Retention of Escherichia coli by house fly and stable fly (Diptera: Muscidae) during pupal metamorphosis and eclosion

Populations of Escherichia coli obtained by feeding larval house flies, Musca domestica L. and stable flies, Stomoxys calcitrans (L.), persisted through the pupal stage. The abundance of E. coli in house fly pupae increased initially then declined before adult emergence. Abundance of E. coli in stable fly pupae increased through pupal development and remained high. Infected stable fly pupal cases typically contained more E. coli than house fly pupal cases. A greater proportion of emerging adult house flies were infected with E. coli compared with stable flies; however, the abundance of E. coli on infected flies was similar between species. Adult flies contained 0.04-0.19% of the E. coli in the pupal cases. The proportion of infected house fly adults and the amount of E. coli on the infected flies were related to the levels of E. coli in the pupal cases; however, these relationships did not occur with the stable fly. Results suggest that retention of E. coli from larval to adult house flies could play a role in the transmission and spread of E. coli, whereas stable fly adults probably play a minor role in E. coli spread. However, pupae of both species have potential to act as reservoirs for E. coli.     

Horizontal transfer of Shiga toxin and antibiotic resistance genes among Escherichia coli strains in house fly (Diptera: Muscidae) gut

Whether the house fly, Musca domestica L., gut is a permissive environment for horizontal transfer of antibiotic resistance and virulence genes between strains of Escherichia coli is not known. House flies were immobilized and force fed suspensions of defined, donor strains of E. coli containing chloramphenicol resistance genes on a plasmid, or lysogenic, bacteriophage-born Shiga toxin gene stx1 (bacteriophage H-19B::Ap1). Recipient strains were E. coli lacking these mobile elements and genes but having rifampicin as a selectable marker. Plasmid transfer occurred at rates of 10(-2) per donor cell in the fly midgut and 10(-3) in the fly crop after 1 h of incubation postfeeding. Bacteriophage transfer rate was approximately 10(-6) per donor cell without induction, but induction with mitomycin C increased rates of transfer to 10(-2) per donor cell. These findings show that genes encoding antibiotic resistance or toxins will transfer horizontally among bacteria in the house fly gut via plasmid transfer or phage transduction. The house fly gut may provide a favorable environment for the evolution and emergence of pathogenic bacterial strains through acquisition of antibiotic resistance genes or virulence factors.     

Dewatered sewage biosolids provide a productive larval habitat for stable flies and house flies (Diptera: Muscidae)

Species diversity and seasonal abundance of muscoid flies (Diptera: Muscidae) developing in biosolid cake (dewatered biosolids) stored at a wastewater treatment facility in northeastern Kansas were evaluated. Emergence traps were deployed 19 May through 20 October 2009 (22 wk) and 27 May through 18 November 2010 (25 wk). In total, 11,349 muscoid flies were collected emerging from the biosolid cake. Stable flies (Stomoxys calcitrans (L.)) and house flies (Musca domestica (L.)), represented 80 and 18% of the muscoid flies, respectively. An estimated 550 stable flies and 220 house flies per square-meter of surface area developed in the biosolid cake annually producing 450,000 stable flies and 175,000 house flies. Stable fly emergence was seasonally bimodal with a primary peak in mid-July and a secondary peak in late August. House fly emergence peaked with the first stable fly emergence peak and then declined gradually for the remainder of the year. House flies tended to emerge from the biosolid cake sooner after its deposition than did stable flies. In addition, house fly emergence was concentrated around midsummer whereas stable fly emergence began earlier in the spring and continued later into the fall. Biosolid age and temperature were the most important parameters affecting emergence for house flies and stable flies, whereas precipitation was not important for either species. This study highlights the importance of biosolid cake as a larval developmental habitat for stable flies and house flies.     

Retention of Campylobacter (Campylobacterales: Campylobacteraceae) in the house fly (Diptera: Muscidae)

The house fly (Musca domestica L.) may transmit Campylobacter to broiler flocks. We assessed the retention time of house flies for Campylobacter jejuni at five temperatures and three doses. Flies were inoculated individually at their proboscis with 1.6 x 10(7) CFU (colony forming units) of C. jejuni and incubated at 15, 20, 25, 30, and 35 degrees C. Furthermore, a dose experiment was conducted at 25 degrees C where individual flies were inoculated in three series: 6.5 x 10(6), 6.0 x 10(4), and 8.2 x 10(2) C.jejuni CFU. Whole flies were tested for C. jejuni carriage at 0, 6, 12, 18, and 24 h by initial preenrichment in Bolton broth, which afterwards was streaked on modified mCCDA agar plates and incubated under micro-aerobic conditions. The results showed that the time C. jejuni remained in flies declined over time with ascending temperatures and when reducing the inoculation dose. All flies stayed Campylobacter positive 24 h postinoculation at 15 degrees C whereas only one-third of the flies were positive at 20 degrees C and few to none at 25, 30, and 35 degrees C. When combinations of temperature and retention time were expressed as accumulated day-degrees, data could be adequately fitted using a generalized linear mixed model that included a linear effect of day-degrees and the difference between the lowest and the two highest doses. Based on model predictions of selected combinations of temperature and dose, the time for 50% and 1% of flies containing Campylobacter was calculated. It is suggested that house flies are mainly short distance carriers of C. jejuni.     

Pyriproxyfen and house flies (Diptera: Muscidae): effects of direct exposure and autodissemination to larval habitats

Pyriproxyfen is an insect growth regulator with juvenile hormone-like activity that has potential uses for dipterans that are difficult to manage with conventional insecticides, such as house flies (Musca domestica L.). The objectives of this study were to determine the efficacy of this insect growth regulator against house flies using variety of delivery systems and target life stages, including an evaluation of the potential for autodissemination by female flies to larval development sites. Adult female house flies exposed to filter paper (3.75% active ingredient) or sugar treated with pyriproxyfen (0.01-0.1%) produced significantly fewer F1 pupae than untreated flies. Adult emergence from pupae was unaffected. In contrast, treatment of larval rearing medium with 0.35 ml/cm2 of a 12 mg pyriproxyfen/liter preparation had no effect on the number of pupae developing from eggs but markedly inhibited adult emergence from those pupae. There was little difference in susceptibility between an insecticide-susceptible and a wild strain of house fly. The LC50 for inhibiting fly emergence of dust formulations in diatomaceous earth incorporating commercial pyriproxyfen products ranged from 8 to 26 mg/liter, with little difference among products. Compared with untreated flies, significantly fewer pupae were produced at concentrations > 0.5% and no adults were produced at concentrations > 0.05% pyriproxyfen. When gravid females were exposed for 1 h to treated fabric (6 mg pyriproxyfen/cm2) and allowed to oviposit in rearing media containing eggs, sufficient pyriproxyfen was autodisseminated to reduce adult emergence from those eggs by > 99%. Intermittent contact with treated fabric over 2 d reduced adult emergence by 63-76%.     

Expressed cDNAS from embryonic and larval stages of the horn fly (Diptera: Muscidae)

We used an expressed sequence tag approach to initiate a study of the genome of the horn fly, Hematobia irritans (L.) (Diptera: Muscidae). Two normalized cDNA libraries were synthesized from RNA isolated from embryos and first instars from a field population of horn flies. Approximately 10,000 clones were sequenced from both the 5' and 3' directions. Sequence data from each library was assembled into a database of tentative consensus sequences (TCs) and singletons and used to search public protein databases and annotate the sequences. Additionally, the sequences from both the egg and larval libraries were combined into a single database consisting of 16,702 expressed sequence tags (ESTs) assembling into 2886 TCs and 1,522 singleton entries. Several sequences were identified that may have roles in the horn fly's resistance to insecticides. The availability of this database will facilitate the design of microarray and other experiments to study horn fly gene expression on a larger scale than previously possible. This would include studies designed to investigate metabolic-based insecticide resistance, identify novel antigens for vaccine-based control approaches, and discover new proteins to serve as targets for new pesticide development.     

Relationship between rainfall and stable fly (Diptera: Muscidae) abundance on California dairies

Populations of adult stable flies, Stomoxys calcitrans (L.), were visually estimated by counting flies on the front legs of cattle on southern and central California confined dairy feedlots between late April and mid-June (encompassing the peak stable fly activity period). Fly counts on 45-90 animals (three to six dairies) per weekly sample date were conducted in 1985, 1986, 1993, 2002, and 2003. Average biting intensity (flies per front leg) for the peak fly season was not significantly related to early winter (December-January), late winter (February-March), or total period (December-March) rainfall, but it was strongly related (r2 = 0.726) to March rainfall. March rains probably moistened outside decaying manure habitats and similar substrates that are particularly suitable at that time for stable fly oviposition and larval development. Degree-day accumulations link the timing of significant (> or = 1.3 cm) late winter or early spring rains to peak adult stable fly activity two generations later in May and early June.     

Assessment of parasitism of house fly and stable fly (Diptera: Muscidae) pupae by pteromalid (Hymenoptera: Pteromalidae) parasitoids using a polymerase chain reaction assay

The internal transcribed spacer (ITS) regions of the ribosomal DNA of house flies, Musca domestica L., the stable flies, Stomoxys calcitrans (L.), and four parasitoid species in the genus Muscidifurax (Hymenoptera: Pteromalidae) were characterized to develop a method based on the polymerase chain reaction (PCR) to better define the role of pteromalid parasitism of pupae of the house fly and stable fly. Two parasitoid-specific primers were designed to anneal to the 5' end of the 5.8S rRNA gene in the parasitoid species. When paired with a universal primer at the 3' end of the 18S rRNA, the primers amplified the target ITS1 region in 10 pteromalid species. PCR allowed detection of parasitoid DNA within 24 h after females of Spalangia endius Walker oviposited into house fly puparia. PCR failed to amplify parasitoid DNA or detect parasitism in puparia that were exposed to parasitoid oviposition, allowed to develop 7 d, then killed by freezing and held at 20-24 degrees C for 4 d to allow DNA degradation. Digestion of the PCR products with restriction enzymes produced restriction fragment length polymorphisms that allowed identification of individual parasitoid species. Significantly greater levels of parasitism (P < 0.05) were detected by PCR for two of the five field collection dates in 1997. On the dates when PCR detected higher levels of parasitism than estimates provided by emergence of adult insects from samples taken at Feedlot M in 1997, more than 65% of all puparia in the emergence samples failed to produce an adult insect. Three puparia collected in 1997 produced double PCR bands that corresponded to PCR band sizes of Muscidifurax spp. and Spalangia sp., possibly indicating multiple parasitism or hyperparasitism.     

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.     

Vector competence of the stable fly (Diptera: Muscidae) for West Nile virus

In 2006-2007, stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae), were suspected of being enzootic vectors of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) during a die-off of American white pelicans (Pelecanus erythrorhynchos Gmelin) (Pelecanidae) in Montana, USA. WNV-positive stable flies were observed feeding en masse on incapacitated, WNV-positive pelicans, arousing suspicions that the flies could have been involved in WNV transmission among pelicans, and perhaps to livestock and humans. We assessed biological transmission by infecting stable flies intrathoracically with WNV and testing them at 2-d intervals over 20 d. Infectious WNV was detected in fly bodies in decreasing amounts over time for only the first 6 d postinfection, an indication that WNV did not replicate within fly tissues and that stable flies cannot biologically transmit WNV. We assessed mechanical transmission using a novel technique. Specifically, we fed WNV-infected blood to individual flies by using a cotton swab (i.e., artificial donor), and at intervals of 1 min-24 h, we allowed flies to refeed on a different swab saturated with WNV-negative blood (i.e., artificial recipient). Flies mechanically transmitted viable WNV from donor to recipient swabs for up to 6 h postinfection, with the majority of the transmission events occurring within the first hour. Flies mechanically transmitted WNV RNA to recipient swabs for up to 24 h, mostly within the first 6 h. Given its predilection to feed multiple times when disturbed, these findings support the possibility that the stable fly could mechanically transmit WNV.     

Rearing stable fly larvae (Diptera: Muscidae) on an egg yolk medium.

The growth and survival of Stomoxys calcitrans (L.) larvae on egg yolk medium inoculated with bacteria isolated from a colony of stable flies was evaluated. Five species of bacteria--Acinetobacter sp., Aeromonas sp., Empedobacter breve (Holmes & Owen), Flavobacterium odoratum Stutzer, and Serratia marcescens Bizio--were identified according to fatty acid profiles using a microbial identification system. Larvae failed to develop on uninoculated plates, confirming that bacteria are required to complete development. Larvae also failed to complete development on plates inoculated with Aeromonas sp. and S. marcescens, and died during the 1st instar. Larvae completed development on the remaining 3 bacterial species as well as on Escherichia coli (Migula). Survival was generally higher when larvae were reared on Acinetobacter sp. and F. odoratum compared with E. coli and E. breve. Egg density did not influence larval survival, although the variability in survival was lowest using 20 and 40 eggs per plate. Larval survival in mixed cultures of Acinetobacter and Flavobacterium averaged 22.7% lower than survival in the pure cultures, and averaged 21.6% higher in mixed cultures of Empedobacter and Flavobacterium compared with pure cultures. Larval survival in mixed cultures did not differ significantly from mean survival in pure cultures for combinations of Acinetobacter and E. coli, Acinetobacter and Empedobacter, E. coli and Empedobacter, and E. coli and Flavobacterium. Larval developmental time was faster on all mixed bacterial cultures compared with developmental time on pure bacterial cultures. Optimal sample sizes and egg numbers are presented for detecting specified differences in larval survival. This rearing procedure will be useful for studying insect-microbe interactions and evaluating mortality using bacterial agents.     

Mitochondrial diversity in new world house flies (Diptera: Muscidae)

Mitochondrial diversity in house flies was examined by using the single-strand conformation polymorphism method in house flies, Musca domestica L. sampled in six zoogeographical subregions in the New World. The number of haplotypes and haplotype diversities were homogeneous among subregions, but a strong spatial component was found in the distribution of particular haplotypes. Nei's differentiation index among subregions, GRT, was 0.53 and that among populations within subregions, GPR, was 0.31. Greater genetic differentiation was found among populations in the Nearctic than in the Neotropics. Haplotype frequency distributions in two of three Nearctic subregions deviated from that expected under the neutral infinite allele model, suggesting the existence of differential selection patterns.     

Laboratory evaluation of novaluron as a development site treatment for controlling larval horn flies, house flies, and stable flies (Diptera: Muscidae)

A granular formulation of novaluron (Novaluron 0.2G, 0.2% [AI]), a newer benzoylphenyl urea insecticide, was evaluated for its efficacy in controlling the larval stage of horn flies, Haematobia irritans (L.); house flies, Musca domestica L.; and stable flies, Stomoxys calcitrans (L.), in cow manure. Various rates and insecticide placement locations (top, middle, and bottom of manure) were evaluated in this study and all combinations of these variables reduced adult emergence of all three species when compared with the untreated controls. The presence of deformed pupae indicated that novaluron had an insect growth regulator effect on the developing fly larvae. Top, middle, or bottom application rates of 0.125, 0.195, 0.25, and 0.375 g novaluron onto manure samples, reduced adult horn fly emergence by > 90%. Middle and bottom application rates of 0.195, 0.25, and 0.375 g novaluron reduced adult house fly emergence >93%. All rates and placement combinations resulted in >98% reduction of adult stable fly emergence. The level of control efficacy observed against these three fly species along with the ease of use of a granular formulation, make this product an ideal candidate for use in an integrated livestock pest management program.     

Novel growth media for rearing larval horn flies,Haematobia irritans (Diptera: Muscidae)

Experiments were conducted to develop an agar-based medium for rearing immature horn flies, Hematobia irritans (L.). Larval survival was determined on sterilized manure inoculated with pure and mixed cultures of Acinetobacter sp., Bacillus pumilus Meyer & Gottheil, Comamonas acidovorans den Dooren de Jong, Pseudomonas mendocina Palleroni, Flavobacterium sp. and Empedobacter breve (Holmes & Owen). Rearing larvae on mixed cultures enhanced pupal weight but not survival. Horn fly larvae failed to survive when reared on standard bacteriological media inoculated with pure and mixed cultures of Acinetobacter sp., P. mendocina, and C. acidovorans. Larvae completed development on a minimal medium supplemented with alfalfa, egg proteins, and vitamins. Medium with low alfalfa content (30 g alfalfa/500 ml minimal medium) had enhanced survival when supplemented with egg yolk protein and vitamins. Medium with high alfalfa content (130 g alfalfa/500 ml minimal medium) had enhanced survival when supplemented with whole egg protein and vitamins. Survival was also favored when media were inoculated with pure cultures of Acinetobacter or Acinetobacter mixed with either Pseudomonas or Comamonas. Individual plates could support larvae developing from up to 40 eggs, and survival was least variable when plates were inoculated with greater numbers of eggs. This rearing system shows promise as a means for conducting standardized bioassays on a meridic diet.     

Effect of livestock manures on the fitness of house fly, Musca domestica L. (Diptera: Muscidae)

The house fly, Musca domestica L. (Diptera: Muscidae) is one of the major pests of confined and pastured livestock worldwide. Livestock manures play an important role in the development and spread of M. domestica. In the present study, we investigated the impact of different livestock manures on the fitness and relative growth rate of M. domestica and intrinsic rate of natural increase. We tested the hypotheses by studying life history parameters including developmental time from egg to adult's eclosion, fecundity, longevity, and survival on manures of buffalo, cow, nursing calf, dog, horse, poultry, sheep, and goat, which revealed significant differences that might be associated with fitness costs. The maggots reared on poultry manure developed faster compared to any other host manure. The total developmental time was the shortest on poultry manure and the longest on horse manure. The fecundity by females reared on poultry, nursing calf, and dog manures was greater than on any other host manures. Similarly, percent survival of immature stages, pupal weight, eggs viability, adults' eclosion, survival and longevity, intrinsic rate of natural increase, and biotic potential were significantly higher on poultry, nursing calf, and dog manures compared to any other livestock manures tested. However, the sex ratio of adult flies remained the same on all types of manures. The low survival on horse, buffalo, cow, sheep, and goat manures suggest unsuitability of these manures, while the higher pupal weight on poultry, nursing calf, and dog manures suggest that these may provide better food quality to M. domestica compared with any other host manures. Our results point to the role of livestock manures in increasing local M. domestica populations. Such results could help to design cultural management strategies which may include sanitation, moisture management, and manure removal.     

Horn fly (Diptera: Muscidae) saliva targets thrombin action in hemostasis

The horn fly, Hematobia irritans (L.), is an important pest of livestock because the adult stage of both sexes are aggressive blood-feeders. Remarkably, even though horn fly adults feed recurrently on their hosts as ectoparasites, these flies lack the ADP-responsive antiplatelet aggregation and vasodilatory antihemostatic systems described for other blood-feeding Diptera. Horn fly salivary gland extracts do interfere with the normal coagulation process as demonstrated by the recalcification time assay. Using this as a baseline, the effects of saliva on recalcification time, activated partial thromboplastin time, prothrombin time, and thrombin time were measured to determine which arm(s) of the coagulation cascade might be impacted. Factor-deficient plasma assays also were used to measure possible perturbations in clotting. Gland-free saliva delayed the recalcification time as well as the activated partial thromboplastin time, prothrombin time, and thrombin time. Saliva also further delayed clotting times of plasmas deficient in factor V, factor VIII, and factor XIII, indicating that other factors in the coagulation cascade were inhibited. Although horn fly saliva did not alter the ability of deficient plasma reconstituted with factor X to clot, it did inhibit deficient plasma reconstituted with factor II (thrombin). Antithrombin activity in saliva was confirmed by its ability to interfere with thrombin hydrolysis of fibrinogen, its normal substrate, and by its inhibition of thrombin action on a chromagenic substrate that mimics the hydrolytic site of fibrinogen. Thus, horn fly saliva contains a factor that specifically targets thrombin, a key component in the coagulation cascade. While the biochemical mechanisms of inhibition may vary, this antihemostatic characteristic is shared with other zoophilic Diptera such as black flies, Simulium spp., and tsetse, Glossina morsitans morsitans Westwood, that feed on ungulates.