Enhancement of the Musca domestica hytrosavirus infection with orally delivered reducing agents.

House flies (Musca domestica L.) throughout the world are infected with the salivary gland hypertrophy virus MdSGHV (Hytrosaviridae). Although the primary route of infection is thought to be via ingestion, flies that are old enough to feed normally are resistant to infection per os, suggesting that the peritrophic matrix (PM) is a barrier to virus transmission. Histological examination of the peritrophic matrix of healthy flies revealed a multilaminate structure produced by midgut cells located near the proventriculus. SEM revealed the PM to form a confluent sheet surrounding the food bolus with pores/openings less than 10nm in diameter. TEM revealed the PM to be multilayered, varying in width from 350 to 900 nm, and generally thinner in male than in female flies. When flies were fed on the reducing agents dithiothetriol (DTT) or tris (2-caboxyethyl)phosphine hydrochloride (TCEP) for 48 h before per os exposure to the virus, infection rates increased 10- to 20-fold compared with flies that did not receive the reducing agent treatments. PM's from flies treated with DTT and TCEP displayed varying degrees of disruption, particularly in the outer layer, and lacked the electron-dense inner layer facing the ectoperitrophic space. Both drugs were somewhat toxic to the flies, resulting in>40% mortality at doses greater than 10mM (DTT) or 5 mM (TCEP). DTT increased male fly susceptibility (55.1% infected) more than that of females (7.8%), whereas TCEP increased susceptibility of females (42.9%) more than that of males (26.2%). The cause for the sex differences in response to oral exposure the reducing agents is unclear. Exposing flies to food treated with virus and the reducing agents at the same time, rather than pretreating flies with the drugs, had no effect on susceptibility to the virus. Presumably, the reducing agent disrupted the enveloped virus and acted as a viricidal agent. In summary, it is proposed that the reducing agents influence integrity of the PM barrier and increase the susceptibility of flies to infection by MdSGHV.

Observations on house fly larvae midgut ultrastructure after Brevibacillus laterosporus ingestion.

The pathological and histopathological course caused by Brevibacillus laterosporus on house fly larvae has been investigated conducting observations on insect behavior and midgut ultrastructure. After dissection and fixation, gut tissues were analyzed under transmission electron microscopy (TEM) in order to compare in vivo-treated and non-treated (control) fly specimens. Treated larvae showed extensively reduced feeding and growth rate, then became sluggish and died within 72 h. A progressive midgut epithelium deterioration was observed in treated larvae, compared to the control. Ultrastructural changes consisted of microvilli disruption, cytoplasm vacuolization and general disorganization, endoplasmic reticulum deformation, mitochondria alteration. Deterioration became progressively more dramatic until the infected cells released their content into the gut lumen. Disruption was associated also with midgut muscular sheath and connective tissue. These ultrastructural changes are similar to those widely described for other entomopathogenic bacteria, such as Bacillus thuringiensis, against different insect species. The rapid disruption of cellular fine structure supports a hypothesis based on an interaction of toxins with the epithelial cell membranes reminiscent of the specific B. thuringiensis δ-endotoxins mechanism of action on other insect targets.

Insecticidal evaluation of essential oils of Citrus sinensis L. (Myrtales: Myrtaceae) against housefly, Musca domestica L. (Diptera: Muscidae).

The housefly, Musca domestica L., is one of the most common insects, associated with vectoring of various etiological agents. In order to search for effective control agent, the essential oil of sweet orange [Citrus sinensis (L.) Osbeck] was evaluated for its insecticidal activity against the larvae and pupae of housefly using contact toxicity and fumigation bioassays. In the contact toxicity assay, lethal concentration, LC(50) of C. sinensis essential oil against housefly larvae, varied between 3.93 and 0.71 µl/cm(2) for different observation days, while lethal time, LT(50), varied between 5.8 to 2.3 days. Mortality of larvae were significant with different concentrations (F = 2.79, df = 4, P < 0.05) and time (F = 6.69, df = 3, P < 0.01). In fumigant assay for housefly larvae, LC(50) of 71.2 and 52.6 µl/l was obtained in 24 and 48 h, respectively. Scanning electron microscopy of oil treated larvae revealed extreme dehydration and surface distortion while control larvae were free from any of the above symptoms and presented smooth surface, conforming effect of essential oil on housefly larvae. Percentage inhibition rate of oil against housefly pupae was 27.3-72.7% for contact toxicity and 46.4-100% for fumigation assay. Compositional analysis of C. sinensis essential oil using gas chromatography/mass spectrometry (GC-MS) revealed D: -limonene (73.24%), α-pinene (5.86%) and myrcene (4.45%) as major components whereas its vapour profile (solid-phase micro extraction-GC/MS) was dominated by D: -limonene at 92.57%. Significant activity of C. sinensis essential oil against larvae and pupae of housefly, pave the way for its use as eco-friendly housefly control measure.

Two hytrosaviruses, MdSGHV and GpSGHV, induce distinct cytopathologies in their respective host insects.

Recently, a new virus family (Hytrosaviridae) was proposed for double-stranded DNA viruses that cause salivary gland hypertrophy in their dipteran hosts. The two type species, MdSGHV and GpSGHV, induce similar gross pathology and share several morphological, biological, and molecular characteristics. This histological study revealed profound differences in the cytopathology of these viruses supporting their previously proposed placement in different genera.

Ultrastructure of the Immature Stages of Musca domestica (Diptera: Muscidae: Muscinae).

Musca domestica (Linnaeus, 1758) is a muscoid species that is widespread throughout the world and acts as a mechanical vector of different enteropathogens primarily in underdeveloped countries. The adult and its immature forms are associated with decaying organic matter and can be seen visiting human corpses and animal carcass, and the larvae can also cause an infestation on human and animal wounds, feeding on the tissues. These characteristics make them have a forensic significance and, mainly, a great sanitary importance. This study aimed to analyze and describe morphological aspects of their immature stages of M. domestica, including the eggs, the first-, second-, and third-instar larvae, and the puparium using scanning electron microscopy (SEM). The eggs have standard format that all muscoid flies shared. The exochorion has some modified cells that were used for embryonic respiration. The first-instar larvae present two openings on the posterior spiracle as in the second-instar larvae, but with the last one, we can observe the anterior spiracle structure. The third-instar larvae, as in some other Muscidae species, have a posterior spiracular opening with a sinuous form that is located near the edge of the spiracular plate. The puparium morphology is equal to the third instar with a respiratory structure that helps the breathing during the metamorphosis process. This article supports the need for knowledge over the morphological characteristics of the immature forms of the muscoid Diptera (Linnaeus, 1758) at the same time helping with the correct identification of this insect phase.

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 10(6) MdSGHV copies per feeding event. Excreta samples collected overnight from individual infected flies at 5 dpi contained an average 6.5 x 10(5) 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.

[How many neuroblasts build mushroom bodies in Lucilia caesar L. and Musca domestica L. (Diptera, Brachycera Cyclorrhapha)?].

The green-bottle fly Lucilia caesar and the housefly Musca domestica differ greatly in the number of neuroblasts producing mushroom bodies. Four neuroblasts were found in each mushroom body of Lucilia pupae, and its calyx has a quadruple structure. In the housefly, the number of mushroom body neuroblasts rises up 20 in each brain hemisphere. This leads to a more complicated calyx structure. The neuroblast number observed in Lucilia and Musca is compared with that found in other Diptera.

Combination of active behaviors and passive structures contributes to the cleanliness of housefly wing surfaces: A new insight for the design of cleaning materials.

Evolutionary pressure has pushed many extant plants and animals to develop micro/nanostructures on their surfaces to keep them clean. These structures have become ideal models for bio-inspired design. Although microstructures on biological surfaces have been widely studied, little attention has been paid to the combined role of microstructures and animal's active cleaning behaviors in keeping their surfaces clean. In this study, we explored the relationship between these micro/nanostructures and wettability as well as the role of the housefly cleaning behaviors in keeping their wings clean. Hierarchical structures consisting of microscale macrotrichias with nanoscale grooves on the wings were observed under scanning electron microscope. The wings were hydrophobic (CA = 133.3°) but with high adhesion to water (CAH = 87.5°), indicating that they were non-self-cleaning surfaces. Macroscale droplets standing on the wings could be best described as being in a transitional wetting state between Wenzel and Cassie-Baxter states due to the presence of the nanoscale grooves, which increased the resistance to water penetration. The hydrophobicity decreased (CA = 109.9°) when the nanostructures were removed by coating the wings with a thick layer of polydimethylsiloxane (PDMS). The houseflies could highly efficiently remove the microscale droplets atop the macrotrichias, and reduce bacterial contamination on their wings through grooming and flutter activities. These active cleaning behaviors could offset the absence of self-cleaning properties and play a key role in keeping the wings clean. The results indicate that housefly wings could be used as a template for the design of special functional surfaces. The present findings not only improve our understanding of the wettability and cleaning properties of natural surfaces, but also provide important insights into the design of bio-inspired materials.

Amorphous calcium phosphate in the pupal cuticle of Bactrocera dorsalis Hendel (Diptera: Tephritidae): A new discovery for reconsidering the mineralization of the insect cuticle.

It is now widely accepted that Hexapoda emerged from Crustacea. Compared to the ubiquitous calcified exoskeleton in crustaceans, a mineralized cuticle in insects is extremely rare. Catecholamine-driven protein cross-links play a leading role in the sclerotization of insect cuticle. In this study, mineralization was discovered in the pupal cuticle of Bactrocera dorsalis (Diptera: Tephritidae), a common pest of fruit farms. We mainly profiled the features of mineralized pupal cuticles from B. dorsalis and its white mutant B. dorsaliswh and unmineralized cuticle from Musca domestica using high-resolution field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM) combined with structural analysis involving infrared (IR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and synchrotron X-ray absorption near-edge structure (XANES) spectroscopy. We also compared the thermodynamic and mechanical properties of different pupal cuticles. The results showed that the pupal cuticles of B. dorsalis contain a phase of stable amorphous calcium phosphate (ACP) with a high level of magnesium, which is mainly distributed in the exocuticle and assists in the formation of a graded, stiffened cuticle structure. Unexpectedly, this ACP possesses a very low Ca/P ratio and has a composition similar to that of CaHPO4·2H2O. The degree of mineralization in the pupal cuticle of B. dorsaliswh (approximately 22 wt%) is significantly greater than that of wild-type B. dorsalis (approximately 12 wt%), which indicates that there may be a connection between the biomineralization and tyrosine-mediated tanning pathways. These findings provide new evidence for the mineralization of the insect cuticle, which may shed new light on the evolutionary mechanism underlying the divergence of cuticle sclerotization between insects and crustaceans.

Morphology of the first instar of the house fly Musca domestica (Diptera: Muscidae).

Scanning electron microscopy documentation of the first instar of Musca domestica L. is presented for the first time. The following morphological structures are documented: antenna, maxillary palpus, facial mask, spinulation, posterior spiracles, anal pad, and integumental micropores. Morphology of the first-instar larva of M. domestica is discussed in light of existing knowledge about early larval instars of Calyptrata flies.

Freeze-fracture analysis of the effects of age and 2,4-dinitrophenol on the morphology of flight muscle mitochondria of Musca domestica L.

Morphometric comparison of freeze-fractured mitochondria in flight muscles of adult (37-day-old) and old (68-day-old) houseflies revealed a 28% decrease of cristae in the old flies. The major membrane change with age was an increase in the 90-120-A particles in the inner membrane external face concomitant with a loss of particle clusters associated with the openings of the cristae on to the inner membrane. In vitro treatment of flight muscle with 2,4-dinitrophenol, and uncoupler of mitochondrial respiration, did not produce this change but resulted in the formation of smooth particle-free vesicular swellings in the mitochondria. Such swelling were infrequent in the old muscle. The cause for the aging change is not clear, but a reduction in the ability of the intramembranous particles to aggregate, either through modification of altered synthesis, is indicated.

Relationship between lipofuscin granules and polyunsaturated fatty acids in the housefly, Musca domestica.

The fractional volume occupied by lipofuscin granules in epithelial cells of the midgut or oenocytes of abdominal fat body of 3-day-old and 13-day-old male houseflies was determined in two groups of flies by electron microscopic morphometry. One group had developed from larvae reared on diets containing no added polyunsaturated fatty acids and the second from larvae reared on diets containing added linoleic acid. No polyunsaturated fatty acids could be detected in the lipids of the first group of flies using a method which would have detected their presence in amounts greater than 0.1% of the total esterified fatty acids. The second group contained at least two hundred times more than this minimal level. The volume of lipofuscin granules increased significantly (p less than 0.01) (about threefold for the fat body and twofold in midgut cells) between 3 days and 13 days of age but no statistically significant difference was seen between the two groups of flies at the same age. The results show that if lipofuscin formation depends on the oxidation of polyunsaturated fatty acids in the housefly, then extremely small amounts of the acids are involved which lie below the detection limit of the methods employed. The age-associated small increase of extractable fluorescence seen previously in the linoleic acid group of flies is not associated with an increase in the lipofuscin granules.

Invagination of presynaptic ribbons in the fly's optic lobe following loss of their target neuron.

In the first optic neuropile of the housefly Musca, photoreceptor terminals innervate fixed clusters of interneurons, one of which is the monopolar cell L2; L2's synapses in turn feed back upon the terminals. We examined the ultrastructure of these feedback synapses following degeneration of their normal targets, the receptor terminals; this was accomplished by photo-ablating the receptor cells after intraretinal injections of sulforhodamine. Even when all the terminals degenerated, their deafferentated target cells, including L2, remained structurally intact for at least 14 d. Despite this lack of obvious trans-synaptic degeneration, L2's synaptic connections did alter. Presynaptic organelles of the feedback synapses, synaptic ribbons and associated synaptic vesicles, soon appeared in L2's cytoplasm, separating from their site of attachment at the presynaptic membrane by invagination. Similar free-floating organelles and vesicles also occurred in another monopolar cell, L4. They were also occasionally encountered in L2, in normal, newly emerged flies at a time when a naturally occurring loss of feedback synapses is greatest. We interpret the process of internalization that forms these floating ribbons to be the first step in synaptic loss which occurs spontaneously, and that the rate is enhanced in L2 when its main synaptic targets, the receptor terminals, degenerate.

Houseflies: not simple mechanical vectors of enterohemorrhagic Escherichia coli O157:H7.

An epidemic of enterohemorrhagic colitis caused by Escherichia coli O157:H7 (EHEC-O157) occurred in a nursery school in a rural area of Japan in September 1996. The EHEC-O157 were isolated both from patients and houseflies collected at the school. The flies were suspected to be mechanical vectors of the pathogen. Feeding experiments of EHEC-O157 to houseflies showed that the ingested bacteria were harbored in the intestine of flies and continued to be excreted at least for 3 days after feeding. Scanning electron microscopy showed that a large number of EHEC-O157 adhered to the surface of the housefly mouthparts and actively proliferated in the minute spaces of the labellum. Food masses containing EHEC-O157 in the fly intestine were completely surrounded by a peritrophic membrane during digestion and discharged rapidly. The persistence of bacteria in the intestine and feces is mainly a result of proliferation in the mouthparts and accumulation in the crop. Our results strongly suggest that houseflies are not simple mechanical vectors of EHEC. The epidemiologic potential of houseflies to disseminate EHEC-O157 may be greater than initially suspected.

Morphological differentiation of the central visual cells R7/8 in various regions of the blowfly eye.

The central rhabdomeres in the retina of the blowfly Calliphora erythrocephala and the house fly Musca domestica are not structurally uniform. In Calliphora, four classes of central rhabdomeres were found; they are formed by a total of seven types of central visual cells, clearly distinguished by the following structural features: length of the rhabdomeres R7 or R8, position of the nucleus, rhabdomere twist, fine structure in the R7/R8 transition region, and cross-sectional area of the rhabdomeres. In the lateral part of the eye only the most common central-rhabdomere class, 'sl,' is present, whereas in the frontal and dorsal parts classes 'sl' and 'ls' are found in a particular numerical ratio. Near the frontal eye margin the rare class 'per' also appears, with two separate rhabdomeres, R7per and R8s; the morphological properties of R7per are midway between those of peripheral and central visual cells. The special ommatidia at the dorsal margin of the eye are characterized by the central rhabdomeres 'marg'. The known functional properties of the visual cells in the fly eye can be readily assigned to these classes (Table 1, Fig. 12). The non-uniform distribution of the various kinds of central rhabdomeres suggests functional differentiation of the eye region.

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.

Orientation of the backbone structure of myosin filaments in relaxed and rigor muscles of the housefly: evidence for non-equivalent crossbridge positions at the surface of thick filaments.

The orientation of the backbone structure of myosin filaments of relaxed and rigor fibers of the flight muscles of the housefly, Musca domestica, relative to the actin filaments has been investigated. In relaxed muscles 23% of the myosin filaments have gaps in the wall of their shaft located opposite the surrounding actin filaments, while in 77% the subfilament pairs of the wall are thus located. These are the expected values if the backbone orientation is random. In rigor muscles 40% of the thick filaments have their gaps opposite the actins and 60%, the subfilament pairs are opposite the actins. This increase in the percentage of filaments with gaps opposite the actins therefore results from binding of the crossbridges in rigor with change in rotational orientation of the backbone. The findings are related to a model of Beinbrech et al. (1988) in which two populations of crossbridges have been postulated: one originating at the surface of the thick filaments, the other coming from within the gap between the subfilament pairs.

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 microalnalytic and histochemical techniques in order to study the mode of genesis, chemical nature and age-associated distribution of dense bodies. Dense bodies contain high concentration of phosphorus sulphur, chlorine, calcium, iron and copper; they are therfore 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.

An improved method for preparation, stabilization, and storage of house fly (Diptera: Muscidae) microsomes.

An improved method for preparation and storage of insect microsomes from house fly, Musca domestica L., abdomens was developed. Microsomes were prepared in phosphate buffer fortified with glycerol, dithiothreitol, ethylenediaminetetraacetic acid, phenylmethylsulfonyl fluoride, and 1-phenyl-2-thiourea. No cytochrome P-420 was observed when abdomens were isolated by our method. No measurable loss of cytochrome P-450, cytochrome b5 or NADPH-cytochrome c (P-450) reductase levels, or methoxyresorufin O-demethylation, 7-ethoxycoumarin O-deethylation, or aryl hydrocarbon hydroxylation activities occurred when a diluted suspension (protein concentration of 2 mg/ml) of microsomes was stored at -80 degrees C for at least 2 mo.

Some ultrastructural superficial changes in house fly (Diptera: Muscidae) and blow fly (Diptera: Calliphoridae) larvae induced by eucalyptol oil.

The ultrastructural superficial changes in third instar house fly (Musca domestica) and blow fly (Chrysomya megacephala) induced by eucalyptol oil were observed using scanning electron microscopy. Dipped in 0.902 g/ml eucalyptol for 30 sec, the larvae integument of both species showed significant aberrant appearance of the body surface, particularly swelling integument, bleb formation, partial breach and deformation of spines.