Field- and laboratory-based studies on correlates of Chlamydia trachomatis transmission by Musca sorbens: Determinants of fly-eye contact and investigations into fly carriage of elementary bodies.

Musca sorbens (Diptera: Muscidae) flies are thought to be vectors of the blinding eye disease trachoma, carrying the bacterium Chlamydia trachomatis (Ct) between the eyes of individuals. While their role as vectors has been convincingly demonstrated via randomised controlled trials in The Gambia, studies of fly-borne trachoma transmission remain scant and as such our understanding of their ability to transmit Ct remains poor. We examined fly-eye contact and caught eye-seeking flies from 494 individuals (79% aged ≤9 years) in Oromia, Ethiopia. Ct-carrying flies (harbouring Ct DNA) were found to cluster spatially in and nearby to households in which at least one resident had Ct infection. Fly-eye contact was positively associated with the presence of trachoma (disease), lower human body weight and increased human body temperature. Studies of laboratory-reared M. sorbens indicated that Ct is found both externally and internally following feeds on Ct culture, with scanning electron microscopy revealing how Ct bodies can cling to fly hairs (setae). Testing for Ct on field-caught M. sorbens found fly 'bodies' (thorax, wings and abdomen) to consistently test positive for Ct while legs and heads were infrequently Ct-positive. These studies strongly support the role of M. sorbens as vectors of trachoma and highlight the need for improved understanding of fly-borne trachoma transmission dynamics and vector competence.

Stable flies are bona fide carriers of mastitis-associated bacteria.

Hematophagous Stomoxys (stable) fly populations in dairy barns are sustained by a constant availability of cattle hosts and manure, which serve as major reservoirs of both zoonotic and opportunistic bacterial pathogens. However, the composition of the Stomoxys fly microbiota, the mechanisms by which flies acquire their microbiome, and the ability of potentially pathogenic bacteria to colonize and persist in fly hosts remain to be investigated. Here, we longitudinally collected fly and manure samples from two connected dairy facilities. High throughput 16S rRNA gene amplicon sequencing was then used to characterize and compare bacterial communities present on or within flies and in manure collected from the same facility, while culture-dependent methods were used to verify the viability of clinically relevant bacteria. Bacterial alpha diversity was overall higher in manure samples as compared to fly samples, with manure-associated bacterial communities being dominated by members of the Bacteroidales, Eubacteriales, and Oscillospirales. In contrast, flies harbored relatively low-complexity communities dominated by members of the Enterobacterales, Staphylococcales, and Lactobacillales. Clinically relevant bacterial strains, including Escherichia spp. and other taxa associated with mastitic cows housed in the same facilities, were detected in paired fly and manure samples but exhibited dramatically elevated abundances in fly samples as compared to manure samples. Viable colonies of Escherichia, Klebsiella, and Staphylococcus spp. were also readily isolated from fly samples, confirming that flies harbor culturable mastitis-associated bacteria. This study identifies biting flies as bona fide carriers of opportunistically pathogenic bacterial taxa on dairy farms. IMPORTANCE: Disease prevention on dairy farms has significant implications for cattle health, food security, and zoonosis. Of particular importance is the control of bovine mastitis, which can be caused by diverse bacteria, including Klebsiella, Escherichia coli, Streptococcus, and Staphylococcus spp. Despite being one of the most significant and costly cattle diseases worldwide, the epidemiology of bovine mastitis is not well understood. This study provides parallel culture-independent and culture-dependent evidence to support the carriage of opportunistically pathogenic bacteria by Stomoxys flies on dairy farms. We further show that the fly microbiota is enriched in clinically relevant taxa-the vast majority of which can be traced to the manure habitats in which flies breed. Altogether, our results identify biting flies as underrecognized carriers of bacterial taxa associated with environmental bovine mastitis and other opportunistic infections in vertebrates and offer important insights into mechanisms of microbial acquisition by these and other medically important insects.

Unveiling the hidden dangers: enteropathogens carried by flies in Pudong New Area.

BACKGROUND: Flies are acknowledged as vectors of diseases transmitted through mechanical means and represent a significant risk to human health. The study aimed to determine the prevalence of enteropathogens carried by flies in Pudong New Area to inform strategies for preventing and controlling flies. METHODS: Samples were collected from various locations in the area using cage trapping techniques between April and November 2021, encompassing various habitats such as parks, residential areas, restaurants, and farmers' markets. The main fly species were identified using cryomicrography and taxonomic enumeration, with 20 samples per tube collected from different habitats. Twenty-five enteropathogens were screened using GI_Trial v3 TaqManTM microbial arrays. RESULTS: A total of 3,875 flies were collected from 6,400 placements, resulting in an average fly density of 0.61 flies per cage. M. domestica were the most common species at 39.85%, followed by L. sericata at 16.57% and B. peregrina at 13.14%. Out of 189 samples, 93 tested positive for enteropathogens, with nine different pathogens being found. 12.70% of samples exclusively had parasites, a higher percentage than those with only bacteria or viruses. The study found that M. domestica had fewer enteropathogens than L. sericata and B. peregrina, which primarily harbored B. hominis instead of bacteria and viruses such as E. coli, Astrovirus, and Sapovirus. During spring testing, all three fly species exhibited low rates of detecting enteropathogens. M. domestica were found in residential areas with the highest number of pathogen species, totaling six. In contrast, L. sericata and B. peregrina were identified in farmers' markets with the highest number of pathogen species, totaling six and seven, respectively. CONCLUSIONS: Flies have the potential to serve as vectors for the transmission of enteropathogens, thereby posing a substantial risk to public health.

Surveillance and screening of Stomoxyinae flies from Mallorca Island (Spain) reveal the absence of selected pathogens but confirm the presence of the endosymbiotic bacterium Wolbachia pipientis.

Adult brachycera biting flies can significantly impact livestock through both direct effects (reduction of food intake, disturbance, painful bites, and blood loss) and indirect effects (pathogen transmission), leading to substantial economic losses and production damage. This study aimed to assess the presence of blood-sucking flies in six mixed-animal farm environments on the island of Mallorca (Balearic Islands, Spain) by employing multiple trapping methods. Additionally, distribution maps of brachycera biting fly species recorded in Spain were created, based on data extracted thorough review of scientific literature and citizen digital databases. Investigation of several pathogens, including equine infectious anemia virus (EIAV), Anaplasmataceae bacteria, and piroplasm protozoa, was carried out using different PCR targets (18S rRNA, 16S rRNA, groESL, and tat genes). Citizen science databases and literature review corroborated the consistent distribution trend for two Stomoxyinae species, underscoring the importance of citizen collaboration as a complement to traditional entomological surveillance. Our study confirmed the presence of two biting Stomoxyinae species: the prevalent stable fly Stomoxys calcitrans across all sampled farms, and the horn fly Haematobia irritans, which turned out to be less abundant. DNA barcoding techniques validated the identification of the two species. Neither EIAV nor bacterial/protozoan pathogens were detected using the selected PCR targets in either fly species. However, Wolbachia pipientis (clustered in the supergroup A together with the only sequence of W. pipientis from the USA) was identified through PCR targeting 16S rRNA, groESL and wsp genes in all pools of H. irritans (n = 13) collected from two of the examined farms. This study represents the first attempt to investigate pathogens in Stomoxyinae biting flies in Spain. The discovery of the endosymbiotic Wolbachia organism in H. irritans represents the first record in Spain and the second from Europe. This finding holds significant implications for future research on the applications of this bacterium in biocontrol programs.

Role of Staphylococcus agnetis and Staphylococcus hyicus in the Pathogenesis of Buffalo Fly Skin Lesions in Cattle.

Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas, most commonly on the medial canthus of the eye, along the lateral and ventral neck, and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus has been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilized homogenized flies. Bacterial identification was conducted by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and strain typing by repetitive sequence-based PCR (rep-PCR) and DNA sequencing to determine species similarity and virulence factors. Of 50 bacterial isolates collected from lesions, 38 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas four isolates from normal skin were S. hyicus and one was Mammaliicoccus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to those of the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions. IMPORTANCE Skin lesions in cattle associated with feeding by Haematobia fly species are a significant welfare issue in Australia, North and South America, and Europe. The development of these lesions has been attributed to a number of causal factors, but the exact etiology and pathogenesis were unclear. This study characterized Staphylococcus agnetis and Staphylococcus hyicus strains from cattle skin lesions and in vector flies and demonstrated their role in the pathogenesis of these lesions. These findings will aid the development of targeted and more effective treatment and control strategies for lesions associated with fly infestation in cattle.

First report of the presence of Anaplasma marginale in different tissues of the stable-fly Stomoxys calcitrans (Linnaeus, 1758) in Rio de Janeiro state, Brazil.

Stomoxys calcitrans is a cosmopolitan hematophagous insect with significant veterinary importance. Besides causing great stress with its bites, the fly is a mechanical vector of several pathogens of the most diverse phylogenetic groups to their hosts. The objective of this study was to test for the presence of Anaplasma marginale in stable flies collected at three points inside the campus of the Federal Rural University of Rio de Janeiro (UFRRJ), Seropédica, Rio de Janeiro, Brazil. The collected flies were dissected and separated into three parts - heads, carcass and guts - which were individually submitted to A. marginale specific semi nested PCR gene amplification. A total 150 samples of 50 flies were assayed and 11.33% were positive with predominant presence in guts. In parallel, 6 F1 colony S. calcitrans flies fed with bovine blood were also investigated, being all positive for the presence of the bacteria. This is the first report of the presence of A. marginale in S. calcitrans at Rio de Janeiro state, actually in Brazil, indicating that the epidemiological importance of this vector in the transmission of diseases with great economic impact must not be ignored.

Livestock-associated MRSA survival on house flies (Musca domestica) and stable flies (Stomoxys calcitrans) after removal from a Danish pig farm.

We caught stable- and house flies on a Danish LA-MRSA positive pig farm. Stable- and house flies were housed together and culled over time to test for the presence of live LA-MRSA bacteria at 24 h intervals to establish the length of time for which LA-MRSA can persist on flies. On average, 7% of stable flies and 27% of house flies tested positive for LA-MRSA immediately upon removal from the farm. LA-MRSA prevalence decreased over time and estimates based on a Kaplan-Meier time-to-event analysis indicated that the probability of a stable- or house fly testing positive for LA-MRSA was 5.4% and 7.8% after 24 h, 3.5% and 4.3% after 48 h, 3.1% and 2.2% after 72 h and 0.4% and 0% after 96 h of removal from the pig farm, respectively. Simultaneously, we found that caged cultivated house flies became carriers of LA-MRSA, without direct contact with pigs, in the same proportions as wild flies inside the farm. We provide distance distributions of Danish pig farms and residential addresses as well as the calculated maximum dispersal potentials of stable- and house flies, which suggest that there is a potential for stable- and house flies dispersing live LA-MRSA bacteria into the surrounding environment of a pig farm. This potential should therefore be considered when modelling the spread between farms or the risk posed to humans living in close proximity to LA-MRSA pig farm sources.

Prevalence of Field-Collected House Flies and Stable Flies With Bacteria Displaying Cefotaxime and Multidrug Resistance.

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.

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.

Strongwellsea tigrinae and Strongwellsea acerosa (Entomophthorales: Entomophthoraceae), two new species infecting dipteran hosts from the genus Coenosia (Muscidae).

Two new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) are described: Strongwellsea tigrinae from adult Coenosia tigrina (Diptera: Muscidae) and Strongwellsea acerosa from adult Coenosia testacea. The descriptions are based on pathobiological, phenotypical and genotypical characters. Further, the circumscription of the genus Strongwellsea is emended. Our findings suggest that Strongwellsea harbors a high number of species, of which now only five have been described.

Transinfection of buffalo flies (Haematobia irritans exigua) with Wolbachia and effect on host biology.

BACKGROUND: Buffalo flies (Haematobia irritans exigua) (BF) and closely related horn flies (Haematobia irritans irritans) (HF) are invasive haematophagous parasites with significant economic and welfare impacts on cattle production. Wolbachia are intracellular bacteria found widely in insects and currently of much interest for use in novel strategies for the area wide control of insect pests and insect-vectored diseases. In this paper, we report the transinfection of BF towards the development of area-wide controls. METHODS: Three stages of BF; embryos, pupae and adult female flies, were injected with different Wolbachia strains (wAlbB, wMel and wMelPop). The success of transinfection and infection dynamics was compared by real-time PCR and FISH and fitness effects were assessed in transinfected flies. RESULTS: BF eggs were not easily injected because of their tough outer chorion and embryos were frequently damaged with less than 1% hatch rate of microinjected eggs. No Wolbachia infection was recorded in flies successfully reared from injected eggs. Adult and pupal injection resulted in higher survival rates and somatic and germinal tissue infections, with transmission to the succeeding generations on some occasions. Investigations of infection dynamics in flies from injected pupae confirmed that Wolbachia were actively multiplying in somatic tissues. Ovarian infections were confirmed with wMel and wMelPop in a number of instances, though not with wAlbB. Measurement of fitness traits indicated reduced longevity, decreased and delayed adult emergence, and reduced fecundity in Wolbachia-infected flies compared to mock-injected flies. Effects varied with the Wolbachia strain injected with most marked changes seen in the wMelPop-injected flies and least severe effects seen with wAlbB. CONCLUSIONS: Adult and pupal injection were the most suitable methods for transinfecting BF and all three strains of Wolbachia successfully replicated in somatic tissues. The Wolbachia-induced fitness effects seen in transinfected BF suggest potential for use of the wMel or wMelPop strains in Wolbachia-based biocontrol programmes for BF.

Wolbachia Endosymbiont of the Horn Fly (Haematobia irritans irritans): a Supergroup A Strain with Multiple Horizontally Acquired Cytoplasmic Incompatibility Genes.

The horn fly, Haematobia irritansirritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between $1 billion and $2.5 billion (U.S. dollars) annually in North America and Brazil. The endosymbiotic bacterium Wolbachia pipientis is a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, TX, and Alberta, Canada, but it has also been identified in wild-caught samples from Canada, the United States, Mexico, and Hungary. Reassembly of PacBio long-read and Illumina genomic DNA libraries from the Kerrville H. i. irritans genome project allowed for a complete and circularized 1.3-Mb Wolbachia genome (wIrr). Annotation of wIrr yielded 1,249 coding genes, 34 tRNAs, 3 rRNAs, and 5 prophage regions. Comparative genomics and whole-genome Bayesian evolutionary analysis of wIrr compared to published Wolbachia genomes suggested that wIrr is most closely related to and diverged from Wolbachia supergroup A strains known to infect Drosophila spp. Whole-genome synteny analyses between wIrr and closely related genomes indicated that wIrr has undergone significant genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes of wIrr suggested two phylogenetically distinct CI loci and acquisition of another cifB homolog from phylogenetically distant supergroup A Wolbachia strains, suggesting horizontal acquisition of these loci. The wIrr genome provides a resource for future examination of the impact Wolbachia may have in both biocontrol and potential insecticide resistance of horn flies.IMPORTANCE Horn flies, Haematobia irritans irritans, are obligate hematophagous parasites of cattle having significant effects on production and animal welfare. Control of horn flies mainly relies on the use of insecticides, but issues with resistance have increased interest in development of alternative means of control. Wolbachia pipientis is an endosymbiont bacterium known to have a range of effects on host reproduction, such as induction of cytoplasmic incompatibility, feminization, male killing, and also impacts vector transmission. These characteristics of Wolbachia have been exploited in biological control approaches for a range of insect pests. Here we report the assembly and annotation of the circular genome of the Wolbachia strain of the Kerrville, TX, horn fly (wIrr). Annotation of wIrr suggests its unique features, including the horizontal acquisition of additional transcriptionally active cytoplasmic incompatibility loci. This study provides the foundation for future studies of Wolbachia-induced biological effects for control of horn flies.

Molecular detection of Streptococcus equi subspecies equi in face flies (Musca autumnalis) collected during a strangles outbreak on a Thoroughbred farm.

The objective of this study was to detect Streptococcus equi subspecies equi (S. equi) (Lactobacillales: Streptococcaceae) using quantitative polymerase chain reaction (qPCR) in flies collected from a farm with a documented outbreak of strangles. A total of 1856 face flies [Musca autumnalis (Diptera: Muscidae)] were collected using conventional fly traps. The flies were processed for nucleic acid purification and tested for the presence of S. equi by qPCR. A total of 10/1856 flies (0.54%) tested qPCR-positive for S. equi. The results may implicate the presence of face flies as a risk factor for the transmission of S. equi and highlight the need to institute proper husbandry measures, biosecurity protocols and fly control in order to reduce the potential for infection in at-risk horses.

Occurrence of Escherichia coli O157:H7 in Pest Flies Captured in Leafy Greens Plots Grown Near a Beef Cattle Feedlot.

Leafy greens are leading vehicles for Escherichia coli O157:H7 foodborne illness. Pest flies can harbor this pathogen and may disseminate it to produce. We determined the occurrence of E. coli O157:H7-positive flies in leafy greens planted up to 180 m from a cattle feedlot and assessed their relative risk to transmit this pathogen to leafy greens. The primary fly groups captured on sticky traps at the feedlot and leafy greens plots included house flies (Musca domestica L.), face flies (Musca autumnalis L.), stable flies (Stomoxys calcitrans L.), flesh flies (family Sarcophagidae), and blow flies (family Calliphoridae). E. coli O157:H7 carriage rates of house, face, flesh, and blow flies were similar (P > 0.05), ranging from 22.3 to 29.0 flies per 1,000 flies. In contrast, the carriage rate of stable flies was lower at 1.1 flies per 1,000 flies (P < 0.05). Differences in carriage rates are likely due to the uses of fresh bovine feces and manure by these different pest fly groups. E. coli O157:H7 carriage rates of total flies did not differ (P > 0.05) by distance (ranging from 0 to 180 m) from the feedlot. Most fly isolates were the same predominant pulsed-field gel electrophoresis types found in feedlot surface manure and leafy greens, suggesting a possible role for flies in transmitting E. coli O157:H7 to the leafy greens. However, further research is needed to clarify this role and to determine set-back distances between cattle production facilities and produce crops that will reduce the risk for pathogen contamination by challenging mechanisms like flies.

An Examination of Flying Insects in Seven Hospitals in the United Kingdom and Carriage of Bacteria by True Flies (Diptera: Calliphoridae, Dolichopodidae, Fanniidae, Muscidae, Phoridae, Psychodidae, Sphaeroceridae).

Insects are efficient vectors of bacteria and in the hospital environment may have a role in spreading nosocomial infections. This study sampled the flying insect populations of seven hospitals in the United Kingdom and characterized the associated culturome of Diptera, including the antibiotic resistance profile of bacterial isolates. Flying insects were collected in seven U.K. hospitals between the period March 2010 to August 2011. The bacteria carried by Diptera were isolated using culture-based techniques, identified and characterized by antimicrobial susceptibility testing. A total of 19,937 individual insects were collected with Diptera being the most abundant (73.6% of the total), followed by Hemiptera (13.9%), Hymenoptera (4.7%), Lepidoptera (2.9%), and Coleoptera (2%). From Diptera, 82 bacterial strains were identified. The majority of bacteria belonged to the Enterobacteriaceae (42%), followed by Bacillus spp. (24%) and Staphylococcus spp. (19%). Less abundant were bacteria of the genus Clostridium (6%), Streptococcus (5%), and Micrococcus (2%). A total of 68 bacterial strains were characterized for their antibiotic resistance profile; 52.9% demonstrated a resistant phenotype to at least one class of antibiotic. Staphylococcus spp. represented the highest proportion of resistant strains (83.3%), followed by Bacillus spp. (60%) and Enterobacteriaceae (31.3%). Diptera were the predominant flying insects present in the U.K. hospital environments sampled and found to harbor a variety of opportunistic human pathogens with associated antimicrobial resistance profiles. Given the ability of flies to act as mechanical vectors of bacteria, they present a potential to contribute to persistence and spread of antimicrobial-resistant pathogenic bacteria in the hospital environment.

Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli Isolated from Flies in the Urban Center of Berlin, Germany.

Background: The monitoring of antimicrobial resistance (AMR) in microorganisms that circulate in the environment is an important topic of scientific research and contributes to the development of action plans to combat the spread of multidrug-resistant (MDR) bacteria. As a synanthropic vector for multiple pathogens and a reservoir for AMR, flies can be used for surveillance. Methods: We collected 163 flies in the inner city of Berlin and examined them for extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli genotypically and phenotypically. Results: The prevalence of ESBL-producing E. coli in flies was 12.9%. Almost half (47.6%) of the ESBL-positive samples showed a co-resistance to ciprofloxacin. Resistance to carbapenems or colistin was not detected. The predominant ESBL-type was CTX-M-1, which is associated with wildlife, livestock, and companion animals as a potential major source of transmission of MDR E. coli to flies. Conclusions: This field study confirms the permanent presence of ESBL-producing E. coli in an urban fly population. For continuous monitoring of environmental contamination with multidrug-resistant (MDR) bacteria, flies can be used as indicators without much effort.

Host-specific associations affect the microbiome of Philornis downsi, an introduced parasite to the Galápagos Islands.

The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact on affected hosts. Here, we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galápagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data, we characterize the bacterial microbiota associated with P. downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp., respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harboured similar microbiomes across islands. Our results suggest that the microbiome of P. downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unravelling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies.

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.

Effects of four commercial fungal formulations on mortality and sporulation in house flies (Musca domestica) and stable flies (Stomoxys calcitrans).

The house fly Musca domestica L. (Diptera: Muscidae) and stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae) are major pests of livestock. Biological control is an important tool in an integrated control framework. Increased mortality in filth flies has been documented with entomopathogenic fungi, several strains of which are commercially available. Three strains of Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) and one strain of Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) were tested in commercial formulations for pathogenicity against house flies and stable flies. There was a significant increase in mortality of house flies with three of the formulations, BotaniGard® ES, Mycotrol® O, and Met52® EC, during days 4-9 in comparison with balEnce™ and the control. In stable flies, mortality rates were highest with Met52® EC, followed by Mycotrol® O, BotaniGard® ES and, finally, balEnce™. There was a significant fungal effect on sporulation in both house flies and stable flies. Product formulation, species differences and fungal strains may be responsible for some of the differences observed. Future testing in field situations is necessary. These commercial biopesticides may represent important tools in integrated fly management programmes.

Identification and Optimization of Microbial Attractants for Philornis downsi, an Invasive Fly Parasitic on Galapagos Birds.

We investigated the role of olfactory cues from actively fermenting yeast (Saccharomyces cerevisiae) in attraction of adult Philornis downsi and identified two synergistically attractive yeast volatiles. Larvae of this invasive fly parasitize the hatchlings of passerines and threaten the Galapagos avifauna. Gas chromatography coupled with electroantennographic detection (GC-EAD), coupled gas chromatography-mass spectrometry (GC-MS), and field trapping experiments were used to identify volatile compounds from a yeast-sugar solution. EAD responses were consistently elicited by 14 yeast volatiles. In a series of field trapping experiments, a mixture of the 14 EAD-active compounds was similarly attractive to P. downsi when compared to the yeast-sugar solution, and we found that acetic acid and ethanol were essential for attraction. A mixture of 0.03 % acetic acid and 3 % ethanol was as attractive as the 14-component blend, but was not as attractive as the yeast-sugar solution. Philornis downsi showed positive and negative dose-responses to acetic acid in the ranges of 0.01 ~ 0.3 % and 0.3 ~ 9 %, respectively. Further optimization showed that the mixture of 1 % acetic acid and 3 % ethanol was as attractive as the yeast-sugar solution. Both mixtures of acetic acid and ethanol were more selective than the yeast-sugar solution in terms of non-target moths and Polistes versicolor wasps captured. These results indicate that acetic acid and ethanol produced by yeasts are crucial for P. downsi attraction to fermented materials on which they feed as adults and can be used to manage this invasive fly in Galapagos.