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.     

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.     

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.     

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.     

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.     

Purification and characterization of a thermal stable antimicrobial protein from housefly larvae, Musca domestica, induced by ultrasonic wave

This work describes the induction, purification and partial biochemical characterizations of an antimicrobial protein from the housefly larvae induced by ultrasonic wave. It has been purified to apparent homogeneity by ammonium sulfate precipitation followed by Sephadex G-75, Bio-gel P6 gel filtration, and CM-Sepharose Fast Flow cation exchange chromatography. The protein is a cationic protein with an apparent molecular weight of 16315 Da determined by no-denaturing electrophoresis and SDS-PAGE, respectively. Biochemical profile assays show that this protein has good thermal stability, and repeatedly frozen and defrosted durability. The optimum pH for antimicrobial activity is around pH5. The antimicrobial range of the protein includes Gram-positive, Gram-negative bacteria and some fungi. Results of the membrane permeability assays suggest that the probable mode of action of this protein is membrane-disrupting mechanism.     

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.     

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.     

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.     

Endonuclease activity of phenol oxidase from Musca domestica larvae

Phenol oxidase (PO), a copper-containing enzyme with oxygenase activity, can convert mono- or diphenol into quinone and plays an important role in the arthropod melanization reaction. Here, we report a new property of PO from Musca domestica larvae: a thermotolerant endonuclease activity, by which PO can degrade plasmid DNA even after being heated to 80 degrees C for 20 min. We cloned PO cDNA, constructed the expression vector pVAX1-PO, and expressed it in HeLa cells. The expression product showed the same properties as purified PO. Our data indicate that PO is a bifunctional enzyme, exhibiting both oxygenase and endonuclease activity, suggesting new roles for this important molecule in the innate responses of M. domestica.