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.     

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.     

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.     

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.

     

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.     

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.     

A complex glutathione transferase gene family in the housefly Musca domestica

In most metazoans, the glutathione S-transferases (GST) are encoded by gene families, and are used to detoxify xenobiotics. We describe the structure of genomic loci coding for the GSTs in the housefly that have been implicated, by both genetic and biochemical means, in mediating insecticide resistance. In earlier work, we showed that one of the theta-class enzymes, MdGST-3, is overproduced in resistant flies and degrades certain insecticides. We used a fragment from a cDNA clone of MdGST-3 as a probe to screen a housefly genomic DNA bank in phage λ. This probe detected multiple gst loci. Genes for GSTs were found in five different, nonoverlapping λ clones, three of which carry multiple, closely linked gsts. Multiple genes for both MdGST-3 and MdGST-4 were found; some of which have introns in their 5′ untranslated regions. In adults, the only MdGST-3 enzymes that are expressed are encoded by the intron-free genes. A new theta-class GST (called MdGST-5) was also discovered. Fusion genes comprising 5′ MdGST-3 sequences and either MdGST-4 or MdGST-5 sequences in their 3′ halves were encountered at three separate loci. The genes described here are found in both the ancestral sensitive strain and the insecticide-resistant strains.     

Fenitroxon insensitive acetylcholinesterases of the housefly, Musca domestica associated with point mutations

The cDNA of AChE in the housefly, Musca domestica, was sequenced and individual flies were genotyped by this gene in an inhibition assay of AChE activity with an organophaspate, fenitroxon. Mutations at Gly(342) and Tyr(407), which are reportedly conserved in resistant strains of Drosophila, were associated with the insensitivity to fenitroxon. Two other mutations, Ile(162) and Val(260), did not have an apparent effect on insensitivity. However, the four mutations are located in the active site of the enzyme, and therefore the non-neutral mutations in this gene are considered to cause the insensitivity of AChE in the development of insecticide resistance of the housefly.     

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.     

Immunocytochemical localisation and biological activity of diuretic peptides in the housefly, Musca domestica

The distribution of a CRF-related diuretic peptide (Musca-DP) and the diuretic/myotropic insect myokinins in the central nervous system of larval and adult houseflies was investigated using antisera raised against Locusta-DH and leucokinin-I, respectively. Two separate, small populations of immunoreactive neurons are present in the brain and fused thoracic-abdominal ganglion mass. There is no evidence for these immunoreactivities being colocalised either within single neurons or at neurohaemal release sites. Crude extracts of tissues containing immunoreactive material increase fluid secretion by isolated Malpighian tubules from adult flies. Diuretic activity is highest in tissues containing myokinin-immunoreactive material. Consistent with this observation, myokinin analogues produce a four- to five-fold increase in fluid secretion, which is more than twice the response to Musca-DP. These effects are mimicked by treatments that increase intracellular calcium and cyclic AMP, respectively. When tested at threshold concentrations, the two classes of diuretic peptide act synergistically to accelerate tubule secretion, and their separate localisation may be important for the precise control of diuresis.