Disease dynamics and persistence of Musca domestica salivary gland hypertrophy virus infections in laboratory house fly (Musca domestica) populations

Past surveys of feral house fly populations have shown that Musca domestica salivary gland hypertrophy virus (MdSGHV) has a worldwide distribution, with an average prevalence varying between 0.5% and 10%. How this adult-specific virus persists in nature is unknown. In the present study, experiments were conducted to examine short-term transmission efficiency and long-term persistence of symptomatic MdSGHV infections in confined house fly populations. Average rates of disease transmission from virus-infected to healthy flies in small populations of 50 or 100 flies ranged from 3% to 24% and did not vary between three tested geographical strains that originated from different continents. Introduction of an initial proportion of 40% infected flies into fly populations did not result in epizootics. Instead, long-term observations demonstrated that MdSGHV infection levels declined over time, resulting in a 10% infection rate after passing through 10 filial generations. In all experiments, induced disease rates were significantly higher in male flies than in female flies and might be explained by male-specific behaviors that increased contact with viremic flies and/or virus-contaminated surfaces.     

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.     

Parasites that attack stable fly and house fly (Diptera: Muscidae) puparia during the winter on dairies in northwestern Florida

Throughout the winter and early spring months, stable fly, Stomoxys calcitrans (L.), and house fly, Musca domestica L., puparia were collected from silage, hay, and manure from six dairies in northwestern Florida and evaluated for parasitism. Of the puparia producing flies or parasites, 23% of the stable flies and 46% of the house flies were parasitized. The predominant parasite observed attacking muscoid flies (76% for stable flies and 58% for house flies) was Spalangia cameroni Perkins. Muscidifurax sp. was recovered from 11 and 36% of the stable fly and house fly pupae, respectively. Other parasite species encountered were Spalangia endius Walker and S. nigroaenea Curtis. Significantly more parasitized fly pupae were collected from silage than from hay residues or manure. Winter and early spring parasite populations in northwestern Florida appear to be present as long as viable fly pupae are available to support the developing parasites.     

Characterization and description of a virus causing salivary gland hyperplasia in the housefly,Musca domestica

Abstract. A double-stranded DNA virus was isolated from hyperplasic salivary glands of male and female houseflies, Musca domestica L. (Diptera: Muscidae), collected from a dairy in Alachua County, Florida, U.S.A. Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) of this housefly salivary gland hyperplasia (SGH) virus revealed the presence of two major and eight minor structural polypeptides. Restriction endonuclease analysis indicated that the c. 137 kilobase pair DNA was double-stranded. Weekly sweep-net sampling of the fly population throughout the season (May-October, 1991) showed that 1.5-18.5% of the dissected flies possessed hyperplasic salivary glands. The virus replicated within the nuclei of the salivary gland cells and was transmitted per os to newly-emerged healthy adult flies.     

Coevolution of hytrosaviruses and host immune responses

Background

Hytrosaviruses (SGHVs; Hytrosaviridae family) are double-stranded DNA (dsDNA) viruses that cause salivary gland hypertrophy (SGH) syndrome in flies. Two structurally and functionally distinct SGHVs are recognized; Glossina pallidipes SGHV (GpSGHV) and Musca domestica SGHV (MdSGHV), that infect the hematophagous tsetse fly and the filth-feeding housefly, respectively. Genome sizes and gene contents of GpSGHV (~ 190 kb; 160–174 genes) and MdSGHV (~ 124 kb; 108 genes) may reflect an evolution with the SGHV-hosts resulting in differences in pathobiology. Whereas GpSGHV can switch from asymptomatic to symptomatic infections in response to certain unknown cues, MdSGHV solely infects symptomatically. Overt SGH characterizes the symptomatic infections of SGHVs, but whereas MdSGHV induces both nuclear and cellular hypertrophy (enlarged non-replicative cells), GpSGHV induces cellular hyperplasia (enlarged replicative cells). Compared to GpSGHV’s specificity to Glossina species, MdSGHV infects other sympatric muscids. The MdSGHV-induced total shutdown of oogenesis inhibits its vertical transmission, while the GpSGHV’s asymptomatic and symptomatic infections promote vertical and horizontal transmission, respectively. This paper reviews the coevolution of the SGHVs and their hosts (housefly and tsetse fly) based on phylogenetic relatedness of immune gene orthologs/paralogs and compares this with other virus-insect models.

Results

Whereas MdSGHV is not vertically transmitted, GpSGHV is both vertically and horizontally transmitted, and the balance between the two transmission modes may significantly influence the pathogenesis of tsetse virus. The presence and absence of bacterial symbionts (Wigglesworthia and Sodalis) in tsetse and Wolbachia in the housefly, respectively, potentially contributes to the development of SGH symptoms. Unlike MdSGHV, GpSGHV contains not only host-derived proteins, but also appears to have evolutionarily recruited cellular genes from ancestral host(s) into its genome, which, although may be nonessential for viral replication, potentially contribute to the evasion of host’s immune responses. Whereas MdSGHV has evolved strategies to counteract both the housefly’s RNAi and apoptotic responses, the housefly has expanded its repertoire of immune effector, modulator and melanization genes compared to the tsetse fly.

Conclusions

The ecologies and life-histories of the housefly and tsetse fly may significantly influence coevolution of MdSGHV and GpSGHV with their hosts. Although there are still many unanswered questions regarding the pathogenesis of SGHVs, and the extent to which microbiota influence expression of overt SGH symptoms, SGHVs are attractive ‘explorers’ to elucidate the immune responses of their hosts, and the transmission modes of other large DNA viruses.

     

Stable fly, Stomoxys calcitrans, mouthpart removal influences stress and anticipatory responses in mice

Abstract Biting fly attack induces a variety of stress and anxiety related changes in the physiology and behaviour of the target animals. Significant reductions in pain, or more appropriately, nociceptive sensitivity (latency of a foot-lifting response to an aversive thermal stimulus), are evident in laboratory mice after a 1 h exposure to stable flies, Stomoxys calcitrans. The role of the various components of biting fly attack in the development of this stress-induced reduction in pain sensitivity (analgesia) is, however, unclear. This study demonstrates that fly-naive mice do not exhibit a stress-induced analgesia when exposed to stable flies whose biting mouthparts have been removed. In contrast, mice that have been previously exposed to intact stable flies exhibit significant analgesia when exposed to flies that are incapable of biting. However, the level of analgesia induced is lower than that elicited by exposure to intact stable flies. Exposure to non-biting house flies, Musca domestica , has no effect on nociceptive sensitivity. It appears that the actual bite of the stable fly is necessary for the induction of analgesia and probably other stress and anxiety associated responses in fly naive mice. However, mice rapidly learn to recognize biting flies and exhibit significant, possibly anticipatory analgesic responses to the mere presence of biting flies.     

Relative attractiveness of paired BL and BLB fluorescent bulbs for house and stable flies (Diptera: Muscidae)

Blacklight (BL) and blacklight blue (BLB) fluorescent bulbs were combined in an electrocuting fly trap and compared with BL or BLB bulbs alone for fly attraction. Combinations of BL and BLB bulbs did not attract more house flies, Musca domestica, or stable flies, Stomoxys calcitrans than were attracted by BL bulbs used alone.     

Biological control of <Emphasis Type="Italic">Musca domestica</Emphasis> and <Emphasis Type="Italic">Stomoxys calcitrans</Emphasis> by mass releases of the parasitoid <Emphasis Type="Italic">Spalangia cameroni</Emphasis> on two Norwegian pig farms

House flies (Musca domestica L.) and stable flies (Stomoxys calcitrans (L.)) (Diptera: Muscidae) are nuisance pests on livestock farms. In the present study we tested the effect of biweekly mass release of Spalangia cameroni Perkins (Hymenoptera: Pteromalidae), the most common parasitoid of house flies and stable flies in southern Norway, on pig premises with scattered fly breeding sites. The study spanned two successive summer periods, with two control and two release units each year. Spalangia cameroni suppressed both house flies and stable flies in one release unit during the first year, and stable flies in one release unit the second year. The apparent lack of success in fly control in the other release units was likely due to immigration of flies in two of the trials and high temperatures in one trial. Recommendations concerning release of S. cameroni in similar pig production premises are given. Handling editor: Torsten Meiners.     

Exposure to stable flies reduces spatial learning in mice: involvement of endogenous opioid systems

Abstract. Biting flies influence both the physiology and behaviour of domestic and wild animals. This study demonstrates that relatively brief (60min) exposure to stable flies, Stomoxys calcitrans (L.), affects the spatial abilities of male mice. Stable fly exposure resulted in poorer subsequent performance in a water maze task in which individual mice had to learn the spatial location of a submerged hidden platform using extramaze visual cues. Determinations of spatial acquisition and retention were made with mice that had been previously exposed for 60min to either stable flies or house flies, Musca domestica (L.). Mice exposed to stable flies displayed over one day of testing (six blocks or sets of four trials) significantly poorer acquisition and retention of the water maze task than either mice that had been exposed to house flies or fly-naive mice. This attenuation of spatial learning occurred in the absence of any evident sensorimotor or motivational impairments. The reduction in spatial abilites involved endogenous opioid systems, as the decreased performance resulting from stable fly exposure was blocked by pre-treatment with the prototypic opiate antagonist, naltrexone. These results indicate that relatively brief exposure to biting flies can lead to a decrease in spatial abilities which is associated with enhanced endogenous opioid activity. These results support the involvement of endogenous opioid systems in the mediation of the behavioural and physiological effects of biting fly exposure. They further suggest that decreases in spatial abilities and performance may be part of the behavioural consequences of biting fly exposure in domestic and wild animals.     

Evaluation of commercial and field‐expedient baited traps for house flies,Musca domestica L. (Diptera: Muscidae)

A comparison of nine commercial baited fly traps on Florida dairy farms demonstrated that Terminator traps collected significantly more (13,323/trap) house flies (Musca domestica L.) than the others tested. Final Flight, Fly Magnet, and FliesBeGone traps collected intermediate numbers of flies (834‐2,166), and relatively few were caught with ISCA, Advantage, Fermone Big Boy, Squeeze & Snap, or OakStump traps (<300). Terminator traps collected about twice as many flies (799.8/trap) as FliesBeGone traps (343.8) when each trap was baited with its respective attractant, but when the attractants were switched between the two trap types, collections were significantly lower (77‐108) than was observed with traps baited with their respective attractant. Solutions of molasses were significantly more attractive to house flies than honey, maple syrup, or jaggery (date palm sugar). Field‐expedient traps constructed from discarded PET water bottles were much less effective than commercial traps, but painting the tops of such traps with black spray paint resulted in a six‐fold increase in trap capture.     

Stable fly, house fly (Diptera: Muscidae), and other nuisance fly development in poultry litter associated with horticultural crop production

Poultry litter usage in horticultural crop production is a contributor to nuisance fly populations, in particular stable flies (Stomoxys calcitrans L.) and house flies (Musca domestica L.). Extrapolation of adult emergence data suggests that approximately 1.5 million house flies and 0.2 million stable flies are emerging on average from every hectare of poultry litter applied as a preplant fertilizer for vegetable production in Perth, Western Australia. To a lesser extent, sideband applications to established crops may allow for the development of 0.5 million house flies and 45,000 stable flies per hectare. However, up to 1 million house flies, 0.45 million lesser house flies, Fannia cannicularis L., and 11,000 stable flies per hectare may be produced from surface dressings of poultry litter associated with turf production. Other nuisance flies present in poultry litter included the false stable fly, Muscina stabulans (Fallén), bluebodied blowfly, Calliphora dubia Hardy, black carrion fly, Hydrotaea rostrata Robineau-Desvoidy, Australian sheep blowfly, Lucilia cuprina Wiedemann, and flesh flies (Sarcophagidae). Only house flies developed in poultry litter for the first 4 d after application in the field. Stable flies were not present in poultry litter until 4-7 d after application, and were the only fly species developing in litter > 9 d after application.     

Biological control of house flies Musca domestica and stable flies Stomoxys calcitrans(Diptera: Muscidae) by means of inundative releases of Spalangia cameroni(Hymenoptera: Pteromalidae)

The efficacy of the pupal parasitoid Spalangia cameroni Perkins as a biological control agent was tested against house flies Musca domestica Linnaeus and stable flies Stomoxys calcitrans (Linnaeus) in one dairy cattle and two pig installations in Denmark. Weekly releases of S. cameroni from April through to September-October 1999 and 2000 resulted in significant suppressions of house fly populations to below nuisance level, whereas no effect on stable flies was found. Parasitism was significantly higher in the release years compared to the control years, but was below 25% averaged over the fly season for each farm. A statistical model based on a functional relationship between the innate capacity of increase of the two fly species and three explanatory variables (air temperature, fly density and parasitism) provided a fairly good fit to data with the abundances of house flies and stable flies explained mostly by temperature, but intra- and interspecific competition, and parasitism had a significant effect as well. Overall, the model was capable of explaining 14% and 6.6% of the total variation in data for house fly and stable fly, respectively. Spalangia cameroni was the predominant parasitoid to emerge from exposed house fly pupae, but from mid summer onwards Muscidifurax raptor Girault & Sanders (Hymenoptera: Pteromalidae) was also quite common. The study indicated that biological control of house flies can be an efficient alternative to chemical control.     

Effect of the entomopathogenic fungus,Entomophthora muscae (Zygomycetes: Entomophthoraceae), on sex pheromone and other cuticular hydrocarbons of the house fly,Musca domestica

House fly (Musca domestica) males are highly attracted to dead female flies infected with the entomopathogenic fungus Entomophthora muscae. Because males orient to the larger abdomen of infected flies, both visual and chemical cues may be responsible for the heightened attraction to infected flies. Our behavioral assays demonstrated that the attraction is sex-specific-males were attracted more to infected females than to infected males, regardless of cadaver size. We examined the effect of E. muscae on the main component of the house fly sex pheromone, (Z)-9-tricosene, and other cuticular hydrocarbons including n-tricosane, n-pentacosane, (Z)-9-heptacosene, and total hydrocarbons of young (7 days old) and old (18 days old) virgin females. Young E. muscae-infected female flies accumulated significantly less sex pheromone and other hydrocarbons on their cuticular surface than uninfected females, whereas the cuticular hydrocarbons of older flies were unaffected by fungus infection. These results suggest that chemical cues other than (Z)-9-tricosene, visual cues other than abdomen size, or a combination of both sets of cues might be responsible for attraction of house fly males to E. muscae-infected females.     

Selection for resistance to imidacloprid in the house fly (Diptera: Muscidae)

The house fly, Musca domestica L. (Diptera: Muscidae), continues to be a primary pest of livestock facilities worldwide. This pest also has shown a propensity for pesticide resistance development when under high selection pressures. In this study the house fly strain FDm was created by a 20% contribution from each of five colonies collected from dairies in Florida with known imidacloprid resistance. The FDm strain was used to evaluate the level ofimidacloprid resistance after five selections near the LC70 value of each selected generation. Overall, the mean selection mortality was 72.7, with males being considerably more susceptible than females. The unselected (F0) FDm strain showed considerable susceptibility to imidacloprid after its creation, compared with the five parental strains. Between 9500 and 14,000 virgin house flies were used in each selection. After the fifth and final selection, a 331-fold increase in imidacloprid resistance at the LC70 was observed over the parental FDm strain. In parallel studies, the FDm strain showed increasing tolerance of the commercial imidacloprid product QuickBayt. These results suggest that livestock producers should use caution when choosing pesticides and consider rotating fly baits, as is encouraged with other pesticide treatment regimes on farms.     

The antiviral drug valacyclovir successfully suppresses salivary gland hypertrophy virus (SGHV) in laboratory colonies of Glossina pallidipes

Many species of tsetse flies are infected with a virus that causes salivary gland hypertrophy (SGH) symptoms associated with a reduced fecundity and fertility. A high prevalence of SGH has been correlated with the collapse of two laboratory colonies of Glossina pallidipes and colony maintenance problems in a mass rearing facility in Ethiopia. Mass-production of G. pallidipes is crucial for programs of tsetse control including the sterile insect technique (SIT), and therefore requires a management strategy for this virus. Based on the homology of DNA polymerase between salivary gland hypertrophy virus and herpes viruses at the amino acid level, two antiviral drugs, valacyclovir and acyclovir, classically used against herpes viruses were selected and tested for their toxicity on tsetse flies and their impact on virus replication. While long term per os administration of acyclovir resulted in a significant reduction of productivity of the colonies, no negative effect was observed in colonies fed with valacyclovir-treated blood. Furthermore, treatment of a tsetse colony with valacyclovir for 83 weeks resulted in a significant reduction of viral loads and consequently suppression of SGH symptoms. The combination of initial selection of SGHV-negative flies by non-destructive PCR, a clean feeding system, and valacyclovir treatment resulted in a colony that was free of SGH syndromes in 33 weeks. This is the first report of the use of a drug to control a viral infection in an insect and of the demonstration that valacyclovir can be used to suppress SGH in colonies of G. pallidipes.     

Azadirachtin as a larvicide against the horn fly, stable fly, and house fly (Diptera: Muscidae)

Effects of azadirachtin, a triterpenoid extracted from neem seed, Azadirachta indica A. Juss., were similar to those of insect growth regulators against the immature stages of the born fly, Haematobia irritans (L.), the stable fly, Stomoxys calcitrans (L.), and the house fly, Musca domestica L. When an ethanolic extract of ground seed was blended into cow manure, LC50 and LC90's for larval horn flies were 0.096 and 0.133 ppm azadirachtin, respectively. An emulsifiable concentrate (EC) had an LC50 for larval horn flies of 0.151 ppm and an LC90 of 0.268 ppm. For larval stable flies, the EC formulation had an LC50 of 7.7 ppm and an LC90 of 18.7 ppm azadirachtin in manure. Against larval house flies, the LC50 and LC90 were 10.5 and 20.2 ppm, respectively. When the EC formulation was administered orally to cattle at a rate of greater than or equal to 0.03 mg azadirachtin per kg of body weight per day or when ground neem seed was given as a daily supplement of greater than or equal to 10 mg seed per kg body weight, horn fly development in the manure was almost completely inhibited. In contrast, ground seed mixed in cattle feed at the rate of 100-400 mg seed per kg of body weight per day caused less than 50% inhibition of stable flies in the manure.     

Commercial and naturally occurring fly parasitoids (Hymenoptera: Pteromalidae) as biological control agents of stable flies and house flies (Diptera: Muscidae) on California dairies

Filth fly parasites reared by commercial insectaries were released on two dairies (MO, DG) in southern California to determine their effect on populations of house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.). Spalangia endius Walker, Muscidifurax raptorellus Kogan and Legner, and Muscidifurax zaraptor Kogan and Legner were released on the MO dairy from 1985 to 1987 in varying quantities. Parasitism by Muscidifurax zaraptor on the MO dairy was significantly higher (P less than 0.05) from the field-collected stable fly (4.4%) and house fly (12.5%) pupae, compared with a control dairy (0.1%, stable fly; 1.3%, house fly). Muscidifurax zaraptor, released from April through October during 1987 on the DG dairy (350,000 per month), was not recovered in a significantly higher proportion from either fly species relative to the corresponding control dairy. No specimens of Muscidifurax raptorellus were recovered from the MO dairy. Parasite treatments had no apparent effect on adult populations of either fly species or on overall parasitism rate of field-collected stable fly (16.8%, MO; 17.2%, DG) and house fly (23.3%, MO; 20.9%, DG) pupae. Spalangia spp. were the predominant parasites recovered from field-collected stable fly and house fly pupae on all four dairies. Sentinel house fly pupae placed in fly-breeding sites on both release dairies were parasitized at a significantly higher rate, as compared with sentinel pupae on control dairies. The generic composition of parasites emerging from sentinel house fly pupae was 20.6% Spalangia spp. and 73.2% Muscidifurax spp., whereas in field-collected house fly pupae, Spalangia spp. and Muscidifurax spp. constituted 74.3 and 19.6% of the parasites, respectively.     

Tsetse Salivary Gland Hypertrophy Virus: Hope or Hindrance for Tsetse Control?

MANY SPECIES OF TSETSE FLIES (DIPTERA: Glossinidae) are infected with a virus that causes salivary gland hypertrophy (SGH), and flies with SGH symptoms have a reduced fecundity and fertility. The prevalence of SGH in wild tsetse populations is usually very low (0.2%-5%), but higher prevalence rates (15.2%) have been observed occasionally. The successful eradication of a Glossina austeni population from Unguja Island (Zanzibar) using an area-wide integrated pest management approach with a sterile insect technique (SIT) component (1994-1997) encouraged several African countries, including Ethiopia, to incorporate the SIT in their national tsetse control programs. A large facility to produce tsetse flies for SIT application in Ethiopia was inaugurated in 2007. To support this project, a Glossina pallidipes colony originating from Ethiopia was successfully established in 1996, but later up to 85% of adult flies displayed symptoms of SGH. As a result, the colony declined and became extinct by 2002. The difficulties experienced with the rearing of G. pallidipes, epitomized by the collapse of the G. pallidipes colony originating from Ethiopia, prompted the urgent need to develop management strategies for the salivary gland hypertrophy virus (SGHV) for this species. As a first step to identify suitable management strategies, the virus isolated from G. pallidipes (GpSGHV) was recently sequenced and research was initiated on virus transmission and pathology. Different approaches to prevent virus replication and its horizontal transmission during blood feeding have been proposed. These include the use of antiviral drugs such as acyclovir and valacyclovir added to the blood for feeding or the use of antibodies against SGHV virion proteins. In addition, preliminary attempts to silence the expression of an essential viral protein using RNA interference will be discussed.