Americana Midgut Research Articles

Midgut Transcriptome of the Cockroach Periplaneta americana and Its Microbiota: Digestion, Detoxification and Oxidative Stress Response.

The cockroach, Periplaneta americana, is an obnoxious and notorious pest of the world, with a strong ability to adapt to a variety of complex environments. However, the molecular mechanism of this adaptability is mostly unknown. In this study, the genes and microbiota composition associated with the adaptation mechanism were studied by analyzing the transcriptome and 16S rDNA pyrosequencing of the P. americana midgut, respectively. Midgut transcriptome analysis identified 82,905 unigenes, among which 64 genes putatively involved in digestion (11 genes), detoxification (37 genes) and oxidative stress response (16 genes) were found. Evaluation of gene expression following treatment with cycloxaprid further revealed that the selected genes (CYP6J1, CYP4C1, CYP6K1, Delta GST, alpha-amylase, beta-glucosidase and aminopeptidase) were upregulated at least 2.0-fold at the transcriptional level, and four genes were upregulated more than 10.0-fold. An interesting finding was that three digestive enzymes positively responded to cycloxaprid application. Tissue expression profiles further showed that most of the selected genes were midgut-biased, with the exception of CYP6K1. The midgut microbiota composition was obtained via 16S rDNA pyrosequencing and was found to be mainly dominated by organisms from the Firmicutes phylum, among which Clostridiales, Lactobacillales and Burkholderiales were the main orders which might assist the host in the food digestion or detoxification of noxious compounds. The preponderant species, Clostridium cellulovorans, was previously reported to degrade lignocellulose efficiently in insects. The abundance of genes involved in digestion, detoxification and response to oxidative stress, and the diversity of microbiota in the midgut might provide P. americana high capacity to adapt to complex environments.

Periplaneta americana midgut proteases differentially expressed against dietary components from different plant seeds

Insect midgut proteases are known to be regulated by plant protease inhibitors. In the present study, the antinutritional effects of a variety of seed extracts against Periplaneta americana (Linnaeus) (Dictyoptera: Blattidae) midgut proteases are assessed in vitro and in vivo. Bioassays are conducted by allowing P. americana to feed on diets incorporated with the tested seed extracts. Low survival rates are recorded on a diet incorporated with seed powders of Ricinus communis (10%), Glycine max (30%), Datura alba (50%) and Mucuna pruriens (50%). Proteolytic and residual proteolytic activities are highly inhibited by the four seed extracts. Electrophoretic analysis shows that the majority of P. americana midgut protease isoforms are inhibited by the four seed extracts in vivo as well as in vitro. The midgut physiology of P. americana is affected by the seed extracts of D. alba, M. pruriens and Sapindus laurifolius, by over synthesising or changing mobilities of existing protease isoforms. Furthermore, some key proteases in P. americana midgut are involved in the regulation of other protease isoforms. These results suggest that seed extracts from the above plants are potential sources of plant protease inhibitors for managing economically important crop pests.

Aminopeptidase a from Rhynchosciara americana (Diptera) larval midguts: Properties and midgut distribution

AbstractL‐aspartic acid α‐(β‐naphthylamide) (AspβNA) hydrolase activity is restricted mostly to the midgut caeca of Rhynchosciara americana larvae. The membrane‐bound activity is solubilized in detergent and, after electrophoretic separation, proved to be identical to leucine p‐nitroanilide (LpNA) hydrolases previously described. Differential centrifugation of midgut caeca homogenates, followed by assays of enzyme markers and aminopeptidase, suggests that the soluble AspβNA hydrolase is associated with the cell glycocalyx. Soluble aminopeptidases from R. americana midgut caeca are resolved into three fractions by gel electrophoresis. The slow migrating fraction hydrolyzes AspβNA well and displays a low activity on LpNA and proline β‐naphthylamide (ProβNA). Thus, this enzyme is an aminopeptidase A (EC 3.4.11.7). It has a pH optimum of 7.5, Mr 117,000 (gel filtration), and is competitively inhibited by aspartate hydroxamate (Ki 0.1 mM). Nevertheless, this enzyme, in contrast to the vertebrate enzyme, is not activated by calcium ions. The aminopeptidase A seems to have a charge variant that displays an intermediate migration and is not resolved from an aminopeptidase N (enzyme very active on LpNA). These two activities are not resolved by either gel filtration or ion‐exchange chromatography. The aminopeptidases N with intermediate and high migration, previously reported to be charge variants, were shown in this paper to differ in substrate specificities and in the strength with which they associate to the cell glycocalyx. © 1994 Wiley‐Liss, Inc.

The detergent form of the major aminopeptidase from the plasma membrane of midgut caeca cells of Rhynchosciara americana (Diptera) larva

1. 1. Plasma membrane-bound aminopeptidases from Rhynchosciara americana midgut caeca cells may be solubilized by Triton X-100 or may be released by papain treatment. 2. 2. The major detergent solubilized aminopeptidase was purified by electrophoresis and was shown to have M r 169,000 and pI 7.8. 3. 3. K m values for two substrates ( l-arginine -p- nitroanilide and l-leucine -p- nitroanilide ) and K i values for four linear inhibitors (arginine hydroxamate, leucine hydroxamate, hydroxyl amine and isoamyl alcohol) corresponding to the major detergent-solubilized aminopeptidase are identical to those of the major papain released aminopeptidase, which was previously purified and characterized. 4. 4. Both aminopeptidase forms display the same pH optimum (7.2 in Tris-HCl buffer) and follow the same thermal inactivation kinetics (half-lives of 23 min at 50°C). 5. 5. Treatment of the major detergent form of the aminopeptidase with papain results in the appearance of a M r 97,000 aminopeptidase. 6. 6. The data support the proposal that the papain-form of the aminopeptidase (M r 207,000) results from the dimerization of the products of the papain action upon the major native (M r 169,000) aminopeptidase.

Minor aminopeptidases purified from the plasma membrane of midgut caeca cells of an insect ( Rhynchosciara americana) larva

Rhynchosciara americana midgut caecal cells display in their plasma membranes, as judged by electrophoresis, a major (T 1) and three minor (T 2, T 3 and T 4) aminopeptidases which are solubilized by Triton X-100, and a major (P 1) and two minor (P 2 and P 3) aminopeptidases which are released by papain treatment. Previous work showed that T 1 corresponds to P 1. Aminopeptidases T 1 and T 3 have the same M r value as determined by density-gradient centrifugation and by electrophoresis, and display different pI values, as judged by isoelectric focusing and electrophoresis. Furthermore, T 1 and T 3 purified by electrophoresis display identical K m values for arginine p- nitroanilide and leucine p- nitroanilide and the same K i values for leucine hydroxamate, hydroxyl amine and isoamyl alcohol. The data suggest that T 1 and T 3 differ only in net charge, implying that T 2 and T 4 should be related to P 2 and P 3. P 2 and P 3 purified by electrophoresis display identical K i values for arginine hydroxamate, leucine hydroxamate, hydroxylamine and isoamyl alcohol and also identical substrate specificities. P 2 and P 3 show a broad specificity in relation to the N-terminal aminoacyl residue and hydrolyses tetrapeptides more efficiently than tripeptides and much more efficiently than dipeptides. The data suggest that T 2 and T 4 as well as P 2 and P 3 are respectively different aggregation states of a minor native membrane bound aminopeptidase solubilized by detergent and released by papain. The native aminopeptidase is probably a tetramer of identical (or similar) subunits, which, due to its substrate specificities, produces short oligopeptides (mainly tripeptides) which are the preferred substrates for the major membrane-bound aminopeptidase.

Soluble aminopeptidases from cytosol and luminal contents of Rhynchosciara americana midgut caeca : Properties and phenanthroline inhibition

Rhynchosciara americana midgut caecal cells contain two major cytosolic aminopeptidases which are resolved by electrophoresis but which have the same M r value (115,700), as determined by gradient ultracentrifugation, and have pI values of 8.7 and 7.8. Electrophoretic migration of the two aminopeptidases in polyacrylamide gels of different concentration suggests they differ only in net charge. Thermal inactivation of both cytosolic aminopeptidases follow apparent first-order kinetics with identical half-lives. The two cytosolic aminopeptidases have the same K m values when either leucine-p- nitroanilide or arginine-p- nitroanilide are substrates. Midgut luminal fluid displays two major aminopeptidases resolved by electrophoresis which have the same properties as the two cytosolic aminopeptidases. The cytosolic aminopeptidases purified by electrophoresis have the same pH optimum of 8.0 and Tris ( K i 107 mM) and 1,10-phenanthroline ( K i 14 μM) both act as simple linear competitive inhibitors. The enzymes are true aminopeptidases with a broad specificity towards aminoacyl-β-naphthylamides and are more active on tetra and tripeptides than on dipeptides. The data support the assumption that the cytosolic aminopeptidases from caecal cells, which are similar to those in luminal fluid, are enzymes en route to their being secreted and that they differ only in net charge. Furthermore, the properties of the aminopeptidases are in accordance to their proposed role of oligopeptide digestion in the ectoperitrophic fluid.

Inhibition of an insect midgut trehalase by dioxane and δ-gluconolactone: Enzyme pK a values and geometric relationships at the active site

1. 1. Inhibition of the Rhynchosciara americana midgut trehalase (α, α'-trehalose glucohydrolase, EC 3.2.1.28) by the competitive inhibitor dioxane have been studied. 2. 2. Determinations of the K i of dioxane at different pH provided the true pK a values of the prototropic groups of the trehalase active site (pK a 5.2 and 8.2), which are in agreement with those determined previously from kinetic and chemical modification data. 3. 3. Dioxane only changes the enzyme pK a values if bound at the enzyme active site. 4. 4. Gluconic acid does not inhibit the trehalase, in contrast to δ-gluconolactone which is a simple intersecting linear inhibitor. 5. 5. Multiple inhibition analyses showed that δ-gluconolactone and Tris bind at the same site in the trehalase active center, whereas dioxane binds closer to δ-gluconolactone than to Tris. 6. 6. The data support the assumption that dioxane binds at the middle portion of the trehalase active site.

Properties of arylamidases found in cytosol, microvilli and in luminal contents of Rhynchosciara americana midgut caeca

Differential centrifugation of homogenates of midgut caecal cells obtained by mild and by vigorous homogenizing have been carried out and hydrolase activities using aminoacyl-β-naphthylamides and phenylalanylglycine (Phe-Gly) as substrates have been determined in the isolated fractions. The data suggest that arylamidases and Phe-Gly hydrolase occur in the microvilli and in the cytosol of caecal cells. Tris buffer activates microvillar arylamidases and slightly inhibits hydrolases from the caecal cytosol and lumen, whereas p- hydroxymercuribenzoate inhibits the microvillar enzymes and has little effect on the soluble enzymes. This, as well as the relative activity upon several different substrates, shows that arylamidases from the caecal cytosol and lumen are similar and that both differ from caecal microvillar arylamidases. Electrophoretic data showed that 4 of the 5 intracellular soluble midgut caecal arylamidases occur in the luminal fluid. The data support the assumption that the majority of the soluble arylamidases from the caecal cells are enzymes en route to their being secreted and only a minor amount would have a typical intracellular function.

Intracellular distribution of hydrolases in midgut caeca cels from an insect with emphasis on plasma membrane-bound enzymes

1. 1. Differentially centrifugated Rhynchosciara americana midgut caeca homogenates were prepared in mild and vigorous conditions, and enzyme assays were accomplished in the isolated fractions. 2. 2. α- and β-glucosidases, α- and β-galactosidases and cellobiases are plasma membrane bound. 3. 3. Maltases occur in plasma membrane and in lysosomes (as soluble enzymes). 4. 4. Aminopeptidases, carboxypeptidases and dipeptidases are found in plasma membrane and cytosol. 5. 5. A successful purification based on differential centrifugation data. 6. 6. The results lead to the proposal that the majority of the terminal digestion of proteins and carbohydrates, with the exception of trehalose, occurs on the surface of the microvili of the insect midgut caeca cells.

Carbodiimide-reactive carboxyl groups at the active site of an insect midgut trehalase

Carbodiimide modification of the Rhynchosciara americana midgut trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) at different pH values revealed the existence of two essential groups (p K a) 5.28 and (p K a 7.74) for the trehalose activity. Those groups must be carboxyl groups since the alternative possibilities (sulfhydryl and phenol groups) have been discarded by selective modification and attempts to reactivate the modified enzyme with hydroxylamine. Furthermore, the increase of the p K a values of carbodiimide-reactive groups in the presence of dioxane supports further evidence that they are carboxyls. The results suggest the p K a 5.28 carboxyl is in the active site, while the p K a 7.74 carboxyl is in its neighborhood buried in the enzyme molecule. The possible role for the carbodiimide-reactive carboxyl groups in catalysis is discussed.

Action pattern, kinetical properties and electrophoretical studies of an alpha-amylase present in midgut homogenates from rhynchosciara americana (diptera) larvae

1. 1. Rhynchosciara americana midgut amylase binds chloride ( K d = 1.6 mM at 37°C ) resulting in a 34-fold increase in the K cat without affecting the K m, but causing a shift in the optimum pH of the enzyme from 6.8 to 8.0. 2. 2. The ions Br −, NO 3 − and I − also activate the amylase but with a decreasing efficiency. 3. 3. An Arrhenius plot of activity versus temperature show that the amylase display only one slope with a corresponding apparent energy of activation of 4.8 Kcal/mole. 4. 4.|The amylase is a calcium-dependent metalloenzyme which shows a pure competitive inhibition by Hg 2+ and a pure non-competitive inhibition by Cu 2+ with K i respectively of 0.21 mM and 0.23 mM. 5. 5.|The resistance to the sulfhydryl reagents p-mercuribenzoate and iodoacetate together with the high Hg 2+ K i suggest that sulfhydryl groups are not essential for the midgut amylase activity. 6. 6. Curves of blue-reducing values showed the midgut amylase is an α-amylase with a multiple attack degree between 1.7 and 6. 7. 7. Polyacrylamide gel electrophoresis of midgut homogenates shows the existence of two major isoamylases one of which is largely predominant. 8. 8. The results are compared with α-amylases from several sources.