Molecular detection and genotyping of Dientamoeba fragilis and Blastocystis sp. in housefly Musca domestica (Diptera: Muscidae): first report for Dientamoeba fragilis.

Dientamoeba fragilis and Blastocystis sp. are single-celled protozoan parasites of humans and animals. Although they are found in the intestines of healthy hosts, the pathogenicity of them is still unclear. To date, there is no report on D. fragilis and only two studies (without subtyping) on the occurrence of Blastocystis sp. in Musca domestica. In this study, fly samples were collected from livestock farms and their surroundings in the Kirsehir province (Central Anatolia Region) of Türkiye from May to August 2023. A total of 150 microscopically identified M. domestica samples were analyzed for the detection of D. fragilis and Blastocystis sp. molecularly. The overall prevalence of Blastocystis sp. and D. fragilis in M. domestica was determined to be 3.3% (5/150) and 8.0% (12/150), respectively. The SSU rRNA gene sequences of the isolates indicated genotype 1 of D. fragilis. Eleven isolates were identical and represented a single isolate (KAU-Dfrag1). BLAST analysis of KAU-Dfrag1 indicated identity with the isolates reported from humans, cattle, sheep, and budgerigars. The other isolate (KAU-Dfrag2) was polymorphic at two nucleotides from KAU-Dfrag1 and three nucleotides from known genotypes from GenBank and represented a variant of genotype 1. The Blastocystis sp. isolates were found to be identical and represent a single genotype (KAU-Blast1). BLAST analysis revealed that the KAU-Blast1 genotype belonged to the potentially zoonotic subtype 5 (ST5) and exhibited the highest genetic identity (ranging from 99.4 to 99.6%) with pigs, cattle, and sheep from different countries. Our study provides the first data on the molecular prevalence, epidemiology, and genotypic characterization of D. fragilis and Blastocystis sp. in M. domestica.

Long-term impact of rearing substrates on susceptibility to insecticides and metabolic enzyme activities in the house fly Musca domestica.

Musca domestica Linnaeus is a devastating insect pest of medical and veterinary importance with reports of resistance development to commonly used insecticides worldwide. Rearing substrates usually play a crucial role in determining susceptibility to insecticides and control of insect pests. The aim of the present study was to investigate the effect of five rearing substrates of M. domestica on its susceptibility to different insecticides and activities of metabolic enzymes. After 30 generations of rearing, susceptibility of M. domestica to tested insecticides, viz., malathion, pirimiphos-methyl, alpha-cypermethrin, deltamethrin, methomyl, propoxur, spinetoram, and chlorfenapyr had evident differences. Musca domestica reared on hen liver exhibited reduced susceptibility to all insecticides followed by the strain reared on poultry manure. However, M. domestica reared on milk-based diet showed the highest susceptibility to tested insecticides followed by the strain reared on manures of buffalo and horse. In addition, M. domestica reared on different substrates exhibited significant differences (p < 0.01) in the activities of glutathione S-transferase (GST), cytochrome P450-dependent monooxygenase, and carboxylesterase (CarE). Overall, hen liver and poultry manure strains exhibited higher activities of metabolic enzymes than those of the milk-based diet, buffalo, and horse manure strains. In conclusion, the data of the present study exhibited a significant effect of rearing substrates on the susceptibility to insecticides and activities of metabolic enzymes in M. domestica. These results could be helpful for the sustainable management of M. domestica on different hosts by selecting appropriate insecticides.

Transgenerational effects of pyriproxyfen in a field strain of Musca domestica L. (Diptera: Muscidae).

Musca domestica L. (Muscidae: Diptera) is a human and livestock pest especially in tropical and sub-tropical areas. Different insecticides have been used to control this pest that pose serious harmful effects on humans and the environment. The current study was planned to investigate the effects of two concentrations (LC25 and LC50) of pyriproxyfen on biological and population parameters of a field strain of M. domestica. The exposed parents (F0) and their progeny (F1) were studied to examine the transgenerational effects. The results indicated that preadult duration was higher in control (13.68 days) compared to LC50 treated individuals (12.44 days). The male and female longevity was relatively lower in the LC25 treated population i.e. 24.62 and 26.62 days, respectively. The adult pre-oviposition period (APOP) and total pre-oviposition period (TPOP) values were higher in the LC25 treated individuals than those of control. Moreover, oviposition days and fecundity were reduced in the treated individuals as compared to the control treatment. A gradual decrease in the net reproductive rate (R0) was observed (8.46-14.07 per day) while the value of R0 was significantly higher in control. The results suggested that pyriproxyfen can be effectively utilized and incorporated in the management programs of M. domestica.

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.

Resistance risk assessment, cross-resistance potential and realized heritability of resistance to methomyl in Musca domestica Linnaeus.

The use of insecticides in agricultural settings often exerts negative effects on nontarget species. Methomyl, a broad-spectrum carbamate insecticide, is recommended to manage a number of insect pests of the cotton crop. Recently, Musca domestica, which is a nontarget insect species in cotton fields, has shown resistance to methomyl in Pakistan. The present study tried to assess resistance-risk assessment, rapidity of resistance development to methomyl, cross-resistance potential to other insecticides, resistance heritability and to forecast the projected rate of resistance development under field conditions. For this purpose, a field strain of M. domestica with 186 fold resistance to methomyl was re-selected in the laboratory for eight consecutive generations. Consequently, LD50 values increased rapidly (126.64 ng/fly to 3112.79 ng/fly) compared to those before selection experiments. Similarly, RR values increased from 186 to 3113 fold as a result of the selection process. However, resistance to methomyl did not remain stable when the selected strain (Meth-SEL) reared for the next five generations in a pesticide free environment. The Meth-SEL strain also developed cross-resistance to permethrin. The realized heritability (h2) value for the Meth-SEL strain was 0.39 with 27% average mortality of M. domestica. Assuming the standard deviation (σp) value 0.27 and the h2 value 0.39 for eight generations of continuous exposure to methomyl, then five, seven, eight, ten and twelve generations at 90, 80, 70, 60 and 50% selection intensity, respectively, would be required for a tenfold increase in the LD50 value of methomyl. In conclusion, the Meth-SEL strain of M. domestica exhibited a high risk of resistance development to methomyl under continuous selection pressure. Resistance increased rapidly during selection experiments that reflect the probability of resistance development under field conditions if M. domestica receive exposures to methomyl during its applications for the management of target pest species.

kdr mutations and deltamethrin resistance in house flies in Abu Dhabi, UAE.

BACKGROUND: The house fly, Musca domestica, is a significant carrier of diseases that can impact public health. Repeated use of pyrethroid insecticides may act as a selection pressure for mutations and amino acid substitutions in the house fly voltage-sensitive sodium channel (VSSC), which ultimately confers resistance. The objectives of this study were to determine the presence of knockdown resistance (kdr) mutations using molecular tools and to set up a CDC bottle bioassay specific for house flies in the United Arab Emirates (UAE) to screen for deltamethrin resistance. METHODS: Adult flies were collected from 19 locations in Abu Dhabi, UAE, and DNA was extracted, followed by PCR amplification of specific alleles (PASA) and conventional PCR using several primers to amplify regions of the VSSC gene. Sanger sequencing was performed on PCR products. We also designed primers that detect four kdr mutations using complementary DNA (cDNA) in reverse transcriptase (RT)-PCR followed by Sanger sequencing. Additionally, a CDC bottle bioassay was set up for detecting deltamethrin resistance in adult house flies. RESULTS: In PASA, the primers successfully amplified the target bands (480, 280 and 200 bp). The kdr allele was found in flies collected from 18 of the 19 locations, at the highest and lowest prevalence of 46.9% and 9.4%, respectively. Resistant homozygous (RR) insects constituted 5.0% of the tested populations, and heterozygous (RS) insects accounted for 36.5%. The RR genotype was prevalent in house flies collected at 10 of 19 sampling locations. House fly populations were mostly in Hardy-Weinberg equilibrium, except in three locations. In addition to verifying the presence of the previously identified kdr mutation L1014F, in this study we detected two kdr mutations, L1014H and T929I, that have not previously been reported in the UAE. Also, for the first time in the UAE, a CDC bottle bioassay for deltamethrin resistance was used, which found that 60 min and 4.5 µg/ml were the diagnostic time and dose, respectively. Using this assay, we detected deltamethrin resistance in house flies from two of 16 locations, with a resistance level of 12.5%. CONCLUSIONS: Using DNA sequencing, we confirmed the presence of a known kdr mutation and uncovered two new kdr mutations in house flies from Abu Dhabi. Additionally, we detected deltamethrin resistance in these flies using a CDC bottle bioassay. Further research is recommended to comprehensively identify more kdr mutations in UAE house fly populations and assess their impacts on control strategies.

Application of bacteria and bacteriophage cocktails for biological control of houseflies.

BACKGROUND: Houseflies, Musca domestica L., are an ubiquitous pest that can transmit numerous diseases and threaten human health. Increasing insecticide resistance shown by houseflies necessitates the develop new control alternatives. The housefly gut is densely colonized with microorganisms that interact with each other dynamically and benefit the host's health. However, the impact of multiple symbiotic bacteria on the composition of housefly gut microbiota and the host's activities remains unclear. METHODS: We isolated and cultured 12 bacterial species from the intestines of housefly larvae. We also isolated seven bacteriophages to precisely target the regulation of certain bacterial species. Using 16S rRNA high-throughput gene sequencing, we analyzed the bacterial diversity after orally administering bacteria/phage cocktails to houseflies. RESULTS: Our results showed that larval growth was promoted, the abundance of beneficial bacteria, such as Klebsiella and Enterobacter, was increased and the abundance of harmful bacteria, such as Providencia, Morganella and Pseudomonas, was decreased in housefly larvae fed with the beneficial bacteria cocktail. However, oral administration of both beneficial and harmful bacterial phage cocktails inhibited larval growth, probably due to the drastic alteration of gut flora. Untargeted metabolomics using liquid chromatography-mass spectrometry showed that disturbances in gut microbiota changed the larval metabolite profiles. Feeding experiments revealed that disrupting the intestinal flora suppressed the beneficial bacteria and increased the harmful bacteria, causing changes in the metabolites and inhibiting larval growth. CONCLUSIONS: Based on our results, bacteria/phage cocktails are effective tools for regulating the intestinal flora of insects and have a high potential as a biological control agent for incorporation into an integrated pest management program.

Genetics, genomics and mechanisms responsible for high levels of pyrethroid resistance in Musca domestica.

Insecticide resistance is both economically important and evolutionarily interesting phenomenon. Identification of the mutations responsible for resistance allows for highly sensitive resistance monitoring and allows tools to study the forces (population genetics, fitness costs, etc.) that shape the evolution of resistance. Genes coding for insecticide targets have many well-characterized mutations, but the mutations responsible for enhanced detoxification have proven difficult to identify. We employed multiple strategies to identify the mutations responsible for the extraordinarily high permethrin resistance in the KS17-R strain of house fly (Musca domestica): insecticide synergist assays, linkage analysis, bulk segregant analyses (BSA), transcriptomics and long read DNA (Nanopore) sequencing. The >85,100-fold resistance in KS17-R was partially suppressed by the insecticide synergists piperonyl butoxide and S,S,S-tributylphosphorothionate, but not by diethyl maleate nor by injection. This suggests the involvement of target site insensitivity, CYP-mediated resistance, possibly hydrolase mediated resistance and potentially other unknown factors. Linkage analysis identified chromosomes 1, 2, 3 and 5 as having a role in resistance. BSA mapped resistance loci on chromosomes 3 and 5. The locus on chromosome 3 was centered on the voltage sensitive sodium channel. The locus on chromosome 5 was associated with a duplication of multiple detoxification genes. Transcriptomic analyses and long read DNA sequencing revealed overexpressed CYPs and esterases and identified a complex set of structural variants at the chromosome 5 locus.

Persistence of Marine Bacterial Plasmid in the House Fly (Musca domestica): Marine-Derived Antimicrobial Resistance Genes Have a Chance of Invading the Human Environment.

The house fly is known to be a vector of antibiotic-resistant bacteria (ARB) in animal farms. It is also possible that the house fly contributes to the spread of ARB and antibiotic resistance genes (ARGs) among various environments. We hypothesized that ARB and ARGs present in marine fish and fishery food may gain access to humans via the house fly. We show herein that pAQU1, a marine bacterial ARG-bearing plasmid, persists in the house fly intestine for 5 days after fly ingestion of marine bacteria. In the case of Escherichia coli bearing the same plasmid, the persistence period exceeded 7 days. This interval is sufficient for transmission to human environments, meaning that the house fly is capable of serving as a vector of marine-derived ARGs. Time course monitoring of the house fly intestinal microflora showed that the initial microflora was occupied abundantly with Enterobacteriaceae. Experimentally ingested bacteria dominated the intestinal environment immediately following ingestion; however, after 72 h, the intestinal microflora recovered to resemble that observed at baseline, when diverse genera of Enterobacteriaceae were seen. Given that pAQU1 in marine bacteria and E. coli were detected in fly excrement (defined here as any combination of feces and regurgitated material) at 7 days post-bacterial ingestion, we hypothesize that the house fly may serve as a vector for transmission of ARGs from marine items and fish to humans via contamination with fly excrement.

Lack of fitness costs associated with resistance to permethrin in Musca domestica.

Resistance to permethrin has been reported in Pakistani strains of Musca domestica. The present study explored the performance of biological traits and analyzed life tables to determine whether there is any detrimental effect of permethrin resistance on the fitness of permethrin-resistant strains [an isogenic resistant strain (Perm-R) and a field strain (Perm-F)] compared to a susceptible strain (Perm-S). Perm-R and Perm-F exhibited 233.93- and 6.87-fold resistance to permethrin, respectively. Life table analyses revealed that the Perm-R strain had a significantly shorter preadult duration, longer longevity, shorter preoviposition period, higher fecundity, finite rate of increase, intrinsic rate of increase, net reproductive rate and a shorter mean generation time, followed by the Perm-F strain when compared to the Perm-S strain. Data of the performance of biological traits reveled that permethrin resistance strains had a better fit than that of the Perm-S strain. The enhanced fitness of resistant strains of M. domestica may accelerate resistance development to permethrin and other pyrethroids in Pakistan. Some possible measures to manage M. domestica and permethrin resistance in situations of fitness advantage are discussed.

Evaluation of the stability of physiological and behavioral resistance to imidacloprid in the house fly (Musca domestica L.) (Diptera: Muscidae).

BACKGROUND: The house fly (Musca domestica L.) is a synanthropic fly species commonly associated with confined animal facilities. House fly control relies heavily on insecticide use. Neonicotinoids are currently the most widely used class of insecticide and have been formulated into granular fly baits since 2002. Physiological resistance to imidacloprid in house flies has been observed to be unstable and decline over time without continual selection pressure, indicating that resistance has a fitness cost to individuals in the absence of exposure to insecticides. The stability of behavioral resistance to imidacloprid in the house fly has not been evaluated. In the current study, we assess the stability of physiological and behavioral resistance in house flies to imidacloprid over time. RESULTS: Physiological susceptibility to imidacloprid varied significantly among three house fly strains examined, with WT-15 exhibiting the greatest susceptibility to imidacloprid with an LC50 and LC95 of 109.29 (95.96-124.49) µg g-1 and 1486.95 (1097.15-2015.23) µg g-1 , respectively. No significant differences in survival were observed across 30 generations of a house fly strain (BRS-1) previously selected for behavioral resistance to imidacloprid with percentage survival ranging from 93.20% at F0 in 2020 to 96.20% survival at F30 in 2022. CONCLUSION: These results have significant implications for the management of house flies exhibiting behavioral resistance in field settings. It appears that standard resistance management tactics deployed to reduce the prevalence of physiological resistance, such as rotating or temporarily discontinuing the use of specific insecticides, may not lead to reduced behavioral resistance to imidacloprid. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Necrophagous flies as bioindicators in Cd and Zn co-contaminated areas of Tak Province, Thailand.

Necrophagous flies may be effective bioindicators of chemical substances within polluted locations, as they are sensitive to environmental changes, have large populations, and thrive in a single location over their lifespan. Diversity and abundance of necrophagous flies were determined at livestock farms contaminated with potentially toxic elements (PTEs) in Tak Province and Nakhon Sawan Province, Thailand. Substantial soil zinc (Zn) concentrations (> 1100 mg kg-1) were detected at a cattle farm at Khaothong, Nakhon Sawan Province, and soil cadmium (Cd) values were significantly elevated (> 3 mg kg-1) at a cattle farm in Pha De, Tak Province. Anthropogenic inputs including Zn mining, domestic wastewater, and certain materials used in local agriculture were point sources of PTEs at the livestock farms in the Pha De and Khaothong subdistricts. Lower temperatures and humidity during the rainy season may have resulted in increased numbers of necrophagous flies, which was 1.5 times greater compared to the dry season. However, the dry season exhibited a higher PTE buildup in fly tissue. The order of important value index (IVI) values of the necrophagous flies were: Chrysomya megacephala (56.80), Musca domestica (27.21), C. rufifacies (25.40) and Sarcophaga spp. (17.54), respectively. These necrophagous flies may play a significant role in PTE-contaminated ecosystems based on their high IVI values, suggesting that they could be used as bioindicators of PTEs. Principal component analysis (PCA) results for necrophagous flies associated with each sampling site during the dry season were consistent with flies having substantial IVI values. Musca domestica and C. megacephala of both sexes displayed substantial correlations with Cr, Al, and Mn, while females of Sarcophaga spp. displayed strong associations with Cd. At the cattle farm in Khaothong, males of M. domestica showed a significant relationship with Zn, Cu, Pb, and Ni. When considering PTE accumulation capacity in flies commonly found at field sites, C. megacephala and M. domestica are the most suitable bioindicators of PTEs. This study confirms that necrophagous flies serve as reliable bioindicators of PTE pollution.

Mechanistic insights into the selectivity of bicyclophosphorothionate antagonists for housefly versus rat GABA receptors.

BACKGROUND: γ-Aminobutyric acid (GABA) receptors (GABARs) are validated targets of insecticides. Bicyclophosphorothionates are a group of insecticidal compounds that act as noncompetitive antagonists of GABARs. We previously reported that the analogs exhibit various degrees of selectivity for housefly versus rat GABARs, depending on substitutions at the 3- and 4-positions. We here sought to elucidate the unsolved mechanisms of the receptor selectivity using quantitative structure-activity relationship (QSAR), molecular docking, and molecular dynamics approaches. RESULTS: Three-dimensional (3D)-QSAR studies using Topomer comparative molecular field analysis quantitatively demonstrated how the introduction of a small alkyl group at the 3-position of bicyclophosphorothionates contributes to the housefly versus rat GABAR selectivity. To investigate the molecular mechanisms of the selective action, bicyclophosphorothionates were docked into housefly Resistance to dieldrin (RDL) GABAR and rat α1ß2γ2 GABAR homology models built using the published 3D-structures of human GABARs as templates. The results of molecular docking and molecular dynamics simulations revealed that the 2'Ala and 6'Thr residues of the RDL subunit within the channel are the key amino acids for binding to the housefly GABARs, whereas the 2'Ser residue of γ2 subunit plays a crucial role in binding to rat GABARs. CONCLUSION: We revealed the molecular mechanisms underlying the selective antagonistic action of bicyclophosphorothionates on housefly versus rat GABARs. The information presented should help design and develop novel, safe GABAR-targeting insecticides. © 2023 Society of Chemical Industry.

Characterization of intrapuparial development of Musca domestica (Diptera: Muscidae), under different temperatures in laboratory conditions.

Musca domestica (Linnaeus, 1758) (Diptera: Muscidae), popularly known as "housefly", is a highly synanthropic species, with economic, medical-sanitary, veterinary, and forensic importance. It is able to cause damage to health, transmit pathogenic agents, cause infection in domestic animals, and, in its immature stage, cause secondary myiasis. The scavenging habit of its immature stages makes these flies pioneers in colonizing both human and animal carcasses, from the initial stages of corpse decomposition to the final stages. Intrapuparial development studies of all stages of the biological cycle of these insects help estimate pupal age, being useful to forensic entomology to aid in determining the minimum postmortem interval (minPMI). This study describes, morphologically, the external structures of the pupae, under temperatures of 23, 27, and 30 ±â€…1 °C aiming to identify the characteristics that define their developmental stages and estimation of the pupae age of M. domestica. The whole experiment was carried out under laboratory conditions, with relative humidity 60 ±â€…10% and 12 hours of photoperiod. The process of pupariation and pupation including pre-pupae phases were observed; larvae pupae apolysis; early cryptocephalic pupae; late cryptocephalic pupae; phanerocephalic pupae; pharate adult, discriminated by eye color (transparent eyes, pink eyes, and red eyes); and the emergency of adults, which occurred in the intervals of 162-180; 138-144, and 96-102 hr, respectively, being described throughout the metamorphosis of the external morphological characteristics of the pupal stage of M. domestica.

Material flow analysis and global warming potential assessment of an industrial insect-based bioconversion plant using housefly larvae.

The significant increase in the demand for biomass waste treatment after garbage classification has led to housefly larvae treatment becoming an attractive treatment option. It can provide a source of protein while treating biomass waste, which means that nutrients can be returned to the natural food chain. However, the performance of this technology in terms of its environmental impacts is still unclear, particularly with regards to global warming potential (GWP).This study used a life cycle assessment (LCA) approach to assess a housefly larvae treatment plant with a treatment capacity of 50 tons of biomass waste per day. The LCA results showed that the 95% confidence intervals for the GWP in summer and winter were determined to be 24.46-32.81 kg CO2 equivalent (CO2-eq)/ton biomass waste and 5.37-10.08 kg CO2-eq/ton biomass waste, respectively. The greater GWP value in summer is due to the longer ventilation time and higher ventilation intensity in summer, which consumes more power. The main GWP contributions are from (1) electricity needs (accounting for 78.6% of emissions in summer and 70.2% in winter) and (2) product substitution by mature housefly larvae and compost (both summer and winter accounting for 96.8% of carbon reduction).

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.

Preparation of housefly (Musca domestica) larvae protein hydrolysates: Influence of dual-sweeping-frequency ultrasound-assisted enzymatic hydrolysis on yield, antioxidative activity, functional and structural attributes.

Dual-sweeping-frequency ultrasound (DSFU) was utilized in the preparation of polypeptides from housefly (Musca domestica) larvae protein (HLP). Results indicated that ultrasonication (20 ± 2/28 ± 2 kHz, 42 W/L, 25 min) significantly increased peptide yield and DPPH scavenging capacity by 8.25 % and 14.83 %, respectively. Solubility, foaming and emulsification properties of polypeptides were improved by 19.89 %, 33.33 % and 38.74 % over the control; along with notable reduction in particle size and increase in zeta potential. Tertiary structural changes of the sonicated hydrolysates were illustrated by UV and fluorescence spectra. FTIR showed that ultrasonication increased α-helix, ß-turn, and random coil by 38.23 %, 46.35 % and 16.36 %, respectively, but decreased ß-sheet by 48.03 %, indicating partial unfolding in HLP hydrolysate conformation and reduction in intermolecular interactions. The research results demonstrated that dual-sweeping-frequency ultrasonication has a great prospect in industry application for the purpose of improving enzymolysis efficiency and product quality for housefly larvae protein hydrolysates production.

Function analysis and characterisation of a novel chitinase, MdCht9, in Musca domestica.

Insect chitinases have been proposed as potential targets for pest control. In this work, a novel group IV chitinase gene, MdCht9, from Musca domestica was found to have multiple functions in the physiological activity, including chitin regulation, development and antifungal immunity. The MdCht9 gene was cloned and sequenced, its phylogeny was analysed and its expression was determined in normal and 20E treated larvae. Subsequently, RNA interference (RNAi)-mediated MdCht9 knockdown was performed, followed by biochemical assays, morphological observations and transcriptome analysis. Finally, the recombinant protein MdCht9 (rMdCht9) was purified and tested for anti-microbial activity and enzyme characteristics. The results showed that MdCht9 consists of three domains, highly expressed in a larval salivary gland. RNAi silencing of MdCht9 resulted in significant down-regulation of chitin content and expression of 15 chitin-binding protein (CBP) genes, implying a new insight that MdCht9 might regulate chitin content by influencing the expression of CBPs. In addition, more than half of the lethality and partial wing deformity appeared due to the dsMdCht9 treatment. In addition, the rMdCht9 exhibited anti-microbial activity towards Candida albicans (fungus) but not towards Escherichia coli (G-) or Staphylococcus aureus (G+). Our work expands on previous studies of chitinase while providing a potential target for pest management.

Klebsiella pneumoniae in the intestines of Musca domestica larvae can assist the host in antagonizing the poisoning of the heavy metal copper.

BACKGROUND: Musca domestica larvae are common saprophytes in nature, promoting the material-energy cycle in the environment. However, heavy metal pollution in the environment negatively affects their function in material circulation. Our previous research found that some intestinal bacteria play an important role in the development of housefly, but the responses of microbial community to heavy metal stresses in Musca domestica is less studied. RESULTS: In this study, CuSO4, CuSO4-Klebsiella pneumoniae mixture and CuSO4-K. pneumoniae phage mixture were added to the larval diet to analyze whether K. pneumoniae can protect housefly larvae against Cu2+ injury. Our results showed that larval development was inhibited when were fed with CuSO4, the bacterial abundance of Providencia in the intestine of larvae increased. However, the inhibition effects of CuSO4 was relieved when K. pneumoniae mixed and added in larval diets, the abundance of Providencia decreased. Electron microscope results revealed that K. pneumoniae showed an obvious adsorption effect on copper ion in vitro. CONCLUSIONS: Based on the results we assume that K. pneumoniae could adsorb Cu2+, reduce Cu2+ impact on gut community structure. Our study explains the role of K. pneumoniae antagonizing Cu2+, which could be applied as a probiotic to saprophytic bioantagonistic metal contamination.

Performance evaluation of protocols for Taenia saginata and Ascaris suum egg recovery from the house fly's gastrointestinal tract and exoskeleton.

BACKGROUND: The synanthropic house fly (Musca domestica) can potentially contribute to the mechanical spread of eggs of Taenia and Ascaris spp. in the environment and between hosts. However, the absence of validated protocols to recover eggs hampers an in-depth analysis of the house fly's role in parasite egg transmission. METHODS: The gastrointestinal tract and exoskeleton of euthanized house flies were spiked with Taenia saginata eggs. The performance of several recovery protocols, in terms of both the recovery rate and ease-of-use, was (microscopically) evaluated and compared. These protocols employed steps such as washing, maceration, filtration, flotation and both passive and centrifugal sedimentation. The final validated protocols were subsequently evaluated for the recovery of Ascaris suum eggs. RESULTS: The final protocol validated for the recovery of T. saginata eggs from the house fly's gastrointestinal tract involved homogenization in phosphate-buffered saline and centrifugation at 2000 g for 2 min, yielding a recovery rate of 79.7%. This protocol required 6.5 min to perform (which included 1.5 min of hands-on time) and removed large debris particles that could hinder the differentiation of eggs from debris. Similarly, the final protocol validated for the recovery of T. saginata eggs from the fly's exoskeleton involved washing by vortexing for 2 min in Tween 80 (0.05%), 15 min of passive sedimentation and centrifugation at 2000 g for 2 min, yielding a recovery rate of 77.4%. This protocol required 20.5 min to perform (which included 3.5 min of hands-on time) and successfully removed debris. The same protocols yielded recovery rates of 74.2% and 91.5% for the recovery of A. suum eggs from the fly's gastrointestinal tract and exoskeleton, respectively. CONCLUSIONS: Effective, simple and easy-to-use protocols were developed and validated for the recovery of T. saginata and A. suum eggs from the house fly's gastrointestinal tract and exoskeleton. These protocols can be applied to investigate the importance of flies as parasite egg transmitters in laboratory and field settings.