Bacterial communities and prevalence of antibiotic resistance genes carried within house flies (Diptera: Muscidae) associated with beef and dairy cattle farms.

House flies (Musca domestica Linnaeus) are vectors of human and animal pathogens at livestock operations. Microbial communities in flies are acquired from, and correlate with, their local environment. However, variation among microbial communities carried by flies from farms in different geographical areas is not well understood. We characterized bacterial communities of female house flies collected from beef and dairy farms in Oklahoma, Kansas, and Nebraska using 16S rDNA amplicon sequencing and PCR. Bacterial community composition in house flies was affected by farm type and location. While the shared number of taxa between flies from beef or dairy farms was low, those taxa accounted >97% of the total bacterial community abundance. Bacterial species richness was 4% greater in flies collected from beef than in those collected from dairy farms and varied by farm type within states. Several potential pathogenic taxa were highly prevalent, comprising a core bacterial community in house flies from cattle farms. Prevalence of the pathogens Moraxella bovis and Moraxella bovoculi was greater in flies from beef farms relative to those collected on dairy cattle farms. House flies also carried bacteria with multiple tetracycline and florfenicol resistance genes. This study suggests that the house flies are significant reservoirs and disseminators of microbial threats to human and cattle health.

Development and first evaluation of an attractant impregnated adhesive tape against blood-sucking flies.

Stable flies are one of the most important arthropod pests of livestock that reduce cattle weight gain and milk production leading to annual economic losses in excess of $2 billion to the US cattle industry. The host-seeking behavior is primarily mediated by associated odors from stable fly larval development environments and host animals. The present paper reports the development and evaluation of attractant-impregnated adhesive tapes to reduce stable fly attacks on cattle. Laboratory bioassays showed that only m-cresol impregnated adhesive tapes caught significantly more stable flies (16 ± 1) than the control tape without attractant added (7 ± 1), with a 77% fly recapture rate. Attractant-impregnated adhesive tapes deployed in cattle feedlots showed significant impacts in reducing fly population, with a total of one million stable flies captured over a period of three weeks (mean catches from 57 596 to 102 088 stable flies per trap per week). It further relieved cattle stress with a significant reduction of biting fly avoidance behavior, (6 ± 0.4 cows observed with tail wagging in control vs. 3 ± 0.4 from the trap-deployed). The efficacy of the developed tapes lasted up to 1-week longevity, although 70% of m-cresol was released starting from the second day. The m-cresol impregnated adhesive tape provided an 80% reduction in cattle stress due to stable fly attack. This is the first report of a technology developed by integrating an attractant compound into an adhesive material on a plastic film with demonstrated effectiveness in trapping biting flies that attack livestock animals.

The genome of the stable fly, Stomoxys calcitrans, reveals potential mechanisms underlying reproduction, host interactions, and novel targets for pest control.

BACKGROUND: The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. RESULTS: This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. CONCLUSIONS: The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.

Retention of lumpy skin disease virus in Stomoxys spp (Stomoxys calcitrans, Stomoxys sitiens, Stomoxys indica) following intrathoracic inoculation, Diptera: Muscidae.

Lumpy skin disease (LSD) is an emerging disease of cattle in Kazakhstan and the means of transmission remains uncertain. In the current study, retention of Lumpy Skin Disease Virus (LSDV) by three Stomoxys species following intrathoracic inoculation was demonstrated under laboratory conditions. A virulent LSDV strain was injected into the thorax of flies to bypass the midgut barrier. The fate of the pathogen in the hemolymph of the flies was examined using PCR and virus isolation tests. LSDV was isolated from all three Stomoxys species up to 24h post inoculation while virus DNA was detectable up to 7d post inoculation.

The combined effects of temperature and relative humidity parameters on the reproduction of Stomoxys species in a laboratory setting.

In this study, Stomoxys species (S. calcitrans, S. sitiens and S. indica) were examined to improve on the current technique for mass rearing using a method of combined incubation parameters. Moreover, the reproductive potential of immature forms at various stages of development was defined. Immature forms of stable flies were incubated according to species. There was no significant difference in the number of immature forms obtained among species incubated under the same conditions. Six incubation parameters were used in combination, at temperatures (T) of 32°C, 27°C and 22°C and relative humidity (RH) of 90% and 70% RH. The combined method resulted in a higher number of eggs hatching at 32°C and 90% humidity as well as an increase in the number of larva pupated and emergence of imago at 27°C and 70% humidity.

Improved Sentinel Method for Surveillance and Collection of Filth Fly Parasitoids.

Parasitoids are important natural enemies of house flies and other muscoid flies. The two most commonly used methods for collecting fly parasitoids from the field have distinct advantages and disadvantages. Collections of wild puparia depend on the ability to find puparia in sufficient numbers and are prone to localized distortions in relative species abundance because of the overrepresentation of samples from hot spots of fly larval activity. Placement and retrieval of sentinel puparia is convenient and allows consistent sampling over time but is strongly biased in favor of Muscidifurax spp. over Spalangia spp. An improved sentinel method is described that combines some of the advantages of these two methods. Fly medium containing larvae is placed in containers, topped with a screen mesh bag of puparia, and placed in vertebrate-proof wire cages. Cages are placed at sites of actual or potential fly breeding and retrieved 3-7 d later. The modified method collected species profiles that more closely resembled those of collections of wild puparia than those from sentinel pupal bags. A method is also described for isolating puparia individually in 96-well tissue culture plates for parasitoid emergence. Use of the plate method provided a substantial saving of time and labor over the use of individual gelatin capsules for pupal isolation. Puparia from the collections that were housed individually in the wells of tissue culture plates had a higher proportion of emerged Spalangia species than puparia that were held in groups.

Methods for Surveying Stable Fly Populations.

Stable flies are among the most important pests of livestock throughout much of the world. Their painful bites induce costly behavioral and physiological stress responses and reduce productivity. Stable flies are anthropogenic and their population dynamics vary depending on agricultural and animal husbandry practices. Standardized sampling methods are needed to better identify the factors controlling stable fly populations, test novel control technologies, and determine optimal management strategies. The current study reviewed methods used for a long-term study of stable fly population dynamics in the central Great Plains. An additional study compared the relative size of flies sampled from the general population with that of flies sampled emerging from substrates associated with livestock production. Flies developing in livestock associated substrates are significantly larger than those in the general population indicating that other types of developmental sites are contributing significant numbers of flies to the general population. Because efforts to identify those sites have yet to be successful, we speculate that they may be sites with low densities of developing stable flies, but covering large areas such as croplands and grasslands. The stable fly surveillance methods discussed can be used and further improved for monitoring stable fly populations for research and management programs.

Spatial repellency, antifeedant activity and toxicity of three medium chain fatty acids and their methyl esters of coconut fatty acid against stable flies.

BACKGROUND: Stable flies are one of the most detrimental arthropod pests to livestock. With changing climates and agronomic practices, they expand their roles as pests and disease vectors as well. Their painful bites reduce livestock productivity, annoy companion animals, and interfere with human recreational activities. Current management technologies are unable to effectively control stable flies. The present study reports new results concerning the contact, spatial repellency, and toxicity of a bio-based product, coconut fatty acid and their methyl ester derivatives of free fatty acids of C8:0 , C10:0 and C12:0 to stable flies. RESULTS: Three medium chain fatty acid methyl esters (C8:0 , C10:0 and C12:0 ) showed strong antifeedant activity against stable flies and their strengths were dose-dependent. Only the C8:0 acid, C8:0 - and C10:0 methyl esters elicited significant antennal responses. Laboratory single cage olfactometer bioassays revealed that coconut fatty acid and C8:0 methyl ester displayed active spatial repellency. All three methyl esters showed strong toxicity against stable flies. CONCLUSION: Antifeedant activity is the main method through which coconut fatty acid deters stable fly blood-feeding. The C8:0 , C10:0 and C12:0 methyl esters act not only as strong antifeedants, but also possess strong toxicity against stable fly adults. Limited spatial repellency was observed from coconut fatty acid and C8:0 methyl ester. © 2019 Society of Chemical Industry.

Effects of Diet Quality and Temperature on Stable Fly (Diptera: Muscidae) Development.

The effects of diet quality and temperature on the development time and size of stable flies, Stomoxys calcitrans (L.), was evaluated. Both development time and size varied relative to diet quality and temperature, and their effects were additive. Diet quality and temperature made similar contributions to the variance in size whereas temperature was responsible for >97% of the variance in development time. Regression analysis predicted the shortest development time, egg to adult, to be 12.7 days at 32 °C and 70% nutrients. Egg to adult development varied curvilinearly relative to diet quality and temperature on the degree day 10 (DD10) scale taking 261 DD10 at 30 °C and 50% nutrients. The thermal threshold was 11.5 °C with a thermal constant of 248. Very few stable flies developed to adult on the poorest diet (12.5% nutrients) and adults emerged from fewer than 1% of the puparia at 35 °C. The heaviest pupae (15.4 mg) were produced with the 100% diet at 15 °C and adults had a higher probability of emerging successfully from heavier puparia. The length of the discal-medial cell of adult wings had a cubic relationship with puparia weight and peaked at 21 °C. Egg to pupariation survival was predicted to peak at 27 °C and 71% diet whereas puparia to adult survival peaked at 24 °C and 100% diet. Diet quality and temperature had no effect on sex ratio and the rate of development did not differ between the sexes. Female stable flies were ≈5% larger than males. Composite metrics for egg to pupariation and egg to adult fitness were developed. The optimum for puparia fitness was 29 °C and 78% diet quality and for adult fitness 25 °C and 83% diet quality. Diet accounted for 31% of the variance in pupal fitness and 24% of the variance in adult fitness whereas temperature accounted for 17% and 20%, respectively.

Frequency of kdr and kdr-his Alleles in Stable Fly (Diptera: Muscidae) Populations From the United States, Costa Rica, France, and Thailand.

Anecdotal evidence of pyrethroid insecticide product failure for the control of stable fly [Stomoxys calcitrans (L.)] populations in the United States and worldwide prompted us to evaluate the frequency of knockdown resistance (kdr)-type polymorphisms within the voltage-sensitive sodium channel (Vssc) gene of field collected specimens from the United States, France, Costa Rica, and Thailand. The kdr-his allele (L1014H), associated with permethrin resistance, was detected in stable flies from the 10 states sampled in the United States, as well as from Costa Rica and France (Toulouse). Field collections of stable flies from California (Modesto) and New York (Cliffton Springs) exhibited reduced susceptibility upon exposure to a diagnostic permethrin concentration of 10× LC99, but survival did not appear to strictly associate with frequency of the kdr-his allele. This suggests that there are additional resistance mechanisms contributing to the phenotype in these states. The kdr allele (L1014F) was detected for the first time in stable flies originating in France and Thailand, and an improved, DNA-based diagnostic assay was developed and validated for use in future screens for kdr and kdr-his allele frequencies from field collections. The absence of kdr in United States and Costa Rica populations suggests that the allele is currently restricted to Europe and Asia.

Cow-Calf Producers' Willingness to Pay for Bulls Resistant to Horn Flies (Diptera: Muscidae).

Horn flies (Haematobia irritans (L.)) have long posed animal health and welfare concerns. Economic losses to the cattle and dairy industries from their blood-feeding behavior include decreased weight gain, loss in milk productivity, and transmission of bacteria causing mastitis in cattle. Horn fly management strategies are labor intensive and can become ineffective due to the horn fly's ability to develop insecticide resistance. Research indicates that for some cattle herds, genetically similar animals consistently have fewer flies suggesting those animals are horn fly resistant (HFR) and that the trait is heritable; however, it is currently unknown if cattle producers value this trait. Tennessee and Texas cow-calf producers were surveyed to estimate their willingness to pay for HFR bulls and to identify the factors affecting their decision to adopt a HFR bull in their herds. Results indicate that Tennessee and Texas cow-calf producers were willing to pay a premium of 51% and 59% above the base price, respectively, for a HFR bull with the intent to control horn flies within their herd. Producer perceptions of horn fly intensities and the HFR trait, along with their pest management practices, were factors that affected Tennessee and Texas producer willingness to adopt a HFR bull. In Texas, demographics of the producers and their farms also had a role. Knowing producers are willing to pay a premium for the HFR bull indicates that producers value the HFR trait and warrants additional research on the development, implementation, and assessment of the trait.

Better than DEET Repellent Compounds Derived from Coconut Oil.

Hematophagous arthropods are capable of transmitting human and animal pathogens worldwide. Vector-borne diseases account for 17% of all infectious diseases resulting in 700,000 human deaths annually. Repellents are a primary tool for reducing the impact of biting arthropods on humans and animals. N,N-Diethyl-meta-toluamide (DEET), the most effective and long-lasting repellent currently available commercially, has long been considered the gold standard in insect repellents, but with reported human health issues, particularly for infants and pregnant women. In the present study, we report fatty acids derived from coconut oil which are novel, inexpensive and highly efficacious repellant compounds. These coconut fatty acids are active against a broad array of blood-sucking arthropods including biting flies, ticks, bed bugs and mosquitoes. The medium-chain length fatty acids from C8:0 to C12:0 were found to exhibit the predominant repellent activity. In laboratory bioassays, these fatty acids repelled biting flies and bed bugs for two weeks after application, and ticks for one week. Repellency was stronger and with longer residual activity than that of DEET. In addition, repellency was also found against mosquitoes. An aqueous starch-based formulation containing natural coconut fatty acids was also prepared and shown to protect pastured cattle from biting flies up to 96-hours in the hot summer, which, to our knowledge, is the longest protection provided by a natural repellent product studied to date.

Laboratory Rearing of Stable Flies and Other Muscoid Diptera.

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in agronomic practices resulted in serious outbreaks of stable flies in several countries. These outbreaks disrupted livestock production and human recreation resulting in public demands for increasing research and management efforts for this pest. A simple and inexpensive procedure for rearing stable flies for laboratory studies is presented. The procedure uses locally available diet components, equipment and supplies. The procedure can be adapted for rearing other muscoid flies including face fly (Musca autumnalis), horn fly (Haematobia irritans), and house fly (Musca domestica). The procedure produces stable fly puparia averaging 12.5 mg and ~35% egg to adult survival. Approximately 3000 flies are produced in each pan.

Precipitation and Temperature Effects on Stable Fly (Diptera: Muscidae) Population Dynamics.

The dynamics of stable fly, Stomoxys calcitrans (L.), populations relative to temperature and precipitation were evaluated in a 13-yr study in eastern Nebraska. During the course of the study, >1.7 million stable flies were collected on an array of 25 sticky traps. A log-normal model using degree-days with a 15 °C threshold and weekly lags 0-4 for temperature and 2-7 for precipitation provided the best fit with the observed data. The relationships of temperature and precipitation to stable fly trap catches were both curvilinear, with maxima at 6.6 degree-day-15 (≈22 °C) and 7.4 mm precipitation per day, respectively. The temperature and precipitation model accounted for 72% of the variance in seasonal trap catches.

Augmenting Laboratory Rearing of Stable Fly (Diptera: Muscidae) Larvae With Ammoniacal Salts.

Stable flies are blood feeding parasites and serious pests of livestock. The immature stages develop in decaying materials which frequently have high ammonium content. We added various ammonium salts to our laboratory stable fly rearing medium and measured their effect on size and survival as well as the physical properties of the used media. The addition of ammonium hydroxide, ammonium phosphate and ammonium sulfate reduced larval survival. These compounds decreased pH and increased ammonium content of the used media. Ammonium bicarbonate had no effect on pH and marginally increased ammonium while increasing survival twofold. The optimal level of ammonium bicarbonate was 50 g (0.63 mol) per pan. Larval survival decreased when pH was outside the range of 8.5 to 9.0.

Environmental Parameters Associated With Stable Fly (Diptera: Muscidae) Development at Hay Feeding Sites.

Substrates composed of hay residues, dung, and urine accumulate around winter hay feeding sites in cattle pastures, providing developmental habitats for stable flies. The objective of this study was to relate physiochemical and microbial properties of these substrates to the presence or absence of stable fly larvae. Properties included pH, temperature, moisture, ammonium concentration, electrical conductivity, and numbers of coliform, fecal coliform, Escherichia coli, and Enterococcus bacteria. Each physiochemical sample was classified as a function of belonging to one of the three 2-m concentric zones radiating from the feeder as well as presence or absence of larvae. In total, 538 samples were collected from 13 sites during 2005-2011. Stable fly larvae were most likely to be found in moist, slightly alkaline substrates with high levels of ammonium and low temperature. The probability of larvae being present in a sample was the highest when the moisture content was 347% relative to dry weight and the average pH was 8.4. Larvae were recovered within all zones, with a nonsignificant, but slightly higher, percentage of samples containing larvae taken 2-4 m from the center. All methods used to enumerate bacteria, except total coliform, indicated decreasing concentrations in hay bale residue throughout the summer. In addition to the environmental parameters, cumulative degree day 10°C had a significant effect on the probability of observing stable fly larvae in a sample, indicating that unidentified seasonal effects also influenced immature stable fly populations.

Visual and olfactory enhancement of stable fly trapping.

BACKGROUND: Stable flies are considered to be one of the major blood-feeding pests in the US livestock industry, causing losses running into billions of dollars annually. Adult stable flies are highly attracted to Alsynite traps; however, Alsynite is becoming increasingly difficult to obtain and is expensive. RESULTS: Here, we report on the development of a less expensive and more efficacious trap based upon a white panel with the option to add visual and olfactory stimuli for enhanced stable fly trapping. White panel traps caught twice as many stable flies than Alsynite traps. Baiting the traps with synthetic manure volatiles increased catches 2-3-fold. Electroretinographic recordings of stable flies showed strong peaks of visual sensitivities occurring at 330-360 nm, 460-525 nm and 605-635 nm. A laboratory study indicated that young stable flies are more responsive to white, whereas gravid females prefer blue; in the field, white traps caught more stable flies than patterned or blue-black traps. CONCLUSION: Stable fly control can be enhanced by developing more efficient trapping systems with added visual and olfactory stimuli. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Biology and trapping of stable flies (Diptera: Muscidae) developing in pineapple residues (Ananas comosus) in Costa Rica.

Pineapple production in Costa Rica increased nearly 300-fold during the last 30 yr, and >40,000 hectares of land are currently dedicated to this crop. At the end of the pineapple cropping cycle, plants are chopped and residues incorporated into the soil in preparation for replanting. Associated with increased pineapple production has been a large increase in stable fly, Stomoxys calcitrans (L.), populations. Stable flies are attracted to, and oviposit in, the decomposing, chopped pineapple residues. In conjunction with chemical control of developing larvae, adult trapping is an important control strategy. In this study, four blue-black fabric traps, Nzi, Vavoua, Model H, and Ngu, were compared with a white sticky trap currently used for stable fly control in Costa Rica. Overall, the white sticky trap caught the highest number of stable flies, followed by the Nzi, Vavoua, Model H, and Ngu. Collections on the white sticky trap increased 16 d after residues were chopped; coinciding with the expected emergence of flies developing in the pineapple residues. During this same time period, collections in the blue-black fabric traps decreased. Sex ratio decreased from >7:1 (females:males) 3-7 d after chopping to 1:1 at 24-28 d. White sticky, Nzi and Vavoua traps collected similar numbers of colonizing flies 3-7 d after residues were chopped. However, white sticky traps collected more flies once emergence from the pineapple residues began. Although white sticky traps collected more flies than fabric traps, they remain labor intensive and environmentally unsound because of their disposable and nonbiodegradable nature.

External Morphology of Stable Fly (Diptera: Muscidae) Larvae.

Scanning electron microscopy was used to examine the external morphology of first-, second-, and third-instar stable flies (Stomoxys calcitrans (L.)). In the cephalic region, the antennae, labial lobe, and maxillary palpi are morphologically similar among instars. Antennae comprise a prominent anterior dome that is the primary site of olfaction, while the maxillary palpi are innervated with mechano- and chemosensilla and scolopodia. The ventral organ and facial mask, also located in the pseudocephalon, are not well-developed in first instars, but become progressively more so in the subsequent instars. When the pseudocephalon is partially retracted, anterior spines cusp around the oral ridges of the facial mask. This indicates the anterior spinose band may be used in conjunction with the facial mask in predigestion. Functional anterior spiracles are absent on first instars, but become evident as a pair of palmate spiracular processes with five to seven lobes in second and third instars. A pair of Keilin's organs, functioning as hygroreceptors, is located on each thoracic segment. Abdominal segments are marked with ventral creeping welts, the anal pad, anus, papillae, and posterior spiracles. Ventral creeping welts are thought to aid in locomotion, while the anal pad acts as an osmoregulatory structure. Posterior spiracles are modified from round spiracular discs with two straight slits in the first instar to triangular discs with two and three sinuous slits in the second and third instars, respectively.