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.     

A morphometric study of the midgut in resistant and non-resistant Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae to its nucleopolyhedrovirus (AgMNPV)

In this investigation, the anterior and posterior regions of the midgut of resistant (RL) and non-resistant (SL) Anticarsia gemmatalis larvae were analyzed morphometrically to characterize different regions along their length. Also, this investigation compares the results between SL and RL to improve the understanding of the resistance mechanisms to the virus. Histological sections were analyzed in a computerized system and the data were statistically analyzed by the Kruskal-Wallis test and by multivariate analysis. The midguts are morphometrically different in the two larval populations; we observed higher values in RL. The morphometric analysis of the epithelial cells showed that only columnar and goblet cells were distinct along the midgut, in both larvae, with the higher values found in the anterior region. Comparing the results between the two larval populations, all the epithelial cells presented significant differences, with RL showing the higher morphometric values. We concluded that there are regional differences along the length of midgut in SL and RL that confirm the idea of two morpho-functional distinct regions. The consistently morphometric superior values in RL indicate that this variability can be related with the resistance of A. gemmatalis to its AgMNPV.     

Density‐dependent prophylaxis in primary anti‐parasite barriers in the velvetbean caterpillar

1. Organisms rely on a set of primary barriers to prevent invasion by parasites, and secondary defences to fight parasites that breach the primary barriers. However, maintaining these defences to be active and effective is costly. Thus, hosts increase investment in anti‐parasite defences under situations of high risk of infection and reduce defences when the risk is reduced (the ‘Density‐Dependent Prophylaxis’ hypothesis). 2. In the present study, it was tested whether the midgut primary defences of the velvetbean caterpillar Anticarsia gemmatalis Hübner present density‐dependent plasticity, and also whether these defences could be induced by a viral pathogenic challenge. The aim was to examine whether morphometry and the structure of the midgut and peritrophic matrix (PM) change in accordance with colour transition in caterpillars, and whether such changes may provide the caterpillars a more protective barrier against invasion by Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). 3. It was found that PM and the midgut epithelium of the velvetbean caterpillar change plastically according to phenotype, itself a response to changes in population density. Caterpillars reared at high densities (black phenotype) had a considerably thicker midgut epithelia and peritrophic matrices than those reared individually (green phenotype), and there was also more chitin in the PM of the former. 4. This was interpreted as the first demonstration of increased investment in primary, barrier, defences against parasites, in response to increased conspecific density and an increased risk of infection. The possibility that this arises as a positive result of pleiotropy is discussed further, wherein the biochemical pathways responsible for the up‐regulation of the immune system are also involved in midgut properties.     

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。     

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.     

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 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.     

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.     

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.     

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.     

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.     

棉铃虫围食膜肠粘蛋白基因HM72的克隆、表达及其蛋白组织定位

围食膜是昆虫中肠上皮细胞分泌形成一层特有的非细胞性半透膜, 肠粘蛋白是其重要的组成成分。本研究利用棉铃虫Helicoverpa armigera围食膜蛋白多克隆抗体免疫筛选棉铃虫中肠cDNA表达文库,共获得385个阳性克隆, 经DNA测序和序列对比, 确认其中之一为编码棉铃虫中肠围食膜肠粘蛋白的cDNA克隆HM72。序列分析显示,该cDNA全长为2 888 bp (GenBank登录号： HM017910), 其中ORF长2 469 bp, 编码823个氨基酸, 包含起始密码子ATG和终止密码子TAA,在poly A 末端上游19 bp处有一个多聚腺苷酸信号序列AATTAA。氨基酸序列分析表明, 其N-端含有16个氨基酸的信号肽, 预测分子量为84.2 kDa,等电点3.63,为酸性蛋白质。结构域分析表明,该蛋白具有5个几丁质结合功能域,一个粘蛋白结构域和两个甘氨酸-天冬氨酸富集区,该基因成功表达100 kDa的目的蛋白。Western blot分析表明,HM72蛋白存在于棉铃虫中肠、围食膜、粪便及蜕中,并由整个中肠分泌,而在棉铃虫脂肪体、体壁、马氏管、唾腺、消化液、血淋巴中没有检测到HM72蛋白。本研究为棉铃虫生物防治相关功能基因的深入研究以及完善昆虫围食膜理论等提供了依据。     

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 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.     

Assessment of Arsenite,Arsenate, and Chromate Phytotoxicity Based on the Activity of Seed Germination and Growth (Root & Shoot) of Various Plant Seeds

Effects of different concentrations of arsenite, arsenate, and chromate on seed germination, root length (RL), and shoot length (SL) in four seed types, chosen from preliminary tests with eight seed types, were investigated to assess the toxicity of the tested metals. The sensitivities of the four different seed types toward germination, relative RL (RRL), and relative SL (RSL) varied with each metal. In a comparison, the germination of the seeds was more sensitive to the tested metals than the other chosen endpoints (RL and SL). Arsenite was generally more restrictive to all the endpoints (germination, root, and shoot growth) than arsenate and chromate. Lactuca (garden lettuce) was also generally more sensitive to the tested metals than the other seed types. The correlation between RRL and RSL varied depending on the seed type and metal tested. However, significant correlations (r² > 0.85) of these were observed with Lactuca seeds, which appeared to be an optimal plant with respect to the tolerance of the tested metals. The differences in the toxicities of metals toward different plant species should be taken into account in the bioassessment of metals-contaminated sites. Thus, this study encourages the need to combine the three endpoints of various seeds in the evaluation of toxicities of metals.     

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.     

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.     

Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551]