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.

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).

Pseudomonas aeruginosa Y12 play positive roles regulating larval gut communities when housefly encountered copper stress.

Heavy metal contamination has become a global concern that threatens the lives of animals and insects throughout the food chain. Pseudomonas is a commonly found genus of bacteria that colonizes the intestines of insects and constitutes a necessary part of the insect gut microbiota. This research analyzed the influence of different concentrations of Cu2+ on housefly larval development, gut microbial structure and antioxidant defense system, and investigated the regulatory mechanism of P. aeruginosa Y12 on the gut microbiota when houseflies were exposed to Cu2+. We found that adding Cu2+ to the larval diet inhibited larval growth, while the mixed addition of P. aeruginosa Y12 and Cu2+ to the diet reduced the inhibitory effects of Cu2+ on larval growth. Oral administration of Cu2+ significantly changed the gut community structure and increased larval gut bacterial diversity. In vitro analysis revealed that P. aeruginosa Y12 showed Cu2+ adsorption effects and increased Cu2+ aggregation. The mixed addition of low concentrations of P. aeruginosa Y12 and Cu2+ to the larval diet caused a dynamic shift in the gut microbiota and resulted in a novel gut community structure with an increase in beneficial bacteria and a decrease in pathogenic bacteria. Furthermore, P. aeruginosa Y12 treatment influenced the activity of antioxidant enzymes in housefly larvae, indicating that the addition of P. aeruginosa Y12 to the larval diet could further influence the antioxidant system through P. aeruginosa Y12-Cu2+ interactions. In conclusion, our research revealed that intestinal flora dysbiosis was the essential reason why copper inhibits housefly larval growth. However, proper supplementation with P. aeruginosa Y12 played positive roles in regulating larval gut communities and protecting insects from toxic heavy metals.

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.

Feasibility of housefly larvae-mediated vermicomposting for recycling food waste added digestate as additive.

The development of methods for the efficient treatment and application of food waste digestate is an important research goal. Vermicomposting via housefly larvae is an efficient way to reduce food waste and achieve its valorization, however, studies on the application and performance of digestate in vermicomposting are rarely. The present study aimed to investigate the feasibility of the co-treatment of food waste and digestate as an additive via larvae. Restaurant food waste (RFW) and household food waste (HFW) were selected to assess the effects of waste type on vermicomposting performance and larval quality. Waste reduction rates of 50.9%-57.8% were observed in the vermicomposting of food waste mixed with digestate at a ratio of 25%, which were slightly lower than those for treatments without the addition of digestate (62.8%-65.9%). The addition of digestate increased the germination index, with a maximum value of 82% in the RFW treatments with 25% digestate, and decreased the respiration activity, with a minimum value of 30 mg-O2/g-TS. The larval productivity of 13.9% in the RFW treatment system with a digestate rate of 25% was lower that without digestate (19.5%). Materials balance shows that larval biomass and metabolic equivalent had decreasing trends as the amount of digestate increased and HFW vermicomposting exhibited lower bioconversion efficiency than that of RFW treatment system regardless of the addition of digestate. These results suggest that mixing digestate at a low ratio (25%) during vermicomposting of food waste especially RFW could lead to considerable larval biomass and generate relatively stable residues.

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.

Genes from Carboxypeptidase A, glutathione S-transferase, and cytochrome b families were found involved in lead transport in insect Musca domestica.

Lead (Pb) is a typical toxic contamination source all over the world. In this research, larvae of the housefly (Musca domestica) were fed a Pb-contaminated diet at different Pb doses of 0, 20 and 5000 mg/kg. RNA sequencing was used to identify genes that were differentially expressed in relation to lead transport or detoxification. RNA interference (RNAi) was carried on 12 candidate genes. The results showed that three luminal pH regions of mid-gut were at pH values of 6.33, 3.10, and 7.80. With increasing Pb concentration, the pH of the middle mid-gut decreased by one unit. The expression levels of carboxypeptidase A (CPA1), glutathione S-transferase (GST), and cytochrome b (Cyt b) were linked to Pb treatments, particularly high Pb concentration of 5000 mg/kg. RNAi-mediated down expression of CPA1, GST2, and CYTb-c1 resulted in low Pb accumulation in the larvae of 5000 mg/kg Pb group. These proteins played key roles in Pb transport and detoxification in M. domestica larvae.

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.

Integrated genome-wide investigations of the housefly, a global vector of diseases reveal unique dispersal patterns and bacterial communities across farms.

BACKGROUND: Houseflies (Musca domestica L.) live in intimate association with numerous microorganisms and is a vector of human pathogens. In temperate areas, houseflies will overwinter in environments constructed by humans and recolonize surrounding areas in early summer. However, the dispersal patterns and associated bacteria across season and location are unclear. We used genotyping-by-sequencing (GBS) for the simultaneous identification and genotyping of thousands of Single Nucleotide Polymorphisms (SNPs) to establish dispersal patterns of houseflies across farms. Secondly, we used 16S rRNA gene amplicon sequencing to establish the variation and association between bacterial communities and the housefly across farms. RESULTS: Using GBS we identified 18,000 SNPs across 400 individuals sampled within and between 11 dairy farms in Denmark. There was evidence for sub-structuring of Danish housefly populations and with genetic structure that differed across season and sex. Further, there was a strong isolation by distance (IBD) effect, but with large variation suggesting that other hidden geographic barriers are important. Large individual variations were observed in the community structure of the microbiome and it was found to be dependent on location, sex, and collection time. Furthermore, the relative prevalence of putative pathogens was highly dependent on location and collection time. CONCLUSION: We were able to identify SNPs for the determination of the spatiotemporal housefly genetic structure, and to establish the variation and association between bacterial communities and the housefly across farms using novel next-generation sequencing (NGS) techniques. These results are important for disease prevention given the fine-scale population structure and IBD for the housefly, and that individual houseflies carry location specific bacteria including putative pathogens.

Growth performance, carcass characteristics, meat quality and serum profile of broiler chicks fed on housefly maggot meal as a replacement of soybean meal.

A study was conducted to invesstigate the housefly maggot meal (HMM) as an alternative protein source to replace the soybean meal in broiler chick's diet. A total of 720 1-day-old male broiler chicks were divided into three groups and fed diets formulated with HMM to replace soybean meal at the rate of 0%, 4% and 8%. The study lasted for 42 days in two phases. Results showed that HMM addition did not markedly affect body weight, average daily body weight gain and average daily feed intake of the broiler chicks. Feed conversion ratio increased linearly (1-21 days) in starter or quadratically (22-42 days) in the grower phase. HMM non-significantly increased the feed intake and body weight during the grower phase. Slight changes were observed for decrease of blood biochemical indices in the platelets (day 21), and alkaline phosphatase and lysozyme (day 42), and increase for red blood cells, packed cell volume, total protein and uric acid on day 42; however, the fluctuations were within the physiological range. Non-significant effects were observed for carcass composition and meat quality, except that HMM numerically reduced the shear force of breast muscle (linear, p = .058). These results are the strong evidence that HMM can be used as an alternative protein source at 8% in broiler chick's diet without any adverse effect on chick's performance.

Spatiotemporally different expression of alternatively spliced GABA receptor subunit transcripts in the housefly Musca domestica.

Insect γ-aminobutyric acid (GABA) receptors are important as major inhibitory neurotransmitter receptors and targets for insecticides. The housefly GABA receptor subunit gene MdRdl is alternatively spliced at exons 3 (a or b) and 6 (c or d) to yield the variants of ac, ad, bc, and bd combinations. In the present study, the expression of the MdRdl transcript in the body parts and in the developmental stages of the housefly Musca domestica was examined by quantitative polymerase chain reaction using specific primers that amplify the combinations of alternative exons. The results indicated that the transcripts of MdRdl, including four combinations, were highly expressed in the adult stage. MdRdlbd was the most abundant in the adult head. The expression pattern did not change in the adult stage over 7 days after eclosion. The expression level of the MdRdl bd transcript in the female head was similar to that of the male head. In contrast, MdRdl bc was the predominant transcript in the pupal head and the adult leg. Because the homomeric Rdl bc GABA receptor has a high affinity for GABA, our results provide grounds for designing agonist or competitive-antagonist insecticides that target the orthosteric site of the GABA receptor containing this Rdl variant.

Housefly larvae (Musca domestica) significantly accelerates degradation of monensin by altering the structure and abundance of the associated bacterial community.

Vermicomposting of livestock manure using housefly larvae is a promising biotechnology for waste reduction and control of antibiotic pollution. Monensin (MON), an ionophore polyether antibiotic (IPA), is widely used in broiler feed to control coccidiosis. However, MON residues in litter have become a major source of pollution in the environment. In this work, we studied the efficiency of housefly larvae (Musca domestica) on monensin attenuation during a 12-day laboratory scale vermicomposting experiment. We observed a 94.99% reduction in MON concentration after four days in treatment groups, while it took twelve days to remove more than 94.71% of MON in the control group. We found that the bacterial community composition of the substrate was reshaped by housefly larvae. From the treatment groups, three MON-degrading bacterial strains were isolated and identified as Acinetobacter sp., Stenotrophomonas sp. and Alcaligenes sp. based on 16 S rRNA gene sequence analysis. These three strains were among dominant the bacteria in treated substrates, showing between 52.80% and 89.25% degradation of MON in mineral salt medium within 28 days. Furthermore, two MON-degrading bacteria (Stenotrophomonas sp. and Alcaligenes sp.) were more abundant in treatment groups and larvae gut groups compared with those in control groups. The abundance enhancement of MON-degrading bacteria was related to the change in ambient temperature and pH in the substrates, which were affected by housefly larvae activities. Our results confirm that housefly larvae can significantly accelerate degradation of MON in chicken manure by increasing the abundance of MON-degrading bacteria.

Housefly Pupae-Derived Antioxidant Peptides Exerting Neuroprotective Effects on Hydrogen Peroxide-Induced Oxidative Damage in PC12 Cells.

In this study, two antioxidant peptides were identified and characterized from the alcalase-hydrolysate of housefly (Musca domestica L.) pupae guided by ABTS cation radical scavenging activity. Peptides sequences were identified as DFTPVCTTELGR (DR12, 1338.48 Da) and ARFEELCSDLFR (AR12, 1485.66 Da) using nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both DR12 and AR12 exert strong ABTS cation radical scavenging ability with EC50 values of 0.39 and 0.35 mM, respectively. Moreover, AR12 can effectively protect PC12 cells from oxidative damage induced by hydrogen peroxide (H2O2) by decreasing intracellular reactive oxygen species (ROS) and malonaldehyde (MDA), recovering cellular mitochondrial membrane potential (MMP), and increasing the activity of intracellular superoxide dismutase (SOD). Stability tests suggest that AR12 is competent for the challenge of heating, acid, alkali or simulated gastrointestinal (GI) digestion and exhibits great activity to remove ABTS cation radical. DR12 shows a great stability against heating, but its antioxidative ability declines after being treated with acid, alkali or simulated GI digestion. In general, both DR12 and AR12 identified from housefly pupae hydrolysate stand a chance of being potential antioxidants or precursors to antioxidants and AR12 might be applied in the field of neuroprotection.

Identification and characterization of a novel antimicrobial protein from the housefly Musca domestica.

Antimicrobial peptides/proteins are immune-related molecules that are widely distributed in bacteria, fungi, plants, invertebrates and higher animals. They have exhibited great potential to be developed into antimicrobial drugs. The housefly, Musca domestica, lives in a highly contaminated environment and has adapted a robust immune system against various pathogens. As an effort to search for new antimicrobial molecules in the housefly, we investigated the function of an uncharacterized gene firstly by confirming that its expression was induced by infection in M. domestica. The corresponding protein was then shown to have potent antimicrobial activity. Scanning Electron Microscopy data showed that treatment of C. albicans cells with the protein caused cell size decreasing and cell elongation. The results here suggest the protein a novel class of antimicrobial protein and provide new insights into the immunological mechanisms by which M. domestica combats invading C. albicans.

Antioxidant activities of protein hydrolysates obtained from the housefly larvae.

The housefly is an important resource insect and the housefly larvae are ideal source of food additives. The housefly larvae protein hydrolysates were obtained by enzymatic hydrolysis by alcalase and neutral proteinase. Their antioxidant activities were investigated, including the superoxide and hydroxyl radicalscavenging activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, reducing power and metal chelating activity. The antioxidant activities of both hydrolysates increased with their increasing concentrations. The alcalase hydrolysate (AH) showed higher scavenging activities against hydroxyl radical and superoxide anion radical at low concentrations and higher metal-chelating activity than the neutral proteinase hydrolysate (NPH). The NPH exhibited higher scavenging activity against DPPH free radical and higher reducing power than the AH. Both hydrolysates showed more than 50% superoxide anion radical-scavenging activity at 10 µg/mL. These results indicate that both housefly larvae protein hydrolysates display high antioxidant activities and they could serve as potential natural antioxidant food additives.

Binary combinations of organophosphorus pesticides exhibit differential toxicity under oxidised and un-oxidised conditions.

Acetylcholinesterase (AChE) inhibition has been demonstrated to be useful as a biomarker for exposure to organophosphorus (OP) insecticides in many environments. The objective of this study was to investigate the response of housefly (Musca domestica) head AChE (HF-AChE) exposed to five OPs as individual compounds and their binary mixtures under in vitro conditions. To examine the effects of oxidation on OP potency in the HF-AChE system, bromine water was used as an oxidisng agent. With oxidation, the sensitivity of HF-AChE to chlorpyrifos (CPF), malathion (MLT) and triazophos (TRZ) increased significantly. Monocrotophos (MCP) and profenofos (PRF) did not exhibit any significant differences in toxicity under oxidised and un-oxidised conditions. The toxicological interaction of five organophosphorus pesticides was evaluated using the concentration addition model, the combination index-isobologram equation and the toxic unit approach. All three models provided similar predictions for the 10 binary combinations of OPs under oxidised and un-oxidised conditions. In the present study, the antagonistic effects of the binary combination of OPs (CPF+PRF, CPF+MLT, MCP+MLT, PRF+MLT, MLT+TRZ and PRF+TRZ) were observed under oxidised conditions. This may be due to dispositional and/or receptor antagonism. Most of the binary combinations assayed under un-oxidised conditions exhibited synergistic responses. Triazophos showed very strong synergism in binary combinations with CPF, MCP and PRF un-oxidised conditions. In contrast, under oxidised conditions, only CPF+TRZ exhibited synergism. The results obtained indicate differential toxicity of binary combinations of OPs under oxidised and un-oxidised conditions. This information could be a valuable tool in understanding the mechanisms of OPs interactions and the interpretation of future in vivo studies with mixtures of OP insecticides.

Horizontal Transmission of Beauveria bassiana (Hypocreales: Cordycipitaceae) and Metarhizium anisopliae (Hypocreales: Clavicipitaceae) in Musca domestica (Diptera: Muscidae).

Beauveria bassiana Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin are fungi with potential for controlling Musca domestica L. However, the impact on this dipteral may vary depending on the fungal isolates and the methodology used. This study evaluated the pathogenicity of direct application and horizontal transmission of B. bassiana (CG240) and M. anisopliae (CG34) on adult M. domestica individuals. The impact of B. bassiana and M. anisopliae on M. domestica was evaluated at the concentrations 2 × 10(4), 2 × 10(5), 2 × 10(6), and 2 × 10(7) conidia/ml. Horizontal transmission was also estimated between sexes at different infection periods of the vector insect. The mortality of adult M. domestica individuals directly infected with B. bassiana was above 90%, and the mortality of those infected with M. anisopliae ranged from 25.50 to 97.78%. Horizontal transmission of B. bassiana caused the death of 100% of individuals, in turn, that of M. anisopliae killed 55% of male and 100% of female individuals. Horizontal transmission of fungi was negatively influenced by time. This study shows the potential of these fungi for controlling M. domestica, both with the direct implementation strategy and horizontal transmission. However, field studies are needed to evaluate the capacity to decrease the M. domestica population using these alternatives.

Cloning, expression, and purification of a new antibacterial substance gene from larvae of Musca domestica (Diptera: Muscidae).

Musca domestica L. (Diptera: Muscidae), the housefly, exhibits unique immune defenses and can produce antibacterial substances upon stimulation with bacteria. On the basis of the cDNA library constructed using the suppression subtractive hybridization method, a 1188-bp antibacterial substance gene, which we named AS566, was amplified by rapid amplification of cDNA ends from M. domestica larva stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In this study, the full-length AS566 gene was cloned and inserted into a His-tagged Escherichia coli (Enterobacteriaceae: Escherichia) prokaryotic expression system to enable production of the recombinant protein. The recombinant AS566 protein was purified in denatured form from inclusion bodies and renatured to obtain functionally active AS566 protein. The bacteriostatic activity of the recombinant purified AS566 protein was assessed using the Oxford plate assay system and the results indicated that AS566 had antibacterial activity against six bacteria, including an E. coli clinical isolate, S. pullorum, Streptococcus bovis (Streptococcaceae: Streptococcus), Streptococcus suis, and Staphylococcus aureus (Staphylococcaceae: Staphylococcus) in vitro. The antibacterial activity of AS566 toward Gram- bacteria was two times greater than that against Gram+ bacteria. The sequencing results and BLAST analysis showed that the antibacterial substance gene AS566 was not homologous to any other antibacterial substance genes in GenBank. The antibacterial mechanisms of the newly discovered AS566 protein warrant further study.

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.

Adulticidal synergy of two plant essential oils and their major constituents against the housefly Musca domestica and bioassay on non-target species.

Single and mixture formulations of lemongrass (Cymbopogon citratus (DC.) Stapf.) and star anise (Illicium verum (J. Presl.)) essential oils (EOs) and their major constituents were assayed for their adulticidal activities against housefly, Musca domestica L., and two non-target species, stingless bee (Tetragonula pegdeni Schwarz) and guppy (Poecilia reticulata Peters). The efficacies of the mixture formulations were compared against those of the single formulations and 1.0% α-cypermethrin, a common synthetic insecticide. GC-MS analysis found that the major constituent of lemongrass EO was geranial (45.23%), and that of star anise EO was trans-anethole (93.23%). Almost all mixture formulations were more effective in adulticidal activity against housefly adults than single formulations and 1.0% α-cypermethrin. A mixture of 1.0% lemongrass EO + 1.0% trans-anethole exhibited the strongest synergistic insecticidal activity with a 100% mortality rate (KT50 of 3.2 min and LT50 of 0.07 h). The relative percentage increase in mortality rate over single formulations was between 1.6 and 91.9%. In addition, it was three times more effective than 1.0% α-cypermethrin. To find the mechanism of adulticidal action, scanning electron microscopy (SEM) was done to find morphological aberrations, such as antennal and mouthpart aberrations, after the houseflies were treated with 1.0% lemongrass EO + 1.0% trans-anethole. The aberrations included deformed and abnormal shape of arista and flagellum, change in labellum pigmentation, and damage to pseudotracheae. Regarding toxicity against non-target species, all single and mixture formulations were not toxic to the two non-target species, while 1.0% α-cypermethrin was highly toxic. To conclude, a mixture of 1.0% lemongrass EO + 1.0% trans-anethole can be an excellent, natural, sustainable housefly adulticidal agent.