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.     

Peritrophic membrane structure and secretion in European corn borer larvae (Ostrinia nubilalis)

Peritrophic membrane (PM) secretion and formation occur primarily in the anterior region of the mesenteron in the European corn borer (Ostrinia nubilalis) as determined by light and electron microscopy. Nascent PM first became visible as fibrous linear chitin-containing structures stained with gold-labeled wheat germ agglutinin between and at the tips of the microvilli. No formed PM was visible at the foregut-midgut junction, but a thin single PM appeared first in the lumen between the stomodeal valves and the midgut epithelium. Just posterior to the stomodeal valves, multiple PMs were observed that became progressively thicker and more numerous in the mid and posterior regions of the mesenteron. The PM consists of an orthogonal chitin meshwork with openings slightly larger than the diameters of the microvilli. As it delaminates from the microvilli, the meshwork becomes embedded in proteinaceous matrix that greatly reduces the pore size of the PM.     

Normal and cocoon-forming peritrophic membrane in larvae of the beetle Gibbium psylloides

Larvae of Gibbium psylloides secrete a peritrophic membrane (PM) which has a mainly orthogonal fibrillar structure, but this merges freely with hexagonal and random arrangements of microfibres. The random arrangement predominates in PM used for cocoons. Shortly before the cocoon is constructed the PM no longer forms a tube but collapses and is compressed in the gut and emerges from the anus as a flat thread. This thread is wound around the larva and forms the cocoon ‘silk’. Both normal and cocoon-forming PMs are produced mainly in the posterior midgut and their constituent microfibres appear first at the tips of the microvilli which form the brush border of the midgut cells.     

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.     

Peritrophic membrane structure and formation of larvalTrichoplusia niwith an investigation on the secretion patterns of a PM mucin

Peritrophic membrane or matrix (PM) secretion and formation patterns were examined in the cabbage looper larvae (Trichoplusia ni[Hubner]) by transmission and scanning electron microscopy (SEM). PM first became visible in the lumen between tips of the microvilli and the stomodeal valves as a single layered fibrous structure that became more compact in appearance in the middle and posterior mesenteron. In the anterior mesenteron, nascent PM was visible within the brush border as a fibrous linear structure that contained both the major PM matrix protein, invertebrate intestinal mucin (IIM) and chitin-containing structures. Even though delamination events were confined to the anterior mesenteron, IIM was secreted by columnar epithelial cells throughout the length of the mesenteron. SEM of the midgut epithelium revealed PM covering individual epithelial cells.     

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.     

Peritrophic membrane structure and formation in the larva of a moth, Heliothis

The peritrophic membrane (PM) in tobacco budworm larvae (Heliothis virescens, Lepidoptera: Noctuidae), is a continuous sac which encloses the food bolus in the midgut and hindgut. The PM is a single-walled structure 3-5 mum thick which is comprised of two main layers or laminae. The laminae may be fused into a single structure or remain separated by a space which may contain additional thin strands of matrix. Staining with an anti-PM antibody and wheat germ agglutinin (WGA) illustrate the laminar nature of the PM and suggest that protein and chitin have co-incident spatial distributions within the matrix. By transmission electron microscopy, the PM is composed of a loose network of fibrils and small granules, the only structural difference among laminae being a compaction of the matrix along the edges of the two limiting laminae facing the endoperitrophic and ectoperitrophic spaces. By scanning electron microscopy, the PM surface has a wrinkled, felt-like texture without pores or slits. Contrary to the classical view that lepidopterans are Type I insects with respect to PM formation in which the PM forms along the full length of the midgut, the PM in the tobacco budworm forms primarily from secretions of specialized midgut epithelial cells at the junction of the foregut and midgut. The secretory cells, their secretions and the nascent PM stain intensely with the anti-PM antibody but not with WGA suggesting that chitin is added more posteriorly. The PM may be supplemented by the addition of minor amounts of matrix material along the length of the midgut. PM synthesis begins during embryogenesis prior to the initiation of feeding. The PM in neonates is only about 0.1 mum thick but otherwise is structurally similar to that in older larvae.     

The ultrastructure of the midgut and the formation of peritrophic membranes in a harvestman,Phalangium opilio (Chelicerata Phalangida)

Summary The ultrastructure of the midgut epithelium of Phalangium opilio was examined. In the anterior part of the midgut the epithelium consists of three different types of cells, called resorption, digestion, and excretion cells according to their presumed functions. Excretion cells may represent old digestion cells. The relation between resorption and digestion cells needs further investigation. The epithelium of the posterior part of the midgut consists of two types, transport and secretion cells, which seem to serve mainly for the resorption of water and the secretion of peritrophic membranes, respectively.Peritrophic membranes are secreted by the anterior midgut epithelium mainly in a period between 2 and 4 h after feeding. Chitin or chitin precursors could be localized in vesicles and in the brush border of midgut cells, and in the peritrophic membranes, using colloidal gold labelled with wheat germ agglutinin. Two different textures of chitin-containing microfibrils were found in the peritrophic membranes, either a random or a hexagonal texture. The latter results if the microfibrils polymerize between the basal parts of the microvilli. Irregularities of the hexagonal texture can be correlated with an irregular pattern of the microvilli. In the posterior midgut peritrophic membranes with a random texture, chitin-containing microfibrils are continuously secreted in the form of patches.     

The fine structure of the digestive system of Daphnia pulex (Crustacea: Cladocera).

The alimentary canal of Daphnia pulex consists of a tube-shaped foregut, a midgut (mesenteron) with an anterior pair of small diverticula, and a short hindgut. The foregut and hindgut are structurally similar. Each is formed by a low cuboidal epithelium 5 mum tall and lined with a chitinous intima. The midgut wall consists of a simple epithelium resting on a thick beaded basal lamina which is surrounded by a spiraling muscularis. Anteriorly the midgut cells are columnar in shape being 30 mum in height each having a basal nucleus, anteriorly concentrated mitochondria and in apical border of long thin microvilli. Posteriorly the midgut cells become progressively shorter so that in the posteriormost region of the midgut the cells are 5 mum tall and cuboidal in shape. The microvilli concomitantly become shorter and thicker. All mesenteron cells contain the usual cytoplasmic organelles. The paired digestive diverticula are simple evaginations of the midgut. The wall of each consists of a simple epithelium of cuboidal cells 25 mum in height, each with a brushed border of long thin microvilli. Enzyme secretion appears to be holocrine in mode and not confined to any one region of the mesenteron though definitely polarized anteriorly. The thin gut muscularis encircles the entire length of the midgut and caeca. Thick and thin filaments appear to be in a 6:1 ratio.     

The peritrophic membrane of the gastropod Megathura crenulata: structure,composition, and site of formation

Peritrophic membranes (PMs) are acellular layered structures secreted around ingested materials by the gut epithelium. Most studies on PMs have focused on those of insects and crustaceans due to their potential ability to block the movement of pathogens from ingested materials into the body, and their possible use as unique targets relevant to pest management. While PMs are known to occur in other taxa, their distribution is spotty and little is known about their role in these other species. The gastropod Megathura crenulata produces a true PM, which has a chitinous matrix that makes up nearly half its wet weight. Unlike arthropod PMs, which are released by delamination from the microvilli of their gut cells, the chitinous matrix of the M. crenulata PM is secreted from epithelial cells lining most regions of its gut. Although its mode of synthesis is unique, it may serve the same functions as proposed for other PMs, including regulating diffusion, binding metabolites, restricting protease activity, blocking pathogens, and providing lubrication. In arthropods, numerous proteins with chitin‐binding specificities have been identified, consistent with the proposed functions. Analysis of PMs in M. crenulata showed several integral proteins associated with the membrane, suggesting that the PM in this mollusc may be involved in complex functions like those seen in the arthropods.     

Regulation of chitin synthesis in the larval midgut of Manduca sexta

In insects, chitin is not only synthesized by ectodermal cells that form chitinous cuticles, but also by endodermal cells of the midgut that secrete a chitinous peritrophic matrix. Using anti-chitin synthase (CHS) antibodies, we previously demonstrated that in the midgut of Manduca sexta, CHS is expressed by two cell types, tracheal cells forming a basal tracheal network and columnar cells forming the apical brush border [Zimoch and Merzendorfer, 2002, Cell Tissue Res. 308, 287-297]. Now, we show that two different genes, MsCHS1 and MsCHS2, encode CHSs of midgut tracheae and columnar cells, respectively. To investigate MsCHS2 expression and activity in the course of the larval development, we monitored chitin synthesis, enzyme levels as well as mRNA amounts. All of the tested parameters were significantly reduced during molting and in the wandering stage when compared to the values obtained from intermolt feeding larvae. By contrast, MsCHS1 appeared to be inversely regulated because its mRNA was detectable only during the molt at the time when tracheal growth occurs at the basal site of the midgut. To further examine midgut chitin synthesis, we measured enzyme activity in crude midgut extracts and different membrane fractions. When we analysed trypsin-mediated proteolytic activation, a phenomenon previously reported for insect and fungal systems, we recognized that midgut chitin synthesis was only activated in crude extracts, but not in the 12,000 g membrane fraction. However, proteolytic activation by trypsin in the 12,000 g membrane fraction could be reconstituted by re-adding a soluble fraction, indicating that limited proteolysis affects an unknown soluble factor, a process that in turn activates chitin synthesis.     

Isolation and characterization of an apically sorted 41-kDa protein from the midgut of tobacco hornworm (Manduca sexta)

Immunocytochemical localization and sorting properties of a newly purified 41-kDa protein (MsM41) were investigated in an insect, the tobacco hornworm Manduca sexta. The protein purified from midgut homogenates of feeding fifth-stadium larvae was found exclusively in this tissue on Western blots. Presence of MsM41 protein was indicated in both anterior and posterior regions of the midgut during the whole fifth stadium. However, in the posterior region an additional 39-kDa protein was also detected during the feeding period of the last larval stage. Upon light-microscopic examination immunoreactivity was localized in the columnar cells, while the goblet, endocrine and regenerative cells remained unlabeled. Distribution of the label during the feeding period was different in the anterior and posterior regions. In the anterior region immunoreactivity was localized only to the brush border membrane of columnar cells, while in the posterior region some cytoplasmic structures identified as large trans-Golgi vesicles, endoplasmic reticulum and small secretory vesicles were also labeled. Large, apical extrusions remained immunonegative. In vitro translation confirmed that our protein was expressed only in the posterior region of the midgut. The primary translation product was a 39-kDa protein. Putative post-translational modifications yielded the 41-kDa form, which was then secreted apically. Its presence in the region of the anterior part microvilli was probably due to the countercurrent flux of the ectoperitrophic fluid.     

Subcellular effects and localization of binding sites of phytohemagglutinin in the potato leafhopper, Empoasca fabae (Insecta: Homoptera: Cicadellidae)

To identify the means by which phytohemagglutinin (PHA) exerts its toxicity on the potato leafhopper, four different methods (thick and semi-thin sectioning combined with immunofluorescent staining, in vitro receptor autoradiography, and immunoelectron microscopy) were used to elucidate the PHA target tissue, binding site, and its effects on this tissue. Sixteen 1- or 2-day-old female potato leafhoppers were fed for 36 h on each of three treatments: a control, diet or a diet containing either the PHA-E subunit or the PHA-L subunit. The PHA-E subunit, but not PHA-L, had previously been shown to be lethal. The insects were then prepared for both light and confocal microscopy. Analysis of images showed that PHA bound only to the surface of midgut epithelial cells of the potato leafhopper. PHA-E caused severe disruption, disorganization, and elongation of the brush border microvilli, and swelling of the epithelial cells into the lumen of the gut, leading to complete closure of the lumen. Furthermore, PHA-E stimulated the division of midgut epithelial cell nuclei, leading to two nuclei in each cell. Nuclei later elongated and degraded. In contrast, PHA-L had little effect on the epithelial cells of the midgut. It did not strongly bind to the surface of epithelial cells and caused much less disruption of brush-border microvilli, less disorganization of the cells and less elongation of nuclei. Strong binding of PHA occurred solely on the cell membrane of the brush border microvilli of epithelial cells. In contrast, the controls (i.e., midgut tissue, blocking agent, PHA, and antibodies) showed that midgut tissue was not autofluorescent and showed no fluorescent binding signal. Analysis of both bright- and dark-field images obtained by autoradiography and immunoelectron microscopy confirmed these findings.     

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.     

Disintegration of the peritrophic membrane of silkworm larvae due to spindles of an entomopoxvirus

Mode of action by which entomopoxvirus (EPV) spindles enhance nucleopolyhedrovirus (NPV) infection remains unclear. Spindles of Anomala cuprea entomopoxvirus (AcEPV), a coleoptran EPV, are known to enhance Bombyx mori NPV (BmNPV) infection in silkworm larvae. AcEPV spindles were orally administered to silkworm larvae with or without BmNPV polyhedra, and the peritrophic membranes (PMs) were observed using a binocular microscope. Soon after the larvae's access to spindles with or without the polyhedra had been terminated, some PMs disappeared wholly and some were observed in partial form. Some of the partial PMs observed were very fragile. The disintegration of the PM due to spindles also was observed by the histological sectioning of the midgut. However, a day after the larvae had terminated their access to the spindles, the PM regenerated partially or wholly. In contrast, the administration of AcEPV spheroids caused neither the disintegration of PMs nor the enhancement of BmNPV infection in silkworm larvae. These findings strongly suggest that the enhancement of NPV infection occurs due to that a greater number of NPV virions reaching the microvilli of midgut susceptible to NPV, since spindles lead to the disintegration of the PM as a barrier against NPV virions.     

Snowdrop lectin (GNA) has no acute toxic effects on a beneficial insect predator,the 2-spot ladybird (Adalia bipunctata L.)

Two-spot ladybird (Adalia bipunctata L.) larvae were fed on aphids (Myzus persicae (Sulz.)) which had been loaded with snowdrop lectin (Galanthus nivalis agglutinin; GNA) by feeding on artificial diet containing the protein. Treatment with GNA significantly decreased the growth of aphids. No acute toxicity of GNA-containing aphids towards the ladybird larvae was observed, although there were small effects on development. When fed a fixed number of aphids, larvae exposed to GNA spent longer in the 4th instar, taking 6 extra days to reach pupation; however, retardation of development was not observed in ladybird larvae fed equal weights of aphids. Ladybird larvae fed GNA-containing aphids were found to be 8-15% smaller than controls, but ate a significantly greater number of aphids (approx. 40% to pupation). GNA was shown to be present on the microvilli of the midgut brush border membrane and within gut epithelial cells in ladybird larvae fed on GNA-dosed aphids, although disruption of the brush border was not observed. It is hypothesised that GNA does not have significant direct toxic or adverse effects on developing ladybird larvae, but that the effects observed may be due to the fact that the aphids fed on GNA are compromised and are thus a suboptimal food.     

Spindles of an entomopoxvirus facilitate its infection of the host insect by disrupting the peritrophic membrane

The mode of action by which entomopoxvirus (EPV) spindles, proteinaceous crystalline bodies produced by EPVs, enhance EPV infection has not been clarified. We fed Anomala cuprea EPV (AcEPV) spindles to host insects; subsequent scanning electron microscopy revealed the disruption of the peritrophic membranes (PMs) of these insects. The PM is reportedly a barrier against the infection of some insects by viruses. Quantitative PCR of AcEPV DNA in the ectoperitrophic area revealed that PM disruption facilitated the passage of EPVs through the PM toward the initial infection site, the midgut epithelium. These results indicate that EPV spindles enhance infection by EPVs by disrupting the PM in the host insects. Fusolin is almost exclusively the constituent protein of the spindles and is the enhancing factor of the infectivity of nucleopolyhedroviruses (NPVs) and possibly that of EPVs. Spheroid is another type of proteinaceous crystalline structure produced by EPVs. Pseudaletia separata EPV (PsEPV) spheroids reportedly contain considerable amounts of fusolin and enhance NPV infection. We assessed the ability of AcEPV spheroids to enhance EPV infectivity and their effect on the PM and carried out immunological experiments; these experiments showed that AcEPV spheroids contain little or no fusolin and are biologically inactive, in contrasts to the situation in PsEPV.     

The role of the mosquito peritrophic membrane in bloodmeal digestion and infectivity of Plasmodium species.

Secretion and luminal formation of the peritrophic membrane (PM) were induced in female Anopheles stephensi and Aedes aegypti by feeding the mosquitoes on a warmed suspension of latex particles in Ringer's solution. The PM in A. stephensi was produced from apical secretion vesicles stored in the midgut epithelial cells and secreted into the lumen during feeding. In A. aegypti, the PM was formed de novo. When the latex feeding was followed 24 hr later by a meal of lyophilized pig blood, the 2 mosquito species exhibited very different modifications to their PM structure; in A. stephensi no PM was formed around the blood meal, whereas de novo synthesis of the PM in A. aegypti continued during the blood meal, with the resulting PM greatly thickened compared to the normal feeding. This artificial induction of PM formation was used as the basis to study the role of the PM in blood meal digestion and in infectivity of mosquitoes by the appropriate species of Plasmodium. The feeding of a latex suspension alone had no stimulatory effect on the 2 major midgut proteases, trypsin and aminopeptidase, in either species. After a blood meal alone, proteases rose to maximum activity at 30 hr and 24 hr after feeding in A. stephensi and A. aegypti, respectively. After double feeding, protease activities in both species were almost identical to those in blood-fed mosquitoes. Neither the absence of a PM (in A. stephensi) nor the presence of a thickened PM (in A. aegypti), therefore, has any effect on the ability of mosquitoes to digest a blood meal. Malaria infectivity, measured by oocyst counts, also was compared after normal and double feeding using infective blood meals. Infectivity of A. stephensi by Plasmodium berghei was unaffected by the presence or absence of the PM. The thickened PM produced by double feeding in A. aegypti caused a reduction of midgut infectivity by Plasmodium gallinaceum. These results suggest that the PM may act as a partial, but not an absolute, barrier to invasion of the midgut by the ookinete.     

Brush border membrane and amino acid transport

Fundamental differences in midgut structure, physiology, brush border proteins, and transporters among Leptinotarsa decemlineata, lepidopteran caterpillars, other insect taxa, and vertebrates are reviewed. The effects of dietary protein concentration on Manduca sexta midgut amino acid transport and brush border membrane proteins are reported. M. sexta fed diet with reduced protein had elevated levels of leucine aminopeptidase in the brush border membrane. No changes in amino acid transport or alkaline phosphatase activity due to dietary differences were detected. Changes in brush border proteins could affect the toxicity and pathogenicity of microbial agents. © 1996 Wiley-Liss, Inc.