Isolation, characterization and biological activity of a diuretic myokinin neuropeptide from the housefly, Musca domestica

A competitive ELISA employing a polyclonal antiserum raised against leucokinin-I was used to isolate and purify a myokinin (muscakinin) from 1.05 kg of adult houseflies (Musca domestica). Following solid-phase purification, seven HPLC column steps were used to purify 4.8 nmol of leucokinin-immunoreactive material. Sequence analysis and mass spectrometry were consistent with the structure Asn-Thr-Val-Val-Leu-Gly Lys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly NH2. This peptide was synthesized and co-eluted with the natural peptide on three different HPLC columns. The activities of natural and synthetic muscakinin were identical, with both producing a 4-5 fold increase in fluid secretion by housefly Malpighian tubules at nanomolar concentrations. The presence of a pair of basic residues (Lys-Lys) suggested muscakinin might be processed further, with the peptide pGlu-Arg-Phe-His-Ser-Trp-Gly NH2 being produced by conversion of an N-terminal glutamine to pyroglutamic acid. However, this analog was 1000-fold less active than the intact peptide, comparable to the activity of AK-V which shares the same C-terminal pentapeptide sequence. The diuretic activity of muscakinin is more than double that of a previously identified CRF-related diuretic peptide (Musca-DP) from the housefly, and the two peptides act synergistically in stimulating fluid secretion. Muscakinin also increased the frequency and amplitude of contractions by housefly hindgut which might further contribute to the excretory process.     

Long-term light adaptation in photoreceptors of the housefly, Musca domestica

The photoreceptors of many animals adapt, when illuminated, by reducing their sensitivities to light and improving their response speeds. Light adaptation is usually considered to be rapid and complete within minutes. However, under bright light conditions, I show that functionally significant improvements in impulse response amplitude and speed continue over the course of an hour in photoreceptors of the fly, Musca domestica. After sustained illumination, the average information rate, a measure of signalling performance, improved by 28% in a sample of sixteen photoreceptors. This long-term light adaptation is a robust phenomenon across animals and is repeatable within the same cell when light-adapting sessions are separated by a period of darkness. White-noise analysis of voltage responses to light and current stimuli indicate that much of the long-term changes observed are attributable to an improvement in the reliability with which photoreceptors register the timing of photon absorptions. It is also found that the impedance amplitude of the photoreceptor increases during long-term adaptation, suggesting that the area of the photoreceptor's membrane is reduced.     

Circadian rhythm gene regulation in the housefly Musca domestica

The circadian mechanism appears remarkably conserved between Drosophila and mammals, with basic underlying negative and positive feedback loops, cycling gene products, and temporally regulated nuclear transport involving a few key proteins. One of these negative regulators is PERIOD, which in Drosophila shows very similar temporal and spatial regulation to TIMELESS. Surprisingly, we observe that in the housefly, Musca domestica, PER does not cycle in Western blots of head extracts, in contrast to the TIM protein. Furthermore, immunocytochemical (ICC) localization using enzymatic staining procedures reveals that PER is not localized to the nucleus of any neurons within the brain at any circadian time, as recently observed for several nondipteran insects. However, with confocal analysis, immunofluorescence reveals a very different picture and provides an initial comparison of PER/TIM-containing cells in Musca and Drosophila, which shows some significant differences, but many similarities. Thus, even in closely related Diptera, there is considerable evolutionary flexibility in the number and spatial organization of clock cells and, indeed, in the expression patterns of clock products in these cells, although the underlying framework is similar.     

Selenium-independent glutathione peroxidase activity associated with glutathione S-transferase from the housefly, Musca domestica

1. A glutathione S-transferase having Se-independent glutathione peroxidase activity was isolated from 100,000 g supernatant from housefly homogenate. 2. The specific activity of the partially purified Se-independent glutathione peroxidase was 1776 nmol NADPH oxidized/min/mg protein, representing an 87-fold purification. 3. The Mr of this enzyme was estimated to be 37,000 and 26,000 by gel filtration chromatography and gel electrophoresis, respectively. 4. Selenium-dependent glutathione peroxidase activity could not be detected in this same supernatant. 5. Se-independent glutathione peroxidase activity should be considered in future studies of the insect antioxidant defense system.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

Lipoid content and resistance in the housefly, Musca domestica L.

The lipoid content of several resistant and normal strains of houseflies was determined. A Swiss extremely DDT-resistant strain had a much higher fat content than a normal Swiss reference strain and several Italian strains. Among the Italian strains, a chlordane-resistant strain had a significantly higher fat content than a diazinon-resistant and a normal strain. This difference was no longer significant however, when the normal Swiss strain was included in the calculations. Iodine numbers, specific gravity and physical consistence showed no consistent difference between the fats of resistant and normal strains, except that the syphoned-off oils from fats of resistant flies seemed to be less viscous than those of normal flies of the same provenience.In a further series of experiments, the lipids were extracted from wheat bran, to which were added instead fats extracted from normal and from DDT-resistant housefly pupae; normal larvae were reared in a medium containing the fat from resistant pupae and vice versa. Neither this procedure nor that of offering cholesterol as sole larval lipid source, had any influence on resistance level in the adult state.

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Zusammenfassung Es wurde der Lipoidgehalt der Puppen resistenter und sensibler Hausfliegenstämme bestimmt. Ein hoch DDT-resistenter Schweizer Stamm hatte einen bedeutend höheren Fettgehalt als ein normaler Schweizer und verschiedene italienische Stämme. Unter den italienischen Stämmen, hatte ein Chlordan-resistenter Stamm einen signifikant höheren Fettgehalt als ein Diazinon-resistenter und ein normaler Stamm. Wenn jedoch bei der statistischen Auswertung dieser Resultate die Zahlen für den Schweizer normalen Vergleichstamm mit in Betracht gezogen wurden, waren die Unterschiede für den Chlordanstamm nicht mehr signifikant.Die Feststellung Wiesmanns, dass resistente Hausfliegen einen höheren Fettgehalt aufweisen, konnte also in unseren Versuchen nur in einem von drei resistenten Stämmen bestätigt werden. Es ist daher anzunehmen, dass die Tatsache vorhandenen höheren Fettgehaltes nicht für alle resistenten Stämme zutrifft. Die Wiesmann'sche Lipoidtheorie der Insektizidresistenz, die übrigens auch von ihm selbst nur als Teilmechanismus des Resistenzgeschehens aufgefasst wird, ist vielleicht für gewisse Stämme anwendbar, so z.B. für den Schweizer Stamm K₁, für den unsere Resultate mit den Ergebnissen Wiesmanns übereinstimmen. Die Theorie ist jedoch sicher nicht für jeglichen resistenten Hausfliegenstamm gültig.Wenn chemische und physikalische Eigenschaften der Fette, z.B. Jodzahl, Dichte und Konsistenz verglichen wurden, konnten keine klaren Unterschiede zwischen dem Fett resistenter und normaler Stämme gefunden werden. Vielleicht könnte man nur von einem Unterschied in der Viskosität der abgeheberten flüssigen Ölphase resistenter und normaler Fliegen gleicher Provenienz sprechen.Extrahierung der im Larvenfutter vorhandenen Fette und ihr Ersatz durch Fliegenpuppefette, oder Verabreichung von Cholesterin als einzige larvale, Fettquelle, hatte keinen Einfluss auf die Resistenz der Adulttiere.

     

Segmentation gene expression in the housefly Musca domestica.

Drosophila and Musca both belong to the group of higher dipteran flies and show morphologically a very similar early development. However, these two species are evolutionary separated by at least 100 million years. This presents the opportunity for a comparative analysis of segmentation gene expression across a large evolutionary distance in a very similar embryonic background. We have analysed in detail the early expression of the maternal gene bicoid, the gap genes hunchback, Krüppel, knirps and tailless, the pair-rule gene hairy, the segment-polarity gene engrailed and the homoeotic gene Ultrabithorax. We show that the primary expression domains of these genes are conserved, while some secondary expression aspects have diverged. Most notable is the finding of hunchback expression in 11-13 stripes shortly before gastrulation, as well as a delayed expression of terminal domains of various genes. We conclude that the early developmental gene hierarchy, as it has been defined in Drosophila, is evolutionary conserved in Musca domestica.     

Developmental patterns in the antioxidant defenses of the housefly, Musca domestica

The activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione S-transferases, GSSG reductase, thiol transferases, gamma glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase, and the concentrations of H2O2 and reduced and oxidized glutathione were determined in the various developmental stages of houseflies. Housefly development was correlated with a progressive increase of cellular oxidizing equivalents and a loss of cellular reducing capacity. The loss of reducing equivalents appeared to result from a decrease in the activity of enzymes involved in glutathione and NADPH synthesis and a concomitant increase in glutathione-oxidizing enzymes. Relatively little change was observed in SOD activity during housefly development; however, the electrophoretic pattern of MnSOD varied in a manner specific to developmental stage. A striking increase in H2O2 concentration occurred prior to pupation possibly due to changes in substrate catabolism. These results support the hypothesis that the cellular environment becomes progressively more oxidizing during development.     

Dietary cholesterol requirements of housefly, Musca domestica, larvae.

Quantitative analyses have been made of the dietary cholesterol requirement for the growth of the larvae of Musca domestica. The larvae will not grow on diets to which no cholesterol is added, a few pupae and adults are obtained when the concentration of cholesterol is 0·05 μmol/g of diet, but the concentration has to be raised to 0·36 μmol/g of diet before the maximum numbers of pupae and adults are obtained. Further addition of cholesterol above 0·36 μmol/g diet did not have any significant effect on the weight and growth of the larvae. However, the ratios of the cholesterol to phospholipid fractions recovered from the larvae increased rapidly when the concentration of cholesterol in the diet was raised from 0·05 to 0·56 μmol/g of diet. Above this concentration only a slight increase in the ratios was observed. Larvae reared on diets containing 0·05 μmol cholesterol/g of diet contain only 25 per cent of the cholesterol content of the larvae reared on the diets containing more than 0·28 μmol of cholesterol/g of diet, the cholesterol content being expressed relative to the weight of the larvae,The absence of cholesterol synthesis has been demonstrated in the larvae by feeding [4-¹⁴C] cholesterol. The specific activity of the cholesterol recovered from the larvae is the same as that of cholesterol added to the diet. Irrespective of the cholesterol concentration of the larval diet, approximately 97 per cent of the radioactivity recovered from the larvae behaved as free cholesterol, less than 1 per cent as cholesterol esters and the rest as unidentified ‘polar sterols’. The results are compared with those from similar studies on other insects.     

Preliminary studies on the zinc-induced metallothionein protein with antibacterial activity in housefly larvae, Musca domestica

Three isoforms of metallothionein protein induced with Zinc were isolated and purified from housefly larvae, Musca domestica, by gel filtration on Sephadex G-75, G-25 and anion exchange on DEAE-52 chromatography. Among them, one was found to possess antibacterial activity, and was further characterized by SDS-polyacrylamide gel electrophoresis, sulphydryl group determination, enzyme hydrolysis, and spectra property. Our results showed that the novel protein has the characteristics of heat-stable, low-molecular weight (6 kDa), rich-cysteine (approximately 12 cysteine residues in one molecule), metal affinity, and antibacterial activity. This paper was the first to report that metallothionein had antibacterial activity. We expect that this characteristic would give some help to investigate definite physiological functions of metallothionein.     

Distribution and substrate specificity of esterases in the housefly,Musca domestica L.

Housefly homogenates perform high cholinesterase and ali-esterase activity. Warburg-manometric studies show that acetylcholine, acetyl-β-methylcholine, butyrylcholine, and benzoylcholine are exclusively hydrolysed by a cholinesterase, the properties of which are more or less comparable to those of true vertebrate cholinesterase. Methylbutyrate and tributyrin are hydrolysed by one or possibly several ali-esterases but not by the cholinesterase. The cholinesterase activity is mainly present in the head and the ali-esterase activity in the thorax and the abdomen. Various other substrates, e.g. methylacetate, ethylacetate, amylacetate, triacetin, phenylacetate, and o-nitrophenylacetate are hydrolysed by both the cholinesterase and the ali-esterases. There is some evidence that o-nitrophenylacetate hydrolysis is partly due to an organophosphate-resistant enzyme distinct from the cholinesterase and ali-esterases, which are both inhibited by low concentrations of organophosphates. The ali-esterase activity is rapidly destroyed at 37°C in slightly alkaline media, whereas the cholinesterase is not affected by these conditions.     

Occurrence and metabolism of prostaglandins in the housefly,Musca domestica (L.)

Prostaglandins (PGs) of the E and F series were quantified in the housefly by radioimmunoassay (RIA). Whole insects and reproductive tissues from both sexes contained PGE₍₁₊₂₎ and PDG_2α which increased in amount with age. PGF_2α levels were higher than PGE series in extracts of whole male and female insects and in ovaries. Male reproductive tissues contained higher amounts of PGE₍₁₊₂₎ than PGF_2α. Gas chromatographic-mass spectrometric (GC-MS) analyses of the products formed after injection of arachidonic acid (20:4) and eicosatrienoic acid (20:3, n-6) into male and femal insects demonstrated the conversion of 20:4 to PGF_2α and 20:3 to PGF_1α. Radiolabeled 20:4 injected into houseflies was rapidly converted to PGE₂ and PGF_2α. The catabolism of PGE₂ was more rapid than PGF_2α in males, whereas in females, PGE₂ and PGF_2α were converted to more polar products at similar rates. Radiolabeled 20:4 injected in the hemolymph was incorporated into the reproductive tracts of male insects. About 2.1% of the total radioactivity from [³H]20:4 injected into males just prior to mating was transferred to females during mating. Thus, PG are formed from 20:4 in male and female houseflies. During mating, 20:4 is transferred from males to females where it can be metabolized to PGF_2α.     

Glutathione depletion associated with rose bengal-photosensitized mortality in the housefly,Musca domestica

Glutathione, pyridine nucleotides, and lipid peroxides were measured in adult houseflies following various regimens of dye treatment and light exposure. Comparisons were made between dark control and light control flies to judge the effect of light exposure alone; between dark control and dark, dye-treated flies to evaluate the effects of dye-feeding in the dark; and between dark, dye-treated and light, dye-treated flies to measure the effect of photodynamic action. No significant effect was observed in levels of NAD⁺, NADH, or NADP⁺. However, a decrease (～ 16.7%) in NADPH during photodynamic treatment was measured. Relatively small inductions of glutathione were observed in light controls and dark, dye-treated flies. Depletion of both GSH and total glutathione (the sum of GSH and GSSG, expressed as GSH equivalents) occurred in light, dye-treated flies as compared to dark, dye-treated flies. Depletion of NADPH, when related to GSH depletion, suggested that GSH is being utilized to conjugate some products of photooxidation or that it is being directly oxidized to GSSG. However, the observation of a reduction in total glutathione also suggests that a fraction of GSH is being either oxidized to a product other than GSSG or irreversibly conjugated. No significant effects from photodynamic treatment on peroxidative potential or lipid hydroperoxides were observed.