Antagonistic effect of the beneficial bacterium Enterobacter hormaechei against the heavy metal Cu2+ in housefly larvae.

Vermicomposting via housefly larvae can be used to efficiently treat manure and regenerate biofertilizer; however, the uptake of heavy metals could negatively influence the growth and development of larvae. Intestinal bacteria play an important role in the development of houseflies, but their effects on resistance to heavy metal damage in houseflies are still poorly understood. In this study, the life history traits and gut microbiota of housefly larvae were evaluated after exposure to an environment with Cu2+ -Enterobacter hormaechei. The data showed that exposure to 300 µg/mL Cu2+ significantly inhibited larval development and locomotor activity and reduced immune capacity. However, dietary supplementation with a Cu2+ -Enterobacter hormaechei mixture resulted in increased body weight and length, and the immune capacity of the larvae returned to normal levels. The abundances of Providencia and Klebsiella increased when larvae were fed Cu2+ -contaminated diets, while the abundances of Enterobacter and Bacillus increased when larvae were exposed to a Cu2+ -Enterobacter hormaechei mixture-contaminated environment. In vitro scanning electron microscopy analysis revealed that Enterobacter hormaechei exhibited obvious adsorption of Cu2+ when cultured in the presence of Cu2+, which reduced the damage caused by Cu2+ to other bacteria in the intestine and protected the larvae from Cu2+ injury. Overall, our results showed that Enterobacter hormaechei can absorb Cu2+ and increase the abundance of beneficial bacteria, thus protecting housefly larvae from damage caused by Cu2+. These results may fill the gaps in our understanding of the interactions between heavy metals and beneficial intestinal bacteria, offering valuable insights into the interplay between housefly larvae and metal contaminants in the environment. This approach could enhance the efficiency of converting manure contaminated with heavy metals to resources using houseflies.

Ecotoxicity effect of aspirin on the larvae of Musca domestica through retinol metabolism.

Aspirin is a widely used multi-efficiency pharmaceutical, and its environmental residues are frequently detected. However, limited information is available on its effects on the development of the public health pest and saprophytic insect Musca domestica. In this study, it was demonstrated that aspirin inhibits the larval growth of house flies in a concentration-dependent manner. Microbiome analysis indicated that the composition of larval intestinal bacteria was influenced by aspirin but not greatly. The dominant bacterial genus in the aspirin group was still Klebsiella, as in the control group. Transcriptome sequencing and gene set enrichment analysis showed that retinol metabolism was activated after aspirin treatment. High performance liquid chromatography indicated that the content of retinol in larvae was decreased and that of retinoic acid was increased. The addition of ß-carotene, a precursor substance of retinol, in feeding promotes larval development and alleviates the inhibitory effect caused by aspirin. In contrast, retinoic acid delayed the larval development of house flies as well as aspirin. Gene expression analysis after aspirin exposure demonstrated that genes involved in the transformation from retinol to retinoic acid were upregulated. Overall, aspirin exposure impairs larval development by activating retinol metabolism in house flies and can be utilized as an effective pesticide. This work uncovers the mechanism underlying the larval development inhibition induced by aspirin in terms of metabolism and genetics, and provides novel functional exploration of a traditional drug for pest management.

Bacterial communities and prevalence of antibiotic resistance genes carried within house flies (Diptera: Muscidae) associated with beef and dairy cattle farms.

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.

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

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

Presence of the toxigenic fungi Aspergillus spp. and Fusarium spp. in Musca domestica L. (Diptera: Muscidae) collected from dairy farms.

The objective of the study was to identify the presence of toxigenic fungi Aspergillus spp. and Fusarium spp. in domestic flies collected from dairy farms. We selected 10 dairy farms distributed in the central valley of the state of Aguascalientes, México. The flies were trapped using entomological traps with an olfactory attractant in 7 sites of the farm (silo-cutting surface, feed store, milking parlor, 3 feeders, and the rearing room). The fungi were cultivated in Sabouraud agar through direct sowing by serial dilutions to obtain the isolates, and a taxonomical identification was carried out under the microscope. The aflatoxins and zearalenone production capacity of the pure isolates were quantified using the ELISA test. The flies were present in all of the capture sites (45.3 flies, 567 mg, trap per day). We obtained 50 isolates of Aspergillus spp. genus, 12 of which produced aflatoxins (327 ± 143 µg/kg), whereas from 56 of the Fusarium spp. isolates, 10 produced large quantities of zearalenone (3,132 ± 665 µg/kg). These results suggest that the presence of domestic flies on dairy farms can constitute a source of dissemination for toxigenic fungi that can eventually contaminate grains and forage that are part of the daily cattle diet.

Isolation of Multidrug-Resistant Helicobacter pylori from Wild Houseflies Musca domestica with a New Perspective for the Treatment.

Background: High frequency of Helicobacter pylori infection and the unknown mode of transmission prompted us to investigate H. pylori-wild housefly relationship. H. pylori causes chronic gastritis, peptic ulcers, and stomach cancer. H. pylori persists in the gut of the experimentally infected houseflies. The existence of H. pylori strains isolated from wild houseflies, on the other hand, has never been documented. Materials and Methods: In this study, 902 wild houseflies from different sites were identified as Musca domestica, then 60 flies were screened by traditional microbiological techniques and H. pylori-specific 16S rRNA gene. The antibiotic resistance (ART) was investigated phenotypically. Wild housefly gut bacterial isolates were further evaluated genotypically to have 23S rRNA gene mutation related to clarithromycin resistance. To find efficient therapeutic alternatives, the potency of three plant extracts (garlic, ginger, and lemon) and the wasp, Vespa orientalis venom was evaluated against H. pylori. The cytotoxic effect of the crude wasp venom, the most potent extract, against Vero and Colon cancer (Caco2) cell lines was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: All isolates from houseflies were positive. The isolated bacteria have variable resistance to frequently used antibiotics in all isolates. Minimum inhibitory concentration values of 15.625 mg/mL for both ginger and lemon extracts, 7.8125 mg/mL for garlic extract, and 0.0313 mg/mL for wasp venom were recorded. Wasp venom has the most potent antibacterial activity compared with the four antibiotics that are currently used in therapies against H. pylori. Conclusion: We conclude that wild houseflies can play a role in disseminating H. pylori. The housefly gut may be a suitable environment for the horizontal transfer of ART genes among its associated microbiome and H. pylori. Wasp venom proved its potential activity as a new and effective anti-H. pylori drug for both therapeutic and preventative usage.

Beneficial Bacteria in the Intestines of Housefly Larvae Promote Larval Development and Humoral Phenoloxidase Activity, While Harmful Bacteria do the Opposite.

The gut microenvironment of houseflies provides unique conditions for microbial colonization. Some gut microorganisms provide benefits for the development of the host by regulating the interaction between the host and intestinal pathogens. Gut microbial alterations can stimulate the host's immune mechanism to resist pathogen invasion and affect the development of insects. In this study, we isolated 10 bacterial strains from housefly larval intestines. The isolated bacteria were added to the larval diet to analyze the effects of microecological regulation of gut bacteria on larval development. Dynamic changes in gut flora composition after oral administration of specific bacteria were analyzed although 16S rRNA gene high-throughput sequencing technology. To explore the interaction between gut bacteria and the host, the immune response of larvae against the invasion of foreign microorganisms was observed through a phenoloxidase activity experiment. Our results showed that the oral administration of various isolated bacteria had different effects on larval development. Oral administration of beneficial bacteria, including Enterobacter hormaechei, Klebsiella pneumoniae, Acinetobacter bereziniae, Enterobacter cloacae, Lysinibacillus fusiformis and Bacillus safensis, promoted larval development by increasing gut community diversity and the humoral immunity of larvae, while harmful bacteria, including Pseudomonas aeruginosa, Providencia stuartii and Providencia vermicola, influenced larval development by inhibiting the growth of beneficial bacteria and reducing the humoral immunity of larvae. The beneficial bacteria isolated in our research could be applied as good probiotic additives for the intensive feeding of larvae, while isolation of the harmful bacteria provides a basis for the development of pest inhibitors. Furthermore, our research revealed the immune response of housefly phenoloxidase to exogenous microorganism stimulation, providing richer and more comprehensive knowledge of the larval innate immune response.

Houseflies harbor less diverse microbiota under laboratory conditions but maintain a consistent set of host-associated bacteria.

The housefly (Musca domestica) is a wide-ranging insect, often associated with decaying matter from livestock and humans. The septic environments in which houseflies live are believed to be a rich source for microbial acquisition. Although the housefly can harbor a wide range of microorganisms, it is not yet well known which microbes are always recurrent, which are dispensable and which environmentally dependent. In the present study, we aim at identifying which microbes are recurrently associated with the housefly gut throughout the species' life cycle and whether their acquisition relies on the fly's living environment. We surveyed three housefly strains-two of them kept under standard laboratory conditions for a long time and one wild-caught. To track any shifts happening throughout the lifecycle of the housefly and to test the consistency of the revealed microbial communities, we sampled houseflies at five developmental stages over the course of four consecutive generations. Both the bacterial and fungal microbiota of five developmental stages were studied for all samples, using amplicon sequencing for the 16S and ITS1 rRNA gene, respectively. Results revealed diverse microbial communities yet consistent for each of the two distinct sampling environments. The wild-caught population showed a more diverse and more distinct gut microbiota than the two laboratory strains, even though the strain was phylogenetically similar and shared geographic origin with one of them. Two bacterial genera, Myroides and Providencia, and two yeasts, Trichosporon and Candida tropicalis, were present in all sampled larvae and pupae, regardless of the strain. Analysis of the provided diet revealed that the flies acquired the yeasts through feeding. Our main findings show that houseflies might lose microbial diversity when reared in controlled environments, however they can maintain a consistent set of bacteria. We conclude that although the environment can facilitate certain microbial transmission routes for the housefly, and despite the fungal microbiota being largely acquired through diet, the larval bacterial gut microbiome remains relatively consistent within the same developmental stage.

Pathogenic fungus uses volatiles to entice male flies into fatal matings with infected female cadavers.

To ensure dispersal, many parasites and pathogens behaviourally manipulate infected hosts. Other pathogens and certain insect-pollinated flowers use sexual mimicry and release deceptive mating signals. However, it is unusual for pathogens to rely on both behavioural host manipulation and sexual mimicry. Here, we show that the host-specific and behaviourally manipulating pathogenic fungus, Entomophthora muscae, generates a chemical blend of volatile sesquiterpenes and alters the profile of natural host cuticular hydrocarbons in infected female housefly (Musca domestica) cadavers. Healthy male houseflies respond to the fungal compounds and are enticed into mating with female cadavers. This is advantageous for the fungus as close proximity between host individuals leads to an increased probability of infection. The fungus exploits the willingness of male flies to mate and benefits from altering the behaviour of uninfected male host flies. The altered cuticular hydrocarbons and emitted volatiles thus underlie the evolution of an extended phenotypic trait.

Role of the Housefly as a Biological Vector for Bacteria and Fungi at Some Slaughterhouses.

<b>Background and Objective:</b> The housefly, <i>Musca domestica</i> L. is the most widespread species of fly in the world. It is in close association with many human pathogens that which can cause serious and life-threatening diseases are known to be carried by house flies, including bacteria, viruses, fungi and parasites, Therefore, this study came intending to determine the role of house flies isolated from some slaughterhouses in the city of Al-Diwaniyah/Iraq in the spread of bacterial and fungal pathogens to humans. <b>Materials and Methods:</b> The 120 insects of house flies were collected randomly from some slaughterhouses for the period September to November, 2021 by special networks prepared for this purpose and transferred to the laboratory and then the bacteria and fungi were isolated and identified from the external surface of house flies by using appropriate culture media. <b>Results:</b> In this study, 148 bacterial isolates belonging to 15 species of bacteria were isolated and diagnosed from the external body of house flies that isolated, 91 samples had given positive growth of bacteria and <i>Escherichia coli</i> was the most occurrence and frequent from the other bacterial isolates, the occurrence rate of it was 16/91 (17.582%) with a frequency rate of 27/148 (18.243%). The 154 fungal isolates belonging to 16 species of fungi were isolated and diagnosed from the external body of house flies, 87 samples had given a positive growth of fungi and <i>Aspergillus niger</i> was the most occurrence and frequent from the other fungal isolates, as its occurrence rate reached 14/87 (16.091%) and the frequency rate of 21/154 (13.636%). <b>Conclusion:</b> House flies transmit many pathological microorganisms such as bacteria, fungi, viruses, etc., which causes various diseases for humans and animals and it may help the spread of antibiotic-resistant species and there is an urgent need to conduct many studies to know the new types of microorganisms that house flies can transmit.

Effects of habitat and sampling time on bacterial community composition and diversity in the gut of the female house fly, Musca domestica Linnaeus (Diptera: Muscidae).

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.

The Expansion of a Single Bacteriophage Leads to Bacterial Disturbance in Gut and Reduction of Larval Growth in Musca domestica.

The housefly larvae gut microbiota influences larval health and has become an important model to study the ecology and evolution of microbiota-host interactions. However, little is known about the phage community associated with the housefly larval gut, although bacteriophages are the most abundant members of the microbiota and have the potential to shape gut bacterial communities. Changes to bacteriophage composition are associated with disease, but how phages impact insect health remains unclear. We noticed that treating 1-day-old housefly larvae with ~107, ~109, and ~1011 phage particles per ml of bacteriophages led to changes in the growth and development of housefly larvae. Additionally, treating housefly larvae with bacteriophages led to bacterial composition changes in the gut. Changes in the compositions of these gut bacteria are mainly manifested in the increase in harmful bacteria, including Pseudomonas and Providencia and the decrease in beneficial bacteria, including Enterobacter and Klebsiella, after different growth and development periods. The alterations in gut microbiota further influenced the larval growth and development. Collectively, these results indicate that bacteriophages can perturb the intestinal microbiome and impact insect health.

Potential antitumor and anti-inflammatory activities of an extracellular polymeric substance (EPS) from Bacillus subtilis isolated from a housefly.

Bacillus subtilis, a probiotic, has been applied in the medical, food, and feed industries among others. However, the mechanisms of its benefits to hosts are not yet fully understood. Here the characterization and bioactivities of an extracellular polymeric substance (EPS) from Bacillus subtilis were investigated to reveal its partial mechanisms and provide the theoretical basics for further development and utilization of Bacillus subtilis. In this study, the novel strain Bacillus subtilis xztubd1 (GenBank: MG458322.1) was isolated from a housefly's body, identified according to phenotypical and genotypical analyses, and found to produce large amounts of an EPS. Through ultraviolet spectroscopy and Fourier transform infrared spectroscopy (FTIR spectroscopy), the EPS was found to contain a variety of chemical functional groups, such as O-H groups, C=C, C=O, CH3, C-O-H and C-O-C bonds, and alpha-type pyranose. Furthermore, the in vitro antioxidant activity of the EPS on DPPH radicals at a concentration of 90 µg/ml was 62%; on the superoxide radical at a concentration of 90 µg/ml, this value was 75%; and on hydroxyl radicals at a concentration of 90 µg/ml, the activity was 54%. EPS also enhanced significantly phagocytosis, lysozyme activity in macrophages, IL-2 content in mice and inhibited dramatically the growth of HeLa cells. These results showed that the EPS with reductive groups have the strong capacity to scavenge reactive oxygen species (ROS), reinforce the immune system and inhibit the growth of cancer cell, which helps theirs hosts defence against many diseases, including inflammation and cancer. The EPS from Bacillus subtilis has the potential to be an anticancer and anti-inflammatory drug candidate in the pharmaceutical industries, which provide scientific evidence for the development and utilization of probiotic-derived medicines.

Flies as a potential vector of selected alert pathogens in a hospital environment.

Multi-drug resistant pathogens are a global problem. Flies are a potential vector of multi-drug resistant pathogens, which can be particularly dangerous in the hospital environment. This study aimed to evaluate flies as vectors of alert pathogens. The research material consisted of 100 flies (Musca domestica (46.0%), Lucilia sericata (28.0%), and Calliphora vicina (26.0%)) collected at the University Hospital No. 1 dr. A. Jurasz in Bydgoszcz (Poland) in 2018-2019 (summer months). The presence of bacteria of the genera: Enterococcus, Staphylococcus, Escherichia, Leclercia, Citrobacter, Hafnia, Providencia, Proteus, Enterobacter, Klebsiella, Raoultella, Morganella, Moellerella, Bordetella, Pantoea, Serratia, Plesiomonas, Wohlfahrimonas, and Lelliottia was confirmed. The most frequently isolated species included: Enterococcus faecalis (n = 64), Escherichia coli (n = 43) and Moellerella wisconsensis (n = 24). The infection rate and antibiotic resistance of bacteria were assessed. One strain of Proteus mirabilis (isolated from Calliphora vicina) produced ESBLs (extended-spectrum beta-lactamases). The infection rate was 0.38%, 0.26%, and 0.20% for Musca domestica, Lucilia sericata, and Calliphora vicina, respectively. The flies from a hospital area were not a vector of alert pathogens. Monitoring flies as potential vectors of pathogens is an important aspect of public health, especially for hospitalized patients.

Enterobacter hormaechei in the intestines of housefly larvae promotes host growth by inhibiting harmful intestinal bacteria.

BACKGROUND: As a pervasive insect that transmits a variety of pathogens to humans and animals, the housefly has abundant and diverse microbial communities in its intestines. These gut microbes play an important role in the biology of insects and form a symbiotic relationship with the host insect. Alterations in the structure of the gut microbial community would affect larval development. Therefore, it is important to understand the mechanism regulating the influence of specific bacteria on the development of housefly larvae. METHODS: For this study we selected the intestinal symbiotic bacterium Enterobacter hormaechei, which is beneficial to the growth and development of housefly larvae, and used it as a probiotic supplement in larval feed. 16S rRNA gene sequencing technology was used to explore the effect of E. hormaechei on the intestinal flora of housefly larvae, and plate confrontation experiments were performed to study the interaction between E. hormaechei and intestinal microorganisms. RESULTS: The composition of the gut microflora of the larvae changed after the larvae were fed E. hormaechei, with the abundance of Pseudochrobactrum, Enterobacter and Vagococcus increasing and that of Klebsiella and Bacillus decreasing. Analysis of the structure and interaction of larval intestinal flora revealed that E. hormaechei inhibited the growth of harmful bacteria, such as Pseudomonas aeruginosa, Providencia stuartii and Providencia vermicola, and promoted the reproduction of beneficial bacteria. CONCLUSIONS: Our study has explored the influence of specific beneficial bacteria on the intestinal flora of houseflies. The results of this study reveal the important role played by specific beneficial bacteria on the development of housefly larvae and provide insight for the development of sustained biological agents for housefly control through interference of gut microbiota.

Persistence and Significance of Chlamydia trachomatis in the Housefly, Musca domestica L.

Trachoma is the leading cause of infectious blindness worldwide. Ocular infection by the obligate intracellular pathogen, Chlamydia trachomatis, causes the eyelashes to turn in and scratch the cornea, leading to blindness if left untreated. The disease is most prevalent in poor, rural communities that lack the infrastructure for basic hygiene, clean water, and proper sanitation. Infection is often spread through infected clothes, contaminated hands, and face seeking flies. The goal of this research was to understand the biological role of Musca domestica flies in the transmission of C. trachomatis. PCR, tissue culture, and immunofluorescence microscopy were used to determine the presence, viability, and the anatomical location of C. trachomatis within the digestive tract of M. domestica. Flies were fed with C. trachomatis and then harvested at various time intervals after feeding. The data confirmed the presence of C. trachomatis DNA and viable elementary bodies (EBs) in fly crops, up to 24 h postfeeding. C. trachomatis DNA was also isolated from the upper portions of the alimentary tract of flies up to 48 h postfeeding. In addition, DNA was isolated from the regurgitation material from fly crops up to 12 h postfeeding. The viability of isolated C. trachomatis EBs was repeatedly confirmed between 12 and 48 h and up to 7 days in ex vivo crops stored at room temperature. Our data suggest that eye-seeking flies such as M. domestica can ingest C. trachomatis during regular feeding. Because M. sorbens does not occur in continental United States, we did not use it in any of our studies. These data also confirm, for the first time, that ingested chlamydia remains viable inside the flies for 24-48 h postfeeding. We further show that these flies can regurgitate and transmit the trachoma agent at their next feeding. We believe that these findings reveal an opportunity for efficient intervention strategies through fly vector control, especially as we near new target date for global elimination of trachoma.

Bacteriophage: A Useful Tool for Studying Gut Bacteria Function of Housefly Larvae, Musca domestica.

Beneficial symbiotic bacteria have positive effects on some insects' (such as mosquitoes, cockroaches, and flies) biological activities. However, the effects of a lack of one specific symbiotic bacterium on the life activities of some insects and their natural gut microbiota composition remain unclear. Bacteriophages are viruses that specifically target and kill bacteria and have the potential to shape gut bacterial communities. In previous work, Pseudomonas aeruginosa that naturally colonized the intestines of housefly larvae was shown to be essential to protect housefly larvae from entomopathogenic fungal infections, leading us to test whether a deficiency in Pseudomonas aeruginosa strains in housefly larvae that was specifically caused using bacteriophages could remold the composition of the intestinal bacteria and affect the development of housefly larvae. Our research revealed that the phage, with a titer of 108 PFU/ml, can remove 90% of Pseudomonas aeruginosa in the gut. A single feeding of low-dose phage had no effect on the health of housefly larvae. However, the health of housefly larvae was affected by treatment with phage every 24 h. Additionally, treating housefly larvae with bacteriophages every 24 h led to bacterial composition changes in the gut. Collectively, the results revealed that deficiency in one symbiotic gut bacteria mediated by precise targeting using bacteriophages indirectly influences the intestinal microbial composition of housefly larvae and has negative effects on the development of the host insect. Our results indicated the important role of symbiotic gut bacteria in shaping the normal gut microbiota composition in insects. IMPORTANCE The well-balanced gut microbiota ensures appropriate development of the host insect, such as mosquitoes, cockroaches, and flies. Various intestinal symbiotic bacteria have different influences on the host gut community structure and thus exert different effects on host health. Therefore, it is of great importance to understand the contributions of one specific bacterial symbiont to the gut microbiota community structure and insect health. Bacteriophages that target certain bacteria are effective tools that can be used to analyze gut bacterial symbionts. However, experimental evidence for phage efficacy in regulating insect intestinal bacteria has been little reported. In this study, we used phages as precision tools to regulate a bacterial community and analyzed the influence on host health after certain bacteria were inhibited by bacteriophage. The ability of phages to target intestinal-specific bacteria in housefly larvae and reduce the levels of target bacteria makes them an effective tool for studying the function of gut bacteria.

PAP1 activates the prophenoloxidase system against bacterial infection in Musca domestica.

We previously identified three putative prophenoloxidase-activating proteinase (mdPAP1, mdPAP2, and mdPAP3) genes from housefly Musca domestica by transcriptomic analysis. In this study, mdPAP1 cDNA was cloned, and the function of its encoded protein was analyzed. The cDNA of mdPAP1 was 1358 bp, and it contained a single open reading frame of 1122 bp encoding a predicted MdPAP1 protein of 373 amino acids. The estimated molecular weight of MdPAP1 was 41267.08 Da with an isoelectric point of 6.25. The deduced amino acid sequence of MdPAP1 exhibited high similarity to known PAPs of insects. mdPAP1 was detected in larvae, pupae, and adult housefly, and the expression level of mdPAP1 was upregulated in bacterial challenged larvae. The recombinant protein of MdPAP1 expressed in Escherichia coli could cleave the prophenoloxidase into phenoloxidase in M. domestica hemolymph infected by bacteria and result in a significant increase of the total phenoloxidase activity. In addition, RNA interference-mediated gene silencing of mdPAP1 significantly increased the mortality of M. domestica larvae. Results indicated that mdPAP1 was involved in the activation of the prophenoloxidase against bacterial infection in M. domestica.

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

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

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.