Walter R. Terra

Distribution of digestive enzymes among the endo- and ectoperitrophic spaces and midgut cells of Rhynchosciara and its physiological significance

Determinations of carbohydrases, proteases, carboxylesterases and phosphatases in the midgut cells and in the luminal spaces outside and inside the peritrophic membrane of Rhynchosciara americana larvae have been carried out. The data show that alpha-amylase, cellulase and proteinases are present in cells, ecto- and endoperitrophic spaces; aminopeptidases and trehalase in cells and ectoperitrophic space; and finally disaccharidases (except trehalase), carboxypeptidases, dipeptidases, carboxylesterases and phosphatases only in cells. The results support the conclusion that digestion takes place in three spatially organized steps. The first one occurs inside the peritrophic membrane and comprises the dispersion and/or decrease in molecular weight of the food molecules. The second is the hydrolysis of the polymeric food molecules in the ectoperitrophic space to dimers and/or small oligomers. Finally, terminal digestion occurs in the midgut caeca and posterior ventriculus cells by enzymes presumed to be plasma membrane bound. The existence of two extracellular sites for digestion in R. americana is considered to be an adaptation to conserve secreted enzymes, since only those penetrating the endoperitrophic space are lost quickly in the faeces.

Midgut adaptation and digestive enzyme distribution in a phloem feeding insect, the pea aphid Acyrthosiphon pisum.

Transmission electron micrographs of the pea aphid midgut revealed that its anterior region has cells with an apical complex network of lamellae (apical lamellae) instead of the usual regularly-arranged microvilli. These apical lamellae are linked to one another by trabeculae. Modified perimicrovillar membranes (MPM) are associated with the lamellae and project into the lumen. Trabeculae and MPM become less conspicuous along the midgut. The most active A. pisum digestive enzymes are membrane-bound. An aminopeptidase (APN) is described elsewhere. An alpha-glucosidase (alpha-Glu) has a molecular mass of 72 kDa, pH optimum 6.0 and catalyzes in vitro transglycosylations in the presence of an excess of the substrate sucrose. There is a major cysteine proteinase activity (CP) on protein substrates that has a molecular mass of 40 kDa, pH optimum 5.5, is inhibited by E-64 and chymostatin and is activated by EDTA+cysteine. The enzyme is more active against carbobenzoxy-Phe-Arg-4-methylcoumarin-7-amide (ZFRMCA) than against ZRRMCA. These features identify the purified CP as a cathepsin-L-like cysteine proteinase. Most CP is found in the anterior midgut, whereas alpha-Glu and APN predominate in the posterior midgut. With the aid of antibodies, alpha-Glu and CP were immunolocalized in cell vesicles and MPM, whereas APN was localized in vesicles, apical lamellae and MPM. The data suggest that the anterior midgut is structurally reinforced to resist osmotic pressures and that the transglycosylating alpha-Glu, together with CP and APN are bound to MPM, thus being both distributed over a large surface and prevented from excretion with honeydew. alpha-Glu frees glucose from sucrose without increasing the osmolarity, and CP and APN may process toxins or other proteins occasionally present in phloem.

Apocrine secretion of amylase and exocytosis of trypsin along the midgut of Tenebrio molitor larvae.

Amylase and trypsin were purified from Tenebrio molitor midgut larvae and used to raise antibodies in a rabbit. A Western blot of T. molitor midgut homogenates, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using amylase and trypsin antisera, showed only bands co-migrating with the purified enzymes. The antisera were used to localize the enzymes by immunofluorescence and immunogold labeling. Amylase occurs in a few regularly disposed anterior midgut cells. Non-amylase-secreting anterior midgut cells are proposed to be water-absorbing cells based on morphology and dye movements. Amylase is found inside vesicles originating from Golgi areas that seem to fuse together before their release along with the now disorganized apical cytoplasm (apocrine secretion). Trypsin precursors are observed inside small vesicles near the apical plasma membrane of posterior midgut cells, suggesting an exocytic mechanism of secretion, followed by putative trypsin activation. Apocrine secretion is thought to be an adaptation to enhance the dispersion of secretory vesicle contents released from a water-absorbing epithelium, whereas exocytosis is an efficient secretory mechanism in a water-secreting epithelium.

Physiological adaptations for digesting bacteria. Water fluxes and distribution of digestive enzymes in Musca domestica larval midgut

Abstract Dye experiments with Musca domestica larvae suggest that in each passage of food (104 min) water is secreted into the gut lumen in the fore-midgut (1.5 μl) and in the hind-midgut (3.9 μl), and water is absorbed from the gut lumen in the mid-midgut (2.3 μl) and hind-gut (3.6 μl). Hydrolases found to be active mainly in luminal contents were amylase and trypsin (fore- and hind-midgut), and lysozyme and pepsin (mid-midgut), whereas those mainly active in midgut cells were aminopeptidase and trehalase (fore- and hind-midgut). Maltase was found in both contents and cells of hind-midgut. Less than 20% of hind-midgut amylase and trypsin are excreted, after a time identical to the passage time of the food bolus, suggesting that there exists an endo-ectoperitrophic circulation of enzymes, by which these enzymes are recovered from the undigested food before it is excreted. The data led to the proposal that bacteria are killed at mid-midgut through the combined action of low pH, lysozyme and pepsin. Digestion of proteins and starch is largely accomplished in the hind-midgut lumen, whereas the resulting oligopeptides and oligosaccharides are hydrolyzed down to monomers at the surface of proximal hind-midgut cells. The adaptive features of the digestion of housefly maggots are thought to be derived characters evolved from a putative Diptera ancestor.

Molecular adaptation of Drosophila melanogaster lysozymes to a digestive function.

A lysozyme (pI 5.5) was purified to homogeneity from heated acid extracts of Drosophila melanogaster larvae, using gel filtration in a Superose column and ion-exchange chromatography in a Mono Q column. The final yield was 67%. The purified lysozyme with Mr 13,700 (determined by SDS-polyacrylamide gel electrophoresis) decreases in activity and has its pH optimum displaced towards acidic values and Km increases as the ionic strength of the medium becomes higher. The lysozyme is resistant to a cathepsin D-like proteinase present in cyclorrhaphous Diptera and displays a chitinase activity which is 11-fold higher than that of chicken lysozyme. Microsequencing of an internal peptide of the purified lysozyme showed that this enzyme is the product of the previously sequenced Lys D gene. The results suggest that the product of the Lys P gene has pI 7.2, a pH optimum around 5 and is not a true digestive enzyme. The most remarkable sequence convergence of D. melanogaster lysozyme D and lysozymes from vertebrate foregut fermenters are serine 104 and a decrease in the number of basic amino acids, suggesting that these features are necessary for digestive function in an acid environment. Adaptive residues putatively conferring stability in an acid proteolytic environment differ between insects and vertebrates, probably because they depend on the overall three-dimensional structure of the lysozymes. A maximum likelihood phylogeny and inferences from insect lysozyme sequences showed that the recruitment of lysozymes as digestive enzymes is an ancestral condition of the flies (Diptera: Cyclorrhapha).

Phylogenetic considerations of insect digestion: Disaccharidases and the spatial organization of digestion in the Tenebrio molitor larvae

Amylase, cellobiase, cellulase, trehalase and trypsin are found in major amounts in the midgut lumen, whereas aminopeptidase (which is membrane bound) occurs mainly in the midgut tissue of Tenebrio molitor larvae. Cellulase and a minor aminopeptidase seem to be derived from fungi contaminating the wheat bran used as food. The data suggest that the majority of carbohydrate digestion should take place in the lumen of anterior midgut, whereas protein digestion should occur partly in the lumen and partly at the cell surface of the posterior midgut. The finding that less than 5% of the total amylase, cellobiase, maltase and trypsin are excreted, after a time identical to the passage time of the food bolus, leads to the proposal that there exists an endo-ectoperitrophic circulation of enzymes by which these enzymes are recovered from the undigested food before it is excreted. There is only one molecular species of cellobiase (pH optimum 5.3, Km 1.1 mM, pI 3.7, Mr 75,000), maltase (pH optimum 5.3, Km 3.7 mM, pI 3.6, Mr 60,000) and trehalase (pH optimum 5.0, Km 0.40 mM, pI 4.0, Mr 60,000) in T. molitor larval midguts as judged by electrophoretic, isoelectric focusing and density-gradient centrifugation data. The data led to the proposal that dimer and oligomer hydrolases are small in Coleoptera ancestors and large in Diptera and Lepidoptera ancestors. For this, the majority of T. molitor larval digestion takes place inside the peritrophic membrane, whereas in Rhynchosciara americana (Diptera) and Erinnyis ello (Lepidoptera) larvae initial digestion occurs inside and intermediate and final digestion outside the peritrophic membrane.

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin (M(r) 26000) and four trypsins (T1-T4) with the following properties: T1, K(m) 0.3 microM, M(r) 70000; T2, K(m) 0.4 microM, M(r) 67000; T3, K(m) 2.4 microM, M(r) 29000; T4, K(m) 15 microM, M(r) 17000. Diet-fed larvae have a chymotrypsin (M(r) 26000) and a major trypsin (K(m) 2.9 microM, M(r) 29000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K(m) values) and a putative decreased affinity for PIs.

Properties of the digestive enzymes and the permeability of the peritrophic membrane of Spodoptera frugiperda (Lepidoptera) larvae

Abstract The physical and kinetic properties of several soluble and detergent-solubilized membrane-bound midgut hydrolases of Spodoptera frugiperda (Lepidoptera) were investigated. The following techniques were employed: isoelectric focusing and electrophoresis in polyacrylamide gels, density-gradient ultracentrifugation and gel filtration in Superose columns. In vivo molecular weights of the hydrolases were considered to correspond with those determined by centrifugation, which is the more gentle of the procedures employed. Using this criterion, the soluble hydrolases display the following number of sub-units: acetylglucosaminidase [pHo5 (pH optimum), Mr 123,000], 2; aminopeptidase (pHo 7.4, Mr 107,000), 2; amylase (pHo9.6, Km for starch 0.38%, Mr 87,000), 1; carboxypeptidase A (pHo 8.0, Mr 45,000), 2; cellobiase (pHo 7.0, Mr 124,000), 2; dipeptidase (pHo 8.0, Mr 95,000), 2; maltase (pHo 5.0, Mr 150,000), 2; trypsin (pHo 7.9, Mr 51,000), 2. Amylase is not activated by chloride. A comparison between the diameters of the enzymes which pass through the peritrophic membrane (amylase, carboxypeptidase and trypsin) with that which is secreted into the lumen but do not pass through the peritrophic membrane (acetylglucosaminase) suggests that the pores of S. frugiperda larval peritrophic membrane have diameters of 7.5–8 nm.

Further evidence that enzymes involved in the final stages of digestion by Rhynchosciara do not enter the endoperitrophic space

Abstract Chitinase, lysozyme, β-glucuronidase, pectinase and xylanase activities are absent from the midguts of Rhynchosciara americana larvae. Glycoprotein glycosidases are restricted to gastric and posterior ventricular cells (α-mannosidase and β-fucosidase) or in both cells and in the ectoperitrophic space ( β-N- acetylglucosaminidase ). Differential centrifugation data and data from purification of gastric caecal microvilli suggested that α-mannosidase and β-fucosidase are always bound to the plasma membrane, whereas β-N- acetylglucosaminidase (which also has a β-N- acetylgalactosaminidase activity), seems to be bound to plasma membrane and also in the cytosol. Molecular weights of midgut hydrolases determined by electrophoresis in acrylamide gels of different concentrations and/or by ultracentrifugation in linear glycerol gradients were as follows: β-N- acetylglucosaminidase : 141,000; aminopeptidase: 111,000; amylase: 64,500; cellulase 1: 42,000; cellulase 2: 64,500; trypsin: 47,200. The data support the hypothesis that enzymes involved in intermediate and final stages of digestion do not penetrate the endoperitrophic space either because of their large size (e.g., β-N- acetylglucosaminidase and aminopeptidase) or because they are bound to the plasma membrane (e.g., α-mannosidase and β-fucosidase).

Digestion of bacteria and the role of midgut lysozyme in some insect larvae

1. Lysozyme is absent from tissues other than the midgut in the drug-feeding larvae of Musca domestica (Diptera, Cyclorrhapha, Muscidae) and in the fruit-feeding larvae of Anastrepha fraterculus (Diptera, Cyclorrhapha, Tephritidae), whereas in the detritus-feeding larvae of Trichosia pubescens (Diptera, Nematocera, Sciaridae) lysozyme is only found in the hemolymph and in the fat body. 2. A. fraterculus larvae have a midgut region with a luminal pH of 3.4, and display a pepstatin-inhibited acid proteolytic activity which has a spec. act. (7.2 U/mg protein) similar to that of M. domestica. 3. The midgut lysozyme from M. domestica and A. fraterculus is more active (high ionic strength) at pH 3.5 than at pH 6.0, the contrary being true for a midgut chitinase. 4. The results suggest that the adaptations to digest bacteria in insects are similar to those in vertebrate foregut fermenters, and that these characteristics were probably present in the Cyclorrhapha ancestor, but not in the Diptera ancestor.