Response Surface Methodology (RSM) for Optimizing Protein Extraction from Housefly (Musca domestica) Larvae Fed with Toad and Its Structural Characterization.

With the global population on the rise, an escalating interest exists in environmentally sustainable and friendly protein sources. Insects have emerged as multifaceted resources, viewed not only as potential food items, but also as sources of traditional medicines and proteins. This study utilized response surface methodology (RSM) to ascertain the optimal extraction conditions for proteins from Musca domestica used in toad feeding, denoted as MDPs-T. The yield of MDPs-T was elevated to 18.3% ± 0.2% under these optimized conditions. Subsequently, the particle size, ζ-potentials, and structures of MDPs-T were analyzed and compared with the proteins derived from Musca domestica fed on a normal diet (MDPs-ND). This comparative analysis utilized a range of advanced techniques, involving UV spectroscopy, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), high-performance gel permeation chromatography (HPGPC), and scanning electron microscopy (SEM). The outcomes have revealed a marginal disparity in the physical and chemical properties between MDPs-T and MDPs-ND. Derosination led to a reduction in the particle size of the MDPs by 10.98% to 62.81%. MDPs-T exhibited a higher proportion of low-molecular-weight components relative to MDPs-ND. Additionally, in a comparative analysis of amino acids, MDPs-T displayed a greater abundance of essential and total amino acids relative to MDPs-ND. Consequently, MDPs-T holds potential as a valuable food supplement for human consumption or as a nutrient-rich feed supplement for animals.

The role of larvae of black soldier fly and house fly and of feed substrate microbes in biotransformation of aflatoxin B1.

Over the past few years, there has been growing interest in the ability of insect larvae to convert various organic side-streams containing mycotoxins into insect biomass that can be used as animal feed. Various studies have examined the effects of exposure to aflatoxin B1 (AFB1) on a variety of insect species, including the larvae of the black soldier fly (BSFL; Hermetia illucens L.; Diptera: Stratiomyidae) and the housefly (HFL; Musca domestica L.; Diptera: Muscidae). Most of these studies demonstrated that AFB1 degradation takes place, either enzymatic and/or non-enzymatic. The possible role of feed substrate microorganisms (MOs) in this process has thus far not been investigated. The main objective of this study was therefore to investigate whether biotransformation of AFB1 occurred and whether it is caused by insect-enzymes and/or by microbial enzymes of MOs in the feed substrate. In order to investigate this, sterile and non-sterile feed substrates were spiked with AFB1 and incubated either with or without insect larvae (BSFL or HFL). The AFB1 concentration was determined via LC-MS/MS analyses and recorded over time. Approximately 50% of the initially present AFB1 was recovered in the treatment involving BSFL, which was comparable to the treatment without BSFL (60%). Similar patterns were observed for HFL. The molar mass balance of AFB1 for the sterile feed substrates with BSFL and HFL was 73% and 78%, respectively. We could not establish whether non-enzymatic degradation of AFB1 in the feed substrates occurred. The results showed that both BSFL and substrate-specific MOs play a role in the biotransformation of AFB1 as well as in conversion of AFB1 into aflatoxin P1 and aflatoxicol, respectively. In contrast, HFL did not seem to contribute to AFB1 degradation. The obtained results contribute to our understanding of aflatoxin metabolism by different insect species. This information is crucial for assessing the safety of feeding fly larvae with feed substrates contaminated with AFB1 with the purpose of subsequent use as animal feed.

Transmission of viable Haemophilus ducreyi by Musca domestica.

Haemophilus ducreyi was historically known as the causative agent of chancroid, a sexually-transmitted disease causing painful genital ulcers endemic in many low/middle-income nations. In recent years the species has been implicated as the causative agent of nongenital cutaneous ulcers affecting children of the South Pacific Islands and West African countries. Much is still unknown about the mechanism of H. ducreyi transmission in these areas, and recent studies have identified local insect species, namely flies, as potential transmission vectors. H. ducreyi DNA has been detected on the surface and in homogenates of fly species sampled from Lihir Island, Papua New Guinea. The current study develops a model system using Musca domestica, the common house fly, as a model organism to demonstrate proof of concept that flies are a potential vector for the transmission of viable H. ducreyi. Utilizing a green fluorescent protein (GFP)-tagged strain of H. ducreyi and three separate exposure methods, we detected the transmission of viable H. ducreyi by 86.11% ± 22.53% of flies sampled. Additionally, the duration of H. ducreyi viability was found to be directly related to the bacterial concentration, and transmission of H. ducreyi was largely undetectable within one hour of initial exposure. Push testing, Gram staining, and PCR were used to confirm the identity and presence of GFP colonies as H. ducreyi. This study confirms that flies are capable of mechanically transmitting viable H. ducreyi, illuminating the importance of investigating insects as vectors of cutaneous ulcerative diseases.

Design, Synthesis, and Bioactivity of Trifluoroethylthio-Substituted Phenylpyrazole Derivatives.

As the first marketed phenylpyrazole insecticide, fipronil exhibited remarkable broad-spectrum insecticidal activity. However, it poses a significant threat to aquatic organisms and bees due to its high toxicity. Herein, 35 phenylpyrazole derivatives containing a trifluoroethylthio group on the 4 position of the pyrazole ring were designed and synthesized. The predicted physicochemical properties of all of the compounds were within a reasonable range. The biological assay results revealed that compound 7 showed 69.7% lethality against Aedes albopictus (A. albopictus) at the concentration of 0.125 mg/L. Compounds 7, 7g, 8d, and 10j showed superior insecticidal activity for the control of Plutella xylostella (P. xylostella). Notably, compound 7 showed similar insecticidal activity against Aphis craccivora (A. craccivora) compared with fipronil. Potential surface calculation and molecular docking suggested that different lipophilicity and binding models to the Musca domestica (M. domestica) gamma-aminobutyric acid receptors may be responsible for the decreased activity of the tested derivatives. Toxicity tests indicated that compound 8d (LC50 = 14.28 mg/L) induced obviously 14-fold lower toxicity than fipronil (LC50 = 1.05 mg/L) on embryonic-juvenile zebrafish development.

Long-term trends in housefly (Musca domestica L.) insecticide resistance in China.

Controlling housefly populations relies on the use of insecticides, which inevitably leads to the development of resistance. A better and more comprehensive understanding of the spatial and temporal distribution of resistance could guide the control of houseflies. However, most studies on housefly resistance in China are scattered and poorly coordinated. We collected resistance data from houseflies in the published literature and from the vector biomonitoring system of the Chinese Center for Disease Control and Prevention. A 5- or 10-year resolution was used to study the temporal dynamics of resistance to five commonly used insecticides: deltamethrin, permethrin, beta-cypermethrin, dichlorvos, and propoxur. ArcGIS was used to visualize their spatial distributions. The correlation between year and resistance coefficient was determined using SPSS 26.0 and RStudio to explore the changes in resistance over the years. A total of 2128 data were included in this study, ranging from 1982 to 2022, based on which we found significant increases in resistance over the past forty years for the five studied insecticides. Among them, pyrethroids had the most strikingly elevated resistance level and were mainly distributed in the northern and southeastern coastal areas. Dichlorvos and propoxur had intermediate increases in resistance, and most of these increases were identified in North China and the Yangtze River. Housefly resistance to commonly used insecticides in China is increasing and spatially heterogeneous. This finding also highlights the necessity of continuous routine surveillance of housefly resistance, which could guide future housefly control operations and slow the development of resistance.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.