Effect of cholesterol deficiency on the composition of phospholipids and fatty acids of the housefly, Musca domestica

The housefly larvae were grown in the aseptic diet containing 0.56 μmole cholesterol/g wet weight of diet (control) and 0.05 μmole cholesterol/g wet weight of diet (deficient). The effects of cholesterol deficiency upon the phospholipid composition and fatty acids of the total phospholipid and triglyceride fractions from the lipid extract of the various larval tissues, whole larva, and in both sexes of adults 4 days after eclosion were examined. The total sterol and phospholipid contents (expressed relative to the wet weight of the insect) of the control and deficient insects at the larval and adult stages were analysed and molar ratios compared. The results suggest that cholesterol deficiency reduced the free sterol content of the larvae and adult insects to approximately 25% of the content of the control insects. However, cholesterol deficiency did not effect the phospholipid content during larval and adult stages when compared to that of control insects. Though the larvae reared on the cholesterol deficient diets did not show a profound alteration in the phospholipid composition, a marked increase in the ratio of phosphatidylcholine to phosphatidylethanolamine of the larval fat body and composite gut fraction were noticed. The cholesterol deficiency induced significant changes in the fatty acid composition of the phospholipid fraction of the insect. The ratio of unsaturated fatty acids to saturated fatty acids of the phospholipid fractions decreased significantly due to cholesterol deficiency in the whole larvae and in both sexes of adult flies. The data indicates that cholesterol deficient insects compensated for the lack of cholesterol by increasing saturated fatty acids preferentially in the phospholipid fraction of the lipids for the maintenance of proper membrane fluidity.     

Radiation-induced dominant lethal mutations in oocytes of Musca domestica

Dominant lethal mutations induced by γ-radiation were measured in stage-7 and stage-14 oocytes of Musca domestica. At both stages the data are consistent with the multi-hit theory on radiation induction of dominant lethals. This conclusion is supported by fractionation experiments which indicate that both] S7 and S14 oocytes are capable of repairing, in defferent periods of time, a similar amount of dominant lethal damage.     

Efficacy of entomopathogenic bacteria for control of Musca domestica

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.     

Properties of olfactory conditioning in the housefly, Musca domestica

The housefly, Musca domestica, was conditioned to odours using the proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response and the properties of conditioning were investigated. Among trials including forward pairing of the conditioned stimulus (CS) with the unconditioned stimulus (US), backward pairing and isolated presentations of CS and US, only forward pairing is effective on the acquisition of conditioning. Backward pairing combined with forward pairing does not influence the effectiveness of the forward pairing. CS given overlapping with a US presentation permits only weak conditioning. The acquisition of conditioning decreases with increase of the CS-US interval. In the differential conditioning situation to two odours, discriminative responses are observed. In the flies conditioned with one antenna, the conditioned response is elicited not only by stimulation of the antenna used for conditioning but also by stimulation of the antenna not used for conditioning, although the response using the former is higher than with the latter. The ability to be conditioned is reduced immediately after fastening on a clay bed and increases with time. Ability can also be improved by transection of the ventral nerve cord.     

Storage-excretion of metallic cations in the adult housefly, Musca domestica

The objective of this investigation was to elucidate further the phenomenon of storage-excretion of metallic cations in the housefly. The sites for deposition of zinc, calcium and copper have been identified in this study. The metal intake of the flies was altered by raising one group on sucrose and tap water while in the experimental groups sucrose was supplemented with either 0.05% zinc sulphate, 0.05% calcium phosphate or 0.03% copper sulphate. There were no significant differences in the average life spans of the flies in different groups indicating physiological tolerance to the higher mineral intake. The Malpighian tubules, the midgut and the remainder of the body were analyzed for mineral content in houseflies ranging from 1 to 25 days posteclosion by atomic absorption spectrophotometry. There was a progressive age-associated increase in the total metal content of the flies with age. Zinc and calcium were primarily stored in the Malpighian tubules whereas copper was sequestered in the midgut. Microscopic examination of the epithelial cells of the Malpighian tubules and the midgut revealed a corresponding age-associated increase in mineralized concretions.     

Synthesis of fatty acids, in vitro by choline-deficient and normal larvae of the housefly, Musca domestica

The synthesis of long chain fatty acids from acetate by a de novo pathway and by direct elongation of endogenous fatty acids has been demonstrated in homogenates of 4-day-old housefly larvae. The distribution of the synthesized fatty acids among the main classes of lipid has been studied. Addition of coenzyme-A to the medium inhibited the de novo synthesis pathway and made elongation the main synthetic route by which the radioactive acetate was incorporated into fatty acids. Direct elongation of palmitoleic to vaccenic acid has been demonstrated to occur in the homogenates. No consistent differences could be observed in the amount and distribution of the radioactivity incorporated into the fatty acids of homogenates prepared from larvae reared on a choline-deprived or a choline-sufficient diet. Addition of phosphatidylcholine to such homogenates also produced no changes in the labelling patterns. It was concluded that the changes seen, in vivo, in the fatty acids of the phospholipids, which accompany alteration of the amount of lipid-choline in the larvae, were unlikely to be due to any direct effect of the phosphatidylcholine on the enzymes involved in fatty acid synthesis.     

Effect of dietary cholesterol deficiency upon its distribution in the larvae of the housefly, Musca domestica

The free sterol, total phospholipids and protein content of the various tissues and haemolymph lipoproteins obtained from the larvae of Musca domestica, reared on the diets containing 0.56 μmole cholesterol/g wet weight of diet (normal) and 0.05 μmole cholesterol/g wet weight of diet (deficient) have been determined. The cholesterol in the diet was found to be taken up by the larvae and distributed between all the tissues examined. About 60% of the free sterol in the larvae was recovered from the composite gut fraction and muscle. Cholesterol deficiency reduced both the growth of larvae and the free sterol content of the various tissues and haemolymph when compared to that of normal larvae. Cholesterol deficiency resulted in a slightly higher proportion of sterol and protein of the larval haemolymph being associated with the lipoproteins having slower electrophoretic mobility. Most of the different tissues from the cholesterol deficient larvae contained a much smaller proportion of their normal free sterol content than of their phospholipid or protein; the brain tissue however contained a higher percentage of free sterol and the haemolymph a much lower percentage than would be expected from the lowering of phospholipid and protein content as a result of the deficiency. When the sterol content was expressed relative to the protein, the ratio was higher in the brain tissue of both the normal and deficient larvae than the ratio present in the remaining tissues, apart from the composite gut fraction of the normal larvae. The results suggest that a disproportionate amount of available cholesterol was being concentrated into the nervous system of the cholesterol deficient insect.A rather higher proportion of the total sterol fraction recovered from the various tissues and haemolymph lipoproteins of cholesterol deficient larvae behaved as ‘polar metabolites’ of cholesterol when compared with that of normal larvae.     

Local search in the housefly Musca domestica after feeding on sucrose

Local search behaviour of the housefly, Musca domestica, released by ingestion of sucrose, was recorded on a bitpad digitizer. Local search is characterized by an initial increase in turning rate and a decrease in locomotory rate followed by an exponential return to control (unfed) levels for both functions. Greater variation in the slope of the exponential return to control levels was observed between runs of different flies than between runs of an individual fly, suggesting a possible internal basis for locomotory and turning functions. The local search pattern, based on decrease in relative turning rate generates a spiral configuration. A zig-zag component increases path width and increases the chances of a fly relocating the drop residue. Looping patterns correlate with successive recontacts with the drop residue and associated changes in turn direction and stopping. The results are discussed with respect to sources of internal and external orientation information controlling local search.     

Classical conditioning to visual stimuli in the housefly, Musca domestica

In the housefly, Musca domestica, classical conditioning to monochromatic light was demonstrated by using a proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response. Sequential and temporal relationships between the conditioned stimulus (CS) and unconditioned stimulus (UCS) to form conditioning were studied; conditioning occurred most readily when the CS was presented before the UCS without an interstimulus interval. Spectral response curves, which were obtained by examining the responsiveness of flies conditioned for one colour to other colours, were divided into two groups; one was that of 462 nm-conditioned flies and the other those of 516 nm-, 579 nm-, and 642 nm-conditioned flies.     

Multiplicity of cytochrome P-450 in microsomal membranes from the housefly, Musca domestica

The heterogeneity of cytochrome P-450 in abdominal microsomes from the CSMA, SBO, Fc, Rutgers and Baygon strains of the housefly was examined by three different methods. Examination of ‘apparent absolute absorption spectra’ indicated at least two types of cytochrome in all strains, one with an absorption maximum at about 394 nm, being present in greater quantity in the insecticide-resistant strains, while the other, with an absorption maximum at about 412 nm, predominates in the insecticide-susceptible strains.Controlled tryptic digestion of microsomes followed by spectral examination at various time intervals indicated a heterogeneous population of cytochromes P-450 in CSMA, Fc and Rutgers strains.Subfractionation of microsomes from houseflies of the CSMA and Fc strains by a two-step discontinuous sucrose gradient centrifugation method provided evidence for cytochromes P-450 of different spectral characteristics. The concentration of cytochrome P-450, as well as its spectral characteristics varied between fractions and strains.     

Changes in fatty acid composition of peripheral nerve myelin in essential fatty acid deficiency

Severe essential fatty acid deficiency (EFAD) was induced by feeding weanling rats a diet free of essential fatty acids 8 months after weaning. The fatty acid compositions of phospholipids and glycosphingolipids in peripheral nerve myelin were compared in rats with and without EFAD. With the deficient diet, 20:3ω9 was found in the major myelin phospholipids. The level of 18:1 was increased and the levels of 18:2ω6, 20:4ω6, and 22:4ω6 were decreased. Both sphingomyelin and cerebroside showed higher proportion of 24:1 and lower proportions of 24:0 in EFA-deficient rats than in control rats. The fatty acid chain elongating system in myelin cerebroside was also depressed by EFAD. A two- to sevenfold increase of the ratio 20:4ω6 to 20:3ω6 was found in myelin phospholipids of regenerated nerve from rats fed control diet. However, this ratio was suppressed by EFAD diet. The biochemical index (20:3ω9/20:4ω6) for EFAD was not affected by crush injury. These results suggest that dietary EFAD in postweaning rats can induce fatty acid alterations in peripheral nerve myelin without resulting in detectable changes in function or structure and that myelin lipids may be sequestered and reused during nerve degeneration and regeneration.     

Fluid secretion by isolated Malpighian tubules of the housefly Musca domestica

The Malpighian tubules of Musca domestica secrete a fluid with a high concentration of potassium and low concentrations of sodium, calcium, magnesium and chloride compared with the isolating medium.Low secretion rates are produced by low medium potassium concentrations (< 7 mM), with low sodium concentrations (up to 5 mM) increasing secretion; higher potassium concentrations produce higher secretion rates whilst higher sodium concentrations have no further effect. Calcium and magnesium are essential for secretion.The rate of tubule secretion is inversely proportional to the osmotic pressure of the isolating medium and the osmotic pressure of the secreted fluid is slightly hyper-osmotic to the medium over a range of medium osmotic pressures.The metabolic inhibitors cyanide, iodoacetate and 2,4-dinitrophenol inhibit secretion: Cu²⁺ ions, arsenate and ouabain have no effect whereas ethacrynic acid abolishes secretion. 5-hydroxytryptamine, cycle AMP and theophylline have no effect on secretion. Sodium thiocyanate stimulates fluid secretion and increases the osmotic pressure and the concentration of sodium and chloride, but not potassium, in the secreted fluid.     

Effect of habitat and motor activity of molluscs on fatty acid composition of triglycerides and phospholipids

A comparative analysis of fatty acids (FA) in neutral lipids and phospholipids of digestive gland and pedal muscle has been performed in molluscs from various ecological groups differing by belonging to sea or fresh water, trophic types or the associated motor activity. In freshwater pulmonary gastropods Lymnaea stagnalis and Lymnaea ovalis and marine prosobranchial molluscs Buccinum undatum and Littorina littorea the total content of ω3-acids in phospholipids of the studied tissues differed more than twice, predominantly due to the combined effect of temperature and salinity of the habitat. The lower viscosity of cell membranes in marine species (ω3/ω6 < 1) is determined to the greatest degree by the presence of eicosapentaenoic acid that accounts for 22–25% of the FA sum in marine species. Comparison of the molluscs by their trophic belonging has revealed the presence of linoleic acid in triglycerides in digestive glands of phytophages (8–12%), but the practically complete absence of this acid in the predator B. undulatum (< 0.8%). By mobility, L. littorea inhabiting the high-low tide littoral was inferior to freshwater pulmonary gastropods and to the marine predator, as it stops moving twice a day during the low tide. In phospholipids of pedal muscle of this mollusc the amount of long-chain polyunsaturated C: 22 FA was 3–6 times lower than that in other studied species, which might possibly indicate the role of these acids in functioning of the pedal muscle contractile tissue. On the whole, use of the FA characteristics as the parameters determining belonging to certain ecological group requires a certain caution due to a complex action of biotic and abiotic factors on the animal metabolism. The exception is the ω3/ω6 ratio in total phospholipids of fresh water and marine gastropods.     

Changes in fatty acid composition of sulfolipid and phospholipids during maturation of alfalfa

Lipids were extracted from alfalfa samples collected at intervals over the growing season and were fractionated to yield pure sulfolipid. In the sulfolipid and in a phospholipid fraction the major fatty acids were palmitic, linolenic, and linoleic, of which the palmitic acid increased in proportion during the season while the proportion of linolenic acid dropped. The sulfolipid contained more linolenic acid and less palmitic and linoleic acids than the phospholipids, and had a greater rate of change of fatty acid composition.     

Effects of zinc on programmed cell death of Musca domestica and Drosophila melanogaster blood cells

Programmed cell death (PCD) and phagocytotic activity of immune cells play a pivotal role in insect development. We examined the influence of Zn²⁺, an important element to fundamental biological processes, on phagocytosis and apoptosis of hemocytes in two fly species: Musca domestica and Drosophila melanogaster. Hemocytes were isolated from the third instar larvae of both species and treated for 3 h with zinc chloride solutions, containing 0.35 mM or 1.7 mM of Zn²⁺, and untreated as control. Phagocytotic activity of hemocytes was examined by flow cytometry after adding latex fluorescent beads to the medium, while apoptosis was evaluated by application of annexinV-FITC and pan-caspase-FITC inhibitor. Mitochondrial viability was determined by measuring resazurin absorbancy in the cell medium. The obtained results showed that Zn²⁺ increases phagocytosis and affects PCD of both species hemocytes but each in a different way. Zinc decreases fraction of annexin-positive hemocytes in M. domestica but increases it in D. melanogaster. The pan-caspase analysis revealed low and high activity of caspases in hemocytes of M. domestica and D. melanogaster, respectively. Zn²⁺ also decreased the viability of hemocyte mitochondria but only in D. melanogaster. It suggests that flies use different pathways of PCD, or that Zn plays a different role in this process in M. domestica than in D. melanogaster.     

A new virus disease in the housefly, Musca domestica (Diptera)

Reovirus particles were isolated from adults in laboratory colonies of the housefly, Musca domestica. These particles were spherical in outline, 57–76 nm in diameter, and were found only in hemocyte cytoplasm, where virions have been disclosed by a new technique. Virions were present in large numbers, and viral inclusion bodies were identified. The virus particles had pentagonal and hexagonal shapes resembling a simple icosahedral structure. The virus was shown to be infectious and pathogenic to adult flies through injection or by feeding them suspensions from flies that had died of the virus. Electron micrographs of midgut sections from infected flies showed that the midgut cells were packed with dark undulating threads which were not present in uninfected flies. However, no virus particles or inclusion bodies could be seen in these cells. On the basis of their association with infected flies, and the similarity to results from other studies on reoviruses and insect viruses, it is suggested that these threads are an alternative replicative form of the reovirus. When the virus suspensions from heavily infected flies were dialyzed against weak alkaline solutions, the threads showed an inner component of coiled material, 12 nm in diameter, inside an envelope with a diameter of 50–83 nm, mean 60.3 ± 7.5, composed of subunits 7–8 nm long and 7–8 nm across.     

Cloning,expression, and purification of a new antimicrobial peptide gene from Musca domestica larva

Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram − bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.