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.     

A baculovirus enhancin alters the permeability of a mucosal midgut peritrophic matrix from lepidopteran larvae

The peritrophic matrix (PM) in lepidopterous larvae may function as a defensive barrier against ingested viral pathogens. PMs isolated from Trichoplusia ni and Pseudaletia unipuncta larvae, were treated with a baculovirus-encoded metalloprotease (enhancin) from Trichoplusia ni granulosis virus (TnGV) and their in vitro permeability to blue dextran and fluorescent-labelled Autographa californica nuclear polyhedrosis virus (AcMNPV) was determined using a dual chamber permeability apparatus. Incubation of T. ni PMs with 0.0, 0.5, 1.0, and 2.0mg/ml enhancin resulted in a blue dextran 2000 flux of 4.4, 6.3, 9.9, and 15.6&mgr;g/mm(2)/h, respectively. In addition, T. ni PMs treated with enhancin were found to be significantly more permeable to fluorescent-labelled AcMNPV than non-treated control PMs. The permeability of T. ni PMs treated with 3.0mg/ml enhancin was 0.017 cumulative percent crossing/mm(2)/h, whereas the permeability of the control PM was below the detectable limit. Similarly, enhancin treatment greatly increased the permeability of P. unipuncta PMs to AcMNPV. These results provide evidence that the PM from two lepidopteran species can block the passage of baculovirions across this matrix thus reducing the probability of larval infection. Furthermore, these results support the hypothesis that enhancin facilitates NPV infection of larvae by altering the permeability of the PM.     

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.     

A novel chitin-binding protein identified from the peritrophic membrane of the cabbage looper, Trichoplusia ni

A novel midgut peritrophic membrane (PM) protein, TnPM-P42, was identified from the cabbage looper, Trichoplusia ni. TnPM-P42 was shown as a 42kDa protein by SDS-PAGE analysis and appeared to be associated with the PM throughout its entire length. In T. ni larvae, the midgut is the only tissue where TnPM-P42 could be detected during the feeding period of the larvae. TnPM-P42 has chitin-binding activity and is strongly associated with the PM, which is similar to the currently known peritrophin type PM proteins. However, TnPM-P42 represents a unique family of proteins distinctly different from the peritrophin type PM proteins in its sequence characteristics. TnPM-P42 does not contain the peritrophin domain which is present in all the currently known PM proteins, but instead has a chitin deacetylase-like domain. Sequence similarity search of the GenBank database did not result in identification of any known proteins with a significant overall sequence similarity to the TnPM-P42. However, expressed sequence tags (ESTs) from various arthropods were identified to code for proteins with high sequence similarities to TnPM-P42, indicating the presence of TnPM-P42 homologs in other arthropods. Consistent with the identification of various ESTs from arthropods, Western blot analysis demonstrated the presence of a TnPM-P42-like protein in the PMs from Heliothis virescens and Helicoverpa zea larvae. The sequence characteristics of TnPM-P42 indicate that TnPM-P42 represents a novel family of insect proteins. However, its biochemical and physiological functions require further investigation.     

Molecular structure of the peritrophic membrane (PM): identification of potential PM target sites for insect control

Peritrophic membranes (PMs) are an invertebrate-unique structure that lines the digestive tract, playing important roles in facilitating food digestion and providing protection to the gut epithelium. The importance of PMs in insects has been recognized ever since its presence was identified 200 years ago. In the last 5 years, significant progress towards understanding the PM molecular structure and the mechanism for PM formation has been made. Recent studies on Type 1 PMs from lepidopteran larvae have suggested a model for the PM molecular structure and formation. The important physiological functions of the PM suggest that PMs can be a significant structural target for insect control and the current understanding of the structure of lepidopteran larval PMs has provided us with potential opportunities for targeting the PM by various mechanisms.     

Dynamic role of microvilli in peritrophic membrane formation

Although the peritrophic membrane (PM) is a common extracellular construction in many invertebrate groups, evidence of the location of its secretion has never been reported. In this study a specific marker for chitin has been developed, enabling a separate examination of secretion of the chitinous and proteinaceous components of the PM in the millipede, Glomeris marginata. Chitin appears first at the base of the microvilli (MV), synchronized in adjacent cells along the entire length of the midgut. Evidence showing that it originates at the plasma membrane is discussed. Proteinaceous components appear to be added from the MV to the chitinous sheet as it moves along the MV toward the lumen. Precedence for such a dynamic role for MV in formation of extracellular structures is reviewed. The completed PM extends around individual items in the gut contents as well as forming a multilayered envelope; this may enhance both its digestive and protective functions.     

Roles of peritrophic membranes in protecting herbivorous insects from ingested plant allelochemicals

Four mechanisms by which peritrophic membranes (PMs) potentially protect herbivorous insects from ingested allelochemicals are reviewed: adsorption, ultrafiltration, polyanion exclusion, and the capacity of PMs to act as antioxidants. Most of the research on the protective roles of PMs against ingested allelochemicals has focused on their impermeability to tannins. Adsorption of tannins by the PMs in grasshoppers may limit their permeability, but ultrafiltration of tannin complexes in the caeca is an alternative explanation. Polyanion exclusion does not explain the impermeability of caterpillar PMs to tannins (polyphenolate anions). Ultrafiltration remains the most likely mechanism by which tannins, and other tested allelochemicals, are retained in the endoperitrophic space. Although the pores in PMs are too large to impede the passage of most free allelochemicals, large allelochemical complexes are retained. Such complexes form in the gut fluid of caterpillars between tannic acid, proteins, lipids, and polyvalent metal cations, and also in the gut fluid of grasshoppers (Melanoplus sanguinipes) between some amphiphilic allelochemicals (digitoxin) and surfactant micelles. Further work is needed to examine the role of PMs as antioxidants in vivo, such as their potential to bind catalytically-active metal ions.     

Plant-mediated alteration of the peritrophic matrix and baculovirus infection in lepidopteran larvae

The peritrophic matrix (PM) lines the midgut of most insects, providing protection to the midgut epithelial cells while permitting passage of nutrients and water. Herein, we provide evidence that plant-mediated alteration of the PM contributes to the well-documented inhibition of fatal infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) of Heliothis virescens F. larvae fed cotton foliage. We examined the impact of the PM on pathogenesis using a viral construct expressing a reporter gene (AcMNPV-hsp70/lacZ) orally inoculated into larvae with either intact PMs or PMs disrupted by Trichoplusia ni granulovirus occlusion bodies containing enhancin, known to degrade insect intestinal mucin. Larvae possessing disrupted PMs displayed infection foci (lacZ signaling) earlier than those with intact PMs. We then examined PMs from larvae fed artificial diet or plant foliage using electron microscopy; foliage-fed larvae had significantly thicker PMs than diet-fed larvae. Moreover, mean PM width was inversely related to both the proportion of larvae with lacZ signaling at 18 h post-inoculation and the final percentage mortality from virus. Thus, feeding on foliage altered PM structure, and these foliage-mediated changes reduced baculoviral efficacy. These data indicate that the PM is an important factor determining the success of an ingested pathogen in foliage-fed lepidopteran larvae.     

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     

Antioxidant defense of the midgut epithelium by the peritrophic envelope in caterpillars

The peritrophic envelope (PE) is an extracellular matrix that is secreted by the midgut epithelium in most arthropods. In addition to protecting the midgut epithelium from abrasive food particles and microbial pathogens, in vitro experiments have suggested that the PE functions as a radical-scavenging antioxidant in caterpillars. This study tested the hypothesis that the PE is a "sacrificial antioxidant" in vivo in caterpillars. As a sacrificial antioxidant, the PE would (1) bind catalytic metal ions, (2) become oxidized itself, and (3) protect the midgut epithelium from oxidative damage. Each of these functions was supported by our results: the PE in Malacosoma disstria adsorbed increased amounts of iron as the concentration of iron was increased in its diet. Iron adsorption by the PE helped protect the midgut epithelium of M. disstria from oxidative damage over a wide range of ingested iron concentrations. Secondly, while the midgut epithelium was protected, protein oxidation in the PE increased 108% when tannic acid was oxidized in the endoperitrophic space. Finally, when the formation of the PE was inhibited by Calcofluor, protein carbonyls in the midgut epithelia of M. disstria and Orgyia leucostigma increased by two- to threefold. We conclude that the PE functions as an effective iron-binding and radical-scavenging antioxidant that protects the midgut epithelia of caterpillars.     

围食膜: 害虫生物防治的潜在靶标

围食膜是昆虫中肠细胞分泌的一层特有的非细胞结构,由蛋白质、粘多糖和几丁质组成,是昆虫中肠细胞抵御随食物摄入的病原微生物入侵的第一道天然屏障。昆虫病毒增效蛋白、几丁质酶、荧光增白剂和外源凝集素等生物防治促进因子通过与围食膜上特异位点的结合,可破坏围食膜结构,改变其通透性,促进病原微生物对害虫的感染。该文综述了与昆虫围食膜密切相关的生防促进因子的增效活性及其作用机理,阐明了以围食膜为害虫生物防治靶标的应用前景。     

Obstructor-A is required for epithelial extracellular matrix dynamics, exoskeleton function, and tubulogenesis

The epidermis and internal tubular organs, such as gut and lungs, are exposed to a hostile environment. They form an extracellular matrix to provide epithelial integrity and to prevent contact with pathogens and toxins. In arthropods, the cuticle protects, shapes, and enables the functioning of organs. During development, cuticle matrix is shielded from premature degradation; however, underlying molecular mechanisms are poorly understood. Previously, we identified the conserved obstructor multigene-family, which encodes chitin-binding proteins. Here we show that Obstructor-A is required for extracellular matrix dynamics in cuticle forming organs. Loss of obstructor-A causes severe defects during cuticle molting, wound protection, tube expansion and larval growth control. We found that Obstructor-A interacts and forms a core complex with the polysaccharide chitin, the cuticle modifier Knickkopf and the chitin deacetylase Serpentine. Knickkopf protects chitin from chitinase-dependent degradation and deacetylase enzymes ensure extracellular matrix maturation. We provide evidence that Obstructor-A is required to control the presence of Knickkopf and Serpentine in the extracellular matrix. We propose a model suggesting that Obstructor-A coordinates the core complex for extracellular matrix protection from premature degradation. This mechanism enables exoskeletal molting, tube expansion, and epithelial integrity. The evolutionary conservation suggests a common role of Obstructor-A and homologs in coordinating extracellular matrix protection in epithelial tissues of chitinous invertebrates.     

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.     

Efficient preparation and site‐directed immobilization of VHH antibodies by genetic fusion of poly(methylmethacrylate)‐binding peptide (PMMA‐Tag)

A PMMA‐binding peptide (PMMA‐tag) was genetically fused with the C‐terminal region of an anti‐human chorionic gonadotropin (hCG) single‐domain antibody (VHH). It was over‐expressed in an insoluble fraction of E. coli cells, and recovered in the presence of 8 M urea via one‐step IMAC purification. Monomeric and denatured PMMA‐tag‐fused VHH (VHH‐PM) was successfully prepared via the reduction and oxidation of VHH‐PM at a concentration less than 1 mg/mL in the presence of 8 M of urea. Furthermore, the VHH‐PM was refolded with a recovery of more than 95% by dialysis against 50 mM TAPS at pH 8.5, because the genetic fusion of PMMA‐tag resulted in a decrease in the apparent isoelectric point (pI) of the fusion protein, and its solubility at weak alkaline pH was considerably increased. The antigen‐binding activities of VHH‐PM in the adsorptive state were 10‐fold higher than that of VHH without a PMMA‐tag. The density of VHH‐PM on a PMMA plate was twice that of VHH, indicating that the site‐directed attachment of a PMMA‐tag resulted in positive effects to the adsorption amount as well as to the orientation of VHH‐PM in its adsorptive state. The preparation and immobilization methods for VHH‐PM against hCG developed in the present study were further applied to VHH‐PMs against four different antigens, and consequently, those antigens with the concentrations lower than 1 ng/mL were detected by the sandwich ELISA. Thus, the VHH‐PMs developed in the present study are useful for preparation of high‐performance and economical immunosorbent for detection of biomarkers. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1563–1570, 2015     

昆虫体内的天然屏障——围食膜

昆虫围食膜是由昆虫中肠上皮细胞分泌的非细胞薄膜状结构,主要成份是几丁质、蛋白质和多糖,是昆虫抵御外界侵害的第一道天然屏障,能够保护中肠上皮细胞不受机械损伤并且能够抵御病毒、细菌及其他有害物质,防止化学损伤.昆虫病毒增效蛋白、荧光增白剂和几丁质酶等生物防治促进因子通过与围食膜上特异位点的结合,能够破坏围食膜结构,加速病原微生物对害虫的感染进程.就围食膜组分、结构、功能以及与害虫防治的关系等方面的研究进展进行综述,并且论述了以围食膜为害虫生物防治靶标的应用前景.     

Keeping good friends close – The surface and secreted proteomes of a probiotic bacterium provide candidate proteins for intestinal attachment and communication with the host

Bacteria use cell surface proteins and secreted proteins to interact with host tissues. Several dozen previously published proteomics studies have identified cell surface proteins for pathogens. In this issue, Celebioglu and Svensson (Proteomics 2017, 17, 1700019) use 2D gel electrophoresis and mass spectrometry to identify secreted and cell surface proteins of a commensual gut bacterium, Lactobacillus acidophilus NCFM. Some of the proteins are known to have functions in the cytoplasm, and their presence on the cell surface suggests they might be moonlighting proteins. In addition, comparisons of proteins used by pathogenic and probiotic species to interact with their hosts could lead to improved treatments of infections and chronic diseases that are associated with an imbalance of pathogenic and probiotic gut bacteria.     

Penetration of the peritrophic membrane of the tick by Babesia microti

Summary A peritrophic membrane (PM) has been demonstrated in the gut of feeding larvae, nymphs, and adults of the tick Ixodes dammini. This is the first report of a PM in ticks. This temporary structure divides the lumen of the gut into two compartments, an endoperitrophic space, the lumen proper, and an ectoperitrophic space located between the PM and the epithelial cells of the gut wall. The PM is a mechanical barrier and even such small particles as ribosomes derived from ingested reticulocytes are retained in the lumen proper; they are never found in the ectoperitrophic compartment. In Ixodes dammini fed on hamsters infected with Babesia microti some of the parasites are found in the ectoperitrophic space. This passage is accomplished by a highly specialized organelle, the arrowhead, which develops in some Babesia during their metamorphosis in the gut of the vector. The arrowhead, while passing through the PM, changes its fine structure and loses its internal organization as if releasing some of its contents. Its disintegration continues and it disappears shortly after the Babesia have entered the epithelial cells. Only Babesia equipped with the arrowhead structure are able to cross the PM. This is the first documented case of a parasite traversing a solidified PM.Supported by Research Grant AI-15886 from the United States Public Health ServiceIt is a great pleasure to acknowledge the excellent technical assistance of Mrs. Ellen Lanners. Special thanks are extended to Dr. Alex S. Raikhel for the courtesy of translating from Russian to English his Chapter 3, Intestine, from the book, An Atlas of Electron Microscopical Anatomy of Ixodid Ticks. We are indebted to Mrs. Liubov Lyandvert for the translation from Russian of selected other parts of the same book     

Fate of bacteria, Aeromonas caviae, in the midgut of the housefly, Musca domestica

Abstract. The role of houseflies as agents in the spread of bacterial diseases has been thoroughly investigated, yet the fate of bacteria ingested by flies has not. We examined the physical location of the bacterial enteropathogen Aeromonas caviae in the midgut of laboratory‐reared adult houseflies. Food ingested by houseflies was separated from the midgut epithelium by a double‐layered peritrophic matrix (PM). The inner PM intimately enveloped the food as fecal pellets (food boluses), while the outer PM appeared as a long continuous tube. In flies fed a suspension of A. caviae, live bacteria were not observed within the inner PM, but were compartmentalized between folds of the PM in the inter‐PM space. Similar observations were made for flies fed a suspension of Serratia liquefaciens and for highly contaminated feral flies. Isolates of both A. caviae and S. liquefaciens were chitinolytic (as demonstrated by clearing zones on chitin agar), but the potential role of bacterial enzymes in the alteration of PM morphology or formation needs further investigation.