Peritrophic membrane role in enhancing digestive efficiency. Theoretical and experimental models.
ABSTRACT 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.
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ABSTRACT: The peritrophic matrix (PM) in the midgut of insects consists primarily of chitin and proteins and is thought to support digestion and provide protection from abrasive food particles and enteric pathogens. We examined the physiological roles of 11 putative peritrophic matrix protein (PMP) genes of the red flour beetle, Tribolium castaneum (TcPMPs). TcPMP genes are differentially expressed along the length of the midgut epithelium of feeding larvae. RNAi of individual PMP genes revealed no abnormal developmental phenotypes for 9 of the 11 TcPMPs. However, RNAi for two PMP genes, TcPMP3 and TcPMP5-B, resulted in depletion of the fat body, growth arrest, molting defects and mortality. In situ permeability assays after oral administration of different-sized FITC-dextran beads demonstrated that the exclusion size of the larval peritrophic matrix (PM) decreases progressively from >2 MDa to <4 kDa from the anterior to the most posterior regions of the midgut. In the median midguts of control larvae, 2 MDa dextrans were completely retained within the PM lumen, whereas after RNAi for TcPMP3 and TcPMP5-B, these dextrans penetrated the epithelium of the median midgut, indicating loss of structural integrity and barrier function of the larval PM. In contrast, RNAi for TcPMP5-B, but not RNAi for TcPMP3, resulted in breakdown of impermeability to 4 and 40 kDa dextrans in the PM of the posterior midgut. These results suggest that specific PMPs are involved in the regulation of PM permeability, and that a gradient of barrier function is essential for survival and fat body maintenance.Insect biochemistry and molecular biology 03/2014; · 3.25 Impact Factor
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ABSTRACT: Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism. PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS. Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM. To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse.PLoS Neglected Tropical Diseases 04/2014; 8(4):e2691. · 4.57 Impact Factor
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ABSTRACT: The effect of dietary protein concentration on the spatial distribution of digestive proteinases in the shrimp Litopenaeus vannamei indicates the existence of endo-ectoperitrophic enzyme circulation in this species. Samples recovered from the midgut gland tissues, stomach contents, three different portions of the midgut and feces were used for quantitative and qualitative analysis of the composition and distribution of the digestive proteinases. Animals were divided into three different groups: (1) animals (controls) fed a commercial 35% protein diet, (2) animals fed a commercial diet supplemented with ovalbumin to a final protein concentration of 60%; (3) animals fed a 80% protein diet. Quantitative determinations using different substrates and zymograms showed that increasing protein concentration in the diet alters the distribution of proteinases along the digestive tract. Composition of proteinases in the midgut gland, stomach contents, midgut sections and feces were similar, but not identical. Chymotrypsin and trypsin paralogues were identified in all enzyme sources in a concentration gradient along the midgut in the control shrimp, the expected distribution supporting the existence of a recycling mechanism. The occurrence of a peritrophic membrane in other Decapoda suggests that endo-ectoperitrophic circulation of digestive enzymes and nutrients may also occur in other crustaceans and also extends beyond the Insecta.Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 05/2014; · 1.61 Impact Factor