Cross resistance to brevetoxin-3 by kdr and super-kdr mutations in house flies.

The dinoflagellate Karenia brevis is a causative agent of red tides in the Gulf of Mexico and generates a potent family of structurally related brevetoxins that act via the voltage-sensitive Na+ channel. This project was undertaken to better understand the neurotoxicology and kdr cross-resistance to brevetoxins in house flies by comparing the susceptible aabys strain to ALkdr (kdr) and JPskdr (super-kdr). When injected directly into the hemocoel, larvae exhibited rigid, non-convulsive paralysis consistent with prolongation of sodium channel currents, the known mechanism of action of brevetoxins. In neurophysiological studies, the firing frequency of susceptible larval house fly central nervous system preparations showed a > 200% increase 10 min after treatment with 1 nM brevetoxin-3. This neuroexcitation is consistent with the spastic paralytic response seen after hemocoel injections. Target site mutations in the voltage-sensitive sodium channel of house flies, known to confer knockdown resistance (kdr and super-kdr) against pyrethroids, attenuated the effect of brevetoxin-3 in baseline firing frequency and toxicity assays. The rank order of sensitivity to brevetoxin-3 in both assays was aabys > ALkdr > JPskdr. At the LD50 level, resistance ratios for the knockdown resistance strains were 6.9 for the double mutant (super-kdr) and 2.3 for the single mutant (kdr). The data suggest that knockdown resistance mutations may be one mechanism by which flies survive brevetoxin-3 exposure during red tide events.

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.

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.

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.

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.

Frequencies and distribution of kdr and Ace alleles that cause insecticide resistance in house flies in the United States.

House flies (Musca domestica L) are nuisances and vectors of pathogens between and among humans and livestock. Population suppression has been accomplished for decades with pyrethroids and acetylcholinesterase (AChE) inhibitors, but recurrent selection has led to increased frequency of alleles conferring resistance to those two classes of active ingredients (Geden et al., 2021). A common mechanism of resistance to both classes involves an altered target site (mutations in Voltage gated sodium channel (Vgsc) for pyrethroids or in Ace for AChE inhibitors). As part of ongoing efforts to understand the origin, spread and evolution of insecticide resistance alleles in house fly populations, we sampled flies in 11 different US states, sequenced, and then estimated frequencies of the Vgsc and Ace alleles. There was substantial variation in frequencies of the four common knockdown resistance alleles (kdr (L1014F), kdr-his (L1014H), super-kdr (M918T + L10414F) and 1B (T929I + L1014F) across the sampled states. The kdr allele was found in all 11 states and was the most common allele in four of them. The super-kdr allele was detected in only six collections, with the highest frequencies found in the north, northeast and central United States. The kdr-his allele was the most common allele in PA, NC, TN and TX. In addition, a novel super-kdr-like mutation in mutually exclusive exon 17a was found. The overall frequencies of the different Ace alleles, which we name based on the amino acid present at the mutation sites (V260L, A316S, G342A/V and F407Y), varied considerably between states. Five Ace alleles were identified: VAGF, VAVY, VAGY, VAAY and VSAY. Generally, the VSAY allele was the most common in the populations sampled. The susceptible allele (VAGF) was found in all populations, ranging in frequency from 3% (KS) to 41% (GA). Comparisons of these resistance allele frequencies with those previously found suggests a dynamic interaction between the different alleles, in terms of levels of resistance they confer and likely fitness costs they impose in the absence of insecticides.

Molecular analysis of acetylcholinesterase gene in field-collected populations of Musca domestica (Diptera: Muscidae) in Northwestern Iran.

Nowadays, pyrethroid (Py) insecticides are commonly used against household insect pests and housefly. The combination of Py and organophosphates (OP) are also utilized to combat these insects. The resistance status of Iranian housefly populations to them and carbamate (CB) insecticides is uncertain. This study investigates the presence of acetylcholinesterase (AChE) mutations related to the resistance of Musca domestica to OP and/or CB insecticides in Northwestern Iran. Nucleotides 1041-1776, based on their positions in the ACE gene of aabys strain, were amplified and sequenced in houseflies collected from West Azerbaijan, Gilan, and Ardebil Provinces, Iran. Among 12 single-nucleotide polymorphisms detected, 3 mismatches were found at nucleotides 1174 (T/A, G), 1473 (G/T, C), and 1668 (T/A), leading to amino acid substitutions in V260L, G342A/V, and F407Y positions with various combinations. Genotyping results showed that 85% of specimens had at least one of these substitutions. In addition, the Iranian housefly population was composed of 5 insensitive and sensitive alleles. For the first time, the current study reports the presence of V260L, G342A, G342V, and F407Y substitutions in M. domestica specimens collected from Northwestern Iran. The selection of multiple alleles in field populations might be due to the application of various pesticides/insecticides during extended periods in the region. These molecular levels signify the presence of control problems in the area and the need for developing effective control strategies for such populations.

First Identification of bla NDM-1 Producing Escherichia coli ST 9499 Isolated from Musca domestica in the Urban Center of Rio de Janeiro, Brazil.

The present study presents phenotypic and molecular characterization of a multidrug-resistant strain of Escherichia coli (Lemef26), belonging to sequence type ST9499 carrying a blaNDM-1 carbapenem resistance gene. The bacterium was isolated from a specimen of Musca domestica, collected in proximity to a hospital in Rio de Janeiro City, Brazil. The strain was identified as E. coli by matrix-assisted laser desorption-ionization time of flight mass spectrometry (Maldi-TOF-MS) and via genotypic analysis (Whole-Genome Sequencing-WGS), followed by phylogenetic analysis, antibiotic resistance profiling (using phenotypic and genotypic methods) and virulence genotyping. Interestingly, the blaNDM-1 was the only resistance determinant detected using a panel of common resistance genes, as evaluated by PCR. In contrast, WGS detected genes conferring resistance to aminoglycosides, fluoroquinolones, quinolones, trimethoprim, beta-lactams, chloramphenicol, macrolides, sulfonamide, tetracycline, lincosamide and streptogramin B. Conjugation experiments demonstrated the transfer of carbapenem resistance, via acquisition of the blaNDM-1 sequence, to a sensitive receptor strain of E. coli, indicating that blaNDM-1 is located on a conjugative plasmid (most likely of the IncA/C incompatibility group, in association with the transposon Tn3000). Phylogenetic analyses placed Lemef26 within a clade of strains exhibiting allelic and environment diversity, with the greatest level of relatedness recorded with a strain isolated from a human source suggesting a possible anthropogenic origin. Analysis of the virulome revealed the presence of fimbrial and pilus genes, including a CFA/I fimbriae (cfaABCDE), common pilus (ecpABCDER), laminin-bind fimbrae (elfADG), hemorrhagic pilus (hcpABC) and fimbrial adherence determinants (stjC) indicates the ability of strain Lemef26 to colonize animal hosts. To the best of our knowledge, this study represents the first report of blaNDM-1 carbapenemase gene in an E. coli strain isolated from M. domestica. In concordance with the findings of previous studies on the carriage of MDR bacteria by flies, the data presented herein provide support to the idea that flies may represent a convenient means (as sentinel animals) for the monitoring of environmental contamination with multidrug-resistant bacteria.

Serratia marcescens in the intestine of housefly larvae inhibits host growth by interfering with gut microbiota.

BACKGROUND: The structure of gut microbiota is highly complex. Insects have ubiquitous associations with intestinal symbiotic bacteria, which play essential roles. Thus, understanding how changes in the abundance of a single bacterium interfere with bacterial interactions in the insect's gut is important. METHODS: Here, we analyzed the effects of Serratia marcescens on the growth and development of housefly larvae using phage technology. We used 16S rRNA gene sequencing technology to explore dynamic diversity and variation in gut bacterial communities and performed plate confrontation assays to study the interaction between S. marcescens and intestinal microorganisms. Furthermore, we performed phenoloxidase activity assay, crawling assay, and trypan blue staining to explore the negative effects of S. marcescens on housefly larvae's humoral immunity, motility, and intestinal organization. RESULTS: The growth and development of housefly larvae were inhibited after feeding on S. marcescens, and their intestinal bacterial composition changed with increasing abundance of Providencia and decreasing abundance of Enterobacter and Klebsiella. Meanwhile, the depletion of S. marcescens by phages promoted the reproduction of beneficial bacteria. CONCLUSIONS: In our study, using phage as a tool to regulate the abundance of S. marcescens, we highlighted the mechanism by which S. marcescens inhibits the growth and development of housefly larvae and illustrated the importance of intestinal flora for larval development. Furthermore, by studying the dynamic diversity and variation in gut bacterial communities, we improved our understanding of the possible relationship between the gut microbiome and housefly larvae when houseflies are invaded by exogenous pathogenic bacteria.

Bacterial genome sequencing tracks the housefly-associated dispersal of fluoroquinolone- and cephalosporin-resistant Escherichia coli from a pig farm.

The regular use of antimicrobials in livestock production selects for antimicrobial resistance. The potential impact of this practice on human health needs to be studied in more detail, including the role of the environment for the persistence and transmission of antimicrobial-resistant bacteria. During an investigation of a pig farm and its surroundings in Brandenburg, Germany, we detected abundant cephalosporin- and fluoroquinolone-resistant Escherichia coli in pig faeces, sedimented dust, and house flies (Musca domestica). Genome sequencing of E. coli isolates revealed large phylogenetic diversity and plasmid-borne extended-spectrum beta lactamase (ESBL) genes CTX-M-1 in multiple strains. [Correction added on 28 February 2023, after first online publication: In the preceding sentence, 'and TEM-1' was previously included but has been deleted in this version.] Close genomic relationships indicated frequent transmission of antimicrobial-resistant E. coli between pigs from different herds and across buildings of the farm and suggested dust and flies as vectors for dissemination of faecal pathogens. Strikingly, we repeatedly recovered E. coli from flies collected up to 2 km away from the source, whose genome sequences were identical or closely related to those from pig faeces isolates, indicating the fly-associated transport of diverse ESBL-producing E. coli from the pig farm into urban habitation areas. The observed proximity of contaminated flies to human households poses a risk of transmission of antimicrobial-resistant enteric pathogens from livestock to man.

Multiple-P450 Gene Co-Up-Regulation in the Development of Permethrin Resistance in the House Fly, Musca domestica.

This paper reports a study conducted at the whole transcriptome level to characterize the P450 genes involved in the development of pyrethroid resistance, utilizing expression profile analyses of 86 cytochrome P450 genes in house fly strains with different levels of resistance to pyrethroids/permethrin. Interactions among the up-regulated P450 genes and possible regulatory factors in different autosomes were examined in house fly lines with different combinations of autosomes from a resistant house fly strain, ALHF. Eleven P450 genes that were significantly up-regulated, with levels > 2-fold those in the resistant ALHF house flies, were in CYP families 4 and 6 and located on autosomes 1, 3 and 5. The expression of these P450 genes was regulated by trans- and/or cis-acting factors, especially on autosomes 1 and 2. An in vivo functional study indicated that the up-regulated P450 genes also conferred permethrin resistance in Drosophila melanogaster transgenic lines. An in vitro functional study confirmed that the up-regulated P450 genes are able to metabolize not only cis- and trans-permethrin, but also two metabolites of permethrin, PBalc and PBald. In silico homology modeling and the molecular docking methodology further support the metabolic capacity of these P450s for permethrin and substrates. Taken together, the findings of this study highlight the important function of multi-up-regulated P450 genes in the development of insecticide resistance in house flies.

Metagenomic analysis of microbiological risk in bioaerosols during biowaste valorization using Musca domestica.

Bioconversion using insects has gradually become a promising technology for biowaste management and protein production. However, knowledge about microbiological risk of insect related bioaerosols is sparse and conventional methods failed to provide higher resolved information of environmental microbe. In this study, a metagenomic analysis including microorganisms, antibiotic resistance genes (ARGs), virulence factor genes (VFGs), mobile gene elements (MGEs), and endotoxin distribution in bioaerosols during biowaste conversion via Musca domestica revealed that bioaerosols in Fly rearing room possess the highest ARGs abundances and MGEs diversity. Through a metagenome-assembled genomes (MAGs)-based pipeline, compelling evidence of ARGs/VFGs host assignment and ARG-VFG co-occurrence pattern were provided from metagenomic perspective. Bioaerosols in Bioconversion and Maggot separation zone were identified to own high density of MAGs carrying both ARGs and VFGs. Bacteria in Proteobacteria, Actinobacteriota, and Firmicutes phyla were predominate hosts of ARGs and VFGs. Multidrug-Motility, Multidrug-Adherence, and Beta lactam-Motility pairs were the most common ARG-VFG co-occurrence pattern in this study. Results obtained are of great significance for microbiological risk assessment during housefly biowaste conversion process.

Accelerated degradation of cellulose in silkworm excrement by the interaction of housefly larvae and cellulose-degrading bacteria.

The environmental pollution caused by silkworm (Bombyx mori) excrement is prominent, and rich in refractory cellulose is the bottleneck restricting the efficient recycling of silkworm excrement. This study was performed to investigate the effects of housefly larvae vermicomposting on the biodegradation of cellulose in silkworm excrement. After six days, a 58.90% reduction of cellulose content in treatment groups was observed, which was significantly higher than 11.5% of the control groups without housefly larvae. Three cellulose-degrading bacterial strains were isolated from silkworm excrement, which were identified as Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis based on 16S rRNA gene sequence analysis. These three bacterial stains had a high cellulose degradation index (HC value ranged to between 1.86 and 5.97 and FPase ranged from 5.07 U/mL to 7.31 U/mL). It was found that housefly larvae increased the abundance of cellulose-degrading bacterial genus (Bacillus and Pseudomonas) by regulating the external environmental conditions (temperature and pH). Carbohydrate metabolism was the bacterial communities' primary function during vermicomposting based on the PICRUSt. The results of Tax4Fun indicated that the abundance of endo-ß-1,4-glucanase and exo-ß-1,4-glucanase increased rapidly and maintained at a higher level in silkworm excrement due to the addition of housefly larvae, which contributed to the accelerated degradation of cellulose in silkworm excrement. The finding of this investigation showed that housefly larvae can significantly accelerate the degradation of cellulose in silkworm excrement by increasing the abundance of cellulose-degrading bacterial genera and cellulase.

Functional characterization of CYP6G4 from the house fly in propoxur metabolism and resistance.

The house fly (Musca domestica L.) (Diptera: Muscidae) is a global vector that can transmit >250 human and animal diseases. The control of house flies has heavily relied on the application of various chemical insecticides. The carbamate insecticide propoxur has been widely used for the control of house flies, and resistance to propoxur has been documented in many house fly populations worldwide. Previous studies have identified several propoxur resistance-conferring mutations in the target protein acetylcholinesterase; however, the molecular basis for metabolic resistance to propoxur remains unknown. In this study, we investigated the involvement of CYP6G4, a cytochrome P450 overexpressed in many insecticide resistant populations of Musca domestica, in propoxur metabolism and resistance by using combined approaches of recombinant protein-based insecticide metabolism and the Drosophila GAL4/UAS transgenic system. The recombinant CYP6G4 and its redox partners (NADPH-dependent cytochrome P450 reductase and cytochrome b5) were functionally expressed in Escherichia coli. Metabolism experiments showed that CYP6G4 was able to transform propoxur with a turnover rate of around 0.79 min-1. Six metabolites were putatively identified, suggesting that CYP6G4 could metabolize propoxur via hydroxylation, O-depropylation and N-demethylation. Moreover, bioassay results showed that ectopic overexpression of CYP6G4 in fruit flies significantly increased their tolerance to propoxur. Our in vivo and in vitro data convincingly demonstrate that CYP6G4 contributes to propoxur metabolism and resistance.

ERIC-PCR-based molecular typing of multidrug-resistant Escherichia coli isolated from houseflies (Musca domestica) in the environment of milk and meat shops.

The emergence and spread of antimicrobial resistance have become a major global public health concern. A component of this problem is the spread of antibiotic-resistant bacteria. Flies move freely between habitats of food-producing animals and human beings and thus have great potential for dissemination of antimicrobial-resistant bacteria from a contaminated environment to milk and meat markets, posing potential hazards for consumers. During the present study, a total of 150 houseflies were captured from milk and meat shops located in Durg and Raipur city of Chhattisgarh, India. The Escherichia coli were isolated from houseflies and characterized on the basis of cultural and molecular tests. Further, the isolates were subjected to antimicrobial susceptibility testing against frequently used antibiotics using the disk diffusion method. The antibiotic resistance genes and int1 gene were detected using polymerase chain reaction (PCR). A total of 45 E. coli isolates were obtained from the fly samples with an overall prevalence rate of 30·0%. Antibiogram results confirmed that E. coli isolates were resistant to multiple antibiotics. Out of the (45) isolates of E. coli, 17 (37·8%) isolates were extended-spectrum beta-lactamase (ESBL) producer and multi-drug-resistant (MDR). Out of the ESBL and MDR E. coli isolates, blaCTX-M (24·4%), blaTEM (11·1%), tetA (28·8%), tetB (26·7%), gyrA (26·7%), parC (31. 1%) and int1 genes (15·5%) were detected but none of the isolates were found positive for blaSHV gene. Findings of the present study confirm that MDR E. coli are widely distributed in houseflies and play an important role in the transmission of antibiotic-resistant bacteria from contaminated environments to milk and meat shop environment.

Constitutively-expressed and induced immune effectors in the house fly (Musca domestica) and the transcription factors that may regulate them.

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.

Risk assessment of resistance to diflubenzuron in Musca domestica: Realized heritability and cross-resistance to fourteen insecticides from different classes.

The Musca domestica L. is a well-known vector for a number of livestock and human diseases. One major challenge for maintaining effective control of this pest is its propensity to develop resistance to insecticides. This study utilized laboratory selection and realized heritability methods to examine the risk of resistance development to diflubenzuron in Musca domestica L. Cross-resistance (CR) to fourteen other insecticides was measured in diflubenzuron-selected (Diflu-SEL) strain which was selected for 20 generations. The resistance ratio (RR) of Diflu-SEL larvae to diflubenzuron increased from 30.33 in generation five (G5) to 182.33 in G24 compared with the susceptible strain, while realized heritability (h2) was 0.08. The number of needed generations (G) for a tenfold increase in the median lethal concentration (LC50) for diflubenzuron ranged from 4 to 45 at h2 values of 0.08, 0.18, and 0.28, at a slope of 1.51. At h2 = 0.08 and slopes of 1.51, 2.51, and 3.51, the number of needed G for a tenfold increase in the LC50 ranged from 9 to 104. The level of CR shown by the Diflu-SEL strain to all other fourteen tested insecticides (insect growth regulators, organophosphates, and pyrethroids) was either absent or very low compared to the field population. The value of h2 and the absent or low CR indicate potential successful management of resistance to diflubenzuron and recommend the use of the tested insecticides in rotation with diflubenzuron to control M. domestica.