ABSTRACT Zoonotic and other livestock-associated pathogens move readily within animal production systems, yet their dispersal into surrounding environments is poorly characterized. We evaluated whether synanthropic filth flies act as spatially integrating sentinels of livestock-associated pathobiomes. From 30 roadside sites in six Texas counties stratified by livestock production intensity, we collected 3,373 Calliphoridae spp. and 12 Musca domestica , together with co-located soil, water, plant, and fecal samples, and characterized 195 metagenomes (122 insect, 73 environmental). Using diversity, differential-abundance, machine-learning, indicator-species, and network analyses, we found three robust patterns. First, fly metagenomes form a microbial compartment compositionally distinct from co-located environmental matrices (PERMANOVA R² = 0.094, F = 19.81, P = 0.001) and carry markedly higher relative abundances of pathogen-associated taxa, so insect sampling is complementary to, not redundant with, environmental sampling. Second, the dominant landscape signal is categorical rather than graded: cattle presence, not a Low–Medium–High density gradient, most strongly correlated with community structure, with the ruminant-associated anaerobe Peptostreptococcus russellii as the most reproducible biomarker. Third, intensification produced taxon-level enrichments (notably the feedlot pathogen Trueperella pyogenes ) and progressive co-occurrence-network fragmentation, without a monotonic dose-response. A curated 12-pathogen panel failed to classify landscape context, whereas whole-community analysis succeeded. Filth flies thus provide a pragmatic, spatially integrated readout that distinguishes low-impact from livestock-impacted landscapes, offering a defensible tool for One Health pathogen surveillance. IMPORTANCE Zoonotic and other livestock-associated pathogens emerging from animal production are among the most pressing threats to global health but tracking how they spill into the wider environment is difficult and expensive. We show that common filth flies, abundant, easy to trap, and free ranging across farms, fields, and human spaces, act as living samplers that concentrate and report the microbial signature of nearby livestock. Sampling flies captured information that soil and water sampling missed, and a single binary signal, the presence or absence of cattle, was associated with the microbial community more strongly than any measure of animal density. Narrow tests targeting a short curated list of pathogens were not successful in differentiating sites, while analyzing the whole microbial community provided more discriminating power. These findings support a practical, low-cost strategy in which insects serve as biological sentinels for the detection of livestock-associated pathogens across agricultural landscapes.

The entry of drug-resistant Salmonella enterica into the food supply is a challenge to public health and food safety. One emerging concern is the role of synanthropic insects for moving microbial pathogens throughout poultry production systems, where insects commonly thrive. We investigated the presence and phenotypic antimicrobial susceptibility of S. enterica from insect and environmental samples from broiler farms. Insects were collected throughout the broiler house and adjacent compost barn. Environmental samples (poultry feed, drinking water, fresh litter, and feces) were collected simultaneously (n = 80). Insect gastrointestinal tracts were dissected and pooled (n = 57). Recovered Salmonella isolates were serotyped and subjected to antimicrobial susceptibility testing against 14 medically important antimicrobials. Overall, six isolates were recovered from 137 total samples (4.4%): 3.5% (2/57) from adult house flies (Musca domestica), 15% (3/20) from poultry feed, and 4.8% (1/21) from litter. Salmonella Montevideo (16.7%; 1/6), Typhimurium (33.3%; 2/6), and Kentucky (50%, 3/6) were identified. All but one Salmonella isolate (83.3%; 5/6) demonstrated resistance to at least one antimicrobial. Further research should investigate movement patterns between broiler operations and food processing facilities to establish efficient biosecurity measures to prevent any instances of foodborne pathogen transmission into human food systems.

The genus Rickettsia contains species that are important in human health as they cause several serious diseases such as Rocky Mountain spotted fever. This publication summarizes the human-disease-causing pathogens in the genus Rickettsia in the United States and delivers general knowledge of their vectors and the diseases they cause. Prevention and management of disease-causing Rickettsia species, mainly for vector control agencies and entities, are also discussed. This publication is written for audiences of all ages and with any educational background and serves as a resource for public education.

Background: Soft ticks (Family: Argasidae) are vectors of relapsing fever Borrelia in the United States and are potential vectors of African swine fever virus, a pathogen that could have a devastating effect on the U.S. swine industry if introduced to the U.S. mainland. Much of the tick-borne disease research in the U.S. focuses on hard ticks, and less is known about the ecology of soft ticks. Some soft tick species found in the southern U.S. have a wide host range and may feed on cattle, swine, native and exotic ungulates, small mammals, reptiles, and humans. Because the feeding habit of most soft tick species involves taking short, repeated blood meals that may include multiple host species, pathogen transmission among hosts is a concern both for human and animal health. Materials and Methods: Sampling was carried out at four locations in south Texas using dry ice traps placed in or near animal burrows and other sheltering cracks and crevasses that may provide refuge for soft ticks. Collected ticks were identified and subsequently screened for Rickettsia and Borrelia species and for host bloodmeal detection using conventional polymerase chain reaction and Sanger sequencing for pathogen and host species identification. Results: In total, 256 ticks of two Ornithodorinae species were screened. Borrelia species were identified in three samples. Bloodmeal detections were made in 22 tick specimens, representing eight vertebrate host species. Conclusions: Results demonstrate that the soft tick species detected herein feed on a range of wildlife hosts in south Texas and are associated with agents of human disease.

Volatile pyrethroids are effective in reducing mosquito populations and repelling vectors away from hosts. However, many gaps in knowledge exist for the sublethal impacts of volatile pyrethroids on mosquitoes. To that end, transfluthrin exposures were conducted on a field strain of Aedes albopictus (Skuse) held as a laboratory colony. Dose-response analysis was conducted on both sexes at either 1-4 days old or 5-10 days old. Resultant concentration data were used to evaluate the LC20 and LC50 values in various mate pairings of treatments and controls in which either the male or female was from a selectively treated group and mated with a counterpart that was treated independently. Blood feeding proportion, delayed mortality after a 24-h recovery period, egg collection totals, and F1 larval survival were determined following transfluthrin treatment in the F0, but outcomes were not significant. In contrast, sterility was predicated on male treatment, with treated females resulting in higher overall egg viability. Treated males in the mating pair resulted in significantly lower egg viability and accelerated larval hatch in the F1. Additionally, the presence of sperm in female spermathecae was significantly diminished in test groups containing treated male mosquitoes. Male sublethal effects may be a critical determinant of a mixed population's reproductive success.

Aedes albopictus, also known as the Asian tiger mosquito, is indigenous to the tropical forests of Southeast Asia. Ae. albopictus is expanding across the globe at alarming rates, raising concern over the transmission of mosquito-borne diseases, such as dengue, West Nile fever, yellow fever, and chikungunya fever. Since Ae. albopictus was reported in Houston (Harris County, Texas) in 1985, this species has rapidly expanded to at least 32 states across the United States. Public health efforts aimed at controlling Ae. albopictus, including surveillance and adulticide spraying operations, occur regularly in Harris County. Despite rotation of insecticides to mitigate the development of resistance, multiple mosquito species including Culex quinquefasciatus and Aedes aegypti in Harris County show organophosphate and pyrethroid resistance. Aedes albopictus shows relatively low resistance levels as compared to Ae. aegypti, but kdr-mutation and the expression of detoxification genes have been reported in Ae. albopictus populations elsewhere. To identify potential candidate detoxification genes contributing to metabolic resistance, we used RNA sequencing of field-collected malathion-resistant and malathion-susceptible, and laboratory-maintained susceptible colonies of Ae. albopictus by comparing the relative expression of transcripts from three major detoxification superfamilies involved in malathion resistance due to metabolic detoxification. Between these groups, we identified 12 candidate malathion resistance genes and among these, most genes correlated with metabolic detoxification of malathion, including four P450 and one alpha esterase. Our results reveal the metabolic detoxification and potential cuticular-based resistance mechanisms associated with malathion resistance in Ae. albopictus in Harris County, Texas.

BACKGROUND:Metabolic detoxification is one of the major mechanisms contributing to the development of resistance in mosquitoes, including the southern house mosquito, Culex quinquefasciatus. The three major detoxification supergene families, cytochrome P450s, glutathione S-transferases and general esterases, have been demonstrated to play an important role in metabolic resistance. In this study, we performed differential gene expression analysis based on high-throughput transcriptome sequencing on samples from four experimental groups to give insight into key genes involved in metabolic resistance to malathion in Cx. quinquefasciatus. We conducted a whole transcriptome analysis of field captured wild Cx. quinquefasciatus from Harris County (WI), Texas and a malathion susceptible laboratory-maintained Sebring colony (CO) to investigate metabolic insecticide resistance. Field captured mosquitoes were also phenotypically classified into the malathion resistant and malathion susceptible groups following a mortality response measure conducted using a Centers for Disease Control and Prevention (CDC) bottle assay. The live (MR) and dead (MS) specimens from the bottle assay, along with an unselected WI sample and a CO sample were processed for total RNA extraction and subjected to whole-transcriptome sequencing.RESULTS:We demonstrated that the genes coding for detoxification enzymes, particularly cytochrome P450s, were highly up-regulated in the MR group compared to the MS group with similar up-regulation observed in the WI group compared to the CO group. A total of 1,438 genes were differentially expressed in comparison between MR and MS group, including 614 up-regulated genes and 824 down-regulated genes. Additionally, 1,871 genes were differentially expressed in comparison between WI and CO group, including 1,083 up-regulated genes and 788 down-regulated genes. Further analysis on differentially expressed genes from three major detoxification supergene families in both comparisons resulted in 16 detoxification genes as candidates potentially associated with metabolic resistance to malathion. Knockdown of CYP325BC1 and CYP9M12 using RNA interference on the laboratory-maintained Sebring strain significantly increased the mortality of Cx. quinquefasciatus after exposure to malathion.CONCLUSION:We generated substantial transcriptomic evidence on metabolic detoxification of malathion in Cx. quinquefasciatus. We also validated the functional roles of two candidate P450 genes identified through DGE analysis. Our results are the first to demonstrate that knockdown of CYP325BC1 and CYP9M12 both significantly increased malathion susceptibility in Cx. quinquefasciatus, indicating involvement of these two genes in metabolic resistance to malathion.

BackgroundRhipicephalus sanguineus s.l. (Latreille, 1806) can establish indoor populations, which increases the risk of pathogen transmission to humans and companion dogs. Rhipicephalus sanguineus s.l. ticks spend most of their life cycle off the host, which subjects developmental timescale to abiotic factors. Previous studies showed that both temperature and relative humidity (RH) influenced Rhipicephalus sanguineus s.l. survival time across all life stages. However, quantified relationships between environmental factors and Rhipicephalus sanguineus s.l. mortality is not currently available. Here, three Rhipicephalus sanguineus s.l. strains were evaluated for mortality under 20 combinations of five temperatures and four RHs. The data obtained were analyzed to quantify the relationship between environmental factors and Rhipicephalus sanguineus s.l. survival. ResultsMortality probabilities did not show a consistent pattern between the three tick strains. Temperature, RH, and their interaction influenced Rhipicephalus sanguineus s.l. mortality probabilities across all life stages, with mortality probability generally increasing with temperature but decreasing with RH. With 50% and lower RH, larvae cannot survive for more than 1 week. However, mortality probabilities in all strains and stages were more sensitive to temperature than to RH. ConclusionThis study identified the predictive relationship between environmental factors and Rhipicephalus sanguineus s.l. survival, which enables estimations of tick survival time under varied residential situations, allows parameterization of population models, and provides guidance for pest control professionals to develop efficient management strategies. (c) 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Rhipicephalus sanguineus s.l. (Latreille), is a vector of multiple disease-causing pathogens to humans and dogs. Permethrin and fipronil are two acaricides used to manage R. sanguineus s.l. infestations. Homeowners have reported treatment failures in managing brown dog ticks using permethrin and fipronil based products. Previous studies demonstrated that high permethrin resistance in some R. sanguineus s.l. populations was due to metabolic detoxification and target site insensitivity. In this study, three R. sanguineus s.l. strains, one from a laboratory colony (NC) and two colonies originally collected from Florida (FL) and California (CA), were evaluated for resistance expression against permethrin and fipronil. Metabolic detoxification mechanisms were evaluated in the FL strain using three synergists, while a polymerase chain reaction assay was used to detect a resistance mutation in all strains. The NC strain was susceptible to both permethrin and fipronil, while both the FL and CA strains exhibited high resistance to permethrin and tolerance to fipronil. The synergist tests and PCR results indicated that the FL strain utilized both metabolic resistance and target site insensitivity against permethrin, while the CA strain was documented to have the target-site insensitivity resistant allele. This study confirmed permethrin resistance in both California and Florida populations and its persistence in Florida populations, although its susceptibility can potentially be increased by adding a synergist. Fipronil resistance was not detected suggesting this acaricide may provide suitable tick control.

Invasive wild pigs are distributed across much of the U.S. and are hosts to tick vectors of human disease. Herein, adult ticks were collected from 157 wild pigs in 21 northern and central Florida counties from 2019-2020 during removal efforts by USDA-APHIS Wildlife Services personnel and evaluated for their potential to be used as a method of tick-borne disease surveillance. Collected ticks were identified, screened for pathogens, and the effects of landscape metrics on tick community composition and abundance were investigated. A total of 1415 adult ticks of four species were collected. The diversity of tick species collected from wild pigs was comparable to collections made throughout the state with conventional surveillance methods. All species collected have implications for pathogen transmission to humans and other animals. Ehrlichia, Anaplasma-like, and Rickettsia spp. were detected in ticks collected from wild pigs. These results suggest that tick collection from wild pigs is a suitable means of surveillance for pathogens and vectors. The strongest drivers of variation in tick community composition were the developed open space and mixed forest landcover classes. Fragmented shrub/scrub habitat was associated with increased tick abundance. Similar models may be useful in predicting tick abundance and distribution patterns.

The adaptation of wildlife species to urban environments can drive changes in the ecology of ectoparasites and vector-borne disease. To better understand ectoparasite dynamics in an urban environment, we investigated the ectoparasite communities of 183 sylvatic and urban opossums and raccoons captured across four seasons at a rural research station and within the city of Gainesville, FL, and of 115 community cats from the Gainesville, FL area. Amblyomma americanum (L.) (Acari: Ixodidae), Dermacentor variabilis (Say), and Ixodes texanus Banks were collected from raccoons, A. americanum, D. variabilis, and Ixodes scapularis Say from opossums, and A. americanum from cats. Few ticks were collected from urban animals, although species richness of ectoparasites was similar between urban and sylvatic habitats. Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae) was collected from all sampled host species, but was particularly abundant on opossums. Additionally, Orchopeas howardi (Baker) (Siphonaptera: Ceratophyllidae) was collected from raccoons, and O. howardi and Xenopsylla cheopis (Rothschild) (Siphonaptera: Pulicidae) from opossums. Only raccoons were infested with raccoon lice, and only cats were infested with cat lice. Primarily opossums were infested with mites. Ectoparasite community composition varied by habitat, host species, and season; seasonal variation in ectoparasite communities differed between the sylvatic and urban habitats. While urban mesomammals did not appear to play an important role in supporting tick populations in an urban habitat, urban opossums appear to serve as an alternate host for large numbers of cat fleas, which may be an important consideration for treatment and control efforts against ectoparasites of companion animals.

Introduction:Culex quinquefasciatus is a mosquito species of significant public health importance due to its ability to transmit multiple pathogens that can cause mosquito-borne diseases, such as West Nile fever and St. Louis encephalitis. In Harris County, Texas, Cx. quinquefasciatus is a common vector species and is subjected to insecticide-based management by the Harris County Public Health Department. However, insecticide resistance in mosquitoes has increased rapidly worldwide and raises concerns about maintaining the effectiveness of vector control approaches. This concern is highly relevant in Texas, with its humid subtropical climate along the Gulf Coast that provides suitable habitat for Cx. quinquefasciatus and other mosquito species that are known disease vectors. Therefore, there is an urgent and ongoing need to monitor the effectiveness of current vector control programs.Methods: In this study, we evaluated the impact of vector control approaches by estimating the effective population size of Cx. quinquefasciatus in Harris County. We applied Approximate Bayesian Computation to microsatellite data to estimate effective population size. We collected Cx. quinquefasciatus samples from two mosquito control operation areas; 415 and 802, during routine vector monitoring in 2016 and 2017. No county mosquito control operations were applied at area 415 in 2016 and 2017, whereas extensive adulticide spraying operations were in effect at area 802 during the summer of 2016. We collected data for eighteen microsatellite markers for 713 and 723 mosquitoes at eight timepoints from 2016 to 2017 in areas 415 and 802, respectively. We also investigated the impact of Hurricane Harvey’s landfall in the Houston area in August of 2017 on Cx. quinquefasciatus population fluctuation.Results: We found that the bottleneck scenario was the most probable historical scenario describing the impact of the winter season at area 415 and area 802, with the highest posterior probability of 0.9167 and 0.4966, respectively. We also detected an expansion event following Hurricane Harvey at area 802, showing a 3.03-fold increase in 2017.Discussion: Although we did not detect significant effects of vector control interventions, we found considerable influences of the winter season and a major hurricane on the effective population size of Cx. quinquefasciatus. The fluctuations in effective population size in both areas showed a significant seasonal pattern. Additionally, the significant population expansion following Hurricane Harvey in 2017 supports the necessity for post-hurricane vector-control interventions.

Abstract Rhipicephalus sanguineus s.l. (Latreille, 1806) can establish populations in residences and may lead to severe domestic and peridomestic infestations. Detection in the early infestation stage is challenging because of their small body size and the lack of visibility when ticks stay in sheltered refugia. The residents may believe that the infestation has been eliminated when no ticks are observed until ticks reappear when seeking hosts. Thus, it is necessary to improve our understanding of tick phenology to achieve more effective infestation management. In this study, the relationships between environmental conditions and tick development were explored in laboratory and using linear and nonlinear models. Three R. sanguineus s.l. strains, from one colony of the temperate lineage and two of the tropical lineage, were evaluated for the development of all life stages and conversion efficiency index (CEI) under five temperatures and four relative humidities (RHs). The development times differed between the three tick strains across stages and were primarily dependent on temperature. The CEIs had little variance explained by temperature, RH, or strains. Compared with the linear and exponential models with temperature as the only variable, the Brière-1 model was the best approximating model for most of the developmental rates. The developmental temperature thresholds for R. sanguineus s.l. development estimated by the Brière-1 model varied inconsistently across strains and life stages. We developed a more predictive relationship between environmental factors and R. sanguineus s.l. development, which can be utilized to predict tick development using temperature and develop appropriate control strategies.

This EDIS publication is an alternate version of a page published first on the Featured Creatures website. The Featured Creatures collection provides in-depth profiles of insects, nematodes, arachnids and other organisms relevant to Florida. These profiles are intended for the use of interested laypersons with some knowledge of biology as well as academic audiences. This 6-page fact sheet that discusses the stable fly was written by Phillip E. Kaufman and Emma N. I. Weeks, and published by the UF Entomology and Nematology Department, January 2016. EENY642/IN1114: Stable Fly Stomoxys calcitrans (L.) (Insecta: Diptera: Muscidae) (ufl.edu)

Amblyomma americanum Linnaeus (Ixodida: Ixodidae) is ubiquitously present throughout the southeastern United States and is capable of vectoring several pathogens. White-tailed deer are the main host for adult A. americanum. However, this tick species is a generalist that will feed on most vertebrates, including humans, deer, livestock, and pets. Management of this species can be challenging due to a lack of cost-effective strategies. Acaricides are often utilized, however, this may lead to pesticide resistance. The Food and Agriculture Organization of the United Nations (FAO) larval packet test (LPT) was performed on susceptible A. americanum to determine the lethal concentration (LC) and discriminating concentration (DC) values for permethrin. The FAO LPT was used at these pre-established values to compare levels of resistance in ticks collected from a captive deer farm and wild areas representing high and low permethrin exposure settings, respectively. Resistance ratios (RR) calculated from the LC values for the ticks collected from farmed and wild deer ranged between 1 and 2. A. americanum collected from farmed and wild deer were not found to be resistant, however, some samples had slightly elevated RRs as compared to the susceptible laboratory strain, which may suggest tolerance development. Although the A. americanum sampled in this study were not resistant to permethrin, the DC calculated in this study will allow for rapid evaluation of resistance in a permethrin resistance monitoring program such that alternate management strategies can be adopted if resistance is detected.

Abstract Field development for optimizing the use patterns of spatial repellents are an ongoing research need. To investigate volatile pyrethroids in blends containing multiple active ingredients, metofluthrin was formulated into a vegetation spray for application to foliage and mosquito harborage. Metofluthrin was compared by itself and in combination with Sector (permethrin), Hyperion (sumithrin), Onslaught Fast Cap (s-fenvalerate, prallethrin), and against a benchmark for comparison, OneGuard (λ-cyhalothrin, prallethrin, pyriproxyfen). Field sites with established Aedes albopictus (Skuse) (Diptera: Culicidae) populations were allocated as control and treatment and surveilled with both adult traps and oviposition cups before and after treatment. Adult mosquito reductions were consistently higher over 4 wk in the combination treatments and were comparable to OneGuard. In contrast, efficacy provided by metofluthrin alone or the standalone existing product (Sector, Hyperion, Onslaught) by were consistently less effective than OneGuard and the comparative blends. Additionally, poorly melanized and collapsing eggs were identified from mosquito adults collected at treated field sites. A 20–50% significant decrease in nonviable mosquito eggs, a 50% or more reduction in total eggs and an 80% or more reduction in adult mosquitoes collected at the local field sites was demonstrated across all metofluthrin-containing treatments. Metofluthrin applications both supplemented the adulticidal control of the selected pyrethroid-containing products and provided significant reduction in egg laying frequency and viability during monitoring of areas adjacent to the treated vegetation. Metofluthrin, and possibly other volatile ingredients, appears to have complex interactions for mosquito control in the field and could be unique as a supporting ingredient to other insecticides.

Antibiotic use in livestock accounts for 80% of total antibiotic use in the United States and has been described as the driver for resistance evolution and spread. As clinical infections with multidrug-resistant pathogens are rapidly rising, there remains a missing link between agricultural antibiotic use and its impact on human health. In this study, two species of filth flies from a livestock operation were collected over the course of 11 mo: house flies Musca domestica (L.) (Diptera: Muscidae), representing a generalist feeder, and stable flies Stomoxys calcitrans (L.) (Diptera: Muscidae), representing a specialist (blood) feeder. The prevalence of flies carrying cefotaxime-resistant (CTX-R) bacteria in whole bodies and dissected guts were assayed by culturing on antibiotic-selective media, with distinct colonies identified by Sanger sequencing. Of the 149 flies processed, including 81 house flies and 68 stable flies, 18 isolates of 12 unique bacterial species resistant to high-level cefotaxime were recovered. These isolates also showed resistance to multiple classes of antibiotics. The CTX-R isolates were predominantly recovered from female flies, which bore at least two resistant bacterial species. The majority of resistant bacteria were isolated from the guts encompassing both enteric pathogens and commensals, sharing no overlap between the two fly species. Together, we conclude that house flies and stable flies in the field could harbor multidrug-resistant bacteria. The fly gut may serve as a reservoir for the acquisition and dissemination of resistance genes.

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.

Abstract 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.