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.     

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.     

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.     

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.     

Fine structure and X-ray microanalysis of mineralized concretions in the Malpighian tubules of the housefly, Musca domestica

The epithelium and the lumen of the Malpighian tubules of the housefly contains mineralized dense bodies called concretions. The morphological characteristics, mode of origin, nature of the sequestered elements and the age-associated changes in the distribution of concretions are reported. There are three types of concretions in the cytoplasm, which have been designated as type A, type B, and type C. Type A concretions are membrane-bound spherical structures which may arise by the gradual intravacuolar accumulation of dense material. Type B concretions appear to be related to multivesicular bodies. Type C concretions are heteromorphic and morphologically resemble the residual bodies. They show a positive localization of acid phosphatase reaction product. X-ray microanalysis of intracytoplasmic and intraluminal concretions revealed the presence of phosphorus, sulphur, chlorine, calcium, iron, zinc and copper. There was no evidence suggesting the extrusion of the intracytoplasmic concretions into the lumen of the Malpighian tubules. There is an age-associated increase in the distribution of type C concretions. It is hypothesized that the sequestration of metal ions within the concretions may provide a means for the effective excretion of these elements.     

Origin,structure, composition and age-dependence of mineralized dense bodies (concretions) in the midgut epithelium of the adult housefly, Musca domestica

The epithelial cells of the midgut in 1–40 day old adult female houseflies were examined by electron microscopic, X-ray microanalytic and histochemical techniques in order to study the mode of genesis, chemical nature and age-associated distribution of dense bodies. Dense bodies contain high concentrations of phosphorus, sulphur, chlorine, calcium, iron and copper; they are therefore termed concretions. Concretionary material is initially deposited within Golgi vesicles, lamellar bodies and residual bodies. The average size of the concretion granules and the concentration of the sequestered material increases with age, while new concretions are continually formed throughout life. With advancing age, concretions accumulate in the epithelial cells and occupy a considerable proportion of the cytoplasm in old flies. It is postulated that the concretions sequester superfluous minerals and may play an important role in the excretory system of the adult housefly.     

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.     

Growth of Musca domestica (Diptera: Muscidae) and Sarcophaga dux (Diptera: Sarcophagidae) larvae in poultry and livestock manures: Implication for animal waste management

Natural diets commonly exploited by the flies are animal manures including production from the poultry and livestock facilities. The larvae of the common filth flies such as Musca domestica and Sarcophaga dux are known as voracious feeders and may thus be used to convert manures into non-polluted residue. This study was conducted to observe the impact on flies' growth rate and capability of the larvae to process animal manures using chicken, goat and cow manures. One hundred newly hatched larvae of M. domestica and S. dux were introduced separately into 150?g manures under laboratory conditions. The initial wet mass and larvae length were recorded while mortality rate and dry mass were measured after the larvae were placed into the manures. The results showed that the manure types give significant effects (p?<?.05) on the growth of M. domestica and S. dux larvae. Cow manures and chicken manures contributed the highest growth for M. domestica and S. dux respectively. This result confirmed by the mean increment in wet mass and larvae length. In contrast, M. domestica greatly reduced 59.9?±?4% chicken manures while 25.0?±?1.8% goat manures reduced by S. dux. The potential of M. domestica and S. dux larvae to reduce animal waste products were further discussed in this study.     

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.     

Transmission of MdSGHV among adult house flies, Musca domestica (Diptera: Muscidae), occurs via oral secretions and excreta

The MdSGHV is a double-stranded DNA virus that replicates in the salivary glands of infected adult house flies. Transmission of this non-occluded, enveloped virus is believed to be mediated orally via deposition and consumption of oral secretions composed of salivary gland secretions and crop contents. In this study, transmission electron micrographs of crops from infected flies showed numerous enveloped virions in the crop lumen adjacent to the cuticular intima, as well as on the hemocoel side in close vicinity to muscle cells. Oral treatments of newly emerged flies with viremic salivary gland homogenates, crop homogenates, or gradient-purified virus resulted in an average 44% infection. Virus released via oral secretion was infectious when ingested by newly emerged adult flies, resulting in an average 66% infection. Using quantitative real-time PCR, MdSGHV DNA was quantified in oral secretions and excreta obtained from viremic flies. Between 2 and 4 days post-infection (dpi), viral copy numbers in oral secretions increased exponentially and from 5 to 21 dpi each infected fly released an average 10⁶ MdSGHV copies per feeding event. Excreta samples collected overnight from individual infected flies at 5 dpi contained an average 6.5 × 10⁵ viral copies. Low but detectable infection rates were produced when newly emerged flies were challenged with excreta samples. In summary, evaluation of the quantity and infectivity of MdSGHV released by individual infected house flies clearly showed that deposition of oral secretions and excreta onto a shared food substrate is the main route of natural MdSGHV transmission among adult house flies.     

The calcified puparium of the face fly, Musca autumnalis (Diptera: Muscidae)

The general composition of the calcified puparium of Musca autumnalis was determined. Ash-weight analyses show that 62% of the puparium is inorganic material. The major components of the puparium are calcium, magnesium, phosphate and carbonate. Calcium and magnesium phosphate are the predominant salts in the puparium, with a lesser contribution by their respective carbonates. Scanning electron microscopy revealed that the exocuticle was the site of calcification in the puparium. Inorganic analyses of the insoluble salt isolated from face fly larvae showed it to be compositionally identical with the puparial salt. Major organic components of the puparium are lipid, chitin and protein. The presence of carbonic anhydrase and alkaline phosphatase activity in post-feeding larvae was confirmed.     

Acetylcholine in the choline-deficient housefly, Musca domestica

The distribution of choline metabolites has been studied in the nervous system of housefly larvae reared on diets containing various amounts of choline or on diets containing either β-methylcholine (β-MCh) or N-dimethylethylcholine (DMECh). Adult houseflies were obtained from larvae reared on diets containing at least 4·6 times the level of choline in the basic diet or on diets containing the two choline analogues. The distribution of choline metabolites has been studied in the heads and thoraces plus abdomina of these adults.

Acetylcholine was found to be concentrated in the larval nervous system where it was synthesized preferentially to other choline metabolites when the amount of choline available to the insect was severely restricted. The acetylcholine content of the adult was between six and nine times that of the larval nervous system and approximately 70 per cent was concentrated in the head. The amount of acetylcholine present in flies obtained from larvae fed on diets containing the lowest amount of choline which allowed adults to develop was 230 pmole/insect. Flies obtained from larvae fed on diets with added β-MCh contained 150 pmole of acetylcholine/insect but no detectable acetyl-βMCh. Flies obtained from larvae fed on diets with added DMECh contained 43 pmole of acetylcholine and 127 pmole of acetyl-DMECh/insect.

It is concluded that the two choline analogues spare the choline requirement of the housefly by two different mechanisms. β-MCh displaces choline from the lipids of non-nervous tissue making more available for uptake into the nervous system where sufficient acetylcholine is synthesized for adult development. DMECh is a complete replacement for choline; the acetyl-DMECh acting as a neurotransmitter in place of acetylcholine.     

Development and physiology of follicular atresia during ovarian growth in the house fly, Musca domestica

Atretic follicles regularly occur in the ovary of the house fly, Musca domestica. The frequency of ovarian follicular atresia and the proportion of atretic follicles per ovary are related to the stage of oögenesis and to the age of the females. Only vitellogenic follicles may become atretic. The atresia may occur at any stage of vitellogenesis, though most follicles become atretic in mid-vitellogenesis. Atretic follicles are completely resorbed within 24–36 hr. The follicle cells may play a synthesizing role during growth and disintegrating one during follicle resorption. The induction of glycogen synthesis by the cessation of RNA and protein synthesis and by vitellogenesis in normal follicles is discussed. The same processes occur prematurely in the atretic follicle which can be thus distinguished by a high content of glycogen.     

Baculovirus-like particles in the midgut epithelium of the phantom midge, Chaoborus crystallinus (Diptera,Chaoboridae)

A rod-shaped microorganism of presumed baculoviral nature is described from larvae of Chaoborus crystallinus collected in Sweden. The infection was restricted to the midgut epithelium, causing strong nuclear hypertrophy. Sectioned rods measured 75–106 × 260–300 nm. Their envelope, similar to a unit membrane, was approximately 10 nm thick. The electron-dense center, interpreted as the nucleocapsid, measured 38–43 × 210–226 nm. The rods had a longitudinal unilateral evagination. Individual rods were connected to chains and arranged as paracrystalline structures, most prominent at the nuclear periphery. Infected midguts had a yellow discoloration, but showed no iridescence. The particles were similar in size and shape to nonoccluded baculoviruses, and the microorganism of C. crystallinus is compared to other nonoccluded microorganisms of verified or suspected baculoviral nature.     

A new cytoplasmic polyhedrosis virus from the salt-marsh mosquito, Aedes cantator (Diptera: Culicidae)

The ultrastructure, development, and histopathology of a new cytoplasmic polyhedrosis virus of Aedes cantator are described. Virus particles measure 70 nm in diameter, are icosahedral in shape, and consist of a central electron-dense core surrounded by a capsid with six projections. Occlusion bodies are irregular in size (0.5–3.0 μm) and shape and contain several virus particles. Virus particles are assembled within an interconnecting network of fine filaments and are occluded by the deposition of a proteinaceous crystal around groups of mature virus particles within a virogenic stroma. Infections are confined to cells of the cardia, gastric ceca, and posterior portion of the midgut, which hypertrophy and frequently lyse. Infected larvae die during the fourth larval instar or as pupae. The prevalence of infection in natural field populations is less than 1%.     

A new species of Nosema from Hylobittacus apicalis (Insecta: Mecoptera: Bittacidae)

An undescribed Nosema was found infecting adults of the mecopteran Hylobittacus apicalis. This microsporidium is described herein as the first record of a microsporidium from the order Mecoptera. The slightly pyriform spores measured 4.5 × 2.4 μm. Mature spores had 9.5–10 polar filament coils irregularly grouped in the posterior end. The life cycle and ultrastructure of the developmental stages were described, and were typical of other Nosema spp. This microsporidium was regularly recorded from adult Hylobittacus apicalis populations over a 10-year period and tbe incidence of infection increased during the summer.     

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. Received: 28 June 1996 / Accepted: 23 April 1997