Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.     

昆虫围食膜的研究进展

围食膜是大多数昆虫中肠内的半透性薄膜 ,主要由几丁质、蛋白质构成。依据其形成的方式分 :Ⅰ型围食膜 ,由整个中肠细胞分泌形成多层管状膜 ;Ⅱ型围食膜由中肠前端特殊的细胞分泌成连续的套筒管状膜。由于位于食物与中肠上皮细胞间而在中肠生理中起重要作用 ,围食膜保护中肠上皮免于机械损伤以及病原菌、毒素的入侵 ;作为半透膜以及将中肠分为不同的区室而在营养物质的消化和吸收中具有重要作用。该文综述了有关围食膜结构、组分、功能、通透性以及与害虫防治的关系等方面的研究进展。     

Aedes aegypti peritrophic matrix and its interaction with heme during blood digestion

A large amount of heme is produced upon digestion of red cell hemoglobin in the midgut of mosquitoes. The interaction between heme and the peritrophic matrix (PM) was studied in Aedes aegypti. By light microscopy, the PM appeared as a light brownish layer between the intestinal epithelium and the alimentary bolus. This natural color can be attributed to the presence of heme bound to the matrix. In histochemical studies, a diffuse peroxidase activity of the heme molecules was clearly observed between the erythrocytes and the PM at 14 h after the blood meal. This activity tends to increase and concentrate in the PM reaching its maximum thickness at 24 h after feeding. Most of the heme of the PM was found associated to with enormous number of small electron-dense granules. The amount of heme bound to the PM increased in parallel with the progression of digestion, reaching a maximum at 48 h after feeding, when 18 nmol of heme were found in an individual matrix. The association of heme with PM from insects fed with plasma is saturable, suggesting the existence of specific binding sites for hemin in the PM. Taken all together, our data indicate that the PM performs a central role in heme detoxification in this insect.     

Peritrophic membrane role in enhancing digestive efficiency. Theoretical and experimental models

The peritrophic membrane (PM) is an anatomical structure surrounding the food bolus in most insects. Rejecting the idea that PM has evolved from coating mucus to play the same protective role as it, novel functions were proposed and experimentally tested. The theoretical principles underlying the digestive enzyme recycling mechanism were described and used to develop an algorithm to calculate enzyme distributions along the midgut and to infer secretory and absorptive sites. The activity of a Spodoptera frugiperda microvillar aminopeptidase decreases by 50% if placed in the presence of midgut contents. S. frugiperda trypsin preparations placed into dialysis bags in stirred and unstirred media have activities of 210 and 160%, respectively, over the activities of samples in a test tube. The ectoperitrophic fluid (EF) present in the midgut caeca of Rhynchosciara americana may be collected. If the enzymes restricted to this fluid are assayed in the presence of PM contents (PMC) their activities decrease by at least 58%. The lack of PM caused by calcofluor feeding impairs growth due to an increase in the metabolic cost associated with the conversion of food into body mass. This probably results from an increase in digestive enzyme excretion and useless homeostatic attempt to reestablish destroyed midgut gradients. The experimental models support the view that PM enhances digestive efficiency by: (a) prevention of non-specific binding of undigested material onto cell surface; (b) prevention of excretion by allowing enzyme recycling powered by an ectoperitrophic counterflux of fluid; (c) removal from inside PM of the oligomeric molecules that may inhibit the enzymes involved in initial digestion; (d) restriction of oligomer hydrolases to ectoperitrophic space (ECS) to avoid probable partial inhibition by non-dispersed undigested food. Finally, PM functions are discussed regarding insects feeding on any diet.     

The origin and functions of the insect peritrophic membrane and peritrophic gel

There is a a fluid (peritrophic gel) or membranous (peritrophic membrane, PM) film surrounding the food bolus in most insects. The PM is composed of chitin and proteins, of which peritrophins are the most important. It is proposed here that, during evolution, midgut cells initially synthesized chitin and peritrophins derived from mucins by acquiring chitin-binding domains, thus permitting the formation of PM. Since PM compartmentalizes the midgut, new physiological roles were added to those of the ancestral mucus (protection against abrasion and microorganism invasion). These new roles are reviewed in the light of data on PM permeability and on enzyme compartmentalization, fluid fluxes, and ultrastructure of the midgut. The importance of the new roles in relation to those of protection is evaluated from data obtained with insects having disrupted PM. Finally, there is growing evidence suggesting that a peritrophic gel occurs when a highly permeable peritrophic structure is necessary or when chitin-binding molecules or chitinase are present in food.     

Shotgun analysis on the peritrophic membrane of the silkworm Bombyx mori

The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. We used gel electrophoresis and mass spectrometry to identify the extracted proteins from the silkworm PM to obtain an in-depth understanding of the biological function of the silkworm PM components. A total of 305 proteins, with molecular weights ranging from 8.02 kDa to 788.52 kDa and the isoelectric points ranging from 3.39 to 12.91, were successfully identified. We also found several major classes of PM proteins, i.e. PM chitin-binding protein, invertebrate intestinal mucin, and chitin deacetylase. The protein profile provides a basis for further study of the physiological events in the PM of Bombyx mori. [BMB Reports 2012; 45(11): 665-670]     

Time and site of assembly of the peritrophic membrane of the mosquito Aedes aegypti

Summary We determined the time and site of secretion of the precursors of the peritrophic membrane (PM) in Aedes aegypti and when the structure is assembled. The fine structure of the developing membrane of blood-feed females was described, and the pattern of secretion of injected tritiated glucosamine analyzed autoradiographically. Immediately following blood feeding, ingested red cells rapidly become compressed, such that the surrounding plasma is extruded to the margin of the midgut contents. Thereby, ingested fluids form a narrow margin separating the blood mass from the midgut epithelium. By electron microscopy, the PM first becomes evident at about 4 to 8 h after blood is ingested, and the membrane attains mature texture by 12 h. The compacted mass of ingested erythrocytes seems to serve as a template for the forming structure. In contrast, tritiated glucosamine, injected into freshly engorged mosquitoes, begins to concentrate on the midgut microvilli by 2 h after feeding. By 8 h the label assumes the layered appearance that characterizes the fine structure of the mature membrane. In contrast to the prevailing concept that the PM of mosquitoes first assumes texture anteriorly immediately after blood is ingested, we find that this potential barrier to pathogen development forms no earlier than 4 h after feeding and that it is formed from precursors secreted along the entire length of the epithelium overlying the food mass.     

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.     

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.     

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.     

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.     

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.     

Morphology and ultrastructure of the midgut in Piscicola geometra (Annelida, Hirudinea)

This paper presents information on the organization of the midgut and its epithelium ultrastructure in juvenile and adult specimens of Piscicola geometra (Annelida, Hirudinea), a species which is a widespread ectoparasite found on the body and gills and in the mouth of many types of fish. The analysis of juvenile nonfeeding specimens helped in the explanation of all alterations in the midgut epithelium which are connected with digestion. The endodermal portion (midgut) of the digestive system is composed of four regions: the esophagus, the crop, the posterior crop caecum, and the intestine. Their epithelia are formed by flat, cuboidal, or columnar digestive cells; however, single small cells which do not contact the midgut lumen were also observed. The ultrastructure of all of the regions of the midgut are described and discussed with a special emphasis on their functions in the digestion of blood. In P. geometra, the part of the midgut that is devoid of microvilli is responsible for the accumulation of blood, while the epithelium of the remaining part of the midgut, which has a distinct regionalization in the distribution of organelles, plays a role in its absorption and secretion. Glycogen granules in the intestinal epithelium indicate its role in the accumulation of sugar. The comparison of the ultrastructure of midgut epithelium in juvenile and adult specimens suggests that electron-dense granules observed in the apical cytoplasm of digestive cells take part in enzyme accumulation. Numerous microorganisms were observed in the mycetome, which is composed of two large oval diverticles that connect with the esophagus via thin ducts. Similar microorganisms also occurred in the cytoplasm of the epithelium in the esophagus, the crop, the intestine, and in their lumen. Microorganisms were observed both in fed adult and unfed juvenile specimens of P. geometra, which strongly suggests that vertical transmission occurs from parent to offspring.     

The establishment of Plasmodium berghei in mosquitoes of a refractory and a susceptible line of Anopheles atroparvus

The events between the ingestion of Plasmodium berghei-infected mouse blood and the establishment of the ookinetes in the epithelium of the midgut in refractory (R) and susceptible (S) Anopheles atroparvus are described. Simultaneously fed, fully engorged female mosquitoes were randomly assigned to dissection at 22, 28, 32, 48 h and 10 days (controls) after the infective feed (post-infection: p.i.). Serial transverse sections of 6 micron were cut. Every 10th section was studied. The maturation of ookinetes was monitored at 16, 19 and 22 h p.i. The infections in R and S mosquitoes developed similarly with regard to the maturation of ookinetes and the number of mature ookinetes in the lumen of the midgut. The semiquantitative evaluation of the envelopment of the food bolus by the peritrophic layer showed that this layer cannot function as a physical barrier against migrating ookinetes. In the midgut epithelium the number of ookinetes decreased significantly with time in both R and S mosquitoes, but a similar number of penetrations was recorded for both types of mosquito. In S mosquitoes maximal 1% of the ookinetes present in the midgut formed an oocyst. In both R and S mosquitoes a substantial loss of parasites was found, first in the lumen of the midgut and second after penetration of the midgut epithelium by the mature ookinetes. Relatively few parasites develop into oocysts in S, but hardly any do so in R individuals. The factors in control of refractoriness are likely to operate on early oocyst development.     

The effect of adipokinetic hormone on midgut characteristics in Pyrrhocoris apterus L. (Heteroptera)

Digestive processes and the effect of adipokinetic hormone (Pyrap-AKH) on the amount of nutrients (lipids, proteins, and carbohydrates), and on the activity of digestive enzymes (lipases, peptidases, and carbohydrases) were studied in the midgut of the firebug, Pyrrhocoris apterus. The analyses were performed on samples of anterior (AM), middle (MM) and posterior (PM) midgut parts. The results revealed that the digestion of lipids, carbohydrates and proteins take place in the acidic milieu. The Pyrap-AKH treatment increased significantly the level of lipids and proteins in the midgut, and also the level of triacylglycerols (TGs) predominantly in the AM, and the level of diacylglycerols (DGs) in the MM. The increase was not uniform for all present TG and DG species - those containing the linoleic fatty acid were predominant. No hormonal effect on lipase activity was recorded, while peptidase and glucosidase activity was increased in the MM and PM. All these facts indicate that the Pyrap-AKH probably stimulates digestion by more intensive food ingestion or turnover, and perhaps by the stimulation of metabolite absorption; the activation of digestive enzymes seems to be secondary or controlled by other mechanisms.     

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.     

Plasmodium vivax: impaired escape of Vk210 phenotype ookinetes from the midgut blood bolus of Anopheles pseudopunctipennis

The site in the midguts of Anopheles pseudopunctipennis where the development of Plasmodium vivax circumsporozoite protein Vk210 phenotype is blocked was investigated, and compared to its development in An. albimanus. Ookinete development was similar in time and numbers within the blood meal bolus of both mosquito species. But, compared to An. pseudopunctipennis, a higher proportion of An. albimanus were infected (P=0.0001) with higher ookinete (P=0.0001) and oocyst numbers (P=0.0001) on their internal and external midgut surfaces, respectively. Ookinetes were located in the peritrophic matrix (PM), but neither inside epithelial cells nor on the haemocoelic midgut surface by transmission electron microscopy in 24h p.i.-An. pseudopunctipennis mosquito samples. In contrast, no parasites were detected in the PM of An. albimanus at this time point. These results suggest that P. vivax Vk210 ookinetes cannot escape from and are destroyed within the midgut lumen of An. pseudopunctipennis.     

Ultrastructural changes in the midgut epithelium of the first larva of Allacma fusca (Insecta, Collembola, Symphypleona)

Abstract. In the newly hatched larva in Allacma fusca , the midgut epithelium was fully developed and formed by flattened epithelial cells surrounding the yolk mass in the midgut lumen. Immediately after hatching, the first larva began to feed; the migut lumen was filled with the yolk mass and food (mainly algae). Regenerative cells typical of the developing midgut epithelium of many insects were not observed. Initially, midgut cells of the larva were cuboidal but became columnar in shape with distinct regionalization in the distribution of cell organelles. Furthermore, urospherites appeared in the midgut cell cytoplasm, i.e., structures characteristic for the midgut epithelium of insects having no Malpighian tubules. As a result, cells with the capacity for digestion, absorption, and excretion were observed to be completely formed in the first larval stage.     

Morphological aspects of blood digestion in a parasitic mite Bakericheyla chanayi

All life stages of B. chanayi (Acariformes: Cheyletidae) are characterized by occasional bloodsucking and a long period of digestion. No newly engorged mites were found during the period of their host birds' migration. The fine structure of the digestive tract of a blood-feeding acariform mite is described for the first time. The anterior midgut (AMG) is a place of blood digestion, while the posterior midgut (PMG) is involved in nitrogen metabolism forming guanine crystals as the main end-product. The AMG epithelium consists of digestive cells that probably arise from mitotically active basal cells with high synthesizing activity.As observed in ticks, blood digestion is accompanied by the formation of huge endosomes that serve as places of storage and sorting of ingested material. Digestive cells show different types of endocytotic activity as well as various late endosomes, which implies different subcellular pathways for different blood components. In both midgut regions, elimination of the excretory material occurs by apocrine secretion or by discharging of apical cell fragments (loaded with lysosomes) into the gut lumen. The formation of guanine granules occurs inside the lysosomes of PMG epithelial cells thus having much in common with intracellular digestion. Peculiarities of intracellular blood digestion were analyzed according to the modern hypothesis of endocytosis and compared to what is known in ticks.