A.G. de Bianchi

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.

Haemolymph amino acids and related compounds during cocoon production by the larvae of the fly, Rhynchosciara americana

Abstract A qualitative and quantitative analysis of free amino acids and related compounds in the haemolymph of Rhynchosciara americana was carried out for different periods of the fourth larval instar. Threonine, serine, proline, and glutamic acid make up 50 per cent of the total free amino acids in R. americana haemolymph just before the larvae start spinning the communal cocoon; after this the titre of most of the amino acids declines continuously. There are few peptides but these are present in high titres; they consist of two to three amino acid residues, of which the most important are histidine and aspartic acid. The fall in the haemolymph amino acid and peptide titres is insufficient to account for the silk protein which accumulates on the communal cocoon during the same period. The results are consistent with a silk protein origin from haemolymph proteins and haemolymph free amino acids. The origin and metabolic role of some haemolymph ninhydrin-positive compounds are 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 (Kd = 1.6 mM at 37°C) resulting in a 34-fold increase in the Kcat without affecting the Km, but causing a shift in the optimum pH of the enzyme from 6.8 to 8.0. 2. 2. The ions Br−, NO3− 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 Hg2+ and a pure non-competitive inhibition by Cu2+ with Ki 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 Hg2+Ki 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.

Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion.

The adult female Culex quinquefasciatus midgut comprises a narrow anterior and a dilated posterior region, with epithelia composed of a monolayer of adjacent epithelial cells joined at the apical portion by septate junctions. Densely packed apical microvilli and an intricate basal labyrinth characterise each cell pole. Our morphological studies suggest that, during blood digestion, the anterior midgut region also participates in an initial absorptive stage which is probably related to the intake of water, salts and other small molecules. This activity peaked by 6h after bloodmeal feeding (ABF) and ended approximately 18 h ABF, when the peritrophic membrane was already formed. After this time, absorption only occurred in the posterior region, with morphologic and biochemical evidence of high synthetic activity related to the secretion of proteases. Chymotrypsin, elastase, aminopeptidase, and trypsin reached their maximum activity at around 36 h ABF. Digestion products were apparently absorbed and transported to the basal labyrinth, from where they should be released to the hemolymph. At 72 h ABF, proteolysis had already ended and protein levels had returned to those observed before blood meal. The epithelium of the posterior region, however, did not return to its initial morphology, appearing quite disorganised. Additionally, from 48 h ABF onwards some epithelial cells showed morphological signals of apoptosis.

Vitellogenin and vitellin of Musca domestica Quantification and synthesis by fat bodies and ovaries

Abstract Vitellogenin and vitellin of Musca domestica are composed of about five subunits with apparent molecular weights of 54, 52, 51, 48 and 46 K. The native vitellogenin and vitellin were not studied and therefore the relationship between the subunits and the native proteins is not known. At the beginning of vitellogenesis the fat bodies are probably the main site of synthesis of vitellogenin, while at the end of vitellogenesis, this role is taken over by the ovaries. Vitellogenin is first detected in the haemolymph at stage 2 and increases to attain its maximal amount at the stage 7, the middle of the gonotrophic cycle. The activation of vitellogenin synthesis in the fat bodies must occur soon after adult ecdysis since vitellogenin is present in the haemolymph of flies at stage 2. Intake of protein is unnecessary for activation of vitellogenin synthesis.

The giant DNA puffs of Rhynchosciara americana code for polypeptides of the salivary gland secretion

Abstract It was found that the polypeptides correlated with the appearance of DNA puffs B2 and C3 in the salivary gland of Rhynchosciara americana are present in the salivary secretion. This makes it very likely that these polypeptides are used in spinning the communal cocoon.

Vitellogenins and other haemolymph proteins involved in the oogenesis of Rhynchosciara americana

Abstract The electrophoretical analysis of haemolymph proteins from Rhynchosciara americana , in the presence of sodium dodecyl sulphate revealed the occurrence of two vitellogenic polypeptides, V 1 and V 2 , with molecular weights of 240,000 and 220,000 respectively. These polypeptides are synthesized by the adult female fat bodies and are found in the yolk granules. The vitellin molecule is formed at least by the polypeptides V 1 , V 2 and two others, V 4 and V 5 with molecular weights of 67,000 and 55,000 respectively. All these polypeptides can be stained by PAS and by methyl green, suggesting that they are phosphoglycopeptides. Haemolymph proteins, other than vitellogenin, participate in egg formation, the most prominent being a monomeric larval protein of molecular weight 47,000. These larval proteins are found in the soluble fraction of the eggs and make a massive contribution to the total egg protein.

Expression patterns of the larval and adult hexamerin genes of Musca domestica

Hexamerins are proteins found in high abundance in the haemolymph of larval and adult insects. The expression patterns of the genes encoding the house fly, Musca domestica, hexamerins were determined by Northern analyses using cDNAs as probes. A cDNA, A1, hybridized to a fat body‐specific messenger RNA (mRNA) which is detectable in larvae until pupation. Antibodies raised to the larval‐specific hexamerin, Hex‐L, bind recombinant protein encoded by a 5′ rapid amplification of cDNA ends ( race) product of A1, A2, indicating that the A cDNAs likely represent the genes encoding Hex‐L. The F1, F2 and F3 cDNAs, corresponding to genes encoding an adult, female‐enriched hexamerin, Hex‐F, hybridized with an mRNA isolated from protein‐fed females which has a temporal expression profile similar to that observed for the accumulation of Hex‐F. Furthermore, expression of the mRNAs hybridizing to the F cDNAs is correlated with the abundance of Hex‐F protein during the gonotrophic cycles. The mRNA transcription profiles indicate that the Hex‐L and Hex‐F genes are regulated in a sex‐, tissue‐ and developmental phase‐dependent manner. This stage‐specific expression of hexamerins contrasts with the expression patterns of hexamerins seen in other insects. The conceptual translation products of larval hexamerin cDNAs showed identity with larval serum protein 1 (LSP1)‐type hexamerins while the deduced products of the female hexamerin cDNAs showed the highest identity with LSP2‐type hexamerins. Genomic analyses showed that the larval hexamerin and female hexamerin genes from M. domestica belong to two distinct multigenic families.

Aedes aegypti lipophorin

The purified lipophorin of Aedes aegypti (Diptera) is composed of two apolipoproteins: apolipophorin I (M(r)=224,000) and apolipophorin II (M(r)=73,000). The density of lipophorin is constant during the Aedes life-cycle and equal to 1.11 +/- 0.01 g/ml. The amount of lipophorin per animal, during the gonotrophic cycles, increases until 48 hr after blood-feeding and then decreases until there is a new blood intake. The density values and quantification of lipophorin during Aedes aegypti gonotrophic cycle suggest that the adaptation to a higher lipid transport demand during oogenesis in Aedes aegypti is accomplished by increasing the amount of lipophorin in the hemolymph. This response is different from that observed in Musca domestica (Diptera) that does not involve changes in hemolymph lipophorin levels.

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

Carbodiimide modification of the Rhynchosciara americana midgut trehalase (alpha, alpha-trehalose glucohydrolase, EC 3.2.1.28) at different pH values revealed the existence of two essential groups (pKa 5.28 and pKa 7.74) for the trehalos 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 pKa values of carbodiimide-reactive groups in the presence of dioxane supports further evidence that they are carboxyls. The results suggest the pKa 5.28 carboxyl is in the active site, while the pKa 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.