) to the A. gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV), in the presence of viral infection. Also, in this investigation the results between SL and RL were compared to improve the understanding of the resistance mechanisms to the virus. The PM of SL of A. gemmatalis was less efficient as a barrier against the viral infection since it was found to be more fragile than the PM of RL. The lower chitin content as seen from weaker fluorescent staining in SL as well as the abundance of non-solubilized vesicular materials in the ectoperitrophic space, would cause the malformation of this membrane, facilitating the passage of the virus toward the epithelium of the midgut. On the other hand, in RL, the intensity of WGA (wheat germ agglutinin)-conjugated FITC (fluorescein) reaction of the PM was greater than in SL, making this insect more resistant to infection. We can conclude that the effectiveness of the PM in protecting against pathogens is dependent on the integrity of the epithelial cells of the midgut and of the structural preservation of the PM, being directly implicated in the resistance of A. gemmatalis larvae to AgMNPV.     

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.     

Modeling the structure of the type I peritrophic matrix: characterization of a Mamestra configurata intestinal mucin and a novel peritrophin containing 19 chitin binding domains

Twelve to fourteen integral proteins were found to reside in the Type I peritrophic matrix (PM) of Mamestra configurata (bertha armyworm) larvae. Several methods were employed, including de novo peptide sequencing, the generation of a midgut-specific EST database and immunological screening, which led to the isolation of cDNAs encoding two integral PM proteins. McPM1, the largest PM protein described to date at 202 kDa, was comprised of a concatamer of 19 chitin binding domains (CBD), 12 of which resided within a central repetitive region consisting of six iterations of a two CBD module. The protein was found to reside within the PM primarily as several lower molecular weight, presumably proteolytically processed, forms. McMUC1 was similar in structure to other insect intestinal mucins (IIM) and was highly glycosylated. The expression of both proteins was restricted to the larval midgut. Lower molecular weight proteins that may represent non- and partially glycosylated forms of McMUC1 were also recognized by an anti-McMUC1 antiserum. These were preferentially degraded upon ingestion of M. configurata multi-capsid nucleopolyhedrovirus by larvae, possibly by a viral-encoded metalloprotease. A molecular model of PM structure is presented featuring the interaction of McPM1 with chitin inter-fibril junctions and McMUC1 with the extended chains in the internodal regions. The potential for interaction between PM proteins via intermolecular disulfide bond formation and through association of CBD with N-linked glycans is discussed.     

The peritrophic membrane of Spodoptera frugiperda: secretion of peritrophins and role in immobilization and recycling digestive enzymes

A peritrophin from the Spodoptera frugiperda peritrophic membrane (PM) and microvillar proteins from S. frugiperda anterior midgut cells were isolated and used to raise antibodies in a rabbit. These antibodies, as well as a Tenebrio molitor amylase antibody that cross-reacts with S. frugiperda amylases, and wheat-germ aglutinin were used in immunolocalization experiments performed with the aid of confocal fluorescence and immunogold techniques. The results showed that the peritrophin was secreted by anterior midgut columnar cells in vesicles pinched-off the microvilli (microapocrine secretion). The resulting double membrane vesicles become single membrane vesicles by membrane fusion, releasing peritrophin and part of the amylase and trypsin. The remaining membranes still containing microvillar proteins and membrane-bound amylase and trypsin are incorporated into a jelly-like material associated with PM. Calcofluor-treated larvae lacking a PM were shown to lose the decreasing gradient of trypsin and chymotrypsin observed along the midgut of control larvae. This gradient is thought to be formed by a countercurrent flux of fluid (in the space between PM and midgut cells) that powers enzyme recycling.     

Sequences of cDNAs and expression of genes encoding chitin synthase and chitinase in the midgut of Spodoptera frugiperda

The focus of this study was on the characterization and expression of genes encoding enzymes responsible for the synthesis and degradation of chitin, chitin synthase (SfCHSB) and chitinase (SfCHI), respectively, in the midgut of the fall armyworm, Spodoptera frugiperda. Sequences of cDNAs for SfCHSB and SfCHI were determined by amplification of overlapping PCR fragments and the expression patterns of these two genes were analyzed during insect development by RT-PCR. SfCHSB encodes a protein of 1523 amino acids containing several transmembrane segments, whereas SfCHI encodes a protein of 555 amino acids composed of a catalytic domain, a linker region and a chitin-binding domain. SfCHSB is expressed in the midgut during the feeding stages, whereas SfCHI is expressed during the wandering and pupal stages. Both genes are expressed along the whole midgut. Chitin staining revealed that this polysaccharide is present in the peritrophic membrane (PM) only when SfCHSB is expressed. There is little or no chitin in the midgut when SfCHI is expressed. These results support the hypothesis that SfCHSB is responsible for PM chitin synthesis during the larval feeding stages and SfCHI carries out PM chitin degradation during larval-pupal molting, suggesting mutually exclusive temporal patterns of expression of these genes.     

Insect resistance of transgenic tobacco expressing an insect chitinase gene

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control     

Formation,properties and degradation of the peritrophic membranes of larval and adult fleshflies,Parasarcophaga argyrostoma (Insecta,Diptera)

Summary Parasarcophaga argyrostoma larvae continuously secrete a single, tube-like peritrophic membrane (PM), which has an electron-dense layer on the lumen side and a thicker chitin-containing electron-lucent part on the epithelium side. In the adult fleshfly, the secretion of PMs starts immediately after emergence. The initial part of the PMs is twisted and tight. The formation zone is folded with two separate secretory pads in which two tube-like PMs are formed continuously. The PMs are different, morphologically and with respect to their peripheral carbohydrate residues. The latter could be demonstrated with lectin gold conjugates. PM 1 consists of an electron-dense, chitin-free layer on the lumen side and a thicker part which contains chitin microfibrils in the matrix. PM 2 appears fluffy and has chitin microfibrils in its matrix, too. Chitin could be localized with WGA gold. Incubation of isolated PM 1 with lectin gold resulted in a peculiar pattern of bound lectins and gaps on the electron dense layer which otherwise appeared to be homogenous. Degradation of peritrophic membranes takes place in the hindgut. The cuticle of the anterior hindgut is studded with small teeth, which seem to be responsible for mechanical degradation of the peritrophic membranes into frayed pieces. This may be completed by the teeth on the rectal pads. From the appearance of the remnants of the peritrophic membranes it can be inferred that chemical degradation takes place in the hindgut.Supported by the Deutsche Forschungsgemeinschaft     

Digestive enzyme compartmentalization and recycling and sites of absorption and secretion along the midgut of Dermestes maculatus (Coleoptera) larvae

Bostrichiformia is the less known major series of Coleoptera regarding digestive physiology. The midgut of Dermestes maculatus has a cylindrical ventriculus with anterior caeca. There is no cell differentiation along the ventriculus, except for the predominance of cells undergoing apocrine secretion in the anterior region. Apocrine secretion affects a larger extension and a greater number of cells in caeca than in ventriculus. Ventricular cells putatively secrete digestive enzymes, whereas caecal cells are supposed to secrete peritrophic gel (PG) glycoproteins. Feeding larvae with dyes showed that caeca are water-absorbing, whereas the posterior ventriculus is water-secreting. Midgut dissection revealed a PG and a peritrophic membrane (PM) covering the contents in anterior and posterior ventriculus, respectively. This was confirmed by in situ chitin detection with FITC-WGA conjugates. Ion-exchange chromatography of midgut homogenates, associated with enzymatic assays with natural and synthetic substrates and specific inhibitors, showed that trypsin and chymotrypsin are the major proteinases, cysteine proteinase is absent, and aspartic proteinase probably is negligible. Amylase and trypsin occur in contents and decrease along the ventriculus; the contrary is true for cell-membrane-bound aminopeptidase. Maltase is cell-membrane-bound and predominates in anterior and middle midgut. Digestive enzyme activities in hindgut are negligible. This, together with dye data, indicates that enzymes are recovered from inside PM by a posterior-anterior flux of fluid outside PM before being excreted. The combined results suggest that protein digestion starts in anterior midgut and ends in the surface of posterior midgut cells. All glycogen digestion takes place in anterior midgut.