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.

Protein-enriched extracts from housefly (Musca domestica) maggots alleviates atherosclerosis in apolipoprotein E-deficient mice by promoting bile acid production and consequent cholesterol consumption.

Currently, atherosclerosis control is important to prevent future heart attacks or strokes. Protein-enriched extract (PE) from housefly maggots (Musca domestica) can inhibit the development of atherosclerosis partially through its antioxidant effects. Whether PE exerts other anti-atherosclerosis functions remains unclear. Here, PE was found to simultaneously promote cholesterol metabolism effects in apolipoprotein E knockout (ApoE-/- ) mice. Bile acid synthesis plays a key role in regulating cholesterol homeostasis in atherosclerosis. Whether PE alleviates atherosclerosis by promoting bile acid production and consequent cholesterol consumption was further explored. First, 8-week-old male ApoE-/- mice were recruited and fed on a cholesterol-enriched diet. After 8 weeks, these mice were divided into three groups and received gavage administration of PE, simvastatin, and saline for another 8 weeks. Atherosclerosis severity was then assessed. Real-time quantitative polymerase chain reaction and western blot were employed to determine the expression of hepatic ATP-binding cassette transporter A1 (ABCA1), liver X receptor α (LXRα), and peroxisome proliferator-activated receptor-γ (PPAR-γ). Serum levels of high-density lipoprotein-cholesterol (HDL), low-density lipoprotein-cholesterol (LDL), and total cholesterol (TC) were determined by enzyme-linked immunoassay. Results revealed that PE reversed the formation of atherosclerotic lesion; increased the expression of PPAR-γ, LXRα, and ABCA1; increased the amount of bile flow and total bile acid; reduced the serum level of LDL and TC; and increased the level of HDL. In conclusion, enhancement on bile acid production and consequent cholesterol consumption may partially contribute to the anti-atherosclerotic effects of PE. The reversal of PPARγ-LXRα-ABCA1 signaling pathway may be involved in this process.

Colon tissue-accumulating mesoporous carbon nanoparticles loaded with Musca domestica cecropin for ulcerative colitis therapy.

Ulcerative colitis (UC) is a modern refractory disease with steadily increasing incidence worldwide that urgently requires effective and safe therapies. Therapeutic peptides delivered using nanocarriers have shown promising developments for the treatment of UC. We developed a novel colon-accumulating oral drug delivery nanoplatform consisting of Musca domestica cecropin (MDC) and mesoporous carbon nanoparticles (MCNs) and investigated its effects and mechanism of action for the treatment of UC. Methods: An optimized one-step soft templating method was developed to synthesize MCNs, into which MDC was loaded to fabricate MDC@MCNs. MCNs and MDC@MCNs were characterized by BET, XRD, and TEM. MDC and MDC@MCNs resistance to trypsin degradation was measured through Oxford cup antibacterial experiments using Salmonella typhimurium as the indicator. Uptake of MDC and MDC@MCNs by NCM460 cells was observed by fluorescence microscopy. The biocompatibility of MDC, MCNs, and MDC@MCNs was evaluated in three cell lines (NCM460, L02, and NIH3T3) and C57BL/6 mice. Dextran sulphate sodium was used to establish models of NCM460 cell injury and UC in mice. MTT assay, flow cytometry, and mitochondrial membrane potential assay were applied to determine the effects of MDC@MCNs on NCM460 cells injury. Additionally, a variety of biological methods such as H&E staining, TEM, ELISA, qPCR, Western blotting, and 16s rDNA sequencing were performed to explore the effects and underlying mechanism of MDC@MCN on UC in vivo. Colonic adhesion of MCNs was compared in normal and UC mice. The oral biodistributions of MDC and MDC@MCNs in the gastrointestinal tract of mice were also determined. Results: MDC@MCNs were successfully developed and exhibited excellent ability to resist destruction by trypsin and were taken up by NCM460 cells more readily than MDC. In vitro studies showed that MDC@MCNs better inhibited DSS-induced NCM460 cells damage with lower toxicity to L02 and NIH3T3 cells compared with MDC. In vivo results indicated that MDC@MCNs have good biocompatibility and significantly improved colonic injury in UC mice by effectively inhibiting inflammation and oxidative stress, maintaining colonic tight junctions, and regulating intestinal flora. Moreover, MDC@MCNs were strongly retained in the intestines, which was attributed to intestinal adhesion and aggregation of MCNs, serving as one of the important reasons for its enhanced efficacy after oral administration compared with MDC. Conclusion: MDC@MCNs alleviated DSS-induced UC by ameliorating colonic epithelial cells damage, inhibiting inflammation and oxidative stress, enhancing colonic tight junctions, and regulating intestinal flora. This colon-accumulating oral drug delivery nanoplatform may provide a novel and precise therapeutic strategy for UC.

Housefly Phormicin inhibits Staphylococcus aureus and MRSA by disrupting biofilm formation and altering gene expression in vitro and in vivo.

The increasing drug resistance of pathogenic bacteria is a crisis that threatens public health. Antimicrobial peptides (AMPs) have been suggested to be potentially effective alternatives to solve this problem. Here, we tested housefly Phormicin-derived peptides for effects on Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) infections in vitro and in vivo. A decreased bacterial load of MRSA was observed in the mouse scald model after treatment with Phormicin and in the positive control group (vancomycin). A mouse scrape model indicated that Phormicin helps the host fight drug-resistant MRSA infections. The protective effect of Phormicin on MRSA was confirmed in the Hermetia illucens larvae model. Phormicin also disrupted the formation of S. aureus and MRSA biofilms. Furthermore, this effect coincided with the downregulation of biofilm formation-related gene expression (agrC, sigB, RNAIII, altA, rbf, hla, hld, geh and psmɑ). Notably, virulence genes and several regulatory factors were also altered by Phormicin treatment. Based on these findings, housefly Phormicin helps the host inhibit MRSA infection through effects on biofilm formation and related gene networks. Therefore, housefly Phormicin potential represents a candidate agent for clinical MRSA chemotherapy.

Design, Synthesis, and Insecticidal Activity of 5,5-Disubstituted 4,5-Dihydropyrazolo[1,5-a]quinazolines as Novel Antagonists of GABA Receptors.

To control the development of resistance to conventional insecticides acting as γ-aminobutyric acid (GABA) receptor antagonists (e.g., fipronil), new GABAergic 5,5-disubstituted 4,5-dihydropyrazolo[1,5-a]quinazolines were designed via a scaffold-hopping strategy and synthesized with a facile method. Among the 50 target compounds obtained, compounds 5a, 5b, 7a, and 7g showed excellent insecticidal activities against a susceptible strain of Plutella xylostella (LC50 values ranging from 1.03 to 1.44 µg/mL), which were superior to that of fipronil (LC50 = 3.02 µg/mL). Remarkably, the insecticidal activity of compound 5a was 64-fold better than that of fipronil against the field population of fipronil-resistant P. xylostella. Electrophysiological studies against the housefly GABA receptor heterologously expressed in Xenopus oocytes indicated that compound 5a could act as a potent GABA receptor antagonist, and IC50 was calculated to be 32.5 nM. Molecular docking showed that the binding poses of compound 5a with the housefly GABA receptor can be different compared to fipronil, which explains the effectiveness of compound 5a against fipronil-resistant insects. These findings have suggested compound 5a as a lead compound for a novel GABA receptor antagonist controlling field-resistant insects and provided a basis for further design, structural modification, and development of 4,5-dihydropyrazolo[1,5-a]quinazoline motifs as new insecticidal GABA receptor antagonists.

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.

Antifungal Activity and Physicochemical Properties of a Novel Antimicrobial Protein AMP-17 from Musca domestica.

Antimicrobial peptides (AMPs) are cationic small peptide chains that have good antimicrobial activity against a variety of bacteria, fungi, and viruses. AMP-17 is a recombinant insect AMP obtained by a prokaryotic expression system. However, the full antifungal activity, physicochemical characteristics, and cytotoxicity of AMP-17 were previously unknown. AMP-17 was shown to have good antifungal activity against five pathogenic fungi, with minimum inhibitory concentrations (MIC) of 9.375-18.75 µg/ml, and minimum fungicidal concentrations (MFC) of 18.75-37.5 µg/ml. Notably, the antifungal activity of AMP-17 against Cryptococcus neoformans was superior to that of other Candida spp. In addition, the hemolytic rate of AMP-17 was only 1.47%, even at the high concentration of 16× MIC. AMP-17 was insensitive to temperature and high salt ion concentration, with temperatures of 98°C and -80°C, and NaCl and MgCl2 concentrations of 50-200 mmol/l, having no significant effect on antifungal activity. However, AMP-17 was sensitive to proteases, trypsin, pepsin, and proteinase K. The elucidation of antifungal activity, physicochemical properties and cytotoxicity of AMP-17 provided an experimental basis for its safety evaluation and application, as well as indicated that AMP-17 might be a promising drug.Antimicrobial peptides (AMPs) are cationic small peptide chains that have good antimicrobial activity against a variety of bacteria, fungi, and viruses. AMP-17 is a recombinant insect AMP obtained by a prokaryotic expression system. However, the full antifungal activity, physicochemical characteristics, and cytotoxicity of AMP-17 were previously unknown. AMP-17 was shown to have good antifungal activity against five pathogenic fungi, with minimum inhibitory concentrations (MIC) of 9.375­18.75 µg/ml, and minimum fungicidal concentrations (MFC) of 18.75­37.5 µg/ml. Notably, the antifungal activity of AMP-17 against Cryptococcus neoformans was superior to that of other Candida spp. In addition, the hemolytic rate of AMP-17 was only 1.47%, even at the high concentration of 16× MIC. AMP-17 was insensitive to temperature and high salt ion concentration, with temperatures of 98°C and ­80°C, and NaCl and MgCl2 concentrations of 50­200 mmol/l, having no significant effect on antifungal activity. However, AMP-17 was sensitive to proteases, trypsin, pepsin, and proteinase K. The elucidation of antifungal activity, physicochemical properties and cytotoxicity of AMP-17 provided an experimental basis for its safety evaluation and application, as well as indicated that AMP-17 might be a promising drug.

Amorphous calcium phosphate in the pupal cuticle of Bactrocera dorsalis Hendel (Diptera: Tephritidae): A new discovery for reconsidering the mineralization of the insect cuticle.

It is now widely accepted that Hexapoda emerged from Crustacea. Compared to the ubiquitous calcified exoskeleton in crustaceans, a mineralized cuticle in insects is extremely rare. Catecholamine-driven protein cross-links play a leading role in the sclerotization of insect cuticle. In this study, mineralization was discovered in the pupal cuticle of Bactrocera dorsalis (Diptera: Tephritidae), a common pest of fruit farms. We mainly profiled the features of mineralized pupal cuticles from B. dorsalis and its white mutant B. dorsaliswh and unmineralized cuticle from Musca domestica using high-resolution field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM) combined with structural analysis involving infrared (IR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and synchrotron X-ray absorption near-edge structure (XANES) spectroscopy. We also compared the thermodynamic and mechanical properties of different pupal cuticles. The results showed that the pupal cuticles of B. dorsalis contain a phase of stable amorphous calcium phosphate (ACP) with a high level of magnesium, which is mainly distributed in the exocuticle and assists in the formation of a graded, stiffened cuticle structure. Unexpectedly, this ACP possesses a very low Ca/P ratio and has a composition similar to that of CaHPO4·2H2O. The degree of mineralization in the pupal cuticle of B. dorsaliswh (approximately 22 wt%) is significantly greater than that of wild-type B. dorsalis (approximately 12 wt%), which indicates that there may be a connection between the biomineralization and tyrosine-mediated tanning pathways. These findings provide new evidence for the mineralization of the insect cuticle, which may shed new light on the evolutionary mechanism underlying the divergence of cuticle sclerotization between insects and crustaceans.

Antidiarrheal effects of a thermostable protein fraction obtained from the larvae of Musca domestica.

Our objective was to investigate whether a thermostable protein fraction (TPF) obtained from the larvae of Musca domestica, which contains cecropin family AMPs, is effective in treating senna leaf (Folium Sennae)-induced diarrhea in mice and its possible underlying mechanism. We did the experiments both in vitro and in vivo. Firstly, lipopolysaccharide (LPS) was used to induce inflammation in RAW 264.7 macrophages. The expression level of nitric oxide (NO) and tumor necrosis factor (TNF)-α was assessed using kits and immunofluorescence assay. A mice model of total diarrhea was established using a decoction of Folium Sennae. Levels of interleukin (IL)-6 and IL-1ß in mice serum and of TNF-α in the supernatant of jejunal tissue homogenate were measured using commercially available ELISA kits. Hematoxylin-eosin staining was employed to evaluate pathological lesions, and immunohistochemistry was used for determining IL-1ß, IL-6, and TNF-α expression levels. Results display that TPF markedly inhibited NO and TNF-α production in LPS-stimulated RAW 264.7 macrophages in vitro. Moreover, TPF significantly lowered the diarrhea index (DI) in diarrheic mice; when TPF was administered at a high dose (120 mg/kg) to mice, in comparison with mice in the model group, DI was markedly reduced. TPF could also decrease the expression levels of some pro-inflammatory factors, high dose TPF treated mice were with the reduction of (202.29 ± 18.58) pg/ml (tumor necrosis factor alpha, TNF-α), (53.69 ± 7.83) pg/ml (interleukin (IL)-1ß, IL-1ß), (48.44 ± 3.77) pg/ml (IL-6I, L-6) to the model separately. In comparison with berberine hydrochloride, which was used as the positive control in this study, TPF could confer better intestinal protection. To conclude, our results demonstrate that TPF has potent anti-inflammatory activities in vitro and antidiarrheal effects on mice with Folium Sennae-induced diarrhea.

Bioaccumulation and health risk assessments of trace elements in housefly (Musca domestica L.) larvae fed with food wastes.

This study aimed to use food waste to culture housefly larvae, which serve as the major source of protein in fish feeds, to evaluate copper (Cu), zinc (Zn), iron (Fe), nickel (Ni), cadmium (Cd), and chromium (Cr) bioaccumulation and trophic transfer in the food chain. In addition, the potential health risk to humans of exposure to these metal elements via dietary intake of tilapia fed with housefly larvae feeds was also evaluated. The results showed the bioavailability of trace elements in dish waste to housefly larvae was lower than that in staple food waste. Trace element concentrations in housefly larvae fed with food waste met the animal feed standards in China and the European Union (EU). The highest concentrations of Cu, Zn, Ni, Cr, and Cd in residue did not exceed the limits specified for fertilizer in China, Canada, and Germany. The tilapia fed with dried housefly larvae presented a greater final weight and protein content than those fed with commercial feed and fresh housefly larvae (p<0.05). The Cu, Cr, and Cd concentrations in tilapia fed with commercial feed were higher than in those fed with commercial housefly larvae or dried housefly larvae (p<0.05). The highest bioaccumulation of Cu, Zn, and Ni was found in tilapia fed with fresh housefly larvae feed. The results of the health risk assessment showed that the tilapia fed with the housefly larvae feed pellets or fresh housefly larvae were safe for consumption from the perspective of trace elements.

Cardiovascular and respiratory safety evaluation of Musca Domestica larvae low molecular weight peptide in beagle dogs.

Background: Many studies have demonstrated that the water extracts and low-molecular-weight peptide (LMWP) of the Musca domestica larvae contain significant biological activity. However, the cardiovascular and respiratory safety evaluations of LMWP are yet to be sufficiently investigated. Aim: This study focused on the cardiovascular and respiratory safety evaluations of the M. domestica larvae LMWP in beagle dogs. Methods: Direct cardiovascular and respiratory effects of three different doses of the M. domestica larvae LMWP were investigated following only once oral administration in conscious telemetered dogs, whereby ECG, arterial pressure, and respiratory data were collected using the Data Science International telemetric system. Results: The PR, QT, and QTcf intervals were significantly shortened in the medium-dose LMWP treatment group at 3 h after drug administration. Furthermore, no significant differences were observed in any of the corresponding indexes of other treatment groups at different time points compared to those of the control group. P wave, ST segment, R wave, systolic pressure, diastolic pressure, and mean pressure were significantly different, although these differences had no significant dose-effect relationship. Respiratory frequency significantly increased in the medium-dose LMWP treatment group at 8 h after drug administration compared to that of the control group. Respiratory rate and tidal volume showed no significant differences at varying time points among all LMWP treatment groups. Conclusions: No toxicological effects related to cardiovascular and respiratory safety in beagle dogs were observed at any dose level of the M. domestica larvae LMWP.

Housefly (Musca domestica) larvae powder, preventing oxidative stress injury via regulation of UCP4 and CyclinD1 and modulation of JNK and P38 signaling in APP/PS1 mice.

Housefly (Musca domestica) Larvae powder (HL) is rich in antioxidants. As oxidative stress is considered as one of the main pathogenesis in Alzheimer's Disease (AD), this study was designed to explore the protective effects of HL as an antioxidant on APP/PS1 mice. 2-Month-old APP/PS1 mice were divided into a model control (MC) group, a Donepezil group and a HL group, and C57BL/6 mice were used as the normal control (NC) group. After 180 days of treatment, the memory ability was measured by Morris Water Maze (MWM). The presence of Aß and the expression of Uncoupling Protein 4 (UCP4) and CyclinD1 were detected by immunohistochemistry. The expressions of Superoxide Dismutase 1 (SOD1), Catalase (CAT) and Mitogen-activated Protein Kinase (MAPK) signal pathways were measured by western blotting. Compared with untreated APP/PS1 mice, the memory abilities of the HL-treated mice were significantly improved. Furthermore, the HL treatment not only down-regulated the deposition of Aß and the expression of CylinD1, but also increased both the mRNA and protein levels of SOD, CAT, and UCP4, and enhanced the phosphorylation of JNK and P38 MAPK activation. In conclusion, these results suggest that HL may have a protective effect against memory impairment and prevent oxidative stress-induced injury via the regulation of UCP4 and CyclinD1 and the modulation of JNK and P38 MAPK signaling in AD.

Protein-rich extract of Musca domestica larvae alleviated metabolic disorder in STZ-induced type 2 diabetic rat model via hepatoprotective and pancreatic ß-cell protective activities.

The study was designed to explore the beneficial effect of Musca domestica larvae extract (MDLE) on a metabolic disorder using a diabetic rat model. Streptozotocin-induced diabetic rats were treated with or without MDLE. Blood glucose, insulin levels, lipid profiles, and oxidative stress markers were measured. The morphological changes in the pancreas and liver were determined, as well as insulin expression. The expression of glucose transporter 4 (GLUT4), phospho-adenosine monophosphate-activated protein kinase (p-AMPK)/total AMPK, superoxide dismutase 1 (SOD1), catalase (CAT), and peroxisome proliferator-activated receptor gamma (PPARγ) were detected. Compared with untreated diabetic rats, MDLEtreated rats had decreased urine volume, food intake, and water intake, along with significantly lower levels of blood glucose, malondialdehyde (MDA), plasma triglycerides, low-density lipoprotein (LDL), and total cholesterol. MDLEtreated rats also had higher levels of SOD activity, high-density lipoprotein (HDL), and insulin. MDLE treatment partially restored the ß-cell population, improved the liver necrosis and islet cell damage, reversed the decreased expression of GLUT4, phospho-AMPK, SOD1, and CAT in the liver, skeletal muscle and pancreatic tissue, and also increased the expression of PPARγ in the liver and adipose tissue in diabetic rats. In conclusion, the obtained results suggest that MDLE could possibly be used pharmacologically as an adjuvant for the treatment of diabetes.

Biological characteristics of a new antibacterial peptide and its antibacterial mechanisms against Gram-negative bacteria.

MDAP-2 is a new antibacterial peptide with a unique structure that was isolated from house- flies. However, its biological characteristics and antibacterial mechanisms against bacteria are still poorly understood. To study the biological characteristics, antibacterial activity, hemolytic activi- ty, cytotoxicity to mammalian cells, and the secondary structure of MDAP-2 were detected; the results showed that MDAP-2 displayed high antibacterial activity against all of the tested Gram-negative bacteria. MDAP-2 had lower hemolytic activity to rabbit red blood cells; only 3.4% hemolytic activity was observed at a concentration of 800µg/ml. MDAP-2 also had lower cytotoxicity to mammalian cells; IC50 values for HEK-293 cells, VERO cells, and IPEC-J2 cells were greater than 1000 µg/ml. The circular dichroism (CD) spectra showed that the peptide most- ly has α-helical properties and some ß-fold structure in water and in membrane-like conditions. MDAP-2 is therefore a promising antibacterial agent against Gram-negative bacteria. To deter- mine the antibacterial mechanism(s) of action, fluorescent probes, flow cytometry, and transmis- sion electron microscopy (TEM) were used to study the effects of MDAP-2 on membrane perme- ability, polarization ability, and integrity of Gram-negative bacteria. The results indicated that the peptide caused membrane depolarization, increased membrane permeability, and destroyed membrane integrity. In conclusion, MDAP-2 is a broad-spectrum, lower hemolytic activity, and lower cytotoxicity antibacterial peptide, which is mainly effective on Gram-negative bacteria. It exerts its antimicrobial effects by causing bacterial cytoplasm membrane depolarization, increas- ing cell membrane permeability and disturbing the membrane integrity of Gram-negative bacte- ria. MDAP-2 may offer a new strategy to for defense against Gram-negative bacteria.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica.

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene MdαE7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (MdαE7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of MdαE7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of MdαE7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Amino acid digestibility of larval meal (Musca domestica) for broiler chickens.

Work was undertaken to investigate the potential use of housefly (Musca domestica) larvae reared on broiler manure as a source of nutrition for poultry production in the United Kingdom. Nutritional analysis showed that larvae have a high (>45% dry wt.) protein content and a favorable amino acid profile that is rich in key amino acids, such as lysine and methionine. A broiler digestibility trial was carried out to determine the apparent ileal digestibility coefficients (AIDC) and true ileal digestibility coefficients (TIDC) of amino acids (AA) from insect larval meal (ILM) from M. domestica and fishmeal (FM) in broiler chickens. This was calculated using multiple linear regression technique based upon 3 inclusions of each protein source in a semisynthetic diet. One-hundred-forty-four day-old male (Ross 308) broilers were fed from hatch on a commercial starter diet for 20 days. Experimental diets were fed from d 21 to 28, and feed intakes were measured daily. On d 28, the trial was terminated, ileal digesta were collected for the determination of AIDC and TIDC of AA, and inflammatory responses (gizzard erosion and eye discharge) were measured. No significant differences were observed in digestibilities between protein sources for any AA. Furthermore, ILM feeding did not induce gizzard erosion or eye discharge at any inclusion. These results provide strong evidence to suggest that ILM of the common housefly can provide a successful alternative protein source to FM in broiler diets.

The crop-residue of fiber hemp cv. Futura 75: from a waste product to a source of botanical insecticides.

In the attempt to exploit the potential of the monoecious fiber hemp cv. Futura 75 in new fields besides textile, cosmetics and food industry, its crop-residue given by leaves and inflorescences was subjected to hydrodistillation to obtain the essential oils. These are niche products representing an ideal candidate for the development of natural insecticides for the control and management of mosquito vectors, houseflies and moth pests. After GC-MS analysis highlighting a safe and legal chemical profile (THC in the range 0.004-0.012% dw), the leaf and inflorescence essential oils were investigated for the insecticidal potential against three insect targets: the larvae of Culex quinquefasciatus and Spodoptera littoralis and the adults of Musca domestica. The essential oil from inflorescences, showing (E)-caryophyllene (21.4%), myrcene (11.3%), cannabidiol (CBD, 11.1%), α-pinene (7.8%), terpinolene (7.6%), and α-humulene (7.1%) as the main components, was more effective than leaf oil against these insects, with LD50 values of 65.8 µg/larva on S. littoralis, 122.1 µg/adult on M. domestica, and LC50 of 124.5 µl/l on C. quinquefasciatus larvae. The hemp essential oil moderately inhibited the acetylcholinesterase (AChE), which is a target enzyme in pesticide science. Overall, these results shed light on the future application of fiber hemp crop-residue for the development of effective, eco-friendly and sustainable insecticides.

Tracing heavy metals in 'swine manure - maggot - chicken' production chain.

With the development of large-scale livestock farming, manure pollution has drawn much attention. Conversion by insects is a rapid and cost-effective new method for manure management. Swine manure conversion with maggots (Musca domestica larvae) has developed, and the harvested maggots are often used as animal feed. However, the flow of heavy metals from manure to downstream processes cannot be ignored, and therefore, heavy metal content was measured in untreated raw manure, maggot-treated manure, harvested maggots and maggot-eating chickens (chest muscle and liver) to evaluate potential heavy metal risks. The levels of zinc, copper, chromium, selenium, cadmium and lead had significant differences between untreated raw manure and maggot-treated manure. The concentrations of all detected heavy metals, except for cadmium and selenium, in maggots met the limits established by the feed or feed additive standards of many countries. The bioaccumulation factor (BAF) of heavy metals decreased with the increase of the maggot instar, indicating that heavy metals were discharged from the bodies of maggots with the growth of maggots. Also, the contents of overall heavy metals in chickens fed harvested maggots met the standards for food. In conclusion, regarding heavy metals, it is eco-safe to use maggots in manure management.

Anti-inflammatory activity of cecropin-A2 from Musca domestica.

This study aimed to investigate the anti-inflammatory activity of Musca domestica cecropin-A2 (Mdc-A2) toward Staphylococcus aureus (S. aureus) to learn more about their immunological functions. RAW264.7 cells were transfected with recombinant lentiviruses introduce pLEX-Mdc-A2into the RAW264.7 cell line (RAW-Mdc-A2). The RAW264.7 cell line with empty pLEX (RAW-pLEX) was produced in the same manner as a negative control. Real-time quantitative reverse transcription PCR (RT-PCR) was performed to analyze the mRNA expression of TNF-a, IL-1ß, NFκB-1 and NFκB-2 in S. aureus-stimulated RAW-Mdc-A2 cells and RAW-pLEX cells in untreated cells and cells treated for 3 h, 6 h, 12 h and 24 h. RT-PCR was performed to analyze the mRNA expression of TNF-a, NFκB-1 and NFκB-2 stimulated by Lipoteichoic acid (LTA). Production of TNF-a was detected by enzyme-linked immunosorbent assay (ELISA). Colony counts were used to calculate the number of CFU per mL of cell culture supernatants. The results showed that compared to RAW-pLEX cells, stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of TNF-a transcript variant 1 (TNF-a-tv-1) at 6 h and 12 h and the mRNA expression of TNF-a transcript variant 2 (TNF-a-tv-2) at 3 h, 6 h and 12 h. Compared to RAW-pLEX cells, stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of IL-1ß-T at 3 h, 6 h and 12 h as well as the mRNA expression of IL-1ß at 3 h and 6 h. The expression and production of TNF-a and bacterial burden of cell culture supernatants were significantly down-regulated in RAW-Mdc-A2 cells stimulated by S. aureus, and the expression and production of TNF-a were significantly down-regulated in RAW-Mdc-A2 cells stimulated by LTA. Compared to RAW-pLEX cells, stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of NFκB-1 at 3 h, 6 h and 12 h as well as the mRNA expression of NFκB-2 at 6 h. Additionally, stable transfection of Mdc-A2 in RAW264.7 cells stimulated by LTA significantly down-regulated the mRNA expression of NFκB-1. In conclusion, Mdc-A2 possesses potent anti-inflammatory activity and potent antimicrobial activity. Additionally, Mdc-A2 may interact with LTA and execute strong anti-inflammatory activity by blocking the activation of NF-κB signaling pathways.

Enzymatic single-step preparation and antioxidant activity of hetero-chitooligosaccharides using non-pretreated housefly larvae powder.

A novel chitinolytic enzyme-producing bacterium Chitiniphilus sp. LZ32 was isolated. Non-pretreated Housefly larvae powder (HLP) was used as an adsorbent to purify chitinolytic enzymes. The optimal conditions for hydrolysis of HLP by purifying enzymes chitinolytic enzymes were investigated. HPLC and MALDI-TOF analyses indicated that HLP enzymatic hydrolyzates mainly contain N-acetylglucosamine (GlcNAc) and hetero-chitooligosaccharides (COS) composed of GlcN and GlcNAc. The hetero-chitooligosaccharides (COS) had a degree of polymerization (DP) in the 2-6 range. The maximum production of COS was 158.3µg/mL after 72h of incubation. Maximum pentamer (51.2µg/mL) and hexamer concentrations (36.1µg/mL) were achieved at hydrolysis times of 72 and 84h, respectively. Antioxidant activities of purified COS products (PCOS) from different hydrolysis times were investigated in vitro. PCOS produced by hydrolysis times of 72h (PCOS-72) exhibited the strongest hydroxyl-scavenging ability and reducing power. These results indicate the potential of Chitiniphilus sp. LZ32 for COS production using HLP.