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.     

Reservoir competence of lesser mealworm (Coleoptera: Tenebrionidae) for Campylobacter jejuni (Campylobacterales: Campylobacteraceae)

The lesser mealworm, Alphitobius diaperinus (Panzer), is a carrier of Campylobacter spp. in poultry facilities; however, the beetle's importance in the epidemiology of campylobacteriosis is not known. A series of laboratory experiments were designed to test the vector and reservoir competence of the lesser mealworm for Campylobacter jejuni. In the first experiment, C. jejuni was swabbed onto the outer surface of adult and larval beetles to determine how long bacteria can survive on the beetles' exterior. Next, adult and larval mealworms were allowed to drink from a solution containing C. jejuni and the duration of internal carriage was monitored. For the third experiment, beetles drank from a Campylobacter suspension and the duration of fecal shedding of bacteria was determined. In the last experiment, 3-d-old chickens were fed either one or 10 infected beetles, and cloacal swabs were tested periodically for Campylobacter. C. jejuni was detected on the exterior of larval beetles for 12 h, from the interior of larvae for 72 h, and from the feces of larvae for 12 h after exposure. Ninety percent of the birds that consumed a single adult or larval beetles became Campylobacter-positive, whereas 100% of the birds that consumed 10 adults or larvae became positive. These experiments demonstrated that the lesser mealworm could acquire and harbor Campylobacter from an environmental source. We found that the lesser mealworm was capable of passing viable bacteria to chickens that consumed the beetle. The beetle should be included in attempts to maintain Campylobacter-free poultry facilities.     

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.     

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.     

Persistence of Escherichia coli in immature house fly and stable fly (Diptera: Muscidae) in relation to larval growth and survival

The persistence of Escherichia coli in artificially fed larvae was examined for up to 48 h after ingestion by house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.). The rate of change in the E. coli load was similar for both species for up to 5 h after ingestion. Up to 48 h after ingestion, abundance of E. coli declined in immature house flies but remained constant in immature stable flies. When different E. coli concentrations were fed to larvae, the abundance of E. coli increased in stable fly larvae regardless of the initial concentration. The E. coli load in house fly larvae increased when larvae were fed a low concentration of bacteria, but it declined when larvae were fed a high concentration of bacteria. Survival of house fly and stable fly larvae averaged 62 and 25%, respectively, when reared on pure E. coli cultures. These observations suggest that house fly larvae digest E. coli and use it as a food source but stable fly larvae do not.     

Ovipositional behavior of the horn fly (Diptera: Muscidae) in the field

In the field, the female horn fly obtained blood meals from the bovine host before oviposition. The female flies moved to the lower portion of the hind legs as the legs were spread, and the tail was raised before excretion. Females deposited their eggs on the manure after excretion. The fly flew onto the manure pat for oviposition as the cow walked forward after excretion. It appeared that flies located on the belly received some stimuli for oviposition from the cow just before excretion. The horn fly deposited most of its eggs during the day with occasional deposition at night. Egg deposition increased at 10 degrees C. Fewer flies were observed on manure pats that contained >90% or <84% water. The location of horn flies on the cow and environmental factors associated with oviposition in the field are discussed.     

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.     

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.     

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.     

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.     

Prey- and density-mediated dispersal in Carcinops pumilio (Coleoptera: Histeridae), a predator of house fly (Diptera: Muscidae) eggs and larvae

Carcinops pumilio (Erichson) were collected using two trapping methods: a black light pitfall trap and a mesh-bottomed trap placed on poultry manure. C. pumilio collected with black lights and subsequently starved had a significantly higher dispersal rate during days 1-3 than fully fed groups. When densities of < 500 Caloglyphus berlesei (Michael) (Acarina: Acaridae) per 50 C. pumilio were provided, mite availability had a significant effect on dispersal of beetles captured with both black light traps and the mesh-bottomed trap during the day 1-3 period. Our results indicate that the availability of acceptable food sources can delay and possibly prevent dispersal by C. pumilio. Black light-captured beetles appeared to be in a state of dispersal when captured. A subset of dispersing beetles was present in groups captured with the mesh-bottomed trap.     

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.     

Feeding deterrent activity of synthesized silver nanoparticles using Manilkara zapota leaf extract against the house fly, Musca domestica (Diptera: Muscidae)

With a greater awareness of the hazards associated with the use of synthetic organic insecticides, there has been an urgent need to explore suitable alternative products for pest control. Musca domestica is ubiquitous insect that has the potential to spread a variety of pathogens to humans and livestock. They are mechanical carriers of more than hundred human and animal intestinal diseases and are responsible for protozoan, bacterial, helminthic, and viral infections. The present work aimed to investigate the feeding deterrent activity of synthesized silver nanoparticles (Ag NPs) using leaf aqueous extract of Manilkara zapota against M. domestica. The synthesized Ag NPs were recorded from UV–vis spectrum at 421 nm and scanning electron microscopy confirm the biosynthesis and characterization of Ag NPs with spherical and oval in shape and size of 70–140 nm. The FTIR analysis of the purified nanoparticles showed the presence of bands 1,079, 1,383, 1,627, 2,353, and 2,648 cm^?1, which were complete synthesis of AgNPs; the XRD pattern of AgNPs showed diffraction peaks at 2θ values of 38.06°, 44.37°, 64.51°, and 77.31° sets of lattice planes were observed (111), (200), (220), and (311) facts of silver, respectively. Adult flies were exposed to different concentrations of the aqueous extract of synthesized Ag NPs, 1 mM silver nitrate (AgNO₃) solution and aqueous extract of M. zapota for 1, 2, and 3 h; however, AgNPs showed 72% mortality in 1 h, 89% mortality was found in 2 h, and 100% mortality was found in 3 h exposure at the concentration of 10 mg/mL and the leaf aqueous extract showed 32% mortality in 1 h, 48% mortality was found in 2 h, and 83% mortality was found in 3 h exposure at concentration of 50 mg/mL. The most efficient activity was observed in synthesized Ag NPs against M. domestica (LD₅₀?=?3.64 mg/mL; LD₉₀?=?7.74 mg/mL), the moderate activity reported in the aqueous extract of M. zapota (LD₅₀?=?28.35 mg/mL; LD₉₀?=?89.19 mg/mL) and nil activity were observed in AgNO₃ solution at 3 h exposure time at 10 mg/mL. Dimethyl 2, 2-dichlorovinyl phosphate (DDVP) was used as a positive control and showed the LD₅₀ value of 3.38 mL/L. These results suggest that the synthesized Ag NPs have the potential to be used as an ideal eco-friendly approach for the control of the adult of M. domestica. This method is considered as a new approach to control sanitary pest. Therefore, this study provides first report on the feeding deterrent activity of synthesized Ag NPs against housefly.