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 The bioassays and analyses are described using previously unpublished data from bioassays on house flies, Musca domestica Linnaeus (Diptera: Muscidae), but can be used on a wide range of pest species.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.

Discovery, Development, and Evaluation of a Horn Fly-Isolated (Diptera: Muscidae) Beauveria bassiana (Hypocreales: Cordyciptaceae) Strain From Florida, USA.

The horn fly, Haematobia irritans (L.) is an important cattle pest and traditionally has been managed using insecticides; however, many horn fly populations are insecticide-resistant in United States. Use of alternative control techniques has been limited because of the challenges of managing a fly pest on pastured cattle. After the discovery of a wild horn fly infected with Beauveria bassiana in Florida, the fungus was cultured and evaluated for efficacy against laboratory-reared horn flies. This fungal strain was selected for increased virulence by passage through laboratory-reared horn fly hosts to shorten interval from infection to fly death and subsequent conidia formation, properties important to future use of the fungus as a biological control agent against horn flies. After seven passages through horn fly hosts, fly mortality was not significantly accelerated as evaluated through LT50 values, but conidia were readily produced from these killed flies. Although further development is needed to improve fungal efficacy, this fungal strain holds promise as a biological control agent for inclusion in horn fly integrated pest management programs.

Comparative susceptibility of adult house fly (Diptera: Muscidae) and an important predator, Carcinops pumilio (Coleoptera: Histeridae), to Beauveria bassiana (Hypocreales: Cordycipitaceae).

House flies (Musca domestica L.) (Diptera: Muscidae) are challenging pests to control. Biological control using Carcinops pumilio beetles may help to reduce house fly populations. However, it is unknown if C. pumilio beetles are compatible with Beauveria bassiana, another house fly biological control option. Five strains of commercially available (GHA, HF23, and L90) and newly discovered (NFH10 and PSU1) strains of B. bassiana were used to test the comparative susceptibility of adult house flies and adult C. pumilio using different laboratory exposure methods. Adult house flies were susceptible to B. bassiana in contact filter paper assays (89%-98% mortality) and immersion assays (100% mortality) at the same 108 conidia suspension using 0.1% CapSil as an aqueous surfactant. Carcinops pumilio were less susceptible than flies to B. bassiana infection using the contact and immersion assays at the same 108 conidial concentration, with 4.4%-12.2% and 8.3%-24.6% mortality, respectively. Immersion in an aqueous conidial suspension resulted in higher mortality compared to contact with treated filter papers at the same 108 concentration with house flies and beetles. We conclude that C. pumilio can safely be used as a biological control agent for house flies with B. bassiana in animal production systems.

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.

Muscavirus (MdSGHV) disease dynamics in house fly populations--how is this virus transmitted and has it potential as a biological control agent?

The newly classified family Hytrosaviridae comprises several double-stranded DNA viruses that have been isolated from various dipteran species. These viruses cause characteristic salivary gland hypertrophy and suppress gonad development in their hosts. One member, Muscavirus or MdSGHV, exclusively infects adult house flies (Musca domestica) and, owing to its massive reproduction in and release from the salivary glands, is believed to be transmitted orally among feeding flies. However, results from recent experiments suggest that additional transmission routes likely are involved in the maintenance of MdSGHV in field populations of its host. Firstly, several hours before newly emerged feral flies begin feeding activities, the fully formed peritrophic matrix (PM) constitutes an effective barrier against oral infection. Secondly, flies are highly susceptible to topical virus treatments and intrahemocoelic injections. Thirdly, disease transmission is higher when flies are maintained in groups with infected conspecifics than when flies have access to virus-contaminated food. We hypothesize that interactions between flies may lead to cuticular damage, thereby providing an avenue to viral particles for direct access to the hemocoel. Based on our current knowledge, two options seem plausible for developing Muscavirus as a sterilizing agent to control house fly populations: The virus may either be formulated with PM-disrupting materials to facilitate oral infection from a feeding bait system, or amended with abrasive materials to enhance infection through a damaged cuticle after topical aerosol applications.

Insecticide resistance in house flies from the United States: resistance levels and frequency of pyrethroid resistance alleles.

Although insecticide resistance is a widespread problem for most insect pests, frequently the assessment of resistance occurs over a limited geographic range. Herein, we report the first widespread survey of insecticide resistance in the USA ever undertaken for the house fly, Musca domestica, a major pest in animal production facilities. The levels of resistance to six different insecticides were determined (using discriminating concentration bioassays) in 10 collections of house flies from dairies in nine different states. In addition, the frequencies of Vssc and CYP6D1 alleles that confer resistance to pyrethroid insecticides were determined for each fly population. Levels of resistance to the six insecticides varied among states and insecticides. Resistance to permethrin was highest overall and most consistent across the states. Resistance to methomyl was relatively consistent, with 65-91% survival in nine of the ten collections. In contrast, resistance to cyfluthrin and pyrethrins + piperonyl butoxide varied considerably (2.9-76% survival). Resistance to imidacloprid was overall modest and showed no signs of increasing relative to collections made in 2004, despite increasing use of this insecticide. The frequency of Vssc alleles that confer pyrethroid resistance was variable between locations. The highest frequencies of kdr, kdr-his and super-kdr were found in Minnesota, North Carolina and Kansas, respectively. In contrast, the New Mexico population had the highest frequency (0.67) of the susceptible allele. The implications of these results to resistance management and to the understanding of the evolution of insecticide resistance are discussed.

High-throughput mosquito and fly bioassay system for natural and artificial substrates treated with residual insecticides.

A high-throughput bioassay system to evaluate the efficacy of residual pesticides against mosquitoes and muscid flies with minimal insect handling was developed. The system consisted of 4 components made of readily available materials: 1) a CO2 anaesthetizing chamber, 2) a specialized aspirator, 3) a cylindrical flat-bottomed glass bioassay chamber assembly, and 4) a customized rack.

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.

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.

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.

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

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

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%.

Salivary gland hypertrophy viruses: a novel group of insect pathogenic viruses.

Salivary gland hypertrophy viruses (SGHVs) are a unique, unclassified group of entomopathogenic, double-stranded DNA viruses that have been reported from three genera of Diptera. These viruses replicate in nuclei of salivary gland cells in adult flies, inducing gland enlargement with little obvious external disease symptoms. Viral infection inhibits reproduction by suppressing vitellogenesis, causing testicular aberrations, and/or disrupting mating behavior. Historical and present research findings support a recent proposal of a new virus family, the Hytrosaviridae. This review describes the discovery and prevalence of different SGHVs, summarizes their biochemical characterization and taxonomy, compares morphological and histopathological properties, and details transmission routes and the influence of infection on host biology and reproduction. In addition, the potential use of SGHVs as sterilizing agents for house fly control and the deleterious impact of SGHVs on colonized tsetse flies reared for sterile insect technique are discussed.

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.

Seasonal abundance of stable flies and filth fly pupal parasitoids (Hymenoptera: Pteromalidae) at Florida equine facilities.

Beginning in November 2007 and continuing until December 2009, weekly stable fly, Stomoxys calcitrans (L.), surveillance was conducted at four equine facilities near Ocala, FL, by using alsynite sticky traps for adults and by searching immature developmental sites for pupae. Adult stable fly trap captures were highly variable throughout the year, ranging from 0 to 1,400 flies per trap per farm. The greatest adult stable fly activity was observed during the spring months of March and April, with weekly three-trap means of 121 and 136 flies per farm, respectively. The importance of cultural control measures was most apparent on the only farm with no reported insecticide use and the lowest stable fly trap captures, where an intense daily sanitation and composting program was conducted. A survey of on-site filth fly pupae revealed that 99.9% of all parasitoids recovered were Spalangia spp., consisting of Spalangia cameroni Perkins (56.5%), Spalangia nigroaenea Curtis (34.0%), Spalangia endius Walker (5.8%), and Spalangia nigra Latreille (3.7%). The implications of these findings are discussed.

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.