RESUMO
Animals encounter diverse microbial communities throughout their lifetime, which exert varying selection pressures. Antimicrobial peptides (AMPs), which lyse or inhibit microbial growth, are a first line of defense against some of these microbes. Here we examine how developmental variation in microbial exposure has affected the evolution of expression and amino acid sequences of Defensins (an ancient class of AMPs) in the house fly (Musca domestica). The house fly is a well-suited model for this work because it trophically associates with varying microbial communities throughout its life history and its genome contains expanded families of AMPs, including Defensins. We identified two subsets of house fly Defensins: one expressed in larvae or pupae, and the other expressed in adults. The amino acid sequences of these two Defensin subsets form distinct monophyletic clades, and they are located in separate gene clusters in the genome. The adult-expressed Defensins evolve faster than larval/pupal Defensins, consistent with different selection pressures across developmental stages. Our results therefore suggest that varied microbial communities encountered across life history can shape the evolutionary trajectories of immune genes.
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Insects possess both infection-induced and constitutively expressed innate immune defences. Some effectors, such as lysozymes and antimicrobial peptides (AMPs), are constitutively expressed in flies, but expression patterns vary across tissues and species. The house fly (Musca domestica L.) has an impressive immune repertoire, with more effector genes than any other flies. We used RNA-seq to explore both constitutive and induced expression of immune effectors in flies. House flies were fed either Pseudomonas aeruginosa or Escherichia coli, or sterile control broth, and gene expression in the gut and carcass was analysed 4 h post-feeding. Flies fed either bacterium did not induce AMP expression, but some lysozyme and AMP genes were constitutively expressed. Prior transcriptome data from flies injected with bacteria also were analysed, and these constitutively expressed genes differed from those induced by bacterial injection. Binding sites for the transcription factor Myc were enriched upstream of constitutively expressed AMP genes, while upstream regions of induced AMPs were enriched for NF-κB binding sites resembling those of the Imd-responsive transcription factor Relish. Therefore, we identified at least two expression repertoires for AMPs in the house fly: constitutively expressed genes that may be regulated by Myc, and induced AMPs likely regulated by Relish.
Assuntos
Moscas Domésticas , Animais , Bactérias , Regulação da Expressão Gênica , Moscas Domésticas/genética , Pseudomonas aeruginosa , Fatores de Transcrição/genéticaRESUMO
Adult house flies feed and breed in a variety of microbe-rich habitats and serve as vectors for human and animal pathogens. To better understand their role in harbouring and disseminating bacteria, we characterized the composition and diversity of bacterial communities in the gut of female house flies collected from three different habitats in Kansas: agricultural (dairy farm), urban (business area dumpsters) and mixed (business located between residential and animal agriculture areas). Bacterial community composition and diversity were influenced more by the house flies' habitat than by sampling time. The most abundant taxa were also highly prevalent in the house flies collected from all three habitats, potentially representing a 'core microbiome' attributable to the fly's trophic and reproductive associations with substrates and food sources comprised of decaying matter and/or animal waste. Bacterial taxa associated with vertebrate guts/faeces and potential pathogens were highly abundant in agricultural fly microbial communities. Interestingly, taxa of potential pathogens were highly abundant in flies from the mixed and urban sites. House flies harboured diverse bacterial communities influenced by the habitat in which they reside, including potential human and animal pathogens, further bolstering their role in the dissemination of pathogens, and indicating their utility for pathogen surveillance.
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Moscas Domésticas , Microbiota , Muscidae , Feminino , Humanos , Animais , Moscas Domésticas/microbiologia , Bactérias , Manejo de Espécimes/veterináriaRESUMO
BACKGROUND: The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. RESULTS: This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. CONCLUSIONS: The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.
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Genoma de Inseto , Interações Hospedeiro-Parasita/genética , Controle de Insetos , Muscidae/genética , Animais , Reprodução/genéticaRESUMO
BACKGROUND: House fly larvae (Musca domestica L.) require a live microbial community to successfully develop. Cattle manure is rich in organic matter and microorganisms, comprising a suitable substrate for larvae who feed on both the decomposing manure and the prokaryotic and eukaryotic microbes therein. Microbial communities change as manure ages, and when fly larvae are present changes attributable to larval grazing also occur. Here, we used high throughput sequencing of 16S and 18S rRNA genes to characterize microbial communities in dairy cattle manure and evaluated the changes in those communities over time by comparing the communities in fresh manure to aged manure with or without house fly larvae. RESULTS: Bacteria, archaea and protist community compositions significantly differed across manure types (e.g. fresh, aged, larval-grazed). Irrespective of manure type, microbial communities were dominated by the following phyla: Euryarchaeota (Archaea); Proteobacteria, Firmicutes and Bacteroidetes (Bacteria); Ciliophora, Metamonanda, Ochrophyta, Apicomplexa, Discoba, Lobosa and Cercozoa (Protists). Larval grazing significantly reduced the abundances of Bacteroidetes, Ciliophora, Cercozoa and increased the abundances of Apicomplexa and Discoba. Manure aging alone significantly altered the abundance bacteria (Acinetobacter, Clostridium, Petrimonas, Succinovibro), protists (Buxtonella, Enteromonas) and archaea (Methanosphaera and Methanomassiliicoccus). Larval grazing also altered the abundance of several bacterial genera (Pseudomonas, Bacteroides, Flavobacterium, Taibaiella, Sphingopyxis, Sphingobacterium), protists (Oxytricha, Cercomonas, Colpodella, Parabodo) and archaea (Methanobrevibacter and Methanocorpusculum). Overall, larval grazing significantly reduced bacterial and archaeal diversities but increased protist diversity. Moreover, total carbon (TC) and nitrogen (TN) decreased in larval grazed manure, and both TC and TN were highly correlated with several of bacterial, archaeal and protist communities. CONCLUSIONS: House fly larval grazing altered the abundance and diversity of bacterial, archaeal and protist communities differently than manure aging alone. Fly larvae likely alter community composition by directly feeding on and eliminating microbes and by competing with predatory microbes for available nutrients and microbial prey. Our results lend insight into the role house fly larvae play in shaping manure microbial communities and help identify microbes that house fly larvae utilize as food sources in manure. Information extrapolated from this study can be used to develop manure management strategies to interfere with house fly development and reduce house fly populations.
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Moscas Domésticas/metabolismo , Esterco/microbiologia , Microbiota , Animais , Archaea/classificação , Archaea/genética , Archaea/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Carbono/análise , Bovinos , Eucariotos/classificação , Eucariotos/genética , Eucariotos/isolamento & purificação , Moscas Domésticas/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/metabolismo , Esterco/análise , Nitrogênio/análise , RNA Ribossômico/genéticaRESUMO
Salmonella enterica serovar Typhimurium is a pathogen harbored by livestock and shed in their feces, which serves as an acquisition source for adult house flies. This study used a green fluorescent protein (GFP) expressing strain of Salmonella Typhimurium to assess its acquisition by and survival within house flies, and transmission from and between flies in the presence or absence of cantaloupe. Female house flies were exposed to manure inoculated with either sterile phosphate-buffered saline or GFP-Salmonella Typhimurium for 12 h, then used in four experiments each performed over 24 h. Experiment 1 assessed the survival of GFP-Salmonella Typhimurium within inoculated flies. Experiment 2 determined transmission of GFP-Salmonella Typhimurium from inoculated flies to cantaloupe. Experiment 3 assessed fly acquisition of GFP-Salmonella Typhimurium from inoculated cantaloupe. Experiment 4 evaluated transmission of GFP-Salmonella Typhimurium between inoculated flies and uninoculated flies in the presence and absence of cantaloupe. GFP-Salmonella Typhimurium survived in inoculated flies but bacterial abundance decreased between 0 and 6 h without cantaloupe present and between 0 and 6 h and 6 and 24 h with cantaloupe present. Uninoculated flies acquired GFP-Salmonella Typhimurium from inoculated cantaloupe and bacterial abundance increased in cantaloupe and flies from 6 to 24 h. More uninoculated flies exposed to inoculated flies acquired GFP-Salmonella Typhimurium when cantaloupe was present than when absent. We infer that the presence of a shared food source facilitated the transfer of GFP-Salmonella Typhimurium from inoculated to uninoculated flies. Our study demonstrated that house flies acquired, harbored, and excreted viable GFP-Salmonella Typhimurium and transferred bacteria to food and each other. Understanding the dynamics of bacterial acquisition and transmission of bacteria between flies and food helps in assessing the risk flies pose to food safety and human health.
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Cucumis melo/microbiologia , Moscas Domésticas/microbiologia , Salmonella typhimurium/patogenicidade , Animais , Feminino , Contaminação de Alimentos/análise , Microbiologia de Alimentos/métodos , Proteínas de Fluorescência Verde/metabolismoRESUMO
BACKGROUND: The new genomic technologies have provided novel insights into the genetics of interactions between vectors, viruses and hosts, which are leading to advances in the control of arboviruses of medical importance. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV. RESULTS: Our analyses reveal that the genome is 189 Mb, assembled in 7974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,612 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study. CONCLUSIONS: The reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a valuable resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and together with the transcriptomic data can yield potential targets for transgenic modification in vector control and functional studies.
Assuntos
Vírus Bluetongue/fisiologia , Bluetongue/transmissão , Ceratopogonidae/genética , Ceratopogonidae/virologia , Genoma de Inseto , Insetos Vetores , Animais , Bluetongue/imunologia , Bluetongue/virologia , Vírus Bluetongue/imunologia , Ceratopogonidae/imunologia , Evolução Molecular , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Insetos Vetores/genética , Insetos Vetores/fisiologia , Anotação de Sequência Molecular , Análise de Sequência de DNA , Transcriptoma/genéticaRESUMO
House flies (Diptera: Muscidae; Musca domestica L.) associate with microbe-rich substrates throughout life history. Because larvae utilize bacteria as a food source, most taxa present in the larval substrate, e.g., manure, are digested or degraded. However, some species survive and are present as third-instar larvae begin pupation. During metamorphosis, many bacteria are again lost during histolysis of the larval gut and subsequent remodeling to produce the gut of the imago. It has been previously demonstrated that some bacterial species survive metamorphosis, being left behind in the puparium, present on the body surface, or in the gut of the emerged adult. We used a combined culture-molecular approach to identify viable microbes from managed manure residue and a wild population of house fly larvae, pupae, puparia, and adults to assess transstadial carriage. All larval (10/10), pupal (10/10), and puparial (10/10) cultures were positive for bacteria. Several bacterial species that were present in larvae also were present either in pupae or puparia. Four viable bacterial species were detectable in 6 of 10 imagoes reared from manure. Of note is the apparent transstadial carriage of Bacillus sonorensis, which has been associated with milk spoilage at dairies, and Alcaligenes faecalis, which can harbor numerous antibiotic resistance genes on farms. The potential of newly emerged flies to harbor and disseminate bacteria from managed manure on farms is an understudied risk that deserves further evaluation.
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Moscas Domésticas/microbiologia , Esterco/microbiologia , Animais , Microbioma Gastrointestinal/genética , Moscas Domésticas/fisiologia , Larva/microbiologia , Metamorfose Biológica , Pupa/microbiologia , RNA Ribossômico 16S/genética , Gerenciamento de ResíduosRESUMO
Organisms produce antimicrobial peptides (AMPs) either in response to infection (induced) or continuously (constitutively) to combat microbes encountered during normal trophic activities and/or through pathogenic infections. The expression of AMPs is tightly regulated often with specificity to particular tissues or developmental stages. As a result, AMPs face varying selective pressures based on the microbes the organism's tissue or developmental stage encounters. Here, we analyzed the evolution and developmental-specific expression of Defensins, which are ancient AMPs in insects, in the stable fly (Stomoxys calcitrans). Stable fly larvae inhibit microbe-rich environments, whereas adult flies, as blood-feeders, experience comparatively fewer encounters with diverse microbial communities. Using existing RNA-seq datasets, we identified six Defensins that were only expressed in larvae (larval Defensins) and five that were not expressed in larvae (non-larval Defensins). Each of the non-larval Defensins was expressed in at least one adult tissue sample. Half of the larval Defensins were induced by mating or feeding in adults, and all three of the induced Defensins were located downstream of canonical binding sites for an Imd transcription factor involved in the highly conserved NF-κB signaling that regulates induction of AMPs. The larval and non-larval Defensins were located in distinct genomic regions, and the amino acid sequences of the larval Defensins formed a monophyletic clade. There were more amino acid substitutions across non-larval Defensins, with multiple genes losing a highly conserved furin cleavage site thought to be required for the removal of the amino terminus from the mature Defensin domain. However, larval Defensins had a higher proportion of radical amino acid substitutions, altering amino acid size and polarity. Our results reveal insights into the developmental stage-specific regulation of AMPs, and they suggest different regulatory regimes impose unique selection pressures on AMPs, possibly as a result of variation in exposure to microbial communities across development.
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From egg to adult, all life history stages of house flies associate with septic environments teeming with bacteria. House fly lysozyme was first identified in the larval midgut, where it is used for digestion of microbe-rich meals because of its broad-spectrum activity against gram-positive and gram-negative bacteria as well as fungi. This study aimed to determine the temporal expression of lysozyme in the life history of house flies (from egg through adults) on both the mRNA and protein level, and to determine the tissue-specific expression of lysozyme in adult flies induced by feeding Staphylococcus aureus. From 30-min postoviposition through adulthood, all life history stages of the house fly express lysozyme on the mRNA level. In adult flies, lysozyme is expressed both locally in the alimentary canal and systemically in the fat body. Interestingly, we found that during the normal life history of flies, lysozyme protein was only detected in larval stages and older adults, likely because of ingestion of immune-stimulating levels of bacteria, not experienced during egg, pupa, and teneral adult stages. Constitutive expression on the mRNA level implies that this effector is a primary defense molecule in all stages of the house fly life history, and that a mechanism for posttranscriptional control of mature lysozyme enzyme expression may be present. Lysozyme active enzyme primarily serves both a digestive and defensive function in larval and adult flies, and may be a key player in the ability of Musca domestica L. to thrive in microbe-rich environments.
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Moscas Domésticas/genética , Muramidase/genética , Animais , Corpo Adiposo/metabolismo , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Regulação da Expressão Gênica , Moscas Domésticas/metabolismo , Larva/genética , Larva/metabolismo , Muramidase/metabolismo , Óvulo/metabolismo , Pupa/genética , Pupa/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Staphylococcus aureus/fisiologiaRESUMO
House flies disseminate numerous species of bacteria acquired during feeding and breeding activities in microbe-rich habitats. Previous house fly surveys have detected the pathogen Staphylococcus aureus Rosenbach 1884, which causes cutaneous and septic infections in mammals, and enterotoxic food poisoning. We assessed the fate of GFP-expressing S. aureus (GFP-S. aureus) in the house fly alimentary canal with microscopy and by culture of whole flies and excreta. Furthermore, the concurrent expression of the antimicrobial peptide gene defensin was measured in the crop, proventriculus, midgut, and fat body. As soon as 4 h postingestion (PI), GFP-S. aureus were visualized as cocci or diplococci in the hindgut and rectum of flies fed approximately equal 10(5) colony forming units. Bacteria persisted up to 6 h PI but significantly decreased. Excretion of viable GFP-S. aureus peaked at 2 h PI and, although significantly less, continued up to 4 h PI. defensin was highly upregulated locally in the alimentary canal and systemically in fat body at 2, 4, and 6 h PI making this study the first to report, to our knowledge, an epithelial and systemic response to a bacterium with lysine-type peptidoglycan in flies exposed via feeding. While flies harbored S. aureus for up to 6 h PI, the highest probability of vectoring biologically relevant amounts of bacteria occurred 0-2 h PI. The combined effects of excretion, digestion and antimicrobial effectors likely contribute to loss of ingested bacteria. Nonetheless, house flies are relevant vectors for S. aureus up to 2 h PI and environmental reservoirs up to 6 h PI.
Assuntos
Defensinas/metabolismo , Moscas Domésticas/microbiologia , Staphylococcus aureus/fisiologia , Animais , Defensinas/genética , Trato Gastrointestinal/microbiologia , Proteínas de Fluorescência Verde , Moscas Domésticas/genética , Moscas Domésticas/imunologia , Insetos Vetores/microbiologiaRESUMO
BACKGROUND: Biting midges (Culicoides spp.) are important vectors of diverse microbes such as viruses, protozoa, and nematodes that cause diseases in wild and domestic animals. However, little is known about the role of microbial communities in midge larval habitat utilization in the wild. In this study, we characterized microbial communities (bacterial, protistan, fungal and metazoan) in soils from disturbed (bison and cattle grazed) and undisturbed (non-grazed) pond and spring potential midge larval habitats. We evaluated the influence of habitat and grazing disturbance and their interaction on microbial communities, diversity, presence of midges, and soil properties. RESULTS: Bacterial, protistan, fungal and metazoan community compositions were significantly influenced by habitat and grazing type. Irrespective of habitat and grazing type, soil communities were dominated by phyla Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria (Bacteria); Apicomplexa, Cercozoa, Ciliophora, Ochrophyta (Protists); Chytridiomycota, Cryptomycota (Fungi) and Nematoda, Arthropoda (Metazoa). The relative abundance of Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, Verrucomicrobia (Bacteria); Apicomplexa, Lobosa (Protists); Ascomycota, Blastomycotina, Cryptomycota (Fungi); and Platyhelminthes (Metazoa) were significantly affected by grazing type. Of note, midge prevalence was higher in grazed sites (67-100%) than non-grazed (25%). Presence of midges in the soil was negatively correlated with bacterial, protistan, fungal and metazoan beta diversities and metazoan species richness but positively correlated with protistan and fungal species richness. Moreover, total carbon (TC), nitrogen (TN) and organic matter (OM) were negatively correlated with the presence of midges and relative abundances of unclassified Solirubrobacterales (Bacteria) and Chlamydomonadales (Protists) but positively with Proteobacteria and unclassified Burkholderiales (Bacteria). CONCLUSIONS: Habitat and grazing type shaped the soil bacterial, protistan, fungal and metazoan communities, their compositions and diversities, as well as presence of midges. Soil properties (TN, TC, OM) also influenced soil microbial communities, diversities and the presence of midges. Prevalence of midges mainly in grazed sites indicates that midges prefer to breed and shelter in a habitat with abundant hosts, probably due to greater accessibility of food (blood meals). These results provide a first glimpse into the microbial communities, soil properties and prevalence of midges in suspected midge larval habitats at a protected natural prairie site.
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House flies, Musca domestica L., (Diptera: Muscidae) mechanically vector diverse disease-causing microorganisms while foraging for food in agricultural and urban habitats. Although flies are diverse feeders, nutrient composition of food is important for both fly longevity and reproduction, especially for anautogenous females who require protein for egg production. We investigated whether fly sex and/or mating status influenced their preference for foods with varying macronutrient composition. Presumably mated or unmated male and female flies were separated by sex and offered four food, each in 10% solution offered on cotton wicks: sugar (carbohydrate-rich), fat-free milk (protein-rich, moderate carbohydrate), egg-yolk (protein and lipid-rich), and water (no macronutrients). Foods were colored with nontoxic dyes, which were rotated between replicates. After 4h exposure, flies were dissected to determine the type of food(s) ingested. The interaction of house fly sex and food type significantly influenced food preference, where females preferred milk (protein and carbohydrate-rich food), and males preferred mainly sugar (carbohydrate-rich). Furthermore, 32.8% of females and 10.6% of males foraged on multiple foods. While interaction of sex and mating status had no effect on food preference, milk preference was significantly higher in presumably mated than unmated females. We also tested whether food color influenced fly feeding preference, and found that color was most significant when flies were offered one food type, but negligible when multiple food types were present. This study suggests that bait-based fly control strategies should consider sex-specific preferences for various food attractants if aiming to target and control both male and female house flies.
Assuntos
Dípteros , Moscas Domésticas , Muscidae , Feminino , Masculino , Animais , Comportamento Alimentar , Carboidratos , AçúcaresRESUMO
Viruses that are transmitted by arthropods, or arboviruses, have evolved to successfully navigate both the invertebrate and vertebrate hosts, including their immune systems. Biting midges transmit several arboviruses including vesicular stomatitis virus (VSV). To study the interaction between VSV and midges, we characterized the transcriptomic responses of VSV-infected and mock-infected Culicoides sonorensis cells at 1, 8, 24, and 96 h post inoculation (HPI). The transcriptomic response of VSV-infected cells at 1 HPI was significant, but by 8 HPI there were no detectable differences between the transcriptome profiles of VSV-infected and mock-infected cells. Several genes involved in immunity were upregulated (ATG2B and TRAF4) or downregulated (SMAD6 and TOLL7) in VSV-treated cells at 1 HPI. These results indicate that VSV infection in midge cells produces an early immune response that quickly wanes, giving insight into in vivo C. sonorensis VSV tolerance that may underlie their permissiveness as vectors for this virus.
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Arbovírus , Ceratopogonidae , Estomatite Vesicular , Animais , Transcriptoma , Ceratopogonidae/genética , Estomatite Vesicular/genética , Insetos Vetores , Vesiculovirus/genética , Arbovírus/genética , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
House flies (Musca domestica Linnaeus) are vectors of human and animal pathogens at livestock operations. Microbial communities in flies are acquired from, and correlate with, their local environment. However, variation among microbial communities carried by flies from farms in different geographical areas is not well understood. We characterized bacterial communities of female house flies collected from beef and dairy farms in Oklahoma, Kansas, and Nebraska using 16S rDNA amplicon sequencing and PCR. Bacterial community composition in house flies was affected by farm type and location. While the shared number of taxa between flies from beef or dairy farms was low, those taxa accounted >97% of the total bacterial community abundance. Bacterial species richness was 4% greater in flies collected from beef than in those collected from dairy farms and varied by farm type within states. Several potential pathogenic taxa were highly prevalent, comprising a core bacterial community in house flies from cattle farms. Prevalence of the pathogens Moraxella bovis and Moraxella bovoculi was greater in flies from beef farms relative to those collected on dairy cattle farms. House flies also carried bacteria with multiple tetracycline and florfenicol resistance genes. This study suggests that the house flies are significant reservoirs and disseminators of microbial threats to human and cattle health.
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Dípteros , Moscas Domésticas , Muscidae , Humanos , Bovinos , Feminino , Animais , Dípteros/microbiologia , Moscas Domésticas/microbiologia , Fazendas , Antibacterianos/farmacologia , Prevalência , Bactérias/genética , Resistência Microbiana a MedicamentosRESUMO
House flies are well recognized as filth-associated organisms and public nuisances. House flies create sanitation issues when they bridge the gap between microbe-rich breeding environments and animal/human habitations. Numerous scientific surveys have demonstrated that house flies harbor bacterial pathogens that pose a threat to humans and animals. More extensive and informative surveys incorporating next-generation sequencing technologies have shown that house fly carriage of pathogens and harmful genetic elements, such as antimicrobial resistance genes, is more widespread and dangerous than previously thought. Further, there is a strong body of research confirming that flies not only harbor but also transmit viable, and presumably infectious, bacterial pathogens. Some pathogens replicate and persist in the fly, permitting prolonged shedding and dissemination. Finally, although the drivers still have yet to be firmly determined, the potential range of dissemination of flies and their associated pathogens can be extensive. Despite this evidence, the house flies' role as reservoirs, disseminators, and true, yet facultative, vectors for pathogens have been greatly underestimated and underappreciated. In this review, we present key studies that bolster the house fly's role both an important player in microbial ecology and population biology and as transmitters of microbial threats to animal and human health.
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Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, ß-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.
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Ceratopogonidae , Animais , Feminino , Ceratopogonidae/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , RNA de Cadeia Dupla/metabolismo , Interferência de RNA , Larva , Perfilação da Expressão Gênica/métodos , Padrões de ReferênciaRESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged coronavirus that is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. COVID-19 in humans is characterized by a wide range of symptoms that range from asymptomatic to mild or severe illness including death. SARS-CoV-2 is highly contagious and is transmitted via the oral-nasal route through droplets and aerosols, or through contact with contaminated fomites. House flies are known to transmit bacterial, parasitic and viral diseases to humans and animals as mechanical vectors. Previous studies have shown that house flies can mechanically transmit coronaviruses, such as turkey coronavirus; however, the house fly's role in SARS-CoV-2 transmission has not yet been explored. The goal of this work was to investigate the potential of house flies to mechanically transmit SARS-CoV-2. For this purpose, it was determined whether house flies can acquire SARS-CoV-2, harbor live virus and mechanically transmit the virus to naive substrates and surfaces. METHODS: Two independent studies were performed to address the study objectives. In the first study, house flies were tested for infectivity after exposure to SARS-CoV-2-spiked medium or milk. In the second study, environmental samples were tested for infectivity after contact with SARS-CoV-2-exposed flies. During both studies, samples were collected at various time points post-exposure and evaluated by SARS-CoV-2-specific RT-qPCR and virus isolation. RESULTS: All flies exposed to SARS-CoV-2-spiked media or milk substrates were positive for viral RNA at 4 h and 24 h post-exposure. Infectious virus was isolated only from the flies exposed to virus-spiked milk but not from those exposed to virus-spiked medium. Moreover, viral RNA was detected in environmental samples after contact with SARS-CoV-2 exposed flies, although no infectious virus was recovered from these samples. CONCLUSIONS: Under laboratory conditions, house flies acquired and harbored infectious SARS-CoV-2 for up to 24 h post-exposure. In addition, house flies were able to mechanically transmit SARS-CoV-2 genomic RNA to the surrounding environment up to 24 h post-exposure. Further studies are warranted to determine if house fly transmission occurs naturally and the potential public health implications of such events.
Assuntos
COVID-19/transmissão , Moscas Domésticas/virologia , Insetos Vetores/virologia , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Animais , Chlorocebus aethiops , Feminino , Células VeroRESUMO
Adult house flies frequent microbe-rich sites such as urban dumpsters and animal facilities, and encounter and ingest bacteria during feeding and reproductive activities. Due to unique nutritional and reproductive needs, male and female flies demonstrate different interactions with microbe-rich substrates and therefore dissemination potential. We investigated culturable aerobic bacteria and coliform abundance in male and female flies (n = 107) collected from urban (restaurant dumpsters) and agricultural (dairy farm) sites. Whole-fly homogenate was aerobically cultured and enumerated on nonselective (tryptic soy agar; culturable bacteria) and selective (violet-red bile agar, VRBA; coliforms) media. Unique morphotypes from VRBA cultures of agricultural flies were identified and tested for susceptibility to 14 antimicrobials. Female flies harbored more bacteria than males and there was a sex by site interaction with sex effects on bacterial abundance at the urban site. Coliform abundance did not differ by sex, site or sex within site. Both male and female flies carried antimicrobial-resistant (AMR) bacteria: 36/38 isolates (95%) were resistant to ≥1 antimicrobial, 33/38 were multidrug-resistant (≥2), and 24/38 isolates were resistant to ≥4 antimicrobials. Our results emphasize the role of house flies in harboring bacteria including AMR strains that pose a risk to human and animal health.