Point Protection with Transfluthrin against Musca domestica L. in a Semi-Field Enclosure.

House flies are notoriously difficult to control, owing to their tendency to live in close relationships with humans and their livestock, and their rapid development of resistance to chemical controls. With this in mind, we explored an alternative chemical control, a spatial repellent to deter Musca domestica L. from points we wanted to protect (i.e., a baited trap). Our results demonstrated that the synthetic spatial repellent, transfluthrin, is effective in preventing M. domestica adults from entering protected traps for both a susceptible strain (CAR21) and a field-acquired permethrin-resistant strain (WHF; 24 h LD50 resistance ratio of 150), comprising 22% and 28% of the total number of flies collected, respectively. These results are promising and demonstrate that transfluthrin can be an effective spatial repellent to protect points of interest where needed.

Effects of larval diets on some biological parameters and morphometric and biochemical analysis of ovaries of Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae).

The effects of three larval diets (beef meat, chicken meat, and beef liver) on development of Lucilia cuprina (Wiedemann) were evaluated. Egg hatching rates were higher on chicken meat and beef meat (99.5%) than on beef liver (96.5%). Pupation success was higher on chicken meat (99.0%) and beef meat (98.0%) than on beef liver (87.1%). Adult emergence rates were higher in flies reared on chicken meat (99.0%) and beef meat (98.5%) than on beef liver (93.5%). Proportions of female flies were somewhat higher in flies reared on chicken meat or beef meat (54-56% female) than on beef liver (52.5%). Flies that were reared on chicken meat and beef meat had shorter egg-adult development times (16.3 days) than flies reared on beef liver (18.5 days), and the generation time of flies reared on chicken meat or beef meat (51-52 days) was several days shorter than flies reared on beef liver (54 days). Flies reared on chicken meat had larger ovaries and more ovarioles per ovary when larvae were reared on chicken than flies reared on beef meat or beef liver. The ovaries of flies reared on chicken meat had significantly higher amounts of protein (21 mg/g body weight) than flies reared on beef liver (12 mg). Analysis of the larval diets showed that beef liver had the lowest amounts of protein and carbohydrate of the three tested diets, whereas the amounts of lipids did not differ among the diets.

Susceptibility of the adult house fly (Diptera: Muscidae) and 3 of its principal parasitoids (Hymenoptera: Pteromalidae) to the GHA strain of Beauveria bassiana and 4 isolates from field-collected muscid flies.

House fly (Musca domestica L.) (Diptera: Muscidae) populations can negatively impact poultry layer facilities, posing a risk to human and animal health and egg food safety. House flies quickly develop resistance to traditional chemical control methods; therefore, improved biological control may provide opportunities for improved integrated pest management (IPM) programs. Biological control methods currently used include augmentative releases of pteromalid pupal parasitoids and application of the fungal entomopathogen Beauveria bassiana (Balsamo) Vuillemin. This study used bioassays to compare the impact of different B. bassiana strains on survival of house flies and of 3 species of filth fly parasitoids. The B. bassiana that were compared were 3 new field-collected isolates, an older field-collected isolate (L90), and a common commercially available strain (GHA). Flies and parasitoids were exposed to filter paper treated with 1.5 × 109 spores of each strain and a control. All field-isolated strains induced lower mean survival times in house flies than GHA did. The results for all species of parasitoids demonstrated less difference among the treatment groups and the control than in-house flies. Although there was some effect of B. bassiana exposure on parasitoid mortality, the expected spatial separation of parasitoids from areas of application may offer some protection. Using the most effective tested strains of B. bassiana and filth fly parasitoids jointly could be a biological component of an IPM plan for fly control in poultry facilities.

Rescuing the Inhibitory Effect of the Salivary Gland Hypertrophy Virus of Musca domestica on Mating Behavior.

Infection with salivary gland hypertrophy virus (MdSGHV) of Musca domestica prevents female flies from accepting copulation attempts by healthy or virus-infected males. This study focused on supplemental hormonal rescue therapy for mating behavior in virus-infected female house flies. The inhibitory effect of the virus on mating behavior in females injected with MdSGHV was reversed by hormonal therapy in the form of octopamine injections, topical application of methoprene, or both therapies combined along with 20-hydroxyecdysone. Infected females whose mating responsiveness had been restored continued to have other viral pathologies associated with infection such as hypertrophy of the salivary glands and a lack of ovarian development.

New insights into the genome and transmission of the microsporidian pathogen Nosema muscidifuracis.

Introduction: Nosema is a diverse genus of unicellular microsporidian parasites of insects and other arthropods. Nosema muscidifuracis infects parasitoid wasp species of Muscidifurax zaraptor and M. raptor (Hymenoptera: Pteromalidae), causing ~50% reduction in longevity and ~90% reduction in fecundity. Methods and Results: Here, we report the first assembly of the N. muscidifuracis genome (14,397,169 bp in 28 contigs) of high continuity (contig N50 544.3 Kb) and completeness (BUSCO score 97.0%). A total of 2,782 protein-coding genes were annotated, with 66.2% of the genes having two copies and 24.0% of genes having three copies. These duplicated genes are highly similar, with a sequence identity of 99.3%. The complex pattern suggests extensive gene duplications and rearrangements across the genome. We annotated 57 rDNA loci, which are highly GC-rich (37%) in a GC-poor genome (25% genome average). Nosema-specific qPCR primer sets were designed based on 18S rDNA annotation as a diagnostic tool to determine its titer in host samples. We discovered high Nosema titers in Nosema-cured M. raptor and M. zaraptor using heat treatment in 2017 and 2019, suggesting that the remedy did not completely eliminate the Nosema infection. Cytogenetic analyses revealed heavy infections of N. muscidifuracis within the ovaries of M. raptor and M. zaraptor, consistent with the titer determined by qPCR and suggesting a heritable component of infection and per ovum vertical transmission. Discussion: The parasitoids-Nosema system is laboratory tractable and, therefore, can serve as a model to inform future genome manipulations of Nosema-host system for investigations of Nosemosis.

Laboratory Evaluation of Pupal Parasitoids for Control of the Cornsilk Fly Species, Chaetopsis massyla and Euxesta eluta.

Cornsilk flies are serious pests of sweet corn through damage to cobs and secondary fungal establishment. As pupae are generally outside the infested cob on the ground, there can be potential for use of pupal parasitoids for control. Two species of gregarious parasitoids, Muscidifurax raptorellus and Nasonia vitripennis, and three species of solitary parasitoids, Spalangia endius, Spalangia cameroni and Muscidifurax raptor, were evaluated against pupae of the two cornsilk fly species, Euxesta eluta and Chaetopsis massyla. House fly pupae, the most common host for most of the parasitoids, were included for comparison. All of the parasitoids killed and successfully parasitized pupae of the two cornsilk fly species at rates that were similar to house fly pupae. Adult parasitoids that emerged from cornsilk fly hosts were somewhat smaller than parasitoids reared from house flies and had proportionally fewer females. These parasitoids, which are widely and commercially available for filth fly control, warrant further consideration for their potential against cornsilk flies in the field.

Filth Fly Parasitoid (Hymenoptera: Pteromalidae) Monitoring Techniques and Species Composition in Poultry Layer Facilities.

Muscid flies, especially house flies (Musca domestica L.) (Diptera: Muscidae), are a major pest of poultry layer facilities. Augmentative biological control of muscid flies with pteromalid wasps has gained increased attention in recent years. Knowing which pteromalid species are present in a specific area could produce more effective filth fly control. The purpose of this project was to survey parasitoid populations in poultry layer facilities in central and southeastern Pennsylvania from June through September. Two genera of parasitoids, Spalangia and Trichomalopsis, were collected over the course of the survey. Overall, out of 3,724 parasitized pupae the species collected in order of most to least common were Spalangia cameroni Perkins, Spalangia nigroaenea Curtis, Trichomalopsis spp., and Spalangia endius Walker. House fly parasitism overall and by each parasitoid species varied by location and over the four study months. A second objective was to evaluate a new parasitoid trap for surveying parasitoid wasp populations. This device uses a combination of house fly third instars and development media. This was compared to a more traditional method, the sentinel bag, which uses only fly pupae. A higher proportion of Spalangia spp. emerged from the new trap design and more Trichomalopsis spp. emerged from the sentinel bag. This suggests that using this new device alongside the traditional collection method may result in more accurate sampling of pteromalid populations.

Comparative Virulence of Metarhizium anisopliae and Four Strains of Beauveria bassiana Against House Fly (Diptera: Muscidae) Adults With Attempted Selection for Faster Mortality.

Entomopathogenic fungi such as Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae/brunneum (Metchnikoff)/Petch have shown promising results for managing the house fly, Musca domestica L. A primary challenge of using these biological control agents (BCAs) in field situations is the time required to induce high adult house fly mortality, typically 6-7 d post-exposure. In this study, virulence of M. anisopliae (strain F52) and four B. bassiana strains were compared. The B. bassiana strains GHA and HF23 are used in commercial products and those were compared with two strains that were isolated from house flies on dairy farms (NFH10 and L90). Assays were conducted by exposing adult house flies to fungal-treated filter paper disks for 2 h. The lethal time to 50% mortality (LT50) at the high concentration of 1 × 109 conidia ranged from 3.8 to 5.2 d for all five strains. GHA, NFH10, and L90 killed flies faster than M. anisopliae strain F52; HF23 did not differ from either the M. anisopliae or the other B. bassiana strains. Attempts with the NFH10 strain to induce faster fly mortality through selection across 10 fungal to fly passages did not result in shorter time to fly death of the selected strain compared with the unselected strain.

Exposure Timing and Method Affect Beauveria bassiana (Hypocreales: Cordycipitaceae) Efficacy Against House Fly (Diptera: Muscidae) Larvae.

House flies, Musca domestica L., are widely recognized for their ability to develop resistance to chemical insecticides so alternative control strategies are desired. The use of entomopathogenic fungi such as Beauveria bassiana (Balsamo) Vuillemin to manage house fly populations has shown promising results; however, the success of using this fungus against larval house flies varies widely. The overall objective of this study was to examine factors that may influence efficacy of B. bassiana treatments against larvae. When a high concentration (4 × 1011 conidia/ml) was applied to first- and second-instar larvae in rearing medium, there was a significant reduction in pupation and adult emergence rates. Treating third-instar larvae at the same concentration did not result in a significant reduction of pupation or adult emergence. Temperature (22 versus 32°C) and media composition (diets with- and without propionic acid) did not affect the B. bassiana treatment efficacy against house fly larvae. The narrow time window of vulnerability of larvae and the high doses required to infect them indicate that B. bassiana has little potential as an operational biocontrol agent for house fly larvae.

Insecticide Resistance Development in the Filth Fly Pupal Parasitoid, Spalangia cameroni (Hymenoptera: Pteromalidae), Using Laboratory Selections.

Filth flies remain one of the most prevalent pest groups affecting the animal production industry. Spalangia spp. and Muscidifurax spp. are beneficial parasitic wasps that often are utilized to manage filth fly populations such as house flies, Musca domestica L. (Diptera: Muscidae), and stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae). These wasps search for filth fly pupae as hosts in areas potentially treated with insecticides, which may result in nontarget insecticide selection effects. However, research regarding resistance development in parasitic wasps such as S. cameroni Perkins (Hymenoptera: Pteromalidae) is limited. Therefore, a study was conducted to determine the potential of S. cameroni to develop resistance to the commonly used insecticide permethrin, as well as compare permethrin susceptibility among several S. cameroni strains. After 10 selected generations, susceptibility was significantly lower for the selected strain when compared with that of its unselected parent strain. A comparison of several parasitoid strains collected from different U.S. states indicated that permethrin susceptibility was not significantly different between a baseline strain and more recently established field strains. The potential implications of this previously unrecognized nontarget insecticide exposure effect on filth fly parasitoids are discussed.

Oral and Topical Insecticide Response Bioassays and Associated Statistical Analyses Used Commonly in Veterinary and Medical Entomology.

Veterinary and medical entomologists who are involved in research on pest control often need to perform dose-response bioassays and analyze the results. This article is meant as a beginner's guide for doing this and includes instructions for using the free program R for the analyses. The bioassays and analyses are described using previously unpublished data from bioassays on house flies, Musca domestica Linnaeus (Diptera: Muscidae), but can be used on a wide range of pest species. Flies were exposed topically to beta-cyfluthrin, a pyrethroid, or exposed to spinosad or spinetoram in sugar to encourage consumption. LD50 values for beta-cyfluthrin in a susceptible strain were similar regardless of whether mortality was assessed at 24 or 48 h, consistent with it being a relatively quick-acting insecticide. Based on LC50 values, spinetoram was about twice as toxic as spinosad in a susceptible strain, suggesting a benefit to formulating spinetoram for house fly control, although spinetoram was no more toxic than spinosad for a pyrethroid-resistant strain. Results were consistent with previous reports of spinosad exhibiting little cross-resistance. For both spinosad and spinetoram, LC50 values were not greatly different between the pyrethroid-resistant strain and the susceptible strain.

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.

Beauveria bassiana Culturing and Harvesting for Bioassays With House Flies.

The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) has been widely studied against a wide range of arthropod pests, including many of medical and veterinary importance. New investigators must sort through a wide array of published methods for the production, harvest, storage, and bioassay methods for this pathogen. Simplified methods for production of conidia using Sabouraud dextrose agar with yeast (SDYA) plates and two conidial harvesting methods are described. Dry harvesting yields conidia that are ready to incorporate into dusts and food baits, but the fungal product includes mycelial debris that can hamper quantification and introduces variable amounts of unwanted bulk. Wet harvesting with filtration produces a cleaner product that is immediately ready for testing in liquid formulations. Examples of bioassays with house flies are presented that include conidia applied topically to the dorsal thorax for dose-mortality assays and conidial suspensions applied to filter paper disks for concentration mortality assays.

Toxicity of fluralaner, a companion animal insecticide, relative to industry-leading agricultural insecticides against resistant and susceptible strains of filth flies.

Filth flies cause billions of dollars of losses annually to the animal production industry. Fluralaner is a relatively new pesticide currently sold for control of fleas, ticks, and mites on companion animals and poultry. We examined the efficacy of fluralaner against three species of filth flies. Insecticide-susceptible horn flies and stable flies were tested topically. Fluralaner outperformed permethrin by > 2-fold for the horn flies but underperformed permethrin by > 45-fold for stable flies at 24 h. House flies were tested topically with fluralaner in comparison to permethrin at 48 h and orally with fluralaner in comparison to imidacloprid at 24 h. Topical fluralaner was 6- to 28-fold as toxic as permethrin in four pyrethroid-resistant strains and not significantly less toxic than permethrin in a susceptible strain and a mildly pyrethroid-resistant strain. There was slight cross-resistance between topically applied fluralaner and permethrin in all five insecticide-resistant strains tested. Oral fluralaner was more toxic than imidacloprid in all four house fly strains tested, 9- to 118-fold as toxic. Oral cross-resistance between imidacloprid and fluralaner was not detected, but imidacloprid resistance was not high in any of the tested strains. Fluralaner shows promise for control of horn flies and house flies.

Heat Adaptation of the House Fly (Diptera: Muscidae) and Its Associated Parasitoids in Israel.

Insects are ectothermic organisms; hence, all aspects of their biology are strongly influenced by ambient temperatures. Different insect species respond differently with phenotypic plasticity and/or genetic adaptation to changing temperatures. Here, we tested the thermal adaptation of the house fly and three of its parasitoids species by comparing life-history parameters in populations from a hot climate region (Jordan Valley) and from a moderate-climate region (Galilee). No significant differences were found between the two house fly populations, both under hot and moderate experimental conditions. Life-history parameters of the parasitoids (Muscidifurax raptor Girault & Sanders, Spalangia endius Walker, and Spalangia cameroni Perkins [Hymenoptera: Pteromalidae]) varied markedly between origins, species, sexes, and experimental conditions. Of the three species tested, only M. raptor collected in the Jordan Valley proved better adapted to experimental heat conditions, compared to its counterpart population that was collected in the Galilee. Additionally, we tested the effect of elevating temperatures on a house fly lab population for 17 consecutive generations and found no evidence for heat adaptation. We discuss our results in the context of house fly control and global warming.

Efficacy and repellency of some essential oils and their blends against larval and adult house flies, Musca domestica L. (Diptera: Muscidae).

House flies are global pests and notoriously difficult to control. Essential oils of vetiver, cinnamon, and lavender and their blends were tested for toxic and repellent effects against larval and adult flies. All of the oils had moderate toxicity for eggs. Mortality of 2nd instar larvae was 57-78% in dipping assays, 38-100% in contact assays, and 94-100% in treated media. Lavender was less effective (38% mortality) than the others (91-100%) in contact bioassays. Oil blends were not more effective against larvae than individual oils. Vetiver and cinnamon oils were strongly repellent (84 and 78%, respectively) for larvae in treated media. None of the oils were repellent for adult house flies in olfactometer assays, but testing of additional products demonstrated significant repellency for neem oil, p-menthane-3,8-diol (PMD), and vanillin. Contact/fumigant toxicity of vetiver, cinnamon, and lavender oils was 100%, significantly higher than mortality from sunflower oil (67%). Blends of oils were not more effective against adults than the individual oils, but blends diluted with sunflower oil were as effective as the individual oils. Essentials oils of vetiver and cinnamon may have potential for fly management in situations where conventional insecticides cannot be used.

Effect of Fluctuating High Temperatures on House Flies (Diptera: Muscidae) and Their Principal Parasitoids (Muscidifurax spp. and Spalangia spp. [Hymenoptera: Pteromalidae]) From the United States.

Colonies of house flies (Musca domestica L. [Diptera: Muscidae]) and four species of parasitoids (Muscidifurax raptor Girault and Sanders, Muscidifurax zaraptor Kogan and Legner, Spalangia cameroni Perkins and Spalangia endius Walker) were established by making collections from dairy farms near Bell, FL, Beatrice, NE, Minneapolis, MN, and San Jacinto, CA. Colonies were assessed for heat tolerance by comparing life history parameters at 25-27°C and fluctuating hot (26.7-41.7°C) temperatures. Muscidifurax raptor, S. cameroni, and S. endius produced 24-28% as many progeny under hot conditions as at 25°C. Colonies of M. zaraptor were more heat-tolerant and produced an average 46.9% as many progeny under the hot regime compared with moderate conditions. There was little evidence for higher heat tolerance in parasitoid populations from historically hot locations (CA desert and FL). Colonies of M. raptor and S. endius that had been in culture for 24 yr were the least heat-tolerant with regard to progeny production. House flies collected from the same locations varied little in longevity, fecundity, or egg-to-adult survival under either hot or moderate regimes. Flies reared under hot conditions laid about half as many eggs (89/female) and had about half the egg-adult survival rate (47.3%) under hot compared with moderate conditions, indicating that heat stress had less effect on flies than on all of the parasitoids except M. zaraptor. An attempt to select for heat tolerance in flies by subjecting them to incremental increases in rearing temperatures for 20 generations resulted in little change in tolerance among the selected flies.

Larvicidal potential of the polyol sweeteners erythritol and xylitol in two filth fly species.

The house fly, Musca domestica (L.) (Diptera: Muscidae), and the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), are two filth flies responsible for significant economic losses in animal production. Although some chemical control products target adults of both species, differences in mouthpart morphology and behavior necessitates distinct modalities for each. For these reasons, larvicides are an attractive means of chemical control. We assessed the potential of the polyol sweeteners erythritol and xylitol as larvicides to the house fly and stable fly. LC50 values of erythritol against 2nd instar larvae were 34.94 mg/g media (house fly) and 22.10 mg/g media (stable fly). For xylitol, LC50 values were 74.91 mg/g media (house fly) and 41.58 mg/g media (stable fly). When given a choice, neither species showed a preference for ovipositing in media treated with either sweetener at various concentrations or in media without sweetener. Significantly lower development from egg to adult was observed when the 2nd instar LC50 equivalent of each sweetener was present in the media compared to controls. Erythritol and xylitol both have larvicidal qualities, however their effective concentrations would necessitate creative product formulation and deployment methods to control all stages of developing flies.

Mortality Effects of Three Bacterial Pathogens and Beauveria bassiana When Topically Applied or Injected Into House Flies (Diptera: Muscidae).

The house fly, Musca domestica L., is a global pest of public health and agricultural importance. The efficacy of conventional management has been waning due to increasing insecticide resistance. A potential management tool is the entomopathogenic fungus, Beauveria bassiana Vuillemin (Hypocreales: Cordycipitaceae) (strain L90), although time-to-death is slower than desired by potential users. This research investigated the effectiveness of three gram-negative bacteria (Pseudomonas protegens Ramette (Psuedomonadales: Pseudomonadaceae) pf-5, Photorhabdus temperata Fischer-Le Saux (Enterobacteriales: Enterobacteriaceae) NC19, and Serratia marcescens Bizio (Enterobacteriales: Enterobacteriaceae) DB11) on house fly mortality when topically applied, compared to B. bassiana. Each pathogen's virulence was measured by injection into adult female house flies or by topical applications to their thorax. All bacterial strains were highly virulent after injection with 1 × 104 colony forming units (cfu), causing fly mortality within 24 h. Beauveria bassiana resulted in high mortality, 3 d postinjection at the high dose of 1 × 104 conidia/µl. Mortality due to topical treatments of P. temperata and S. marcescens was low even at the highest dose of 1 × 106 cfu/µl. Mortality after topical treatments with P. protegens was evident 4 d after application of 1 × 106 cfu/µl. Mortality from B. bassiana was low at 4 d but increased at 5 d. These results imply that P. protegens holds great potential as a biological control agent for incorporation into an integrated pest management program against adult house flies.

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies.

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds. Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts. This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.